Sharing Of The Genetic Toolkit Among Animals

Hox genes

One of the most exciting and unexpected discoveries that occurred soon after the cloning of the Hox genes was the detection of genes with related sequences in all sorts of animals. Using the homeobox to search for similar sequences in other genomes by hybridization, researchers isolated Hox-related genes from a broad sample of other animals. The similarity between the sequences of the homeodomains of genes isolated from frogs, mice, and humans and the original Drosophila Hox sequences was surprisingly extensive given the vast evolutionary distances between these animals. As many as 59 of the 60 amino acid residues were shared between the most similar homeodomains (Fig. 2.21).

An even greater surprise emerged with the physical mapping of vertebrate Hox genes. The map revealed that these Hox genes occurred in four large, linked complexes and that the order of the Hox genes within these complexes paralleled the order of their most related counterparts in the insect Hox complexes (Fig. 2.22).The vertebrate complexes define 13 groups of Hox genes, compared with the eight genes in Drosophila, although not every Hox gene is represented in each vertebrate complex (Fig. 2.22). Furthermore, the relative order of expression of vertebrate Hox genes along the anteroposterior (rostrocaudal) axis of vertebrate embryos correlates with gene position in each complex (Fig. 2.22).

With the invention of techniques for knocking out gene function in mice, it became possible to analyze the functions of the 39 Hox genes in the four mouse Hox complexes. This analysis has been complicated by genetic redundancy—that is, the expression and function of

Fly Dfd PKRQRTAYTRHQILELEKEFHYNRYLTRRRRIEIAHTLVLSERQIKIWFQNRRMKWKKDN KLPNTKNVR

AmphiHox4 TKRSRTAYTRQQVLELEKEFHFNRYLTRRRRIEIAHSLGLTERQIKIWFQNRRMKWKKDN RLPNTKTRS

Mouse HoxB4 PKRSRTAYTRQQVLELEKEFHYNRYLTRRRRVEIAHALCLSERQIKIWFQNRRMKWKKDH KLPNTKIRS

Human HoxB4 PKRSRTAYTRQQVLELEKEFHYNRYLTRRRRVEIAHALCLSERQIKIWFQNRRMKWKKDH KLPNTKIRS

Chick HoxB4 PKRSRTAYTRQQVLELEKEFHYNRYLTRRRRVEIAHSLCLSERQIKIWFQNRRMKWKKDH KLPNTKIRS

Frog HoxB4 AKRSRTAYTRQQVLELEKEFHYNRYLTRRRRVEIAHTLRLSERQIKIWFQNRRMKWKKDH KLPNTKIKS

Fugu HoxB4 PKRSRTAYTRQQVLELEKEFHYNRYLTRRRRVEIAHTLCLSERQIKIWFQNRRMKWKKDH KLPNTKVRS Zebrafish HoxB4 AKRSRTAYTRQQVLELEKEFHYNRYLTRRRRVEIAHTLRLSERQIKIWFQNRRMKWKKDH KLPNTKIKS

Figure 2.21

The similarities of Drosophila and vertebrate Hox protein sequences

The sequence of the Drosophila Dfd homeodomain and C-terminal flanking region and the sequences of several members of the vertebrate Hox 4 genes are shown. Note the great sequence similarity between the Drosophila and vertebrate proteins, and among the vertebrate Hox proteins.

Figure 2.22

Hox gene complexes and expression in vertebrates

(a) In the mouse, four complexes of Hox genes, comprising 39 genes in all, occur on four different chromosomes. Not every gene is represented in each complex, however. (b) The Hox genes are expressed in distinct rostrocaudal domains of the mouse embryo. Source: Carroll SB. Nature 1995; 376: 479-485.

Figure 2.22

Hox gene complexes and expression in vertebrates

(a) In the mouse, four complexes of Hox genes, comprising 39 genes in all, occur on four different chromosomes. Not every gene is represented in each complex, however. (b) The Hox genes are expressed in distinct rostrocaudal domains of the mouse embryo. Source: Carroll SB. Nature 1995; 376: 479-485.

Figure 2.23

Hox genes regulate vertebrate axial morphology

The morphologies of different regions of the vertebral column are regulated by Hox genes. (a) In the mouse, normally six lumbar vertebrae arise just anterior to the sacral vertebrae. (b) In mice lacking the function of the posteriorly acting Hoxd11 gene, and possessing one functional copy of the Hoxd11 gene, seven lumbar vertebrae form and one sacral vertebra is lost. (c) In mice lacking both Hoxall and Hoxd11 function, eight lumbar vertebrae form and two sacral vertebrae are lost. The anterior limit of Hoxd11 expression is at the first sacral vertebrae. Loss of these Hox gene functions transforms the sacral vertebrae into lumbar vertebrae. Source: Photographs courtesy of Dr Anne Boulet, HHMI, University of Utah.

wild-type Hoxall+ ¡Hoxall' ; HornII" !Hoxall";

HoxdiriHoxdir Hoxdir/Hoxdir

Figure 2.23

Hox genes regulate vertebrate axial morphology

The morphologies of different regions of the vertebral column are regulated by Hox genes. (a) In the mouse, normally six lumbar vertebrae arise just anterior to the sacral vertebrae. (b) In mice lacking the function of the posteriorly acting Hoxd11 gene, and possessing one functional copy of the Hoxd11 gene, seven lumbar vertebrae form and one sacral vertebra is lost. (c) In mice lacking both Hoxall and Hoxd11 function, eight lumbar vertebrae form and two sacral vertebrae are lost. The anterior limit of Hoxd11 expression is at the first sacral vertebrae. Loss of these Hox gene functions transforms the sacral vertebrae into lumbar vertebrae. Source: Photographs courtesy of Dr Anne Boulet, HHMI, University of Utah.

two or more similar Hox genes in overlapping domains. In some cases, loss of function of a specific Hox gene causes the homeotic transformation of the identity of particular repeated structures, such as vertebrae, and, in other cases, the loss of particular organs (Fig. 2.23). Conversely, the expression of Hox genes in more anterior sites often causes the reciprocal transformations. Similar results have been obtained in birds, amphibians, and fish, which indicates that in vertebrates, as well as Drosophila, Hox genes act as region-specific selector genes.

Hox genes also affect the development of unsegmented animals. In the nematode Caenorhabditis elegans, for example, Hox genes regulate the differentiation of cell types and certain structures along the main body axis. As Hox genes have been found on all branches of the metazoan tree and play such important roles in body patterning, we will devote considerable attention to their evolution and function in later chapters.

Field- and cell-type-specific selector genes

The discovery of homologs of Drosophila Hox genes in vertebrates and other animal phyla inspired the search for homologs of other Drosophila selector and developmental genes.

Figure 2.24

The Pax6/small eye gene controls vertebrate eye development

(a) Normal late-stage mouse embryo showing the head and developing eye. (b) Sey mutant embryo lacks the eye entirely. (c) In situ hybridization of a Pax6/Sey gene probe to mouse embryos reveals that the gene is expressed throughout the region from which the eye will develop. Source: Photographs courtesy of Nadean Brown.

Figure 2.24

The Pax6/small eye gene controls vertebrate eye development

(a) Normal late-stage mouse embryo showing the head and developing eye. (b) Sey mutant embryo lacks the eye entirely. (c) In situ hybridization of a Pax6/Sey gene probe to mouse embryos reveals that the gene is expressed throughout the region from which the eye will develop. Source: Photographs courtesy of Nadean Brown.

Through cross-hybridization between insect genes and vertebrate genes, families of vertebrate genes were isolated that included homologs of the Distal-less (Dlx), tinman (Nkx2.5), and AS-C (MASH) genes. Vertebrate homologs of eyeless(Pax6), scalloped(TEF-1), apterous (Lmx), engrailed (En1 and En2), nautilus (myoD), and Dmef (mef), genes are also known (Table 2.1).

Thus homologs of most identified field- and cell-type-specific selector genes have been found in vertebrates. Of even greater interest, however, are the structures and cell types in which these vertebrate homologs function. For example, the vertebrate eyeless homolog, Pax6, is involved in the development of the vertebrate eye. Mutations that reduce the activity of the mouse Pax6gene, called small eye (Sey), result in loss of eye tissue, including loss of the retina, lens, and cornea in homozygous mutants that lack all Sey function (Fig. 2.24). Mutations in the human Pax6gene, Aniridia, similarly affect eye development. Furthermore, when a version of the Sey gene is introduced into and expressed in flies, it behaves just like the eyeless gene in terms of its ability to induce new eye tissue. The Pax6 gene has been found to be associated with eye development across the metazoan tree, despite the differences in the architectures and optic principles of animal eyes.

Similar results have been found for vertebrate homologs of the Distal-less, tinman, and achaete-scute genes. In vertebrates and other phyla, Distal-less-related genes are expressed in an enormous variety of appendages with very distinct morphologies and functions.

Tinman/Nkx2.5 homologs are also expressed in, and required for, the development of the vertebrate heart. Similarly, AS-C homologs are expressed in neural precursors in vertebrates.

Signaling pathways: classical organizers, and morphogens

The components of all the major signaling pathways in Drosophila also have vertebrate counterparts (Table 2.2). These pathways operate in many tissues throughout vertebrate development. Importantly, some widely shared signaling proteins play important roles in the classical organizers defined in vertebrate embryos. For example, searches for molecules with activities associated with the Spemann organizer in the Xenopus embryo revealed that several of the secreted signaling proteins have potent inducing activities. One protein, dubbed Chordin because of its activity in inducing dorsal derivatives (the notochord), is a homolog of the Drosophila Short gastrulation protein. Both the vertebrate and Drosophila proteins interact with members of the TGF-P signaling protein family, and both are involved in dorsoventral axis formation.

Signaling proteins have also been found that account for the activities of the ZPA and AER in the vertebrate limb bud. For example, Sonic hedgehog (Shh), a homolog of the Drosophila Hedgehog signaling protein, is expressed in the posterior mesenchyme of the limb bud, precisely where the ZPA is localized (Fig. 2.25). Consistent with Shh carrying out the organizing

Figure 2.25

Organizers and signaling proteins in the vertebrate limb bud

In situ hybridization of probes for the FGF8and Shh transcripts reveal that these genes are expressed in regions corresponding to the AER and ZPA, respectively, as defined in transplantation and ablation experiments. Source: Photograph courtesy of Cliff Tabin.

Figure 2.25

Organizers and signaling proteins in the vertebrate limb bud

In situ hybridization of probes for the FGF8and Shh transcripts reveal that these genes are expressed in regions corresponding to the AER and ZPA, respectively, as defined in transplantation and ablation experiments. Source: Photograph courtesy of Cliff Tabin.

activity of the ZPA, expression of Shh in the anterior of the limb induces the same ectopic mirror-image duplications of digits as does transplantation of the ZPA. Similarly, members of the fibroblast growth factor signaling protein family are expressed in the distal tip of the limb bud in the AER and promote its activity (Fig. 2.25).

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