Question

Explain the following in terms a highschooler would understand (from article Expression of HoxD Genes in Developing and Regenerating Axolotl Limbs, 1998):

DISCUSSION:

Expression of HoxD genes in developing axolotl limb buds. We have examined the expression of Hoxd-8, Hoxd10, and Hoxd-11 genes in axolotl limb buds and find many similarities between the patterns of expression in axolotls and those described for other vertebrates. Similarities are most pronounced in phases I and II (Nelson et al., 1996; Shubin et al., 1997) and include spatial and temporal colinearity and equivalence of expression domains. The most obvious differences between axolotls and higher vertebrates become evident in phase III and concern the expression of Hoxd-11. First, although the zeugopodial domain of Hoxd-11 is strong, the distal–posterior domain is much weaker than in other tetrapods, and it does not show the anterior expansion characteristic of all other vertebrates except fish (Sordino et al., 1995). It is not unreasonable to consider that the failure of the distal–posterior to exhibit an anterior expansion is related to the reversal in the sequence of digit differentiation in urodeles relative to that of other tetrapods. Rather than form a handplate prior to differentiation, in axolotls the distal tip of the limb bud remains unexpanded as differentiation of the most anterior digit begins.

Digit 1 forms from the anterior half of the limb bud, from cells that are not expressing Hoxd-11. The remaining digits are formed from Hoxd-11-expressing cells. Hence, the anterior border of the weak distal domain is in a conserved position relative to limb anatomy, such that in all tetrapods this border falls between digits 1 and 2. Based on these observations, we suggest that the anterior expansion of Hoxd-11 and other 59 Hox D genes across the distal limb bud is not indispensable for hand and foot development. Similar conclusions can be drawn from studies of mutant mice (Davis et al., 1995). Rather, our studies suggest that the expansion could be related to the formation of a handplate prior to digit differentiation and may be a necessary feature of limbs in which the sequence of digit formation is from posterior to anterior.

Reexpression of HoxD genes during regeneration. Given the conserved expression of HoxD genes in development, it would be expected that they would also be reexpressed during regeneration, as shown for HoxA genes by Gardiner et al. (1995). We have examined reexpression of HoxD genes during regeneration and find that the temporal pattern of reexpression is similar to that in development, with Hoxd-8 and Hoxd-10 being expressed earlier in the process than Hoxd-11. Interesting because this is in contrast to the findings with HoxA genes, where both 39 and 59 members of the complex are expressed simultaneously (Gardiner et al., 1995). Hoxd-11 is not expressed until a blastema has formed, and the earliest expression is confined to the posterior half of the early bud blastema. At medium bud, expression is very strong in a limited region of the posterior–distal tip of the blastema and is weak in more proximal regions. It is possible to interpret the expression of Hoxd-11 in early regenerates as a modified version of the pattern seen in early development. However, in light of previous findings that make it very likely that the order in which limb parts are specified in regeneration is not the same as in development, another interpretation is possible. The data suggest that it is the distal tip of the new pattern that is specified first, followed later by intercalation of forearm regions between the newly respecified tip and the stump at the base of the regenerate (Gardiner et al., 1995). Hence, the early distal posterior expression of Hoxd-11 would occur at a time when only the hand region is represented in the blastema. At later times when HoxA expression patterns indicate the emergence of the zeugopod, expression that extends across the proximal base of the regenerate develops, and the distal expression domain is downregulated. A more thorough understanding of the relationship between Hox expression patterns and the order in which parts are specified might be possible by combining cell lineage and gene expression data.

The rapid upregulation of Hoxd-8 and Hoxd-10 in regenerates suggests that these genes are induced in response to wounding of the limb and might therefore be expressed in non-regenerating wounds. We found that both of these genes are rapidly induced (within 24 h) in non-regenerating wounds, as they are in amputated limbs. However, Hoxd-11, which is not detected until a blastema has formed, is not expressed in non-regenerating wounds.

It is possible that the early upregulation of Hoxd-8 and Hoxd-10, as part of wound healing, may be a prerequisite for the establishment of the signal to form a regeneration blastema. It will be interesting to find out whether activation of any Hox genes occurs during wound healing in non-regenerating animals and, if not, whether loss of expression correlates with decline in regenerative ability during development.

The later time of Hoxd-11 induction during regeneration relative to other Hox genes and the absence of expression in response to wounding suggest that a secondary signal is necessary to upregulate Hoxd-11 expression. To test the relationship between Hoxd-11 expression and limb patterning, we analyzed induced lateral supernumerary limbs for expression of the transcripts. Our data show that Hoxd-11 transcripts are associated with cells on the posterior half of limbs, whether in supernumerary limb outgrowths, blastemas, or limb buds in normal development. Since we have shown that superficial wounding is insufficient to induce Hoxd-11 expression, the induction of expression in lateral supernumerary limbs is correlated with pattern formation events.

Comparing the limited data available so far for axolotls with that for other tetrapods, we suggest that patterns of Hox gene expression are not consistent with a direct and conserved role in either the timing or the sequence of differentiation of limb elements. On the other hand, the expression domains are consistent with a role in the formation of the conserved features of limb anatomy. For example, in both the forearm and the hand, where the order of skeletal differentiation is A-P-reversed relative to that of other tetrapods, Hoxd-11 expression is nevertheless restricted to the same posterior expression domain relative to the skeletal elements themselves. Additional evidence from the expression patterns of HoxA genes in developing and regenerating axolotl limbs (Gardiner et al., 1995) also suggests a conserved role relative to morphology, but lends no support for a conserved role in either the timing or sequence of differentiation. Hence, in development, Hoxa-13, expressed in the hand region, is the last gene to be activated in the HoxA complex and the hand is the last part of the pattern to differentiate. In contrast, Hoxa-13 is expressed precociously during regeneration such that the hand region is established prior to that of more proximal domains. However, the distal part of the pattern still differentiates last.

Despite the forgoing, there remains an obvious relationship between pattern formation and growth control (French et al., 1976; Bryant et al., 1981), and it is likely that there is at some level a functional interaction between Hox gene expression, pattern formation, and the control of growth and differentiation (Duboule, 1994; Morgan and Tabin, 1994). One possibility is that the patterns of growth and timing of differentiation are downstream consequences of the activities of Hox genes in the specification of morphological identity. This would allow for variation in the response to expression of Hox genes whose expression with respect to morphology is conserved. Such variation could result in changes in the rate and timing of growth and differentiation and thus generate the heterochrony upon which much evolutionary change has relied (Gould, 1977; McKinney and McNamara, 1991).

We conclude that the underlying patterning mechanisms of all vertebrate limbs are homologous, despite differences in the sequence of developmental events. The AP asymmetry in structural design that is a major feature of both tetrapod limbs and sarcopterygian fins is underlain by a conserved pattern of asymmetry in gene expression, as exemplified by the expression of Hoxd-10, Hoxd-11, and shh (Imokawa et al., 1997; Torok et al., submitted for publication).

Answer 1

Ans: Homeobox genes are important in the directive of outgrowth and pattern formation during limb development. Hox genes help in the development of the vertebrate axis and limbs and alsocontrols the growth and the timing of differentiation. Axolotl limbs distinguish these alternatives because the sequence of skeletal differentiation is reversed along the anterior-posterior axis relative to that of other tetrapods. HoxD genes in axolotls look a lot like with the amniotes and anuran amphibians.The anterior boundary of Hoxd-11 expression is conserved with respect to morphological landmarks, but there is no anterior-distal expansion of the posterior domain of Hoxd-11 expression. The expression patterns of two 5′ members of the complex, HoxA13 and HoxA9. The expression area of HoxA13 is more distally restricted than that of HoxA9. These genes are expressed in cells of developing limb buds and regenerating blastemas. As axolotls do not form a prolonged paddle like handplate prior to digit differentiation. Therefore, the anterior expansion of expression in higher vertebrates is linked to the formation of the handplate but is clearly not necessary for digit differentiation. HoxD genes are reexpressed during limb regeneration. While the change in the expression pattern of Hoxd-11 during the course of regeneration is constant and the distal tip of the regenerate is specified first this is then followed by intercalation of intermediate levels of the pattern. Both Hoxd-8 and Hoxd-10 are expressed in non-regenerating wounds but Hoxd-11 is specific for regeneration. It is also expressed in the posterior half of nerve-induced supernumerary outgrowths.