BIL 250 Chapter 13 The Genetic Control of Development review part 1

Dorsal mutants are

"dorsalized" and lack ventral structures (such as the mesoderm and nervous system).

Morgan also said that

"if the mystery that surrounds embryology is ever to come within our comprehension, we must ...have recourse to other means than description of the passing show."

Two principal technologies for the visualization of gene expression in embryos or other tissues are

(1) the expression of RNA transcripts visualized by in situ hybridization and (2) the expression of proteins visualized by immunological methods.

For any toolkit gene, three pieces of information are key toward understanding gene function

(1) the mutant phenotype, (2) the pattern of gene expression, and (3) the nature of the gene product.

By saturating each of a fly's chromosomes

(except the small fourth chromosome) with chemically induced mutations, the researchers were able to identify most genes that were required for the building of the fly.

Animal genomes typically contain about

13,000 to 22,000 genes.

Each cluster contains from

9 to 11 Hox genes, a total of 39 Hox genes altogether.

The great geneticist Thomas Hunt Morgan was not immune to its aesthetic appeal:

A transparent egg as it develops is one of the most fascinating objects in the world of living beings. The continuous change in form that takes place from hour to hour puzzles us by its very simplicity. The geometric patterns that present themselves at every turn invite mathematical analysis.... This pageant makes an irresistible appeal to the emotional and artistic sides of our nature.1

But how are the spatial coordinates of the developing embryo conveyed as instructions to genes, to turn them on and off in precise patterns?

As described in Chapters 11 and 12, the physiological control of gene expression in bacteria and simple eukaryotes is ultimately governed by sequence-specific DNA-binding proteins acting on cis-acting regulatory elements (for example, operators and upstream-activation-sequence, or UAS, elements).

A key member of this class is the

Bicoid gene.

The mechanisms that we will see for controlling toolkit-gene expression in the

Drosophila embryo have emerged as models for the spatial regulation of gene expression in animal development in general.

We will first focus on the genetic toolkit of

Drosophila melanogaster because its identification was a source of major insights into the genetic control of development; its discovery catalyzed the identification of the genetic toolkit of other animals, including humans.

We will begin our inventory of the

Drosophila toolkit by examining the genes that control the identity of segments and appendages.

Subsequently, several more homeotic mutants were identified in

Drosophila, such as the dramatic Antennapedia Mutant in which legs develop in place of the antennae (Figure 13-1c).

gap genes

Each of these genes affects the formation of a contiguous block of segments; mutations in gap genes lead to large gaps in segmentation (Figure 13-14, left).

Bicoid gene.

Embryos from Bicoid mutant mothers are missing the anterior region of the embryo (Figure 13-13), telling us that the gene is required for the development of that region.

The scientific fascination with homeotic mutants stems from three properties.

First, it is amazing that a single gene mutation can alter a developmental pathway so dramatically. Second, it is striking that the structure formed in the mutant is a well-developed likeness of another body part. And, third, it is important to note that homeotic mutations transform the identity of serially reiterated structures.

This correspondence indicates that the

Hox complexes of insects and vertebrates are related and that some form of Hox complex existed in their distant common ancestor.

The fifth class includes the

Hox genes already discussed; Hox mutants do not affect segment number, but they alter the appearance of one or more segments.

First, we must understand how

Hox genes exert their dramatic effects on fly development.

It is crucial to distinguish the role of

Hox genes in determining the identity of a structure from that governing its formation.

We will examine how

Hox proteins and other toolkit proteins orchestrate gene expression in development a little later.

The long drought in embryology lasted well beyond

Morgan's heyday in the 1910s and 1920s, but it was eventually broken by geneticists working very much in the tradition of Morgan-style genetics and with his favorite, most productive genetic model, the fruit fly Drosophila melanogaster.

For their pioneering efforts

Nüsslein-Volhard, Wieschaus, and Lewis shared the 1995 Nobel Prize in Physiology or Medicine.

Of the millions of living species, which offered the most promise?

The fruit fly Drosophila melanogaster has emerged as the leading genetic model of animal development because its ease of rearing, rapid life cycle, cytogenetics, and decades of classical genetic analysis (including the isolation of many very dramatic mutants) provided important experimental advantages (see the Model Organism box on Drosophila above).

Why would such different animals have these sets of genes in common?

Their deep, common ancestry indicates that Hox genes play a fundamental role in the development of most animals.

How does the turning on and off of toolkit genes build animal form? And how is it choreographed during development?

To answer these questions, we will examine the interactions among fly toolkit proteins and genes in more detail.

For example, the dark blue shading in Figure 13-6 indicates where the

Ubx gene is expressed.

The development of these segments is altered in

Ubx mutants.

From the genetic viewpoint, there are four key questions concerning the number, identity, and function of genes taking part in development:

Which genes are important in development? Where in the developing animal and at what times are these genes active? How is the expression of developmental genes regulated? Through what molecular mechanisms do gene products affect development?

Rather, in this chapter, we will focus on

a few general concepts that illustrate the logic of the genetic control of animal development. We will explore how the information for building complex structures is encoded in the genome.

In 1915, Calvin Bridges, then Morgan's student, isolated

a fly having a mutation that caused the tiny hind wings (halteres) of the fruit fly to resemble the large forewings. He dubbed the mutant bithorax.

Each gene is expressed in

a region that can be mapped to specific coordinates along either axis of the embryo.

We will see that it is also true of many other toolkit genes

a significant fraction of these genes encode transcription factors that control the expression of other genes.

The members of this toolkit constitute only

a small fraction, perhaps several hundred genes, of the roughly 14,000 genes in the fly genome

The product of the dorsal maternal-effect gene is

a transcription factor—the Dorsal protein.

The study of animal and plant development is

a very large and still-growing discipline.

A handful of genes that are

activated in the zygote are also required for the subdivision of the dorsoventral axis.

In vertebrate embryos, adjacent Hox genes are also expressed in

adjacent or partly overlapping domains along the anteroposterior body axis.

They found that

all eight Hox genes of the two complexes were similar enough to hybridize to each other.

Furthermore, their spectacular mutant phenotypes indicate that they are

among the most globally acting genes that affect animal form.

The genes controlling segmental and appendage identity were

among the very first toolkit genes identified.

That is, the dead larva was not an

amorphous carcass but exhibited specific, often striking patterning defects.

That role is apparent from

analyses of how the Hoxgenes are expressed in different animals.

Because the early Drosophila embryo is a syncytium with all nuclei in one cytoplasm

and lacks any cell membranes that would impede the diffusion of protein molecules, the Bicoid protein can diffuse through the cytoplasm.

For example, the transplantation of a part of a developing amphibian embryo to

another site in a recipient embryo was shown to induce the surrounding tissue to form a second complete body axis (Figure 13-2a).

A high concentration means

anterior end, a lower concentration means middle, and so on.

The first class sets up the

anteroposterior axis and consists of the maternal-effect genes.

As such, we do not

attempt a comprehensive overview of embryology.

of all the phenomena in biology, few if any inspire more

awe than the formation of a complex animal from a single-celled egg.

This stretch of DNA sequence similarity,

because of its presence in homeotic genes, was dubbed the homeobox.

Bicoid binds to these sites cooperatively; that is, the

binding of one Bicoid protein molecule to one site facilitates the binding of other Bicoid molecules to nearby sites.

Similarly, the gap proteins are expressed in

blocks of cells that correspond to the future positions of the segments that are missing in respective gap-gene mutants (Figure 13-15b).

Each locus could thus be classified according to the

body axis that it affected and the pattern of defects caused by mutations.

Other genes encode proteins that

carry out the specialized tasks of various organ systems, tissues, and cells of the body such as the globin proteins in oxygen transport or antibody proteins that mediate immunity.

The more interesting mutations are those that

cause some discrete defects in either the embryonic or the adult body pattern or both.

Each gap gene contains

cis-acting regulatory elements with different arrangements of binding sites, and these binding sites may have different affinities for the Bicoid protein.

In vertebrates, such as the laboratory mouse, the Hox genes are also

clustered together in four large gene complexes on four different chromosomes.

The most intriguing feature of Hox genes is that they are

clustered together in two gene complexes that are located on the third chromosome of Drosophila

Edward Lewis, a pioneer in the study of homeotic genes, noted early on that the

clustering of Bithorax complex genes suggested that the multiple loci had arisen by tandem duplication of an ancestral gene.

Development is a

continuum in which every pattern of gene activity has a preceding causal basis.

These spatial aspects of gene expression and gene regulation are

crucial to understanding the logic of the genetic control of development.

Mutants of this class display

defects in segment polarity and number (Figure 13-14, right).

Similarly, transplantation of the posterior part of a

developing chick limb bud to the anterior could induce extra digits, but with reversed polarity with respect to the normal digits (Figure 13-2b).

Ubx is also expressed in the

developing hind wing but not in the developing forewing (Figure 13-7), as one would expect knowing that Ubx promotes hind-wing development and represses forewing development in this appendage.

Clusters of hox genes control

development in most animals

Similarly, the spatial control of gene expression during

development is largely governed by the interaction of transcription factors with cis-acting regulatory elements.

However, the spatial and temporal control of gene regulation in the

development of a three-dimensional multicellular embryo requires the action of more transcription factors on more numerous and more complex cis-acting regulatory elements.

To address these questions, strategies had to be

devised to identify, catalog, and analyze genes that control development.

The similarities in the homeobox sequences from

different species were astounding.

This activation is through

direct binding of the Bicoid protein to three sites 5′ of the promoter of the hunchback gene.

Thus, most toolkit proteins either

directly (as transcription factors) or indirectly (as components of signaling pathways) affect gene regulation.

Consequently, genes are activated in

discrete domains along the dorsoventral axis.

In this spectacular transformation, unseen forces organize the

dividing mass of cells into a form with a distinct head and tail, various appendages, and many organs.

The wild-type sequences 5′ of the hunchback gene are sufficient to

drive reporter expression in the anterior half of the embryo.

The four Hox complexes in the mouse arose by

duplications of entire Hox complexes (perhaps of entire chromosomes) in vertebrate ancestors.

Little was known about the rest of the toolkit until the late 1970s and

early 1980s, when Christine Nüsslein-Volhard and Eric Wieschaus, working at the Max Planck Institute in Tübingen, Germany, set out to find the genes required for the formation of the segmental organization of the Drosophila Embryo and larva.

One of the first tasks following the

execution of a genetic screen for mutations is to sort out those of interest.

Consequently, each gap gene is

expressed in a unique distinct domain in the embryo, in response to different levels of Bicoid and other transcription-factor gradients.

We have seen that toolkit genes are

expressed in reference to coordinates in the embryo.

In each subregion, a different set of zygotic genes are

expressed that contribute to dorsoventral patterning.

Another clue that the

expression of one set of genes might govern the expression of the succeeding set of genes comes from examining the protein products.

Extensive study of a few dozen genes has led to a

fairly detailed picture of how each body axis is established and subdivided into segments or germ layers.

The fifth class of genes determines the

fate of each segment.

Genes with products provided by the

female to the egg are called maternal-effect genes.

We will focus on a

few connections between genes in different levels of the hierarchies that lay out the basic segmental body plan and on nodal points where key genes integrate multiple regulatory inputs and respond by producing simpler gene-expression outputs.

The genes are grouped into

five classes on the basis of their realm of influence on embryonic pattern.

Dominant gain-of-function mutations in Ubx transform the

forewing into a hind wing.

Other genes control the

formation of segments, limbs, and wings and will be described later.

One clue that this progression is indeed the case comes

from analyzing the effects of mutations in toolkit genes on the expression of other toolkit genes.

Many toolkit genes can be classified according to their

function in controlling the identity of body parts (for example, of different segments or appendages), the formation of body parts (for example, of organs and appendages), the number of body parts, the formation of cell types, and the organization of the primary body axes (the anteroposterior, or A-P, and dorsoventral, or D-V, axes).

The genetic control of development, then, is

fundamentally a matter of gene regulation in space and over time.

Although the discovery of a common protein motif in each of the Hox proteins was very exciting

further analysis of the structure of the homeodomain revealed that it forms a helix-turn-helix motif—the structure common to the Lac repressor, the λ repressor, Cro, and the α2 and a1 regulatory proteins of the yeast mating-type loci!

For example, the hunchback gene is a

gap gene activated in the zygote in the anterior half of the embryo.

The second class contains the

gap genes

A case in point is the

gap genes, which must be activated in specific regions along the axis.

Thus, a way to ensure that a

gene is activated in only one location along the axis is to link gene expression to the concentration level.

We will illustrate the

general principles of how the positions of gene expression are specified with three examples.

More than one Bicoid site must be occupied to

generate a sharp boundary of reporter expression, which indicates that a threshold concentration of Bicoid protein is required to occupy multiple sites before gene expression is activated.

It has proved useful to group the

genes affected by mutations into several categories based on the nature of their mutant phenotypes.

So, they came up with a scheme to search for

genes that were required in the zygote (the product of fertilization; Figure 13-12, bottom).

Here, we are interested in a different set of

genes, those concerned with the building of organs and tissues and the specification of cell types—the genetic toolkit for development that determines the overall body plan and the number, identity, and pattern of body parts.

One of the first considerations in the

genetic analysis of animal development was which animal to study.

The long impasse in defining embryology in molecular terms was broken by

genetic approaches—mainly the systematic isolation of mutants with discrete defects in development and the subsequent characterization and study of the gene products that they encoded.

Mutant phenotypes of strict maternal-effect genes depend only on the

genotype of the female (Figure 13-12, top).

The order of gene action further suggests that the expression of one set of genes might

govern the expression of the succeeding set of genes.

Furthermore, clusters of Hox genes have been shown to

govern the patterning of other insects and to be deployed in regions along the anteroposterior axis in annelids, molluscs, nematodes, various arthropods, primitive chordates, flatworms, and other animals.

For example, the maternal-effect Bicoid protein is expressed in a

graded pattern emanating from the anterior pole of the early embryo, the section of the embryo missing in mutants (Figure 13-15a).

This protein is expressed in a

gradient along the dorsoventral axis, with its highest level of accumulation in ventral cells (Figure 13-17a).

Furthermore, the order of the Hox genes in the complexes corresponds to the

head-to-tail order of body regions in which the genes are expressed (Figure 13-10b).

Many mutations are lethal when

hemi- or homozygous because cells cannot survive without products affected by these mutations.

In the former case, the alteration is not

heritable, but homeotic mutants breed true from generation to generation.

The dominant mutations that transform adults are viable in

heterozygotes because the wild-type allele provides normal gene function to the developing animal.

Loss-of-function mutations in Ubx transform the

hind wing into a forewing.

We do so for both

historical and conceptual purposes.

They found many

homeoboxes in each of these animal genomes.

To address this possibility, researchers searched for

homeoboxes in the genomes of other insects, as well as earthworms, frogs, cows, and even humans.

The Bicoid protein is a

homeodomain-type transcription factor that is translated from maternally derived mRNA that is deposited in the egg and localized at the anterior pole.

The transformation in bithorax mutants is called

homeotic (Greek homeos, meaning same or similar) because one part of the body (the hind wing) is transformed to resemble another (the forewing), as shown in Figure 13-1b.

A mutation may cause a loss of

homeotic gene function where the gene normally acts or it may cause a gain of homeotic function where the homeotic gene does not normally act.

In addition to these transformations in appendage identity

homeotic mutations can transform segment identity, causing one body segment of the adult or larva to resemble another.

For example, mutations in the Hoxa11 and Hoxd11 genes cause the

homeotic transformation of sacral vertebrae to lumbar vertebrae (Figure 13-11).

They also developed screens to

identify those genes with products that function in the egg, before the zygotic genome is active, and that are required for the proper patterning of the embryo.

The relation between the structure of the Hox-gene complexes and the phenotypes of Hox-gene mutants was

illuminated by the molecular characterization of the genes.

Among the most fascinating abnormalities to be described in animals are those

in which one normal body part is replaced by another.

Each technology depends on the

isolation of cDNA clones representing the mature mRNA transcript and protein (Figure 13-5).

When the homeobox was discovered in fly Hox genes

it raised the question whether this feature was some peculiarity of these bizarre fly genes or was more widely distributed, in other insects or segmented animals, for example.

These examples should be thought of as

just a few snapshots of the vast number of regulatory interactions that govern fly and animal development.

The genes are also expressed in the

larval and pupal tissues that will give rise to the adult body parts.

Generally, the complete loss of any Hox-gene function is

lethal in early development.

These pathways, shown in generic form in Figure 13-16, mediate

ligand-induced signaling processes between cells, and their output generally leads to gene activation or repression.

These tests require

linking gene regulatory sequences to a reporter gene (an enzyme-encoding gene such as the LacZ gene or the green fluorescent protein of jellyfish), introducing the DNA construct into the fly germ line, and monitoring reporter expression in the embryo offspring of transgenic flies (a general overview of the method is shown in Figure 13-19).

A gap gene with fewer binding sites will not be activated at

locations with lower concentration of Bicoid protein.

Genetic crosses reveal whether the

locus is active in the maternal egg or zygote.

Elegant refinements of the latter approach have

made possible systematic searches for mutants that have identified many members of the fly's genetic toolkit.

First, the geneticist need not

make any assumptions about the number or nature of molecules required for a process.

The key catalysts to understanding the

making of animal forms were the discoveries of genetic monsters—mutant fruit flies with dramatic alterations of body structures (Figure 13-1).

The order of gene expression and the progressive refinement of domains within the embryo reveal that the

making of the body plan is a step-by-step process, with major subdivisions of the body outlined first and then refined until a fine-grain pattern is established.

The same principles apply to the

making of the dorsoventral body axis as apply to the anteroposterior axis.

The genetic approach to studying development presented

many advantages over alternative, biochemical strategies.

The nematode worm Caenorhabditis elegans also presented

many attractive features, most particularly its simple construction and well-studied cell lineages.

Yet, for all its beauty and fascination, biologists were stumped for

many decades concerning how biological form is generated during development.

Whereas only one member of a bilateral pair of structures is commonly altered in

many naturally occurring variants, both members of a bilateral pair of structures are altered in homeotic mutants of fruit flies.

Such homeotic transformations have been observed in

many species in nature, including sawflies in which a leg forms in place of an antenna and frogs in which a thoracic vertebra forms in place of a cervical vertebra (Figure 13-3).

Pair-rule mutants are

missing part of each pair of segments, but different pair-rule genes affect different parts of each double segment.

It was essentially impossible to isolate the

molecules responsible for these activities by using biochemical separation techniques.

Toolkit genes of the fruit fly have generally been identified through the

monstrosities or catastrophes that arise when they are mutated.

The cells in the organizers were postulated to produce

morphogens, molecules that induced various responses in surrounding tissue in a concentration-dependent manner.

Until their efforts

most work on fly development focused on viable adult phenotypes and not the embryo.

Over the 60 amino acids of the homeodomain, some

mouse and frog Hox proteins were identical with the fly sequences at as many as 59 of the 60 positions (Figure 13-9).

The forewings and hind wings, the segments, and antennae, legs, and

mouthparts of insects are sets of serially reiterated body parts.

Through systematic and targeted genetic analysis, as well as comparative genomic studies,

much of the genetic toolkit for the development of the bodies, body parts, and cell types of several different animal species has been defined.

In these screens, genes were identified that were

necessary to make the proper number and pattern of larval segments, to make its three tissue layers (ectoderm, mesoderm, and endoderm), and to pattern the fine details of an animal's anatomy.

Second, the (limited) quantity of a gene product is

no impediment: all genes can be mutated regardless of the amount of product made by a gene

The Drosophila larval body has various features whose

number, position, or pattern can serve as landmarks to diagnose or classify the abnormalities in mutant animals.

A morphogen could be any

one of these molecules but would be present in minuscule quantities—one needle in a haystack of cellular products.

For example, the even-skipped gene affects

one set of segmental boundaries, and the odd-skipped gene affects the complementary set of boundaries (Figure 13-14, middle).

Among vertebrates, the development of targeted gene disruption techniques

opened up the laboratory mouse to more systematic genetic study, and the zebrafish Danio rerio has recently become a favorite model owing to the transparency of the embryo and to advances in its genetic study.

Moreover, the order of the genes in the complexes and on the chromosome corresponds to the

order of body regions, from head to tail, that are influenced by each Hox gene (Figure 13-4).

Furthermore, the order of the genes in the mouse Hox complexes parallels the

order of their most related counterparts in the fly Hox complexes, as well as in each of the other mouse Hox clusters (Figure 13-10a).

The third class comprises the

pair-rule genes, which act at a double-segment periodicity.

Surprisingly, these strange fruit-fly genes turned out to be a

passport to the study of the entire animal kingdom, as counterparts to these genes were discovered that played similar roles in almost all animals.

A similar theme is found in the

patterning of the dorsoventral axis: cis-acting regulatory elements contain different numbers and arrangements of binding sites for Dorsal and other dorsoventral transcription factors.

For many decades, the study of embryonic development largely entailed the

physical manipulation of embryos, cells, and tissues.

This concentration gradient provides

positional information about the location along the anteroposterior axis.

This Hox gene is expressed in the

posterior thoracic and most of the abdominal segments of the embryo.

Yet we will see that the

principles governing the genetic control of development are connected to those already presented in Chapters 11 and 12, governing the physiological control of gene expression in bacteria and single-celled eukaryotes.

Thus, the products of these remarkable genes function through

principles that are already familiar from Chapters 11 and 12—by binding to regulatory elements of other genes to activate or repress their expression.

Note that the domains of gene expression become

progressively more refined as development proceeds: genes are expressed first in large regions (gap proteins), then in stripes from three to four cells wide (pair-rule proteins), and then in stripes from one to two cells wide (segment-polarity proteins).

A few dozen genes are required for

proper organization of the anteroposterior body axis of the fly embryo.

Thus, Bicoid has the

properties of a DNA-binding transcription factor.

Several key concepts were established about the

properties of developing embryos through experiments in which one part of an embryo was transplanted into another part of the embryo.

This diffusion establishes a

protein concentration gradient (Figure 13-18a): the Bicoid protein is highly concentrated at the anterior end, and this concentration gradually decreases as the distance from that end increases, until there is very little Bicoid protein beyond the middle of the embryo.

The homeobox encodes a

protein domain, the homeodomain, containing 60 amino acids.

Many of these genes encode

proteins that function in essential processes in all cells of the body (for example, in cellular metabolism or the biosynthesis of macromolecules). Such genes are often referred to as housekeeping genes.

Molecular cloning of the sequences encompassing each Hox locus

provided the means to analyze where in the developing animal each gene is expressed.

The second source comprises mutations induced at

random by treatment with mutagens (such as chemicals or radiation) that greatly increase the frequency of damaged genes throughout the genome.

The dorsoventral axis also is subdivided into

regions

The sets of zygotic genes expressed define

regions that give rise to particular tissue layers, such as the mesoderm and neuroectoderm (the part of the ectoderm that gives rise to the central nervous system), as shown in Figure 13-17b.

Several zygotic genes, including gap genes, are

regulated by different levels of Bicoid protein.

To define a position in an embryo

regulatory information must exist that distinguishes that position from adjacent regions.

Insect and many animal bodies are made of

repeating parts of similar structure, like building blocks, arranged in a series.

Each class of genes appeared to

represent different steps in the progressive refinement of the embryonic body plan—from those that affect large regions of the embryo to those with more limited realms of influence.

Several maternal-effect genes, such as dorsal, are

required to establish these regions at distinct positions from the dorsal (top) to ventral (bottom) side of the embryo.

This idea led researchers to

search for similarities in the DNA sequences of Hox genes.

The fourth class consists of the

segment-polarity genes, which affect patterning within each segment.

In the absence of function of all Hox genes

segments form, but they all have the same identity; limbs also can form, but they have antennal identity; and, similarly, wings can form, but they have forewing identity.

This similarity suggested immediately (and it was subsequently borne out) that Hox proteins are

sequence-specific DNA-binding proteins and that they exert their effects by controlling the expression of genes within developing segments and appendages.

These transcription factors include representatives of most known families of

sequence-specific DNA-binding proteins; so, although there is no restriction concerning which family they may belong, many early-acting toolkit proteins are transcription factors.

Such results have been obtained in

several classes, including mammals, birds, amphibians, and fish.

This hybridization was found to be due to a

short region of sequence in each gene, 180 bp in length.

Among plants, Arabidopsis thaliana has played a

similar role as Drosophila in illuminating fundamental mechanisms in plant development.

The Hox genes are perhaps the best-known members of the toolkit, but they are just a

small family in a much larger group of genes required for the development of the proper numbers, shapes, sizes, and kinds of body parts.

Subsequent discoveries about their nature were

sources of profound insights into not just how their products work, but also the content and workings of the toolkits of most animals.

In the developing embryo, the Hox genes are expressed in

spatially restricted, sometimes overlapping domains within the embryo (Figure 13-6).

The Hox-gene expression patterns of vertebrates suggested that they also

specify the identity of body regions, and subsequent analyses of Hox-gene mutants have borne this suggestion out.

The first source consists of

spontaneous mutations that arise in laboratory populations.

In the early days of Drosophila genetics, rare mutants arose

spontaneously or as by-products of other experiments with spectacular transformations of body parts.

The pair-rule proteins are expressed in

striking striped patterns: one transverse stripe is expressed per every 2 segments, in a total of 7 stripes covering the 14 future body segments (the position and periodicity of the stripes correspond to the periodicity of defects in mutant larvae), as shown in Figure 13-15c.

Many segment-polarity genes are expressed in

stripes of cells within each segment, 14 stripes in all corresponding to 14 body segments (Figure 13-15d).

The gradient establishes

subregions of differing Dorsal concentration.

The entire process includes

tens of thousands of regulatory interactions and outputs.

These transplanted regions of the amphibian embryo and chick limb bud were

termed organizers because of their remarkable ability to organize the development of surrounding tissues.

Inspection of the Bicoid protein sequence reveals

that it contains a homeodomain, related to but distinct from those of Hox proteins.

The amino acid sequence of the homeodomain is very similar among

the Hox proteins (Figure 13-8).

In vivo experiments can demonstrate that

the activation of hunchback depends on the concentration gradient.

For example, Ultrabithorax (Ubx) gene acts in

the developing hind wing to promote hind-wing development and to repress forewing development.

In regard to the Hox genes and other toolkit genes

the development of technology that made possible the visualization of gene and protein expression was crucial to understanding the relation among gene organization, gene function, and mutant phenotypes.

Similarly, the antenna-to-leg transformations of Antennapedia (Antp) mutants are caused by

the dominant gain of Atp function in the antenna.

For example, in embryos from Bicoid mutant mothers

the expression of several gap genes is altered, as well as that of pair-rule and segment-polarity genes.

In light of the vast evolutionary distances between these animals, more than 500 million years since their last common ancestor,

the extent of sequence similarity indicates very strong pressure to maintain the sequence of the homeodomain.

In contrast to the control of gene regulation in single bacterial or eukaryotic cells,

the genetic control of body formation and body patterning is fundamentally a matter of gene regulation in three-dimensional space and over time.

Systematic searches for homeotic genes have led to

the identification of eight loci, now referred to as Hox genes, that affect the identity of segments and their associated appendages in Drosophila.

Because Hox genes have large effects on the identities of entire segments and other body structures

the nature and function of the proteins that they encode are of special interest.

In addition to what we have learned from the spatial patterns of toolkit-gene expression

the order of toolkit-gene expression over time is logical.

Therefore, despite enormous differences in anatomy

the possession of one or more clusters of Hox genes that are deployed in regions along the main body axis is a common, fundamental feature of at least all bilateral animals.

The patterns of Hox-gene expression (and other toolkit genes) generally correlate with

the regions of the animal affected by gene mutations.

This finding suggests that the Bicoid protein somehow (directly or indirectly) influences

the regulation of gap genes.

Although these experimental results were spectacular and fascinating, further progress in understanding the nature of organizers and morphogens stalled after

their discovery in the first half of the 1900s.

The power of the genetic screens was

their systematic nature.

If we picture a three-dimensional embryo as a globe

then positional information must be specified that indicates longitude (location along the anteroposterior axis), latitude (location along the dorsoventral axis), and altitude or depth (position in the germ layers).

Homeotic mutations transform identities within

these sets.

Although homeotic genes were first identified through spontaneous mutations affecting adult flies

they are required throughout most of a fly's development.

The most striking and telling features of the newly identified mutants were that

they showed dramatic but discrete defects in embryo organization or patterning.

Nüsslein-Volhard and Wieschaus realized that the sorts of genes that

they were looking for were probably lethal to embryos or larvae in homozygous mutants.

Importantly, deletions of Bicoid-binding sites in

this cis-acting regulatory element reduce or abolish reporter expression (see Figure 13-18b).

Embryonic cells make

thousands of substances—proteins, glycolipids, hormones, and so forth.

The Bithoraxcomplex contains

three Hox genes, and the Antennapedia complex contains five Hox Genes.

To understand the relation between genes and mutant phenotype, we must know the

timing and location of gene-expression patterns and the molecular nature of the gene products.

Each gap gene also encodes a

transcription factor, as does each pair-rule gene, several segment-polarity genes, and, as described earlier, all Hox genes.

Those that are not

transcription factors tend to be components of signaling pathways (Table 13-1).

Indeed, the surprising lessons from the Hox genes portended what

turned out to be a general trend among toolkit genes; that is, most toolkit genes are common to different animals.

Toolkit-gene mutations from

two sources have yielded most of our knowledge.

And, third, the genetic approach can

uncover phenomena for which there is no biochemical or other bioassay.

Thus, as in the fly, the loss or gain of function of Hox genes in

vertebrates causes transformation of the identity of serially repeated structures.

The patterns of expression of the toolkit genes turn out to

vividly correspond to their phenotypes, inasmuch as they are often precisely correlated with the parts of the developing body that are altered in mutants.

The existence of Hox genes with homeoboxes throughout the animal kingdom

was entirely unexpected.

Learning about these genes should

whet our appetites for learning more about the whole toolkit that controls the development of animal form

Why different types of animals would possess the same regulatory genes was not obvious

which is why biologists were further surprised by the results when the organization and expression of Hox genes was examined in other animals.

First, there is one more huge discovery to describe,

which revealed that what we learn from fly Hox genes has very general implications for the animal kingdom.

The spectacular effects of homeotic mutants inspired what

would become a revolution in embryology, once the tools of molecular biology became available to understand what homeotic genes encoded and how they exerted such enormous influence on the development of entire body parts.

The maternal-effect Bicoid protein appears before the

zygotic gap proteins, which are expressed before the 7-striped patterns of pair-rule proteins appear, which in turn precede the 14-striped patterns of segment-polarity proteins.

The next three classes are

zygotically active genes required for the development of the segments of the embryo.