500_AN-INTRO DUCTION TO GENETICS_ANALYSIS_711

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546 Chapter 17 • Genetic Regulation of Cell Number: Normal and Cancer Cells

CHAPTER OVERVIEW normally, what happens when these homeostatic mecha-
nisms go awry because of mutations in the genes govern-
The regulation of somatic-cell number is a splendid ing these processes? The consequences in many organ-
example of homeostasis—the sophisticated mecha- isms, including humans, are dramatic: the accumulation
nisms that maintain an organism’s physiology within of multiple mutations accelerating proliferation and
normal limits. Cell proliferation is shut down and cells blocking cell death in the same somatic cell is the under-
die when too many cells of a given type are present, and lying cause of cancer.
cell proliferation accelerates and cell death is inhibited
when there is a deficiency of cells of a particular type. If This chapter will explore the regulation of cell num-
cell proliferation and cell death are carefully balanced ber and the underlying causes of misregulation that lead
to tumor formation and cancer (Figure 17-1). In its sim-

CHAPTER OVERVIEW Figure

(a) Survival Proliferation (b) Survival Proliferation
cues cues
cues cues
Growth-
Death Growth- Death ×
cues inhibition cues × inhibition
cues
cues

×
×Normal Cell Normal Cell
××
Death Division Death Division
mechanisms checkpoints programs checkpoints
activated
off okay blocked

Survival and Cell death
proliferation

(c) Proliferation (d) Survival Proliferation
Survival cues Death cues cues cues
cues
× Growth- ×
Death inhibition
cues cues Growth-
inhibition
Normal Cell
cues
Death Division Cancer Cell
mechanisms checkpoints

off blocked

Survival but
no cell division

Uncontrolled survival
and proliferation

Figure 17-1 Overview of the regulation of cell numbers in normal and cancer
cells. (a) Proper external cues for normal cell survival and proliferation. (b) Proper external cues
for normal cell death or inhibition of proliferation. (c) Proper external cues for normal cell survival
without proliferation. (d) Self-generated survival and proliferation signals in cancer cells.

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17.1 The balance between cell loss and cell proliferation 547

plest form, the normal regulation of cell numbers can be these proteins, such as the formation of complexes of
regarded as the interplay between mechanisms that con- the proteins with allosteric effectors. Much of genetics,
trol cell proliferation and those that control cell death. indeed much of the biology of a cell, depends on such
Cell proliferation is controlled by the mitotic cell cycle, modulations, in which key proteins are toggled between
whereas cell death is achieved through a mechanism active and inactive states.
called programmed cell death, or apoptosis. In both
cases, sequential biochemical events depend on the suc- 17.1 The balance between cell
cessful occurrence of prior events. In the cell cycle, fail- loss and cell proliferation
safe mechanisms — called checkpoints — prevent the cell
cycle from progressing until prior events have been suc- Adult tissues are composed mostly of differentiated
cessfully completed. Similarly, survival factors block ad- cells — nondividing cells that fulfill specialized physio-
vancement of the death pathways. In multicellular ani- logical roles within the tissue. Typically, there is constant
mals, there is community input into the decision-making low-level turnover of these differentiated cell popula-
process for these pathways. Sensor molecules (receptor tions. That is, occasionally cells die and, when they do,
proteins) interact with chemical signaling proteins pre- they are replaced. Some cell loss is accidental — for ex-
sent in the immediate external milieu of the cell. These ample, from a burn or a wound. Other cell loss is
protein signals and their sensors are wired into the ma- through programmed cell death (apoptosis) of a cell that
chinery of the cell cycle or apoptosis to act as either is abnormal in some way (perhaps dividing too rapidly
accelerators or brakes on one of these two pathways. or replicating an infecting virus).

The genetic basis of cancer is the disruption of these Abnormal cells can do considerable harm. A cell
homeostatic mechanisms through mutations. Cancers that is dividing too rapidly may be on its way to becom-
are malignant tumors, exhibiting uncontrolled growth ing cancerous. A cell infected by a virus may shed virus
and, as they become more advanced, acquiring the abil- particles that will circulate through the body, spreading
ity to metastasize — spread throughout the body. the infection. The loss of such a cell would be negligible,
but its continued presence could be catastrophic to the
When we say that cancer has a “genetic basis,” we organism as a whole. Because we are concerned with
mean something different from the way in which we use regulation of somatic cell numbers (not germ-line cells),
the term in standard genetic analysis. In standard genetic cell loss is not a problem for the organism as long as a
analysis, we are referring to the transmission of distinct cell population can be replenished. Thus, one of the
alleles of a gene from parent to offspring. Although roles of the apoptosis machinery is to survey for cellular
some cancers have a heritable form, cancer occurrence is abnormalities and execute a self-destruct mechanism
sporadic in most cases; that is, a particular type of cancer when they are detected.
occurs in one member of a family but not in any of his
or her relatives. In these cases, the underlying mutations If there were only cell loss and no proliferation, then
have arisen within a lineage of somatic cells in that ultimately we would all run out of cells. We do not do
member, after the time early in development when the so, because most mature cell populations have stem cells
germ line and the soma separated from each other. Over in reserve. Stem cells are undifferentiated cells that can
time, several somatic mutations accumulate within the divide in a way that gives rise to a variety of differenti-
same somatic-cell lineage, altering or knocking out the ated cell types — skin cells or hair follicle cells in the epi-
function of several genes, until finally a cancerous cell is dermis, T-cells, B-cells, or mast cells in the immune sys-
produced. The progeny of this cancerous cell expand tem, and so forth. Each tissue has its own kind of stem
into a clone of cancerous cells — the primary tumor. If cells. When surveillance mechanisms detect an under-
left unchecked, this tumor will continue to grow and representation of a specific cell type, nearby stem cells
eventually invade other tissues and organs in the body. are induced to undergo mitosis. Stem-cell mitoses are
asymmetrical, giving rise to two progeny cells of differ-
Given that most cancers are somatic, not germ-line ent size. The larger of the two cells will be like its par-
mutational events, we cannot study them by standard ent — a stem cell — and the other, smaller cell will differ-
genetic analysis. Rather, we need to use other ap- entiate into a specialized cell of the type that had
proaches to discover the mutational alterations and to become underpopulated in the tissue. Because a stem-
understand how they affect their cellular pathways. We cell division always produces another stem cell, there is
will see that there are many pathways to cancer forma- an essentially limitless supply of replacement cells in re-
tion, but all ultimately lead to the creation of cells that serve for tissue repair.
are insensitive to normal regulation of the cell cycle and
apoptosis. How can a stem cell tell that it needs to divide to re-
plenish a particular cell type? It receives commands from
In our exploration of these topics, we will see again neighboring cells to engage the cell-division machinery.
the recurrent theme that cells modulate the activity of How does the cell-death program begin? Often, the cell
key target proteins by relatively minor modifications in

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548 Chapter 17 • Genetic Regulation of Cell Number: Normal and Cancer Cells

itself senses damage and initiates its own death. In other later section will consider how these molecules are inte-
cases, the cell-death program is initiated by signals from grated into the overall biology of the cell.
neighboring cells that tell the cell to die. These signals are
in the form of proteins or other molecules that attach to Cyclins and cyclin-dependent protein kinases
receptors on the surface of the stem cell or the dying
cell. Eukaryotic cells have elaborate intercellular signaling The engines that drive the cell cycle from one step to
pathways as status indicators of the environment. Some the next are a series of protein complexes called CDK-
signals, called mitogens, stimulate proliferation, whereas cyclin complexes. Ultimately, going to the next step re-
others inhibit it. Death signals can activate apoptosis, quires the activation of genes whose protein products
whereas survival signals block activation. are necessary for the next phase of the cell cycle. This
activation occurs through the turning on of transcription
The cell-proliferation and cell-death machinery factors by the CDK-cyclin complexes (Figure 17-2).
must be wired into the cellular systems that are survey- Consider, for example, the CDK-cyclin complex active
ing the cell’s external environment for the presence of during G1, which takes the cell cycle into the S phase,
signaling molecules. Thus intercellular signaling path- when DNA is synthesized. The G1 CDK-cyclin com-
ways typically consist of several components: the signals plex activates multiple cellular components:
themselves, the receptors that receive the signals, and
the transduction systems that relay the signal to various • A transcription factor that turns on the genes
parts of the cell. The various components of the intercel- encoding DNA polymerase subunits
lular signaling systems are modified — by protein phos-
phorylation, allosteric interactions between proteins and • The genes for the enzymes that produce
small molecules, and interaction between protein sub- deoxyribonucleotides
units — to control the activity of these pathways.
• Other proteins with roles in the duplication of the
17.2 The cell-proliferation chromosomes
machinery of the cell cycle
• The gene for one of the subunits of the next required
CDK-cyclin complex

Before we consider the regulatory systems that control Cdk1– cyclin B G0
cell proliferation, it is first important to understand Cdk1– cyclin A
the basic events in the cell cycle. The cell cycle has G1 Cdk4–cyclin D
four main parts: M phase — mitosis, the nuclear division G2 M Cdk6–cyclin D
process described in detail in Chapter 3 — and three
parts that are components of interphase — G1, the S
period between the end of mitosis and the start of DNA
replication; S, the period of DNA synthesis; and G2, the
period that follows DNA replication and precedes the
mitotic prophase. S, G2, and M phases are normally
fixed in duration. G1, however, can be quite variable be-
cause the cell cycle can enter an optional G0 resting
phase.

M G1 Cdk2– cyclin A
G2
Cdk2–cyclin E
S G0
Figure 17-2 Variations in CDK-cyclin activities throughout the
Some cells, such as the cells of the newly fertilized em- cell cycle of a mammalian cell. The widths of the bands
bryo, are rapidly dividing and have no G0 phase. At the indicate the relative kinase activities of the various CDK-cyclin
other extreme, differentiated cells are in G0 for the re- complexes. Note that different CDKs and cyclins can bind to
mainder of their natural lives. Stem cells fluctuate be- one another to form different complexes, increasing the array
tween G0 and the cell-division cycle. This section will of combinations of CDK-cyclin complexes that can form in the
consider the molecular signals that drive the cell cycle. A course of the cell cycle. [After H. Lodish, A. Berk, S. L. Zipursky,

P. Matsudaira, D. Baltimore, and J. Darnell, Molecular Cell Biology,
4th ed. Copyright 2000 by W. H. Freeman and Company.]

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17.2 The cell-proliferation machinery of the cell cycle 549

As different CDK-cyclin complexes appear and disap- ferent cyclins are present at different phases of the cell
pear, the proteins necessary for carrying out the different cycle (see Figure 17-2), each phase is characterized by
phases of the cell-division cycle are transcribed and the phosphorylation of different target proteins.
translated like a well-choreographed ballet. The chore-
ography requires that each active CDK-cyclin complex As the cell cycle proceeds from one stage to the
be “on stage” only for a limited part of the cell cycle. If next, it is just as important that the currently active
an active CDK-cyclin complex is present at the wrong CDK-cyclin complexes be deactivated as that new ones
time, it will cause wholly inappropriate sets of genes to be activated. Both the cyclin mRNA and the cyclin itself
be transcribed or shut off. are highly unstable, and so the existing pool of cyclin
and its mRNA will be quickly eliminated when the tran-
The CDK-cyclin protein complexes are composed scription factor for the cylin’s gene is inactivated. The
of two subunits: a cyclin and a cyclin-dependent protein phosphorylation events are likewise transient and re-
kinase (abbreviated CDK). versible. When a CDK-cyclin complex disappears, its
phosphorylated target proteins are rapidly dephosphory-
• Cyclins. Every eukaryote has a family of structurally lated by other enzymes always present in the cell.
and functionally related cyclin proteins. Cyclins are so
named because each is present in the cell only during CDK targets
one or more defined segments of the cell cycle. The
appearance of a specific cyclin is the result of the How does the phosphorylation of some target proteins
activity of the preceding CDK-cyclin complex, which control the cell cycle? Phosphorylation initiates a chain
leads to the activation of a transcription factor for the of events that culminates in the activation of certain
new cyclin. transcription factors. These transcription factors promote
the transcription of certain genes whose products are re-
• CDKs. Cyclin-dependent protein kinases are another quired for the next stage of the cell cycle.
family of structurally and functionally related
proteins. Kinases are enzymes that add phosphate Much of our knowledge of the cell cycle comes from
groups to target substrates; for protein kinases, such as genetic studies of yeast (see the Model Organism box,
CDKs, the substrates are the side groups of specific Yeast) or from biochemical studies of cultured mam-
amino acids on specific proteins. Each CDK catalyzes malian cells. Indeed, work on the cell cycle was recog-
the phosphorylation of specific serine and threonine nized with the Nobel Prize for Medicine or Physiology in
residues belonging to one or more unique target 2001. A well-understood example of the path connecting
proteins. Becoming phosphorylated changes the the appearance of cyclin to gene transcription is the Rb-
activity of the target protein. E2F pathway in mammalian cells. Rb is the target protein
of a CDK-cyclin complex called Cdk2 – cyclin A, and E2F
CDKs are named “cyclin dependent” because each is the transcription factor that Rb regulates (Figure 17-4).
CDK must be attached to a cyclin to function. The cy- From late M phase through the middle of G1, the Rb and
clin tethers the target protein so that the CDK can E2F proteins are combined in a protein complex that
phosphorylate it (Figure 17-3). The CDKs are present does not promote transcription. In late G1, however, the
throughout the cell cycle, and so which complex is ac- active Cdk2 – cyclin A complex is produced, which phos-
tive is a function of which cyclin is present. Because dif- phorylates the Rb protein. This phosphorylation produces

Target PP
protein

Cyclin Cyclin-CDK Target protein Phosphorylation Figure 17-3 Phosphorylation of
CDK binding binding of target protein target proteins by the CDK-cyclin
complex. The target protein binds
Active cyclin-CDK to the cyclin part of an active
complex CDK-cyclin complex, placing the
target phosphorylation sites close
to the active site of CDK. Once
phosphorylated, the target protein
is no longer able to bind to cyclin
and is released from the complex.

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550 Chapter 17 • Genetic Regulation of Cell Number: Normal and Cancer Cells

Inactive Late G1 Activate that prevents progression to the next stage until the pre-
complex Cdk2 cyclin A transcription. ceding stage has been successfully fulfilled.
Active
Rb How do checkpoints act as brakes on the cell cycle?
E2F E2F They activate proteins that can inhibit the protein ki-
nase activity of one of the CDK-cyclin complexes. In
Genome this way, the cell cycle can be held in check until the
checkpoint monitoring mechanisms give a “green light,”
Phosphorylate Rb. indicating that the cell is properly prepared to proceed
to the next phase of the cycle.
Bind E2F. Inactive Enzymes for
Rb DNA synthesis One example of how this checkpoint system
Active operates begins with damaged DNA (Figure 17-5).
Rb P When DNA is damaged during G1 (for example, by X-
irradiation), CDK-cyclin complexes stop phosphorylat-
Dephosphorylated G1 S phase ing their target proteins. A protein called p53 recognizes
late M phase certain kinds of DNA mismatches and then proceeds to
activate another protein, p21. When its levels are high,
Figure 17-4 Contributions of the Rb and E2F proteins in the p21 binds to the CDK-cyclin complex and inhibits its
regulation of the G1-to-S-phase transition in a mammalian cell. protein kinase activity. Thus CDK cannot phosphorylate
its target proteins, and the cell cycle is unable to
[After H. Lodish, D. Baltimore, A. Berk, S. L. Zipursky, P. Matsudaira, progress from G1 to S. The inhibiting processes are re-
and J. Darnell, Molecular Cell Biology, 3d ed. Copyright 1995 by versed by a drop in p53 levels after DNA repair is com-
Scientific American Books.] plete. In the absence of p53, CDK-cyclin protein kinase
activity is no longer inhibited, and the G1-to-S check-
a change in the shape of Rb so that it can no longer bind point block is removed. A checkpoint is thus a regula-
to the E2F protein. The unattached E2F protein is then tory protein, such as p53, that can act as a brake on the
free to promote the transcription of certain genes that en- protein kinase activity of the CDK-cyclin complexes.
code the enzymes vital for DNA synthesis and other as-
pects of chromosome replication. It also activates gene MESSAGE Fail-safe systems called checkpoints ensure
expression of the next cyclin to be expressed, cyclin B. that the cell cycle does not progress until the cell has
With cyclin B’s appearance, the next phase of the cell completed all prior events necessary to assure its survival
cycle — S phase — can proceed. through the next steps.

Rb and E2F are in fact representatives of two fami- E2F
lies of related proteins. In mammals, different CDK-
cyclin complexes selectively phosphorylate different Enzymes for
proteins of the Rb family, each of which in turn releases DNA synthesis
a specific E2F family member to which it is bound. The Rb
different E2F transcription factors then promote the
transcription of different genes that execute different P
aspects of the cell cycle.

MESSAGE Sequential activation of different CDK-cyclin Rb
complexes ultimately controls progression of the cell cycle. E2F

Checkpoints as brakes DNA p53 p21

on cell-cycle progression damage

Proper choreography of the cell cycle’s events is crucial to G0 phase Cdk2 cyclin A S phase
the production of progeny cells with the proper number G1 phase
of intact chromosomes. For example, attempting to con-
dense chromosomes and move them to metaphase (re- Figure 17-5 Inhibitory control of the progression of the cell
call the stages of mitosis, Figure 3-28) before their DNA cycle in mammals. In the presence of damaged DNA, p53
molecules have completed replication could lead to the protein is induced, which in turn induces p21 protein.
production of chromosome fragments or other kinds of Elevated levels of p21 inhibit the protein kinase activity of the
damage. Attempting to separate the chromosomes to op- Cdk2 – cyclin A complex. E2F remains complexed with Rb,
posite poles at anaphase before all kinetochores are at- and the cell does not progress to S phase. After the damaged
tached to spindle fibers could lead to nondisjunction and DNA has been repaired, p53 and then p21 levels drop.
aneuploidy of the progeny cells. These potentially disas- The inhibition of the Cdk2-cyclin protein kinase activity is
trous problems are prevented by having a series of relieved, permitting the cell to enter S phase. [After C. J. Sherr
checkpoints — circuitry attached to the cell-cycle system
and J. M. Roberts, Genes and Development 9, 1995, 1150.]

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17.2 The cell-proliferation machinery of the cell cycle 551

MODEL ORGANISM Yeast Scanning electron micrograph of S. cerevisiae cells
at different points in the cell cycle, as indicated by
Yeast As a Model for the Cell Cycle different bud sizes. [Courtesy of E. Schachtbach and
I. Herskowitz.]
Work initiated by Leland Hartwell and his associates on
the cell-cycle genetics of the budding yeast Saccha-
romyces cerevisiae revealed a large array of genetic
functions that maintain the proper cell cycle. These
functions were identified as a specific subset of
temperature-sensitive (ts) mutations called cdc (cell
division cycle) mutations. When grown at low tempera-
ture, yeasts with these cdc mutations grew normally.
When shifted to higher, restrictive temperatures,
the yeasts stopped growing. What made these cdc
mutations novel among the more general class of ts mu-
tations was that a particular cdc mutant would stop
growing at a specific time in the cell cycle, and all the
yeast cells would look alike. Consider some examples of
S. cerevisiae, a yeast that divides through budding, a
process in which a mother cell develops a small out-
pocketing, a “bud.” The bud grows and mitosis takes

Nuclear migration Spindle formation place in such a way that one spindle pole is in the
mother cell and the other in the bud. The bud contin-
G2 Chromosome ues to grow until it is as big as the mother cell, and the
S mother cell and bud then separate into two daughter
DNA M segregation; cells. Any run-of-the-mill ts mutations in S. cerevisiae,
replication nuclear when shifted to restrictive temperature, stop growing at
various times in the cycle of bud formation and growth.
division In contrast, after a shift to restrictive temperature, one
S. cerevisiae cdc mutation produces yeast cells with only
Bud G1 tiny buds, whereas another produces yeast cells exclu-
emergence Growth sively with larger buds, half the size of the mother cell.
Such different Cdc phenotypes indicate different de-
Spindle pole Cytokinesis fects in the machinery required to execute specific
body events in the cell cycle.
duplication START
With the sequencing of the S. cerevisiae genome
Cell cycle of S. cerevisiae. [From H. Lodish, D. Baltimore, A. Berk, now complete, we are able to identify the entire array
S. L. Zipursky, P. Matsudaira, and J. Darnell, Molecular Cell Biology, of proteins of the cyclin and CDK families (22 and 5
3d ed. Copyright 1995 by Scientific American Books.] proteins, respectively). Their genes are now being sys-
tematically mutagenized and genetically characterized
to reveal how each contributes to the cell cycle.

It is necessary not only to release the cell-cycle kinases that phosphorylate the appropriate CDK-cyclin
“brake” but also to engage the “transmission” and the complex. The activated complex is now able to phos-
“engine” to advance the cell cycle. In regard to the phorylate its target proteins. Just as an automobile
CDK-cyclin complexes, it is not enough to remove any doesn’t perform unless it has a functional engine,
inhibition and add the correct cyclin; the complexes transmission, steering, acceleration, and braking sys-
must also be activated by phosphorylation. Once the tem, the cell-cycle machinery is ineffective unless all
brake has been released, independent signals from the necessary components for governing its progres-
within or outside the cell induce a cascade of protein sion are present.

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552 Chapter 17 • Genetic Regulation of Cell Number: Normal and Cancer Cells

17.3 The machinery Normal cell Intact
of programmed cell death Cell death mitochondrion

In multicellular organisms, systems have evolved to Intact
eliminate damaged (and hence potentially harmful) cells chromosomal
by a self-destruct and disposal mechanism called pro- DNA
grammed cell death or apoptosis. This self-destruct
mechanism can be activated under many different cir- Nucleus
cumstances, such as cells that are no longer needed for
development. In all cases, however, the events in apopto- Apoptosis initiated
sis seem to be the same (Figure 17-6). First, the DNA of
the chromosomes is fragmented, organelle structure is Mitochondrion
disrupted, and the cell loses its normal shape and be- broken open
comes spherical. Then, the cell breaks up into small frag-
ments called apoptotic bodies, which are phagocytosed Highly
(literally, eaten up) by motile scavenger cells. fragmented
DNA
The engines of self-destruction are a series of
enzymes called caspases, which is short for cysteine- Cell rounded up
containing aspartate-specific proteases (see the Model Cell breaks apart into
Organism box, Caenorhabditis elegans). Proteases are en- several fragments
zymes that cleave other proteins. The general term for
such cleavage of proteins is proteolysis. Each caspase is a Scavenger
protease rich in cysteines: when activated, it cleaves cer- cell
tain target proteins at a specific aspartate. These target
proteins initiate fragmentation of DNA, disruption of Removal of dead Apoptotic
organelles, and other events that characterize apoptosis. cell fragments body

Every multicellular animal has a family of caspase Figure 17-6 Sequence of events in apoptosis. First, the
proteins, related to one another by polypeptide se- membranes of organelles such as mitochondria are disrupted
quence; in humans, for example, 14 caspases have been and their contents leak into the cytoplasm, chromosomal DNA
identified so far. In normal cells, each caspase is in an in- breaks into small pieces, and the cell loses its normal shape.
active state, called the zymogen form. In general, a zy- Then the cell breaks apart into small fragments, which are
mogen is an inactive precursor form of an enzyme hav- disposed of by phagocytotic scavenger cells.
ing a longer polypeptide chain than that of the final
active enzyme. To turn the zymogen form of an enzyme How do the executioner caspases carry out the sen-
into the active form, a part of the polypeptide is re- tence of death? In addition to activating other caspases,
moved by enzyme cleavage. executioner caspases enzymatically cleave other target
proteins in the damaged cell (Figure 17-7). One target is
There are two classes of caspases: initiators and exe- a “sequestering” protein that forms a complex with a
cutioners. The initiator caspases are cleaved in response DNA endonuclease, thereby holding (sequestering) the
to activation signals coming from other classes of pro- endonuclease in the cytoplasm. When the sequestering
teins. They in turn cleave one of the executioner cas- protein has been cleaved, the endonuclease is free to
pases, which in turn cleaves another, and so forth, until
all executioner caspases are active. Unlike the cell cycle,
which as its name implies can be activated repeatedly, a
given cell can only die once. Thus, instead of being
linked to one another in a circular pathway, as in the cell
cycle, the events of programmed cell death need only
proceed unidirectionally. The logic of the programmed
steps in each of these systems fits well with its need to
proceed cyclically or reach a final conclusion.

MESSAGE Programmed cell death is mediated by a
sequential cascade of proteolysis events that activate
enzymes that destroy several key targeted cellular components.

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17.4 Extracellular signals 553

Inactive executioner caspase (zymogen) blocking the release of cytochrome c from mitochondria
H2N COOH (possibly by making it more difficult for mitochondria to
burst) and binding to Apaf, preventing its interaction
Cleavage by another caspase with the initiator caspase.

Cleaved fragments Large subunit Small subunit 17.4 Extracellular signals

Active Aggregation of executioner A cell in a multicellular organism continually assesses its
executioner caspase subunits own internal status to determine whether conditions are
appropriate to initiate the cell cycle or commit cellular
caspase Proteolysis of suicide. Ultimately, however, the fate of a cell must be
target proteins subservient to the needs of the population of cells of
which it is a member (such as the entire early embryo, a
Inactivation of Activation of Other targets tissue, or a body part such as a limb or an organ). Hence,
DNA endonuclease- actin-cleaving when cell numbers are depleted, cells in concert deter-
mine that their normal neighbors are missing and pass
sequestering protein an extracellular signal to the stem cells that they need to
protein divide. To consider how these signals are wired into the
proliferation and apoptosis pathways, we first need to
DNA Loss of normal Breakdown Fragmentation look at the basic circuitry that mediates cross talk be-
fragmentation tween cells, often called cell – cell communication.
cell shape of organelles of cell
Mechanisms for cell – cell communication
Figure 17-7 Role of executioner caspases in apoptosis.
Cleavage of the zymogen precursor leads to the formation of Many kinds of signals are transmitted between cells to
enzymatically active executioner caspases. As the executioner coordinate virtually all aspects of a multicellular organ-
caspases cleave target proteins, the various cellular breakdown ism’s development and physiology. All systems for inter-
events take place, leading to cell death and removal. cellular communication have multiple components. To
initiate communication, a signaling cell secretes a mole-
enter the nucleus and chop up the cell’s DNA. Another cule called a ligand (Figure 17-8). Some ligands, called
target is a protein that, when cut by the caspases, cleaves hormones, are long-range endocrine signals that are
actin, a major component of the cytoskeleton. The dis-
ruption of actin filaments leads to the loss of normal cell Blood vessel
shape. In similar fashion, caspase-activated proteases are
thought to initiate all other events of apoptosis. Hormone secretion Distant target cells
into blood by endocrine gland
What triggers caspase activation? It has been known
for several years that, in some manner, many forms of cel- (a) Endocrine signaling
lular damage trigger leakage of material from mitochon-
dria and that this leakage somehow induces the apoptotic Secretory cell Adjacent target cell
response. Indeed, it now appears that one of the switches
for apoptosis activation is cytochrome c, a mitochondrial ()
protein that normally takes part in cell respiration. Cy-
tochrome c that has leaked into the cytoplasm is thought Figure 17-8 Modes of intercellular signaling. (a) Endocrine
to bind to another protein called Apaf (apoptotic
protease-activating factor). The cytochrome c-Apaf com- signals enter the circulatory system and can be received by
plex then binds to the initiator caspase and activates it.
distant target cells. (b) Paracrine signals act locally and are
Because cell death is irreversible, it is crucial that
healthy cells not be destroyed by apoptosis. The need to received by nearby target cells. [From H. Lodish, D. Baltimore,
preserve healthy cells has probably been the compelling
factor in the evolution of systems to make sure that the A. Berk, S. L. Zipursky, P. Matsudaira, and J. Darnell, Molecular Cell
apoptosis pathway remains “off” under normal condi-
tions. Proteins such as Bcl-2 and Bcl-x in mammals act Biology, 3d ed. Copyright 1995 by Scientific American Books.]
as roadblocks to prevent apoptosis from being triggered.
Among the possible actions of these Bcl proteins is

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554 Chapter 17 • Genetic Regulation of Cell Number: Normal and Cancer Cells

MODEL ORGANISM Caenorhabditis elegans

The Nematode Caenorhabditis elegans As a cell death (see the illustration below). Divisions of this
Model for Programmed Cell Death type are necessary for the progeny cell to fulfill its
proper developmental role.
In the past 15 years, genetic studies by Robert Horvitz
and his associates of the nematode (roundworm) A set of mutations has been identified in the
Caenorhabditis elegans have advanced our understand- worm that block programmed cell death; the progeny
ing of programmed cell death. Researchers have cell that would otherwise die. Some of these mutations
mapped the entire series of somatic-cell divisions that knock out genes that encode caspases — an example is
produce the 1000 or so cells of the adult worm. For ced-3 (cell-death gene number 3) — clearly implicating
some of the embryonic and larval cell divisions, partic- these caspases in the apoptosis process. Other key
ularly those that will contribute to the worm’s ner- players in this process are being uncovered through
vous system, a progenitor cell gives rise to two prog- the analysis of other genes with mutant cell-death
eny cells, one of which then undergoes programmed phenotypes in worms and other experimental systems.

P3,P4,P5,

Egg W P1 P2 P6,P7,P8,P9 P10 P11 P12

L1 (first larval stage) L1

×
P12.pa×
P12.app
P12.apa
P12.aap

P12.aaaa

P11.app
P11.apa

P11.aaaa

P10.app
P10.apa

P10.aaap
Pn.p;vhP10.aaaa

Pn.app
Pn.apa

Pn.aaap
P2.p;vh Pn.aaaa

P2.app
P2.apa
P2.aap

P2.aaap
P2.aaaa
P1.p;vh
P1.app
P1.apa
P1.aaap
P1.aaaa
W.pp
W.pa
W.aap
W.aaa
L2 (second larval stage) L2 ×
××

L3 (third larval stage) L3

L4 (fourth larval stage) L4 P11.ppp
P11.ppap
P11.ppaa

P11.papp
P11.papa
P11.paap
P11.paaa
P11.aapp
P11.aapa

P10.ppppp
P10.ppppa
P10.pppa
P10.ppap
P10.ppaa
P10.papp
P10.papa
P10.paap
P10.paaa
P10.aapp
P10.aapa

Pn.aapp
Pn.aapa

Adult Adult

The life cycle of the nematode Examples of programmed cell death in the development of
C. elegans. C. elegans. A symbolic representation of the cell lineages of
13 cells (the W cell, the P1 cell, and so forth) produced during
embryogenesis. The vertical axis is developmental time,
beginning with the hatching of the egg into the first larval stage
(L1). In each lineage, a vertical blue bar connects the various
division events (horizontal blue bars). The names of the final
cells are shown, such as W.aaa or P1.apa. In several cases, a
cell division produces one viable cell and one cell that
undergoes apoptosis. A cell that undergoes programmed cell
death is indicated with a blue ϫ at the end of a branch of a
lineage. In homozygotes for mutations such as ced-3, these cell
deaths do not occur.

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17.4 Extracellular signals 555

released from endocrine organs into the circulatory sys- Transmembrane
tem, which transmits them throughout the body. Hor- domain
mones can act as master control switches for many dif-
ferent tissues, which can then respond in a coordinated Exterior
fashion. Other secreted ligands act as paracrine signals; Cytoplasm
that is, they do not enter the circulatory system but act
only locally, in some cases only on adjacent cells. Most (a) Extracellular domain
(but not all) endocrine signals are small molecules, such Transmembrane domain
as the mammalian steroid hormones androgen or estro- Exterior
gen that are responsible for male or female sex- Cytoplasmic domain
specific phenotypes. In contrast, most paracrine signals Cytoplasm
are proteins. Because the signals initiating cell prolifera- Kinase
tion and cell death emanate from nearby, this discussion active
will focus on paracrine signaling through protein ligands. site

Protein ligands and (b)

transmembrane receptors Figure 17-9 Examples of transmembrane receptors.
(a) A receptor that passes through the cell membrane seven
Protein ligands act as signals by binding to transmem- times. (b) Receptor tyrosine kinase, which has a single
brane receptors, proteins that are embedded in the transmembrane domain. The extracellular domain binds to
plasma membrane at the surface of the cell. These ligand. The active site of the tyrosine kinase is in the
ligand – receptor complexes release chemical signals into cytoplasmic domain. [After H. Lodish, D. Baltimore, A. Berk,
the cytoplasm just inside the plasma membrane. Such
signals are passed through a series of intermediary mole- S. L. Zipursky, P. Matsudaira, and J. Darnell, Molecular Cell Biology,
cules until they finally alter the structure of transcription 3d ed. Copyright 1995 by Scientific American Books.]
factors in the nucleus, activating the transcription of
some genes and the repression of others. Protein ligands released in a tissue may act as signals
that regulate the cell-division cycle or apoptosis. The
Transmembrane receptors have one part (the extracel- general theme is that there are pathways for controlling
lular domain) outside of the cell, a middle part that passes cell proliferation and self-destruction and that activation
once or several times through the plasma membrane, and of these pathways requires the correct array of positive
another part (the cytoplasmic domain) inside the cell (Fig- inputs and the absence of negative, or inhibitory, inputs.
ure 17-9). The ligand binds to the extracellular domain of
the receptor. The binding of the ligand changes the struc- THE CELL CYCLE: POSITIVE EXTRACELLULAR CON-
ture of the receptor in some way that initiates signaling ac- TROLS Cell division is promoted by the action of mito-
tivity. Many polypeptide ligands are dimers and can simul- gens, protein ligands usually released from a paracrine
taneously bind two receptor monomers. This simultaneous (nearby) source. Mitogens are also called growth factors.
binding brings the cytoplasmic domains of the two recep- Many mitogens activate a receptor tyrosine kinase and
tor subunits into close proximity and activates their signal- initiate a signal-transduction pathway that eventually
ing activity. Some receptors for polypeptide ligands are re- leads to the expression of the G1 cyclin D genes. Mito-
ceptor tyrosine kinases (RTKs, see Figure 17-9b). Their gens include epidermal growth factor (EGF), which sig-
cytoplasmic domains, when activated, have the ability to nals by binding to a receptor tyrosine kinase called the
phosphorylate certain tyrosine residues on target proteins. EGF receptor (EGFR).
Other receptors are serine/threonine kinases. Still others
have no enzymatic activity but, when bound to ligands, THE CELL CYCLE: NEGATIVE EXTRACELLULAR CON-
they undergo conformational changes that in turn cause TROLS Certain secreted protein ligands are known to
conformational changes in (and activation of) proteins inhibit cell division in completely intact tissue, when
bound to their cytoplasmic domains. growth is not necessary. One example is TGF-␤, a ligand

Sequentially, modification of the conformation of
one protein leads to modification of the conformation of
another, ending with the modification of transcriptional
activators and repressors that alter the activities of many
genes in the target cell. Each altered protein acts as one
signal in a chain. The chain of signals leading from the
ligand – receptor complex to the altered gene activity is
called a signal-transduction cascade.

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556 Chapter 17 • Genetic Regulation of Cell Number: Normal and Cancer Cells

that is thought to be secreted in a variety of tissues un- Cytoplasm Cell membrane
der growth-inhibitory conditions. The TGF-␤ ligand of neighboring cell
binds to the TGF-␤ receptor and activates the receptor’s Initiator FasL
serine/threonine kinase activity. The ensuing signal- caspase Fas receptor
transduction cascade eventually blocks the phosphoryla- zymogen Cell membrane
tion and inactivation of the Rb protein. Recall, from ear-
lier in the chapter, the role that Rb plays in regulating Apaf Activated
the cell cycle by preventing activation of the E2F tran- initiator
scription factor. Blocking Rb inactivation thus keeps E2F caspase
expression turned off. Consequently, the ingredients
necessary for DNA replication are not present and the Caspase
cell cycle will progress no farther. activation
cascade
APOPTOSIS: POSITIVE EXTRACELLULAR CONTROLS
Often, the command for self-destruction comes from a Apoptosis
neighboring cell. For example, within the immune sys-
tem, only a small percentage of B cells and T cells ma- Figure 17-10 Positive extracellular control of apoptosis.
ture to make functional antibody or receptor protein, re- Receptor – ligand interaction leads to activation of a molecule
spectively. The nonfunctional immature B cells and T (Apaf), which in turn causes proteolysis and activation of the
cells must be eliminated by induced self-destruction; initiator caspase. A series of caspases are then proteolysed
otherwise, the numbers of these unnecessary cells would and activated in turn, ultimately leading to apoptosis of the
clog the immune system. The self-destruction signal is cell. [After S. Nagata, Cell 88, 1997, 357.]
activated through the Fas system (Figure 17-10) and
other death receptors. A membrane-bound protein Signal-transduction cascades
called FasL (Fas ligand) protrudes from the surface of
the cell; its presence is the signal to an adjacent cell to Perhaps the best understood of the receptors for
self-destruct. The FasL protein binds to transmembrane polypeptide ligands are the receptor tyrosine kinases
Fas receptors on the surface of the adjacent cell. This (Figure 17-11). A receptor tyrosine kinase, or RTK, is a
binding induces three receptors to join to form a trimer single unit, or monomer, “floating” in the plasma mem-
bound to the single FasL protein. The trimerization of brane. When ligand binds to two neighboring RTK units,
their cytoplasmic domains in turn indirectly activates a the two monomers bind to form a dimer. Thus a
molecule such as Apaf. As described earlier in this chap- ligand – RTK complex consists of a single ligand mole-
ter, Apaf activates an initiator caspase, which sets off the cule bound to a dimer of two RTK molecules. RTK
cascade leading to cell death. dimerization activates the protein kinase activity of the
receptor’s cytoplasmic domain. The first phosphoryla-
APOPTOSIS: NEGATIVE EXTRACELLULAR CONTROLS tion targets of the kinase are several tyrosines in the cy-
It appears that self-destruction may in some ways be a toplasmic domain of the RTK itself; this process is called
default state and that cells receive constant signals in- autophosphorylation, because the kinase acts on itself.
structing them to stay alive. The advantage of such a The phosphorylated RTK is conformationally changed
system may be that it is able to eliminate abnormal cells so that its tyrosine kinase active site phosphorylates
rapidly to ward off the dangers that they pose to the or- other target proteins.
ganism as a whole. The signals that can block activation
of the apoptosis pathway are secreted factors called sur- Quite often, the next step in propagating the signal
vival factors. Some survival factors do double duty as is to activate a G-protein. A G-protein cycles between
mitogens, such as EGF. The action of EGF and the being bound by GDP in the inactive state and being
EGFR on survival is largely mediated by a different bound by GTP in the activated state. The autophosphor-
signal-transduction cascade from the usual receptor ylated RTK activates a protein that binds to the inactive
tyrosine kinase pathway. This alternative cascade eventu-
ally leads directly or indirectly to activation of proteins
of the Bcl-2 family.

MESSAGE Intercellular signaling systems communicate
instructions to activate or to arrest the cell cycle and to
initiate or postpone self-destruction.

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17.4 Extracellular signals 557

Ligand Ligand-binding
site
Extracellular Autophosphorylation
domain Ligand on tyrosines
binding
Exterior Kinase Receptor ATP P P Phosphotyrosines
Transmembrane active dimerization ADP P P
domain P
Cytoplasm site ATP

Cytoplasmic ADP
domain P

Figure 17-11 Consequences of ligand binding on RTK activity and the initiation of Target PPP
the signal-transduction cascade. Activated receptor tyrosine kinase directly protein
phosphorylates target proteins, which induces their signal-transduction activities. Tyrosine
phosphorylation
[Adapted from H. Lodish, D. Baltimore, A. Berk, S. L. Zipursky, P. Matsudaira, and by dimerized RTK
J. Darnell, Molecular Cell Biology, 3d ed. Copyright 1995 by Scientific American Books.]

G-protein, changing its conformation so that it binds The activated GTP-bound G-protein then binds
to a molecule of GTP and becomes activated (Figure to a cytoplasmic protein kinase, in turn changing its
17-12). The specific G-protein called Ras is especially conformation and activating its kinase activity. This
important in carcinogenesis, as will be explained later. protein kinase then phosphorylates other proteins, in-
cluding other protein kinases. (In the example in Figure
Inactive Ras 17-13, the protein kinases farther down the cascade
Pi GDP are called Raf, MEK, and MAP kinase.) The targets of
some of these protein kinases are transcriptional activa-
Intrinsic Active Ras GTP tors and repressors. The phosphorylated transcription
GTPase activity GTP factors alter their conformations, leading to the tran-
Sos interaction scription of some genes and the repression of others (see
stimulates Figure 17-13).
GDP– GTP
exchange The steps in ligand – receptor binding and in signal-
ing within the cell depend on conformational changes.
GDP For example, the conformational changes caused by the
binding of ligands to receptors activate the signaling
Activates downstream pathways. Likewise, conformational changes in protein
serine/threonine kinase kinases enable them to phosphorylate specific amino
acids on specific proteins, and other proteins undergo
Figure 17-12 Ras as an example of the G-protein activity cycle. conformational changes when they bind to GTP. Not
When bound to GDP, Ras does not signal. Interaction with a only do these conformational changes permit rapid re-
second protein, Sos, promotes the exchange of GDP for GTP. sponse to an initial signal, but they are readily reversible,
The active Ras – GTP complex interacts with a cytoplasmic enabling signals to be shut down rapidly. Thus the com-
serine/threonine kinase, which transmits the signal to the next ponents of the signaling system can be recycled so that
step in the signal-transduction pathway. When Ras – GTP is they are ready to receive further signals.
released from Sos, it hydrolyzes GTP to GDP and reassumes
the inactive Ras – GDP state. [After J. D. Watson, M. Gilman, An integrated view

J. Witkowski, and M. Zoller, Recombinant DNA, 2d ed. Copyright 1992 of the control of cell numbers
by James D. Watson, Michael Gilman, Jan Witkowski, and Mark Zoller.]
We are learning that there is intimate coupling be-
tween cell-proliferation and cell-death decisions. We
saw the example of the EGF – EGFR signaling system,
which acts both as a mitogenic stimulus and as a sur-
vival factor, inhibiting apoptosis. Similarly, it turns out
that active p53 protein, which is a key intermediary in

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558 Chapter 17 • Genetic Regulation of Cell Number: Normal and Cancer Cells

Ligand from signaling cell 17.5 Cancer: the genetics of
aberrant cell number regulation
Receptor tyrosine kinase (RTK)
A basic article of faith in genetic analysis is that we learn
GDP GTP a great deal about biology, both normal and diseased, by
studying the properties of mutations that disrupt normal
RasиGDP RasиGTP processes. It has certainly been true in regard to cancer. It
(inactive) (active) has become clear that virtually all cancers of somatic cells
arise owing to a series of special mutations that accumu-
Pi late in a cell. Some of these mutations alter the activity of
Raf (serine/threonine kinase) a gene; others simply eliminate the gene’s activity. Cancer-
promoting mutations fall into a few major categories:
MEK (serine/threonine and tyrosine kinase) those that increase the ability of a cell to proliferate, those
that decrease the susceptibility of a cell to apoptosis, or
MAP kinase (serine/threonine kinase) those that increase the general mutation rate of the cell or
its longevity, so that all mutations, including those that
Nuclear transcription factors encourage proliferation or apoptosis, are more likely to
occur. Improvements in diagnosis and treatment of can-
Transcriptional activation cers have been dramatic, but, though some battles have
or repression of target genes been won, we are a very long way from declaring victory
in the war against cancer. The genetic and genomic analy-
Cell proliferation sis of cancer offers important new avenues to explore.

Figure 17-13 One pathway for RTK signaling. Raf, MEK, and How cancer cells differ from normal cells
MAP kinase are three cytoplasmic protein kinases that are
sequentially activated in the signal-transduction cascade. Malignant tumors, or cancers, are aggregates of cells, all
descended from an initial aberrant founder cell. In other
[After H. Lodish, D. Baltimore, A. Berk, S. L. Zipursky, P. Matsudaira, words, the malignant cells are all members of a single
and J. Darnell, Molecular Cell Biology, 3d ed. Copyright 1995 by clone. This is true even in advanced cancers having mul-
Scientific American Books.] tiple tumors at many sites in the body. Cancer cells typi-
cally differ from their normal neighbors by a host of
the S to G2 checkpoint, promotes apoptosis by attack- phenotypic characters, such as rapid division rate, ability
ing the integrity of the mitochondrion as well as by in- to invade new cellular territories, high metabolic rate,
hibiting the expression of genes whose products con- and abnormal shape. For example, when cells from nor-
tribute to survival. Why does it make sense that the cell mal epithelial cell sheets are placed in cell culture, they
cycle and apoptosis are linked? Simply put, the condi- can grow only when anchored to the culture dish itself.
tions that encourage proliferation also demand that the In addition, normal epithelial cells in culture divide
cell survives to duplicate itself. On the other hand, a only until they form a continuous monolayer (Figure
cell with damage to its DNA poses a danger to the or- 17-14a). At that point, they somehow recognize that
ganism itself. Thus, it is in the organism’s interest to they have formed a single epithelial sheet and stop di-
have evolved mechanisms that not only block cell-cycle viding. In contrast, malignant cells derived from epithe-
progression but also remove a mutated cell that might lial tissue continue to proliferate, piling up on one
be on its way to becoming abnormal. another (Figure 17-14b).

Clearly, the factors regulating normal cellular physi-
ology have been altered. What, then, is the underlying
cause of cancer? Many different cell types can be con-
verted into a malignant state. Is there a common theme?
or does each arise in a quite different way? We can think
about cancer in a general way: as being due to the accu-
mulation of multiple mutations in a single cell that
cause it to proliferate out of control. Some of those mu-
tations may be transmitted from the parents through the
germ line. But most arise de novo in the somatic-cell
lineage of a particular cell.

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17.5 Cancer: the genetics of aberrant cell number regulation

(a) (b)

Figure 17-14 Normal cells and cell transformed by an oncogene. Scanning electron
micrographs of (a) normal cells and (b) cells transformed by Rous sarcoma virus, which
infects cells with the src oncogene. (a) A normal cell line called 3T3. Note the organized
monolayer structure of the cells. (b) A transformed derivative of 3T3. Note how the cells are
rounder and piled up on one another. [From H. Lodish, A. Berk, S. L. Zipursky, P. Matsudaira,

D. Baltimore, and J. Darnell, Molecular Cell Biology, 4th ed. Copyright 2000 by W. H. Freeman
and Company. Courtesy of L.-B. Chen.]

The multicellular solution to life that has evolved in A tumor does not arise as a result of a single ge-
higher animals relies on the collaboration and communi- netic event but rather as the result of multiple hits;
cation exhibited by the organized cells of the tissues and that is, several mutations must arise within a single cell
organ systems. In a sense, cancer cells have reverted to for it to become cancerous. Occasionally, a single muta-
an antisocial, isolated state where they operate without tion is powerful enough to guarantee that a cancer will
external constraint. Cancerous cells are “deaf” to signals form. For example, certain cancers are inherited as
from neighboring cells to stop dividing or to activate highly penetrant single Mendelian factors; an example
their self-destruct systems. is familial retinoblastoma, a cancer of the retina further
discussed on page 564. Perhaps more common are less
Mutations in cancer cells penetrant alleles that increase the probability of devel-
oping a particular type of cancer. In some of the best-
Several lines of evidence point to a genetic origin of the studied cases, the progression of colon cancer and as-
transformation of cells from the benign into the cancer- trocytoma (a brain cancer) has been shown to entail
ous state. the accumulation of several different mutations in the
malignant cells (Figure 17-15).
1. Most carcinogenic agents (chemicals and radiation)
are also mutagenic, suggesting that they produce In regard to how mutations act within the cancer
cancer by introducing mutations into genes. cell, two general kinds are associated with tumors:
oncogene mutations and mutations in tumor-suppressor
2. In the past few years, several alleles that increase genes.
susceptibility to cancer have been cloned and mapped.
• Oncogene mutations act in the cancer cell as gain-of-
3. Mutations that are frequently associated with particular function dominant mutations (see Chapter 16 for a
kinds of cancers have been identified. In some cases, discussion of gain-of-function dominant mutations).
investigators have created transgenic or knockout That is, the mutation need be present as only one
mimics of these naturally occurring mutations in cell allele to contribute to tumor formation. When the
lines in culture or in intact experimental models. mutation is present in protein-coding DNA, the
These artificially created mutations produce oncogene causes a structural change in the encoded
cancerous or cancerlike phenotypes. protein. When the mutation is present in a regulatory
element, the oncogene causes a structurally normal
MESSAGE Tumors arise from a sequence of mutational protein to be misregulated. The gene in its normal,
events that lead to uncontrolled proliferation and cellular unmutated form is called a proto-oncogene.
immortality.

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560 Chapter 17 • Genetic Regulation of Cell Number: Normal and Cancer Cells

(a) Colon cancer

Loss of apc gene Oncogenic mutation Loss of p53 gene
(chromosome 5) of a ras gene (chromosome 17)
Loss of a gene (possibly
(chromosome 12) dcc) on chromosome 18

Normal colonic mucosa Benign Benign Malignant invasive carcinoma
(early) adenoma (late) adenoma

(b) Astrocytoma Loss of a cluster of genes Multiplication of gene for
Loss of p53 gene on chromosome 9 epidermal growth factor
receptor (chromosome 7)

Loss of one copy of
chromosome 10

Normal tissue Low-grade tumor Higher-grade tumor Most aggressive form of tumor

Figure 17-15 Multistep progression to malignancy in cancers of the colon and brain. Several
histologically distinct stages can be distinguished in the progression of these tissues from the
normal state to benign tumors to a malignant cancer. (a) A common sequence of mutational
events in the progression to colon cancer. Note that the tissue becomes more disorganized as the
tumor progresses toward malignancy. (b) A different characteristic series of mutations mark the
progression toward a malignant astrocytoma, a form of brain cancer. [Micrographs by E. R. Fearon

and K. Cho. From W. K. Cavanee and R. L. White, Scientific American, March 1995, pp. 78–79.]

• Mutations in tumor-suppressor genes that promote either to increase the rate of cell proliferation or to pre-
tumor formation are loss-of-function recessive vent apoptosis. On the other hand, tumor-suppressor
mutations. That is, for cancer to occur, both alleles of genes encode proteins that arrest the cell cycle or induce
the gene must encode gene products having reduced apoptosis; in these cases, the cell loses a brake that can
or no residual activity (that is, they are null stop cell proliferation.
mutations).
It is obvious why mutations that increase the rate of
MESSAGE The proteins that oncogenes encode are cell proliferation cause tumors. It is less obvious why
activated in tumor cells, whereas the proteins that tumor- mutations that decrease the chances that a cell will un-
suppressor genes encode are inactivated. dergo apoptosis cause tumors. The reason seems to
be twofold: (1) a cell that cannot undergo apoptosis
Many proteins that are altered by cancer-producing has a much longer lifetime in which to accumulate
mutations take part in intercellular communication and proliferation-promoting mutations and (2) the sorts of
the regulation of the cell cycle and apoptosis (Table damage and unusual physiological changes that take
17-1). Genes that become oncogenes are genes encoding place inside a tumor cell would otherwise induce the
proteins that positively control (turn on) the cell cycle self-destruction pathway. Thus tumor cells will not be
or negatively control (block) apoptosis. The mutant pro- able to survive unless they acquire mutations that pre-
teins are now active even in the absence of the appropri- vent the triggering of apoptosis.
ate activation signals. As a consequence, oncogenes act
How have tumor-promoting mutations been identi-
fied? Several approaches have been used. It is well
known that certain types of cancer can “run in families.”

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17.5 Cancer: the genetics of aberrant cell number regulation 561

TABLE 17-1 Functions of Wild-Type Proteins and Properties
of Tumor-Promoting Mutations in the Corresponding Genes

Wild-type protein function Properties of tumor-promoting mutations

Promotes cell-cycle progression Oncogene (gain-of-function)
Inhibits cell-cycle progression Tumor-suppressor mutation (loss-of-function)
Promotes apoptosis Tumor-suppressor mutation (loss-of-function)
Inhibits apoptosis Oncogene (gain-of-function)
Promotes DNA repair Tumor-suppressor mutation (loss of function)

With modern techniques for pedigree analysis, familial MESSAGE Tumor-promoting mutations can be identified
tendencies toward certain kinds of cancer can be in various ways. When located, they can be cloned and
matched with molecular markers such as microsatellites; studied to learn how they contribute to the malignant state.
in several cases, this process has led to the successful
identification of a mutated gene. Alternatively, many Classes of oncogenes
types of tumors have characteristic chromosomal
translocations or deletions of particular chromosomal re- Roughly a hundred different oncogenes have been iden-
gions. In some cases, these chromosomal rearrangements tified (some examples are listed in Table 17-2). How do
are so reliably a part of a particular cancer that they can their normal counterparts, proto-oncogenes, function?
be used for diagnosis. For example, 95 percent of pa- Proto-oncogenes generally encode a class of proteins
tients with chronic myelogenous leukemia (CML) have that are active only when the proper regulatory signals
a characteristic translocation between chromosomes 9 allow them to be activated. As already mentioned,
and 22. This translocation, called the Philadelphia chro- many proto-oncogene products are elements in path-
mosome after the city where the translocation was first ways that induce (positively control) the cell cycle.
described, is a critical part of the CML diagnosis. How- These products include growth-factor receptors, signal-
ever, not all tumor-promoting mutations are specific to a transduction proteins, and transcriptional regulators.
given type of cancer. Rather, the same mutations seem to Other proto-oncogene products act to inhibit (nega-
promote tumors in a variety of cell types and thus are tively control) the apoptotic pathway. In both types of
seen in many different cancers. oncogene mutation, the activity of the mutant protein

Table 17-2 Some Well-Characterized Oncogenes and Functions
of the Corresponding Proteins

Oncogene Location Function

Nuclear transcription regulators Nucleus Transcription factor
jun Nucleus Transcription factor
fos Nucleus Member of steroid-receptor family
erbA
Cytoplasm Protein tyrosine kinase
Intracellular signal transducers Cytoplasm Protein serine kinase
abl Cytoplasm G-protein ␣ subunit
raf Cytoplasm GTP/GDP-binding protein
gsp
ras Extracellular Secreted growth factor

Mitogen Transmembrane Receptor tyrosine kinase
sis Transmembrane Receptor tyrosine kinase

Mitogen receptors Cytoplasm Upstream inhibitor of caspase cascade
erbB
fms

Apoptosis inhibitor
bcl2

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562 Chapter 17 • Genetic Regulation of Cell Number: Normal and Cancer Cells

Inactive Ras

GDP

Amino acid 10 Amino acid 15 GTP

Gly Ala Gly Gly Val Gly × Sos interaction
ras wild-type DNA GGC GCC GGC GGT GTG GGC Active Ras stimulates
GDP– GTP
exchange.

GDP

ras oncogene DNA GGC GCC GTC GGT GTG GGC GTP
Val
Ras oncoprotein
(a) is blocked here.
Signal remains on.
Figure 17-16 Ras oncoprotein. (a) The ras oncogene differs from the wild type
by a single base pair, producing a Ras oncoprotein that differs from the wild Continuously
type in one amino acid, at position 12 in the ras open reading frame. (b) The activates downstream
Ras oncoprotein that cannot hydrolyze GTP to GDP. Because of this defect, serine/threonine kinase
the Ras oncoprotein remains in the active Ras–GTP complex and continuously
activates the downstream serine/threonine kinase (see Figure 17-13). (b)

has been uncoupled from its normal regulatory path- PROTEIN DOMAIN DELETION OF A MITOGEN RE-
way, leading to its continuous unregulated expression. CEPTOR TYROSINE KINASE A growth factor is not
The continuously expressed protein product of an necessary to initiate the cell proliferation pathway if its
oncogene is called an oncoprotein. Several categories of receptor has been altered appropriately. The v-erbB
oncogenes have been identified according to the differ- oncogene is a mutated gene in a tumor-producing virus
ent ways in which the regulatory functions have been that infects birds. It encodes a mutated form of a recep-
uncoupled. tor tyrosine kinase known as the EGFR, a receptor for
the epidermal growth-factor ligand (Figure 17-17). Sev-
MESSAGE The wild-type counterparts of oncogenes act
to positively control the cell cycle or negatively control Ligand-binding No ligand-binding
apoptosis. domain domain

The following examples illustrate some of the ways Plasma
in which oncogenes are mutated and act to produce a membrane
cancerous cell.
Normal EGF receptor Truncated
POINT MUTATION OF AN INTRACELLULAR SIGNAL carboxyl
TRANSDUCER The ras oncogene illustrates a tumor- terminus
promoting mutation of a molecule in a signal-
transduction pathway. As is often the case, the change v-erbB oncogene product
from normal protein to oncoprotein entails structural
modifications of the protein itself, in this case caused by Wild-type H2N Kinase COOH
a simple point mutation. A single base-pair substitution receptor
that converts glycine into valine at amino acid number
12 of the Ras protein, for example, creates the onco- v-erbB-encoded H2N Kinase COOH
protein found in human bladder cancer (Figure 17-16a). receptor
Recall that the normal Ras protein is a G-protein sub-
unit that takes part in signal transduction. It normally Figure 17-17 Oncogenic mutation that affects signaling
functions by cycling between the active GTP-bound between cells. Normally, the kinase activity of the EGFR is
state and the inactive GDP-bound state (see Figure activated only when EGF binds to the ligand-binding domain.
17-12). The missense mutation in the ras oncogene pro- The erythroblastosis tumor virus carries the v-erbB oncogene,
duces an oncoprotein that always binds GTP (Figure which encodes a mutant form of the EGFR. The mutant
17-16b), even in the absence of normal signals. Thus, the protein dimerizes constitutively, leading to continuous
Ras oncoprotein continuously propagates a signal that autophosphorylation and continuous transduction of a signal
promotes cell proliferation. from the receptor. [After J. D. Watson, M. Gilman, J. Witkowski, and

M. Zoller, Recombinant DNA, 2d ed. Copyright 1992 by James D.

Watson, Michael Gilman, Jan Witkowski, and Mark Zoller.]

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17.5 Cancer: the genetics of aberrant cell number regulation 563

eral parts of the normal EGFR are missing in the mu- protein kinase activity, which is responsible for its onco-
tated form. The mutated form of the EGFR lacks the genic effect. The oncoprotein continually propagates its
extracellular, ligand-binding domain as well as some reg- growth signal downstream in the pathway, regardless of
ulatory components of the cytoplasmic domain. As a whether an upstream signal is present.
consequence, the truncated EGFR oncoprotein is able to
dimerize even in the absence of the EGF ligand. The GENE FUSION CAUSING MISEXPRESSION OF AN
EGFR oncoprotein dimer is always autophosphorylated APOPTOSIS INHIBITOR Some oncogenes produce on-
through its tyrosine kinase activity and so continuously coproteins that are identical in structure with the nor-
initiates a signal-transduction cascade that promotes cell mal proteins. In these cases, the mutation induces misex-
proliferation. pression of the protein; that is, the protein is expressed
in cell types from which it is ordinarily absent. Here we
A PROTEIN FUSION INVOLVING AN INTRACELLULAR see how, through gene fusion, an oncogene causes a pro-
SIGNAL TRANSDUCER Perhaps the most remarkable tein that inhibits apoptosis to be overexpressed in an im-
type of structurally altered oncoprotein is caused by a mune system cell.
gene fusion. The classic example emerged from the re-
sults of studies of the Philadelphia chromosome, which, Several oncogenes that cause misexpression are as-
as already mentioned, is a translocation between chromo- sociated with chromosomal translocations diagnostic of
somes 9 and 22 that is a diagnostic feature of chronic various B-lymphocyte tumors. B lymphocytes and their
myelogenous leukemia. Recombinant DNA methods descendants, plasma cells, are the cells that synthesize
have shown that the breakpoints of the Philadelphia antibodies, also called immunoglobulins. In the translo-
chromosome translocation in different CML patients are cations observed in B-cell oncogenes, no protein fusion
quite similar and cause the fusion of two genes, bcr1 and results; rather, the chromosomal rearrangement causes a
abl (Figure 17-18). The abl (Abelson) proto-oncogene gene near one breakpoint to be turned on in the wrong
encodes a tyrosine-specific protein kinase present in tissue. In follicular lymphoma, 85 percent of patients
the cytoplasm that participates in a signal-transduction have a translocation between chromosomes 14 and 18
pathway — specifically, a signal-transduction pathway (Figure 17-19). Near the chromosome 14 breakpoint is
initiated by a growth factor that leads to cell prolifera- a transcriptional enhancer from one of the immuno-
tion. The Bcr1 – Abl fusion oncoprotein has a permanent globulin genes. This translocated enhancer element is
fused with the bcl-2 gene, which is a negative regulator

Centromere Normal bcr1 mRNA

Chromosome 22 bcr1 gene

Chromosome 9 c-abl gene
Recombination

Hybrid bcr1–abl mRNA

Translocation (9; 22)
Philadelphia chromosome

Bcr1–Abl fusion protein

Figure 17-18 Chromosome rearrangement in CML. The Philadelphia chromosome, which
is diagnostic of chronic myelogenous leukemia, is a translocation between chromosomes 9
and 22. The translocation produces a hybrid Bcr1–Abl protein that lacks the normal controls
for repressing c-abl-encoded protein tyrosine kinase activity. Only one of the two rearranged
chromosomes of the reciprocal translocation is shown. [After J. D. Watson, M. Gilman,

J. Witkowski, and M. Zoller, Recombinant DNA, 2d ed. Copyright 1992 by James D. Watson,
Michael Gilman, Jan Witkowski, and Mark Zoller.]

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564 Chapter 17 • Genetic Regulation of Cell Number: Normal and Cancer Cells

Centromere Breakpoint
IgH enhancer
Human
chromosome 14

Immunoglobulin heavy
chain gene (IgH)

Human Not active in B lymphocytes
chromosome 18

bcl-2 gene

Rejoining Breakpoint

of breakpoints

Translocation Active in B lymphocytes
(14; 18)

Figure 17-19 Chromosomal rearrangement in follicular lymphoma. The translocation
fuses the transcriptional enhancer element of an antibody-coding gene, on chromosome
14, with the transcription unit of a gene on chromosome 18 that encodes Bcl-2, a negative
regulator of apoptosis. This gene fusion causes the Bcl-2 protein to be produced in antibody-
producing cells, thus preventing any self-destruction signals from inducing apoptosis in
those cells.

of apoptosis. This enhancer – bcl-2 fusion causes large for the Rb protein must be inactivated, either by the
amounts of Bcl-2 protein to be expressed in B lympho- same mutation or by a different mutation in each. Most
cytes. The plentiful Bc1-2 protein effectively blocks patients have one or a few tumors localized to one site
apoptosis in these lymphocytes, giving them an unusu- in one eye, and the condition is sporadic — in other
ally long lifetime in which to accumulate mutations words, there is no history of retinoblastoma in the family
that promote cell proliferation. and the affected person does not transmit it to his or her
offspring. In these cases, the rb mutations arise in a so-
MESSAGE Dominant oncogenes contribute to the matic cell whose descendants populate the retina (Fig-
oncogenic state by causing a protein to be expressed in an ure 17-20). Presumably, the mutations arise by chance at
activated form or in the wrong cells. different times in the course of development in the same
cell lineage.
Classes of tumor-suppressor genes
A few patients, however, have an inherited form of
The normal functions of tumor-suppressor genes fall the disease, called hereditary binocular retinoblastoma
into categories complementary to those of proto- (HBR). Such patients have many tumors, in the retinas
oncogenes (see Table 17-1). Some tumor-suppressor of both eyes. Paradoxically, even though rb is a recessive
genes encode negative regulators of the cell cycle, such allele at the cellular level, standard genetic analysis
as the Rb protein or elements of the TGF-␤ signaling would describe HBR as transmitted as an autosomal
pathway. Others encode positive regulators of apop- dominant (see Figure 17-20b). How do we resolve this
tosis (at least part of the function of p53 falls into this paradox? A germ-line mutation knocks out one of the
category). Still others are indirect players in cancer, two copies of the RB gene in all cells of the retina in
with a normal role in the repair of damaged DNA or in both eyes. It is virtually certain that the single remaining
controlling cellular longevity. We shall consider two ex- normal RB gene will acquire an rb mutation in at least
amples here. some of the retinal cells. These cells will produce no
functional Rb protein.
THE KNOCKOUT OF A PROTEIN THAT INHIBITS
CELL PROLIFERATION Retinoblastoma is a cancer of Why does the absence of Rb protein promote tumor
the retina that typically affects young children. In growth? Recall from our consideration of the cell cycle
retinoblastoma, the gene that encodes the Rb protein that Rb protein functions by binding the E2F transcrip-
has mutated, and retinal cells that lack a functional RB tion factor. Bound Rb prevents E2F from promoting the
gene proliferate out of control. The cancer is a recessive transcription of genes whose products are needed for
trait at the cellular level: both alleles of the gene coding DNA replication and other S-phase functions. An inac-
tive Rb is unable to bind E2F, and so E2F can promote
the transcription of S-phase genes. In homozygous null
rb cells, Rb protein is permanently inactive. Thus, E2F is

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17.5 Cancer: the genetics of aberrant cell number regulation 565

(b) (a)

HEREDITARY RETINOBLASTOMA

RB /rb RB /RB
Tumors

RB /rb

Zygote Second
mutation
rb´ rb

RB rb

Mitotic
crossover

rb rb

SPORADIC RETINOBLASTOMA Figure 17-20 Origin
of the two types of
RB /RB RB /RB retinoblastoma.
(a) Retinoblastoma, a
Zygote RB /RB Second Tumors cancer of the retina.
mutation rb´ rb (b) The mutational origin
First RB rb rb rb of retinal tumors in
mutation Mitotic hereditary and sporadic
crossover retinoblastoma.Recessive
RB RB rb alleles of the RB gene
lead to tumor development.
First Note that, if one
mutation chromosome in a cell
already has an rb
RB rb mutation, the homologous
chromosome may also
acquire it through either
chance mutation or
crossing-over in the course
of mitosis. [Part a from

Custom Medical Stock.]

always able to promote S phase, and the arrest of normal or RFLPs (see Chapter 12). To think about this from
cells in late G1 does not occur in retinoblastoma cells. the opposite direction, if we were to identify a region
of the genome that consistently exhibited LOH in a
As already noted, the germ-line rb mutation is only particular type of tumor, we would be highly suspi-
one event leading to the loss-of-function rb phenotype. cious that a tumor-suppressor gene normally resided in
The other allele has to be knocked out as well, which that region of the genome.
can occur through mutation or through deletion of the
other allele or through a mitotic abnormality (mitotic THE KNOCKOUT OF A PROTEIN THAT INHIBITS
crossing-over or mitotic nondisjunction). Of these, CELL PROLIFERATION AND PROMOTES APOPTOSIS
deletion and mitotic crossing-over or nondisjunction The p53 gene has been identified as another tumor-
would lead to the loss of one of the two alleles of suppressor gene. Mutations in p53 are associated with
the gene, a condition called loss-of-heterozygosity, or many types of tumors, and estimates are that 50 percent
LOH for short. There are now many ways to identify of human tumors lack a functional p53 gene. The active
loss-of-heterozygosity in tumor cells by comparing p53 protein is a transcriptional regulator that is activated
their DNA content with their wild-type neighbors, by in response to DNA damage. Activated wild-type p53
using molecular polymorphisms such as SNPs, SSLPs,

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566 Chapter 17 • Genetic Regulation of Cell Number: Normal and Cancer Cells

serves double duty: it prevents progression of the cell through its removal from the genome, and the location
cycle until the DNA damage is repaired, and, under of the absent allele can be determined by looking for
some circumstances, it induces apoptosis. If no func- polymorphic markers that also show LOH. Such a poly-
tional p53 gene is present, the cell cycle progresses even morphism can be assumed to map to the vicinity of the
if damaged DNA has not been repaired. The progression tumor-suppressor gene on the sequence map of the hu-
of the cell cycle into mitosis elevates the overall fre- man genome.
quency of mutations, chromosomal rearrangements, and
aneuploidy and thus increases the chances that other A recent study of prostate cancer surveyed LOH in
mutations that promote cell proliferation or block apop- 11 patients, using approximately 1500 SNPs across the
tosis will arise. genome that could be detected by differential hybridiza-
tion on DNA oligonucleotide chips (SNP-chips). The
It is now clear that null mutations able to elevate idea of this approach is to identify heterozygous SNPs in
the mutation rate are important contributors to the pro- normal cells from these patients (only a subset of pa-
gression of tumors in humans. These null mutations are tients will be heterozygous for a given SNP) and then
recessive mutations in tumor-suppressor genes that nor- see if both variants of these SNPs are present in the
mally function in DNA repair pathways and thus inter- prostate tumor cells. If only one SNP variant is present,
fere with DNA repair. They promote tumor growth in- then LOH has occurred. Figure 17-21 shows the distrib-
directly by elevating the mutation rate, which makes it ution of 32 sites in the genome that exhibited LOH, su-
much more likely that a series of oncogene and tumor- perimposed on the human cytogenetic map. Each of
suppressor mutations will arise, corrupting the normal these regions could then be subjected to more intensive
regulation of the cell cycle and programmed cell death. screening for SNPs or other heterozygous polymorphic
Large numbers of such tumor-suppressor-gene muta- markers in the vicinity of the detected LOH sites to de-
tions have been identified, including some associated termine the size of the lost region. After the sizes of
with heritable forms of cancer in specific tissues. Exam- these regions have been determined, they can be com-
ples are the BRCA1 and BRCA2 mutations and breast pared with the known genes on the sequence map that
cancer. fall into these regions. The comparison will allow re-
searchers to focus in on candidate tumor-suppressor
MESSAGE Mutations in tumor-suppressor genes, like genes and to determine which of them are actually
mutations in oncogenes, act directly or indirectly to knocked out in these tumors.
promote the cell cycle or block apoptosis.
Genomic approaches
17.6 Applying genomic
approaches to cancer research, to cancer diagnostics and therapies
diagnosis, and therapies
A goal of cancer diagnostics is to properly classify the
At this point, we have a thematic understanding of the type of cancer that a patient has contracted, often by in-
sorts of mutational lesions that arise in cancers. Most of specting tumor cells under the microscope. However, ge-
this information historically has come from the results nomic approaches have revealed that tumors that look
of studies directed at specific genes, but there is now a the same under a microscope may have very different
great deal of emphasis on applying genomewide analyti- molecular bases. Let’s examine how genomics can be
cal methods to a variety of subjects of inquiry in cancer used to address this diagnostic problem. As an example,
research and medicine. Let’s briefly consider some of we will consider a form of lymphoma.
these approaches.
Lymphomas and leukemias are cancers of the
Genomic approaches white blood cells — the cells that make up the immune
to identifying tumor-promoting mutations system. In these diseases, certain white blood cells mas-
sively overproliferate, leading to an imbalance in im-
Although a couple of hundred genes have already been mune cells and, ultimately, a failure of the immune sys-
demonstrated to mutate to a tumor-promoting state un- tem. One class of lymphoma is called diffuse large
der some circumstances, the hunt for such genes is by no B-cell lymphoma (DLBCL), the most common form of
means over. As one example of a genome-scale approach non-Hodgkin’s lymphoma. In the United States alone,
to finding cancer genes, let’s consider how loss-of- about 25,000 new cases of DLBCL are diagnosed every
heterozygosity could be exploited. Recall that mutations year. The diagnosis is based on finding a characteristic
in tumor-suppressor genes act as recessive alleles in tu- set of symptoms and on the histology (microscopic ex-
mor cells. Often, the knockout of one allele occurs amination of cell and tissue morphology) of affected
lymph nodes (Figure 17-22). Standard chemotherapy
cures about 40 percent of DLBCL patients, but other
patients do not respond to this regimen. Why is there
this dichotomy among the patient population? It could

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17.6 Applying genomic approaches to cancer research, diagnosis, and therapies 567

1p31.2 2p13.3 6p25.1-p25.3
100% 67% 75%

3p12.1-p12.2 8p22-p23.2
56% 71%
67%
5q23.1-q23.2 7q21.11 67%
64% 43%
8p21.1
6q22.33
67% 8q22.2
60%
2q37.3 4q35.1
8
67% 44%

1 23 4 567

9p13.3 10q11.21 12p13.1
50% 100% 100%

10q22.1 13q14.11-14.2 15q13.3
64% 67%
70%

11q23.3 16q23.1-q23.2
33% 100%

9 10 11 12 13 14 15 16
17p13.3 20p12.2
100% 18p11.22 19p13.3 67% 21q21.2 22q11.1-q11.21
56% 30% 100% 63%
17q21.31 50% 20
100% 18q21.31-
q21.32 19q13.12
19q13.12
56%
67%
17 18 19q13.4 21 22

33%
19

Figure 17-21 Genomic SNP analysis of LOH in 11 prostate cancer patients. The blocks indicate
the location of the LOH regions from the sequence map overlaid on the cytogenetic map.
The horizonal bars represent the proportion (in percentage) of prostate cancer patients showing
LOH at the indicated loci. [From C. I. Dumur et al., “Genomic-wide Detection of LOH in Prostate

Cancer Using Human SNP Microarray Technology,” Genomics 81, 2003, 265.]

(a) (b)

Figure 17-22 Histology of (a) a normal lymph node and (b) diffuse large B-cell lymphoma
(DLBCL). The darkly stained cells are lymphoid cells. Note that the lymphocytes in the normal
lymph node are small and uniformly shaped, but the lymphoma cells are large and more
varied in shape. [G. W. Willis, M.D./Visuals Unlimited.]

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568 Chapter 17 • Genetic Regulation of Cell Number: Normal and Cancer Cells

(a) (b)
1

Probability 19 patients, 6 deaths
.5

21 patients, 16 deaths

0
0 2 4 6 8 10 12

Overall survival (years)

GC B-like DLBCL

Activated
B-like DLBCL

Figure 17-23 Functional genomic analysis of DLBCL. (a) Microarray analysis of malignant
cells indicates that patients possess one of two basic RNA-expression profiles, one
termed GC B-like and the other activated B-like. (b) Higher patient survival correlates
with the GC B-like RNA profile. [Reprinted by permission from L. Liotta and E. Petricoin,

Nature Reviews: Genetics 1, 2000, 53. Macmillan Magazines Ltd.]

result from differences in genetic or environmental fac- A challenge: These examples just barely touch the
tors, differences in disease stage, or any of many other surface of how genomic technologies are being applied
factors. to cancer research and medicine. On the basis of tech-
niques discussed earlier in the text, think of other ways
A breakthrough was achieved from profiling gene that you would apply genomic techniques for under-
transcription in malignant cells from 40 patients, with standing the molecular basis of cancer, for diagnostics
the use of microarray technology (Figure 17-23). In and early detection of cancer, and for developing new
this study, two different patterns of gene expression cancer therapies.
were observed. One pattern, called the germinal-center
B-cell-like pattern (GCB), correlates with a much The complexities of cancer
higher probability of patient survival after standard
chemotherapy than does the other pattern, called acti- We have seen that numerous mutations that promote
vated B-cell-like DLBCL. (These patterns are named for tumor growth can arise. These mutations alter the nor-
their similarities in gene expression to certain normal mal processes that govern proliferation and apoptosis
B-lymphocyte cell types.) This finding suggests that (Figure 17-24). The story does not end here, however.
even though DLBCL is diagnosed histologically as one There is evidence that other modes of gene inactivation,
disease, at a molecular level it is more like two different such as epigenetic imprinting, can produce tumor-
diseases. This result raises the hope of more accurate promoting lesions. There is also evidence that the over-
diagnosis and of treatment that can be focused specifi- expression of telomerase is another condition required
cally on each disease. Further, such microarray experi- for cell immortality, a feature of cancer cells. (Normal
ments are beginning to reveal unexpected RNAs that human somatic cells can undergo only a relatively small
differ in level either between normal and tumor cells or number of divisions before their telomeres are reduced
between different stages of malignancy; the protein in size to the point at which the cells can no longer
products of these RNAs are candidate targets for drug grow. In human tumor cells, however, the length of the
therapy. telomeres appears to be substantially extended, proba-

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Key questions revisited 569

GAIN-OF-FUNCTION IN TUMOR-SUPPRESSOR GENES DOMINANT ONCOGENE MUTATIONS Mitogen Survival bly through the overexpression of telomerase.) Even
pathways factor malignant tumors differ in their rates of proliferation
and their ability to invade other tissues, or metastasize.
pathways Undoubtedly, even after a malignant state is achieved,
many more mutations accumulate in the tumor cell
Cell Apoptosis that further promote its proliferation and invasiveness.
proliferation Thus, there is a considerable way to go before we have a
LOSS-OF-FUNCTION MUTATIONS truly comprehensive view of how tumors arise and
Growth p53 pathways Apoptosis progress.
inhibitor activator
pathways pathways Even so, there is light at the end of the tunnel. In the
early 1990s, researchers were able to correlate overex-
Irreparable pression of the Abelson tyrosine kinase, through the
DNA damage Philadelphia chromosome gene fusion, with chronic
myelogenous leukemia. Chemists then developed a com-
Figure 17-24 Main events contributing to tumor formation: pound, called ST1571, that bound to the ATP-
increased cell proliferation and cell survival (decreased apoptosis). binding site of the Abelson tyrosine kinase and thereby
The pathways in red are susceptible to gain-of-function inhibited its ability to phosphorylate tyrosines on target
oncogene mutation. The pathways in blue are susceptible to proteins. ST1571 was shown to inhibit cell proliferation
loss-of-function tumor-suppressor-gene mutation. induced by bcr – abl in cell lines and was then tested as
the drug Gleevec in clinical trials. The treatment had dra-
matic effects; more than 90 percent of treated patients
have recovered normal blood counts. Even more striking,
more than half show no evidence of cells containing the
Philadelphia chromosome in their bloodstreams. No
other treatment for CML has been this effective. This is
the first clear case in which an understanding of the mol-
ecular biology of a malignancy has been translated into a
highly effective, targeted treatment.

KEY QUESTIONS REVISITED well as cells that have only temporary functions in
development. Cell survival requires direct survival
• Why is it important for multicellular organisms signals that inhibit activation of the self-destruction
to have mechanisms for regulating cell program. The cost of replacing discarded cells is far
numbers? less than the cost of compromising the health of the
entire organism.
Multicellular organisms depend heavily on a division
of labor among the various cell types in a tissue. To • How do neighboring cells influence cell proliferation
maintain the proper proportion and number of cells of and cell death?
the various types within a tissue, mechanisms are
needed to replace accidentally lost cells and to elimi- Proteins secreted or presented by nearby cells act as sig-
nate abnormal cells that present a danger to the organ- nals. These proteins bind to transmembrane receptor
ism as a whole. proteins on the cells receiving the signal, activating a
signal-transduction pathway that ultimately changes the
• Why is cell-cycle progression regulated? expression of key genes in the cell-cycle or apoptosis
pathways.
First, regulation at checkpoints ensures that the cell cy-
cle progresses to the next stage only when the proper • Why is cancer considered a genetic disease of somatic
preconditions have been fulfilled. Second, because the cells?
cell cycle is wired into the intercellular signaling system,
proliferation can be halted until the proper signals for it Cancers are caused by the accumulation of a set of mu-
are received. tations in a single somatic cell. These mutations under-
mine the basic control mechanisms that keep a cell from
• Why is programmed cell death necessary? proliferating out of control.

Apoptosis is a normal self-destruction mechanism that
eliminates damaged and potentially harmful cells as

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570 Chapter 17 • Genetic Regulation of Cell Number: Normal and Cancer Cells

• How do mutations promote tumors? • How can genomic methodologies be applied to cancer
research and medicine?
Tumor-producing mutations either release normal in-
hibitory controls on the cell cycle or block cell death. In Microarray technology can be used to search for muta-
addition, mutations that increase the mutation rate of the tions in tumor-suppressor genes and to more precisely
cell are tumor promoting, presumably because mutations diagnose the type of cancer in patients presenting the
increase in the cell-proliferation or apoptosis pathways. same histological tumor phenotype.

SUMMARY Cancer is a genetic disease of somatic cells. In can-
cer, cells proliferate out of control and avoid self-
Somatic cells of higher eukaryotes are integrated into a destruct mechanisms through the accumulation of a se-
variety of tissues and may be of many different types, ries of tumor-promoting mutations in the same somatic
each with a specialized role in the physiology of an or- cell. Many of the genes that promote cancers when they
ganism. Regulation of cell numbers is essential for main- are mutated contribute directly or indirectly to growth
taining proper physiological balance among the various control and differentiation. Genes that normally acceler-
tissues and cell types. Higher eukaryotic organisms have ate survival or proliferation may be mutated to become
evolved mechanisms that control cell survival and the oncogenes. Oncogene mutations behave in the cancer
ability to proliferate. cell as gain-of-function dominant mutations, in which
their tumor-promoting effect is due to altered activity of
Normal cell proliferation is controlled by regulation the mutant gene product. Genes that normally acceler-
of the cell cycle. This regulation achieves multiple ends. ate apoptosis or inhibit mitosis are tumor-suppressor
First, regulation at checkpoints of the cell cycle ensures genes. Mutations in tumor-suppressor genes behave in
that the cell cycle can progress to the next stage only the cancer cell as recessive loss-of-function mutations.
when a set of preconditions that ensure proper chromo- In other words, the abolition of activity of these gene
some replication and segregation are fulfilled. Second, products promotes tumor growth. In addition, mutants
because the cell cycle is wired into the intercellular sig- in repair pathways, as well as those that increase the
naling system, proliferation can be halted until the general mutation rate, increase the likelihood of tumor-
proper signals for it are received. promoting mutations and hence indirectly promote
tumor development.
Apoptosis is a normal self-destruction mechanism
that eliminates damaged and potentially harmful cells as Functional genomics is being used in diagnostic tests
well as cells that have only temporary functions in de- and to identify drug targets in studies to improve the de-
velopment. We now know that cell survival requires di- tection and treatment of cancer. Instead of applying the
rect survival signals that inhibit activation of the execu- former method of screening for specific genetic contri-
tion program and the caspase cascade, which lead to butions to tumor development, we are now able to sur-
self-destruction. vey virtually the entire genome for changes in gene ac-
tivity associated with tumor development. Even if we
Cell proliferation and apoptosis are coordinated in a don’t understand the nature of all the changes, func-
population of cells through intercellular signaling sys- tional genomics reveals gene-expression “fingerprints”
tems. Signaling consists of the secretion of a signal from that allow different disease states to be identified.
sending cells, receipt of that signal by surface receptors
on target cells, and transduction of that signal from the
transmembrane receptor to the interior (usually the nu-
cleus) of the target cells.

KEY TERMS differentiated cell (p. 547) oncoprotein (p. 562)
endocrine signal (p. 553) paracrine signal (p. 555)
apoptosis (p. 552) growth factor (p. 555) programmed cell death (p. 552)
cancer (p. 558) kinase (p. 549) proto-oncogene (p. 559)
caspase (p. 552) ligand (p. 553) signal-transduction cascade (p. 555)
CDK-cyclin complexes (p. 548) loss-of-heterozygosity stem cell (p. 547)
cell – cell communication (p. 553) survival factor (p. 556)
checkpoint (p. 550) (LOH) (p. 565) tumor-suppressor gene (p. 560)
cyclin (p. 549) mitogen (p. 555) zymogen (p. 552)
cyclin-dependent protein kinase oncogene (p. 559)

(CDK) (p. 549)

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Problems 571

SOLVED PROBLEM allele has been passed on to all affected offspring.
That allele is the mutant one.
In the inherited form of retinoblastoma, an affected
child is heterozygous for an rb mutation, which is ei- b. If the trait arose de novo in the sperm or the oocyte,
ther passed on from a parent or has newly arisen in the we can still possibly determine from which parent it
sperm or the oocyte nucleus that gave rise to the child. came, but with considerable difficulty. One way to do
The heterozygous RB/rb cells are nonmalignant, how- so would be to use recombinant DNA cloning tech-
ever. The RB allele of the heterozygote must be niques to isolate each of the two copies of the RB gene
knocked out in the developing retinal tissue to create a from normal cells. Only one of these alleles should be
tumorous cell. Such a knockout can occur through an mutant. When the gene has been cloned, by DNA se-
independent mutation of the RB allele or by mitotic quencing of the two alleles we may be able to identify
crossing-over such that the original rb mutation is now the mutation that inactivates RB. If it arose de novo in
homozygous. the sperm or the oocyte, this mutation would not be
present in the somatic cells of the parents. If sequencing
a. If retinoblastoma is passed on to other siblings as well, also reveals some polymorphisms (for example, in
could we determine whether the original mutation was restriction-enzyme recognition sites) that distinguish
derived from the mother or the father? How? the alleles, we should then be able to go back to the
parents’ DNA to find out whether the mother or the
b. Could we determine whether the rb mutation was father carries the polymorphisms that were found in
maternally or paternally derived if it arose de novo in a the cloned mutant allele. (Whether this approach will
germ cell of one parent? work depends on the exact nature of the parental alleles
and the mutation.)
Solution
If the mutation arose by mitotic crossing-over, addi-
a. If the trait is inherited, we can determine from which tional tools are available. In this case, the entire region
parent it came. The most straightforward approach is to around the rb gene will be homozygous for the mutant
identify DNA polymorphisms, such as restriction frag- chromosome. By examining DNA polymorphisms
ment length polymorphisms (RFLPs), that map within known to map in this region, we should be able to deter-
or near the RB gene. RB has the curious property of be- mine whether this chromosome derived from the
ing inherited as an autosomal dominant, even though mother or from the father. This approach becomes a
it is recessive on a cellular basis. Given the dominant standard exercise in DNA fingerprinting similar to that
pattern of inheritance, by finding DNA differences in described in part a.
the parental genomes that map near each of the four
parental alleles, we should be able to determine which

PROBLEMS 7. How is the activity of caspases controlled?

BASIC PROBLEMS 8. Give an example of an oncogene. Why is the muta-
1. Describe three lines of evidence that cancer is a tion dominant?

genetic disease. 9. Give an example of a tumor-suppressor gene. Why is
the mutation recessive?
2. Describe three mechanisms used to control the ac-
tivities of the proteins in the cell-cycle and cell- 10. How do the following mutations lead to cancer?
death pathways. a. v-erbB
b. ras oncogene
3. What are the two roles of cyclins? How are the lev- c. Philadelphia chromosome
els of cyclins regulated?
11. For each of the following proteins, describe how a
4. What are three major categories of mutations that mutation could lead to the formation of an oncogene.
lead to the development of cancer? a. growth-factor receptor
b. transcriptional regulator
5. What are the roles of Apaf and Bcl proteins in c. G-protein
apoptosis?

6. What are two mechanisms by which translocations
can lead to oncogene formation?

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572 Chapter 17 • Genetic Regulation of Cell Number: Normal and Cancer Cells

CHALLENGING PROBLEMS 17. We now understand that mutations that inhibit
apoptosis are found in tumors. Because prolifera-
12. Cancer is thought to be caused by the accumula- tion itself is not induced by the inhibition of apop-
tion of two or more “hits” — that is, two or more tosis, explain how this inhibition might contribute
mutations that affect cell proliferation and survival to tumor formation.
in the same cell. Many of these oncogenic muta-
tions are dominant: one mutant copy of the gene is 18. Suppose that you had the ability to introduce nor-
sufficient to change the proliferative properties of a mal copies of a gene into a tumor cell in which
cell. Which of the following general types of muta- mutations in the gene caused it to promote tumor
tions have the potential to create dominant onco- growth.
genes? Justify each answer.
a. If the mutations were in a tumor-suppressor
a. a mutation that increases the number of copies gene, would you expect these normal transgenes to
of a transcriptional activator of cyclin A block the tumor-promoting activity of the muta-
tions? Why or why not?
b. a nonsense mutation located shortly after the
beginning of translation in a gene that encodes a b. If the mutations were of the oncogene type,
growth-factor receptor would you expect the normal transgenes to block
their tumor-promoting activity? Why or why not?
c. a mutation that increases the level of FasL
19. Insulin is a protein that is secreted by the pancreas
d. a mutation that disrupts the active site of a cyto- (an endocrine organ) when the level of blood sugar
plasmic tyrosine-specific protein kinase is high. Insulin acts on many distant tissues by bind-
ing and activating a receptor tyrosine kinase, leading
e. a translocation that joins a gene encoding an to a reduction in blood sugar by appropriate storage
inhibitor of apoptosis to an enhancer element for of the products of sugar metabolism. Diabetes is a
gene expression in the liver disease in which the level of blood sugar remains
high because some part of the insulin pathway is de-
13. Many of the proteins that participate in the pro- fective. One kind of diabetes (call it type A) can be
gression pathway of the cell cycle are reversibly treated by giving the patient insulin. Another kind
modified, whereas, in the apoptosis pathway, the of diabetes (call it type B) does not respond to in-
modification events are irreversible. Rationalize sulin treatment.
these observations in relation to the nature and end
result of the two pathways. a. Which type of diabetes is likely to be due to a
defect in the pancreas, and which type is likely to
14. Tumor-promoting mutations are described as being be due to a defect in the target cells? Justify your
either gain-of-function or loss-of-function. Describe answer.
the effects of both classes of tumor-promoting
mutations on the cell cycle and apoptosis. b. Type B diabetes can be due to mutations in any
of several different genes. Explain this observation.
15. Normally, FasL is present on cells only when a
message needs to be sent to neighboring cells in- 20. Irreparable DNA damage can have consequences
structing them to undergo apoptosis. Suppose that for both the cell cycle and apoptosis. Explain what
you have a mutation that produces FasL on the the consequences are, as well as the pathways by
surfaces of all liver cells. which the cell implements them.

a. If the mutation were present in the germ line, 21. Retinoblastomas arise through mutations in the RB
would you predict it to be dominant or recessive? gene. In HBR (hereditary binocular retinoblastoma),
the gene is inherited from parent to offspring as a
b. If such a mutant arose in somatic tissues, would simple autosomal dominant. Nonetheless, RB is
you expect it to be tumor promoting? Why or why thought to be a recessive tumor- suppressor gene. In
not? HBR, there are typically multiple tumors in both
eyes. In contrast, in the form of retinoblastoma that
16. Some genes can be mutated to oncogenes by in- is not inherited (called sporadic retinoblastoma),
creasing the copy number of the gene which, for there are fewer tumors and they are usually re-
example, is true of the Myc transcription factor. On stricted to one eye. Account for all of these obser-
the other hand, oncogenic mutations of ras are al- vations in relation to your understanding of how
ways point mutations that alter the protein struc- tumor-suppressor genes mutate to cause neoplasia.
ture of Ras. Rationalize these observations in rela-
tion to the roles of normal and oncogenic versions
of Ras and Myc.

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Problems 573

22. The wild-type Rb protein functions to sequester b. If a cell were doubly homozygous for loss-of-
E2F in the cytoplasm. At the appropriate time in function mutations in both the RB and E2F genes,
the cell cycle, E2F is released so that it can act as a would you expect tumor growth? Explain.
functional transcription factor.
c. How would a mutation in the RB protein that
a. In cells homozygous for a loss-of-function rb mu- irreversibly binds E2F affect the cell cycle?
tation, where in the cell do you expect to find E2F?

EXPLORING GENOMES A Web-Based Bioinformatics Tutorial

Exploring the Cancer Anatomy Project

The Cancer Anatomy project at NCBI is a specialized database focused on a
particular subset of data. It brings together, in a graphical and searchable
format, all known information about individual genes, map positions, and
chromosomal location for genes associated with cancer. In the Genomics
tutorial at www.whfreeman.com/mga, we explore gene-expression data, gene
mutations, and chromosomal aberrations associated with the various tumor
types.

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18

THE GENETIC BASIS
OF DEVELOPMENT

A homeotic mutation that alters the basic Drosophila body KEY QUESTIONS
plan. Homeosis is the replacement of one body part by another.
In place of the normal antennae (shown at the top), an • What sequence of events produces
Antennapedia mutation causes the antennal precursor cells to the basic body plan of an animal?
develop into a leg. [F. R. Turner/BPS.]
• How are polarities that give rise
to the main body axes created?

• How do cells recognize their locations
along the developing body axes?

• Do cells participate in single or
multiple decision-making processes?

• What role does cell-to-cell communication
play in building the basic body plan?

• Are the pathways for building biological
pattern conserved among distant species?

OUTLINE

18.1 The logic of building the body plan
18.2 Binary fate decisions:

the germ line versus the soma
18.3 Forming complex pattern:

the logic of the decision-making process
18.4 Forming complex pattern:

establishing positional information
18.5 Forming complex pattern: utilizing positional

information to establish cell fates
18.6 Refining the pattern
18.7 The many parallels in vertebrate

and insect pattern formation
18.8 The genetics of sex determination in humans
18.9 Do the lessons of animal development apply

to plants?
18.10 Genomic approaches to understanding

pattern formation

575

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576 Chapter 18 • The Genetic Basis of Development

CHAPTER OVERVIEW CHAPTER OVERVIEW Figure

The general body plan of an animal is a specific pat- (a)
tern of structures common to all members of its
species. Such body plans may be common to many very Embryo
different species: all mammalian species have four limbs,
whereas all insects have six. However, all mammals and (b)
insects must, in the course of their development, differ- Create
entiate the anterior from the posterior end and the dor- asymmetries
sal from the ventral side. Eyes and legs always appear in at poles.
the appropriate places. The basic body plan of a species
appears to be quite robust — that is, the internal genetic (c)
program produces the same body plan within a broad
range of environmental conditions. We should not for- Create a chemical
get, however, that the study of the genetic determina- longitude and
tion of these basic developmental processes does not latitude system.
provide an explanation of the phenotypic differences be-
tween individual members of a species. This chapter (d)
focuses on the processes that underlie pattern forma-
tion, the construction of complex biological form. These Subdivide
processes are dictated by a genetic developmental pro- territory into
gram for the basic body plan. broad domains.

This chapter will show that the production of the (e)
body plan is a step-by-step process in which the outline
of the major body subdivisions is first painted with Create finer
broad strokes and then refined until fine-grained pattern subdivisions and
is finally laid down. The general types of steps in the de- commit them to
velopment of a Drosophila embryo are summarized in memory.
Figure 18-1. Substances are laid down at the poles of the
egg by the maternal parent, and these substances give Figure 18-1 An overview of developmental strategies. The
rise to gradients in both the anterior – posterior and the mother’s tissue deposits material that creates asymmetries,
dorsal – ventral axes. Interaction of these gradients leads which generates gradients that define several broad areas.
to broad fields of gene expression. These patterns are re- Subsequent gene interaction subdivides these areas.
membered through cell division, and so all descendant These commitments are “remembered.”
cells are programmed to that developmental fate. Subse-
quent gene signaling, both within and between cells, sets
up finer regions of gene expression.

This approach to building pattern is sensible, be-
cause it is well set up to compensate for errors. Any ad-
justments needed to correct errors are carried out as the
system progresses. First, a few different populations of
cells are made, and each population is subdivided in
step-by-step fashion into a larger and larger set of dis-
tinct cell types, eventually forming functional tissues and
organs. Often, cell proliferation is going on concomi-
tantly. At each step in the process, there is an opportu-
nity to adjust cell types and cell numbers if the preced-
ing step didn’t work quite right. Only after midcourse
evaluations and corrections have been executed can the
system proceed to the next step.

Understanding the development of a pattern re-
quires understanding all the alternative routes through a
network of developmental pathways, but with particular
focus on the key points — the switches that allow direc-
tion to specific routes through the network. The devel-

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18.1 The logic of building the body plan 577

opment of a pattern includes points at which choices what times in the course of development the gene is
must be made: some are like toggle switches and others transcribed and how much of the gene product is syn-
are analogous to the preset buttons on a radio. Toggle thesized. From that point of view, all developmental
switches have only two states: ON or OFF. Preset but- programming is determined by the regulatory informa-
tons allow the selection of a single option among many tion encoded in DNA. We can look at the genome as a
alternative states. All of the molecular elements that parts list of all the gene products (RNAs and polypep-
constitute choice points have been discussed in earlier tides) that can possibly be produced and as an instruc-
chapters. The challenge of pattern formation is to under- tion manual of when, where, and how much of these
stand how these molecular elements are interconnected products are to be expressed. Thus, one aspect of devel-
in a coherent and reproducible way to build the same opmental genetics is to understand how this instruction
basic body plan in every member of a species. manual operates to send cells down different develop-
mental pathways, ultimately producing a large constella-
In genetic circuits, the ultimate effect of these tion of characteristic cell types (refer to Figure 18-1).
choice points is to modulate the activity of a constella-
tion of genes that together give a cell its defining devel- This is not all that there is to the production of cel-
opmental characteristics — the kinds of cell types to lular diversity during development, however. How are
which it can give rise. A body plan is produced by wiring these different cell types deployed coherently and con-
the gene transcriptional apparatus in a cell in such a structively? In other words,
way that it communicates its decisions to adjacent cells.
In this way, each cell can share with its neighbors infor- • how do they become organized into organs and
mation concerning the status of its developmental deci- tissues?
sions. As a result of this information sharing, the entire
cell population can coordinate a community effort, to • how are those organ systems and tissues organized
ensure that all of the necessary cell types are represented into an integrated, coherently functioning individual
in the proper spatial deployment for building the tissues, organism?
organs, and appendages of the mature body plan. Thus,
this chapter will not present new types of molecular We shall explore these questions by examining the for-
functions. Rather, we will encounter the same cast of mation of the basic animal body plan.
characters that take part in gene regulation and intercel-
lular signaling but in more highly integrated and coordi- In the elaboration of the body plan, at some stage a
nated contexts. This should not be surprising, because cell must commit to a specific cell fate — that is, differ-
one of the basic themes of biology is that natural selec- entiate to become one particular kind of cell. The com-
tion mixes and matches a limited set of existing tools to mitment has to make sense in light of the cell’s location;
solve new problems, such as the construction of com- it would be neither useful for a cell at the body’s tail end
plex body plans. to become a brain cell, for example, nor helpful to have
lens cells develop in the location of the retina. Because
18.1 The logic all organs and tissues are made up of many cells, the
of building the body plan structure of an organ or tissue requires a cooperative di-
vision of labor among the participating cells. Thus, some-
In all higher organisms, life begins as a single cell, the how, cell position must be identified, and fate assign-
newly fertilized egg. It reaches maturity as a population ments must be parceled out within a cooperating group
of thousands, millions, or even trillions of cells combine of cells; such a group of interacting cells is called a devel-
into a complex organism with many integrated organ opmental field.
systems. The goal of developmental biology is to unravel
the fascinating and mysterious processes that achieve A cell’s position is generally established through
the transfiguration of egg into adult. Because we know protein signals that emanate from a specific position
more about development in animal systems than in within the initial one-cell zygote or from one or more
plants or multicellular fungi, we shall focus our atten- cells clustered within a developmental field. Just as we
tion initially on animals. need longitudes and latitudes to navigate on earth, a cell
needs information specifying its location within a devel-
Each cell has a protein profile consisting of the types opmental field. This information is called positional in-
and amounts of proteins that it contains. In a multicellu- formation. By using this information, a cell can execute
lar organism, a cell’s protein profile is the end result of a the developmental program appropriate to that location.
series of decisions that determine the “when, where, and When that positional information has been received,
how much” of gene expression. Thus, for a particular generally a few different precursor cell types are created
gene, a geneticist wants to know in which tissues and at within a field. Through further processes of cell division
and decision making, a population of cells with the nec-
essary final diversity and spatial distribution of fates will
be established.

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578 Chapter 18 • The Genetic Basis of Development

These further processes — fate refinement — can 18.2 Binary fate decisions:
be of two types. Sometimes, through asymmetrical di- the germ line versus the soma
visions of one of the precursor types of cells, descen-
dants are created that have received different regula- The toggle-switch type of decision making results in one
tory instructions and therefore become committed to of two outcomes; hence it is called a binary fate deci-
different fates. This mechanism for partitioning fates sion. How do toggle switches work in cells? We are in-
can be thought of as cell lineage dependent. Alterna- terested in the processes that subdivide the embryo into
tively, such fate decisions are made by “committee”; different cellular populations. One key binary decision
that is, the fate of a cell depends on input from neigh- is between becoming either part of the germ line (the
boring cells and feed-back to them. This mechanism gamete-forming cells) or the soma (everything else).
for partitioning fates can be thought of as neighborhood Once this separation occurs, it is irreversible. Germ cells
dependent. The cell-neighborhood-dependent mecha- do not contribute to somatic structures. Somatic cells
nisms provide for a certain flexibility so that an organ- cannot form gametes, and thus their descendants never
ism can compensate for unscripted events such as the contribute genetic material to the next generation. Let’s
accidental death of a cell. If some cells are lost by acci- examine the toggle switch that creates this decision.
dent, the cell-to-cell communication system is used to
reprogram the surviving neighbors to divide and divert In the cases that are best understood, the germ line
a subset of their descendants to adopt the fates of the versus soma decision takes advantage of underlying cel-
deceased cells. Indeed, the complete regeneration of sev- lular asymmetries. From the time that an oocyte is fertil-
ered limbs, as occurs in some animals such as starfish ized, special regulatory molecules are tethered to one
or amphibians, depends on neighborhood-dependent end of the cell. Hence, after division, only a subset of the
mechanisms. progeny cells acquire these regulatory molecules in their
cytoplasms. That subset of cells becomes the germ line.
MESSAGE Cells within a developmental field must be Cells not acquiring these regulatory molecules develop
able to identify their spatial locations and make as soma. In other words, for this toggle switch, we can
developmental decisions in the context of the decisions being think of soma as the default, or OFF, state, and the germ
made by their neighbors. line as the shunted, or ON, state of the toggle switch.
Thus, we need to consider three questions:
Many developmental decisions have two distin-
guishable phases: (1) establishing the decision and • What is the nature of the underlying asymmetrical
(2) maintaining it. Establishing the decision, as previ- cellular structures?
ously stated, is analogous to flipping a toggle switch or
pressing a button on a radio. Maintenance entails set- • How do regulatory molecules accumulate at one end
ting up a memory system that permanently locks a cell of these structures?
and its descendants into the “on” or “off” position with
regard to a specific decision. Making a developmental • What are these regulatory molecules and how do
decision and subsequently remembering that decision they work?
through subsequent cell divisions are keys to the com-
mitment of cells to their fates. Let’s begin by considering the underlying cellular
asymmetries that are exploited by the germ-line-
The process of commitment to a particular fate is a determination system — the asymmetrical components
gradual one. A cell does not go in one step from being of the cytoskeleton, the “roadways” and “girders” that
totally uncommitted, or totipotent, to becoming ear- traverse the cell’s cytoplasm and give the cell its shape
marked for a single fate. Each major patterning deci- and structure.
sion is a series of events in which cells are commited to
their fates step by step. Thus, if we examine a cell lin- The cytoskeleton of the cell
eage — that is, the family tree of a somatic cell and its
descendants — we see that parental cells in the tree are The cytoskeleton consists of several networks of very
less committed than their descendants. highly organized structural rods that run through each
cell. Each network is made up of one of three types of
MESSAGE As cells proliferate in the developing organism, rod: intermediate filaments, microfilaments, and micro-
decisions are made that specify more and more precisely tubules (Figure 18-2). Each type of rod has its own ar-
the options for the fate of cells of a given lineage. chitecture formed of unique protein subunits that act to
promote polymerization or disassembly of the rods. We
will focus on the last two types. Microfilaments are linear
polymers of the structural protein actin, whereas micro-
tubules are linear polymers of the two closely related

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18.2 Binary fate decisions: the germ line versus the soma

(a) Intermediate filaments (vimentin) (b) Microtubules (tubulin) (c) Microfilaments (actin)

Figure 18-2 Different cytoskeletal systems in the same cell. The distributions of
(a) the intermediate filament protein vimentin, (b) the microtubulin protein tubulin, and
(c) the microfilament protein actin are shown. [Courtesy of V. Small. Reprinted from H. Lodish,

D. Baltimore, A. Berk, S. L. Zipursky, P. Matsudaira, and J. Darnell, Molecular Cell Biology,
3d ed. Copyright Scientific American Books, 1995.]

structural proteins, ␣-tubulin and ␤-tubulin. Each type The cytoskeleton performs several tasks that are im-
of rod participates in higher-order networks by forming portant to the formation of asymmetries: control of the
reversible cross-links with neighboring rods of its type. location of the mitotic cleavage plane within the cell,
The different cytoskeletal systems often play different control of cell shape, and directed transport of molecules
biological roles. For example, actin microfilaments often and organelles within the cell. All of these tasks depend
play a primary role in cellular motility, such as muscle on the fact that the cytoskeletal rods are polar structures
contraction or the “crawling” movement of amebae, exhibiting directionality (Figure 18-3). That is, one end
whereas microtubules are the cytoskeletal elements of a cytoskeletal rod can be chemically distinguished
within the mitotic and meiotic spindles of cell division. from the other.

Figure 18-3 Polarity of subunits in an actin microfilament. An Exploiting the polarity
actin microfilament does not ordinarily have this appearance, of microfilaments and microtubules
but the microfilament has been coated with a protein that
binds in a fashion that reveals the underlying polarity of Microfilaments and microtubules are intracellular high-
the actin microfilament itself. [Courtesy of R. Craig. Reprinted ways, along which other molecules move up and down
throughout the cell. They are multilane two-way high-
from H. Lodish, D. Baltimore, A. Berk, S. L. Zipursky, P. Matsudaira, ways in the sense that different vehicles can travel on
and J. Darnell, Molecular Cell Biology, 3d ed. Copyright Scientific them in each of the two possible directions.
American Books, 1995.]
To understand how directional travel along cyto-
skeletal rods can occur, let’s consider microtubules.
Near the center of most cell types are found all the “Ϫ”
(minus) ends of the microtubules (Figure 18-4). This
location is called the microtubule organizing center
(MTOC). The “ϩ” (plus) ends of microtubules are lo-
cated at the periphery of the cell. Very much as an auto-
mobile uses the combustion of gasoline to create energy
that is then transduced into motion, special “motor”

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580 Chapter 18 • The Genetic Basis of Development

Figure 18-4 Fluorescence micrograph showing the distribution proteins hydrolyze ATP for energy that is utilized to
of tubulin in an interphase animal fibroblast. Notice that the propel movement along a microtubule. For example,
microtubules radiate out from a microtubule organizing center. a protein called kinesin is able to move in a Ϫ to ϩ
The negative (minus) ends of the microtubules are in the direction along microtubules, carrying cargos such as
center, and the positive (plus) ends are at the periphery of the vesicles from the center of the cell to its periphery
cell. [Courtesy of M. Osborn. Reprinted from H. Lodish, D. Baltimore, (Figure 18-5a and b). The motor — the part of the
kinesin protein that directly interacts with the micro-
A. Berk, S. L. Zipursky, P. Matsudaira, and J. Darnell, Molecular Cell tubule rod — is contained in the globular heads of the
protein (Figure 18-5c). The tail of kinesin is thought
Biology, 3d ed. Copyright Scientific American Books, 1995.] to be where the cargo is attached. These cargos might
be individual molecules, organelles, or other subcellular
particles to be towed from one part of the cell to an-
other. (Other motors exist that travel in the ϩ to Ϫ
direction on microtubules, and still other motors can
travel in each of the two directions on actin microfila-
ments.) What is the value of having multiple indepen-
dent trafficking systems? A part of the answer is division
of labor. Different cell components must be moved to
different places and at different rates.

(a) Vesicles

Microtubule

(b) 0.1 ␮m
(c)
Figure 18-5 Movement of vesicles along Vesicle Kinesin
microtubules. (a) A scanning electron tail
micrograph of two small vesicles
attached to a microtubule. (b) A diagram Kinesin
of how kinesin is thought to attach to
cellular cargos such as vesicles and to (–) (+)
move the cargos along the microtubule
in the minus-to-plus direction by using Microtubule stationary
the motor domain in the kinesin heads.
(c) A diagram of the kinesin protein Globular Heavy chain
showing the functions associated with heads Coiled ␣ helix
various parts of the molecule. [Part a
10 nm Light
from B. J. Schnapp et al., Cell 40, 1985, chain
455. Courtesy of B. J. Schnapp, R. D. Valle, ATP hydrolysis;
M. P. Sheetz, and T. S. Reese. All parts: binding to Binding to
Reprinted from H. Lodish, D. Baltimore, microtubules transported
A. Berk, S. L. Zipursky, P. Matsudaira, and vesicle
J. Darnell, Molecular Cell Biology, 3d ed.
Copyright Scientific American Books, 1995.]

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18.2 Binary fate decisions: the germ line versus the soma 581

MESSAGE The cytoskeleton serves as a highway system P0*
for the directed movement of subcellular particles and
organelles. Hypodermis AB P1*
Neurons P2*
Determining germ-line cells Pharynx (anterior) P3*
through cytoskeletal asymmetries
Intestine EMS
In many organisms, a visible particle is asymmetrically Body-wall muscle
distributed to the cells that will form the germ line. Pharynx (posterior)
These particles are called P granules in Caenorhabditis
elegans, polar granules in Drosophila, and nuage in frogs. Hypodermis C
They are thought to be transport vehicles that ride on Neurons
specific cytoskeletal highways to deliver attached regula- Body-wall muscle
tory molecules, which are the germ-cell determinants, to
future germ-line cells. In C. elegans and Drosophila, the Body-wall muscle D P4*
evidence relating germ-line determination to mecha-
nisms that depend on the cytoskeleton is particularly Germ-line
strong. Let’s consider each of these cases in turn and cells
then review the features thay they have in common.
Figure 18-6 Early divisions of the C. elegans zygote. The
DETERMINING THE C. ELEGANS GERM LINE The mature cell types that arise from the various daughter cells of
early cell divisions of the C. elegans zygote provide an the early divisions are indicated. Note that the entire germ line
example of how cytoskeletal asymmetries help form the comes from the P4 cell. Each of the P cell divisions indicated
germ line. One of the favorable properties of C. elegans by an asterisk is asymmetrical, and each of the posterior
as an experimental system is that the same pattern of daughter cells inherits all the P granules, which are thought to
cell divisions is present from one worm to another — and be germ-line determinants in the worm. The letters (for
this pattern can be readily followed under the micro- example, AB, EMS) are symbols for names of daughter cells.
scope. A lineage tree can then be constructed that traces
the descent of each of the thousand or so somatic cells to take advantage of an asymmetrical mark (the point of
of the worm. fertilization) to begin to create pattern.

Because they are traceable, every somatic cell in the The P granules, having been delivered to the zy-
worm is given a name. Fertilization in C. elegans initially gote’s posterior side, end up in the P1 cell. That cell di-
produces a one-cell zygote called the P0 cell. It is an el- vides, also asymmetrically, again producing a larger, ante-
lipsoidal cell that divides asymmetrically across its long rior cell and a smaller, posterior P cell, called P2. This
axis to produce a larger, anterior AB cell and a smaller, pattern of asymmetrical anterior – posterior cell divisions
posterior P1 cell (Figure 18-6). This division is very im- continues, producing a series of P cells as the more pos-
portant in that it sets up specialized roles for the descen- terior of the two progeny cells produced at each succes-
dants of these first two cells. Descendants of the AB cell sive cell division. The posterior descendants of P0 (P1, P2,
will form most of the skin of the worm (the hypoderm) and so forth) each acquire , the P granules. The P4 cell
and most of the nervous system, whereas descendants of becomes the germ line of the worm — all other cells are
the P1 cell will give rise to most of the muscles, all of the somatic. P granules depend on microfilaments to carry
digestive system, and the entire germ line. them to their original location in the P0 cells. When micro-
filaments in the P0 cell are disrupted by drugs, P granules
The key to the formation of the germ line is the end up distributed evenly between the two progeny cells.
presence of fluorescent particles called P granules. Before (Presumably because other fate determinants also are
fertilization, P granules are distributed uniformly through- abnormally distributed owing to the actin disruption,
out the oocyte cytoplasm. The event of sperm entry itself the resulting embryos are quite “confused” and die as
marks the anterior side of the future embryo, causing a masses of cells that look nothing like normal worms.)
reorganization of the actin microfilament network. This
reorganized network delivers the P granules to the poste- DETERMINING THE D. MELANOGASTER GERM LINE
rior side of the cytoplasm of the newly formed zygote. The cytoskeleton is also exploited in germ-line determi-
This phenomenon, in which fertilization causes a reorga- nation in early Drosophila development. Here, the regu-
nization of the actin cytoskeletal network in a manner of latory molecules are the polar granules, which appear under
a few seconds, occurs in many animals including some a light microscope as dense particles. During oogenesis
vertebrates such as amphibians. It is a very powerful way in the ovary of the mother, the polar granules become
tethered to the posterior pole of the oocyte. They re-
main in this location throughout early embryogenesis.
An unusual feature of early Drosophila development is
that the first 13 mitoses are nuclear divisions without

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582 Chapter 18 • The Genetic Basis of Development

Pole cells types. In other words, once metazoan evolution “in-
vented” a basic “dance” to form complex pattern, it ap-
Somatic plied that “innovation” as a solution to a great many
nuclei problems in embryogenesis and organogenesis.

Plasma What are the choreographed steps of this process?
membrane In outline form (Figure 18-8), they are:

Yolk • Create a population of developmentally identical
cells (part a in Figure 18-8).
Syncitium
(a) (b) • Create an asymmetry within that population (part b).

Figure 18-7 Germ-line formation at the syncitial stage of the • Exploit the asymmetry to set up a chemical
early Drosophila embryo. (a) A scanning electron micrograph of concentration gradient within the population of
a Drosophila embryo with the egg shell removed. Note that the cells (part c).
pole cells (the cap of cells on top of the embryo) lie outside
the somatic syncitium. (b) A diagram of a longitudinal section (a) Formation of asymmetries
through the embryo in part a, showing that the germ-line Symmetrical imposed by cytoskeleton
cells — the pole cells — have formed, whereas the soma still precursor
consists of syncitial nuclei. [Modified from F. R. Turner and cell Positional Binary decision
information Soma Germ line
A. P. Mahowald, Developmental Biology 50, 1976, 95.] (b) established

any cell division, making the early embryo a synci- A Chemical
tium — a multinucleate cell. At nuclear division 9, a few (c) gradients
nuclei migrate to the posterior pole where the polar
granules are tethered. The plasma membrane of the P D V
syncitial egg then moves inward at the posterior pole to (d) Primary gene expression
surround each nucleus, incorporating some of the polar
granules into the cytoplasm that surrounds the nucleus. domains established
This action creates the pole cells, the first mononucleate
cells formed in the embryo. The pole cells will uniquely Broad domains
form the fly’s germ line (Figure 18-7). of cardinal
genes
How do the polar granules get tethered to the pos-
terior pole of the oocyte? Again, one of the cytoskeletal Refinement
networks carries the granules to the posterior pole and Cell–cell signaling
provides the rods to which the granules attach. In con-
trast with C. elegans, in which the P granules attach to Fine-grained
actin-based microfilaments, here the tubulin-based expression
microtubules provide the essential trafficking system. (e) domains

18.3 Forming complex pattern: Repeating units Dorsal–ventral Positive
the logic of the (segments) cell layers feedback
decision-making process
ioops
The major pattern-formation decisions within the soma
typically require a choreographed set of steps that lead Figure 18-8 Choreographed steps of pattern formation in
to the establishment of all the necessary cell types with Drosophila. (a) The cytoskeleton imposes an asymmetry on
the appropriate spatial distribution. In the newly fertil- the egg. (b) A germ line is separated off by cell division.
ized animal embryo, this set of steps is used to establish (c) Gradients of soluble chemicals are laid down on the A – P
the overall body plan. Later on in development, nests of and D – V axes, stimulating gene action and (d) leading to
cells are set aside to become the various organs and ap- broad bands. (e) Within these bands more complex gene
pendages of the body. When first set aside, each of these interactions subdivide the broad regions.
nests of cells undergoes a similar choreography to subdi-
vide the initial population into its final pattern of cell

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18.4 Forming complex pattern: establishing positional information 583

• Create a system that can mount a differential sive maternal-effect mutation “m,” in a cross of an m/m
response to that local concentration, thereby creating female with a ϩ/ϩ male, all the embryos are abnormal
multiple cell states (part d). even though they are genetically normal (ϩ/m). See the
Model Organism box, Drosophila, for a description of
• Create mechanisms for cross talk between the cells of how these mutants were recovered and analyzed.
different states, such that cells can take on additional
cell states, refine their positions, and adjust their Cytoskeletal asymmetries
numbers (part e). and the Drosophila anterior – posterior axis

• As key decisions are made, develop systems that allow We have seen that the Drosophila germ line is estab-
these decisions to become fixed in the molecular lished when specific regulatory molecules, anchored to
memory banks of a cell and its descendants. microtubules, are localized to one area of the embryo. The
same is also true for the determination of cell type from
We will focus largely on the construction of the the front to the back of the organism (the anterior –
basic Drosophila body plan in the embryo as a well- posterior, or A – P, axis). Positional information along the
understood application of the logic of this choreography. A – P axis of the syncitial Drosophila embryo is initially
Specifically, we will look at the establishment of the two established through the creation of concentration gradi-
major body axes of the embryo: anterior – posterior ents of two transcription factors: the BCD and HB-M
(head to tail) and dorsal – ventral (back to front). Devel- proteins, which are products of two maternal-effect
opment of these two axes leads to the following events: genes. These factors interact to generate different patterns
of gene expression along the axis. The BCD protein,
• Subdivision of the embryonic anterior – posterior axis encoded by the bicoid ( bcd) gene, is distributed in a
into a series of distinct units called segments, or steeper gradient in the early embryo, whereas the HB-M
metameres, and assignment of distinct roles to each protein, encoded by one of the mRNAs of the hunch-
segment according to its location in the developing back (hb) gene, is distributed in a shallower but longer
animal. Here, a developing cell can adopt one of gradient (Figure 18-9). Both concentration gradients
many possible cell states, corresponding to the 14
different segments, and, even within each segment, Concentration BCD
multiple cell states will be established. Thus, this
decision is much more complex than simply the HB-M
yes – no decision of germ line versus soma.
Anterior A–P location in embryo Posterior
• Subdivision of the embryonic dorsal – ventral axis into pole pole
the outer, middle, and inner sheets of cells, called the
germ layers (not to be confused with germ lines), and BCD P
assignment of distinct roles to each of these layers. Here A
again, we are looking at the establishment of a wide
variety of cell types within each of the three primary HB-M P
germ layers: the cells of the epidermis, nervous, A
circulatory, immune, musculature, respiratory, digestive,
extractory, and reproductive systems, and so forth. Figure 18-9 Concentration gradients of BCD and HB-M proteins.
The BCD gradient is steeper, and the BCD protein is not
18.4 Forming complex pattern: detectable in the posterior half of the early Drosophila embryo.
establishing positional information The HB-M gradient is shallower, and the HB-M protein can be
detected well into the posterior half of the embryo.
The first steps in the process of pattern formation take
place in the oocyte while it is developing in the ovary of
the mother. A genetic circuit is activated that leads to the
maternal expression of the genes responsible for creating
the asymmetries and the chemical concentration gradi-
ents of positional information that the embryo uses for its
spatial coordinates. The existence of the maternal effect
(and many other aspects of Drosophila development) be-
came known from studies on mutants. Specifically, geneti-
cists discovered mutations in maternal-effect genes,
which produce anterior – posterior axis abnormalities only
when expressed in the mother. Hence, if we label a reces-

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584 Chapter 18 • The Genetic Basis of Development

MODEL ORGANISM Drosophila skeleton of such a mutant larva mirrors the mutant
fates assigned to subsets of the epidermal cells and
Mutational Analysis can thus identify genes worthy of detailed analysis.
of Early Drosophila Development
Researchers, most notably Christiane Nüsslein-
The initial insights into the genetic control of pattern Volhard, Eric Wieschaus, and their colleagues, have
formation emerged from studies of the fruit fly performed extensive mutational screens, essentially
Drosophila melanogaster. Drosophila development has saturating the genome for mutations that alter the
proved to be a gold mine to researchers because devel- A – P or D – V patterns of the larval exoskeleton. These
opmental problems can be approached by the use of mutational screens identified two broad classes of
genetic and molecular techniques simultaneously. genes that affect the basic body plan: zygotically act-
Let’s consider the basic genetic and molecular tech- ing genes and maternal-effect genes (see diagrams).
niques that are employed. The zygotically acting genes are those genes whose ex-
pression in the zygote is the sole source of their gene
The Drosophila embryo has been especially im- products. They are part of the DNA of the zygote it-
portant in understanding the formation of the basic self and are the “standard” sorts of genes that we are
animal body plan. One important reason is that an
abnormality in the body plan of a mutant is easily Zygotically acting genes
identified in the larval exoskeleton in the Drosophila P: + / m& × + / m(
embryo. The larval exoskeleton is a noncellular struc-
ture, made of a polysaccharide polymer, called chitin, F1:
that is produced as a secretion of the epidermal cells +/++/m+/m m/m
of the embryo. Each structure of the exoskeleton is
formed from epidermal cells or cells immediately un- Normal Mutant
derlying that structure. With its intricate pattern of phenotype phenotype
hairs, indentations, and other structures, the ex- Maternal-effect genes
oskeleton provides numerous landmarks to serve as
indicators of the fates assigned to the many epider- P: + / m& × + / +( + / m& × m / m( m / m& × + / +( m / m& × m / +(
mal cells. In particular, there are many distinct
anatomical structures along the anterior – posterior F1: +/mm/m +/m+/m m/+m/m
(A – P) and dorsal – ventral (D – V) axes. Furthermore, +/++/m
because all the nutrients necessary to develop to the All normal All mutant All mutant
larval stage are prepackaged in the egg, mutant em- All normal phenotype phenotype phenotype
bryos in which the A – P or D – V cell fates are drasti- phenotype
cally altered can nonetheless develop to the end of
embryogenesis and produce a mutant larva. The exo- The genetic distinction between recessive zygotically acting
and maternal-effect mutations.

have their high points at the anterior pole. In somewhat incorporate a higher concentration of the diffusing BCD
different ways, the gradients of both these proteins de- protein than do those farther away; this difference re-
pend on the diffusion of protein from a localized origin: sults in the steep BCD protein gradient (Figure 18-10b).
each protein is produced through localized translation of
an mRNA species, one tethered to microtubules at the The origin of the HB-M protein gradient is more
anterior pole of the syncitial embryo, and the other teth- complex. Unlike the bcd mRNA, the hb-m RNA is
ered at the posterior pole.
Figure 18-10 Photomicrographs showing the expression of
The origin of the BCD gradient is quite straightfor- localized A – P determinants in the embryo. (a) By in situ
ward. The maternal bcd mRNA, packaged during oogen- hybridization to RNA, the localization of bcd (bicoid) mRNA to
esis into the developing oocyte, is tethered to the Ϫ (mi- the anterior (left) tip of the embryo can be seen. (b) By
nus) ends of microtubules, which are located at the antibody staining, a gradient of Bicoid protein (brown stain)
anterior pole (Figure 18-10a). Translation of BCD pro- can be visualized, with its highest concentration at the anterior
tein begins midway through the early nuclear divisions tip. (c) Similarly, nanos (nos) mRNA localizes to the posterior
of the embryo. The protein diffuses in the common (right) tip of the embryo, and (d) NOS protein is in a gradient
cytoplasm of the syncitium. Because the protein is a with a high point at the posterior tip. [Parts a and b from
transcription factor, it contains signals that direct it to
the nuclei. Those nuclei nearer to the anterior pole C. Nüsslein-Volhard, Development, Suppl. 1, 1991, 1. Parts c and
d provided by E. R. Gavis, L. K. Dickinson, and R. Lehmann,
then of Massachusetts Institute of Technology.]

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18.4 Forming complex pattern: establishing positional information 585

used to thinking about. Recessive mutations in zygoti- transformation in Drosophila (see Chapter 11). A
cally acting genes elicit mutant phenotypes only in ho- cloned pattern-formation gene is mutated in a test
mozygous mutant animals. The alternative category — tube and put back into the fly. The mutated gene is
the maternal-effect genes — has already produced its then analyzed to see how the mutation alters the
gene products in the ovary of the mother. Thus, the gene’s function.
maternal-effect proteins are already present in the
oocyte at fertilization. In maternal-effect mutations, Using Knowledge from One Model Organism to
the phenotype of the offspring depends on the geno- Fasttrack Pattern-Formation Research in Others
type of the mother, not of the offspring, because the With the discovery that there are numerous homeo-
source of the gene products is the mother’s genes. box genes within the Drosophila genome, similarities
A recessive maternal-effect mutation will produce among the DNA sequences of these genes could be
mutant animals only when the mother is a mutant exploited in treasure hunts for other members of the
homozygote. homeotic gene family. These hunts depend on DNA
base-pair complementarity. For this purpose, DNA hy-
Genes that contribute to the Drosophila body plan bridizations were carried out under moderate strin-
can be cloned and characterized at the molecular level gency conditions, in which there could be some mis-
with relative ease. Any Drosophila gene with a well- match of bases between the hybridizing strands
established chromosomal map location can be cloned without disrupting the proper hydrogen bonding of
by using recombinant DNA techniques such as those nearby base pairs. Some of these treasure hunts
described in Chapter 11. The analysis of the cloned were carried out in the Drosophila genome itself, in
genes often provides valuable information on the func- looking for more family members. Others searched for
tion of the protein product — usually by identifying homeobox genes in other animals, by means of zoo
close relatives in amino acid sequence of the encoded blots (Southern blots of restriction-enzyme-digested
polypeptide through comparisons with all the protein DNA from different animals), by using radioactive
sequences stored in public databases. In addition, one Drosophila homeobox DNA as the probe. This ap-
can investigate the spatial and temporal pattern of ex- proach led to the discovery of homologous homeobox
pression of (1) an mRNA, by using histochemically sequences in many different animals, including hu-
tagged single-stranded DNA sequences complementary mans and mice. (Indeed, it is a very powerful ap-
to the mRNA to perform RNA in situ hybridization, or proach for “fishing” for relatives of almost any gene in
(2) a protein, by using histochemically tagged antibod- your favorite organism.) Some of these mammalian
ies that bind specifically to that protein. homeobox genes are very similar in sequence to the
Drosophila genes.
Extensive use is made of in vitro mutagenesis
techniques, as well. P elements are used for germ-line

(a) Bicoid mRNA (b) Bicoid protein
(c) nos mRNA (d) NOS protein

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586 Chapter 18 • The Genetic Basis of Development

uniformly distributed throughout the oocyte and the within the untranslated regions of the mRNA 3Ј to the
syncitial embryo. However, translation of hb-m mRNA translation-termination codon (3Ј UTRs). The 3Ј UTR
is blocked by a repressor protein — the NOS protein localization sequences of bcd mRNA are bound by a
product. This protein is encoded by the nanos (nos) protein that can also bind the minus ends of the micro-
gene. In the mother, nos mRNA is deposited at the pos- tubules. In contrast, the 3Ј UTR localization sequences
terior pole in the oocyte, through its association with of nos mRNA are bound by a protein that can also bind
the ϩ (plus) ends of microtubules (Figure 18-10c). At the plus ends of microtubules.
the time of its translation, NOS protein becomes dis-
tributed by diffusion in a gradient opposite that of How can we demonstrate that the 3Ј UTRs of the
BCD. The NOS gradient has a high point at the poste- mRNAs are where the localization sequences reside? This
rior pole and drops down to background levels in the has been determined in part by “swapping” experiments.
middle of the A – P axis of the embryo (Figure 18-10d). A synthetic transgene is constructed that produces an
NOS protein inhibits translation of hb-m mRNA, and mRNA with the 5Ј UTR and protein-coding regions of
the level of inhibition is proportional to the concentra- the normal nos mRNA glued to the 3Ј UTR of the normal
tion of NOS protein, producing the shallow gradient of bcd mRNA. When inserted into the fly genome, this fused
HB-M protein from the anterior to the posterior of the nos – bcd mRNA will be localized at the anterior pole of
embryo. the oocyte. This localization causes a double gradient of
NOS: one from anterior to posterior (due to the trans-
MESSAGE Localization of mRNAs within a cell is gene’s mRNA) and one from posterior to anterior (due to
accomplished by anchoring the mRNAs to one end of the normal nos gene’s mRNA). This procedure produces a
polarized cytoskeleton chains. very weird embryo, with two mirror-image posteriors and
no anterior (Figure 18-11). This double-abdomen embryo
How do the maternal bcd and nos mRNAs get teth- arises because NOS protein is now present throughout
ered to opposite ends of the polarized microtubules of the embryo and represses the translation of hb-m mRNA
the oocyte and early embryo? The answer is that there (it also represses translation of bcd mRNA, although it is
are specific microtubule-association sequences located not clear that repression of bcd mRNA is its normal func-
tion in wild-type animals).

(a) Translated 3Ј UTR Figure 18-11 Effect of
5Ј UTR region bcd replacement of the 3Ј UTR of
the nanos mRNA with the 3Ј UTR
nos of the bicoid mRNA on mRNA
localization and embryonic
(b) Wild type nos-bcd 3Ј UTR phenotypes. (a) The structure of
transgene the nos – bcd 3Ј UTR transgene.
(b) The effects on embryonic
Exoskeletons development of the transgene.
The embryos and larva in the
nos nos ϩ transgene left-hand column are derived
mRNA mRNAs from wild-type mothers. Those
Probe ϭ nos cDNA in the right-hand column are
bcd ϩ transgene derived from transgenic mothers.
bcd mRNAs All embryos are shown with
mRNA anterior to the left and posterior
Probe ϭ bcd cDNA to the right. The exoskeletons
are shown for comparison. The
transgene causes a perfect
mirror-image double abdomen.
In the embryo from a transgenic
mother, mRNAs coding for NOS
protein are now present at both
poles of the embryo. NOS
protein will inhibit translation
of hb-m mRNA (and, actually,
of bcd mRNA as well).

[From E. R. Gavis and R. Lehmann,

Cell 71, 1992, 303.]

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18.4 Forming complex pattern: establishing positional information 587

MESSAGE The positional information of the Drosophila Maternal BCD protein Position of cephalic furrow
A – P axis is generated by protein gradients. The gradients dosage cf
ultimately depend on diffusion of newly translated protein of bcd +
from localized sources of specific mRNAs anchored by their 3Ј
UTRs to ends of cytoskeletal filaments. gene

Studying the BCD gradient 1X

How do we know that molecules such as BCD and HB-M 2X
contribute A – P positional information? Let’s consider the
example of BCD in detail. 3X

1. Genetic changes in the bcd gene alter anterior fates. 4X
Embryos derived from mothers that are
homozygous for bcd null mutants lack anterior Figure 18-13 Concentration of BCD protein affects A – P cell
segments (Figure 18-12). If, on the other hand, we fates. The amount of BCD protein can be changed by varying
overexpress bcd in the mother, by increasing the the number of copies of the bcdϩ gene in the mother. Embryos
number of copies of the bcdϩ gene from the normal derived from mothers carrying from one to four copies of bcdϩ
two to three, four, or more, we “push” fates that have increasing amounts of BCD protein. (Two copies is the
ordinarily appear in anterior positions into normal diploid gene dosage.) Cells that invaginate to form the
increasingly posterior zones of the resulting cephalic furrow (cf) are determined by a specific concentration
embryos, as shown in Figure 18-13. The position of of BCD protein. In the progression from one maternal copy to
the cephalic furrow (a normal feature of the four copies of bcdϩ, this specific concentration is present
embryo) serves as an example in Figure 18-13. more and more posteriorly in the embryo. Thus, the position of
These observations suggest that BCD protein exerts the cephalic furrow (marked dorsally by the arrow) arises
global control of anterior positional information. farther toward the posterior according to bcdϩ gene dosage.

2. The bcd mRNA can completely substitute for the [After W. Driever and C. Nüsslein-Volhard, Cell 54, 1988, 100.]
anterior determinant activity of the anterior
cytoplasm (Figure 18-14). If the anterior cytoplasm

Figure 18-12 Photomicrographs of the exoskeletons of larvae is removed from a punctured syncitial embryo,
derived from wild-type and bcd maternal-effect-lethal mutant anterior segments (head and thorax) are lost (not
mothers. These photomicrographs are dark-field, and so dense shown). Injection of anterior cytoplasm from
structures appear white, as in a photographic negative. Note another embryo into the anterior region of
the bright, segmentally repeated denticle bands present on the anterior-cytoplasm-depleted embryo restores
the ventral side of the embryo. Maternal genotypes and larval normal anterior segment formation, and a normal
phenotypes (and class of mutation) are as follows: (left) wild larva is produced. Similarly, synthetic bcd mRNA
type, normal phenotype; (right) bcd (bicoid), anterior head and can be made in a test tube and injected into the
thoracic structures missing (anterior). [From C. H. Nüsslein- anterior region of an anterior-cytoplasm-depleted
embryo. Again, a normal larva is produced. In a
Volhard, G. Frohnhöfer, and R. Lehmann, Science 238, 1987, 1678.] control experiment, transplantation of cytoplasm
from middle or posterior regions of a syncitial
embryo does not restore normal anterior formation.
Thus, the anterior determinant should be located
only at the anterior end of the egg. As already
discussed, this is exactly where bcd mRNA is found.

3. As has already been described (see Figure 18-10b),
the BCD protein shows the predicted asymmetrical
and graded distribution required to fulfill its role
of establishing positional information.

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588 Chapter 18 • The Genetic Basis of Development

(a) (b) (c) bcd + (D – V) axis in the early Drosophila embryo by the DL
bcd bcd mRNA protein. The immediate effect of the D – V positional in-
formation is to create a gradient of DL protein activity
bcd + bcd in cells along the D – V axis. The DL protein is a tran-
scription factor encoded by the dorsal (dl) gene. It exists
He He in two forms: (1) active transcription factor located in
the nucleus and (2) inactive protein located in the cyto-
Th Th plasm, where it is sequestered in a complex bound to
Ab the CACT protein encoded by the cactus (cact) gene. A
concentration gradient of active DL protein determines
Ab Ab cell fate along the D – V axis. Both dl mRNA and inac-
tive DL protein are distributed uniformly in the oocyte
Figure 18-14 The bcd “anteriorless” mutant phenotype can be and the very early embryo. However, late in the syncitial
rescued by wild-type cytoplasm or purified bcd؉ mRNA. (a) In embryo stage, there develops a gradient of active DL
embryos derived from bcd mothers, the anterior (head and protein, with its high point in cells at the ventral midline
thoracic) segments do not form, producing the anteriorless of the embryo (Figure 18-15).
phenotype. (b) If anterior cytoplasm from an early wild-type
donor embryo is injected into the anterior of a recipient embryo How is the gradient of active DL protein gener-
derived from a bcd mutant mother, a fully normal embryo and ated? By a rather complex set of gene interactions. The
larva are produced. Cytoplasm from any other part of the donor key events take place in the developing egg (Figure
embryo does not rescue. (c) Injection of bcdϩ mRNA into the 18-16a), well before fertilization. At this time an inter-
anterior of an embryo derived from a bcd mutant mother also action takes place between the oocyte itself and the
rescues the wild-type segmentation pattern. (Ab ϭ abdomen; layer of surrounding somatic cells — the follicle cells,
He ϭ head; Th ϭ thorax.) [After C. H. Nüsslein-Volhard, which also make the eggshell (Figure 18-16b). The fol-
licle cells on the ventral side of the oocyte secrete some
G. Frohnhöfer, and R. Lehmann, Science 238, 1987, 1678.] proteins that activate a secreted precursor of the SPZ
ligand, encoded by the spaetzle (spz) gene. As a conse-
Cell-to-cell signaling quence, the activated SPZ ligands are concentrated on
and the Drosophila dorsal – ventral axis the ventral side of the oocyte, where they form a gradi-
ent that is highest at the ventral midline. On the inner
In the examples considered thus far, products inside the boundary of the eggshell is the vitelline membrane
cell determined the position of future body parts: these (Figure 18-17a). The SPZ ligand is sequestered in the
products were mRNAs or larger macromolecular assem- vitelline membrane until near the end of the syncitial
blies packaged into the oocyte. In many circumstances, stage of early embryogenesis, when it is released. Active
though, positional information depends on proteins out- SPZ ligand (with its highest concentration at the ven-
side the cell that have been secreted from a localized
subset of cells within a developing field. These secreted Dorsal midline
proteins diffuse in the extracellular space to form a con-
centration gradient of ligand that binds to receptors on Ventral midline
target cells. The ligand thus activates the target cells in
a concentration-dependent fashion through a receptor – Figure 18-15 Distribution of DL shown in a cross section of the
signal-transduction system. The level of signal outside cellular blastoderm-stage Drosophila embryo. The dorsal midline
a given target cell determines the level of signal trans- is at the top and the ventral midline is at the bottom. Note
duction and response to the signal inside the cell. The that the DL protein is in the nucleus (where it is active)
level of response inside the cell ultimately determines ventrally, throughout the cell laterally, and in the cytoplasm
the combination of genes whose transcription rates are (where it is inactive) dorsally. [After S. Roth, D. Stein, and
elevated or lowered in the target cell.
C. Nüsslein-Volhard, Cell 59, 1989, 1196.]
An example of such a mechanism for establishing
position is the establishment of the dorsal – ventral

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18.4 Forming complex pattern: establishing positional information 589

(a) Developing oocyte (a)

Follicle cells TOLL Dorsal midline
receptor
Oocyte Nuclei
SPZ
(b) Mature oocyte ligand
DL
protein Yolk
inside
Dorsal Perivitelline space nuclei
Oocyte nucleus
Plasma
Anterior Posterior membrane
Perivitelline
Polar fluid Ventral midline Vitelline
granules membrane
(b)
Oocyte plasma Eggshell
membrane

Ventral

Figure 18-16 Developing (a) and mature (b) oocyte. The follicle SPZ ligand TOLL
cells that built and surrounded the eggshell in part a have been Plasma membrane receptor
discarded in the mature oocyte, and the plasma membrane of TUB
the oocyte is interior to the eggshell. Note the polar granules
at the posterior end of the oocyte. [After C. Nüsslein-Volhard,

Development, Suppl. 1, 1991, 1.]

tral midline) then binds to the TOLL transmembrane re- CACT DL PLL ATP CACT
ceptor, encoded by the Toll gene, present uniformly in the Phosphorylation
oocyte plasma membrane (see Figure 18-17a). In a con- +
centration-dependent manner, the SPZ – TOLL complex Nucleus
triggers a signal-transduction pathway that ends up phos- DL
phorylating the inactive DL and CACT cytoplasmic pro- DL
teins of the DL – CACT complex (Figure 18-17b). Phos- Import
phorylation of DL and CACT causes conformational Regulation of
changes that break apart the cytoplasmic complex. The zygotic
free phosphorylated DL protein is then able to migrate
into the nucleus, where it serves as a transcription factor patterning
that activates genes necessary for establishing the ventral genes
fates. The crucial arrangement is the positioning of the
SPZ ligand near the ventral midline, which ensures that Figure 18-17 Signaling pathway leading to the gradient of
only the DL on the ventral side of the embryo is able to nuclear versus cytoplasmic localization of DL protein shown in
activate the genes that create structures for the ventral Figure 18-15. (a) A cross section of a Drosophila embryo
part of the body. showing the blastoderm cells inside the plasma membrane
and the space (perivitelline space) between the inside
MESSAGE Positional information can be established by boundary of the eggshell (vitelline membrane) and the plasma
cell-to-cell signaling through a concentration gradient of a membrane, where the active SPZ ligand is produced on the
secreted signaling molecule. ventral side of the embryo. (b) The SPZ ligand binds to the
TOLL receptor, activating a signal-transduction cascade
Other roles of the cytoskeleton through two proteins called TUB and PLL, leading to the
in establishing the body axes phosphorylation of DL and its release from CACT. DL then is
able to migrate into the nucleus, where it serves as a
The cytoskeleton can play other key roles in establishing transcription factor for D – V cardinal genes. [After H. Lodish,
the body axes. We will consider one example: establish-
ing the poles in the early Drosophila oocyte. D. Baltimore, A. Berk, S. L. Zipursky, P. Matsudaira, and J. Darnell,

Molecular Cell Biology, 3d ed. Copyright Scientific American

Books, 1995.]

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590 Chapter 18 • The Genetic Basis of Development

There is a certain “which came first, the chicken or ligand. The EGFR, the target of the EGF-like ligand, is
the egg?” aspect to tracing the earliest steps of establish- on the surface of all follicle cells. However, the EGFR
ing the body axes in any organism. For Drosophila axis will have its tyrosine kinase activated only where EGF-
formation, this is certainly the case. Even before the like ligand is concentrated — namely, in the vicinity of
events just described take place to set up gradients along the nucleus.
the A – P and D – V axes, something must distinguish the
front from the back and the top from the bottom of the So, the name of the game is to strategically move
oocyte. That something turns out to be the microtubules, the nucleus around the oocyte. Briefly, this movement
which act in two phases: the first phase gives the follicle is accomplished in a two-phase process (Figure 18-18).
cells overlying the posterior pole of the oocyte a differ- In the first phase, the microtubule network orients
ent identity from that of the follicle cells at the anterior with its plus ends at the prospective posterior pole of
pole; the second phase gives the dorsal follicle cells a dif- the early oocyte, dragging the oocyte nucleus to that
ferent identity from that of their ventral and lateral pole. Then, after EGFR activation in the posterior folli-
counterparts. Later on, instructive signals from these fol- cle cells, instructing them to be different from their an-
licle cells will create the asymmetries that have been terior counterparts, these follicle cells send a signal
outlined in the preceding sections. back to the oocyte that causes the microtubule net-
work to be reoriented perpendicularly, so that the plus
The early oocyte transcribes very few genes, but one ends of the network are at the prospective dorsal side
of these few is a gene that encodes an epidermal growth of the oocyte. The nucleus is dragged along and, be-
factor-like (EGF-like) ligand that acts by binding to a re- cause it is attached to the nucleus, so is the rough en-
ceptor tyrosine kinase called the EGF receptor (EGFR) doplasmic reticulum and hence the EGF-like signal.
and activating it. The EGF-like ligand is secreted to the This secreted signal then activates the EGFR in the fol-
nearest cell surface through the channels of the endo- licle cells overlying the nucleus, instructing them to be-
plasmic reticulum, which are located near the nucleus. come dorsal follicle cells.
Thus, if the nucleus is nearer to one side of the oocyte,
that side will secrete a higher concentration of EGF-like Figure 18-19 summarizes the two types of posi-
tional information that we have covered.

Gene interactions in Drosophila embryogenesis: (a) (b) A–P commitment
GRK Nucleus
www. ANIMATED ART Cell signaling in dorsal – ventral axis formation
EGFR
Signal

Oocyte Follicle
nucleus cell

Figure 18-18 Determination of posterior and anterior ends (c) D–V commitment
in the Drosophila embryo. (a) The cytoskeleton is
reorganized to create populations of posterior and dorsal Microtubules
follicle cells through EGF-like ligand and EGFR signaling Follicle cells
between the oocyte and the overlying follicle cells.
(The EGF-like ligand is called gurken (GRK) because
mutations in the gurken gene produce a phenotype like
little pickles.) (b) Microtubule reorganization delivers the
nucleus to the posterior pole, thereby delivering the EGF-
like signal to the posterior pole and creating the posterior
polar follicle cells. (c) These posterior polar follicle
cells send a signal back to the oocyte that causes the
microtubules to reorganize again, reversing their polarity
and bringing the nucleus and GRK to the dorsal anterior
sector of the oocyte, leading to the establishment of the
dorsal follicle cells. [After A. Gonzalez-Reyes, H. Elliott, and

D. St. Johnston, Nature 375, 1995, 657.]

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18.5 Forming complex pattern: utilizing positional information to establish cell fates 591

(a) Cell membrane (b) Cell sheet
(1) Cytoskeletal element (1) (developmental
field)
(2) (2)
Cell secreting
positional-
information signal

Localized mRNA Receptor for signal
for transcription (3)
factor
(3)

Newly translated (4)
transcription factor
Activated signal-
(4) transduction pathway

Direction of (5) High level Low-level activation
protein diffusion of transcription-
Low-level factor activation
activation

Figure 18-19 The two general classes of positional information. (a) Asymmetrical
organization of the cytoskeletal system permits localization of mRNA encoding a
transcription factor that will provide positional information. Translation of the anchored
mRNA will lead to diffusion of the newly translated transcription factor and the formation
of a transcription-factor gradient with a high point near the site of the mRNA. (b) Secretion
of a positional-information signaling molecule from a localized source (cell) activates the
signal-transduction apparatus and the target transcription factors according to the level
of signaling molecule that binds to its transmembrane receptor.

18.5 Forming complex pattern: field. What elements in the cell interpret the informa-
utilizing positional information tion in the gradient?
to establish cell fates
Given that positional information is given by a gra-
To develop correctly in a specific position, a cell needs dient of active transcription factor, we might expect that
to assess and respond to the local positional cues. To use the receivers are regulatory elements (enhancers and
a geographical analogy, having a system of longitudes silencers) of special genes. Then the protein products of
and latitudes is not sufficient; we also need equipment these special genes can begin the gradual process of
that can receive longitude and latitude information — specifying cell fate. This is exactly what we see. The
special instruments to read the positions of stars or re- genes targeted by the A – P and D – V transcription fac-
ceivers that can triangulate signals transmitted from ra- tors are genes expressed in the zygote and collectively
dio beacons. In the same way, the developmental known as the cardinal genes because they are the first
positional-information system requires that elements genes to respond to the positional information supplied
within the cell be able to interpret transmitted signals. by the oocyte. The cardinal genes of the A – P axis are
listed in (Table 18-1).
The initial interpretation
of positional information To see how these genes work, we need to review a
bit of Drosophila embryology. After 2 – 3 hours of devel-
As described earlier, two very different kinds of posi- opment, all somatic nuclei migrate to the surface of the
tional signal can be produced, one kind generated from egg, and the egg’s plasma membrane invaginates around
within the cell and the other kind diffusing from outside each nucleus and its cytoplasm (Figures 18-20 and
the cell. However, both lead to the same outcome: a gra- 18-21a). The result is in an embryonic stage called the
dient in the concentration of one or more specific tran- cellular blastoderm, a hollow spheroid with a wall that is
scription factors within the cells of the developmental one cell thick. A few hours later, this wall of cells has
undergone numerous foldings, outpocketings, and other
movements and now manifests the first signs of segmen-
tation. At the end of 10 hours of development, the embryo

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592 Chapter 18 • The Genetic Basis of Development

Table 18-1 Examples of Drosophila A – P Axis Genes That Contribute to Pattern Formation

Gene symbol Gene name Protein function Role(s) in early
development

hb-z hunchback-zygotic Transcription factor — zinc-finger protein Gap gene
Kr Krüppel Transcription factor — zinc-finger protein Gap gene
kni knirps Transcription factor — steroid receptor-type protein Gap gene
eve even-skipped Transcription factor — homeodomain protein Pair-rule gene
ftz fushi tarazu Transcription factor — homeodomain protein Pair-rule gene
opa odd-paired Transcription factor — zinc-finger protein Pair-rule gene
prd paired Transcription factor — PHOX protein Pair-rule gene
en engrailed Transcription factor — homeodomain protein Segment-polarity gene
ci cubitus-interruptus Transcription factor — zinc-finger protein Segment-polarity gene
wg wingless Signaling WG protein Segment-polarity gene
hh hedgehog Signaling HH protein Segment-polarity gene
fu fused Cytoplasmic serine/threonine kinase Segment-polarity gene
ptc patched Transmembrane protein Segment-polarity gene
arm armadillo Cell-to-cell junction protein Segment-polarity gene
lab labial Transcription factor — homeodomain protein Segment-identity gene
Dfd Deformed Transcription factor — homeodomain protein Segment-identity gene
Antp Antennapedia Transcription factor — homeodomain protein Segment-identity gene
Ubx Ultrabithorax Transcription factor — homeodomain protein Segment-identity gene

is already externally divided into 14 segments from ante- has developed a unique set of anatomical structures, cor-
rior to posterior — 3 head, 3 thoracic, and 8 abdominal responding to its special identity and role in the biology
segments (Figure 18-21b ). By this time, each segment of the animal. At the end of 12 hours, distinct organs
first form. At 15 hours, the exoskeleton of the larva be-
(a) (c) Syncitium gins to form, with its specialized hairs and other external
structures. Only 24 hours after development began at
Elongated fertilization, a fully formed larva hatches out of the
somatic eggshell (Figure 18-21c). The segmental arrangement of
nucleus spikes, hairs, and other sensory structures on the larval
exoskeleton makes each segment distinct and recogniz-
able under the microscope. Now let’s return to the A – P
cardinal genes.

Figure 18-20 Cellularization of the Drosophila embryo. The

Cellularization embryo begins as a syncitium; cellularization is not completed

(b) until there are about 6000 nuclei. (a) and (b) Scanning
electron micrographs of embryos removed from the eggshell.

Cellular blastoderm (a) A syncitium-stage embryo, fractured to reveal the common
cytoplasm toward the periphery and the central yolk-filled

region. The bumps on the outside of the embryo are the

beginning of cellularization, in which the plasma membrane of

the egg folds inward from the outside. (b) A cellular

blastoderm embryo, fractured to reveal the columnar cells that

Somatic have formed by cell membranes being drawn down between
cell the elongated nuclei to create some 6000 mononucleate

somatic cells. (c) Diagrams of the changes taking place during
Yolk cellularization. [After F. R. Turner and A. P. Mahowald,

Developmental Biology 50, 1976, 95.]

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18.5 Forming complex pattern: utilizing positional information to establish cell fates 593

(a) Figure 18-21 Scanning electron micrographs of (a) a 3-hour
3-hour Drosophila embryo, (b) a 10-hour embryo, and (c) a newly hatched
embryo larva. Note that outlines of individual cells are visible by 3
(b) hours; by 10 hours, the segmentation of the embryo is obvious.
10-hour Lines are drawn to indicate that the segmental identity of cells
embryo along the A – P axis is already fated early in development. The
abbreviations refer to different segments of the head (CL, PC,
(c) O, D, Mx, Ma, Lb), thorax (T1 to T3), and abdomen (A1 to A8).
Newly
hatched larva [T. C. Kaufman and F. R. Turner, Indiana University.]

Gap The A – P cardinal genes are also known as gap
genes, because flies that have mutations in these genes
do not have the proper sequential set of larval segments:
there is a gap in the normal segmentation pattern. (For
examples, look at the phenotypes of mutations in two
gap genes, Krüppel and knirps in Figure 18-22). Through

Figure 18-22 Types of Drosophila mutants. These diagrams
depict representative mutants in each class of mutant larval
phenotypes due to mutations in the three classes of zygotically
acting genes controlling segment number in Drosophila. The
red trapezoids are segmentally repeating swatches of dense
projections on the ventral surface of the larval exoskeleton.
The boundary of each segment is indicated by a dotted line.
The left-hand diagram of each pair depicts a wild-type larva,
and the right-hand diagram shows the indicated A – P mutant.
The pink regions on the wild-type diagram indicate the
A – P domains of the larva that are missing in the mutant.

Pair Rule Segment Polarity

Krüppel even-skipped odd-skipped gooseberry

knirps paired runt patched

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594 Chapter 18 • The Genetic Basis of Development

its expression limited to a restricted region of the A – P Refining fate assignments
axis, each gap gene organizes the formation of a spa-
tially localized set of segments. How is expression through transcription-factor interactions
confined to just the proper location? BCD or HB-M
protein or both bind to enhancer elements of the pro- Ultimately, every one of the cell rows in each of the 14
moters of the gap genes, thereby regulating their tran- segments will establish its own A – P identity during the
scription. For example, transcription of one gene, Krüp- segmentation process. The number of gap genes are
pel (Kr), is repressed by high levels of the BCD clearly too few and their expression patterns too coarse
transcription factor but is activated by low levels of to account for all of the fates necessary to distinguish
BCD and HB-M. In contrast, the knirps (kni) gene is re- each row in each segment. Thus, these gap-gene domains
pressed by the presence of any BCD protein but re- need to be refined to produce the detailed anatomy of
quires low levels of the HB-M transcription factor for the 14 segments of the fully formed larva. The process of
its expression. Thus the gap-gene regulatory sequences refinement begins at about the time when the embryo is
have been exquisitely “tuned” by evolution each to transformed from a mass of cytoplasm into a structure
have a unique sensitivity to the concentrations of the formed of separate cells. The cytoplasm of each cell then
transcription factors of the A – P positional-information contains a particular concentration of one or perhaps two
system. The result of this regulation is that the kni gene adjacent gap-gene-encoded proteins, which enter its nu-
is expressed more posteriorly than is Kr (Figure 18-23a). cleus. Essentially all further decisions are driven by the
In general, through differences in their regulatory element particular A – P gap proteins “captured” in the nucleus of
properties, the gap genes are expressed in a series of a given blastoderm cell at the time of cellularization.
distinct domains. These domains then become different
developmental fields, and the cells in the different do- The A – P developmental pathway downstream of
mains become committed to different A – P fates. Each the gap genes bifurcates. Each of the two branches illus-
gap gene itself encodes a unique transcription factor trates strategies for refining the pattern. One branch
and hence regulates a different set of downstream tar- establishes the correct number of segments. The other
get genes necessary to refine the fate of the segments assigns the proper identity to each segment. (These dif-
arising in that field. ferent identities are manifest in the unique patterns of
spikes and hairs on each segment of the larva, as de-
scribed earlier.) The existence of two branches means

(a) Gap genes (b) Pair-rule genes

Figure 18-23 Photomicrographs showing the early embryonic expression
patterns of gap and pair-rule genes. All embryos are shown with anterior to
the left and posterior to the right. (a) Early blastoderm expression patterns
of proteins from three gap genes: hb-z, Kr, and kni. (b) Late blastoderm
expression patterns of proteins from two pair-rule genes: ftz (stained gray)
and eve (stained brown). Note the localized gap-gene expression patterns
compared with the reiterated pair-rule-gene expression pattern. [Part a from

M. Hulskamp and D. Tautz, BioEssays 13, 1991, 261. Part b from Peter Lawrence,

The Making of a Fly. Copyright 1992 by Blackwell Scientific Publications.]

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18.5 Forming complex pattern: utilizing positional information to establish cell fates 595

that the transcription factors encoded by the gap genes Next, let’s briefly consider the establishment of seg-
regulate two different sets of target genes. mental identity. The gap genes also target a clustered
group of genes known as the homeotic gene complexes.
First, let’s briefly consider the formation of segment They are called gene complexes because several of the
number. (Refer to Figure 18-22 for a description of the genes are clustered together on the DNA. Drosophila has
mutant phenotypes produced by the different classes of two homeotic gene clusters. The ANT-C (Antennapedia
segment-number genes.) The gap genes activate a set of complex) is largely responsible for segmental identity in
A – P patterning genes called the pair-rule genes, each of the head and anterior thorax, whereas the BX-C (Bitho-
which encodes a transcription factor, expressed in a re- rax complex) is responsible for segmental identity in the
peating pattern of seven stripes (see Figure 18-23b). The posterior thorax and abdomen.
different pair-rule genes produce a slightly offset pattern
of stripes, as shown by the gray and reddish bands in Fig- Homeosis, or homeotic transformation, is the devel-
ure 18-23b. Within a given cell, several different pair- opment of one body part with the phenotype of an-
rule proteins are expressed. other. Three examples of body-part-conversion pheno-
types due to homeotic gene mutations are:
There is a hierarchy within the pair-rule-gene class.
Some of them, called primary pair-rule genes, are regulated 1. the loss-of-function bithorax class of mutations that
directly by the gap genes. How do the asymmetrically dis- cause the entire third thoracic segment (T3) to be
tributed gap genes produce the repeating seven stripe ex- transformed into a second thoracic segment (T2),
pression pattern of the primary pair-rule genes? The gap giving rise to flies with four wings instead of the
proteins at work here are expressed in a very different, normal two (Figure 18-24);
asymmetrical pattern. The key is that the regulatory ele-
ments for certain pair-rule genes are quite complex. One 2. the gain-of-function dominant Tab mutation,
primary pair-rule gene is eve (even-skipped). The eve gene described in Chapter 10 (see Figure 10-28), that
contains separate enhancer elements, each of which inter- transforms part of the adult T2 segment into the
acts with different combinations of gap transcription fac- sixth abdominal segment (A6); and
tors to produce the seven eve stripes. For example, the en-
hancer for the first eve stripe is activated by high levels of 3. the gain-of-function dominant Antennapedia (Antp)
the HB-Z gap transcription factor, the enhancer for the mutation that transforms antenna into leg (see the
second eve stripe by low levels of HB-Z but high levels of photograph on the first page of this chapter).
the KR gap transcription factor, and so forth.
Note that, in each of these cases, the number of segments
MESSAGE The complexity of regulatory elements of the in the animal remains the same; the only change is in the
primary pair-rule genes turns an asymmetrical (gap-gene) identity of the segments. The results of studies of these
expression pattern into a repeating one. homeotic mutations have revealed much about how seg-
ment identity is established.
Once the primary pair-rule genes are activated, they
in turn activate the expression of the other pair-rule The various gap proteins activate the target
genes (also encoding transcription factors) to produce homeotic genes to initially produce a series of overlap-
the full seven-stripe pattern. No two pair-rule genes ping domains as shown in Figure 18-25, in which Scr
yield exactly the same pattern of stripes. The products of and Antp are both genes in the Antennapedia complex,
the pair-rule genes then act combinatorially to regulate and Ubx and Abd-B are genes in the Bithorax complex.
the transcription of segment-polarity genes, which are ex- The homeotic genes encode transcription factors of a
pressed in offset patterns of 14 stripes. Thus, the hierar- class called homeodomain proteins. Homeodomain pro-
chy of transcription-factor regulation extends all the way teins interact with the regulatory elements of other
from the positional-information system to the repeating genes in a specific combination so that the expression
pattern of segment-polarity-gene expression. The prod- pattern in each of the different segments is unique. (We
ucts of the segment-polarity genes permit the 14 seg- shall consider the relations of structure and function
ments to form and define the individual A – P rows of within the homeotic gene complexes later in this chap-
cells within each segment. ter, in the context of the evolution of developmental
mechanisms.)
MESSAGE Through a hierarchy of transcription-factor
regulation patterns, positional information leads to the Summary of the cascade of regulatory events
formation of the correct number of segments. In the readout of
positional information, transcription factors act combinatorially As we have seen, A – P patterning of the Drosophila em-
to create the proper segment-number fates. bryo is accomplished through a sequential triggering of
regulatory events. Positional information establishes dif-
ferent concentrations of transcription factors along the
A – P axis, and target regulatory genes are then deployed