Genetic basis of hindlimb loss in a naturally occurring vertebrate model.

Emily K Don,Benjamin Heng,Tanya A De Jong-Curtain,Daniel Hesselson,Garth A Nicholson,Claire Winnick,Nicholas J Cole,Gilles J Guillemin,Peter D Currie,Karen Doggett,Andrew P Badrock,Joan K Heath,Thomas E Hall

doi:10.1242/bio.016295

Abstract

ABSTRACTHere we genetically characterise pelvic finless, a naturally occurring model of hindlimb loss in zebrafish that lacks pelvic fin structures, which are homologous to tetrapod hindlimbs, but displays no other abnormalities. Using a hybrid positional cloning and next generation sequencing approach, we identified mutations in the nuclear localisation signal (NLS) of T-box transcription factor 4 (Tbx4) that impair nuclear localisation of the protein, resulting in altered gene expression patterns during pelvic fin development and the failure of pelvic fin development. Using a TALEN-induced tbx4 knockout allele we confirm that mutations within the Tbx4 NLS (A78V; G79A) are sufficient to disrupt pelvic fin development. By combining histological, genetic, and cellular approaches we show that the hindlimb initiation gene tbx4 has an evolutionarily conserved, essential role in pelvic fin development. In addition, our novel viable model of hindlimb deficiency is likely to facilitate the elucidation of the detailed molecular mechanisms through which Tbx4 functions during pelvic fin and hindlimb development.

Highlights

The study of limb development has relied heavily on mouse and chick embryos as models to understand the genetic mechanisms of limb induction, identity and outgrowth
We show using a unique pelvic finless zebrafish model that is Tbx4 required for pelvic fin development, and that the nuclear localisation signal (NLS) of Tbx4 must be intact for Tbx4 to play its essential role in the induction of the apical ectodermal ridge and the outgrowth of the pelvic fin
Pelvic finless zebrafish initiate pelvic fin development, evident by the 3-4 cell thick mesenchymal bulges that form in the pelvic regions around 3-4 weeks of development; these bulges do not form an apical ectodermal ridge and a subsequent loss of pelvic fin development is observed (Don et al, 2011)

Summary

Introduction

The study of limb development has relied heavily on mouse and chick embryos as models to understand the genetic mechanisms of limb induction, identity and outgrowth. To confirm whether mutations in the Tbx4 NLS are responsible for the developmental defects in pelvic finless zebrafish, we used genetic complementation, protein localization and in-situ hybridisation studies to explore the functional consequences of the naturally occurring A78V; G79A mutations.

Results

Conclusion