Identification of a Tbx1/Tbx2/Tbx3 genetic pathway governing pharyngeal and arterial pole morphogenesis

The 22q11.2 deletion syndrome (22q11.2DS) is the most common microdeletion disorder and is characterized by abnormal development of the pharyngeal apparatus and heart. Cardiovascular malformations (CVMs) affecting the outflow tract (OFT) are frequently observed in 22q11.2DS and are among the most commonly occurring heart defects. The gene encoding T-box transcription factor 1 (Tbx1) has been identified as a major candidate for 22q11.2DS. However, CVMs are generally considered to have a multigenic basis and single-gene mutations underlying these malformations are rare. The T-box family members Tbx2 and Tbx3 are individually required in regulating aspects of OFT and pharyngeal development. Here, using expression and three-dimensional reconstruction analysis, we show that Tbx1 and Tbx2/Tbx3 are largely uniquely expressed but overlap in the caudal pharyngeal mesoderm during OFT development, suggesting potential combinatorial requirements. Cross-regulation between Tbx1 and Tbx2/Tbx3 was analyzed using mouse genetics and revealed that Tbx1 deficiency affects Tbx2 and Tbx3 expression in neural crest-derived cells and pharyngeal mesoderm, whereas Tbx2 and Tbx3 function redundantly upstream of Tbx1 and Hh ligand expression in pharyngeal endoderm and bone morphogenetic protein- and fibroblast growth factor-signaling in cardiac progenitors. Moreover, in vivo, we show that loss of two of the three genes results in severe pharyngeal hypoplasia and heart tube extension defects. These findings reveal an indispensable T-box gene network governing pharyngeal and OFT development and identify TBX2 and TBX3 as potential modifier genes of the cardiopharyngeal phenotypes found in TBX1-haploinsufficient 22q11.2DS patients