Abstract Niemann-Pick disease, type C (NP-C), often associated with Niemann-Pick disease,
Abstract Niemann-Pick disease, type C (NP-C), often associated with Niemann-Pick disease, type C1 (NPC1) mu-tations, is a cholesterol-storage disorder characterized by cellular lipid accumulation, neurodegeneration, and reduced steroid production. and improved neurological function (21, 27), possibly by quenching elevated levels of reactive oxygen species in the brain (28). Moreover, chronic treatment with the sex steroid estradiol in Npc1-deficient mice enhances defective pituitary development and is usually capable of reversing ovarian defects and infertility in Npc1-deficient females (29, 30). These studies suggest that further insight into the pathophysiology of NP-C may be gained by having a better understanding of cholesterol metabolism and steroid hormone action during development and adulthood. The Npc1 gene has been found to be strongly conserved in many experimental model organisms (31), playing a crucial role in some aspect of sterol homeostasis in each species examined to date. Structurally, Npc1 is usually a 13 transmembrane-spanning protein (32), made up of a sterol-sensing domain name (SSD) composed of five transmembrane helices. The SSD is usually found in a number of different protein, some of which are also involved in binding cholesterol and in sterol metabolism [at the.g., sterol regulatory element-binding protein cleavage-activating protein (SCAP)], and others of which are involved in moving and binding to the cholesterol-modified protein Sonic hedgehog (at the.g., Dispatched and Patched) (33). Despite this, the mechanistic role of this family of proteins in vertebrate development has not been widely examined. Our interest in the contribution of Npc1 to vertebrate development stems from our overall interest in the role of SSD-containing protein during development (34). To this point, most studies concerning Npc1 function have focused on either the cell biology of sterol movements within a cell or the neuropathological outcomes that result from disruption of this process. Less is usually known about the contribution of this protein to vertebrate development. Because it is usually a vertebrate animal and genetic morphants can be produced in large figures, the zebrafish embryo is usually an ideal organism to examine the role of Npc1 in development. Additionally, zebrafish closely resemble mammals in their development and in cholesterol metabolism (examined in Ref. 35), making them highly amenable for studying proteins involved in lipid and sterol metabolism. In zebrafish development, early studies have revealed a requirement for cholesterol in promoting cell migration during epiboly, one of the earliest morphogenetic movements of gastrulation that ultimately generate the embryo’s complex body plan (36, 37). The morphogenetic process of epiboly entails coordinated movements of each of the embryonic cell layers that are present p18 during late blastula CYT997 (1): the deep cell layer, which gives rise to the embryo proper (2); the enveloping layer (EVL), CYT997 an extra-embryonic superficial epithelial layer covering the deep cells; and (3) the yolk syncytial layer (YSL), an extra-embryonic cytoplasmic cell layer within the yolk cell (38, 39). Epiboly commences when the yolk cell bulges toward the animal pole and the deep cell blastomeres radially intercalate. This process continues with the thinning and vegetal migration of the blastoderm over the yolk cell until 50% of the yolk surface is usually covered (50% epiboly) (40C42), CYT997 at which time the deep cells begin the second phase of -gastrulation including dorsal convergence and involution movements which form the germ cell layers. Concomitant with this, each of the three cell layers continues to spread over the yolk in the epiboly process until the yolk cell is usually completely covered and internalized (41C44). Reducing the levels of cholesterol metabolites in zebrafish embryos results in an epiboly-delay phenotype (36). In this study we have recognized and cloned the zebrafish gene and found that it is usually widely present during early embryonic development. Using targeted morpholino (MO) antisense oligonucleotides, we have exhibited that loss of prospects to sterol localization defects in early embryos, comparable to defects observed in travel and mammalian cells lacking Npc1. Our gene knockdown studies further revealed that is usually required for normal epiboly movement. Epiboly defects may be in part due to abnormal cytoskeletal structures, as we observed disruptions in CYT997 the actin cytoskeleton in mRNA at the 1-cell stage or into the yolk cell of a 1,000-cell stage embryo, showing conservation of function between the fish and mammalian orthologs. Moreover, two downstream components of steroid synthesis, including the cholesterol derivative pregnenolone (P5) and glucocorticoid dexamethasone (Dex), could partially rescue the epiboly defects, demonstrating that such deficits are likely due, at least in.