Biliary atresia, the most common indication for pediatric liver transplantation, is a fibrotic disease of unknown etiology affecting the extrahepatic bile ducts of newborns. The recently-described toxin biliatresone causes lumen obstruction in mouse cholangiocyte spheroids and represents a new model of biliary atresia. Our aim was to determine the cellular changes caused by biliatresone in mammalian cells that ultimately lead to biliary atresia and extra-hepatic fibrosis. We treated mouse cholangiocytes in 3D spheroid culture and neonatal extra-hepatic duct explants with biliatresone and compounds that regulate glutathione. We examined the effects of biliatresone on SOX17 levels, and determined the effects of Sox17 knockdown on cholangiocytes in 3D culture. We found that biliatresone caused disruption of cholangiocyte apical polarity and loss of monolayer integrity. Spheroids treated with biliatresone had increased permeability as shown by rhodamine efflux within 5 hours compared to untreated spheroids, which retained rhodamine for longer than 12 hours. Neonatal bile duct explants treated with the toxin showed lumen obstruction with increased subepithelial staining for α-smooth muscle actin and collagen, consistent with fibrosis. Biliatresone caused a rapid and transient decrease in glutathione, which was both necessary and sufficient to mediate its effects in cholangiocyte spheroid and bile duct explant systems. It also caused a significant decrease in in cholangiocyte levels of SOX17, and Sox17 knockdown in cholangiocyte spheroids mimicked the effects of biliatresone. Conclusion Biliatresone decreases glutathione and SOX17 in mouse cholangiocytes. In 3D cell systems, this leads to cholangiocyte monolayer damage and increased permeability and in extrahepatic bile duct explants it leads to disruption of the extra-hepatic biliary tree and subepithelial fibrosis. This mechanism may be important in understanding human biliary atresia.

Opacification of the ocular lens, termed cataract, is a common cause of blindness. To become transparent, lens fiber cells undergo degradation of their organelles, including their nuclei, presenting a fundamental question: does signaling/transcription sufficiently explain differentiation of cells progressing toward compromised transcriptional potential? We report that a conserved RNA-binding protein Celf1 post-transcriptionally controls key genes to regulate lens fiber cell differentiation. Celf1-targeted knockout mice and celf1-knockdown zebrafish and Xenopus morphants have severe eye defects/cataract. Celf1 spatiotemporally down-regulates the cyclin-dependent kinase (Cdk) inhibitor p27Kip1 by interacting with its 5’ UTR and mediating translation inhibition. Celf1 deficiency causes ectopic up-regulation of p21Cip1. Further, Celf1 directly binds to the mRNA of the nuclease Dnase2b to maintain its high levels. Together these events are necessary for Cdk1-mediated lamin A/C phosphorylation to initiate nuclear envelope breakdown and DNA degradation in fiber cells. Moreover, Celf1 controls alternative splicing of the membrane-organization factor beta-spectrin and regulates F-actin-crosslinking factor Actn2 mRNA levels, thereby controlling fiber cell morphology. Thus, we illustrate new Celf1-regulated molecular mechanisms in lens development, suggesting that post-transcriptional regulatory RNA-binding proteins have evolved conserved functions to control vertebrate oculogenesis.

Although majority of the genes linked to early-onset cataract exhibit lens fiber cell-enriched expression, our understanding of gene regulation in these cells is limited to function of just eight transcription factors and largely in the context of crystallins. We report on small Maf transcription factors Mafg and Mafk as regulators of several non-crystallin human cataract-associated genes in fiber cells and establish their significance to this disease. We applied a bioinformatics tool for cataract gene discovery iSyTE to identify Mafg and its co-regulators in the lens, and generated various null-allelic combinations of Mafg:Mafk mouse mutants for phenotypic and molecular analysis. By age 4-months, Mafg−/−:Mafk+/− mutants exhibit lens defects that progressively develop into cataract. High-resolution phenotypic characterization of Mafg−/−:Mafk+/− mouse lens reveals severely disorganized fiber cells, while microarrays-based expression profiling identifies 97 differentially regulated genes (DRGs). Integrative analysis of Mafg−/−:Mafk+/− lens-DRGs with 1) binding-motifs and genomic targets of small Mafs and their regulatory partners, 2) iSyTE lens-expression data, and 3) interactions between DRGs in the String database, unravels a detailed small Maf regulatory network in the lens, several nodes of which are linked to cataract. This approach identifies 36 high-priority candidates from the original 97 DRGs. Significantly, 8/36 (22%) DRGs are associated with cataracts in human (GSTO1, MGST1, SC4MOL, UCHL1) or mouse (Aldh3a1, Crygf, Hspb1, Pcbd1), suggesting a multifactorial etiology that includes oxidative stress and mis-regulation of sterol synthesis. These data identify Mafg and Mafk as new cataract-associated candidates and define their function in regulating largely non-crystallin genes linked to human cataract.

Background It was recently demonstrated that deficiency of a conserved RNA binding protein (RBP) and RNA granule (RG) component Tdrd7 causes ocular defects including cataracts in human, mouse and chicken, indicating the importance of post-transcriptional regulation in eye development. Here we investigated the function of a second conserved RBP/RG component Caprin2 that is identified by the eye gene discovery tool iSyTE. Results In situ hybridization, western blotting and immunostaining confirmed highly enriched expression of Caprin2 mRNA and protein in mouse embryonic and postnatal lens. To gain insight into its function, lens-specific Caprin2 conditional knockout (cKO) mouse mutants were generated using a lens-Cre deleter line Pax6GFPCre. Phenotypic analysis of Caprin2cKO/cKO mutants revealed distinct eye defects at variable penetrance. Wheat germ agglutinin staining and scanning electron microscopy demonstrated that Caprin2cKO/cKO mutants have an abnormally compact lens nucleus, which is the core of the lens comprised of centrally located terminally differentiated fiber cells. Additionally, Caprin2cKO/cKO mutants also exhibited at 8% penetrance a developmental defect that resembles a human condition called Peters anomaly, wherein the lens and the cornea remain attached by a persistent stalk. Conclusions These data suggest that a conserved RBP Caprin2 functions in distinct morphological events in mammalian eye development.