GoKawiilAnimals
All animal husbandry and procedures were performed in compliance with Yale University’s Institutional Animal Care and Use Committee and National Institute of Health (NIH) guidelines. Both male and female mice were used for the experiments and no sex-specific differences were observed.
Mouse lines
Wild-type C57BL/6J (000664), Wnt1-cre (022501), Sox10-cre (025807), Baf53b-cre (027826), Phox2b-Flpo (022407), lox-tdTomato (007914), frt-tdTomato (032864), lox-Sun1-sfGFP (021039) and Ascl1-creERT2 (012882) were obtained from the Jackson Laboratory. lox-L10GFP mice were described previously51. The Lox-knockout mice42 were provided by the laboratory of C. M. Halabi. The Itga1-knockout mice38 were provided by the laboratory of A. Pozzi, and were backcrossed onto the C57BL/6J background for nine generations.
Whole-embryo or organ clearing and immunofluorescence staining
Embryos and postnatal mice were euthanized and visceral organs were dissected. Mice older than 10 days were transcardially perfused with 20 ml cold 4% paraformaldehyde (PFA) containing 10 U ml−1 of heparin (Sigma-Aldrich), followed by 10 ml cold PBS (pH 7.4) before tissue dissection. Whole embryos or dissected organs were fixed in 4% PFA 4 °C overnight and kept in cold PBS at 4 °C before clearing. Tissues were cleared with the CUBIC52 method and stained as follows. Dissected tissues were immersed in half-diluted reagent-1 (R1; 25 wt% urea, 25 wt% Quadrol, 15 wt% Triton X-100) at 37 °C with shaking for 3–6 h, then in undiluted R1 at 37 °C until optically cleared. R1 was replaced every 2 days. Cleared tissues were washed (PBS/0.01% NaN 3 ), blocked (5% normal donkey serum, 0.1% Triton X-100, PBS/0.01% NaN 3 ) and incubated with primary antibodies (1:500 in blocking buffer) at room temperature with shaking for 1–2 days. Samples were washed (0.1% Triton X-100, PBS/0.01% NaN 3 ) and incubated with fluorophore-conjugated secondary antibodies diluted in blocking buffer at room temperature with shaking for 1–2 days. After staining, samples were washed overnight in 0.1% Triton X-100, PBS/0.01% NaN 3 . The samples were immersed in half-PBS-diluted reagent-2 (R2; 25 wt% urea, 50 wt% sucrose, 10 wt% triethanolamine) at room temperature overnight for a second clearing, and then in undiluted R2 at room temperature for 2–7 days until optically clear. For whole-embryo and embryonic heart imaging, samples were embedded in 5% low-melting-point agarose in PBS before the second clearing step. Cleared samples were immersed in mineral oil (Sigma-Aldrich) for at least 1 h and imaged on a Leica SP8 confocal microscope with a 16× immersion objective (HC FLUOTAR L 16×/0.8 IMM motCORR VISIR, working distance: 8 mm). Embryonic lungs, pancreas, and intestines and E11.5 embryos were immersed in R2, placed on a glass slide with 1.75 mm concavity and imaged on a Leica SP8 confocal microscope using a 10× objective (HC PL APO 10×/0.40 CS2, working distance: 2.1 mm) or a 40× objective (HC PL FLUOTAR L 40×/0.60 CORR, working distance: 3.3 mm). Postnatal samples were immersed in R2, flattened to approximately 500 μm in a custom-built imaging chamber and imaged using the Leica SP8 confocal microscope with a 10× or 40× objective. A full list of antibodies is provided in Supplementary Tables 1 and 2. All primary antibodies were used at a dilution of 1:500. All secondary antibodies were used at a dilution of 1:1,000.
Image analysis of OINS spatial distribution
Cell segmentation
Individual cells labelled with PHOX2B antibody were manually segmented in Fiji (ImageJ; v2.14.0/1.54f; NIH) and their (x, y, z) coordinates were exported for further analysis.
Distribution variability
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