Lineage and organ signals sequentially build organ intrinsic nervous systems

Animals 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 previously 51 . The Lox -knockout mice 42 were provided by the laboratory of C. M. Halabi. The Itga1 -knockout mice 38 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 CUBIC 52 method and stained as follows. …