INTRODUCTIONMulticellular organisms begins their lives as a fertilized eggFrom this simple organization they proceed step by step to a much more complex arrangement

As this occurs, cells divide, migrate, and change their characteristicsThey become highly specialized units within a multicellular individual

Developmental genetics studies the genes that orchestrate the changes that occur during developmentIt 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 process25-2

25.1 OVERVIEW OF ANIMAL DEVELOPMENTDetails differ widely among various speciesGeneral features and steps are largely conserved

25-3

The Early Stages of Embryonic Development Multicellular development in plants and animals follows a body plan or patternPattern 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:

25-4Cell movementCell differentiationCell divisionCell deathThe coordination of these four events leads to the formation of a body with a particular pattern

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 gradientA key feature of morphogens is that they act in a concentration-dependent mannerA particular concentration range of one or more morphogens restrict a cell into a specific developmental pathway

25-5

25-6Figure 25.2 Three molecular mechanisms of positional informationThis provides positional information that establishes the general polarity of an embryoThe process by which a cell or group of cells governs the developmental fate of neighboring cells Is known as induction

25-7Figure 25.2 Three molecular mechanisms of positional informationIn addition to morphogens, positional information is conveyed by cell adhesionEach cell makes its own collection of cell surface receptorsThese are known as cell adhesion molecules (CAMs)CAMs cause the cell to adhere to the extracellular matrix (ECM) and/or to other cells

Genes that Control DevelopmentMutations in organisms such as Drosophila have contributed to our understanding of developmentMutation in a complex of genes called bithorax causes the fly to have 4 wingsSee Figure 25.3Third thoracic segment forms structures like the secondGenes that specify the final identity of a body region are called homeotic genesThe establishment of the body axes and division into segments involves a few dozen genesSee list in Table 25.125-8

Figure 25.3 The bithorax mutation in Drosophila25-9

25-10Animal DevelopmentMost animals are bilaterians with left-right symmetry

Development in bilaterians generally proceeds in four overlapping stages (see Figure 25.4)Formation of body axesSegmentation of the bodyDetermination of structures within segmentsCell differentiation

Figure 25.4 Four overlapping phases of animal development25-11

In this section we will consider two model organismsDrosophila melanogaster and Caenorhabditis elegans

Why Drosophila melanogaster?It has many mutant strains with altered developmental pathwaysIt is large enough to conduct transplantation experimentsYet small enough to determine the various sites of gene expression

Why Caenorhabditis elegans?It is a rather simple organism, consisting of ~ 1,000 somatic cellsThe pattern of cell division and the fate of each cell is known

25.2 INVERTEBRATE DEVELOPMENT25-12

25-13Portions of the cell membrane surround each nucleusFigure 25.5 Drosophila development This stage involves a great deal of cell migration which forms theZygote goes through a series of nuclear divisions but NOT cytoplasmic divisionsIn the middleOn the insideOn the outsideYolk

25-14Figure 25.5 Drosophila development At the end of embryogenesisIn Drosophila, there are three larval stages separated by moltsDuring molting, the larva sheds its cuticle

After the third larval stage, Drosophila proceeds through a process termed metamorphosisGroups of cells called imaginal disks were produced earlier in developmentThese imaginal disks grow and differentiate into the structures found in the adult flyThe fly then emerges from its pupal caseIn metazoa, the final result of development is an adult body organized along three axesEven before hatching, the embryo develops the basic body plan that will be found in the adult organism

25-15

The Establishment of the Body Axes The first stage in Drosophila embryonic pattern development is the establishment of the body axesDuring oogenesis, certain gene products important in early development are deposited asymmetrically within the eggAfter fertilization, these gene products establish independent developmental programsThese govern the formation of the body axes of the embryoBicoid, Nanos, Torso and Toll are examplesThese gene products act as key morphogens or receptors for morphogensRefer to Figure 25.625-16

25-17Figure 25.6 The establishment of the axes of polarity in the Drosophila embryoNanos is required for the formation of the abdomen

Lets now take a closer look at the molecular mechanism of bicoidThe bicoid gene got its name because a larva whose mother is defective in this gene develops with two posterior ends25-18Figure 25.7Normally found only at the posterior end

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 offspringThis occurs because the bicoid gene product is provided to the oocyte via the nurse cells25-19

In the ovaries of female flies, the nurse cells are localized asymmetrically towards the anterior end of the oocyteThus, maternally encoded gene products enter one side of the oocyteThis side will eventually become the anterior end of the embryoThe bicoid gene is actively transcribed in the nurse cellsBicoid mRNA enters the anterior end of the oocyte and is trapped there25-20Figure 25.8

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 proteinWhen the bicoid mRNA is translated, a gradient of Bicoid protein is established

The Bicoid protein functions as a transcription factorIt influences gene expression based on its concentrationIt stimulates a gene called hunchback in the anterior part of the embryoBut not in the posterior part, where its concentration is low25-21

Figure 25.8 (b) and (c)25-22(b) In situ hybridization of bicoid mRNA(c) Immunostaining of Bicoid proteinCopyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.(b-c): Christiane Nusslein- Volhard, Development, Supplement 1, 1991. The Company of Biologists Limited. dev.biologists.org

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 25.9This pattern of positional information will be maintained or remembered throughout the rest of development25-23

25-24Figure 25.9 A comparison of segments and parasegments in the Drosophila embryoNote that the segments and parasegments are out of registerThe anterior part of a segment coincides with the posterior region of a parasegmentThe posterior part of a segment coincides with the anterior region of the next parasegmentThe pattern of gene expression that occurs in the anterior region of one parasegment and the posterior region of an adjacent parasegmentResults in the formation of the corresponding segment

Early in development the segmentation genes play a role in the formation of body segmentsThe expression of segmentation genes in specific regions of the embryo causes it to become segmented

There are three classes of segmentation genesGap genesPair-rule genes Segment-polarity genes

Figure 25.10 shows a few phenotypic effects observed in Drosophila larvae when a segmentation gene is defective25-25

25-26Figure 25.10Defective geneEight adjacent segments are missing from the larvaDefective geneDefective gene

Sequential expression of genes divides the embryo into segments1. Maternal effect gene products, such as bicoid mRNA are deposited asymmetrically in the oocyteThese will form a gradient that will later influence the formation of axes2. After fertilization, maternal effect gene products activate zygotic genesThe first set to be activated is the gap genes3. The gap genes and maternal effect genes then activate the pair-rule genes4. The pair-rule genes then regulate the segment polarity genesLater in development, the anterior end of one parasegment and the posterior end of another parasegment will develop into a segmentEach segment will have particular morphological characteristics

25-27

25-28Figure 25.11

Homeotic Genes and Segment Phenotype The term cell fate describes the ultimate morphological features of a cell or group of cellsIn 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 anotherIt was coined by the English zoologist William Bateson25-29

Drosophila contains two clusters of homeotic genesAntennapedia complexBithorax complex Both complexes are located on chromosome 3, but a large segment of DNA separates them (Figure 25.12)

The Antennapedia complex contains five geneslab (labial)pb (proboscipedia)Dfd (Deformed)Scr (Sex combs reduced)Antp (Antennapedia)

The bithorax complex contains three genesUbx (Ultrabithorax)abd-A (abdominal A)Abd-B (Abdominal B)25-30

Embryo(10 hours)FlychromosomeAdultAntennapediacomplexbithoraxcomplexlabpbDfdScrAntpUbxabd-AAbd-BCopyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.25-31Figure 25.12 Expression pattern of homeotic genes in DrosophilaThe order of gene expression, from anterior to posterior, parallels the order of genes on the chromosome

The role of homeotic genes has been revealed by mutations that alter their functionFor example, Figure 25.13 shows the Antennapedia mutation in Drosophila This is a gain-of-function mutation in the Antp geneIt causes the gene to be expressed in an additional place in the embryoIn this case, it is also expressed abnormally in the anterior segment that normally gives rise to the antennaeThe 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 genesWhen a particular homeotic gene is defective, its function is replaced by the gene that acts in the adjacent anterior region25-32

(a) Normal fly(b) Antennapedia mutant Juergen Berger/Photo ResearchersF. R. Turner, Indiana University/Visuals UnlimitedCopyright The McGraw-Hill Companies, Inc. Permission required for reproduction or displayy.25-33Figure 25.13 The Antennapedia mutation in Drosophila

The homeotic genes are regulated in a complex wayControlled by gap and pair-rule genes, interactions between different homeotic genes and other genesThe Polycomb genes represses the expression of homeotic genes in regions of the embryo where they should not actThis is done by remodeling chromatin into a closed conformationThis is not compatible with transcriptionThe Trithorax genes promote the expression of homeotic genes in regions of the embryo where they should actThis is done by remodeling chromatin into an open conformationThis facilitates transcription

Overall, the concerted action of many gene products cause the homeotic genes to be expressed in the appropriate regions

25-34

Homeotic genes encode transcription factorsThe coding sequence of homeotic genes contains a 180 bp consensus sequence, termed the homeoboxThis 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 homeodomainThe arrangement of a-helices promotes the binding of the protein to the major groove in DNAIn a sequence-specific manner

In addition, homeotic proteins also contain a transcriptional activation domain

25-35

25-36Figure 25.14The DNA-binding sites are found within genetic regulatory elements (i.e., enhancers)

The transcription factors encoded by homeotic genes activate the next set of genes in the developmental program of the flyThese genes produce the morphological characteristics of each segment

Future research will shed more light on how these genes control morphological changes25-37

The Nematode Caenorhabditis elegans This invertebrate has been the subject of numerous studies in developmental genetics

Life cycleThe embryo develops within the eggshell and hatches when it reaches 550 cellsAfter hatching it passes through four successive moltsIt takes about 3 days for a fertilized egg to develop into an adult worm25-38

With regards to sex, C. elegans can be a1. MaleAn adult male is composed of 1,031 somatic cellsIt produces about 1,000 sperm2. HermaphroditeAn adult hermaphrodite is composed of 959 somatic cellsIt 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 cellsTherefore, researchers can follow cell division step by stepAn illustration that depicts how cell division proceeds is called a cell lineage diagram

25-39

Fertilized eggNervoussystem,skin,musculatureMusculature,Nervoussystem,gonadSkin,NervoussystemGerm lineMusculatureIntestineP1P2ABEMSEMSABaABp010HeadTail1.2 mmCopyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.Time after fertilization (hours)25-40Figure 25.15 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

Experiment 25A: Heterochronic Mutations Disrupt DevelopmentSpatial expression and localization of gene products are crucial in embryonic developmentAnother important issue in development is timingIn C. elegans, the timing of developmental events can be examined carefully at the cellular levelUsing a microscope, a scientist can focus on a particular cell within this transparent wormHe/she can therefore judge whether a cell is behaving as it should during the developmental process25-41

To identify genes involved in the timing of cell fates, researchers have searched for timing mutants

One such mutant had a defect in egg-layingThe hermaphrodite is able to fertilize its own eggs but is unable to lay themThe eggs will then hatch within the hermaphrodites body This ultimately leads to the death of the hermaphroditeIndeed, the hermaphrodite is termed a bag of wormsEventually the newly hatched larva eat their way outThey 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

The HypothesisMutations that cause an egg-laying defective phenotype may affect the timing of cell lineagesTesting the HypothesisRefer to Figure 25.1625-43This experiment was carried out by Victor Ambros, and H. Robert Horvitz in the 1980s

Three mutant lines were used25-44Figure 25.16These were designated n536, n355 and n540Experimental levelConceptual level1. Obtain a large number of C. elegans strains that have a defective egg-laying phenotype. The wild-type strain was also studied as a control.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 division pattern, and the pattern of subsequent daughter cells, during the larval stages. These patterns were examined in both wild-type and defective egg-laying worms.In wild type:IntestineNormal adult worm:Egg-laying mutant:Newly hatched larva:IntestineSingle row of eggsIntestineMany eggscrowded insideT cellL1 (first larvalstage)L2 (second larvalstage)DiesCopyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

The Data 25-45L1L2L3L4T-cell lineages in different strains of C. elegansWild typen536n355n540Larvalstage/hour10203040NeuronsEpidermal cellsProgrammed to dieT.aT.pT.aaT.apT.paT.ppXXXXTTXXXXTTCopyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

L1L2L3L4T-cell lineages in different strains of C. elegansWild typen536n355n540Larvalstage/hour10203040NeuronsEpidermal cellsProgrammed to dieT.aT.pT.aaT.apT.paT.ppXXXXTTXXXXTTCopyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.Interpreting the Data 25-46Follows a particular pattern of cell divisionEach division occurs at a specific time in the L1 and L2 larval stagesThe irregularity in the timing of cell fates produced a worm with several additional cells derived from the T cell lineageThis strain skips the divisions and cell fates of L1It proceeds directly to cell fates that occur during the L2 stage This strain continues to reiterate the normal events of L1 during L2, L3 and L4It is now known that the three mutants are located in a gene called lin-14

The types of mutations just discussed are called heterochronic mutationsThe timing of fates for particular cell lineages is not synchronized with the development of the rest of the organismMore recent molecular data have shown that this is due to an irregular pattern of gene expressionIn wild-type worms, the Lin-14 protein accumulates during the L1 stageIt promotes the T cell division pattern shown for the wild-typeThe n536 and n355 alleles are examples of gain-of-function mutationsIn these alleles, the Lin-14 protein persists during late larval stagesn536 is expressed one cell division too laten355 is expressed for several cell divisionsThe n540 allele is a loss-of-function mutationIt causes Lin-14 to be inactive during L1Therefore, it cannot promote the normal L1 pattern of cell division and cell fate

25-47

25.3 VERTEBRATE DEVELOPMENTHistorically, development was extensively studied in amphibians and birdsEggs are rather large and easy to manipulateVertebrate species that have been studied includeMouseFrog (Xenopus laevis)Small aquarium zebrafish (Brachydanio rerio)

Among mammals, the most extensive genetic analyses have been performed in the mouse25-48

Researchers Have Identified Homeotic Genes in Vertebrates Vertebrates typically have long generation times and produce relatively few offspringTherefore, it is not practical to screen large numbers of embryos or offspring in search of developmental mutantsRather, cloned Drosophila genes are used as probes to identify homologous vertebrate genesUsing this method, researchers have found complexes of homeotic genes in many vertebrate speciesIn the mouse, the groups of adjacent homeotic genes are called Hox complexes25-49

25-50Figure 25.17 A comparison of homeotic genes in Drosophila and the mouse Chromosome 6Chromosome 11Chromosome 15Chromosome 2

Orthologous genesThis suggests that there is a universal body plan for animal development Thirteen different types of homeotic genes are found in the mouseHowever, none of the four Hox complexes contains all 13

The arrangement of Hox genes along the mouse chromosome reflects their pattern of expression from the anterior to the posterior end25-51Figure 25.18This phenomenon is seen in more detail in Figure 25.18bThe expression pattern for a group of HoxB genes is shown

In mice, few natural mutations affect developmentThis 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 genetics1. The Hox genes are first cloned using Drosophila genes as probes2. A mutant version of a Hox gene is created in vitro3. The mutant allele is the re-introduced into a mouse4. 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 mouse25-52

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 vertebratesFor instance, HoxC-6 expression in different species is correlated with the number of neck vertebrae producedSee Figure 25.1925-53

25-54Figure 25.19 Expression of the HoxC-6 gene in different species of vertebratesLine marks the anterior edge of HoxC-6 expression, which defines end of neck vertebraeSnakes have no neck, no expression of HoxC-6

Cell Differentiation Cell determination A cell is destined (predetermined) to become a particular cell typeCell 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 regulationThough 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 typesThese genes trigger undifferentiated cells to differentiate into their proper cell fates

In 1987, Harold Weintraub and his colleagues identified a gene, which they called MyoDMyoD plays a key role in skeletal muscle cell differentiationWhen a cloned copy was introduced into fibroblast cells, they differentiated into skeletal muscle cellsNormally, in vivo they never follow this pathway 25-56

Researchers later found that MyoD belongs to a group of 4 genes that initiate muscle developmentThe 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)25-57Binds DNA and activates skeletal-muscle specific genesNecessary for dimer formation between transcription factor proteins The four genes are called myogenic bHLH proteinsThey are found in all vertebrates and even some invertebrates (Drosophila and C. elegans)They are activated during skeletal muscle cell development

Molecularly, two key features enable myogenic bHLH proteins to promote muscle cell differentiation

1. The basic domain binds specifically to a muscle-cell- specific enhancer sequenceThis is adjacent to genes that are only expressed in muscle cells

2. Their activity is regulated by dimerizationHeterodimers may be activating or inhibitory

Refer to Figure 25.2025-58

25-59Figure 25.20The Id protein is produced during early stages of developmentIt prevents myogenic bHLH from promoting muscle differentiation too soonAt later stages, the levels of the Id protein fallMyogenic bHLH can now combine with the E proteins to induce muscle differentiationInhibitor of differentiation

*