Mutations in vacuolar H+-ATPase subunits lead to biliary developmental defects in zebrafish

Abstract

Summary We identified three zebrafish mutants with defects in biliary development. One of these mutants, pekin (pn), also demonstrated generalized hypopigmentation and other defects, including disruption of retinal cell layers, lack of zymogen granules in the pancreas, and dilated Golgi in intestinal epithelial cells. Bile duct cells in pn demonstrated an accumulation of electron dense bodies. We determined that the causative defect in pn was a splice site mutation in the atp6ap2 gene that leads to an inframe stop codon. atp6ap2 encodes a subunit of the vacuolar H+-ATPase (V-H+-ATPase), which modulates pH in intracellular compartments. The Atp6ap2 subunit has also been shown to function as an intracellular renin receptor that stimulates fibrogenesis. Here we show that mutants and morphants involving other V-H+-ATPase subunits also demonstrated developmental biliary defects, but did not demonstrate the inhibition of fibrogenic genes observed in pn. The defects in pn are reminiscent of those we and others have observed in class C VPS (vacuolar protein sorting) family mutants and morphants, and we report here that knockdown of atp6ap2 and vps33b had an additive negative effect on biliary development. Our findings suggest that pathways important in modulating intracompartmental pH lead to defects in digestive organ development, and support previous studies demonstrating the importance of intracellular sorting pathways in biliary development.

(Pro)renin receptor and V-ATPase: from Drosophila to humans

“…The presence of skin and eye hypopigmentation in pn provided further evidence as this phenotype was previously associated with inhibition of the V-ATPase [21,22,24,25]. In the skin, melanoblast markers decreased with time which suggests that melanocytes developed normally but underwent degeneration [23]. In the eye, striking degradation of various cell layers was also observed.…”

“…These results suggested that (P)RR is important for embryonic development probably linked to V-ATPase activity but no direct evidence was provided by this animal model for years. Recently, Eauclaire et al performed a chemical mutagenesis screen to identify proteins important for biliary development [23]. Among all mutants with abnormalities in hepatic development, one mutant called pekin (pn) was particularly studied.…”

Interferon-Gamma Directly Mediates Developmental Biliary Defects

“…The in situ hybridization studies were performed essentially as described previously 13 , in accordance to standard protocols. PCR primers to generate riboprobes for ifng1-1 and ifng1-2 are shown in Supplementary Table S1.…”

“…Expression QPCR studies were essentially performed as per previous studies, with primers (vhnf1, hprt) also used in previous studies. 13 For the methylation-specific QPCR (MSP), in silico analysis of sequence surrounding the transcription start site (-2.5 to + 0.5 kb) was performed using MethPrimer (http:// www.urogene.org/methprimer/), which identified putative CpG islands and methylated and unmethylated primers. MSP primers are also shown in Supplementary Table S1.…”

“…19 Although this family of proteins has a wide variety of functions, GTPases play an important role in intracellular trafficking and the establishment of cell polarity-processes clearly important in biliary development and disease pathogenesis. 6,9,13,27 IFNc in biliary atresia…”