Limin Hao, Gilbert Lauter, Krishanu Mukherjee, Samuel Liegeois, David Baillie, Michel Labouesse, Shai Shaham, Thomas R. Burglin. The C. elegans genome encodes about 60 hedgehog-related genes in 4 families: quahog, warthog, groundhog, and ground-like. Here we report functional studies for
wrt-6,
wrt-5 and
qua-1, and show how they play essential roles in the extracellular environment.
wrt-6 is primarily expressed in the sensilla socket cells and mutants display Dyf, Osm and Daf-d phenotypes. The cilia structure is normal, as shown by EM and confocal microscopy. However, the sheath cell channel is expanded and the dendrites of the sensory neurons do not form tight bundles. Using the socket cell marker Punc-53::GFP we observe morphological defects in the channel. The Dyf phenotype can be rescued in L4 or young adults by a hsp::WRT-6 construct, suggesting that there are no major developmental defects in cilia or cell fate specification. WRT-6 point mutations have identified several functionally important residues in the WRT domain. The WRT domain alone is not able to rescue the Dyf phenotype, the HOG domain is required. Replacing the WRT domain of WRT-6 with that of WRT-5 is able to rescue the Dyf phenotype, suggesting that the WRT domain structures are similar and can tolerate numerous substitutions. The translational GFP fusions have revealed that WRT-6 is associated with the cuticle in the channel of amphids and phasmids and in the vulval labia. WRT-5 lacks the Hint/Hog domain and is expressed in seam cells, the pharynx, pharyngeal-intestinal valve cells, neurons, neuronal support cells, the excretory cell, and the reproductive system. WRT-5 protein is secreted into the extracellular space during embryogenesis. Furthermore, during larval development WRT-5 protein is secreted into the pharyngeal lumen and the pharyngeal expression changes in a cyclical manner in phase with the molting cycle.
wrt-5 mutations cause a cold-sensitive embryonic lethality. Animals that hatch exhibit variable abnormal morphology, e.g., bagging worms, blistering, molting defects, or Roller phenotypes. Using AJM-1::GFP we observed severe cell boundary abnormalities in the arrested embryos. AJM-1:GFP protein is also misplaced in pharyngeal muscle cells in the absence of WRT-5. WRT-5 may be an essential cofactor for cell fusion at low temperatures.
qua-1 is expressed in hypodermal cells, rectal cells, sensilla support cells.
qua-1::GFP undergoes cyclical changes during development in phase with the molting cycle.
qua-1 mutants display a 100% penetrant Mlt phenotype. EM reveals double cuticles due to defective ecdysis and abnormal alae structures, but no obvious defects in the hypodermis. QUA domain-only::GFP and full-length QUA-1::GFP fusion constructs are secreted and associated with the overlying cuticle. QUA-1::GFP rescues the mutant phenotype, but QUA domain-only::GFP causes dominant Mlt phenotypes in N2.