1C,D), exhibiting few intact ductular structures Immunostaining

1C,D), exhibiting few intact ductular structures. Immunostaining using the bile duct cell marker 2F1131 showed larger cell bodies and shortened ductular processes (Fig. 1E,F). These findings suggested to us that inhibition of methylation leads to developmental biliary defects. To determine whether reduced methylated DNA could account for

the dtp biliary phenotype, we treated wildtype larvae with azaC, a DNA methylation inhibitor.35 The larvae were injected with azaC at 2 dpf to avoid toxicity during early development. As depicted in Omipalisib Fig. 2, azaC treatment of larvae did not affect liver morphology or overall growth or development, but did lead to reduced gallbladder PED6 uptake (insets). Cytokeratin immunostainings demonstrated that azaC treatment led IWR-1 purchase to a dramatic effect on bile duct development, similar to dtp (Fig. 2). Immunostaining with the bile duct cell marker 2F11 demonstrated fewer cells, consistent with the decrease in ducts but distinct from dtp. Unlike in dtp, in which there is global inhibition of methylation, azaC treatment did not lead to hepatic steatosis or degeneration (data not shown). Inhibition of DNA methylation with azaC in the developing liver from 2-4 dpf most likely affects maintenance of methylation via Dnmt1,36 as biliary cells are highly proliferative at this stage.37 Zebrafish dnmt1 is expressed in a pattern similar

to ahcy, and MO-mediated inhibition of dnmt1 has extensive effects on early development.32 To circumvent the early effects of dnmt1 knockdown, we examined 5 dpf larvae injected with dnmt1 MOs at 2 dpf,33, 38 by which point digestive organ anlagen have already formed.39 As noted with azaC treatment, injection of dnmt1 MOs did not affect the overall appearance of the larva, including liver morphology, but did inhibit gallbladder uptake of PED-6, similar to azaC (Fig. 2, insets). Intrahepatic cytokeratin stainings

in dnmt1-deficient larvae were similar to those seen in dtp and azaC-treated larvae, and 2F11 stainings shared features with both dtp and azaC-treated larvae, with find more fewer cells in clusters and with shorter ductular processes. Treatment with azaC or injection with MOs against dnmt1 resulted in inhibition of DNA methylation quantitatively similar to dtp33 (Supporting Information Fig. 1). Thus, inhibition of DNA methylation disrupts intrahepatic bile duct development in zebrafish, and is likely responsible for the biliary phenotype of dtp. To identify candidate genes with altered expression in azaC-treated larvae, we performed expression microarray analysis on livers dissected from 4 dpf azaC-treated fish compared to control (see Supporting Information Table 3). Many of the up-regulated genes were IFN-γ-stimulated genes and other inflammatory pathway genes (≥17 of the top 100; see Table S3). This was intriguing, as IFN-γ is elevated in patients with BA4 and is critical in the generation of experimental biliary atresia in mice.

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