An important question during limb regeneration is how the identity along the proximodistal axis is re-specified. An often-assumed model mainly based on grafting experiments suggested that in axolotls this re-specification occurred mainly by cell intercalation. Under this model the first blastema cells that appear acquire the most distal identity and then the missing identities intercalate between the stump and this most distal part. This intercalation is observed, for instance, when grafting distal leg fragments to more proximal ones in insects, or grafting a head and tail planarian pieces, or grafting a distal limb blastema to an upper arm stump in amphibians. But the question that remains is whether this intercalation model is also followed during normal regeneration.
Now, a recent paper from the laboratory of Elly Tanaka indicates that during axolotl limb regeneration there is a proximal-to-distal sequence of segment specification (http://www.ncbi.nlm.nih.gov/pubmed/24337297). During axolotl limb development (and other vertebrates) HoxA9, HoxA11 and HoxA13 are expressed in a spatial and temporal sequence. HoxA9 is first expressed in the limb bud before HoxA11 and HoxA13. Then, HoxA11 is expressed with a more distal expression boundary compared to HoxA9, whereas HoxA13 is expressed at the most distal part. These results suggest that progenitor cells are specified in a proximal-to-distal sequence. So, here the authors sought to determine whether this spatial and temporal colinearity for HOXA proteins was also observed during regeneration. They used axolotls in which the connective tissue was labelled with GFP. At day 1 of regeneration no blastema cells (derived from the GFP positive connective tissue) expressed any of the HOXA proteins. At day 6 (early-bud stage) HOXA9 and HOXA11 but not HOXA13 were expressed in the blastema. Between days 8 and 12 (medium- and late-bud stages) a nested expression pattern was observed with HOXA9 found broadly in the blastema, HOXA11 in a medial band and HOXA13 at the distal tip. Thus, these results indicate that during regeneration HOXA proteins are expressed colinearly, as it happens during limb development.
The association of HOXA13 expression with hand identity was further confirmed by: i) the observation that after hand amputation HOXA13 was expressed in the blastema cells at 4 days well before the onset of HOXA13 expression in an upper arm blastema, and ii) the fact that treatment with retinoic acid (that convert hand blastemas into upper arm blastemas) silenced the expression HOXA13 at 4 and 6 days after hand amputation. Finally, the authors performed a series of transplantation experiments to assess the order of blastema cell specification. In all those experiments the host was always an unlabelled 6-day upper arm blastema (a stage in which HOXA13 is not yet expressed). The donor cells were blastema cells derived from GFP positive connective cells from different stages and amputation levels. When 8-day hand blastema cells were transplanted into the donor they only contributed to hand structures (as expected based on the rule of distal transformation in which blastema cells derived from connective tissue can only form segments more distal to their original identity). When 8-day upper arm blastema cells from the distal tip were transplanted they contributed also only to hand structures, indicating that at 8 days the transplanted distal blastema cells were already committed to hand identity. On the other side, when 8-day proximal upper arm blastema cells were transplanted they contributed to lower arm and hand structures, corresponding to the HOXA9+HOXA13- upper-arm progenitor cell potential. Finally, when the most distal tip cells of 4-day upper arm blastemas (a stage at which they do not express yet HOXA13) were transplanted they contributed to lower arm and hand in the regenerated limb indicating that those transplanted cells had not been committed yet to hand-identity.
Overall these transplantations indicate that blastema cells are specified in a proximal-to-distal sequence during normal regeneration similarly to what happens during embryonic development.