Posterior Hox Genes in Xenopus Limb Development
We describe the expression patterns of three posterior Hox genes, XHoxc10, XHoxa13, and XHoxd13. Little differences exist between the expression patterns of the latter two genes and published data for other vertebrates, including axolotl (Gardiner et al., 1995; Torok et al., 1998). In contrast, the XHoxc10 pattern diverges somewhat from the one seen in developing axolotl limbs but agrees with the pattern seen in the chick (Nelson et al., 1996). To date, no one has attributed a particular role to Hoxc10 in a functional assay; therefore, it is unclear whether this difference in expression has any significance for later regeneration capacity.
Development vs. Regeneration
Over the years, it has become clear that regeneration and development have a lot in common. Later regeneration events seem to recapitulate development by using the same genetic pathways that have been used to establish a limb or tail initially. Important genes for limb or tail development are reexpressed in regenerating appendices with a strikingly similar expression pattern (Endo et al., 1997, 2000; Christen and Slack, 1998; Torok et al., 1998; Yokoyama et al., 2001). Therefore, it is legitimate to compare different blastema stages with developing limb or tail buds. We propose that a 1-day and a 3-day tail blastema correspond to a stage 30 and stage 32/33 tail bud, respectively. For the limb, a mid-bud blastema is similar to a stage 51 limb bud in appearance and gene expression.
Regeneration-Specific Expression Patterns
Hox genes have long been identified as universal gene products that code for positional information of the primary and secondary axes of the developing vertebrate embryo. This finding makes them good candidates to encode positional information in regenerating systems as well. For this reason, they have been studied in regenerative systems from hydra to axolotl and newts (Shenk et al., 1993; Simon and Tabin, 1993; Torok et al., 1998; Gauchat et al., 2000; Carlson et al., 2001). In axolotl and newts, it has been shown that some of the Hox genes are expressed in the mature adult fore- or hindlimb (Simon and Tabin, 1993; Beauchemin et al., 1994; Savard and Tremblay, 1995). None of these studies, however, addressed the spatial distribution of the Hox genes in the adult limb. We have previously published spatial expression patterns of some of the posterior Hox genes during the stages when Xenopus is able to regenerate its limbs, consistent with the idea that Hox genes are able to provide the positional information needed after a resection at knee level (Lombardo and Slack, 2001).
In this study, we have found some differences in expression in regenerating limb and tail blastemas between Xenopus and axolotl. For Hoxc10 and Hoxa13 where direct comparison is possible (Gardiner et al., 1995; Endo et al., 2000; Carlson et al., 2001), we find differences in the first detectable XHoxc10 expression. In Xenopus, XHoxc10 comes on 24 hr after amputation in the distal limb stump, just proximal to the amputation plane, whereas Hoxc10 in axolotl is not detected until 5 or 6 days after amputation and then accumulates at the amputation plane in a very thin layer. Early and mid-bud expression look identical between the two species, and no differences are seen in mid-bud tail blastema expression. However, although Hoxc10 expression in axolotl seems to recapitulate normal development, the pattern in Xenopus is novel to regeneration and not seen in developing limb buds. In the developing limb bud, XHoxc10 expression is restricted to anterior and then anterior/proximal very early on. In contrast, in regenerating limbs, XHoxc10 expression is seen in the region where cells dedifferentiate and undifferentiated cells are recruited and, later on in the whole blastema, with no obvious anterior restriction. Note, however, that this expression pattern is the same as the pattern seen in developing and regenerating axolotl limbs. The difference in timing between species may arise because Xenopus regenerates its limbs and tail quicker than axolotl. The simplest explanation for this difference is a difference in the water temperature in which the two species are normally cultured. Alternatively, the difference could be attributed to the fact that Hoxc10 in axolotl is reexpressed, whereas in Xenopus it only needs to be up-regulated in stage 54 regenerating limbs. Other people have speculated about a prominent role for the Hoxc genes in regeneration (Simon and Tabin, 1993; Savard and Tremblay, 1995; Carlson et al., 2001). In axolotl, Hoxc10 is only expressed in the hindlimb during normal development but is reexpressed very quickly after amputation in the hindlimb and activated de novo in the forelimb. This novel expression in regenerating forelimbs makes Hoxc10 the only true regeneration-specific gene to date (Carlson et al., 2001). In newts, however, Simon and Tabin (1993) did not detect Hoxc10 expression in forelimb blastemas by Northern hybridisation analysis.
The Hoxa paralogs are expressed very early (1–2 days after resection) in regeneration in axolotl and have been called dedifferentiation markers for that reason (Gardiner et al., 1995). We also see XHoxa13 reactivation early in the regeneration process (tail blastemas express it very strongly after 1 day and half the amputated limbs show expression after 1 day), and no expression in either wounded limbs or tails. However, despite the similar timing, after 1–2 days Xenopus has progressed further in the regeneration process compared with axolotl and is well into early bud stage.
For XHoxd13, no other regeneration data exist. It is not reactivated until mid-bud stage when patterning of the blastema takes place. A similar story is reported for Hoxd11 in axolotl limb blastemas. Hoxd11 too, seems to have only a role in patterning and is not expressed during the first phase of regeneration (Torok et al., 1998). Of interest, the pattern seen in limb blastemas differs from the pattern in normal developing limbs, insofar that it is restricted to a distal/posterior region and does not spread over the entire anteroposterior axis as in normal development. Hence, in regeneration, XHoxd13 expression is more similar to the expression patterns seen in chick and mouse (Nelson et al., 1996; Torok et al., 1998). It is not known what importance this asymmetric expression pattern might have in regeneration.
Two Phases for Hox Genes in Regeneration
It has become increasingly clear that the Hox genes play several distinct roles in regeneration. In the early regenerate, their temporal control and their expression pattern are quite different from their developmental expression patterns. In this first phase, they seem to fulfil a unique role in setting up a regeneration blastema or having a role in wound healing. In the second phase during mid-bud stage, when the blastema gets patterned, their expression patterns become similar to the ones observed during normal development (Gardiner et al., 1995; Carlson et al., 2001). We also see differences in expression patterns between the early phase of regenerates and limb development, whereas the expression at mid-bud stage more closely resembles the developmental pattern. We propose a role as a “first phase Hox gene” for XHoxc10 in the dedifferentiation and cell recruitment process. Several lines of evidence support this idea. First, XHoxc10 has the right expression profile in blastemas to support such a role. Second, Hoxc10 was detected in regenerating axolotl forelimbs, although it is not expressed there during normal limb development and, therefore, must play an early role in regeneration but not in patterning (Gardiner et al., 1995).
Even though gene expression of the Hoxa genes during regeneration seems to be similar in axolotl and Xenopus, our interpretation varies slightly from one presented previously (Gardiner et al., 1995; Endo et al., 2000). Others have proposed a dual role for Hoxa13 during early regeneration, first in dedifferentiation and second in patterning. Because Hoxa13 specifies the autopod in normal development, they argue, the distal part of the regenerated limb is the first part to be respecified during regeneration, whereas intermediate parts are intercalated secondarily. This finding is in contrast to normal development, where it is accepted that the pattern is specified in a proximal-to-distal sequence. Also, in axolotl, the proximal structures of the regenerated limb differentiate before the distal ones. Furthermore, XHoxc10 is coexpressed with XHoxa13 in these distal blastemas while, during normal limb development, coexpression is minimal. For these reasons, we suggest that the early Hoxa13 expression has nothing to do with patterning or with specifying the autopod but more likely with the proliferation of the blastema. Only later in regeneration would Hoxa13 specify the autopod. Up-regulation of posterior Hox genes and, in particular, Hoxa genes has been implicated in proliferation in hematopoiesis and acute myeloid leukaemia (Bjornsson et al., 2001; Calvo et al., 2002). In C. elegans, a two-phase expression of mab-5, an Antennapedia homolog, has been reported to allow for proliferation in the first phase and patterning in the second one (Salser and Kenyon, 1996). In the second phase, by mid-bud blastema stage, Hoxa13 is restricted to the distal part of knee/elbow level blastemas but covers the whole blastema in ankle/wrist amputations in axolotl, which corresponds to the expected pattern if patterning in regeneration recapitulates patterning during limb development (Gardiner et al., 1995; Endo et al., 2000). We did not observe this distal restriction of XHoxa13 in amputations at knee level; however, our interpretation of the mid-bud blastema staining is consistent with the imperfect regeneration in Xenopus limbs. Stage 54 limbs amputated at knee level regenerate with intermediate structures (zeugopod) missing; therefore, even a proximal amputation regenerates mainly an autopod, which would explain XHoxa13 staining in the whole blastema.
The third Hox gene studied, XHoxd13, does not have an early phase expression and, therefore, may fulfil only a patterning role, similar to normal limb and tail development. There are other Hoxd genes in axolotl, however, which are expressed during the first phase of regeneration. Two of the more 3′ Hoxd genes, Hoxd8 and Hoxd10, are expressed very early in regenerating limbs or wounds putting them at the forefront of the regeneration process (Torok et al., 1998).
It does not look like a particular Hox cluster or a Hox paralog group fulfils a specific role during regeneration. Instead, it seems to be very complex with differences between species.