GSE161867
The role of Niemann-Pick type C2 in zebrafish embryonic development
Niemann-Pick disease type C (NPC) is a rare fatal neurodegenerative lysosomal storage disease caused by mutations of either NPC1 or NPC2.  NPC2 is a soluble lysosomal protein that in coordination with NPC1 is responsible for the efflux of unesterified cholesterol from the lysosome.  Mutations of both genes cause a similar cellular pathology, that includes the accumulation of unesterified cholesterol and other lipids in the late endosome/lysosome, while reducing cholesterol bioavailability. Here we describe a npc2 null zebrafish model generated by CRISPR/Cas9 gene targeting.  Zygotic npc2m/m zebrafish from the intercross of npc2+/m individuals showed significant unesterified cholesterol accumulation at larval stages, and a reduction in body size in adulthood.  Additionally, zygotic npc2m/m adults exhibited motor and balance defects shortly before a premature death.  These findings mimic defects found in human and mice, however the phenotype at embryonic stages were milder than expected.  To address the possible dose protection effects of npc2 mRNA present in the oocyte at the time of fertilization we intercrossed npc2m/m zebrafish to generate Maternal-zygotic (MZ) npc2m/m embryos. MZnpc2m/m zebrafish exhibited significant developmental defects including absent otolith, abnormal head/brain development, curved/twisted body axis, no circulating blood cells, and died by 72 hpf.   These developmental defects have not previously been reported in connection with either NPC2 or NPC1.  RNA-seq analysis conducted on 30 hpf npc2+/m and MZnpc2m/m embryos revealed a significant reduction in notch3 expression as well as reduction in other downstream genes in the signally pathway such as hey1 and her12 that could be partially rescued by microinjection of a plasmid containing the constitutively active notch3 ICD at the 1-cell stage, suggesting that impaired Notch3 signaling likely underlies some aspects of the developmental defects observed in MZnpc2m/m zebrafish. Overall design: RNA-seq of homozygous and heterozygous npc2 mutant zebrafish compared to WT; 3 replicates for each genotype