Fig. 6

cdk5r1a (p35) mutant larvae show intrahepatic biliary network defects, which are rescued by LimKi treatment. (A) The TALEN-based cdk5r1a knockout strategy. Sequence-specific TALENs were designed against the first exon of the cdk5r1a gene, which includes the entire ORF. We established the lri58 allele in which 11 bp were deleted from the cdk5r1a gene, truncating the Cdk5r1a protein at the fourteenth amino acid. (B-D) Skeletal representations of the intrahepatic biliary network in DMSO-treated wild-type (B) and in DMSO-treated (C) or LimKi-treated (D) cdk5r1a^lri58 mutant larvae computed based on Tg(Tp1-MmHbb:EGFP)^um14 expression in the liver at 5 dpf. (E-G) Projected colocalization signal images computed based on Tg(Tp1-MmHbb:EGFP)^um14 expression and phalloidin to visualize the actin network in biliary epithelial cells of larvae treated as in B-D. (H) Mean segment density in DMSO-treated or LimKi-treated wild-type or cdk5r1a^lri58 mutant larvae at 5 dpf. (I) Number of connected branches per biliary epithelial cell in the liver of DMSO-treated or LimKi-treated wild-type or cdk5r1a^lri58 mutant larvae at 5 dpf. n=5 for all experiments. Error bars are s.d. *P<0.05, **P<0.01, ***P<0.001; n.s., not significant. (J) Percentage of larvae showing high, low and no PED6 fluorescence in the gallbladder of DMSO-treated or LimKi-treated wild-type or cdk5r1a^lri58 mutant larvae at 5 dpf. (K) Model of the Cdk5-mediated kinase cascade that regulates branching morphogenesis of the intrahepatic biliary network. Cdk5r1a appears to be responsible for Cdk5 activation in biliary epithelial cells. Cdk5 suppresses Pak1 activity, and thus suppressing Cdk5 by olomoucine enhances Pak1 activity. Suppressing Pak1 activity by IPA-3 generated a biliary network phenotype opposite to that of cdk5r1a mutant and olomoucine-treated larvae. Pak1 appears to regulate LimKs, and thus suppressing LimK activity with LimKi generated a similar biliary network phenotype to that of larvae in which Pak1 activity is suppressed. LimKs suppress Cofilin activity by direct phosphorylation. Suppressing Cofilin by cucurbitacin E thus generated a biliary phenotype opposite to that of larvae in which Pak1 or LimK activity was suppressed.