Jag1b and Jag2b are required for all canonical Notch signaling in the developing liver and exocrine pancreas. a–d 3D rendering of the foregut endoderm of 72 hpf embryos in the canonical Notch signaling transgenic reporter tg(Tp1:GFP) (green) background, stained with hepatopancreatic endoderm marker Prox1 antibodies (red) and nuclear marker DAPI (blue). Ventral view, anterior up. Tp1:GFP expression in liver and pancreas of jag1b ^−/− mutants (b, representative sample, n > 15) is comparable to that in wild type (a), but is decreased in jag2b ^−/− mutants (c, representative of 14/18 samples). jag1b ^−/− ;jag2b ^−/− double mutants (d, representative sample, n = 15) show a complete absence of Tp1:GFP expression in Prox1⁺ cells. e Bright-field microscopy of 4 dpf wild type (top) and jag1b ^−/− ;jag2b ^−/− mutant (bottom), showing mutant with grossly normal development with the exception of heart and pronephric edema. Samples from three different jag1b ^−/+ ;jag2b ^−/+ in-cross clutches. Scale bars 50 μM (a–d), and 500 μM e

Jag1b and Jag2b are required for intrahepatopancreatic duct lineage specification. a–h Whole organ immunofluorescence expression analysis of the 72 hpf foregut endoderm of wild type and jag1b ^−/− ;jag2b ^−/− mutant embryos. (a, b Alcam channel removed in bottom panels). Intrahepatic duct cells marked by high Alcama levels (a, green), and by Prox1 (red) but not Hnf4a expression (blue, a), are found in wild type liver (arrowheads, a). These duct cells are not found in the jag1b ^−/− ;jag2b ^−/− mutant liver (b, representative sample, n = 13), which is comprised entirely of hepatocytes expressing both Prox1 and Hnf4a. c–f Intrapancreatic ductal cells expressing Nkx6.1 (red, c, d) or Pdx1 (red, arrow, e, f) are present in wild type but not in jag1b ^−/− ;jag2b ^−/− mutants. Pdx1 expression in Islet1⁺ cells (blue, arrowheads) and the duodenum (asterisk) is comparable to wild-type siblings, as is expression of ptf1a:GFP⁺ (green) acinar cells c–f and Anxa4⁺ (red) extrahepatopancreatic duct cells (red, g, h). g, h Anxa4⁺ (red) extrahepatopancreatic ducts can be found joining the ptf1a:GFP⁺ (green) pancreatic acinar cells to the fabp10a:DsRed⁺ hepatocytes (blue). Representative samples from three different jag1b ^−/+ ;jag2b ^−/+ in-cross clutches. Scale bars, 50 μM

Distinct early and late roles of Notch signaling in regulating duct and endocrine lineages. a, b Pancreas of 60 hpf jag1b ^−/− ;jag2b ^−/− and wt sibling embryos. Neogenic endocrine cells expressing Islet1 (red) are normally found outside the principal islet at the base of the wild type pancreas (arrows, a). These neogenic endocrine cells are not present in the 60hpf jag1b ^−/− ;jag2b ^−/− mutant pancreas (b, representative sample, n = 18, pooled from three different jag1b ^−/+ ;jag2b ^−/+ in-cross clutches), which is entirely ptf1a:GFP⁺ (green) with the exception of the principal islet. c, d Pancreas of 5 dpf-treated embryos. Early inhibition of Notch signaling with gamma secretase inhibitor LY575411 treatment (d, n = 9 and c, n = 5. Samples from 2 different experiments) at 30–72 hpf leads to loss of Nkx6.1⁺ pancreatic duct (red) without excess neuroD:GFP⁺ (green) endocrine cells. e, f In contrast, late LY575411 treatment (f, n = 9, and n = 6 for control, e Samples pooled from 2 different experiments) also leads to loss of nkx6.1⁺ pancreatic duct (red) that is coupled with excess neuroD:GFP⁺ (green) endocrine cells (arrowheads, f). Scale bar, 50 μM

Intrahepatopancreatic ducts do not contribute significantly to the developing acinar and hepatocyte lineages. a–d Immunofluorescence analysis of the pancreas (a, b) and liver (c, d) in 5 dpf wild type (a, c) and jag1b ^−/− ;jag2b ^−/− mutant embryos (b, d, bottom panels with lfabp10:DsRed channel removed). Despite the absence of ductal cells in jag1b ^−/− ;jag2b ^−/− mutants b, d, lumen formation (arrows) and apical localization of ZO-1 (red in b, green in d) remain comparable to wild type siblings (a, c). Normal differentiation indicated by zymogen granule accumulation in the ptf1a:GFP⁺ pancreatic acinar cells (green) and lfabp-DsRed expression (red) in hepatocytes. e Schematic of approach to Notch activity lineage tracing. f–i Notch active labeled H2B-mCherry⁺ cells (red) in the liver are all Prox1⁺ (blue) and Hnf4a⁻ (green) (n = 12 embryos, 1,416 labeled cells counted from two different experiments (f), P < 0.0001). g, h In the pancreas, nearly all H2B-mCherry⁺ cells (red) co-express Nkx6.1 (blue) and are ptf1a:GFP⁻ (green) (n = 24 embryos, 1,586 labeled cells counted from 3 different experiments, P < 0.0001) g Only a few H2B-mCherry⁺ cells that lack Nkx6.1 but are ptf1a:GFP⁺ were observed (arrows, h. n = 4/24 embryos, 12 cells total). i Graph summarizing numbers of Notch active labeled H2B-mCherry⁺ cells found in hepatocytes versus intrahepatic duct (IHD) cells and acinar pancreas versus intrapancreatic duct (IPD) cells. Representative samples; a–d, n = 4 each. Scale bars 25 μM

Endoderm expression of Jagged induces Notch activity and lineage specification. a Schematic of transplant approach to generate endoderm chimeras. At the one-cell stage, wild type Tp1:GFP donor embryos were injected with rhodamine dextran for lineage tracing and sox32 mRNA for conversion to endoderm. At late blastula stage, donor cells were transplanted into Tp1:GFP host embryos that had been injected with antisense MO’s to knockdown Jag1a and Jag1b. (a, bottom panel) Example of endoderm chimeric embryo demonstrating rhodamine dextran labeled donor cells (red) localized primarily to the sox17:GFP⁺ (green) host gut endoderm region. b, c Immunofluorescence analysis of chimeric host pancreas (b) and liver (c) generated by the approach described in a demonstrating a rescue of Notch signaling in Jag1b/Jag2b compromised embryos by endoderm cells with wild type expression of Jag1b/Jag2b. Tp1:GFP⁺ cells (green) are only observed in regions of these organs with wild type donor cells (red) but not in regions without donor cells (yellow asterisks), (d, e) (representative samples, n = 10 embryos. Samples pooled from 2 experiments). Rescued Tp1:GFP⁺ cells found without rhodamine dextran (red, white arrows and arrowhead), are always directly associated with rhodamine dextran + cells. All rescued Tp1:GFP + host cells are Nkx6.1⁺ (blue) in the pancreas (d) and Alcama⁺ (blue) in the liver (e), indicating that they are of the duct lineage. Scale bars 250 μM a, and 25 μM (b–e)

Supplementary Figure 1. jag1b and jag2b expression in the developing pancreas and liver.

Supplementary Figure 2. Canonical Notch activity in liver and pancreas. (a-c)

Supplementary Figure 3. Jag1b and Jag2b are not required for all Notch active endocrine or endothelial cells associated with pancreas. (a-b, Three dimensional renderings, single plane in panels to right) Pancreas of wild type (a, representative sample n=9) and jag1b^-/-;jag2b^-/- mutant (b, representative sample n=4) 72 hpf embryos showing loss of Tp1:GFP from all Prox1+ (blue) cells in mutants. Tp1:GFP expression can still be found in endothelial cells (white arrowheads) and Islet1+ endocrine cells (yellow arrowheads) Scale bar 25μM

Supplementary Figure 4. High magnification of jag1b and jag2b double mutant livers. (a-b) Wild type (a) and jag1b^-/-;jag2b^-/- mutant (b) livers labelled for Prox1 (red), Hnf4a (blue), and Tp1:GFP (green) reveals some double mutant embryos retain 1-2 intrahepatic duct cells (arrow, b, representative sample, 12/17 double mutants) (c, d) Enlargement from Figure 2a-b of wildtype (c) and jag1b^-/-;jag2b^-/- livers (d) labeled for Prox1 (red), Hnf4a (blue), and Alcama (green). Low-level Alcama expression that persists in mutants is otherwise associated with cells expressing both Prox1 and Hnf4a. Scale bars, 25μM

Supplementary figure 5. Apoptosis does not contribute to the lack of intrahepatic duct cells in jag1b and jag2b double homozygous mutants. (a-d) Whole mount immunofluorescent images of 54 hpf sibling (a,b) and double mutant (c,d) embryos labeled for cleaved Caspase3 (*Casp3) expression. Sibling (a) and double mutant (c) livers labeled for Hnf4a (blue) show no signs of apoptosis based on a lack of cleaved Caspase3 (red) expression or blebs of Tp1:GFP+ (green), whereas the ventral forebrains (b,d, with DAPI channel also shown) of the same embryos (from a,c) show normal apoptosis patterns as indicated by variable presence of cleaved Caspase3 (red). Scale bar 50μM. Representative samples, n= 4 siblings, 4 jag1b^-/-;jag2b^-/-

Supplementary figure 6. Specification of intrahepatic duct lineage in the absence of the vasculature. (a-b) Whole organ immunofluorescent analysis of 76 hpf wild type (a) and npas4l mutant (b) livers labelled for Prox1 (red), Hnf4a (blue), and Alcama (green). Green Alcama channel is removed in panels below a and b. Intrahepatic duct cells (arrows), expressing both Prox1 and high Alcama, but lacking Hnf4a, are present in both wild type and mutant livers. Scale bars 20μM