Mouse experimental model and details of other animal experiments
All animal procedures in this study followed experimental protocols approved by the respective institutions.
Mouse procedures in this study followed experimental protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Francisco (UCSF). The Nkx2-1-Cre;Ai14 mouse line is the result of crossing the C57BL/6J-Tg(Nkx2-1-cre)2Sand/J (Nkx2-1-Cre; Jackson Laboratory stock no. 008661) and B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (Ai14; Jackson Laboratory stock no. 007914) strains. Mouse housing and husbandry were performed in accordance with the standards of the Laboratory Animal Resource Center (LARC) at UCSF. Mice were group housed in a 12-h light–dark cycle, with access to food and water ad libitum. Nkx2-1Cre;Ai14 mice were crossed, and the date of a positive vaginal plug was considered as E0. Pregnant dams were euthanized, and the brains of embryos from their litters were extracted and assessed for tdTomato fluorescence at the following developmental stages: E15 (n = 16 embryos from 2 litters), E17 (n = 7 embryos from one litter) and E18 (n = 5 embryos from 2 litters).
Rat procedures in this study followed experimental protocols approved by the IACUC at UCSF. Rat housing and husbandry were performed in accordance with the standards of the LARC at UCSF. Rats were group housed in a 12-h light–dark cycle, with access to food and water ad libitum. Long Evans outbred rats were purchased from Charles River (strain code 006). After rat crossing, the day of positive vaginal plug was considered E0. Pregnant dams were euthanized, the brains of embryos from their litters were extracted at E18 and brains from two litters were pooled for sequencing.
Pig samples (E62, E73 and one year and two months of age) were collected at the Swine Teaching and Research Center at the University of California, Davis. Pig procedures were performed in accordance with the standards of the Animal Welfare Act and under conditions approved by the Association of Assessment and Accreditation of Laboratory Animal Care (AAALAC), and followed experimental protocols approved by the IACUC at the University of California, Davis.
Opossum samples (n = 2, P20) come from a pedigreed, breeding colony of grey short-tailed opossums (Monodelphis domestica) that was established by founder individuals purchased from the Southwest Foundation for Biomedical Research, and is maintained by the laboratory of K.E.S. at the University of California, Los Angeles (UCLA). Opossums were euthanized by CO 2 inhalation, followed by decapitation. These procedures are in accordance with the AVMA Guidelines for the Euthanasia of Animals 2013, and all animal procedures were approved by the IACUC at UCLA.
Sugar glider (n = 2, P40) (Petaurus breviceps) experiments were performed with the approval of the IACUC committee at Princeton University. Captive-born, adult sugar gliders were purchased through the US pet trade and subsequently housed in a breeding colony at Princeton University. Sugar gliders were fed a diet of dried food, fruits and protein daily, and housed in breeding pairs or trios. The colony was kept under a 12-h light–dark cycle (temperature, 20–27 °C; humidity, 30–70%). Adult female sugar gliders were checked for pouch young by manual palpation of the maternal pouch and visual inspection. Pouch young identified during inspection were collected by briefly anaesthetizing the mother with isoflurane and gently everting the pouch to expose the neonate. Joeys were gently detached from the nipple, euthanized and processed in the laboratory. More details about the sugar glider colony and husbandry can be found a previous study35.
Ferret samples (P1, P14, P22 and 33 months of age) were a gift from the University of Iowa National Ferret Resource and Research Center and were collected under experimental protocols approved by the IACUC at the University of Iowa. New Zealand white rabbit embryos (n = 3, PCD23) were obtained from BioIVT (USDA NY-TEC-0004) and procedures were performed in accordance with the standards of the Animal Welfare Act and under the AAALAC.
Harbour porpoise (Phocoena phocoena) brain samples were collected from stranded individuals by the Marine Mammal Center and obtained under the National Marine Fisheries Service (NMFS) permit no. 23922.
Dissections and dissociation
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