RESUMO
We dissect genetically a gene regulatory network that involves the transcription factors Tbx4, Pitx1 and Isl1 acting cooperatively to establish the hindlimb bud, and identify key differences in the pathways that initiate formation of the hindlimb and forelimb. Using live image analysis of murine limb mesenchyme cells undergoing chondrogenesis in micromass culture, we distinguish a series of changes in cellular behaviours and cohesiveness that are required for chondrogenic precursors to undergo differentiation. Furthermore, we provide evidence that the proximal hindlimb defects observed in Tbx4 mutant mice result from a failure in the early differentiation step of chondroprogenitors into chondrocytes, providing an explanation for the origins of proximally biased limb defects.
Assuntos
Membro Posterior/anormalidades , Botões de Extremidades/metabolismo , Proteínas com Domínio T/metabolismo , Animais , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Condrogênese , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Botões de Extremidades/citologia , Botões de Extremidades/crescimento & desenvolvimento , Células-Tronco Mesenquimais/metabolismo , Camundongos , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Proteínas com Domínio T/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The forelimbs and hindlimbs of vertebrates are bilaterally symmetric. The mechanisms that ensure symmetric limb formation are unknown but they can be disrupted in disease. In Holt-Oram Syndrome (HOS), caused by mutations in TBX5, affected individuals have left-biased upper/forelimb defects. We demonstrate a role for the transcription factor Tbx5 in ensuring the symmetric formation of the left and right forelimb. In our mouse model, bilateral hypomorphic levels of Tbx5 produces asymmetric forelimb defects that are consistently more severe in the left limb than the right, phenocopying the left-biased limb defects seen in HOS patients. In Tbx hypomorphic mutants maintained on an INV mutant background, with situs inversus, the laterality of defects is reversed. Our data demonstrate an early, inherent asymmetry in the left and right limb-forming regions and that threshold levels of Tbx5 are required to overcome this asymmetry to ensure symmetric forelimb formation.