Revealing new roles for SSD proteins Disp1 and Npc1 in zebrafish development and disease.

摘要

The vertebrate head skeleton is primarily derived from cranial neural crest cells (CNCC). The loss of Hedgehog (Hh)-signaling in the vertebrate embryo causes severe craniofacial defects, in part due to defects in CNCC-derived tissues. Craniofacial defects are characteristic of the Hh-associated spectral disorder holoprosencephaly in humans. The Hh-signaling pathway is conserved in zebrafish and inactivating the pathway in mutant embryos leads to severe skeletal loss, yet the direct requirements for Hh-signaling in zebrafish craniofacial development are largely unknown.;Dispatched 1 (disp1) is a sterol sensing domain (SSD) protein that positively regulates the Hh-signaling pathway. SSD family members are involved in the intracellular movements of cholesterol and cholesterol-linked molecules and Disp1 primary action is to release cholesterol-linked Hh-ligands from their site of synthesis.;In this dissertation, I show that chameleon mutant zebrafish, lacking a functional Disp1, exhibit severe cranial cartilage and muscle defects and their postmigratory CNCC are defective in both patterning and differentiation. By inhibiting Hh-signaling at different developmental stages, I found that Hh-signaling is required during gastrulation and the late pharyngula stage to promote proper CNCC development. By designing a Gli reporter transgenic fish to determine Hh-responding cells, I determined that the Hh-requirement for CNCC development is likely influencing the surrounding epithelium cells that interact with CNCC mesenchyme.;Npc1 is a SSD protein that is closely related in protein structure to Disp1 and Patched1 (Ptch1), the Hh-signaling receptor, and is involved in sterol trafficking from late endosomes to other cellular compartments. Inactivating mutations in Npc1 lead to increased levels of cellular sterols but fewer amounts of sterol derivatives due to reductions in sterol metabolism. The identification of sterol derivatives and sterol-like compounds that modulate the Hh-signaling pathway has provided new potentials for therapeutics.;By identifying and cloning the zebrafish npc1 gene, I was able to test its function in sterol homeostasis in the zebrafish embryo. Reducing its function with morpholinos led to the mislocalization of cellular cholesterol, but did not impact levels of Hh-signaling in morphants. npc1 morphants exhibited a delay in epiboly, likely a consequence in a reduction of sterol metabolism to hormones in the embryo.