SPLTRAK Abstract Submission

Hedgehog signalling in taste receptor cells and other chemosensory epithelia

Robert Margolskee
Monell Chemical Senses Center, , ,

Hedgehog (Hh) signalling is a principal regulator of cell proliferation and differentiation in many types of epithelial tissues, including gut, lung, taste papillae and taste buds. In adult mice, taste cells survive only a few days to a few weeks, necessitating their regeneration throughout life. In anterior tongue, sonic hedgehog (Shh), released by a subpopulation of basal taste cells and gustatory neurons that innervate taste buds, signals through the integral membrane receptor complex Patched and Smoothened to regulate zinc finger transcription factors Gli2 and Gli3, principal effectors of the Hh pathway in adults. In the absence of Shh signaling, Gli2 and Gli3 are C-terminally truncated to generate transcriptional repressors that are mostly sequestered in the cytoplasm. Shh signaling prevents the proteolysis of Gli2 and Gli3 and promotes their localization to the nucleus, where they regulate the expression of multiple target genes. Shh is known to regulate fungiform papillae development and patterning, but the downstream transcriptional effectors of hedgehog signalling in taste organ maintenance are largely unknown. Gli3 is a key transcriptional effector in the sonic hedgehog signalling pathway, but its distribution in the adult tongue and its role in taste bud maintenance was previously unknown. We used RNA sequencing of single taste cells and bioinformatics to determine that Gli3 is more highly expressed in type II taste cells and stem cells than in type III taste cells. Gli3 is selectively expressed in Tas1r3-expressing type II taste cells and Lgr5-expressing taste stem cells, but not in 5-HT-expressing type III taste cells or Glast-expressing type I taste cells. Behavioral tests showed that in comparison to wildtype Gli3 conditional knockout mice are more sensitive to sweet compounds, less sensitive to bitter and sour compounds, and similarly responsive to umami and salty compounds. Recording from the glossopharyngeal nerve showed that Gli3 conditional knockout mice are much more responsive to sweet and bitter compounds than are wildtype mice. Moreover, in vivo in Gli3 conditional knockout mice, significant increases were observed in the size of the taste buds and the number of taste cells in each taste bud, as well as in the number of taste receptor cells per taste bud. In an ex vivo taste organoid system we found that conditional loss of Gli3 from Lgr5+ stem cells led to increased numbers of Tas1r3+ cells and decreased numbers of Car4+ type III cells. Thus, in adult taste cells Gli3 functions as a negative regulator of differentiation and/or survival of taste stem cells and Tas1r3+ type II taste cells that influences taste receptor cell composition and function. In other organoid experiments we found that the Gli antagonist GANT61 arrested growth of taste organoids, especially in the early stages of culturing. In sum, broadly interrupting Gli mediators of Shh signaling prevents taste stem cells from proliferating in cultured taste organoids, while selective inhibition of Gli3 affects relative numbers of type II vs. III cells.

Acknowledgements: Supported by NIDCD/NIH grant R01DC014105 to RFM.