Collagenase 1

Primitive erythropoiesis was measured by performing colony-forming assays as described previously (Palis et al., 1999 (link)). Briefly, after timed mating of heterozygous Dot1l-MM male and females, E8.5 conceptuses were collected in IMDM. Embryos were used for genomic DNA extraction and genotyping. Yolk sacs were digested with 0.1% collagenase I (StemCell Technologies) in IMDM (Gibco-BRL) at 37°C for 30 min, neutralized with 10% ES grade FBS (Hyclone) and single cell suspensions were prepared by repeated pipetting. Yolk sac cells were washed with serum free IMDM and approximately equal numbers of homozygous Dot1l-MM and wildtype cells were plated in methylcellulose medium (StemCell Technologies) containing 10% ES serum (Hyclone), 5% protein-free hybridoma medium (Gibco-BRL), and cytokines SCF (100 ng/ml), LIF (1000 U/ml), EPO (2 U/mL), VEGF (5 ng/ml), IL-3 (5 ng/ml), IL-6 (5 ng/ml), IL-11 (5 ng/ml), GM-CSF (3 ng/ml), G-CSF (30 ng/ml), and M-CSF (5 ng/ml) (all cytokines were purchased from PeproTech). Colonies were analyzed for identity, size, and number after 7 days of in vitro culture.

Whole bone marrow cells were isolated from 11-week-old C57Bl/6 male mice (Jackson Laboratory). The epiphysis/metaphysis fraction from long bones was collected, crushed, cut into small pieces, and digested using Collagenase I (STEMCELL Technologies) with agitation for 30 min at 37 °C. Bone marrow cells were filtered through a 70-μm filter. Red blood cells were lysed using Ack-lysis (ThermoFisher Scientific) on ice for 5 min, quenched with Media 199 (ThermoFisher Scientific) supplemented with 2% fetal bovine serum (ThermoFisher Scientific), and spun down at 500 g for 5 min. Cells were stained for 30 min with the red blood cell marker TER119 (Biolegend) and cells were sorted using DAPI (ThermoFisher Scientific) as a live/dead viability marker. Live whole bone marrow cells (400,000; negative for TER119) were sorted into medium 199 (ThermoFisher Scientific). Before inDrop encapsulation cells were counted using a Cellometer (Nexcelom Bioscience). Cell viability was over 90%.

To study the architecture of the biliary tree, 5 dpf zebrafish larvae were stained with the 2F11 antibody (Abcam, ab71286). Larvae were fixed with 4% paraformaldehyde overnight and then in 100% ice-cold methanol for long-term storage. Prior to antibody treatment, larvae were rehydrated through decreasing concentrations of methanol in PBS containing 0.2% Triton X-100 (PBSX). The skin was then peeled off the larvae and they were deyolked and treated with collagenase 1 (1 mg/ml, Stem Cell Technologies, 07415) for 15 min at room temperature. Blocking was performed for 1 h with 10% bovine serum albumin with 0.2% Triton X-100. The 2F11 antibody (1:100) was diluted with blocking reagents and incubated overnight at 4°C on a shaker. The next day, larvae were washed with PBSX and incubated with secondary antibody (goat anti-mouse Cy3, 1:500, Jackson ImmunoResearch, 115-166-071) overnight at 4°C on a shaker. Whole larvae were mounted on glass slides with anti-fade reagent (Dako, S3023). Images of immunostained zebrafish larvae were acquired using a Leica SP8 light-sheet confocal microscope. Confocal stack images were processed and analyzed using Imaris software (Bitplane). The length of intrahepatic ducts, liver area and number of BECs were measured using ImageJ software.

To study the architecture of the biliary tree, 5 dpf zebrafish larvae were stained with the 2F11 antibody (Abcam, ab71286). Larvae were fixed with 4% paraformaldehyde overnight and then in 100% ice-cold methanol for long-term storage. Prior to antibody treatment, larvae were rehydrated through decreasing concentrations of methanol in PBS containing 0.2% Triton X-100 (PBSX). The skin was then peeled off the larvae and they were deyolked and treated with collagenase 1 (1 mg/ml, Stem Cell Technologies, 07415) for 15 min at room temperature. Blocking was performed for 1 h with 10% bovine serum albumin with 0.2% Triton X-100. The 2F11 antibody (1:100) was diluted with blocking reagents and incubated overnight at 4°C on a shaker. The next day, larvae were washed with PBSX and incubated with secondary antibody (goat anti-mouse Cy3, 1:500, Jackson ImmunoResearch, 115-166-071) overnight at 4°C on a shaker. Whole larvae were mounted on glass slides with anti-fade reagent (Dako, S3023). Images of immunostained zebrafish larvae were acquired using a Leica SP8 light-sheet confocal microscope. Confocal stack images were processed and analyzed using Imaris software (Bitplane). The length of intrahepatic ducts, liver area and number of BECs were measured using ImageJ software.