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Chapter 25

Lecture Outline
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INTRODUCTION
Multicellular organisms begins their lives as a fertilized egg
From this simple organization they proceed step by step to a much
more complex arrangement

As this occurs, cells divide, migrate, and change their


characteristics
They become highly specialized units within a multicellular individual

Developmental genetics studies the genes that orchestrate


the changes that occur during development
It is currently one of the hottest fields in molecular biology

Here, we will consider several examples in which geneticists


understand how genes govern the developmental process
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25.1 OVERVIEW OF ANIMAL


DEVELOPMENT
Details differ widely among various species
General features and steps are largely
conserved

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The Early Stages of Embryonic


Development
Multicellular development in plants and animals follows a
body plan or pattern
Pattern refers to the spatial arrangement of different body regions
At the cellular level, the body pattern is due to the arrangement of
cells and their specialization

The progressive growth of a fertilized egg into an adult


organism involves four types of cellular events:

Cell division

Cell movement

Cell differentiation

Cell death

The coordination of these four events leads to the formation of a


body with a particular pattern

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Morphogens are molecules that convey positional


information and promote developmental changes
A morphogen influences the developmental fate of a cell

Within an oocyte and during embryonic development


morphogens are distributed along a concentration
gradient
A key feature of morphogens is that they act in a
concentration-dependent manner
A particular concentration range of one or more
morphogens restrict a cell into a specific developmental
pathway

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This provides positional


information that establishes the
general polarity of an embryo

The process by
which a cell or group
of cells governs the
developmental fate
of neighboring cells
Is known as
induction

Figure 25.2 Three molecular mechanisms of positional information

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In addition to morphogens, positional information is conveyed by cell


adhesion
Each cell makes its own collection of cell surface receptors
These are known as cell adhesion molecules (CAMs)

CAMs cause the cell to adhere to the extracellular matrix (ECM) and/or
to other cells

Figure 25.2 Three molecular mechanisms of positional information

25-7

Genes that Control Development


Mutations in organisms such as Drosophila have
contributed to our understanding of development
Mutation in a complex of genes called bithorax causes the
fly to have 4 wings
See Figure 25.3
Third thoracic segment forms structures like the second

Genes that specify the final identity of a body region are


called homeotic genes
The establishment of the body axes and division into
segments involves a few dozen genes
See list in Table 25.1

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Figure 25.3 The bithorax mutation in Drosophila

25-9

Animal Development
Most animals are bilaterians with left-right symmetry
Development in bilaterians generally proceeds in
four overlapping stages (see Figure 25.4)

Formation of body axes


Segmentation of the body
Determination of structures within segments
Cell differentiation

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Figure 25.4 Four overlapping phases of animal development

25-11

25.2 INVERTEBRATE
DEVELOPMENT

In this section we will consider two model organisms

Drosophila melanogaster and Caenorhabditis elegans

Why Drosophila melanogaster?

It has many mutant strains with altered developmental pathways


It is large enough to conduct transplantation experiments

Yet small enough to determine the various sites of gene expression

Why Caenorhabditis elegans?

It is a rather simple organism, consisting of ~ 1,000 somatic cells

The pattern of cell division and the fate of each cell is known

25-12

Yolk

Zygote goes through a series


of nuclear divisions but NOT
cytoplasmic divisions

Portions of the cell membrane


surround each nucleus

In the middle

On the inside

This stage involves a great


deal of cell migration which
forms the

Figure 25.5 Drosophila development

On the outside

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At the end of embryogenesis

In Drosophila, there are


three larval stages
separated by molts
During molting, the
larva sheds its cuticle

After the third larval stage,


Drosophila proceeds
through a process termed
metamorphosis
Groups of cells called
imaginal disks were
produced earlier in
development
These imaginal disks
grow and differentiate
into the structures
found in the adult fly
The fly then emerges from
its pupal case

Figure 25.5 Drosophila development

In metazoa, the final result of


development is an adult body
organized along three axes
Even before hatching, the embryo
develops the basic body plan that
will be found in the adult organism

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25-15

The Establishment of
the Body Axes
The first stage in Drosophila embryonic pattern
development is the establishment of the body axes
During oogenesis, certain gene products important in early
development are deposited asymmetrically within the egg
After fertilization, these gene products establish
independent developmental programs
These govern the formation of the body axes of the embryo
Bicoid, Nanos, Torso and Toll are examples

These gene products act as key morphogens or receptors


for morphogens
Refer to Figure 25.6

25-16

Nanos is required for the


formation of the abdomen

Figure 25.6

The establishment of the axes of polarity in the Drosophila embryo

25-17

Lets now take a closer look at the molecular mechanism


of bicoid
The bicoid gene got its name because a larva whose mother is
defective in this gene develops with two posterior ends

Normally found only


at the posterior end

Figure 25.7

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Bicoid exhibits a maternal effect mode of inheritance


Refer to Chapter 5
Consider a female fly that is
Phenotypically normal (because its mother was heterozygous for
the normal bicoid allele)
But genotypically homozygous for an inactive bicoid allele
(because it inherited the inactive allele from her mother and father)

This fly produces 100% affected offspring even if mated to


a male that is homozygous for the normal bicoid allele
In other words, the genotype of the mother determines the
phenotype of the offspring
This occurs because the bicoid gene product is provided to the
oocyte via the nurse cells

25-19

In the ovaries of female flies, the nurse cells are localized asymmetrically
towards the anterior end of the oocyte
Thus, maternally encoded gene products enter one side of the oocyte
This side will eventually become the anterior end of the embryo

The bicoid gene is actively transcribed in the nurse cells


Bicoid mRNA enters the anterior end of the oocyte and is trapped there

Figure 25.8

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Figure 25.8b is an in situ hybridization experiment


showing the location of bicoid mRNA
Bicoid mRNA is highly concentrated near the anterior end

Figure 25.8c is an immunostaining experiment


showing the location of Bicoid protein
When the bicoid mRNA is translated, a gradient of Bicoid
protein is established
The Bicoid protein functions as a transcription factor
It influences gene expression based on its concentration
It stimulates a gene called hunchback in the anterior part of the
embryo
But not in the posterior part, where its concentration is low

25-21

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

(b) In situ hybridization of bicoid mRNA

(c) Immunostaining of Bicoid protein


(b-c): Christiane Nusslein- Volhard, Development, Supplement 1, 1991.
The Company of Biologists Limited. dev.biologists.org

Figure 25.8 (b) and (c)

25-22

The Establishment of
Segmentation
The next developmental process after axes
formation, is the organization of the embryo into
segments
The segmentation pattern is shown in Figure 25.9
This pattern of positional information will be maintained or
remembered throughout the rest of development

25-23

Note that the segments and parasegments are out of register


The anterior part of a segment coincides with the posterior region of a parasegment
The posterior part of a segment coincides with the anterior region of the next parasegment

The pattern of gene expression that occurs in the anterior region of one parasegment and
the posterior region of an adjacent parasegment

Results in the formation of the corresponding segment

Figure 25.9

A comparison of segments and parasegments in the Drosophila embryo

25-24

Early in development the segmentation genes play a


role in the formation of body segments
The expression of segmentation genes in specific regions
of the embryo causes it to become segmented

There are three classes of segmentation genes


Gap genes
Pair-rule genes
Segment-polarity genes
Figure 25.10 shows a few phenotypic effects observed in
Drosophila larvae when a segmentation gene is defective
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Eight adjacent
segments are
missing from the
larva

Anterior portion
of each segment
is missing from
the larva
Defective
gene

Even-numbered
parasegments
are missing
from the larva
Defective
gene

Defective
gene

Figure 25.10

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Sequential expression of genes


divides the embryo into segments
1. Maternal effect gene products, such as bicoid mRNA are
deposited asymmetrically in the oocyte
These will form a gradient that will later influence the formation of axes

2. After fertilization, maternal effect gene products activate


zygotic genes
The first set to be activated is the gap genes

3. The gap genes and maternal effect genes then activate the
pair-rule genes
4. The pair-rule genes then regulate the segment polarity
genes
Later in development, the anterior end of one parasegment and the
posterior end of another parasegment will develop into a segment
Each segment will have particular morphological characteristics

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Figure 25.11

25-28

Homeotic Genes and


Segment Phenotype
The term cell fate describes the ultimate
morphological features of a cell or group of cells
In Drosophila, the cells in each body segment have their
fate determined very early in embryological development,
long before morphological features become apparent

The term homeotic refers to mutant alleles in which


one body part is replaced by another
It was coined by the English zoologist William Bateson
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Drosophila contains two clusters of homeotic genes


Antennapedia complex
Bithorax complex
Both complexes are located on chromosome 3, but a large segment of
DNA separates them (Figure 25.12)

The Antennapedia complex contains five genes

lab (labial)
pb (proboscipedia)
Dfd (Deformed)
Scr (Sex combs reduced)
Antp (Antennapedia)

The bithorax complex contains three genes


Ubx (Ultrabithorax)
abd-A (abdominal A)
Abd-B (Abdominal B)

25-30

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Antennapedia
complex

bithorax
complex

Fly
chromosome
lab pb Dfd Scr Antp Ubx abd-A Abd-B

The order of gene expression,


from anterior to posterior,
parallels the order of genes on
the chromosome

Embryo
(10 hours)

The expression pattern of


four genes- lab, Dfd, Antp
and Abd-B is shown

Adult

Figure 25.12 Expression pattern of homeotic genes in Drosophila

25-31

The role of homeotic genes has been revealed by


mutations that alter their function
For example, Figure 25.13 shows the Antennapedia
mutation in Drosophila
This is a gain-of-function mutation in the Antp gene
It causes the gene to be expressed in an additional place in the
embryo
In this case, it is also expressed abnormally in the anterior segment
that normally gives rise to the antennae

The abnormal expression of the Antp gene in this region causes


the antennae to be converted into legs!

Investigators have also studied many loss-of-function


alleles in homeotic genes
When a particular homeotic gene is defective, its function is
replaced by the gene that acts in the adjacent anterior region

25-32

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Juergen Berger/Photo Researchers

(a) Normal fly

Figure 25.13

F. R. Turner, Indiana University/Visuals Unlimited

(b) Antennapedia mutant

The Antennapedia mutation in Drosophila

25-33

The homeotic genes are regulated in a complex way


Controlled by gap and pair-rule genes, interactions
between different homeotic genes and other genes
The Polycomb genes represses the expression of
homeotic genes in regions of the embryo where they
should not act
This is done by remodeling chromatin into a closed conformation
This is not compatible with transcription

The Trithorax genes promote the expression of homeotic


genes in regions of the embryo where they should act
This is done by remodeling chromatin into an open conformation
This facilitates transcription

Overall, the concerted action of many gene products cause


the homeotic genes to be expressed in the appropriate
regions
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Homeotic genes encode transcription factors


The coding sequence of homeotic genes contains a 180 bp
consensus sequence, termed the homeobox
This has been found in all homeotic genes and in other genes
affecting pattern development, such as bicoid

The protein domain encoded by the homeobox is called a


homeodomain
The arrangement of -helices promotes the binding of the protein
to the major groove in DNA
In a sequence-specific manner

In addition, homeotic proteins also contain a transcriptional


activation domain

25-35

The DNA-binding sites


are found within genetic
regulatory elements (i.e.,
enhancers)

Figure 25.14

25-36

The transcription factors encoded by homeotic


genes activate the next set of genes in the
developmental program of the fly
These genes produce the morphological
characteristics of each segment

Future research will shed more light on how


these genes control morphological changes

25-37

The Nematode
Caenorhabditis elegans
This invertebrate has been the subject of numerous
studies in developmental genetics
Life cycle
The embryo develops within the eggshell and hatches
when it reaches 550 cells
After hatching it passes through four successive molts
It takes about 3 days for a fertilized egg to develop into an
adult worm
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With regards to sex, C. elegans can be a


1. Male
An adult male is composed of 1,031 somatic cells
It produces about 1,000 sperm

2. Hermaphrodite
An adult hermaphrodite is composed of 959 somatic cells
It produces about 2,000 gametes (both sperm and eggs)

Remarkably, the pattern of cellular development in


this organism is invariant from worm to worm
C. elegans is transparent and has relatively few cells
Therefore, researchers can follow cell division step by step
An illustration that depicts how cell division proceeds is called a cell
lineage diagram

25-39

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Fertilized egg

Time after fertilization (hours)

AB
ABa ABp
Nervous
system,
skin,
musculature

MS

EMS

P1

P2

Musculature,
Nervous
system,
gonad

Skin,
Nervous
system

Germ line

Musculature

10

The EMS cell, E cell,


and the intestinal
cells are all part of
the same cell lineage
A cell lineage refers
to a series of cells
that are derived
from each other by
cell division

Intestine

Head

Tail

1.2 mm

Figure 25.15

25-40

Experiment 25A: Heterochronic


Mutations Disrupt Development
Spatial expression and localization of gene products
are crucial in embryonic development
Another important issue in development is timing
In C. elegans, the timing of developmental events
can be examined carefully at the cellular level
Using a microscope, a scientist can focus on a particular
cell within this transparent worm
He/she can therefore judge whether a cell is behaving as it should
during the developmental process

25-41

To identify genes involved in the timing of cell fates,


researchers have searched for timing mutants
One such mutant had a defect in egg-laying
The hermaphrodite is able to fertilize its own eggs but is
unable to lay them
The eggs will then hatch within the hermaphrodites body
This ultimately leads to the death of the hermaphrodite
Indeed, the hermaphrodite is termed a bag of worms

Eventually the newly hatched larva eat their way out


They can then be saved for further study

The first egg-laying defective organism yielded several


mutant strains that were defective in certain cell lineages
25-42

This experiment was carried out by Victor Ambros,


and H. Robert Horvitz in the 1980s

The Hypothesis
Mutations that cause an egg-laying defective
phenotype may affect the timing of cell lineages

Testing the Hypothesis

Refer to Figure 25.16


25-43

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Experimental level
1. Obtain a large number of C. elegans Normal adult worm:
strains that have a defective egg-laying
phenotype. The wild-type strain was
also studied as a control.
Single row of eggs

Three mutant lines were


used
These were designated
n536, n355 and n540

Conceptual level

Intestine

Egg-laying mutant:
Intestine
Many eggs
crowded inside

2. Right after hatching, observe the fate of


particular cells via microscopy. In this
example, a researcher began watching a
cell called the T cell and monitored its Newly hatched larva:
division pattern, and the pattern of
Intestine
subsequent daughter cells, during the
larval stages. These patterns were
examined in both wild-type and
defective egg-laying worms.

In wild type:
T cell

L1 (first
larval
stage)

Dies

L2 (second
larval
stage)

Figure 25.16

25-44

The Data
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T-cell lineages in different strains of C. elegans


Larval
stage/hour

Wild type
T

L1

n355

T.a

10

n536

n540

T.p
T.ap

T.pa

T.pp

T.aa
X

L2 20
L3 30

L4 40

Neurons
Epidermal cells
Programmed to die

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Interpreting the Data


Follows a particular pattern
of cell division
Each division occurs at a specific
time in the L1 and L2 larval stages

This strain skips the


divisions and cell fates
of L1
It proceeds directly to
cell fates that occur
during the L2 stage

The irregularity in the timing of


cell fates produced a worm with
several additional cells derived
from the T cell lineage

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

T-cell lineages in different strains of C. elegans


Larval
stage/hour

Wild type

n536

n355

n540

T.a

L1
10 T.aa
L2

20

L3

30

L4

40

T.p
T.ap

T.pa

T.pp

Neurons

It is now known
that the three
mutants are
located in a
gene called
lin-14

This strain continues


to reiterate the
normal events of L1
during L2, L3 and L4

Epidermal cells
X

Programmed to die

25-46

The types of mutations just discussed are called


heterochronic mutations
The timing of fates for particular cell lineages is not synchronized with
the development of the rest of the organism

More recent molecular data have shown that this is due to an


irregular pattern of gene expression
In wild-type worms, the Lin-14 protein accumulates during the L1 stage
It promotes the T cell division pattern shown for the wild-type

The n536 and n355 alleles are examples of gain-of-function mutations


In these alleles, the Lin-14 protein persists during late larval stages
n536 is expressed one cell division too late
n355 is expressed for several cell divisions

The n540 allele is a loss-of-function mutation


It causes Lin-14 to be inactive during L1
Therefore, it cannot promote the normal L1 pattern of cell division and
cell fate

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25.3 VERTEBRATE
DEVELOPMENT
Historically, development was extensively studied in
amphibians and birds
Eggs are rather large and easy to manipulate

Vertebrate species that have been studied include


Mouse
Frog (Xenopus laevis)
Small aquarium zebrafish (Brachydanio rerio)
Among mammals, the most extensive genetic analyses
have been performed in the mouse
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Researchers Have Identified


Homeotic Genes in Vertebrates
Vertebrates typically have long generation times
and produce relatively few offspring
Therefore, it is not practical to screen large numbers of
embryos or offspring in search of developmental mutants

Rather, cloned Drosophila genes are used as


probes to identify homologous vertebrate genes
Using this method, researchers have found complexes of
homeotic genes in many vertebrate species
In the mouse, the groups of adjacent homeotic genes are
called Hox complexes
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This suggests that there is


a universal body plan
for animal development
Orthologous genes

Chromosome 6

Chromosome 11
Chromosome 15
Chromosome 2

Thirteen different types of homeotic genes are found in the mouse


However, none of the four Hox complexes contains all 13

Figure 25.17

A comparison of homeotic genes in Drosophila and the mouse

25-50

The arrangement of Hox genes along the mouse


chromosome reflects their pattern of expression from the
anterior to the posterior end
Figure 25.18

This phenomenon is seen in more detail in Figure 25.18b

The expression pattern for a group of HoxB genes is shown

25-51

In mice, few natural mutations affect development


This makes it tough to understand the role genes play in
the development of the mouse and other vertebrates

To circumvent this problem, geneticists are using


reverse genetics
1. The Hox genes are first cloned using Drosophila genes
as probes
2. A mutant version of a Hox gene is created in vitro
3. The mutant allele is the re-introduced into a mouse
4. A gene knockout is generated when the function of the
wild-type gene is eliminated
This allows the geneticist to determine how the mutant allele
affects the phenotype of the mouse

25-52

In recent years, a reverse genetics approach has


been used to understand the role of the Hox genes
Overall, the indication is that they play a key role in
patterning the anteroposterior axis in vertebrates
For instance, HoxC-6 expression in different species is
correlated with the number of neck vertebrae produced
See Figure 25.19

25-53

Snakes have no neck, no


expression of HoxC-6

Figure 25.19

Line marks the anterior


edge of HoxC-6
expression, which defines
end of neck vertebrae

Expression of the HoxC-6 gene in different species of vertebrates

25-54

Cell Differentiation
Cell determination
A cell is destined (predetermined) to become a particular
cell type

Cell differentiation
A cells morphology and function have changed, usually
permanently, into a highly specialized cell type

At the molecular level, the profound difference


between cell types arises from gene regulation
Though different cells contain the same set of genes, they
regulate the expression of their genes in different ways
25-55

Researchers have identified specific genes that


cause cells to differentiate into particular cell types
These genes trigger undifferentiated cells to differentiate
into their proper cell fates

In 1987, Harold Weintraub and his colleagues


identified a gene, which they called MyoD
MyoD plays a key role in skeletal muscle cell differentiation
When a cloned copy was introduced into fibroblast cells,
they differentiated into skeletal muscle cells
Normally, in vivo they never follow this pathway

25-56

Researchers later found that MyoD belongs to a


group of 4 genes that initiate muscle development
The other three are Myogenin, Myf5, and Mrf4

All four genes encode transcription factors that


contain a
basic domain and a helix-loop-helix domain (bHLH)

Binds DNA and


activates skeletalmuscle specific genes

Necessary for dimer formation


between transcription factor proteins

The four genes are called myogenic bHLH proteins

They are found in all vertebrates and even some


invertebrates (Drosophila and C. elegans)
They are activated during skeletal muscle cell development
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Molecularly, two key features enable myogenic bHLH


proteins to promote muscle cell differentiation
1. The basic domain binds specifically to a muscle-cellspecific enhancer sequence
This is adjacent to genes that are only expressed in muscle cells

2. Their activity is regulated by dimerization


Heterodimers may be activating or inhibitory

Refer to Figure 25.20

25-58

At later stages, the levels


of the Id protein fall

Inhibitor of
differentiation

Myogenic bHLH can now


combine with the E
proteins to induce muscle
differentiation

The Id protein is produced


during early stages of
development
It prevents myogenic
bHLH from promoting
muscle differentiation too
soon

Figure 25.20

25-59

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