Additional Supporting Information may be found in the online version of this article.

DVDY_22220_SuppFig-S1.tif998KSupp. Fig. 1. Development of the intrahepatic and extrahepatic biliary system: Confocal projections through the liver of embryos and larvae immunostained with the 2F11 antibody. These images outline liver morphogenesis and development of the gallbladder (g) and extrahepatic ducts (ed) in relation to the intrahepatic ductal network. The 2F11 epitope is also detected in secretory cells on the intestinal epithelium (i), the ductal network of the pancreas (p).
DVDY_22220_SuppFig-S2.tif1831KSupp. Fig. 2. Development of hepatocyte canaliculi and intrahepatic biliary network. A, B: Confocal projections through the liver of an 80-hpf (A) and 96-hpf (B) larva immunostained with Keratin-18 (green) and Mdr (red) antibodies. Compared with double immunostains of 5-dpf larvae (Fig. 2F″), there is much less overlap of the Mdr-1 and Keratin-18 epitopes at these developmental stages (open arrowheads). C–F: Transmission electron micrographs showing the canalicular-terminal duct junction (arrows) in 71-hpf (C), 96-hpf (D), and 120-hpf (E, F) larvae. bd, bile duct; c, canaliculus; arrowhead, tight junctions. Bar = 500 nm in C–E, 100 nm in F
DVDY_22220_SuppFig-S3.tif382KSupp. Fig. 3. Notch inhibition reduces biliary secretion. A–D: Fluorescence microscopy images (lateral view) of live 6-dpf larvae (144 hpf) following ingestion of the fluorescent lipid bodipy-C16. In wild types (wt), strong fluorescence is detected in the intestine (i) and gallbladder (g). Fluorescence emission from both tissues is reduced in larvae treated with DAPT between 50–120 hpf. Each panel shows a unique larva. These data are representative of 10 wild type and 10 DAPT-treated larvae.
DVDY_22220_SuppFig-S4.tif1603KSupp. Fig. 4. Notch signaling is required for expansion of the intrahepatic biliary ductal network. A–C: Confocal projections through the liver of wild type larvae (70, 92, and 120 hpf) and larvae treated with DAPT from 48–70 hpf (E), 48–92 hpf (F), 31–120 hpf (D), and 48–120 hpf (H), fixed immediately after the treatment, followed by 2F11 immunostainings. A larva treated with DAPT from 48–70 hpf and then fixed at 92 hpf after DAPT withdrawal at 70 hpf is also shown (G). DAPT treatment arrests intrahepatic biliary development (compare A with E; B with F; C with D and H). There is expansion of the ductal network after DAPT withdrawal (compare G with F).
DVDY_22220_SuppFig-S5.tif3218KSupp. Fig. 5. Notch reporter expression in developing intrahepatic biliary cells: Whole mount confocal images (63×) through the liver of developing Notch reporter larvae stained with the 2F11 antibody (A–D) and a GFP antibody (A′–D′) with overlap of the two markers (A″–D″). GFP-positive biliary epithelia are first detected at 45 hpf (A′). At this stage, scattered 2F11-positive cells are present in the liver with only partial overlap with GPF. From 50–70 hpf, there is progressive increase in the number of GFP-positive cells. AT 50 hpf, there is significant overlap between the GFP and 2F11 epitope in the biliary cells. There is nearly complete overlap between these markers at 60 and 70 hpf. The gallbladder (g) and extrahepatic duct (ed) remain GPF negative.
DVDY_22220_SuppMovie-S1.mov5699KSupplementary Movie 1
DVDY_22220_SuppMovie-S2.mov5949KSupplementary Movie 2
DVDY_22220_SuppMovie-S3.mov5871KSupplementary Movie 3
DVDY_22220_SuppMovie-S4.mov5520KSupplementary Movie 4

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