Spatiotemporal Twist1 deficiency shifts forelimb to hindlimb expression pattern resulting in multidigit phenotype

Twist1 encodes a transcription factor that plays a vital role in limb development. Conditional Twist1 knockout mouse models show specific skeletal phenotypes with partial penetrance and variable expression in the hindlimb versus forelimb. However, the differential role of Twist1 in the forelimb versus hindlimb is yet to be investigated. By deletion of Twist1 limb enhancers (eTw5-7^∆/∆), we showed that reduction of Twist1 leads to hindlimb-like expression pattern in the developing forelimb that results in autopod forelimb phenotype characterized by polydactyly. A phenotype that was not observed in Twist1+/- embryos. Using transcriptomic analysis, we found 32 differentially expressed genes in eTw5-7^∆/∆ mouse limb when most of them are expressed in the forelimb. In addition to Twist1 that showed reduced expression in both hindlimb and forelimb of eTw5-7^∆/∆ mice, we showed that most of the elevated expressed genes in the forelimb are specific hindlimb markers (Pitx1, Tbx4 and HoxcC9-12). Using whole mount in situ hybridization and qPCR, we showed that these hindlimb specific markers are ectopically expressed in the forelimb of eTw5-7^∆/∆ E11.5 mouse embryos. Thus, these results suggest that Twist1 has a novel regulatory activity on hindlimb specific genes in the developing forelimb and plays a role in limb identity. Further studies are required to decipher the regulatory mechanism of Twist1 in the hind limb versus forelimb for the development of the limb identity.