TBX3 restricts anterior gene expression from the posterior mesenchyme to protect posterior identity that is promoted by HAND2

Embryonic development and organogenesis depend on precise spatial and temporal control of gene expression, which is regulated by cis-regulatory modules (CRMs). The signalling pathways and transcription factors form a robust gene regulatory network (GRN) to control growth and cell differentiation patterns.
The establishment of limb anterior-posterior (A-P) polarity requires the genetic interaction between Tbx3, Hand2, and Gli3. The interaction involves posterior Tbx3 working with Hand2 to genetically antagonise Gli3. This results in the segregation of an anterior-distal Gli3 and a posterior Hand2 and Tbx3 signal. This distinction in A/P patterning will control other transcription factors and establish early A/P polarisation. Hand2 and Tbx3 are required to prevent posterior expansion of Gli3 expression in early limb buds and maintain posterior gene expression. Hand2 has been shown to be important for ulna and autopod formation; in the absence of Hand2, the ulna is completely lost, leaving only a digit that appears to develop independently of SHH signalling. Meanwhile, the deletion of Tbx3 in the limb results in the loss of digit 5 and a mild downregulation of posterior gene expression. Hand2 and Tbx3 maintain each other's expression; deletion of Hand2 results in complete loss of posterior Tbx3, whereas mutation of Tbx3 leads to downregulation of Hand2.
How Tbx3 and Hand2 restrict Gli3 expression and the observed differential roles of Tbx3 and Hand2 in maintaining posterior gene expression remain to be elucidated.
Here, we have identified the role of posterior TBX3 in restricting anterior gene expression in the posterior. In doing so, TBX3 supports HAND2 in promoting posterior gene expression, setting the stage for Shh to be properly expressed.
TBX3 binds to three out of four Gli3 enhancers active in the limb, whereas Hand2 only binds to one. This suggests that TBX3 may be essential to exclude the high expression of Gli3 in the posterior limb buds. Indeed, our results show that mutation of Tbx3 leads to an expansion of the Gli3 enhancers mm1179 and mm-hs1586 activity in the posterior limb buds. TBX3 binds several CRMs associated with anterior genes. We propose that TBX3, by interacting with CRMs of anterior gene, would restrict Irx3/5, Alx4, and Hand1 expression and exclude from the posterior limb bud.
We've also investigated the anterior role of Tbx3 using the genetic interaction between Tbx3 and Gli3. As reported in previous studies, Tbx3 cooperates with Gli3 to restrict digit 1 development and prevent preaxial polydactyly (PPD). The combined mutation of Tbx3 and heterozygous Gli3 increases PPD. Observations of the Hand2-Tbx3 combined mutants versus Gli3-Tbx3 and our late-stage HCR data highlight a digit 1 development that relies only on anterior Tbx3 and Gli3 but would be independent of Hand2. Since Tbx3 has been shown to play a role in both anterior and posterior limb development, the mild downregulation of posterior genes seen in a Tbx3 mutant may be due to a buffering effect caused by the deletion of anterior and posterior Tbx3.
TBXs would also play a role in promoting Gli3 expression, as deletion of the T-box binding motif resulted in a partial loss of spatial Gli3 enhancer activity. By examining the literature and TBXs gene expression concerning Tbx3 mutation, we propose that TBX15, TBX18 and TBX5 could be candidates for the activation and promotion of Gli3 expression in the mouse limb bud. Interestingly, Tbx15 expression largely overlaps with Gli3 and extends to the posterior mesenchyme in Tbx3 deficient limb buds. On the other hand, TBX5 shares phenotypic limb changes and binds Gli3 enhancers, which suggests that TBXs may function in maintaining Gli3 enhancer activities and expression, which raises new questions about how TBXs might compete to regulate the expression of shared target genes.