Inhibition of HH signaling rescues liver morphology in DNA hypomethylated larvae. Cytokeratin-18 (green) labels biliary ducts. DNA methyltransferase (dnmt1) morphants show complete abrogation of biliary structure (B). Incubation of larvae in cyclopamine protects against liver degeneration (C). Numbers represent total livers with normal structure. A′-C′ diagram ductular structure of panels A-C, respectively.

Biliary defects in zebrafish larvae injected with PDGF-AA peptide. (A) Larvae were injected with PDGF-AA, PDGF-AB, or PDGF-BB peptide at 2 dpf. PED-6 gallbladder uptake, showing the proportion of larvae at 5 dpf with normal, faint, and absent gallbladders (GB). Note the effect of PDGF-AA on biliary function, as depicted by the increase in faint and absent gallbladders (**pd .0001), while the effect of PDGF-AB (p≤.04) and PDGF-BB (p≤.02) is milder. (B) Confocal projections of cytokeratin immunostaining of livers from 5 dpf control (cont) and PDGF-AA (PDGF)-injected larvae. The bile ducts are fewer and less complex in the PDGF-injected liver (as quantified in Table 3).

Activation of the HH pathway upregulates PDGF. Larvae were incubated in 30µM purmorphamine, a HH receptor agonist, at 2dpf. (A) PED-6 gallbladder uptake shows decreased gallbladder staining in incubated larvae (p≤.00001) (B) PDGF-AA protein expression of purmorphamine-incubated larvae relative to the control (ctrl) at 5dpf. (B′) PDGF-AA protein quantification of two independent experiments with standard deviations. (C,D) Confocal projections of cytokeratin-18 immunostaining of livers from 5dpf control (ctrl) and purmorphamine (pur) incubated larvae. Numbers refer to individuals exhibiting comparable morphology as representative image.