Actinopterygians

elongate tins pectoral fins only

posterior Hox genes expressed In both appendages

VERTEBRATES paired appendages a/p poiarily of tin structure

Hox genes expressed along body axis pectoral fins only

posterior Hox genes expressed In both appendages

VERTEBRATES paired appendages a/p poiarily of tin structure

Hox genes expressed along body axis

cluster activity must have been involved at this step; i.e., at (c) in Fig. 5.4C. Since forelimb and hindlimb buds develop into structurally homologous appendages, and both express sets of posterior box genes, this expression denotes a discrete patterning cassette. The forelimb buds in fact utilize the posterior box genes "out of register" with their use in the main body axis. Furthermore, a gnathostome common ancestor of teleosts and tetrapods had already coopted tbx4 and tbx5 genes to specification of forelimb and hindlimb (cf. Fig. 4.3), since this usage is also a shared property of amniotes and fish (Ruvinsky et al., 2000). Figure 5.4C shows that the phase 3 expression of the posterior box genes and the development of the curved axis of the autopod in part e are indeed evolutionarily novel features of the limbed tetrapods, additional evolutionary cooptions. This follows from the fact that the extinct close relatives of the tetrapods such as that shown at (d) in Fig. 5.4C (as well as living sarcopterygian relatives such as lungfish) retain the straight axis that is also retained in teleosts (Fig. 5.4B). Therefore the common gnathostome ancestor generated a straight axis of box cluster expression in its limb buds as well, underlying the bony structures of its fins (Coates, 1995; Shubin et al, 1997).

The cooption event which put the posterior box genes to work in paired limb buds occurred no later than the Silurian (~440-410 mya), when the first jawless fish with paired appendages and then the first gnathostomes appear (Janvier, 1996). A "trans-vertebrate" gene transfer experiment suggests how this cooption might have happened. Figure 5.4D shows a mouse embryo expressing a zebrafish boxdll lacz transgene (Beckers et al., 1996). This teleost regulatory element generates expression in the hindlimb and forelimb buds, and also in both the caudal tail and genital regions. The mouse boxdll gene is expressed in the same regions (Dolle et al., 1991; Beckers et al, 1996). The inputs needed to animate the boxdll as-regulatory system apparently survive in both evolutionary branches of the gnathostomes, i.e., teleosts and tetrapods. The implication is that these inputs were originally present at the caudal end of more basal vertebrate forms that did not have paired appendages. So the cooption would have been effected by genomic changes that caused the genes encoding the relevant transcription factors to be expressed as well in the limb bud domains. In mice some mutations that perturb posterior boxd gene function coordinately affect both limb and genital development. The original cooption could have mobilized elements of a genital patterning system to the limb buds (Peichel et al., 1997; Kondo etal., 1997).

Colinear Expression of box Genes in the Somatocoel of a Sea

Urchin Larva

The example of colinear box gene expression most remote from axial A/P patterning in chordates and arthropods is illustrated in Fig. 5.5 (Arenas-Mena etal., 2000). Here we see a sequential set of box gene expression domains that traverse the somatocoels of a microscopic larval sea urchin. The paired somatocoels are the posterior coeloms, which at the stages shown surround the larval gut, and from which various mesenteric elements of the adult body plan will eventually be formed (Arenas-Mena et al., 2000; Pearse and Cameron, 1991). The fivefold radially symmetric structures of the adult echinoderm body plan form within the rudiment (cf. Fig. 5.1), which as Fig. 5.5 illustrates, overlies the left somato-coelar sac. There is only a single box gene cluster in this sea urchin genome (Martinez et al., 1999), and the five, 5'-most box genes known (those related in sequence to vertebrate hox7-13) are all expressed in the somatocoels during early and middle stages of postembryonic development (e.g., Fig. 5.5C). The pattern of box gene expression is affected by the rudiment, so that as development proceeds it becomes different on the left as compared to the right somatocoel (Fig. 5.5D1-4). Though the developmental significance of these somatocoelar box cluster expression patterns are unknown, they have some remarkable features.

The first point is that the expression pattern is colinear with box gene order in the cluster. The colinear pattern describes a curved axis through the somatocoels (white arrows in Fig. 5.5D1-2), but this conforms to the axes of neither adult nor larval body plans. The axes of the larval body plan are shown in Fig. 5-5B. Though it is folded up upon itself, in the adult echinoid the primordial A/P axis runs inward from the mouth (anterior, face down in sea urchins as they crawl along the substrate; Peterson et al., 2000c). In the diagram of Fig. 5.5D3 the future adult axis would run from the oral surface of the rudiment which faces the viewer down into the page. Second, though the body plan of the adult is radially symmetric, the box gene expression patterns in the somatocoels are bilateral and also somewhat asymmetric, as are the fates of the right and left somatocoels (Peterson et al., 2000c). The colinear somatocoelar expression patterns may well have descended from a straight colinear pattern of A/P expression that was parallel to the gut in a remote bilaterian ancestor of echinoderms plus hemichordates, since the most posterior (i.e., 5') of the genes, boxll/13b, is expressed around the anus. That is, the ancestral A/P pattern of coelomic mesoderm expression probably obtained its curvature in the course of echinoderm evolution together with the gut, which the coeloms enclose. So though it looks different, this aspect of the colinear somatocoelar expression pattern can be considered conserved. The somatocoelar box gene expression pattern (Fig. 5-3D) obviously has some downstream functional significance, or it could not have survived. The somatocoels give rise to a unique set of structures, the stacked mesenteries of the adult echinoid body plan (Peterson et al., 2000c). Perhaps the posterior box genes have been coopted for some patterning function required for development of these structures. Third, note the microscopic scale on which this box cluster expression pattern is formulated: the whole somatocoel is only a couple of hundred microns across at these stages. The pattern is also dynamic and transient (Arenas-Mena et al., 2000). This example delivers the warning that there probably remain to be discovered many other colinear box gene cooptions in bilaterians, special uses of the box gene cluster that are different from those with which we are familiar along the A/P body axes of arthropods and chordates.

FIGURE 5.5 (Continued)

^ SpHox11/13b % SpHox9/10 £ SpHox7 SpHox11/13a 0 SpHox8

The feature of hox cluster usage in the bilaterians that emerges is its remarkable ability to be coopted for diverse patterning functions. The box gene cluster has the unusual property of generating a set of transcriptional domains that form an oriented spatial sequence; as remarked at the outset, this has the abstract property of a vectorial patterning system. The individual transcription factors the cluster encodes are useful in multiple contexts, perhaps because they function combin-atorially with diverse cofactors (e.g., Li et al., 1999). These transcription factors provide inputs to czs-regulatory elements that operate at all levels of pattern formation systems. The box cluster system is linked into an amazing variety of

FIGURE 5.5 Domains of expression of posterior hox genes in the larval somatocoels of a sea urchin. (A) Left lateral view of an eight-armed larva of S. purpuratus: m, mouth; ph, pharynx; a, anus; lar, larval arms; ep, epaulettes; c, cilia; pp, the primary podia of the adult rudiment; am, adult mouth (red arrowhead), which will form in the center of the rudiment. At this stage the rudiment has grown to occupy a large region of the larval body alongside the stomach, which lies beneath the rudiment in this view. The pentameral organization of the rudiment is clearly evident. (B) Axial designations for the larva itself; the axis of the rudiment (and adult) runs from the mouth into the page. (C) Examples of somatocoelar hox gene expression patterns in sections oriented as in (B); below, somatocoels (green), larval ectoderm (not included in juvenile at metamorphosis, black); gut (yellow). In these drawings, o, oral; an, anal; abo, aboral; abn, abanal. In sections, Is, left somatocoel; rs, right somatocoel; st, stomach; int, intestine; cb, ciliary band; other abbreviations as in (A). (CI) Expression of hox8 gene; (C2) Expression of hoxll/l3b gene (nomenclature of hox genes as in Martinez et o/., 1999). (D) Summary of patterns of expression of five "posterior" hox genes in the larval somatocoels. Early stages are represented in (Dl) and (D2); and later stages in (D3) and (D4). The color code (bottom) indicates the domains of expression of individual genes; abbreviations as in (C). Domains of overlapping gene expression are represented as bicolored stripes. (DI), (D3) left views, where pentameral rudiment can be seen; (D2), (D4) right views. The most 5' gene of the cluster so far known, hoxllll3b, is expressed in the anal region and consecutively more "anterior" genes are expressed in a sequential set of domains describing a curved pattern along the somatocoels (white arrows). Except for hoxll/l3a the gene expression domains are expanded or are stronger on the left side near the rudiment as development progresses; compare (D3) with (D4). In (D3) the pentameral water vascular system is indicated within the outline of the rudiment in gray, and the dental sacs in white. In (D4) the patterns of expression in the right somatocoel are similar to those in (D2), except for the absence of hoxll/l3b expression around the anus; the transcript levels are also lower (indicated by oblique white lines), compared to those in the left somatocoel (D3), except for hoxll/l3a, which stains similarly in both somatocoels. In (D3) expression of hoxl and hox8 is seen around the canal leading from the center of the rudiment to the outside. None of these five genes is expressed in a pentameral pattern. [From Arenas-Mena et al. (2000) Development, in press.]

morphogenetic functions, some which are not aspects of A/P patterning at all. And even for those which are, a close look at the patterning gene networks in which box genes participate shows that each specific usage is a novel evolutionary cooption (as in the examples of the Drosophila wing and vertebrate hindbrain considered in this and in Chapter 4). Utilization of the box complex in bilaterian evolution has included a great many nonconservative changes.

Nor in this light is it so obvious what were the original functions for which the vectorial box cluster patterning system was utilized. It was indeed a key A/P patterning device in the bilaterian common ancestor, but it could have had many other uses in the precursors and cousins of that animal. The case of the sea urchin larval somatocoel shows that box cluster genes could have evolved to pattern a variety of structures in a microscopically scaled stem group fauna, one branch of which gave rise to the bilaterians (Davidson et al., 1995; Peterson and Davidson, 2000; Runnegar, 2000). This chapter is about evolutionary change in patterning systems, and the box gene system provides some of the most dramatic examples of cooptive change in the evolutionary history of the Bilateria.

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