L M

-¡- ' "Z" normal mutant phenotype from other animals. Thus the systematic inventory of the Drosophila genome is representative of what is generally known about the types of molecules with large-scale effects on animal patterning.

The anteroposterior axis

A few dozen Drosophila genes are required for proper anteroposterior patterning of the embryo and larva. These genes are grouped into five classes based on their realm of influence on embryonic pattern. Each class represents a progressively finer subdivision of the developing embryo.

The first class consists of the maternal effect genes, such as the bicoid gene (which affects the anterior region of the embryo) and the nanos and caudal genes (which affect the posterior region) (Fig. 2.14).

The second class contains the zygotically active gap genes, which include the hunchback, Kruppel, giant, knirps, tailless, and huckebein genes, each of which regulates the formation of a contiguous set of segments. Mutations in gap genes lead to gaps in segmentation (Fig. 2.15a,b).

The third class comprises the pair-rule genes, such as fushi tarazu, even-skipped, hairy, and paired, which act at a double-segment periodicity. Pair-rule mutants display defects in part of each pair of segments (Fig. 2.15c).

Figure 2.14

Maternal effect genes controlling embryonic polarity in Drosophila (top) The cuticle pattern of wild-type Drosophila larvae. Various structures develop at characteristic positions along the anteroposterior axis. Note the mouth hooks at the anterior (left) end of the animal, the presence of triangular organs in each of the three thoracic segments, and the broad bands of denticles marking each abdominal segment. (middle) Embryos from homozygous bicoid/bicoid mutant females lack anterior structures and have duplicated posterior structures. (bottom) Embryos from homozygous nanos/nanos mutant females lack most abdominal structures.

Source: Redrawn from Lawrence PA. The making of a fly. Oxford, UK: Blackwell Scientific Publications, 1992.

The fourth class consists of the segment polarity genes, which affect patterning within each segment. Mutants in this class lack the normal polarity of pattern elements within segments and display polarity reversals and segmentation defects (Fig. 2.15d). The products of two segment polarity genes, wingless and hedgehog, are largely responsible for the polarity-organizing activities identified within insect segments by classical transplantation and ablation techniques.

The fifth class includes the homeotic selector genes, which we discussed earlier in this chapter.

Collectively, these five classes of mutants indicate that the segmental body plan of the Drosophila larva is progressively specified by genes acting over the realm of the whole embryo, in subregions of the embryo, in every other segment, and, ultimately, in each individual segment.

The dorsoventral axis

The dorsoventral axis of the Drosophila embryo is also regionally subdivided. Several maternal effect genes are required to initiate the establishment of dorsoventral polarity. Embryos from

b A5/A6 A7 A8
C T1 Ti A2 A4 A6 A8

Figure 2.15

Segmentation gene mutants

(a) The cuticle of a Drosophila larvae has belts or denticles that form characteristic patterns in each segment (T1, T2, T3, A1, and so on). The position and pattern of these denticles are landmarks for identifying developmental abnormalities.

(b) A Krüppel gap mutant. Loss of Krüppel function prevents the formation of several segments. (c) A fushi tarazu pair-rule mutant. Loss of this gene's function results in loss of every other segment boundary and pairwise fusion of segments. (d) A gooseberry segment polarity mutant. Loss of gooseberry function alters the polarity of each segment. Source: Courtesy of Nipam Patel.

females that lack the activity of any of these genes, such as dorsal, are "dorsalized"—that is, ventral structures do not form in these animals. The zygotically active genes decapentaplegic (dpp), zerknüllt(zen), shortgastrulation (sog), twist(twi), and snail(sna) all play major roles in the subdivision of the dorsoventral axis. In addition, neurogenic genes, such as Delta (Dl) and Notch (N), are required in distinct dorsoventral subregions for the formation of the ectoderm.

Figure 2.16

Expression of maternal morphogens

The maternally derived Bicoid and Nanos proteins form concentration gradients emanating from the anterior (Bcd) and posterior (Nos) poles of the Drosophila embryo.

Source: Photomicrographs courtesy of Ruth Lehmann.

Figure 2.16

Expression of maternal morphogens

The maternally derived Bicoid and Nanos proteins form concentration gradients emanating from the anterior (Bcd) and posterior (Nos) poles of the Drosophila embryo.

Source: Photomicrographs courtesy of Ruth Lehmann.

Expression of toolkit genes

The phenotypes of mutant fruit flies tell only part of the story regarding what developmental genes do. To fully understand the link between these genes and patterning, we must know the timing and location of the genes' expression patterns and the molecular nature of the genes' protein products. The identification of gene classes that, when mutated, cause similar effects in development raises the possibility that a given class of genes may affect the same developmental process or genetic pathway, or that the genes may encode products with similar functions.

Analysis of the expression of toolkit genes has revealed a very informative correlation between the locations at which genes are expressed in development and the pattern of defects caused by mutations. For each of the five classes of anteroposterior axis-patterning genes, a clear correspondence exists between the regions of the embryo in which the gene is transcribed (or the protein product is localized) and the regions affected by mutations in that gene. For example, the bicoid and nanos proteins are expressed in graded patterns emanating from the anterior and posterior poles of the embryo, respectively (Fig. 2.16). These proteins are largely responsible for the organizing activities that classical experiments identified as residing at the two poles of the insect egg.

The gap genes are expressed in blocks of cells that correspond to the future positions of the segments affected by gap gene mutants (Fig. 2.17a). The pair-rule genes are expressed in one transverse stripe per every two segments, for a total of seven stripes that span 14 future body segments; the stripes correspond to the periodicity of defects in mutant embryos (Fig. 2.17b). The segment polarity genes are expressed in each segment, in 14 or 15 transverse stripes (Fig. 2.17c). The various dorsoventral patterning genes are expressed in different

Figure 2.17

Expression of segmentation genes

Drosophila embryos stained with antibodies specific for the (a) Krüppel gap protein, (b) Hairy pair-rule protein, and (c) Engrailed segment polarity protein. Each protein is localized to nuclei in regions of the embryo that are affected by mutations in the respective genes. Source: Photographs by James Langeland.

Figure 2.17

Expression of segmentation genes

Drosophila embryos stained with antibodies specific for the (a) Krüppel gap protein, (b) Hairy pair-rule protein, and (c) Engrailed segment polarity protein. Each protein is localized to nuclei in regions of the embryo that are affected by mutations in the respective genes. Source: Photographs by James Langeland.

domains along the dorsoventral axis that correspond to regions of the embryo that give rise to elements such as the mesoderm, neuroectoderm (the part of the ectoderm from which the ventral nervous system develops), and amnioserosa (a dorsal sheet of extraembryonic cells) (Fig. 2.18).

Toolkit gene products: transcription factors and signaling pathway components

The proteins encoded by selector and axial patterning genes most often belong to one of two categories: transcription factors or components of signaling pathways. Ultimately, these proteins exert their effect through the control of gene expression. Thus developmental

Figure 2.18

Expression of dorsoventral patterning genes

(a) Dorsal protein expression. The concentration of Dorsal in cell nuclei is graded from ventral to dorsal cells. Zygotic genes are expressed at different positions along the dorsoventral axis with (b) snail expression in the most ventral cells, (c) sog expression in lateral cells, and (d) zen expression in the most dorsal cells. Source: Photographs courtesy of Michael Levine.

Figure 2.18

Expression of dorsoventral patterning genes

(a) Dorsal protein expression. The concentration of Dorsal in cell nuclei is graded from ventral to dorsal cells. Zygotic genes are expressed at different positions along the dorsoventral axis with (b) snail expression in the most ventral cells, (c) sog expression in lateral cells, and (d) zen expression in the most dorsal cells. Source: Photographs courtesy of Michael Levine.

processes such as embryonic axis formation and segmentation are organized by regulating gene expression in discrete regions and cell populations of the embryo.

The transcription factors found in the Drosophila toolkit include representatives of most of the known families of sequence-specific DNA-binding proteins. These families are distinguished by the type of secondary structures in the folded protein that are involved in protein subunit interactions and contact with DNA.

Most transcription factors possess either a helix-turn-helix, zinc finger, leucine zipper, or helix-loop-helix (HLH) motif (Fig. 2.19; Table 2.1). The homeodomain superfamily belongs to the helix-turn-helix class of factors, for example (Fig. 2.19a). Three proteins with divergent homeodomain sequences—Bicoid, Fushi tarazu, and Zen—have very different roles in organizing the anteroposterior axis, segmentation, and dorsoventral axis patterning, respectively. All three proteins are also encoded within the Antennapedia Complex, surrounded by Hox genes. Most other homeodomain proteins are encoded by genes dispersed throughout the genome.

Figure 2.19

Structural motifs of major transcription factor families

Figure 2.19

Structural motifs of major transcription factor families

(a) The second and third a-helices of the homeodomain form a helix-turn-helix structure. (b) The zinc finger motif involves a coordination complex of Zn++ with critically positioned cysteine (C) and histidine (H) residues. The "finger" contacts DNA. (c) The leucine zipper structure is formed by association of two subunits with regularly spaced leucine residues. (d) The helix-loop-helix structure is similar to the leucine zipper except that a protein loop interrupts the helices and the association of the subunits.

TABLE 2.1 Transcription factors in the Drosophila genetic toolkit for development, and mouse homologs

Domain Drosophila gene Developmental function Mouse homolog(s)

Homeodomain

TABLE 2.1 Transcription factors in the Drosophila genetic toolkit for development, and mouse homologs

Domain Drosophila gene Developmental function Mouse homolog(s)

Homeodomain

labial

Homeotic

Hox a1, b1, c1

proboscipedia

Homeotic

Hox a2, b2

deformed

Homeotic

Hox a4, b4, c4, d4

Sex combs reduced

Homeotic

Hox a5, b5, c5

Antennapedia

Homeotic

Most similar to Hox6-8

Ultrabithorax

Homeotic

Most similar to Hox6-8

abdominal-A

Homeotic

Most similar to Hox6-8

Abdominal-B

Homeotic

Most similar to Hox9-13

bicoid

Maternal anteroposterior axis organizer

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