developmental genetics

This unusual

four-winged Drosophila

has developed an extra

set of wings as a result of

a mutation in a homeotic

selector gene.

C H A P T E R C O N C E P T S

■ Gene action in development is based on differential transcription of selected genes.

■ Animals use a small number of signaling systems and regulatory networks to construct adult body forms from the zygote. These shared properties make it possible to use animal models to study human development.

■ Differentiation is controlled by cascades of gene action that follow the specification and determination of developmental fate.

■ Plants independently evolved developmental mechanisms that parallel those of animals.

■ In many organisms, cell–cell signaling programs the develop-mental pathway of adjacent and distant cells.

Developmental Genetics

18

452 18 DEVELOPMENTAL GENETICS

Over the last two decades molecular biolo-

gy and genomic analysis have shown that

in spite of wide diversity in the size and

shape of adults, all multicellular organ-

isms share many genes, genetic pathways,

and molecular signaling mechanisms in the developmental

events leading from the zygote to the adult. At the cellular

level, development is marked by three important events:

specification, when genetic and positional cues confer a

spatially discrete identity on cells, determination, the time

when a specific developmental fate for a cell becomes fixed,

and differentiation, the process by which a cell achieves its

final form and function. In addition, comparative genom-

ics has shown that higher organisms share many evolution-

ary and developmental relationships. Genetic analysis has

identified many regulatory genes that control networks of

structural genes involved in developmental processes, and

we are beginning to understand how the action and interac-

tion of these genes control basic developmental processes in

eukaryotes.

In this chapter, the primary emphasis will be on how ge-

netics has been used to study development. This area, called

developmental genetics, has contributed tremendously to

our understanding of developmental processes because ge-

netic information is required for the molecular and cellular

functions mediating developmental events and contributes

to the continually changing phenotype of the newly formed

organism.

18.1

Differentiated States Develop from Coordinated Programs of Gene Expression

Animal genomes contain tens of thousands of genes, but

only a small subset of these control the events that shape the

adult body (Figure 18–1).

Developmental geneticists study mutant alleles of these

genes to ask important questions about development:

■ What genes are expressed?

■ When are they expressed?

■ In what parts of the developing embryo are they expressed?

■ How is the expression of these genes regulated?

■ What happens when these genes are defective?

These questions provide a foundation for exploring the

molecular basis of developmental processes such as deter-

mination, induction, cell–cell communication, and cellular

differentiation. Genetic analysis of mutant alleles is used to

establish a causal relationship between the presence or ab-

sence of inducers, receptors, transcriptional events, and cell

and tissue interactions, and the observable morphological

events that accompany development.

A useful way to define development is to say that it is

the attainment of a differentiated state by all the cells of an

organism (except for stem cells). For example, a cell in a blas-

tula-stage embryo (when the embryo is just a ball of uniform-

looking cells) is undifferentiated, while a red blood cell syn-

thesizing hemoglobin in the adult body is differentiated. How

do cells get from the undifferentiated to the differentiated

state? The process involves progressive activation of different

gene sets in different cells of the embryo. From a genetics per-

spective, one way of defining the different cell types that form

during development in multicellular organisms is to identify

and catalog the genes that are active in each cell type. In other

words, development depends on patterns of differential gene

expression.

(a)

(b)

FIGURE 18–1 (a) A Drosophila embryo and (b) the adult fly

that develops from it.

18.3 GENETIC ANALYSIS OF EMBRYONIC DEVELOPMENT IN DROSOPHILA REVEALS HOW THE ANIMAL BODY AXIS IS SPECIFIED 453

The idea that differentiation is accomplished by activat-

ing and inactivating genes at different times and in different

cell types is called the variable gene activity hypothesis. Its

underlying assumptions are, first, that each cell contains an

entire genome, and, second, that differential transcription

of selected genes controls the development and differentia-

tion of each cell. In multicellular organisms, evolution has

conserved the genes involved in development, the patterns

of differential transcription, and the ensuing developmental

mechanisms. As a result, scientists are able to learn about

development in multicellular organisms in general by dis-

secting these mechanisms in a small number of genetically

well-characterized model organisms.

18.2

Evolutionary Conservation of Developmental Mechanisms Can Be Studied Using Model Organisms

Genetic analysis of development in a wide range of organ-

isms has demonstrated that all animals use a common set

of developmental mechanisms and signaling systems. For

example, most of the differences in shape between zebras

and zebrafish are controlled by different patterns of expres-

sion in a single cluster of genes called the homeotic genes,

and not by expression of many different genes scattered

across the genome. Sequencing projects have confirmed

that genes from a wide range of organisms are highly con-

served at the level of DNA sequence. This homology in

genes and regulatory mechanisms means that many aspects

of normal human embryonic development and associated

genetic disorders can be studied in model organisms such

as Drosophila, which, unlike humans, can be genetically

manipulated (see Chapter 1 for a discussion of model or-

ganisms in genetics).

Although many developmental mechanisms are simi-

lar among all animals, selection has generated new and

alternative pathways for transforming zygotes into adult

organisms. Several genetic mechanisms underlie these evo-

lutionary changes including mutation, gene duplication and

divergence, the assignment of new functions to old genes,

and the recruitment of genes to new developmental path-

ways. The emphasis in this chapter, however, will be on the

similarities among species.

Analysis of Developmental Mechanisms

In this chapter, we cannot survey all aspects of develop-

ment, nor can we explore the genetic analysis of all devel-

opmental mechanisms triggered by the fusion of sperm and

egg. Rather, we will focus on several general processes in

development:

■ how the adult body plan of animals is laid down in the

embryo

■ the program of gene expression that turns undifferenti-

ated cells into differentiated cells

■ the role of cell–cell communication in development

We will use three model systems—Drosophila melano-

gaster, Arabidopsis thaliana, and Caenorhabditis elegans—to

illustrate these developmental processes and related topics.

We will examine the role of differential gene expression in the

progressive restriction of developmental options leading to

the formation of the adult body plan in the model organisms

Drosophila and Arabidopsis. We will then expand the discus-

sion to include the selection of pathways that result in differ-

entiated cells in plants and animals, and consider the role of

cell–cell communication in development in C. elegans.

18.3

Genetic Analysis of Embryonic Development in Drosophila Reveals How the Animal Body Axis Is Specified

How specific cells in an embryo turn gene sets on or off at

precisely timed stages of development is a central question in

developmental biology. The study of model organisms gives

us an insight into the identity of regulatory genes and the

networks they control during development. In particular, the

genetic and molecular analysis of embryonic development in

Drosophila highlights the key role of molecular components

in the oocyte cytoplasm in controlling gene expression.

Overview of Drosophila Development

The development of Drosophila from zygote to adult takes

about 10 days and includes several distinct phases: the em-

bryo, three larval stages, the pupal stage, and the adult stage

(Figure 18–2). Internally, the cytoplasm of the fertilized egg is

organized into a series of maternally constructed molecular

gradients that play key roles in determining the developmen-

tal fates of nuclei located in specific regions of the embryo.

Immediately after fertilization, the zygote nucleus un-

dergoes a series of nuclear divisions without cytokinesis

[Figure 18–3(a) and (b)], forming a syncytial blastoderm (a

syncytium is any cell with more than one nucleus). At about

the tenth division, nuclei migrate to the periphery of the egg

into cytoplasm containing localized gradients of maternally

derived mRNA transcripts and proteins [Figure 18–3(c)].

After several more divisions, the nuclei become enclosed in

plasma membranes [Figure 18–3(d)] and form cells.


Cells that form at the posterior pole of the embryo

[Figure 18–3(c) and (d)] become germ cells. If nuclei from

other regions of the embryo are transplanted into the pos-

terior cytoplasm, they will form germ cells, confirming that

the cytoplasm in this region contains maternal components

that direct nuclei to form germ cells.

Transcriptional programs activated in the non–germ-

cell nuclei form the embryo’s anterior–posterior (head to

tail) and dorsal–ventral (back to front) axes of symmetry,

leading to the formation of a segmented embryo [Figure

18–3(e)]. At a later stage of development, under control of

the Hox gene set (discussed in a later section), these seg-

ments give rise to the differentiated structures of the adult

fly [Figure 18–3(f)].

Genetic Analysis of Embryogenesis

Two different gene sets control embryonic development

in Drosophila: maternal-effect genes and zygotic genes

(Figure 18–4). During development of the egg, products of

maternal-effect genes (mRNA and proteins) are placed in

the egg cytoplasm. Many of these products are distributed

in a gradient or concentrated in specific regions of the egg.

Female flies homozygous for certain recessive mutations of

maternal-effect genes are sterile: none of their embryos re-

ceive wild-type gene products from their mother, so all the

embryos develop abnormally. Maternal-effect genes encode

transcription factors and proteins that regulate gene expres-

sion. At specific stages of embryonic development, these

gene products activate or repress expression of the zygotic

genome in a temporal and spatial sequence.

Zygotic genes are transcribed in the nuclei of the devel-

oping embryo. Flies with certain homozygous mutations in

zygotic genes exhibit embryonic lethality. In a cross between

two flies heterozygous for a recessive zygotic mutation, one-

fourth of the embryos (the recessive homozygotes) there-

fore fail to develop normally and die. In Drosophila, many

zygotic genes are transcribed in specific regions of the

embryo in response to the distribution of maternal-effect

proteins.

Much of our knowledge about the genes that regulate

Drosophila development is based on the work of Chris-

tiane Nüsslein-Volhard, Eric Wieschaus, and Ed Lewis,

who were awarded the 1995 Nobel Prize for Physiology

or Medicine. Ed Lewis initially identified and studied one

of these regulatory genes in the 1970s. In the late 1970s,

Nüsslein-Volhard and Wieschaus devised a screening

strategy to identify all the genes that control develop-

ment in Drosophila. Their method required examining

thousands of offspring of mutagenized flies, looking for

recessive embryonic lethal mutations with phenotypic de-

fects in body segments and other external structures. The

parents were thus identified as heterozygous carriers of

these mutations, which the researchers grouped into three

classes: gap, pair-rule, and segment polarity genes. In 1980,

on the basis of their observations, Nüsslein-Volhard and

Wieschaus proposed a model in which embryonic devel-

opment is initiated by gradients of maternal-effect gene

products. The positional information laid down by these

molecular gradients is interpreted by expression of two

sets of zygotic genes: (1) segmentation genes (gap, pair-

rule, and segment polarity genes) and (2) homeotic selec-

tor genes. Action of the segmentation genes divides the

embryo into a series of stripes or segments and defines the

number, size, and polarity of each segment. The homeotic

genes specify the identity of each segment and the adult

structures formed from the segments (Figure 18–4).

The model developed by Nüsslein-Volhard and

Wieschaus is shown in Figure 18–5. Most maternal-effect

gene products placed in the egg during oogenesis are acti-

vated immediately after fertilization and help establish the

anterior–posterior axis of the embryo [Figure 18–5(a)].

Many maternal gene products encode transcription fac-

tors that activate transcription of the gap genes, whose

expression divides the embryo into a series of regions cor-

responding to the head, thorax, and abdomen of the adult

[Figure 18–5(b)]. Gap proteins, in turn, act as other sets of

transcription factors that activate pair-rule genes, whose

products divide the embryo into smaller regions about

two segments wide [Figure 18–5(c)]. The pair-rule genes

Embryo

1st InstarLarva

2nd InstarLarva

3rd InstarLarva

Pupa

FIGURE 18–2 Drosophila life cycle.

18.3 GENETIC ANALYSIS OF EMBRYONIC DEVELOPMENT IN DROSOPHILA REVEALS HOW THE ANIMAL BODY AXIS IS SPECIFIED 455

in turn activate the segment polarity genes, which divide

each segment into anterior and posterior regions [Figure

18–5(d)]. The collective action of the maternal genes that

form the anterior–posterior axis and the segmentation

genes define the field of action for the homeotic (Hox) genes

[Figure 18–5(e)].

Maternal-effect genes

Posteriorgroup

Zygotic genes

Anteriorgroup

Terminalgroup

Gap genes

Seg

men

tati

on

gen

es

Pair-rule genes

Segment polarity genes

Homeotic genes

FIGURE 18–4 The hierarchy of genes involved in establish-

ing the segmented body plan in Drosophila. Gene products from

the maternal genes regulate the expression of the first three

groups of zygotic genes (gap, pair-rule, and segment polarity,

collectively called the segmentation genes), which in turn control

expression of the homeotic genes.

18–1 Suppose you perform a screen for maternal-effect mu-

tations in Drosophila affecting external structures of the em-

bryo and your screen identifies more than 100 mutations

that affect external structures. From their screening, other

researchers concluded that there are about 40 maternal-

effect genes. How do you reconcile your results with those

of the other researchers?

HINT: This problem involves an understanding of how mutant

screens work. Once mutants are identified, they must be screened

by complementation analysis (Chapter 4). The key to its solution

lies in remembering the differences between genes and alleles.

Nuclei become enclosed inmembranes, forming a single layer of

cells over embryo surface.

(c)

Nine rounds of nuclear divisionsproduce multinucleated syncytium.

Diploid zygote nucleus is produced byfusion of parental gamete nuclei.

(a)

(b)

Pole cells form at posterior pole(precursors to germ cells).

(d)

(e)

(f)

Approximately four furtherdivisions take place at the cell periphery.

Pole cells

T1 A1T3T2 A2 A3 A4 A5 A6A7

A8

Embryo

Adult

A7

T1

T2

T3A1

A2A3

A4A5

A6

A8

FIGURE 18–3 Early stages of embryonic development in Drosophila. (a) Fertilized egg with zygotic nucleus (2n), shortly after

fertilization. (b) Nuclear divisions occur about every 10 minutes. Nine rounds of division produce a multinucleate cell, the syncytial

blastoderm. (c) At the tenth division, the nuclei migrate to the periphery or cortex of the egg, and four additional rounds of nuclear

division occur. A small cluster of cells, the pole cells, form at the posterior pole about 2.5 hours after fertilization. These cells will form

the germ cells of the adult. (d) About 3 hours after fertilization, the nuclei become enclosed in membranes, forming a single layer of

cells over the embryo surface, creating the cellular blastoderm. (e) The embryo at about 10 hours after fertilization. At this stage, the

segmentation pattern of the body is clearly established. Behind the segments that will form the head, T1–T3 are thoracic segments,

and A1–A8 are abdominal segments. (f ) The adult fly showing the structures formed from each segment of the embryo.


18.4

Zygotic Genes Program Segment Formation in Drosophila

The expression or repression of zygotic genes during em-

bryonic development occurs in response to the positional

gradient of maternal-effect gene products in the cytoplasm.

The sequential expression of three subsets of segmentation

genes divides the embryo into a series of segments along its

anterior–posterior axis. These segmentation genes are nor-

mally transcribed in the developing embryo, and mutations

of these genes have embryo-lethal phenotypes.

Over 20 segmentation genes (Table 18.1) have been

identified. They are classified on the basis of their mutant

phenotypes: (1) mutations in gap genes delete a group of

adjacent segments, (2) mutations in pair-rule genes af-

fect every other segment and eliminate a specific part of

each affected segment, and (3) mutations in segment po-

larity genes cause defects in homologous portions of each

segment.

In addition to the genes that determine the anterior–

posterior axis of the developing embryo, the dorsal–ventral

axis of the embryo is organized by a combination of ma-

ternal and zygotic genes and gene products. Our discussion

will be limited to the genes involved in establishing the an-

terior–posterior axis. Let us now examine each member of

this group in greater detail.

Gap Genes

The embryonic gap genes are activated or inactivated by

gene products previously expressed along the anterior–

posterior axis and by other genes of the maternal gradient

system. When mutated, these genes produce large gaps in

the embryo’s segmentation pattern. Mutants of the hunch-

back gene lose head and thorax structures, Krüppel mutants

lose thoracic and abdominal structures, and knirps mutants

lose most abdominal structures. Transcription of wild-type

gap genes (which encode transcription factors) divides the

embryo into a series of broad regions that will form the

head, thorax, and abdomen. Within these regions, specific

patterns of gene expression specify both the type of seg-

ment that will form and the order of segments in the body

of the larva, pupa, and adult. Regional patterns of gap genes

expression in different parts of the embryo correlate roughly

with the location of their mutant phenotypes: hunchback

at the anterior, Krüppel in the middle (Figure 18–6), and

knirps at the posterior. As mentioned earlier, gap genes en-

code transcription factors that control the expression of

pair-rule genes.

Pair-Rule Genes

Pair-rule genes are expressed in a series of seven narrow

bands or stripes that extend around the circumference of

the embryo. Expression of this gene set first establishes the

boundaries of segments and then establishes the develop-

mental fate of the cells within each segment by control-

ling expression of the segment polarity genes. Mutations

Anterior

(a)

(b)

(c)

(d)

(e)

Posterior

Anterior–posteriorgradients formed by

maternal-effect genes.

Zygotic gap genesdivide embryo into

broad regions.

Zygotic pair-rule genesdivide embryo intostripes about two

segments wide. Thecombined action of allpair-rule genes defines

segment borders.

Zygotic segmentpolarity genes divide

segments into anteriorand posterior halves.

Homeotic selectorgenes specify the

identity ofeach segment.

FIGURE 18–5 (a) Progressive restriction of cell fate during

development in Drosophila. Gradients of maternal proteins are

established along the anterior–posterior axis of the embryo. (b),

(c), and (d) Three groups of segmentation genes progressively

define the body segments. (e) Individual segments are given

identity by the homeotic genes.

TABLE 18.1

Segmentation Genes in Drosophila

Gap Genes Pair-Rule Genes Segment Polarity Genes

Krüppel hairy engrailed

knirps even-skipped wingless

hunchback runt cubitis

giant fushi-tarazu hedgehog

tailless paired fused

huckebein odd-paired armadillo

caudal odd-skipped patched

sloppy-paired gooseberry

paired

naked

disheveled

18.4 ZYGOTIC GENES PROGRAM SEGMENT FORMATION IN DROSOPHILA 457

in pair-rule genes eliminate segment-size sections at every

other segment. At least eight pair-rule genes act to divide

the embryo into a series of stripes. However, the bound-

aries of these stripes overlap, so that within each area of

overlap, cells express a different combination of pair-rule

genes (Figure 18–7). The transcription of the pair-rule

genes is mediated by the action of gap gene products and

maternal gene products, but the resolution of this segmen-

tation pattern into highly delineated stripes results from

the interaction among the gene products of the pair-rule

genes themselves (Figure 18–8).

Segment Polarity Genes

Expression of segment polarity genes is controlled by tran-

scription factors encoded by pair-rule genes. Within each

segment created by pair-rule genes, segment polarity genes

become active in a single band of cells that extends around

the embryo’s circumference (Figure 18–9). This divides the

(b)

(a)

No transcription Transcription No transcription

1 2

Overlap

Area of mRNA transcription

1 1 2 2

FIGURE 18–7 New patterns of gene expression can be

generated by overlapping regions containing two different gene

products. (a) Transcription factors 1 and 2 are present in an

overlapping region of expression. If both transcription factors

must bind to the promoter of a target gene to trigger expression,

the gene will be active only in cells containing both factors (most

likely in the zone of overlap). (b) The expression of the target

gene in the restricted region of the embryo.

(a)

(b)

FIGURE 18–8 Stripe pattern of pair-rule gene expression

in Drosophila embryo. This embryo is stained to show patterns

of expression of the genes even-skipped and fushi-tarazu; (a) low-

power view and (b) high-power view of the same embryo.

FIGURE 18–9 The 14 stripes of expression of the segment

polarity gene engrailed in a Drosophila embryo.

Hunchback protein

Yellow stripe contains bothHunchback and Krüppel proteins

Anterior Posterior

Krüppel protein

FIGURE 18–6 Expression of gap genes in a Drosophila

embryo. The Hunchback protein is shown in orange, and

Krüppel is indicated in green. The yellow stripe is created when

cells contain both Hunchback and Krüppel proteins. Each dot in

the embryo is a nucleus.


embryo into 14 segments. The products of the segment po-

larity genes control the cellular identity within each of them

and establish the anterior–posterior pattern (the polarity)

within each segment.

Segmentation Genes in Mice and Humans

We have seen that segment formation in Drosophila de-

pends on the action of three subsets of segmentation genes.

If Drosophila is to be a useful model for understanding gen-

eral principles of animal development, it is logical to ask

whether these gene families are found in humans and other

mammals, and if so, do they control aspects of embryonic

development in these organisms? To answer this question,

let’s examine runt, one of the pair-rule genes in Drosophila.

In late stages of development, it controls aspects of sex de-

termination and formation of the nervous system. The gene

encodes a protein that regulates transcription of its target

genes, and contains a 128-amino-acid DNA-binding region

(called the runt domain) that is highly conserved in mouse

and human proteins. In fact, in vitro experiments show that

the Drosophila and mouse runt proteins are functionally

interchangeable. In mice, runt is expressed early in devel-

opment and controls formation of blood cells, bone, and

the genital system. Although the target gene sets controlled

by runt are different in Drosophila and the mouse, in both

organisms, expression of runt specifies the fate of uncom-

mitted embryonic cells by regulating transcription of target

genes.

In humans, mutation in RUNX2, a human homolog of

runt, causes cleidocranial dysplasia (CCD), an autosomal

dominantly inherited trait. Those affected with CCD have

a hole in the top of their skull because their fontanel does

not close. Their collar bones (clavicles) do not develop,

enabling them to fold their shoulders across their chest

(Figure 18–10). Mice with one mutant copy of the runt ho-

molog have a phenotype similar to that seen in humans;

mice with two mutant copies of the gene have no bones at

all. Their skeletons contain only cartilage, much like sharks

(Figure 18–11), emphasizing the role of runt in these spe-

cies as an important gene controlling the initiation of bone

formation.

18.5

Homeotic Selector Genes Specify Parts of the Adult Body

As boundaries are established by expression of segmenta-

tion genes, the homeotic (from the Greek word for “same”)

genes are activated. Expression of homeotic selector genes

determines which adult structures will be formed by each

body segment. In Drosophila, this includes the antennae,

FIGURE 18–10 A boy affected with cleidocranial dysplasia

(CCD). This disorder, inherited as an autosomal dominant trait,

is caused by mutation in a human runt gene, RUNX2. Affected

heterozygotes have a number of skeletal defects, including a hole

in the top of the skull where the infant fontanel fails to close,

and collar bones that do not develop or form only small stumps.

Because the collar bones do not form, CCD individuals can fold

their shoulders across their chests. Reprinted by permission from

Macmillan Publishers Ltd.: Fig. 1 on p. 244 from: British Dental

Journal 195: 243–248 2003. Greenwood, M. and Meechan, J. G.

“General medicine and surgery for dental practitioners.” Copyright

© Macmillan Magazines Limited.

FIGURE 18–11 Bone formation in normal mice and mutants

for the runt gene Runx2. (a) Normal mouse embryos at day

17.5 show cartilage (blue) and bone (brown). (b) The skeleton

of a 17.5-day homozygous mutant embryo. Only cartilage has

formed in the skeleton. There is complete absence of bone for-

mation in the mutant mouse. Expression of a normal copy of the

Runx2 gene is essential for specifying the developmental fate of

bone-forming osteoblasts.

mouth parts, legs, wings, thorax, and abdomen. Mutants

of these genes are called homeotic mutants because the

structure formed by one segment is transformed into that

formed by another segment. For example, the wild-type

allele of Antennapedia (Antp) specifies formation of a leg

on the second segment of the thorax. Dominant gain-of-

function Antp mutations cause this gene to be expressed in

the head as well, and in mutant flies the antenna is trans-

formed into a leg (Figure 18–12).

Hox Genes in Drosophila

The Drosophila genome contains two clusters of homeotic

selector genes (called Hox genes) on chromosome 3 that

encode transcription factors (Table 18.2). The Antenna-

pedia (ANT-C) cluster contains five genes that specify struc-

tures in the head and the first two segments of the thorax

[Figure 18–13(a)]. The second cluster, the bithorax (BX-C)

(a)

(b)

FIGURE 18–12 Antennapedia (Antp) mutation in Drosophila.

(a) Head from wild-type Drosophila, showing the antenna and

other head parts. (b) Head from an Antp mutant, showing the

replacement of normal antenna structures with legs. This is

caused by activation of the Antp gene in the head region.

TABLE 18.2

Hox Genes of Drosophila

Antennapedia Complex Bithorax Complex

labial Ultrabithorax

Antennapedia abdominal A

Sex combs reduced Abdominal B

Deformed

proboscipedia

ANT-C

lab pb Dfd Scr Antp

A7A8

BX-C

A7A8

Ubx abd-A Abd-B

(a)

(b)

T1

T2

T3

A1A2

A3

A4

A5

A6

T1

T2

T3

A1A2

A3

A4

A5

A6

FIGURE 18–13 Genes of the Antennapedia complex and the

adult structures they specify. (a) In the ANT-C complex, the labial

(lab) and Deformed (Dfd) genes control the formation of head

segments. The Sex comb reduced (Scr) and Antennapedia (Antp)

genes specify the identity of the first two thoracic segments,

T1 and T2. The remaining gene in the complex, proboscipedia

(pb), may not act during embryogenesis but may be required

to maintain the differentiated state in adults. In mutants, the

labial palps are transformed into legs. (b) In the BX-C complex,

Ultrabithorax (Ubx) controls formation of structures in the pos-

terior compartment of T2 and structures in T3. The two other

genes, abdominal A (abdA) and Abdominal B (AbdB), specify the

segmental identities of the eight abdominal segments (A1–A8).

18.5 HOMEOTIC SELECTOR GENES SPECIFY PARTS OF THE ADULT BODY 459


complex, contains three genes that specify structures in the

posterior portion of the second thoracic segment, the en-

tire third thoracic segment, and the abdominal segments

[Figure 18–13(b)].

Hox genes (listed in Table 18.2) have two properties

in common. First, each contains a 180-bp domain known

as a homeobox. (Hox is a contraction of homeobox.) The

homeobox encodes a DNA-binding sequence of 60 amino

acids known as a homeodomain. Second, in most species,

expression of the genes is colinear with the anterior to pos-

terior organization of the body. Genes at the 3¿end of a clus-

ter are expressed at the anterior end of the embryo, those

in the middle are expressed in the middle of the embryo,

and genes at the 5¿ end of a cluster are expressed at the em-

bryo’s posterior region (Figure 18–14). Although first iden-

tified in Drosophila, Hox genes are found in the genomes

of most eukaryotes with segmented body plans, including

nematodes, sea urchins, zebrafish, frogs, mice, and humans

(Figure 18–15).

To summarize, genes that control development in Dro-

sophila act in a temporally and spatially ordered cascade, be-

ginning with the genes that establish the anterior–posterior

Head Thorax Abdomen

(a) Expression domains of homeotic genes

(b) Chromosomal locations of homeotic genes

5‘3‘lab pb Dfd Scr Antp Ubx abd-A Abd-B

FIGURE 18–14 The colinear relationship between the spatial

pattern of expression and chromosomal locations of homeotic

genes in Drosophila. (a) Drosophila embryo and the domains

of homeotic gene expression in the embryonic epidermis and

central nervous system. (b) Chromosomal location of homeotic

selector genes. Note that the order of genes on the chromo-

some correlates with the sequential anterior borders of their

expression domains.

lab

Hox1

HoxA

Hum

an HoxB

HoxC

HoxD

Hox2 Hox3 Hox4 Hox5 Hox6 (central) Hox7 (posterior)

A1 A2 A3 A4 A5 A6 A7

B1 B2 B3 B4 B5 B6 B7 B8 B9

C4 C5 C6 C8 C9 C10 C11 C12 C13

D8D4D2D1

1 2 3

3‘ 5‘

4 5 6 7 8 9 10 11 12 13

D9 D10 D11 D12 D13

B13

A9 A10 A11 A13

Anterior

Drosophila HOM-C

Ancestral Urbilaterian

HOM-C

Posterior

Anterior

Homology group

Transcription

Posterior

pbbcd.zen Dfd Scr ftz Antp Ubx abd-A Abd-B

FIGURE 18–15 Conservation of orga-

nization and patterns of expression in Hox

genes. (Top) The structures formed in adult

Drosophila are shown, with the colors cor-

responding to members of the Hox cluster

that control their formation. (Middle) The

reconstructed Hox cluster of the common

ancestor to all bilateral organisms contains

seven genes. (Bottom) The arrangement and

expression patterns of the four clusters of

Hox genes in an early human embryo. Some

of the posterior genes are expressed in the

limbs. The expression pattern is inferred

from that observed in mice. As in Drosophila,

genes at the 3¿ end of the cluster form an-

terior structures, and genes at the 5¿ end of

the cluster form posterior structures. Genes

homologous to the same ancestral sequence

(because of duplications) are indicated by

brackets.

18.5 HOMEOTIC SELECTOR GENES SPECIFY PARTS OF THE ADULT BODY 461

(and dorsal–ventral) axis of the egg and early embryo.

Gradients of maternal mRNAs and proteins along the an-

terior–posterior axis activate gap genes, which subdivide

the embryo into broad bands. Gap genes in turn activate

pair-rule genes, which divide the embryo into segments.

The final group of segmentation genes, the segment polar-

ity genes, divides each segment into anterior and posterior

regions arranged linearly along the anterior–posterior axis.

The segments are then given identity by the Hox genes.

Therefore, this progressive restriction of developmental po-

tential of the Drosophila embryo’s cells (all of which occurs

during the first third of embryogenesis) involves a cascade

of gene action, with regulatory proteins acting on transcrip-

tion, translation, and signal transduction.

Hox Genes and Human Genetic

Disorders

Although first described in Drosophila, Hox genes with a

high degree of homology are found in the genomes of all

animals where they play a fundamental role in shaping the

body and its appendages. In vertebrates, the conservation of

sequence, the order of genes in the Hox clusters, and their

pattern of expression suggest that, as in Drosophila, these

genes control development along the anterior–posterior axis

and the formation of appendages (Figure 18–16). However,

in vertebrates, including mice and humans, there are four

clusters of Hox genes: HOXA, HOXB, HOXC, and HOXD

instead of a single cluster as in Drosophila. This means that

in vertebrates, not just one, but a combination of 2–4 Hox

genes is involved in forming specific structures. As a result,

homeotic mutations in individual vertebrate Hox genes do

not produce complete transformations as in Drosophila,

where mutation of a single Hox gene can transform a hal-

tere into a wing (see the photo at the beginning of this chap-

ter). In spite of these differences, the role for HOXD genes

in human development was confirmed by the discovery that

several inherited limb malformations are caused by muta-

tions in HOXD genes. For example, mutations in HOXD13

cause synpolydactyly (SPD), a malformation characterized

by extra fingers and toes, and abnormalities in bones of the

hands and feet (Figure 18–17).

Chick Mouse

FIGURE 18–16 Patterns of Hox gene expression control

the formation of structures along the anterior–posterior axis of

bilaterally symmetrical animals in a species-specific manner. In

the chick (left) and the mouse (right), expression of the same set

of Hox genes is differentially programmed in time and space to

produce different body forms.

FIGURE 18–17 Mutations in posterior Hox genes (HOXD13

in this case) in humans result in malformations of the limbs,

shown here as extra toes. This condition is known as synpoly-

dactyly. Mutations in HOXD13 are also associated with abnor-

malities of the bones in the hands and feet.

18–2 In Drosophila, both fushi tarazu (ftz) and engrailed (eng)

genes encode homeobox transcription factors and are ca-

pable of eliciting the expression of other genes. Both genes

work at about the same time during development and in

the same region to specify cell fate in body segments. To

discover if ftz regulates the expression of engrailed genes, if

engrailed regulates ftz, or if both are regulated by another

gene, you perform a mutant analysis. In ftz– embryos (ftz/ftz)

engrailed protein is absent; in engrailed embryos (eng/eng) ftz

expression is normal. What does this tell you about the reg-

ulation of these two genes—does the engrailed gene regulate

ftz, or does the ftz gene regulate engrailed?

HINT: This problem involves an understanding of regulation of

gene expression by trans-acting factors (see Chapter 17). The key

to its solution is analysis of the genetic background of each mutant

strain.


18.6

Plants Have Evolved Developmental Systems That Parallel Those of Animals

Plants and animals diverged from a common unicellular

ancestor about 1.6 billion years ago, after the origin of eu-

karyotes and probably before the rise of multicellular organ-

isms. Genomic analysis of mutants in plants and animals

indicates that basic mechanisms of developmental pattern

formation evolved independently in animals and plants. We

have already examined genetic systems that control devel-

opment and pattern formation in animals, using Drosophila

as a model organism.

In plants, pattern formation has been studied using

flower development in Arabidopsis thaliana (Figure 18–18),

a small plant in the mustard family, as a model organism. A

cluster of undifferentiated cells, called the floral meristem,

gives rise to flowers (Figure 18–19). Each flower consists

of four organs—sepals, petals, stamens, and carpels—that

develop from concentric rings of cells within the meristem

(Figure 18–20). Each organ develops from a different con-

centric ring, or whorl of cells.

Homeotic Genes in Arabidopsis

Three classes of floral homeotic genes control the develop-

ment of these organs (Table 18.3). Class A genes acting alone

specify sepals, class A and class B genes expressed together

specify petals, and together, class B and class C genes control

stamen formation. Class C genes acting alone specify car-

pels. During flower development [Figure 18–21(a)], Class A

genes are active in whorls 1 and 2 (sepals and petals), class B

genes are expressed in whorls 2 and 3 (petals and stamens),

and class C genes are expressed in whorls 3 and 4 (stamens

and carpels). The organ formed depends on the expression

FIGURE 18–18 The flowering plant Arabidopsis thaliana, used

as a model organism in plant genetics.

(a) (b)

FIGURE 18–19 (a) Parts of the Arabidopsis flower. The floral organs are arranged concentrically. The sepals form the outermost

ring, followed by petals and stamens, with carpels on the inside. (b) View of the flower from above.

18.6 PLANTS HAVE EVOLVED DEVELOPMENTAL SYSTEMS THAT PARALLEL THOSE OF ANIMALS 463

pattern of the three gene classes. Expression of class A genes

in whorl 1 causes sepals to form. Expression of class A and

class B genes in whorl 2 leads to petal formation. Expres-

sion of class B and class C genes in whorl 3 leads to stamen

formation. In whorl 4, expression of class C genes causes

carpel formation.

As in Drosophila, mutations in homeotic genes cause

organs to form in abnormal locations. For example, in

APETALA2 mutants (ap2), the order of organs is carpel,

stamen, stamen, and carpel instead of the normal order,

sepal, petal, stamen, and carpel [Figure 18–21(b)]. In

class B loss-of-function mutants (ap3, pi) petals become

sepals, and stamens are transformed into carpels [Figure

18–21(c)] and the order of organs becomes sepal, sepal,

carpel, carpel. Plants carrying a mutation for the class C

gene AGAMOUS will have petals in whorl 3 (instead of

stamens) and sepals in whorl 4 (instead of carpels), and

the order of organs will be sepal, petal, petal, and sepal

[Figure 18–21(d)].

Evolutionary Divergence in Homeotic GenesDrosophila and Arabidopsis use different sets of nonho-

mologous master regulatory genes to establish the body

axis and specify the identity of structures along the axis.

In Drosophila, this task is accomplished in part by the Hox

genes, which encode a set of transcription factors sharing

a homeobox domain. In Arabidopsis, the floral homeotic

genes belong to a different family of transcription fac-

tors, called the MADS-box proteins, characterized by a

common sequence of 58 amino acids with no similarity

in amino acid sequence or protein structure with the Hox

genes. Both gene sets encode transcription factors, both

sets are master regulators of development expressed in a

pattern of overlapping domains, and both specify identity

of structures.

Reflecting their evolutionary origin from a common

ancestor, the genomes of both Drosophila and Arabidop-

sis contain members of the homeobox and MADS-box

genes, but these genes have been adapted for different uses

in the plant and animal kingdoms, indicating that devel-

opmental mechanisms evolved independently in each

group.

In both plants and animals, the action of transcrip-

tion factors depends on changes in chromatin structure

that make genes available for expression. Mechanisms of

transcription initiation are conserved in plants and ani-

mals, as is reflected in the homology of genes in Drosophila

and Arabidopsis that maintain patterns of expression initi-

ated by regulatory gene sets. Action of the floral homeo-

tic genes is controlled by a gene called CURLY LEAF. This

gene shares significant homology with members of a Dro-

sophila gene family called Polycomb. This family of regula-

tory genes controls expression of homeobox genes during

development. Both CURLY LEAF and Polycomb encode

proteins that alter chromatin conformation and shut off

gene expression. Thus, although different genes are used to

control development, both plants and animals use an evo-

lutionarily conserved mechanism to regulate expression of

these gene sets.

C genes

A genes

B genes

(a) (b)

Sepal

Petal

Stamen

Carpel

1

2

3

4

FIGURE 18–20 Cell arrangement in the floral meristem. (a) The four concentric rings, or whorls, labeled 1–4, give rise to (b)

arrangement of the sepals, petals, stamens, and carpels, respectively, in the mature flower.

TABLE 18.3

Homeotic Selector Genes in Arabidopsis*

Class A APETALA1 (AP1)

APETALA2 (AP2)

Class B APETALA3 (AP3)

PISTILLATA (Pl)

Class C AGAMOUS (AG)

*By convention, wild-type genes in Arabidopsis use capital letters.


18.7

Cell–Cell Interactions in Development Are Modeled in C. elegans

During development in multicellular organisms, cell–cell

interactions influence the transcriptional programs and de-

velopmental fate of surrounding cells. Cell–cell interaction

is an important process in the embryonic development of

most eukaryotic organisms, including Drosophila, as well as

vertebrates, including mice and humans.

Signaling Pathways in Development

In early development, animals use a number of signaling

pathways to regulate development; after organ formation

begins, other signal pathways are added to those already in

use. These newly activated pathways act both independently

and in coordinated networks to elicit specific transcriptional

responses. The signal networks establish anterior–posterior

polarity and body axes, coordinate pattern formation, and

direct the differentiation of tissues and organs. The signal-

ing pathways used in early development and some of the de-

velopmental processes they control are listed in Table 18.4.

After an introduction to the components and interactions

of one of these systems—the Notch signaling pathway—

we will briefly examine its role in the development of the

vulva in the nematode, Caenorhabditis elegans.

The Notch Signaling Pathway

The genes in the Notch pathway are named after the Dro-

sophila mutants that were used to identify components

of this signal transduction system. Notch works through

direct cell–cell contact to control the developmental fate

of the interacting cells. The Notch gene (and the equivalent

gene in other organisms) encodes a signal receptor embed-

ded in the plasma membrane (Figure 18–22). The signal

is another membrane protein encoded by the Delta gene

(and its equivalents). Because both the signal and receptor

are membrane proteins, the Notch signal system works be-

tween adjacent cells. When the Delta signal protein binds

to the Notch receptor protein, the cytoplasmic tail of the

Notch protein is cut off and binds to a cytoplasmic pro-

tein encoded by the Su(H) (suppressor of Hairless) gene.

This protein complex moves into the nucleus and binds

to transcriptional cofactors, activating transcription of a

(a) (b) (c) (d)

FIGURE 18–21 (a) The individual and combined action of Class A, B, and C genes form the sepals, petals, stamens, and carpels

of wild-type flowers of Arabidopsis. (b) Homeotic APETALA2 (ap2) mutant flower (a Class A mutant), has carpels, stamens, stamens,

and carpels. (c) Class B mutants (ap3 and pi) have sepals, sepals, carpels, and carpels. (d) Class C mutants (ag) have petals and sepals

at places where stamens and carpels should form.

TABLE 18.4

Signaling Pathways Used in Early Embryonic Development

Wnt Pathway

Dorsalization of body

Female reproductive development

Dorsal–ventral differences

TGF-B Pathway

Mesoderm induction

Left–right asymmetry

Bone development

Hedgehog Pathway

Notochord induction

Somitogenesis

Gut/visceral mesoderm

Receptor Tyrosine Kinase Pathway

Mesoderm maintenance

Notch Signaling Pathway

Blood cell development

Neurogenesis

Retina development

Source: Taken from Gerhart, J. 1999. 1998 Warkany lecture: Signaling pathways in development. Teratology 60: 226–239.

18.7 CELL–CELL INTERACTIONS IN DEVELOPMENT ARE MODELED IN C. ELEGANS 465

gene set that controls a specific developmental pathway

(Figure 18–22).

One of the main roles of the Notch signal system is

specifying the fate of equivalent cells in a population. In its

simplest form, this interaction involves two neighboring

cells that are developmentally equivalent. We will explore

the role of the Notch signaling system in development of the

vulva in C. elegans, after a brief introduction to nematode

embryogenesis.

Overview of C. elegans Development

The nematode C. elegans is widely used to study the ge-

netic control of development. This organism has several

advantages for such studies: (1) the genetics of the organism

are well known, (2) its genome has been sequenced, and (3)

adults contain a small number of cells that follow a highly

deterministic developmental program. Adults are about

1 mm long and develop from a fertilized egg in about two

days (Figure 18–23). The life cycle includes an embryonic

stage (about 16 hours), four larval stages (L1 through L4),

and the adult stage. Adults are either XX self-fertilizing her-

maphrodites that can make both eggs and sperm, or XO

males. Self-fertilization of mutagen-treated hermaphrodites

is used to develop homozygous stocks of mutant strains,

and hundreds of such mutants have been generated, cata-

logued, and mapped.

AB

Zygote

P1

Hypodermis Neurons

Pharyngeal musclesBody muscles

Glands

P2

MS E C P3

D P4

Body muscles Pharyngeal muscles

NeuronsGlands

GutHypodermis

Body muscles Two neurons

Bodymuscles

Z2 Z3

Germcells

EMS

GonadEgg

VulvaSperm

Intestine Nervous system

Pharynx

Cuticle

(a)

(b)

FIGURE 18–23 (a) A truncated cell lineage chart for C. elegans, showing early divisions and the tissues and organs formed from

these lineages. Each vertical line represents a cell division, and horizontal lines connect the two cells produced. For example, the first

division of the zygote creates two new cells, AB and P1. During embryogenesis, cell divisions will produce the 959 somatic cells of the

adult hermaphrodite worm. (b) An adult C. elegans hermaphrodite. This nematode, about 1 mm in length, consists of 959 cells and is

widely used as a model organism to study the genetic control of development.

Notchreceptor

Proteolyticcleavage

Nucleus

Alteredpattern of

gene expression

Deltaprotein Su(H)

NCID

Transcriptionstart site

FIGURE 18–22 Components of the

Notch signaling pathway in Drosophila. The

cell carrying the Delta transmembrane pro-

tein is the sending cell; the cell carrying the

transmembrane Notch protein receives the

signal. Binding of Delta to Notch triggers a

proteolytic-mediated activation of transcrip-

tion. The fragment cleaved from the cyto-

plasmic side of the Notch protein, called

the Notch intracellular domain (NCID),

combines with the Su(H) protein and moves

to the nucleus where it activates a program

of gene transcription.


Genetic Analysis of Vulva Formation

Adult C. elegans hermaphrodites lay eggs through the vulva,

an opening near the middle of the body (Figure 18–23). The

vulva is formed in stages during larval development and in-

volves three sequential rounds of cell–cell interactions.

In C. elegans, two developmentally equivalent adja-

cent cells, Z1.ppp and Z4.aaa, interact with each other so

that one becomes the gonadal anchor cell and the other

becomes a precursor to the uterus (Figure 18–24). The

determination of which cell becomes which occurs during

the second larval stage (L2) and is controlled by the Notch

receptor gene, lin-12. In recessive lin-12(0) mutants (a

loss-of-function mutant), both cells become anchor cells.

The dominant mutation lin-12(d) (a gain-of-function

mutation) causes both to become uterine precursors.

Thus, expression of the lin-12 gene causes the selection

of the uterine pathway, since in the absence of the LIN-12

(Notch) receptor, both cells become anchor cells.

Adult hermaphrodites have 959 somatic cells (and about

2000 germ cells). The lineage of each cell, from fertilized egg to

adult, has been mapped (Figure 18–23) and, is invariant from

individual to individual, with the single exception of the an-

chor cell/ventral uterine cell specification, which is a random

event (see Figure 18–24). Knowing the lineage of each cell, we

can easily follow events caused by mutations that alter cell fate

or by killing specific cells with laser microbeams or ultraviolet

irradiation. In C. elegans hermaphrodites, the developmental

fate of cells in the reproductive system is determined by

cell–cell interaction, illustrating how gene expression and

cell–cell interaction work together to specify developmental

outcomes.

However, the situation is more complex than it first

appears. Initially, the two neighboring cells are develop-

mentally equivalent (Figure 18–24). Each synthesizes low

levels of the Notch signal protein (encoded by the lag-2

gene) and the Notch receptor protein. By chance, one cell

ends up producing more of the signal (LAG-2 protein).

This causes its neighboring cell to increase production

of the receptor (LIN-12 protein). The cell producing

more of the receptor protein becomes the uterine precur-

sor, and the cell producing more signal protein becomes

the anchor cell. The critical factor in this first round of

cell–cell interaction is the balance between the LAG-2

(Delta) signal gene product and the LIN-12 (Notch) gene

product.

A second and third round of cell–cell interactions leads

to formation of the vulva itself (Figure 18–25). The second

round involves the anchor cell (located in the gonad) and

six of its neighboring cells (called precursor cells) located in

the skin. The precursor cells, named P3.p to P8.p, are called

During L2, both cells begin secretingsignal for uterine differentiation

(a)

(b)

By chance, Z1.pppsecretes more signal

In response to signal, Z4.aaaincreases production of LIN-12

receptor protein, triggeringdetermination as uterine

precursor cell

Becomes anchor cell Becomes ventral uterineprecursor cell

Z1.ppp Z4.aaa

Z1.ppp

Signal

Receptor

Z4.aaa

FIGURE 18–24 Cell–cell interaction in anchor cell determi-

nation. (a) During L2, two neighboring cells begin the synthesis

of signal and receptor molecules for the induction of uterine dif-

ferentiation. (b) By chance, cell Z1.ppp produces more of these

signals, causing cell Z4.aaa to increase production of the recep-

tor for signals. The action of increased signals causes Z4.aaa to

become the ventral uterine precursor cell and allows Z1.ppp to

become the anchor cell.

18–3 Vulval development in C. elegans begins when two

neighboring cells (Z1.ppp and Z4.aaa) interact with each

other by cell–cell signaling involving two components: a

membrane-bound signal molecule and a membrane-bound

receptor. By chance, one cell produces more signal, which

in turn, causes its neighbor to produce more receptor. The

cell producing more signal becomes the anchor cell, and the

cell producing more receptor becomes the ventral uterine

cell. This form of cell–cell interaction is called the Notch/

Delta signaling system. Although it is a widely used signal-

ing mechanism in metazoans, this pathway works only in

adjacent cells. Why is this so, and what are the advantages

and disadvantages of such a system?

HINT : This problem involves an understanding of how adjacent

cell–cell signaling systems work. The key to its solution is recogniz-

ing the dynamic interactions between signal and receptor systems in

this form of cell–cell signaling.

18.8 PROGRAMMED CELL DEATH IS REQUIRED FOR NORMAL DEVELOPMENT 467

(Figure 18–26), a process called apoptosis. The genes that

control apoptosis were first identified in C. elegans. In C. el-

egans, as in many other organisms, normal development re-

lies on programmed cell death. The number of cells that die

during the worm’s development is always the same: 131 of

1090 in hermaphrodites and 147 of 1178 in males. In addi-

tion, the time in development at which a given cell dies and

the identity of the cells that die are always the same.

Mutational analysis indicates that, although pro-

grammed cell death occurs in cells in different lineages, all

Pn.p cells. The fate of each Pn.p cell is specified by its posi-

tion relative to the anchor cell.

The anchor cell synthesizes the LIN-3 signal protein

which is received and processed by three adjacent Pn.p pre-

cursor cells (Pn.p 5–7). The cell closest to the anchor cell

(usually Pn.p 6) becomes the primary vulval precursor cell,

and the adjacent cells (Pn.p 5 and 7) become secondary pre-

cursor cells. Once the primary vulval cell has been estab-

lished, in a third round of cell–cell interaction a signal pro-

tein made by the primary vulval cell activates the lin-12 gene

in the secondary cells and prevents them from becoming

primary precursor cells. The other precursor cells (Pn.p 3,

4, and 8) receive no signal from the anchor cell and become

skin cells.

18.8

Programmed Cell Death Is Required for Normal Development

During normal development, programmed cell death is a

genetically controlled event that helps shape tissues and

organs. One well-known example of programmed cell

death is the formation of digits in the vertebrate limb. This

process requires the death of the cells between the digits

Secondarypathway

Primarypathway

Secondarypathway

Vulva Hypodermis

Inhibitionsignal

Inhibitionsignal

Anchor cell produces LIN-3 protein

P6.p P7.p

Tertiarypathway

Tertiarypathway

Hypodermis

Hypodermis

Gonad

Anchor cell

Tertiarypathway

Precursorvulva

(Pn.p) cellsP8.pP5.pP4.pP3.p

FIGURE 18–25 Cell lineage determination in C. elegans vulva formation. A signal from the anchor cell in the form of LIN-3 protein

is received by three precursor vulval cells (Pn.p cells). The cell closest to the anchor cell becomes the primary vulval precursor cell, and

adjacent cells become secondary precursor cells. The primary cell produces a signal that activates the lin-12 gene in secondary cells,

preventing them from becoming primary cells. Flanking precursor cells, which receive no signal from the anchor cell, become skin

(hypodermis) cells, instead of vulval cells.

On

Off

Off

On

Cellsurvival

Celldeath

ced-9

ced-3

ced-4

ced-3

ced-4

FIGURE 18–26 In C. elegans, the genetic pathway control-

ling cell death, the gene ced-9 acts as a binary switch. If ced-9

is active, it represses the action of ced-3 and ced-4, and the cell

remains alive. If ced-9 is inactive, ced-3 and ced-4 are expressed,

initiating a cascade of events that results in cell death, a process

known as apoptosis.


cells use the same genetic pathway. In C. elegans, at least

15 genes are involved in cell death. These genes control four

processes: (1) decisions about cell death, (2) implementa-

tion of decisions, (3) engulfment of dying cells, and (4) deg-

radation of cell debris within the engulfing cells.

Expression of ced-3 and ced-4 is necessary for execution

of the cell death program; mutations that inactivate either

of these genes result in survival of cells that normally die.

Expression of ced-3 and ced-4 is controlled by ced-9. Gain-

of-function mutations that cause constitutive expression or

overexpression of ced-9 prevent cell death. Conversely, loss-

of-function mutants that inactivate ced-9 cause embryonic

lethality, meaning that ced-9 works by inactivating ced-3 and

ced-4 in surviving cells (Figure 18–26). In other words, ced-9

is a binary switch gene for apoptosis. Cells that express ced-9

survive and those that do not, die.

The ced-9 gene of C. elegans has a human homolog,

bcl-2, a proto-oncogene that controls apoptosis in humans.

Mutations that overexpress bcl-2 prevent death in cells that

would normally die. In humans, overexpression of bcl-2 is

found in follicular lymphoma, a form of cancer. Transfer of

a cloned human bcl-2 gene into ced-9 null mutant C. elegans

embryos prevents apoptosis, indicating that nematodes and

mammals share a common pathway for this process. Thus,

the homology in molecules and mechanisms among spe-

cies across the phylogenetic tree, illustrated throughout this

chapter, extends to the molecules and mechanisms required

for cells to die.

G E N E T I C S , T E C H N O L O G Y , A N D S O C I E T Y

Stem Cell Wars

Stem cell research may be the most controversial research area since the beginning of recombi-

nant DNA technology in the 1970s. Al-though stem cell research is the focus of presidential proclamations, media cam-paigns, and ethical debates, few people understand it sufficiently to evaluate its pros and cons.

Stem cells are primitive cells that rep-licate indefinitely and have the capacity to differentiate into cells with special-ized functions, such as the cells of heart, brain, liver, and muscle tissue. All the cells that make up the approximately 200 distinct types of tissues in our bodies are descended from stem cells. Some types of stem cells are defined as totipotent, meaning that they have the ability to dif-ferentiate into any mature cell type in the body. Other types of stem cells are plu-

ripotent, meaning that they are able to dif-ferentiate into any of a smaller number of mature cell types. In contrast, mature, fully differentiated cells do not replicate or undergo transformations into differ-ent cell types.

In the last few years, several research teams have isolated and cultured human pluripotent stem cells. These cells remain undifferentiated and grow indefinitely in culture dishes. When treated with growth factors or hormones, these pluripotent

stem cells differentiate into cells that have the characteristics of neural, bone, kidney, liver, heart, or pancreatic cells.

The fact that pluripotent stem cells grow prolifically in culture and differen-tiate into more specialized cells has cre-ated great excitement. Some foresee a day when stem cells may be a cornucopia from which to harvest unlimited numbers of specialized cells to replace cells in dam-aged and diseased tissues. Hence, stem cells could be used to treat Parkinson dis-ease, type 1 diabetes, chronic heart dis-ease, Alzheimer disease, and spinal cord injuries. Some predict that stem cells will be genetically modified to eliminate transplant rejection or to deliver specific gene products, thereby correcting genetic defects or treating cancers. The excite-ment about stem cell therapies has been fueled by reports of dramatically success-ful experiments in animals. For example, mice with spinal cord injuries regained their mobility and bowel and bladder control after they were injected with hu-man stem cells. Both proponents and critics of stem cell research agree that stem cell therapies could be revolution-ary. Why, then, should stem cell research be so contentious?

The answer to that question lies in the source of the pluripotent stem cells. Until recently, all pluripotent stem cell

lines were derived from five-day-old em-bryonic blastocysts. Blastocysts at this stage consist of 50–150 cells, most of which will develop into placental and supporting tissues for the early embryo. The inner cell mass of the blastocyst consists of about 30 to 40 pluripotent stem cells that can develop into all the embryo’s tissues. In vitro fertilization clinics grow fertilized eggs to the five-day blastocyst stage prior to uterine transfer. Embryonic stem cell (ESC) lines are cre-ated by taking the inner cell mass out of five-day blastocysts and growing the cells in culture dishes.

The fact that early embryos are de-stroyed in the process of establishing human ESC lines disturbs people who believe that preimplantation embryos are persons with rights; however, it does not disturb people who believe that these embryos are too primitive to have the status of a human being. Both sides in the debate invoke fundamental ques-tions of what constitutes a human being.

Recently, scientists have developed several types of pluripotent stem cells without using embryos. One of the most promising types—known as induced plu-

ripotent stem (iPS) cells—uses adult somatic cells as the source of pluripotent stem cell lines. To prepare iPS cells, scientists iso-late somatic cells (such as cells from skin)

C A S E S T U D Y One foot or another

In humans the HOXD homeotic gene cluster plays a critical role in limb development. In one large family, 16 of 36 mem-bers expressed one of two dominantly inherited malforma-

tions of the feet known as rocker bottom foot (CVT) or claw foot (CMT). One individual had one foot with CVT and the other with CMT. Genomic analysis identified a single missense mutation in the HOXD10 gene, resulting in a single amino acid substitution in the homeodomain of the encoded transcription factor. This region is crucial for making contact and binding to the target genes controlled by this protein. All family members

with the foot malformations were heterozygotes; all unaffected members were homozygous for the normal allele.

1. Given that affected heterozygotes carry one normal allele of the HOXD10 gene, how might a dominant mutation in a gene encoding a transcription factor lead to a developmental malformation?

2. How can two clinically different disorders result from the same mutation?

3. What might we learn about the control of developmental pro-cesses from an understanding of how this mutation works?

Summary Points

4. Extensive genetic analysis of embryonic development in Dro-

sophila has led to the identification of maternal-effect genes

whose products establish the anterior–posterior axis of the

embryo. In addition, these maternal-effect genes activate sets

of zygotic segmentation genes, initiating a cascade of gene reg-

ulation that ends with the determination of segment identity

by the homeotic selector genes. These same gene sets control

aspects of embryonic development in all bilateral animals,

including humans.

5. Flower formation in Arabidopsis is controlled by homeotic

genes, but these gene sets are from a different gene family than

the homeotic selector genes of Drosophila and other animals.

6. In C. elegans, the strictly determined lineage of each cell

allows developmental biologists to study the cell–cell sig-

naling required for organogenesis and to determine which

genes are required for the normal process of programmed

cell death.

1. Developmental genetics, which explores the mechanisms by

which genetic information controls development and differ-

entiation, is one of the major areas of study in biology. Ge-

neticists are investigating this topic by isolating developmental

mutations and identifying the genes involved in developmental

processes.

2. During embryogenesis, the activity of specific genes is con-

trolled by the internal environment of the cell, including local-

ized cytoplasmic components. In flies, the regulation of early

events is mediated by the maternal cytoplasm, which then in-

fluences zygotic gene expression. As development proceeds,

both the cell’s internal environment and its external environ-

ment become further altered by the presence of early gene

products and communication with other cells.

3. In Drosophila, both genetic and molecular studies have con-

firmed that the egg contains information specifying the body

plan of the larva and adult.

SUMMARY POINTS 469

and infect them with engineered retrovi-ruses that integrate into the cells’ DNA. These retroviruses contain several cloned human genes that encode products re-sponsible for converting the somatic cells into immortal, pluripotent stem cells.

The development of iPS cell lines has generated renewed enthusiasm for stem cell research, as these cells bypass the ethical problems associated with the use of human embryos. In addition, they may become sources of patient-specific plu-ripotent stem cell lines that can be used for transplantation, without immune sys-tem rejection.

At the present time, it is unknown whether stem cells of any type will be as miraculous as predicted; however, if stem cell research progresses at its current rap-id pace, we won’t have long to wait.

Your Turn

Take time, individually or in groups, to answer the following questions. Investigate the references and links

to help you understand the technologies and controversies surrounding stem cell research.

1. What, in your opinion, are the scien-tific and ethical problems that still surround stem cell research? Are these problems solved by the new methods of creating pluripotent stem cells?

You can find descriptions of some new methods of generating pluripotent stem cell lines, and the ethical issues that accompany these meth-ods, in Kastenberg, Z. J. and Odorico, J. S. 2008. Alternative sources of pluripotency: sci-ence, ethics, and stem cells. Transplantation Rev.22: 215–222.

2. What are the current stem cell re-search laws in your region, and how do these laws compare with national regulations?

A starting point for information about stem cell research regulations can be found on the Stem

Cell Information Web site of the National Insti-

tutes of Health (http://stemcells.nih.gov).

3. Do you oppose or support stem cell research? Why, or why not?

An interesting online poll, along with argu-ments for and against stem cell research, is offered by the Public Broadcasting Corpora-

tion, at http://www.pbs.org/wgbh/nova/

body/stem-cell-poll.html.

4. What, in your opinion, is the most significant development in stem cell research in the last year?

Some ideas to start your search are: the PubMed

Web site (http://www.ncbi.nlm.nih.gov/

sites/entrez? db=PubMed) and the New

York Times online Stem Cell page (http://

topics.nytimes.com/top/news/health/

diseasesconditionsandhealthtopics/

stemcells).

For activities, animations, and review quizzes, go

to the study area at www.masteringgenetics.com

http://www.pbs.org/wgbh/nova/body/stem-cell-poll.html

http://www.pbs.org/wgbh/nova/body/stem-cell-poll.html

http://www.ncbi.nlm.nih.gov/sites/entrez?db=PubMed

http://www.ncbi.nlm.nih.gov/sites/entrez?db=PubMed

http://topics.nytimes.com/top/news/health/diseasesconditionsandhealthtopics/stemcells




http://stemcells.nih.gov

www.masteringgenetics.com


H O W D O W E K N O W ? 1. In this chapter we focused on how differential gene expres-

sion guides the processes that lead from the fertilized egg to

the adult. At the same time, we found many opportunities to

consider the methods and reasoning by which much of this

information was acquired. From the explanations given in the

chapter, what answers would you propose to the following fun-

damental questions?

(a) How do we know how many genes control development in

an organism like Drosophila?

(b) What experimental evidence is available to show that mo-

lecular gradients in the egg control development?

(c) How do we know that a genetic program specifying a body

part can be changed?

(d) What genetic evidence shows that chemical signals be-

tween cells control developmental events?

(e) How do we know whether a signaling system in vulva de-

velopment works only on adjacent cells or uses signals that can

affect more distant cells?

2. Carefully distinguish between the terms differentiation and

determination. Which phenomenon occurs initially during

development?

3. Nuclei from almost any source may be injected into Xenopus

oocytes. Studies have shown that these nuclei remain active in

transcription and translation. How can such an experimental

system be useful in developmental genetic studies?

4. Distinguish between the syncytial blastoderm stage and the cel-

lular blastoderm stage in Drosophila embryogenesis.

5. (a) What are maternal-effect genes? (b) When are gene

products from these genes made, and where are they lo-

cated? (c) What aspects of development do maternal-effect

genes control? (d) What is the phenotype of maternal-effect

mutations?

Problems and Discussion Questions

I N S I G H T S A N D S O L U T I O N S

1. In the slime mold Dictyostelium, experimental evidence sug-

gests that cyclic AMP (cAMP) plays a central role in the de-

velopmental program leading to spore formation. The genes

encoding the cAMP cell-surface receptor have been cloned,

and the amino acid sequence of the protein components is

known. To form reproductive structures, free-living individual

cells aggregate together and then differentiate into one of two

cell types, prespore cells or prestalk cells. Aggregating cells se-

crete waves or oscillations of cAMP to foster the aggregation of

cells and then continuously secrete cAMP to activate genes in

the aggregated cells at later stages of development. It has been

proposed that cAMP controls cell–cell interaction and gene ex-

pression. It is important to test this hypothesis by using several

experimental techniques. What different approaches can you

devise to test this hypothesis, and what specific experimental

systems would you employ to test them?

Solution: Two of the most powerful forms of analysis in biol-

ogy involve the use of biochemical analogs (or inhibitors) to

block gene transcription or the action of gene products in a

predictable way, and the use of mutations to alter genes and

their products. These two approaches can be used to study

the role of cAMP in the developmental program of Dictyo-

stelium. First, compounds chemically related to cAMP, such

as GTP and GDP, can be used to test whether they have any

effect on the processes controlled by cAMP. In fact, both

GTP and GDP lower the affinity of cell-surface receptors for

cAMP, effectively blocking the action of cAMP.

Mutational analysis can be used to dissect components

of the cAMP receptor system. One approach is to use trans-

formation with wild-type genes to restore mutant function.

Similarly, because the genes for the receptor proteins have

been cloned, it is possible to construct mutants with known

alterations in the component proteins and transform them

into cells to assess their effects.

2. In the sea urchin, early development may occur even in the

presence of actinomycin D, which inhibits RNA synthesis.

However, if actinomycin D is present early in development but

is removed a few hours later, all development stops. In fact, if

actinomycin D is present only between the sixth and eleventh

hours of development, events that normally occur at the fif-

teenth hour are arrested. What conclusions can be drawn con-

cerning the role of gene transcription between hours 6 and 15?

Solution: Maternal mRNAs are present in the fertilized sea

urchin egg. Thus, a considerable amount of development can

take place without transcription of the embryo’s genome. Be-

cause development past 15 hours is inhibited by prior treat-

ment with actinomycin D, it appears that transcripts from the

embryo’s genome are required to initiate or maintain these

events. This transcription must take place between the sixth

and fifteenth hours of development.

3. If it were possible to introduce one of the homeotic genes

from Drosophila into an Arabidopsis embryo homozygous

for a homeotic flowering gene, would you expect any of the

Drosophila genes to negate (rescue) the Arabidopsis mutant

phenotype? Why or why not?

Solution: The Drosophila homeotic genes belong to the Hox

gene family, whereas Arabidopsis homeotic genes belong to

the MADS-box protein family. Both gene families are present

in Drosophila and Arabidopsis, but they have evolved different

functions in the animal and the plant kingdoms. As a result,

it is unlikely that a transferred Drosophila Hox gene would

rescue the phenotype of a MADS-box mutant, but only an

actual experiment would confirm this.

For instructor-assigned tutorials and

problems, go to www.masteringgenetics.com

www.masteringgenetics.com

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developmental genetics