Tissue sample collection, preservation and processing
Back skin samples from age-matched adults and one litter of neonatal naked mole rats (Heterocephalus glaber) were maintained at the University of Texas Health Science Center at San Antonio for unrelated studies performed under protocols approved by the University of Texas Health Science Center at San Antonio Institutional Animal Care and Use Committee (IACUC; 20210034AR). Rhesus macaques (Macaca mulatta) were maintained at the Oregon National Primate Research Center (ONPRC) at Oregon Health and Science University for unrelated studies performed under protocols approved by Oregon Health and Science University IACUC (IP03716, IP03276, IP00367). The ONPRC is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (Animal Welfare Assurance D16-00195) and registered with the USDA (92-R-001). Rhesus macaque back skin samples were shared through the ONPRC tissue distribution programme. Common marmosets (Callithrix jacchus) were maintained at the Southwest National Primate Research Center at Texas Biomedical Research Institute for unrelated studies performed under an approved animal use protocol (assurance number D16-00048). Back skin was recovered from the above species at necropsy after euthanization for unrelated studies. Skin samples were fixed in 4% paraformaldehyde (PFA) overnight and stored in 70% EtOH until tissue processing for paraffin embedding or fixed in 4% PFA, shipped to Washington State University in 1× phosphate-buffered saline (PBS) on ice, and embedded in OCT (Fisher) before cryopreservation at −80 °C. Biological replicates were compiled from samples collected across different litters unless otherwise specified. Histological analyses of bottlenose dolphin (Tursiops truncatus), long-beaked common dolphin (Delphinus capensis) and short-beaked common dolphin (Delphinus delphis) trunk skin were performed using trunk skin samples obtained by the Plikus laboratory at the University of California, Irvine, from the NOAA (Southwest Fisheries Science Center, La Jolla, California) under the destructive loan permit. The analysed specimens included: D. capensis (numbers KXD0225, KXD0226, 1741-2023 Dc2301B), D. delphis (numbers BLH0012, KXD0357, 585-2022 Dd2202B) and T. truncatus (numbers KXD0410, KZP0069, 812-2022 Tt2202B). North American grizzly bears (Ursus arctos horribilis), a mix of males and females ranging in age from to 8 to 21 years in the summer of 2023, were housed at the Washington State University Bear Research, Education, and Conservation Center. North American grizzly bears were anaesthetized, and biopsies were collected from the dorsal skin of the rump (lower back) for unrelated studies related to subcutaneous fat performed under Washington State University IACUC-approved protocols (6546). Skin samples used in this study were the whole 6-mm diameters of discarded skin from the biopsies, which were used to sample subcutaneous fat. Representative histology in Fig. 2a and Extended Data Fig. 2b is from a 21-year-old male (haematoxylin and eosin; H&E) and female (Herovici), which were both born in the wild. Mice (Mus musculus) used in this study were of a mixed WT C57BL/6 background housed at Washington State University in a 12-h light/dark cycle with food and water ad libitum in a climate-controlled facility set to approximately 68–73 °F and 40% humidity. K14-Cre;Lef1fl/fl (Lef1-eKO) mice were generated by crossing Tg(KRT14-cre)1Amc/J (Jackson Laboratory, 004782)61 with B6.Cg-Lef1tm1Hhx/J (Jackson Laboratory, 030908)62. Housing and sample collection was conducted in accordance with Washington State University IACUC-approved protocols (6723, 6724). Paraffin-embedded 3mo K14-Noggin and C57BL/6 mouse digits analysed in this study were archived from a previous study4 and shared with the Driskell laboratory upon request. K14-CreERT;Bmpr1afl/fl and K14-CreERT mice53,63,64 used in this study were housed at the University of Warsaw. The animal studies were approved by the First Local Ethics Committee: no. 971/2020 as of 28 January 2020, no. 1669/2025 as of 18 March 2025. The studies were conducted in accordance with local legislation and institutional requirements. K14-CreERT;Bmpr1afl/fl and K14-CreERT mice were all treated with tamoxifen (12.5 mg ml−1 in 10% EtOH) topically to the paws from P1 to P5 and then aged to P56. K14-CreERT;Bmpr1afl/fl were labelled TAMX (tamoxifen-induced knockout group), and K14-CreERT mice were labelled Ctrl (control, tamoxifen-induced without gene knockout) in Fig. 5d and Extended Data Fig. 5i. Collected digits were fixed for 24 h in 4% PFA at 4 °C, then in 0.5 M EDTA (pH 7) for 6 days at room temperature (to soften the bone and nail), before being moved to 30% sucrose at 4 °C and frozen in OCT. Gestational human tissue samples ranging from GW7 to GW20 were obtained by the Birth Defects Research Laboratory at University of Washington under University of Washington institutional review board (IRB)-approved protocols with maternal written consent (University of Washington STUDY00000380; received under Washington State University 19680). Fetal tissues were obtained surgically, and although there is a possibility of samples being damaged by this process, we did not observe damage in the samples analysed in this study. Adult human tissue samples were obtained by Advanced Dermatology in Spokane, Washington, as surgical discard tissue after informed consent and in accordance with IRB-approved protocols (Washington State University 19796). All human tissue samples were deidentified before receipt at Washington State University and analysed in accordance with Washington State University IRB-approved protocols, as specified above. As they were surgical discards, samples came from a variety of anatomical sites, including the trunk skin of the torso and the skin of the face or head. Human tissue samples were processed for paraffin embedding as described above except for the adult samples, which were fixed overnight in 10% neutral buffered formalin (Fisher) instead of PFA. Pigs (Sus scrofa) were housed at Washington State University under approved Washington State University IACUC and USDA protocols in a climate-controlled facility ranging from 70 °F to 80 °F and 30% to 50% humidity. Housing included a 12-h light/dark cycle with regular feeding and ad libitum water. Some fetal pigs were obtained from Biology Products; their age was estimated on the basis of length, and they were exempt from Washington State University IACUC approval. Embryonic day 90 fetal pigs were collected postmortem from a pregnant pig with known date of conception under WSU IACUC-approved protocols (6492). Skin samples from all fetal pigs were collected from the upper back along the dorsal midline. Postnatal pig samples were collected postmortem from pigs either housed at Washington State University, obtained from local farmers, or postmortem from local butchers in accordance with Washington State University IACUC-approved protocols (6492). Skin samples from all postnatal pigs were collected from the upper back along the dorsal midline from multiple litters owing to limited litter sizes and difficulty in obtaining animals. Fetal pigs obtained from Biology Products were generally unpigmented and of unknown background and multiple litters. One litter of known embryonic day 90 pigs (used for histology, immunostaining and scRNA-seq) was collected from an unpigmented pregnant sow raised on a local farm of mixed Yorkshire and Red Duroc background. Postnatal pigs obtained from local farmers or butchers were of mixed backgrounds involving predominantly Yorkshire, Red Duroc and Hampshire breeds, with occasional regional interbreeding with Idaho Pasture pigs and Kunekunes, which have pigmented skin. Notably, Hampshire breeds are known to exhibit banded pigmentation in their skin. As such, most skin we studied was unpigmented, but some piglets or adults had pigmented skin or spots (as visible in Fig. 2b and Supplementary Fig. 5c). Every effort was made to collect skin samples from both males and females across all time points. Adult male skin (6 mo/7 mo) collected from local farmers and butchers was from animals presumably castrated before weaning to avoid boar taint in the meat. Owing to the opportunistic nature of these collections, it is unknown whether postbutchering biological replicates were derived from the same or different litters. Ages were approximated. Postnatal Yucatan miniature hairless pig and Hanford miniature pig skin samples collected from the upper back along the dorsal midline were received from Sinclair Biosciences, and Mangalitsa pigs were raised on farms, with samples collected from the upper back along the dorsal midline following butchering, and were exempt from Washington State University IACUC approval. EDA-KO pig42 samples were collected from the backs of one litter of pigs at P5 or age-matched at 5 mo by the Welsh and Ostedgaard laboratories at the University of Iowa under University of Iowa IACUC-approved protocols (3071121) and shared upon request. All experiments followed relevant guidelines and regulations of the appropriate ethics committees, as detailed where relevant. A visual summary of tissue sample collection sites can be found in Extended Data Fig. 2a. For all animal tissue sample collections, an individual organism was considered to be one biological replicate. Multiple samples may have been collected from one biological replicate, but these were used only as technical replicates. Unless otherwise specified, tissue samples were fixed in 4% PFA overnight and processed for paraffin embedding as described above, or fixed in 4% PFA, washed in PBS and then embedded in OCT (Fisher) before cryopreservation at −80 °C. Epidermal whole mounts were collected using a process similar to that described in previous studies65.
Histological analysis
Paraffin-embedded tissue samples were sectioned at 5 μm (Lef1-eKO back skin) or 10 μm (all others) and stained either with H&E according to standard protocols or using Herovici’s polychrome in a process adapted from previously published protocols66. Coverslips for H&E-stained tissue were mounted using Permount Mounting Medium (Fisher), and those for Herovici-stained tissue were mounted using DPX (Sigma). Slides were bright-field imaged using a Nikon Eclipse E600 fluorescence microscope equipped with a Nikon DS-Fi3 colour camera. Dolphin cryo samples were sectioned at 10 μm, fixed for 30 min at room temperature, washed 3 times with PBS, and rinsed in tap water for several minutes before staining with H&E or Herovici’s polychrome, as above. Dolphin H&E and Herovici slide coverslips were mounted in DPX and imaged using a Keyence BZ-X810 wide-field microscope. K14-CreERT experiment digit samples were sectioned at 13–15 μm using a Leica cryostat and H&E stained using standard protocols.
Hair density assessment
The surface of skin samples was imaged using an AmScope dissecting microscope with an AmScope colour camera before further processing for paraffin embedding or cryopreservation. Multiple distinct images were captured per biological replicate and treated as technical replicates when skin samples were of sufficient size or quantity. Owing to the surgical discard nature of adult human samples collected in this study, many samples were not large enough to allow accurate assessment of hair density or were collected before the beginning of the hair density assessment, hence the smaller sample size in Fig. 2d (n = 8) compared with Fig. 2c. Hair density was also quantified from samples across more anatomical sites, including trunk skin (n = 4), face skin (n = 2), scalp skin (n = 1), and from the base of the nose, which we considered separate from face skin (n = 1).
Porcine wound healing
One litter of neonatal pigs was housed with their mother at Washington State University, were anaesthetized, and received 2.5 × 2.5-cm-square full-thickness wounds under aseptic conditions in accordance with WSU IACUC-approved protocols (6492). Following surgery, piglets were returned to their mother, and the wound site was periodically imaged to assess the size of the wound across the surgery cohort of seven littermates of mixed sexes. Twenty-eight days postwounding (28 dpw), two littermates were euthanized, and the wound site was collected for histological analysis, followed by two more at 43dpw, and the final three at 58dpw. Collected wounds were fixed in 4% PFA for several hours, washed in PBS, then embedded and frozen in OCT at −80 °C.
Porcine BrdU labelling
One litter of neonatal pigs was housed with their mother at Washington State University and injected intraperitoneally twice daily for 3 days from P5 to P7 with 50 mg kg−1 of BrdU (AdipoGen Life Sciences, CDX-B0301-G005) dissolved in sterile saline, in strict compliance with WSU IACUC-approved protocols (6492). One piglet was injected with sterile saline only and was considered a negative control. Three BrdU-injected piglets were euthanized, and tissue was collected from the upper back along the dorsal midline for histological analyses at each of three time points: P8 (1 day postinjection, 1 dpi), P12 (5 dpi) and P16 (9 dpi), along with one negative control individual at P16.
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