INVESTIGADORES
CHARA Osvaldo
congresos y reuniones científicas
Título:
HOXA expression sequence and functional transplantation assays indicate that axolotl limb regeneration does not occur by intercalation
Autor/es:
TAZAKY AKIRA; ROENSCH KATHLEEN; CHARA OSVALDO; TANAKA ELLY M
Lugar:
Mont-Tremblant
Reunión:
Conferencia; 12th International Conference on Limb Development and Regeneration. Mont-Tremblant; 2012
Resumen:
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.