The roles of Tbx5 and Tbx4 in the bilaterallysymmetric initiation of the left and right limbs

Although there is no obvious communication between the left and right limb buds they develop to form bilaterally symmetrical structures of equal size (Summerbell & Wolpert, 1973). The underlying mechanisms that ensure symmetrical limb formation are unknown. Holt-Oram Syndrome (HOS) [OMIM 142900] is a congenital syndrome associated with mutations in TBX5 that lead to heart and upper limb defects. Strikingly, over 70% of HOS patients have left-biased upper limb defects. Using two separate strategies, I show that hypomorphic levels of Tbx5 in both the left and right forelimb buds produces forelimb defects that are consistently more severe in the left limb than the right. Using the INV/INV mutant background, in which the left-right axis is reversed, I show that the laterality of these defects is reversed in Tbx5 hypomorphic mutants with situs inversus. Additionally, I also show that transgenic expression of equal levels of Fgf10 in the forelimb buds of these Tbx5 hypomorphs can partially rescue outgrowth defects but not the left-bias asymmetry of their presentation. Together, this data suggests that Tbx5 has a role in ensuring symmetrical forelimb formation and that this is independent of its transcriptional regulation of Fgf10. Tbx4, the paralogue of Tbx5, is expressed in the hindlimb. I have used a conditional deletion approach to delete Tbx4 expression from the hindlimb area thus avoiding early embryonic lethality. I show that deletion of Tbx4 leads to a loss of proximal hindlimb skeletal elements as well as mispatterning of the distal hindlimb. I also show that Pitx1 partially contributes towards the establishment of the FGF signalling positive feedback loop during initiation of hindlimb bud outgrowth.