Am nun

Figure 2.6

Methods for visualizing gene expression in developing animals

The two most common means of visualizing where a gene is transcribed and its protein product is synthesized are (left) in situ hybridization of complementary RNA probe to mRNA and (right) immunolocalization of protein expression. The procedures for each method are indicated. Gene expression patterns are visualized as the product of enzymatic reactions (left) or with fluorescently labeled compounds (right).

The relationship between the structure of Hox gene complexes and the phenotypes of Hox mutants was illuminated by the molecular characterization of both the Bithorax Complex and the Antennapedia Complex. Cloning of the Hox genes provided the means to uncover when and where each of the eight genes is expressed during development. The ability to visualize Hox and other gene expression patterns during development was crucial to understanding the correlation between gene function and phenotypes. Localization of Hox genes' RNA transcripts by in situ hybridization or of Hox proteins via immunological methods (Fig. 2.6) revealed that all Hox genes are expressed in spatially restricted, sometimes overlapping domains within the embryo. These genes are also expressed in subsets of the developing larval imaginal discs, which proliferate during larval development and differentiate during the pupal stages to give rise to the adult fly.

The patterns of Hox gene expression generally correlate with the regions of the animal affected by homeotic mutations. For example, the Ubx gene is expressed within the posterior thoracic and most anterior abdominal segments of the embryo (Fig. 2.7a). The development of these segments is altered in Ubx mutants. In larvae, Ubx is expressed in the developing haltere, but not in the developing wing (Fig. 2.7b-e). This expression correlates with the requirement for Ubx to promote haltere development and to suppress wing identity.

The boundaries of Hox gene expression in the Drosophila embryo are not segmental, but usually begin in the posterior part of one segment and extend to (or beyond) the anterior portion of the next-most-posterior segment, a unit dubbed a parasegment. In the various imaginal tissues of the developing adult, homeotic genes are often expressed in segmental domains. For example, flies have three pairs of legs, with one pair extending from each of the three thoracic segments. Each pair of adult legs has a distinctive morphology. Indeed, genetic analysis has shown that the morphology of the first legs is largely influenced by the Scr gene, the second legs by the Antp gene, and the third legs by the Ubx gene. These respective genetic requirements correlate with the respective patterns of homeotic gene expression in the developing imaginal legs.

It is crucial to understand the distinction between Hox gene function in determining the identity of a field, as opposed to a requirement for Hox gene function in the formation of the field. The antennae, mouthparts, and walking limbs of flies all develop from serially homologous limb fields. In the absence of homeotic genes, each limb field develops, but with antennal identity. Therefore, Hox genes specify the particular identity, but are not required for the formation of the limb fields. The expression and function of Hox genes are not limited to body segments and their appendages. These genes act as region-specific selectors in all three germ layers (ectoderm, mesoderm, and endoderm) and in diverse structures and tissues.

The homeobox

The large effects of Hox genes on the developmental fates of entire segments and structures made the nature of the proteins encoded by these genes of special interest. The close genetic linkage and similar function of Drosophila Hox genes suggested that they might have evolved through the tandem duplication of one or more ancestral Hox genes. This idea led to the discovery that the DNA sequences of the Hox genes of the Bithorax and Antennapedia Complexes were similar enough to hybridize to each other. This similarity was traced to a 180 base-pair (bp) stretch of DNA, dubbed the homeobox, that encodes a 60 amino acid protein domain (the homeodomain); the sequence of the homeodomain is very similar among the

Figure 2.7

Hox gene expression

Figure 2.7

Hox gene expression

Hox gene expression is restricted to regions of the body and to particular structures. For example, (a) the Ubx protein (shown in green) is expressed in the posterior thoracic (T) and seven anterior abdominal (A) segments of the embryo. (b) The adult wing. (c) Ubx is not expressed in the cells of the wing imaginal disc. (d) The adult haltere. (e) Ubx is expressed in the cells of the haltere imaginal disc.

homeotic proteins (Fig. 2.8). The structure of the homeodomain resembles the DNA-binding domain of many prokaryotic regulatory proteins, suggesting that homeotic gene products exert their effects by controlling gene expression during development and that the homeo-domain binds to DNA in a sequence-specific manner.

The homeobox gene family is large and diverse. In fact, the homeodomain motif is found in approximately 20 other distinct families of homeobox-containing genes, all of which encode DNA-binding proteins.

lab NNSGRTNFTNKQLTELEKEFHFNRYLTRARRIEIANTLQLNETQVKIWFQNRRMKQKKRV

pb PRRLRTAYTNTQLLELEKEFHFNKYLCRPRRIEIAASLDLTERQVKVWFQNRRMKHKRQT

Dfd PKRQRTAYTRHQILELEKEFHYNRYLTRRRRIEIAHTLVLSERQIKIWFQNRRMKWKKDN

Scr TKRQRTSYTRYQTLELEKEFHFNRYLTRRRRIEIAHALCLTERQIKIWFQNRRMKWKKEH

Antp RKRGRQTYTRYQTLELEKEFHFNRYLTRRRRIEIAHALCLTERQIKIWFQNRRMKWKKEN

Ubx RRRGRQTYTRYQTLELEKEFHTNHYLTRRRRIEMAHALCLTERQIKIWFQNRRMKLKKEI

abd-A RRRGRQTYTRFQTLELEKEFHFNHYLTRRRRIEIAHALCLTERQIKIWFQNRRMKLKKEL

abd-B VRKKRKPYSKFQTLELEKEFLFNAYVSKQKRWELARNLQLTERQVKIWFQNRRMKNKKNS

consensus -RRGRT-YTR-QTLELEKEFHFNRYLTRRRRIEIAHALCLTERQIKIWFQNRRMK-KKE-

Helix 1 Helix 2 Helix 3

Figure 2.8

Homeodomains of Drosophila Hox genes

Each of the eight Drosophila Hox genes encodes proteins containing a highly conserved 60 amino acid DNA-binding domain, the homeodomain, composed of three alpha helices. The third helix is most conserved in sequence. Conserved residues are shaded in yellow; divergent residues are shaded in red; those shared among subsets of proteins are shaded in blue or green.

Field-specific selector genes

Another class of selector genes acts within specific developing fields to regulate the formation and/or the patterning of entire structures. Such genes have been identified in Drosophila through spontaneous or induced mutations that selectively abolish or reduce the development of the eye, wing, limbs, and heart. Several of these genes also have the remarkable ability to induce the formation of ectopic organs when they are expressed at different sites in the animal.

Perhaps the best-known Drosophila field-specific selector gene is the eyeless (ey) gene. Flies that lack ey function can reach adulthood, but never develop a compound eye (Fig. 2.9a,b). Molecular characterization of the ey gene revealed that it encodes a member of a particular homeobox gene family (Pax6), suggesting that the Ey protein acts as a DNA-binding transcription factor to regulate the expression of other genes.

The ey gene is expressed in the developing eye field in the embryo, and in the larval eye imaginal disc, before the formation of the units (ommatidia) that make up the compound fly eye (Fig. 2.10b). Most remarkable, however, is the ability of the ey gene when expressed elsewhere in the developing fly, such as in the imaginal wing or leg discs, to induce the formation of eye tissue composed of properly organized, pigmented ommatidia (Fig. 2.9c,d). Its ability to reprogram other developing tissues to form eyes suggests that ey is a major regulatory gene in the genetic program of eye development.

The Distal-less (Dll) gene displays similar properties with respect to the formation of Drosophila limbs. Named for the effect of its mutations on the formation of the proximodistal axis of the limbs, Dll affects the development of all limbs, including the walking legs, mouthparts, antenna, and genitalia. Complete loss of Dll function truncates all limbs (that is, the limbs lack distal elements). The Dll gene is yet another type of homeobox-containing gene, suggesting that Dll also exerts its effects by regulating the expression of other genes. It is expressed in the limb primordia in the embryo, and in the distal portion of all imaginal limb fields

Figure 2.9

The eyeless selector gene controls eye development

(a) Normal fly head with eye. (b) The ey mutant fly lacks the eye. (c) Expression of the ey gene induces the formation of pigmented eye tissue at new sites, including (d) on the wing. Source: Photographs courtesy of Georg Halder.

Figure 2.9

The eyeless selector gene controls eye development

(a) Normal fly head with eye. (b) The ey mutant fly lacks the eye. (c) Expression of the ey gene induces the formation of pigmented eye tissue at new sites, including (d) on the wing. Source: Photographs courtesy of Georg Halder.

(Fig. 2.10c,d). Expressing Dll in places where it is not normally active can induce the outgrowth of ectopic limbs.

Development of the flight appendages of Drosophila depends on the function of a pair of genes, vestigial(vg) and scalloped(sd), whose products act together in a molecular complex. Fruit flies with vg and sd mutations lack wings and halteres altogether. The Vg and Sd proteins are expressed in the wing and haltere primordia in the embryo and in fields of cells within the imaginal discs that will give rise to the flight appendages (see Fig. 2.10c). As is the case with the other field-specific selector genes, expression of vg with sd in developing eyes, legs, antenna, or genitalia can induce the formation of wing tissue. The Vg and Sd proteins form a

Figure 2.10

Field-specific selector genes

(a) Development of parts of the Drosophila adult depend upon the function of the ey(eyes), vg(flight appendages), and Dll(limbs) selector genes. (b-d) These genes are expressed in both the embryonic primordia (left) and larval imaginal discs (right), which will give rise to these structures.

Source: Photomicrographs courtesy of Georg Halder and Grace Panganiban.

complex that binds to DNA, indicating that their selector function is mediated by regulation of gene expression.

The formation of the Drosophila heart depends on still another selector gene, dubbed tinman (tin). Mutants lacking tin function lack a heart. The tin gene is expressed in the developing mesoderm and in all cells that will form the cardiac tissue of the fly. It is a member of a distinct homeobox family, and thus also a DNA-binding protein that acts by controlling gene expression.

Compartment selector genes

Several genes have been identified in Drosophila that act within certain developing fields to subdivide them into separate cell populations, or compartments. The engrailed (en) gene acts in the posterior part of all segments of the embryo; it is expressed continuously such that the posterior portions of all structures that develop from these segments also express en (Fig. 2.11a). The function of the engrailed gene is best understood in the embryo and in the developing wing, where it acts to determine posterior identity. Mutations in this gene cause posterior cells to develop as anterior cells but with reversed segmental polarity, resulting in mirror-image duplications of anterior tissue. The engrailed gene encodes member of a distinct class of homeodomain-containing transcription factors.

A second compartmental selector gene, apterous (ap), subdivides the developing wing imaginal disc into dorsal and ventral compartments (Fig. 2.11b-e). Complete loss of apterous function blocks wing development, whereas loss of apterous function within a subset of dorsal cells transforms their identity to ventral fate. The Apterous protein belongs to yet another class of homeodomain-containing transcription factors.

Cell-type-specific selector genes

Another class of selector genes operates within developing fields to control the differentiation of particular cell types. The formation of neuroblasts and other neural precursor cells in Droso-phila requires the action of members of the Achaete-Scute Complex (AS-C), a gene complex that contains four genes. Loss of AS-C gene function in the embryo prevents formation of the nervous system; loss or reduction of individual AS-C gene functions in particular body regions in the imaginal tissues of the developing adult fly causes loss of particular sensory bristles.

All four AS-C genes encode structurally related transcription factors. The genes are expressed in dynamic and complex patterns that foreshadow the formation of central and peripheral nervous system elements in the larva and adult. The development of neural precursors is initiated within clusters of cells that express AS-C genes, from which a single precursor segregates, divides, and gives rise to neurons and associated cells (Fig. 2.12). A similar process involving a distantly related group of transcription factors specifies muscle development in Drosophila. The twist, nautilus, and Dmef-2genes control the development and differentiation of muscle cells.

Formation of the body axes

Systematic searches for developmental genes in Drosophila

Many of the selector genes described in the previous section were first identified on the basis of the adult phenotypes of spontaneous mutants in Drosophila. Most of those mutations,

Figure 2.11

Compartmental selector genes

Figure 2.11

Compartmental selector genes

(a) The Engrailed protein (shown in blue) is expressed in all cells in the posterior compartment of the wing imaginal disc. (b) The Apterous protein (green) is expressed in all cells in the dorsal compartment of the wing imaginal disc, and subdivides the fields of (c) vestigial-expressing cells (red) into (d) dorsal (yellow; overlap) and ventral (red) populations. (e) The territories marked by expression of the proteins in parts b-d in the larval imaginal disc correspond to future regions of the adult wing.

however, did not completely disrupt the gene's function during development. Complete loss of function of many selector genes is lethal at earlier stages of development. Therefore, to find genes that control other aspects of embryo organization and patterning, genetic screens had to be designed that could identify recessive lethal mutations.

Figure 2.12

A cell-type-specific selector gene

(a) The Achaete protein is expressed in clusters of proneural cells (shown in greater detail in b) that foreshadow the pattern of neural precursors. (c) Single precursor cells within each cluster will segregate and give rise to neuroblasts.

Figure 2.12

A cell-type-specific selector gene

(a) The Achaete protein is expressed in clusters of proneural cells (shown in greater detail in b) that foreshadow the pattern of neural precursors. (c) Single precursor cells within each cluster will segregate and give rise to neuroblasts.

Two types of systematic screens have harvested the lion's share of the Drosophila genetic toolkit. The first searched for all loci that were required in the fertilized egg, or zygote, for proper patterning of the larva. The second type of search sought to identify those genes whose products function in the egg for proper patterning, before the zygotic genome becomes active. Genes whose products are provided by the female to the egg are called maternal effect genes. Mutant phenotypes of strict maternal effect genes depend only on the genotype of the female parent (Fig. 2.13).

The two types of systematic mutagenesis screens revealed that mutations in only a small fraction of all genes in the genome have very specific effects on the organization and patterning of the embryo and larva. In addition to their maternal or zygotic actions, these mutants and the corresponding genetic loci can be classified according to the embryonic axis affected (anteroposterior or dorsoventral), and the type of patterning defect observed. Molecular characterization of these genes identified many of the first known representatives of widely shared transcription factor families and signaling pathways. Indeed, the molecular analysis of many of these genes in Drosophila led to the development of tools to isolate them

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