Tgifs Regulate Ciliogenesis and the Development of Neuroepithelium in Mice.

Holoprosencephaly (HPE) is a severe developmental defect affecting 1 in 250 conceptions and 1 in 8,000 live births. 18-25% of HPE cases are caused by single gene mutations, most commonly targeting the Sonic Hedgehog (Shh) pathway. Loss of function mutations in the transcriptional corepressor TGIF are also associated with HPE. HPE defects can be replicated in a mouse model in which Tgif1 is conditionally deleted within the epiblast and Tgif2 is constitutively deleted (cdKO). The severity of HPE can be partially rescued by decreasing the genetic dose of the Shh-antagonist, Gli3. Decreased Gli3 does not, however, rescue defects cdKO embryos have in addition to those associated with HPE, including disruption of epithelial polarity, failure to establish proper left-right asymmetry, and defective cilia formation. As Tgif inhibits TGFβ signaling, we hypothesized that Nodal, a TGFβ signaling ligand essential for embryonic development, is misregulated in the absence of Tgifs. We demonstrated cdKO embryos lacking one allele of Nodal have rescued epithelial polarity, but not left-right asymmetry. We also hypothesized that the disruption of cilia formation is driven by Tgif target gene overexpression. We performed RNA-seq on whole cdKO and control embryos to identify over-expressed genes that might be TGIF targets. After validating expression by qRT-PCR, several cilia-related genes were identified as differentially regulated in cdKO embryos when compared to wild type embryos. The RabGAP Evi5l, whose overexpression inhibits cilia formation, was found to contain a Tgif binding element in its promoter region. Through knock-down and luciferase experiments we showed that Evi5l is a direct Tgif target and that knockdown of Evi5l in the absence of Tgifs rescues the defect in cilia formation in a TGFβ-independent manner. The knowledge that Tgif directly affects cilia formation not only elucidates defects in cdKO embryos, but also helps explain how Tgif mutations contribute to defects in Shh signaling via defective cilia and produce left-right asymmetry defects due to defective cilia at the posterior notochord.