Identification of genes associated with regenerative success of Xenopus laevis hindlimbs.
BACKGROUND
Epimorphic regeneration is the process by which complete regeneration of a complex structure such as a limb occurs through production of a proliferating blastema. This type of regeneration is rare among vertebrates but does occur in the African clawed frog Xenopus laevis, traditionally a model organism for the study of early development. Xenopus tadpoles can regenerate their tails, limb buds and the lens of the eye, although the ability of the latter two organs to regenerate diminishes with advancing developmental stage. Using a heat shock inducible transgene that remains silent unless activated, we have established a stable line of transgenic Xenopus (strain N1) in which the BMP inhibitor Noggin can be over-expressed at any time during development. Activation of this transgene blocks regeneration of the tail and limb of Xenopus tadpoles.


RESULTS
In the current study, we have taken advantage of the N1 transgenic line to directly compare morphology and gene expression in same stage regenerating vs. BMP signalling deficient non-regenerating hindlimb buds. The wound epithelium of N1 transgenic hindlimb buds, which forms over the cut surface of the limb bud after amputation, does not transition normally into the distal thickened apical epithelial cap. Instead, a basement membrane and dermis form, indicative of mature skin. Furthermore, the underlying mesenchyme remains rounded and does not expand to form a cone shaped blastema, a normal feature of successful regeneration. Using Affymetrix Gene Chip analysis, we have identified genes linked to regenerative success downstream of BMP signalling, including the BMP inhibitor Gremlin and the stress protein Hsp60 (no blastema in zebrafish). Gene Ontology analysis showed that genes involved in embryonic development and growth are significantly over-represented in regenerating early hindlimb buds and that successful regeneration in the Xenopus hindlimb correlates with the induction of stress response pathways.


CONCLUSIONS
N1 transgenic hindlimbs, which do not regenerate, do not form an apical epithelial cap or cone shaped blastema following amputation. Comparison of gene expression in stage matched N1 vs. wild type hindlimb buds has revealed several new targets for regeneration research.
Pearl
Esther J
EJ
Department of Zoology, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand. esther.pearl@ircm.qc.ca
Barker
Donna
D
Day
Robert C
RC
Beck
Caroline W
CW
D000671:Amputation, Surgical; D000818:Animals; D019485:Bone Morphogenetic Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics; D020869:Gene Expression Profiling; D005796:Genes; D006614:Hindlimb / Q000196:embryology / Q000502:physiology*; D018878:Limb Buds; D030781:Organisms, Genetically Modified; D012038:Regeneration; D014945:Wound Healing; D014982:Xenopus laevis / Q000502:physiology*; D000099176:Noggin Protein
D019485:Bone Morphogenetic Proteins; D002352:Carrier Proteins; D000099176:Noggin Protein