Role of patched homologs in development and cellular physiology in Caenorhabditis elegans

Abstract

The Hedgehog (Hh) signaling pathway is essential for animal development and health maintenance. In the canonical Hh pathway, the signaling molecule Hh binds to the membrane cholesterol transporter PTCH, thereby relieving the inhibition of Smoothened (SMO), which in turn activates a signaling cascade required for proper development. Nonetheless, increasing evidence points to the existence of SMO-independent Hh pathways. However, the co-existence with the canonical pathway has undermined the efforts to characterize these novel pathways. C. elegans is an excellent model to study SMO-independent Hh pathways. However, even if this worm lacks SMO, the PTCH homologs are essential for worm development, therefore, indicating that the Hh pathway still operates in worms. Thus, we studied the role of two PTCH proteins in the C. elegans’ SMO-independent context to elucidate their function in an essential non-canonical Hh pathway. Here, we demonstrate that the loss of PTC-3 leads to the accumulation of cholesterol at the plasma membrane in vivo. Further analysis of the lipid metabolism revealed a reduction in acyl-chain length and desaturation, which suggested membrane structure defects. Indeed, we discovered defects in ER structure and lipid droplets. Even more, we show evidence that cholesterol accumulation modulates the function of nuclear hormone receptors such as the PPARalpha homologs NHR-49 and NHR-181. Finally, reduction of dietary cholesterol rescued all described phenotypes, consequently improving development and survival. Therefore, our data uncover a novel SMO-independent pathway which is necessary for lipid homeostasis and fat storage. Concomitant with the evolution of PTCH homologs, Patched-related proteins (PTR), another group of proteins derived from the ancestral PTCH evolved. However, the roles PTRs play in the cell and whether they share functions with other PTCH proteins remains to be determined. Our work indicates that cholesterol regulation is a conserved function of the PTR protein PTR-4. However, we showed that PTC-3 and PTR-4 have different and specific roles in C. elegans development. Interestingly, the analysis of PTR-4 expression pattern revealed that protein levels and localization are dynamic throughout larval growth, suggesting dynamic cholesterol levels during development. Finally, a phenotypical characterization of animals lacking PTR-4 protein or a hypomorph allele of PTR-4 revealed a role of the protein in cuticle stability, which has an impact on locomotion. In summary, through the use of the nematode as a model organism, we have shown how PTCH proteins have a conserved role in cholesterol modulation. Furthermore, we demonstrated that they have essential SMO-independent roles.