Ectopic Expression of T‐Box Transcription Factors Alters Mouse Forelimb Formation

The developing vertebrate forelimb expresses seven T-box transcription factors, with several in overlapping expression domains. All T-box transcription family members share similarity within their DNA binding domain, the T-domain. Outside of the T-domain, these factors share little similarity, allowing family members to have different transcriptional properties and binding partners. Several human T-box genes show haploinsufficiency in the limb, including Tbx5 and Tbx3 that, when mutated, cause Holt-Oram and ulnar-mammary syndrome, respectively. This dosage sensitivity combined with the shared T-domain leads to our hypothesis that when co-expressed a competition between T-box factors at target genes can occur. To test this, we ectopically expressed two exogenous T-box factors, T and Tbx6, in the developing forelimb mesenchyme to examine how artificially changing the relative levels of T-box proteins affects forelimb formation. Skeletal, apoptotic, and gene expression assays were used to characterize the resulting phenotypes. While ectopic T and Tbx6 both affected the size and shape of the forearm bones and ossification, they differentially affected digit formation: T caused loss of digits and Tbx6 led to phalange bone duplications and extra digit formation. These dissimilar phenotypes suggest that these transcriptional activators differentially affect pathways critical for regulating forelimb development.