All animal experiments were performed in accordance with Stockholm’s Norra Djurförsöksetiska nämnd (Stockholm animal research ethics board, ethics approval numbers: N150/14, N61/16, N5253/19, N2987/20) regulations. Animals were maintained with standard day/night cycles, provided with food and water ad libitum, and were housed in cages with enrichment. For postnatal day 15 (P15) experiments, 10 wild type (Jag1+/+) (eight males and two females) and 10 Jagged1 Nodder (Jag1Ndr/Ndr) littermate pups (five males and five females) were used for serum analysis. Within this group, nine Jag1+/+ and seven Jag1Ndr/Ndr mice were injected with resin. All 16 animals were analyzed in 3D, revealing extensive heterogeneity that would necessitate performing DUCT on a large number of animals to obtain significant quantitative data, while the bile duct paucity was obvious. 1 Jag1+/+ and 1 Jag1Ndr/Ndr pair was therefore scanned and rendered in 3D. From this group, four Jag1+/+ and four Jag1Ndr/Ndr left medial lobes were used for 2D liver sections and staining.

Adult animals were between 4.5 and 6.5 months old. In total, 18 Jag1+/+ and 6 Jag1Ndr/Ndr animals were injected with resin for µCT. Quality control of injections (Figure 1—figure supplement 2) was performed on all livers during method development until surgery and injection technique resulted in well-injected livers. Three Jag1+/+ and three Jag1Ndr/Ndr animals were used for the DUCT quantifications in adulthood. For liver histology, two Jag1+/+ and three Jag1Ndr/Ndr mice were used. For ink injections, nine Jag1+/+ mice were used (four males and five females) and for iDISCO+ four Jag1+/+ and four Jag1Ndr/Ndr mice were used (six males and two females). For lung 3D resin casting five Jag1+/+ mice were used (two males and three females). Samples were not blinded for investigation since the phenotype is overt and the genotype is therefore obvious to the experimenter. The animals were maintained on a mixed C57bl6J/C3HeN background. Jag1Ndr/+ (Nodder) mice were bred and genotyped as previously described (Andersson et al., 2018).