Contrary to the widely-held belief that nearly all vertebrate cells have primary cilia, our lab recently discovered that extra-embryonic visceral endoderm cells lack primary cilia. Our lab was also the first to demonstrate that primary cilia are required for all signaling through the Hedgehog pathway in mammals, which is consistent with data that extra-embryonic cells do not respond to Hedgehog ligands. However, extra-embryonic cells do secrete Hedgehog ligands, which are required for normal development of the embryo-derived vasculature of the placenta. I hypothesized that other cell types that make, but do not respond, to Hedgehog ligands may also lack cilia. Consistent with this hypothesis, I find that epithelia of a number of adult endodermal organs, which secrete but do not respond to Hedgehog ligands, lack primary cilia. However, I have also found that endodermal epithelia have primary cilia in early development. This primary cilia are present early in gut tube morphogenesis but are lost before adulthood, suggesting that dynamic signals from adjacent tissues can inhibit formation of primary cilia. In the proposed research, I will determine at what point primary cilia are lost in endodermal epithelium, which will allow me to identify the exogenous signals present at this time point. Expression of candidate factors will be manipulated to test whether primary cilia can be ectopically assembled in cells that would normally lack these organelles. I will also test the functional relevance the lack of autocrine hedgehog signaling in these tissue by expressing a constitutively active mutant of Gli2, the major Hedgehog transcriptional activator, in embryonic and adult endodermal epithelium, and assaying its effects on gut development and recovery from gut injury. To find additional factors responsible for inhibition of ciliogenesis, I propose to perform transcriptome analysis of ciliated and non-ciliated endodermal epithelium. I also propose to perform screens to identify small molecules that can induce cilia on non-ciliated extra embryonic visceral endodermal stem cells (XEN), in an effort to find therapeutic targets capable of inducing ectopic ciliogenesis, which may prove invaluable in treatment of a variety of developmental disorders and cancers.
Hedgehog ligands made in the endoderm signal to the adjacent mesenchyme to regulate mammalian organ development and play a central role in endodermal organ repair, but the endoderm cells that make Hedgehog do not respond to the ligand. Experiments will test the hypothesis that endoderm cells do not respond to Hedgehog because they lack primary cilia and determine the signals that regulate cilia formation in the gut endoderm, ultimately allowing regulation of cilia formation. Because loss of primary cilia correlates with tumor progression, the findings will be important for cancer biology as well as regenerative medicine.
