Theshhlimb enhancer is activated in patterned limb regeneration but not in hypomorphic limb regeneration inXenopus laevis

Abstract

ABSTRACTXenopusyoung tadpoles regenerate a limb with the anteroposterior (AP) pattern, but metamorphosed froglets regenerate a hypomorphic limb after amputation. The key gene for AP patterning,shh, is expressed in a regenerating limb of the tadpole but not in that of the froglet. Genomic DNA in theshhlimb-specific enhancer, MFCS1 (ZRS), is hypermethylated in froglets but hypomethylated in tadpoles:shhexpression may be controlled by epigenetic regulation of MFCS1. Is MFCS1 specifically activated for regenerating the AP-patterned limb? We generated transgenicXenopus laevislines that visualize the MFCS1 enhancer activity with a GFP reporter. The transgenic tadpoles showed GFP expression inhoxd13-andshh-expressing domains of developing and regenerating limbs, whereas the froglets showed no GFP expression in the regenerating limbs despite havinghoxd13expression. Genome sequence analysis and co-transfection assays using cultured cells revealed that Hoxd13 can activateXenopusMFCS1. These results suggest that MFCS1 activation correlates with regeneration of AP-patterned limbs and that re-activation of epigenetically inactivated MFCS1 would be crucial to confer the ability to non-regenerative animals for regenerating a properly patterned limb.