HOXA expression sequence and functional transplantation assays indicate that axolotl limb regeneration does not occur by intercalation

TAZAKY AKIRA; ROENSCH KATHLEEN; CHARA OSVALDO; TANAKA ELLY M

Mont-Tremblant

Conferencia; 12th International Conference on Limb Development and Regeneration. Mont-Tremblant; 2012

The axolotl (Ambystoma mexicanum) has the exceptional capacity to regenerate the appropriate limb segments after amputation. A local proliferative zone called the blastema is formed adjacent to the amputation plane and will give rise to the missing structures. Currently, it is unclear how the regenerating limb regenerates the correct number of limb segments. It has been hypothesized that distal HoxA9/HoxA13 double positive cells are the first blastema cells formed followed by intercalation (Gardiner et al., 1995). To test this possibility we examined the expression pattern of the well-known proximal-distal identity markers, HoxA9, HoxA11 and HoxA13, at the mRNA and protein level during axolotl limb development and regeneration using tissue sections. As part of this study full-length axolotl HoxA11 and HoxA13 were isolated and antibodies against HoxA9, A11 and A13 were generated. During axolotl embryonic limb development, HoxA genes showed sequential, co-linear expression as in other vertebrates. Surprisingly, in limb regeneration HoxA9, HoxA11 and HoxA13 were expressed in a co-linear manner as well, suggesting a proximal to distal order in limb specification. To investigate when the distal-most identity was determined and if this time of determination correlates with the onset of HoxA13 expression we performed hetero-chronic and hetero-topic limb blastema transplantation experiments using animals expressing GFP in lateral plate mesoderm descendents. Our results show that by transplanting early distal blastema (HoxA13 negative) into the proximal blastema, the labeled cells spread all along the proximal-distal limb axis. By transplanting late distal blastema (HoxA13 positive),  the cells give a biased distribution to the hand suggesting that the early blastema does not contain distally determined cells and the distal identity was determined in correlation with HoxA13 at a later stage. This also suggests that normal regeneration occurs in a proximal to distal sequence rather than via intercalation. We are currently performing functional studies of the HoxA genes by knock-down and overexpression approaches.