DEV02140 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Injection of vps33b morpholino affects the later stages of biliary development. Confocal projections through the livers of control (wild type) (A-C) and vps33b MO-injected larvae at 3 dpf (A,D), 4 dpf (B,E) and 5 dpf (C,F), showing cytokeratin staining of intrahepatic bile ducts. The progression to longer ducts and a more complex network occurs over the 2 days, with the most dramatic change between 4 and 5 dpf. The pattern of staining in control and MO-injected livers is indistinguishable at 3 and 4 dpf, while at 5 dpf there is a lack of interconnecting ducts in the vps33b-injected larva.

• Supplemental Figure 2 -

  Fig. S2. Rescue of vps33b morphant phenotype by vps33b mRNA. Confocal projections of whole-mount livers processed for cytokeratin immunohistochemistry from 5 dpf control (A), vps33b MO-injected alone (B) and vps33b MO co-injected with 75 pg zebrafish vps33b mRNA derived from pCS2(+)-vps33b (C). The bile duct patterns in A,B are consistent with Fig. 3, and the pattern of C more closely resembles the wild-type control pattern of ducts.

• Supplemental Figure 3 -

  Fig. S3. hnf6 and vps33b coregulate zebrafish intrahepatic biliary development. (A-D) Confocal projections of whole-mount livers processed for cytokeratin immunohistochemistry. (A) Bile ducts from a 5 dpf larva injected with vehicle (wt). (B,C) Bile ducts from 5 dpf larvae injected with 150 fg (0.1X) hnf-6 (B) or vps33b (C) morpholino. (D) There is pronounced bile duct paucity in this 5 dpf larva that was injected with low-doses of hnf6 and vps33b morpholinos. There is only a mild degree of paucity of B,C compared with A, especially relative to D.

• Supplemental Figure 4 -

  Fig. S4. Mouse Vps33b gene expression is regulated by hnf6 in P3 mouse liver. Real-time quantitative PCR of cDNA derived from two wild-type livers and two hnf6^–/– livers, from E12 to P3, shows decreased Vps33b gene expression only in P3 hnf6^–/– liver. Total RNA was extracted from mouse embryonic liver cells and served as template for cDNA synthesis using random hexamers. Real-time PCR was then performed with qPCR MasterMix Plus SYBR Green 1 No Rox (RT-SN2X-03+NR) (Eurogentec, Liége, Belgium) on a MyiQ cycler (Bio-Rad, Eke, Belgium). Vps33b mRNA was normalized to b-actin mRNA. Sequence of PCR primers: vps33bS , 5¢- CATGTGCCTTTTGTCCATCA-3¢; vps33bAS, 5¢- CGCAGGTTGGAGAAGGTTAG-3¢.