bioRxiv preprint doi: https://doi.org/10.1101/2020.05.07.083287; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 4 5 6 Title: Spiny mice (Acomys) exhibit attenuated hallmarks of aging and rapid cell turnover after UV 7 exposure in the skin epidermis 8 Authors: Wesley Wong1, Austin Kim1, Ashley W. Seifert2, Malcolm Maden3, and Justin D. Crane1 9 Affiliations: 1Department of Biology, Northeastern University, 360 Huntington Avenue, Boston, 10 MA 02115, 2Department of Biology, University of Kentucky, Lexington, KY 40506, 3UF 11 Genetics Institute & Department of Biology, University of Florida, Gainesville, FL 32611 12 13 Keywords: Spiny mouse, skin, epidermis, UV radiation, aging 14 15 16 17 18 19 20 21 22 23 24 25 26 27 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.07.083287; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

28 Abstract 29 The study of long-lived and regenerative models has revealed diverse protective responses 30 to stressors such as aging and tissue injury. Spiny mice (Acomys) are a unique mammalian model 31 of skin regeneration, but their response to other types of physiological skin damage have not been 32 investigated. In this study, we examine how spiny mice skin responds to acute UVB damage or 33 chronological aging compared to non-regenerative C57Bl/6 mice (M. musculus). We find that, 34 compared to M. musculus, the skin epidermis in A. cahirinus experiences a similar UVB-induced 35 increase in basal cell proliferation but exhibits increased epidermal turnover. Notably, A. cahirinus 36 uniquely form a suprabasal layer co-expressing 14 and Keratin 10 after UVB exposure 37 concomitant with reduced epidermal inflammatory signaling and reduced markers of DNA damage. 38 In the context of aging, old M. musculus exhibit typical hallmarks including epidermal 39 thinning, increased inflammatory signaling and senescence. However, these age-related changes 40 are absent in old A. cahirinus skin. Overall, we find that A. cahirinus have evolved novel responses 41 to skin damage that reveals new aspects of its regenerative phenotype. 42 43 44 45 46 47 48 49 50 51 52 53 54 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.07.083287; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

55 Introduction 56 Maintenance and repair of the skin barrier are essential to prevent infection and protect the 57 body from external hazards. However, normal stress and damage during an organism’s lifespan 58 deteriorates skin structure and repair through undefined mechanisms. The skin epidermis is 59 composed primarily of derived from basal stem cells that continually renew the 60 differentiated spinous, granular, and cornified suprabasal layers (Fuchs, 2008). Together, these 61 cells protect against environmental insults such as radiation, physical injury, dehydration, and 62 pathogens. A major environmental hazard to the skin is ultraviolet (UV) solar radiation, which 63 induces potentially mutagenic DNA damage if left unrepaired. UV-damaged cells undergo either 64 apoptosis or cell cycle arrest coupled with DNA repair before re-entry into the cell cycle which 65 results in thickening of the epidermis (El-Abaseri et al., 2006). Aging of the skin induces cellular 66 senescence, thinning of the epidermis and reduced basal stem cell renewal and proliferation (Liu 67 et al., 2019). While we are beginning to understand some of the mechanisms that underlie the slow 68 repair of aged skin (Keyes et al., 2016; Wong et al., 2019), further research is needed. 69 Long-lived or stress-resistant model organisms permit a better understanding of adaptive 70 mechanisms of tissue homeostasis and repair. For example, the Snell dwarf mouse attains a long 71 lifespan through impaired pituitary hormone signaling and its study has informed our 72 understanding of how organismal physiology and longevity are balanced (Brown-Borg, 2011; 73 Flurkey et al., 2002). The naked mole rat Heterocephalus glaber (H. glaber) has evolved 74 distinctive mechanisms of cancer resistance which may be mediated by microenvironment and 75 immune system (Hadi et al., 2018; Seluanov et al., 2009; Tian et al., 2015). In a similar vein, 76 models of vertebrate regeneration such as salamanders and newts have been used extensively to 77 understand the optimal healing of nervous, muscle and connective tissue (Joven et al., 2019), but 78 a critical barrier to deriving clinical therapies from this work has been a lack of mammalian models 79 of tissue repair . The recent discovery that spiny mice (Acomys spp., hereafter referred to as Acomys) 80 can fully regenerate skin and hair following wounding or burn injury (Maden, 2018; Seifert et al., 81 2012), a feature not observed in typical strains of mice ( musculus; Mus), lends well to 82 uncovering novel regenerative mechanisms in skin tissue. However, it is not known how Acomys 83 responds to other types of skin stress, including UV-damage and chronological aging. 84 Here, we examine epidermal responses to acute UVB exposure and chronological aging in 85 the conventional C57Bl/6 M. musculus strain compared to A. cahirinus. We find that A. cahirinus bioRxiv preprint doi: https://doi.org/10.1101/2020.05.07.083287; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

86 has attenuated pathological hallmarks of UVB exposure and aging in conjunction with distinctive 87 differences in epidermal turnover, senescence and inflammation. These processes may underlie 88 the unique skin regeneration phenotype of A. cahirinus, and such mechanisms can inform 89 therapeutic strategies to manage skin diseases such as skin cancer and aging. 90 91 Materials and Methods 92 Mice 93 For all studies, C57Bl/6 (M. musculus) mice were acquired from Jackson Labs (#000664). For the 94 UVB experiments, 5-6 month old male A. cahirinus animals were shipped from the University of 95 Florida and compared to 3-4 month old male C57Bl/6 mice. M. musculus were housed 4-5 to a 96 cage in mouse cages while A. cahirinus were housed 2-3 to a cage in rat cages with a 12h/12h light 97 cycle. Animals were housed at Northeastern University for at least 2 weeks prior to UVB or sham 98 treatments to acclimate. For the aging studies, young, female animals of each at 3-4 months 99 of age were used and old, female animals (M. musculus: 2 years of age; A. cahirinus: 4 years of 100 age) were analyzed. The young and old M. musculus mice were housed at Northeastern University 101 while the young and old A. cahirinus animals were housed at the University of Florida and 102 University of Kentucky. For the UVB studies, all animals were sacrificed by cervical dislocation. 103 For the aging studies, animals were euthanized either by cervical dislocation (M. musculus) or 104 isoflurane overdose (A. cahirinus). All procedures were performed according to IACUC approved 105 procedures from each institution. 106 107 Ultraviolet Irradiation 108 Animals were sedated with intraperitoneal ketamine/xylazine anesthesia (50 mg/kg ketamine; 5 109 mg/kg xylazine) and shaved with clippers prior to UVB exposure. Animals were exposed to a 110 UVB dose of 200 mJ/cm2 using a dosimeter calibrated UV instrument (310 nm peak output) (Tyler 111 Research, Alberta, Canada). Full-thickness dorsal skin samples in telogen phase were collected 112 and fixed at 24- and 48-hours after irradiation in addition to sham controls and paraffin embedded 113 cross-sections were prepared for histologic analysis. BrdU (5-Bromo-2’-doxyuridine, Sigma 114 Aldrich, St. Louis, Missouri, USA) was injected intraperitoneally at 100 mg/kg in sterile PBS 24 115 hours prior to sacrifice. Animals were sacrificed by cervical dislocation. 116 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.07.083287; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

117 Histology 118 Tissues were fixed overnight in 10% neutral buffered formalin and thereafter transferred to 70% 119 ethanol at 4°C until processing. Tissues were processed using an automated tissue processor 120 (Thermo HistoStar, Kalamazoo, Michigan, USA) and embedded in paraffin wax. A microtome 121 (Leica Biosystems, Buffalo Grove, Illinois, USA) was used to cut 4 μm skin cross-sections, which 122 were allowed to dry overnight before de-waxing and further processing. Heat induced epitope 123 retrieval was performed on de-waxed slides with either citrate or Tris-EDTA buffer prior to 124 immunofluorescence staining. Skin cross-sections were blocked for 30 minutes at room 125 temperature in 5% normal goat serum PBS, which was also the primary antibody diluent, unless 126 the target was a phospho- in which case 5% normal goat serum TBS was used. Primary 127 antibodies and their usage were as follows: (chicken, BioLegend #906004, 1:500, San 128 Diego, California, USA), HMGB1 (rabbit, Abcam #AB79823, 1:250, Cambridge, Massachusetts, 129 USA), B1 (rabbit, Abcam #AB16048, 1:1000), Keratin 10 (rabbit, BioLegend #905404, 130 1:500), Loricrin (rabbit, BioLegend #905104, 1:500), BrdU (rat, Abcam #AB6326, 1:200), 131 thymine dimer mouse, Novus #NB600-1141, 1:400, Centennial, Colorado, USA), cleaved 132 caspase-3 (rabbit, Cell Signaling #9661, 1:100, Danvers, Massachusetts, USA), γH2AX (rabbit, 133 Cell Signaling #9718, 1:40). Primary antibody incubations were carried out overnight at 4°C 134 except Keratin 14, Keratin 10, and Loricrin, which were 2 hours at room temperature. Following 135 either PBS or Tris-buffered saline (TBS) wash, secondary detection antibodies conjugated with 136 either AlexaFluor-647 or AlexaFluor-488 (Invitrogen, Carlsbad, California, USA) were diluted in 137 either PBS or TBS at 1:200 were applied for 30 minutes at room temperature. Mounting media 138 with DAPI (4’,6-diamidino-2-phenylindole) used was either ProLong Gold (Invitrogen) or 139 ProLong Diamond (Invitrogen), which were used interchangeably and allowed to cure prior to 140 imaging. For thymine dimer staining, a mouse-on-mouse blocking kit (Vector Labs #BMK-2202, 141 Burlingame, California, USA) was used per the manufacturer’s instructions. 142 Hematoxylin and Eosin staining was performed on 4 um de-waxed sections using standard 143 histology protocols using Gill’s Hematoxylin No.1 and Eosin Y (Sigma Aldrich). Colorimetric 144 stained slides were mounted with Permount (Fisher Scientific, Pittsburgh, Philadelphia, USA). All 145 brightfield imaging was done using an inverted EVOS XL (Life Technologies, Carlsbad, 146 California) microscope. All fluorescence imaging was performed on either an Revolve R4 (Echo 147 Labs, San Diego, California, USA) microscope equipped with Olympus UPlanFL 10x/0.30, and bioRxiv preprint doi: https://doi.org/10.1101/2020.05.07.083287; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

148 UPlanFL 20x/0.50 air objectives using DAPI, FITC, and Cy5 fluorescence channels or an Axio 149 Observer Z1 (Zeiss, White Plains, New York, USA), equipped with Plan Apochromat 5x/0.16, 150 10x/0.45, and 20x/0.8 air objectives and 40x/1.3 and 63x/1.4 oil objectives using DAPI, GFP, 151 DsRed, and Cy5 fluorescence channels. 152 153 Image analysis 154 All image processing and analysis was conducted using ImageJ FIJI (version 2.0.0-rc-69/1.52i, 155 NIH), Photoshop CC 2019 (version 20.0.4, Adobe), and Illustrator CC 2019 (version 23.0.3, 156 Adobe, San Jose, California, USA). Immunofluorescence images first underwent thresholding 157 over entire image set of all samples from the experiment followed by manual counting of positive 158 cells for at least 3 unique fields from each sample. All epidermal analyses only counted 159 interfollicular epidermal cells and excluded hair follicles. Nuclear HMGB1 positive cells were 160 those that had overlapping signal with their nuclear DAPI signal. positive cells were 161 those that exhibited perinuclear signal relative to their DAPI signal. Keratin 14, Keratin 10, and 162 Loricrin positive cells were those that had DAPI-associated cytoplasmic signal. 163 Epidermal thickness measurements were performed on H&E stained intact skin cross-sections as 164 previously described (Crane et al., 2015) by measuring the orthogonal distance from the basement 165 membrane to the outer edge of the cellular epidermis. 166 167 Expression 168 RNA was extracted by homogenizing whole skin in TRIzol (Invitrogen) reagent using a bead mill 169 apparatus (MPBio 5G, Irvine, California, USA) followed by column purification (Zymo Direct- 170 zol RNA mini prep, Irvine, California, USA) with on column DNase treatment and subsequent 171 elution. 600 ng of total RNA was then reversed transcribed to cDNA (ABI HC cDNA synthesis 172 kit, Waltham, Massachusetts, USA), diluted 1:30 in ultrapure water and mRNA expression was 173 assessed using qPCR with SYBR chemistry. CDK-interacting protein 1 (Cdkn1a) was used as a 174 housekeeping gene and its expression did not significantly differ between species or with UVB 175 treatment. Data was analyzed by the delta Ct (ΔCt) method. 176 177 Primers are as follows: bioRxiv preprint doi: https://doi.org/10.1101/2020.05.07.083287; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

178 M. musculus Cxcl1-Forward: ACTCAAGAATGGTCGCGAGG; M. musculus Cxcl1-Reverse: 179 GTGCCATCAGAGCAGTCTGT. A. cahirinus Cxcl1-Forward: 180 CCCATGGTTCGGAAGGTTGT; A. cahirinus Cxcl1-Reverse: GTTGTCAGACGCCAGCATTC. 181 M. musculus Il1b-Forward: TGCCACCTTTTGACAGTGATG; M. musculus Il1b-Reverse: 182 TGATGTGCTGCTGCGAGATT. A. cahirinus Il1b-Forward: CTGGGCTCCAGAGACACAAG; 183 A. cahirinus Il1b-Reverse: GAACCCCTGCATCAACTCCA. M. musculus Cdkn1a-Forward: 184 CGGTGTCAGAGTCTAGGGGA; M. musculus Cdkn1a-Reverse: 185 AGGATTGGACATGGTGCCTG. A. cahirinus Cdkn1a-Forward: 186 TGCACTCTGGTATCTCACGC; A. cahirinus Cdkn1a-Reverse: 187 CAGTCGGCGCTTAGAGTGAT. 188 189 Statistical Analysis 190 All data were analyzed in Prism 8.0 (GraphPad, San Diego, California, USA) using a two-way 191 analysis of variance (ANOVA) on either log or square root transformed data with Tukey’s HSD 192 post-hoc testing for differences between treatments and species. Statistical significance was set as 193 p<0.05. 194 195 Results 196 UV-irradiation induced changes to skin epidermal morphology and epidermal stratification 197 of M. musculus and A. cahirinus. 198 While A. cahirinus can fully regenerate skin wounds and burns without scarring (Maden, 2018; 199 Seifert et al., 2012), it has not been established how this regenerative ability translates to other 200 types of skin damage. Since UVB radiation is a common form of epidermal damage (Brash et al., 201 1991), we sought to compare the morphological changes between A. cahirinus and M. musculus 202 epidermis following an acute dose of UVB known to induce epidermal proliferation and DNA 203 damage in M. musculus (Trevithick et al., 1992). We first measured the total thickness of the 204 cellular epidermis using hematoxylin and eosin staining. As expected, M. musculus had a 205 significantly thicker epidermis at 48 hours post-UVB compared to sham or 24 hours (Fig. 1A). In 206 contrast, there was no appreciable difference in epidermal thickness in response to UVB in A. 207 cahirinus at 24 hours (p=0.21) or 48 hours (p=0.80) compared to sham (Fig. 1B). To understand 208 the processes underlying this differential response, we examined rates of cell division by analyzing bioRxiv preprint doi: https://doi.org/10.1101/2020.05.07.083287; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

209 post-UVB epidermal incorporation of the synthetic nucleoside 5-bromo-2’-deoxyuridine (BrdU). 210 During imaging, we noted species specific patterns of BrdU labeling in basal and suprabasal 211 epidermal compartments (Fig. 1C). To distinguish these changes, we analyzed patterns of label 212 retention in the two compartments separately. In the basal epidermis, BrdU incorporation was 213 significantly lower in M. musculus basal epidermis 24 hours after UVB-exposure, and significantly 214 higher at 48 hours following UVB compared to both sham and 24 hours (Fig 1D). In contrast, A. 215 cahirinus epidermis did not exhibit changes in BrdU incorporation at 24 hours post-exposure but 216 was significantly elevated at 48 hours compared to both the sham and 24 hour groups (Fig. 1D). 217 In the suprabasal epidermis, both M. musculus and A. cahirinus exhibited greater BrdU 218 incorporation at 48 hours compared to sham, however this UV-induced proliferation was greater 219 in M. musculus (Fig. 1E). 220 Since stem cell fate decisions control the balance of cell division and upward transport 221 through the suprabasal layers (Williams et al., 2011), we next stained for keratin 14 (K14) to mark 222 the basal stem/progenitor layer, keratin 10 (K10) to mark the spinous layer, and for the Loricrin 223 (Lor) expressing cornified envelope after UVB in each species. During imaging, we noticed 224 species specific patterns of UVB-induced epidermal differentiation, most notably the unique 225 double-positive K14+/K10+ suprabasal layer in UVB-exposed A. cahirinus (Fig. 1F). We then 226 measured the thicknesses of each of the labeled basal (K14+/K10–), double-positive (K14+/K10+), 227 spinous (K14–/K10+), and cornified (Lor+) epidermal layers. Exposure to acute UVB resulted in a 228 significantly thicker K14+ layer in M. musculus by 48 hours but not in A. cahirinus. However, a 229 double-positive K14+/K10+ suprabasal epidermal layer was uniquely evident only in A. cahirinus 230 at 24 and 48 hours post-UVB (Fig. 1G). The thickness of the Lor+ layer between species was 231 similar at all time points and was not statistically different (p=0.0986) in A. cahirinus at 48 hours 232 (Fig. 1G and Fig. S1A). Overall, A. cahirinus skin exhibits a unique program of epidermal 233 stratification without hyperproliferation. This distinctive pattern of differentiation consists of a 234 transient intermediate layer with both basal and spinous characteristics in response to UVB 235 exposure. 236 237 Differences in UV-induced damage and epidermal cell death between M. musculus and A. 238 cahirinus bioRxiv preprint doi: https://doi.org/10.1101/2020.05.07.083287; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

239 In M. musculus and humans, differentiation and eventual shedding of skin cells via upward 240 transport through the epithelium serves as a method of eliminating damaged and compromised 241 cells (Freije et al., 2014). Since we observed an altered differentiation pattern in A. cahirinus in 242 response to UVB, we reasoned that this could impact the removal of damaged cells. We first 243 assessed this by measuring the UVB-induced DNA photoproduct thymine dimer (T-T dimer) in 244 the epidermis (Fig. 2A). As expected from this dose of UVB, we found significantly higher 245 thymine dimer positive cells in M. musculus basal epidermis by 24 hours with a near return to 246 sham levels by 48 hours (Fig. 2B), yet in the suprabasal layer levels rose significantly by 24 hours 247 and remained elevated at 48 hours (Fig. 2C). In A. cahirinus, while we found an induction of 248 thymine dimer positive cells 24 hours following UVB, this UV-response was reduced compared 249 to M. musculus animals in both the basal and suprabasal epidermis (Fig. 2B-C). To extend our 250 thymine dimer results, we next assessed the DNA repair marker γH2AX (Fig. 2D). M. musculus 251 had greater basal and suprabasal epidermal γH2AX labeling at 24 hours and 48 hours after UVB 252 (Fig. 2E and 2F). A. cahirinus also had more γH2AX-positive cells versus sham at 24 hours in the 253 basal layer, with a similar pattern in the suprabasal layers at 24 and 48 hours (Fig. 2E and 2F). 254 However, similar to thymine dimers, there were more γH2AX-positive cells in M. musculus 255 compared to A. cahirinus at 24 and 48 hours post UVB exposure (Fig. 2E and 2F). 256 To understand the contribution of apoptosis to the post-UVB epidermal response, we 257 measured the abundance of cleaved caspase-3 (CC3) positive cells (Fig. 2G). In the basal layer, 258 there was a similar UVB-induced response between species, with an increase in cleaved caspase- 259 3 expressing cells at 24 hours followed by a return to sham levels 48 hours later (Fig. 2H). This 260 was in contrast to the suprabasal epidermis, where at 24 hours A. cahirinus exhibited a significantly 261 greater number of cleaved caspase-3 expressing cells compared to M. musculus. However, there 262 was a similar abundance of cleaved caspase-3 expressing cells between species at 48 hours (M. 263 musculus significantly elevated vs baseline; p=0.0913 for A. cahrinus vs baseline) (Fig. 2I). Thus, 264 A. cahirinus epidermis have modest changes in cell death responses following UVB exposure 265 compared to M. musculus. 266 267 Attenuated skin epidermal inflammatory response following UV-irradiation in A. cahirinus 268 Inflammation is a major component of epidermal remodeling following UVB exposure (Barker et 269 al., 1991). To gain further insight into the differential UVB responses between M. musculus and bioRxiv preprint doi: https://doi.org/10.1101/2020.05.07.083287; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

270 A. cahirinus, we analyzed the epidermal abundance of the damage associated molecular pattern, 271 HMGB1 (Fig. 3A). Loss of nuclear HMGB1 is a biomarker of cell stress, senescence, 272 inflammation, and autophagic responses particularly in UVB-exposed basal epidermis (Bald et al., 273 2014; Davalos et al., 2013). Quantification of HMGB1 labeling revealed that M. musculus 274 experiences a significant loss of basal nuclear HMGB1 by 48 hours after UVB (Fig. 3B). However, 275 we found that post-UVB A. cahirinus basal epidermis retained nuclear HMGB1 labeling similar 276 to sham levels (Fig. 3B). Suprabasal levels of HMGB1 were similar between species and 277 treatments (Fig. 3C), suggesting that this effect is restricted to basal progenitors. A milieu of pro- 278 inflammatory signaling within the skin also occurs concomitantly to the epidermal hyperplastic 279 response (Ouhtit et al., 2000). To better understand the UV-induced inflammatory response in each 280 species, we measured mRNA levels of the inflammation associated Il1b, Cxcl1, Tgfb1, and 281 Mmp9 in whole skin using species specific primers and qPCR. Increased epidermal expression of 282 Il1b and Cxcl1 mediate inflammatory responses after UV-exposure (Qiang et al., 2017), and 283 knockout mouse studies have shown loss of Tgfb1 and Mmp9 are associated with increased and 284 prolonged inflammation in the skin epidermis (Wang et al., 1999; Yoshinaga et al., 2008). Acute 285 UVB-irradiation resulted in significant increase in Cxcl1 in M. musculus skin at 48 hours, but 286 levels in A. cahirinus were not significantly altered by UVB exposure (Fig. 3D). In contrast, Il1b 287 expression was not significantly changed by acute UVB-irradiation in either species but was 288 consistently lower in A. cahirinus vs. M. musculus (Fig. 3E). Transcript levels of Tgf1b in M. 289 musculus skin significantly decreased at 24 hours but A. cahirinus levels were unchanged (Fig. 290 3F). Similarly, levels of Mmp9 in M. musculus skin significantly decreased at 24 and 48 hours but 291 A. cahirinus levels were not significantly altered by UVB (Fig. 3G). Thus, compared to M. 292 musculus, A. cahirinus epidermis resists UVB-driven inflammatory changes, 293 consistent with their anti-inflammatory tissue damage response seen after wounding and burn 294 injury (Brant et al., 2015; Gawriluk et al., 2019; Maden, 2018). 295 296 Aging associated epidermal thinning, inflammatory signaling, and senescence are absent in 297 A. cahirinus 298 Total organismal aging is the sum of chronological aging and environmental exposure. We sought 299 to further explore the differences in cellular stress response in these two species by examining their 300 epidermal responses to chronological aging. A. cahirinus have a maximal lifespan of 5.9 years, bioRxiv preprint doi: https://doi.org/10.1101/2020.05.07.083287; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

301 versus 3.5 years in M. musculus (C57Bl/6) (Edrey et al., 2012). Thus, to study aging in these 302 species, we used young animals from each species at 3-4 months old as well as aged 2-year old M. 303 musculus and 4-year old A. cahirinus animals. At these ages the species are roughly matched as a 304 fraction of their respective maximum lifespans: approximately 9.4% for young M. musculus, 8.4% 305 for young A. cahirinus, 56% for old M. musculus, and 68% for old A. cahirinus. Using formalin 306 fixed skin cross-sections for both species, we used hematoxylin and eosin staining of the skin to 307 reveal morphological changes due to age (Fig. 4A). This revealed an expected decrease in the 308 thickness of the cellular epidermis of old versus young M. musculus (Fig. 4B), however, the 309 cellular epidermal thickness of A. cahirinus remained unchanged with age (Fig. 4B). To better 310 understand the cellular epidermal thickness differences, we measured the nuclear envelope protein 311 and senescence associated biomarker Lamin B1 (Fig. 4C). Similar to previous research by 312 ourselves (Wong et al., 2019) and others (Freund et al., 2012; Wang et al., 2017), we found a 313 decrease in basal epidermal Lamin B1 with age in M. musculus (Fig. 4D). However, A. cahirinus 314 basal epidermal Lamin B1 labeling was similar to young M. musculus and was not different in old 315 A. cahirinus versus young (Fig. 4D). To corroborate these findings regarding senescence, we also 316 evaluated the proportion of epidermal cells with nuclear HMGB1 staining (Fig. 4E). As previously 317 reported by ourselves (Wong et al., 2019) and others (Davalos et al., 2013), we observed a decrease 318 in epidermal nuclear HMGB1+ cells with age in M. musculus (Fig. 4F), yet HMGB1 in old A. 319 cahirinus remained unchanged from young animals (Fig. 4F). Collectively, these data show that 320 A. cahirinus have reduced hallmarks of epidermal aging. 321 322 Discussion 323 The comparative study of exceptionally long-lived, stress-resistant, or regenerative organisms can 324 be used to provide insight into evolved mechanisms of cellular repair. In this study, we found 325 substantial differences in how the regenerative A. cahirinus responds to UVB skin damage 326 compared to M. musculus. This included altered patterns of proliferation and 327 differentiation, an earlier induction of UVB-induced cell death, and an attenuated inflammatory 328 response. The acanthosis commonly observed in M. musculus after UVB was not evident in A. 329 cahirinus despite similar rates of basal cell proliferation, suggesting an alternative fate of newly 330 formed keratinocytes. Notably, the A. cahirinus epidermis forms a unique middle suprabasal layer 331 of keratinocytes with both basal and spinous characteristics (K14+/K10+) which may facilitate bioRxiv preprint doi: https://doi.org/10.1101/2020.05.07.083287; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

332 increased keratinocyte upward transport and removal. This was supported by the observations of 333 fewer retained damaged cells (thymine dimers, gH2AX) as well as more apoptotic cells transiting 334 the suprabasal epidermis at intermediate stages of repair (24 hours) in A. cahirinus compared to 335 M. musculus. Taken together, these data suggest that A. cahirinus skin epidermis is capable of 336 more rapid removal of damaged and dying cells after UVB-exposure through an enhanced rate of 337 differentiation. 338 The presence of a co-expressed K14+/K10+ suprabasal layer in the epidermis has been 339 reported by others, but usually this is f in the context of skin disorders. For example, in psoriatic 340 human skin biopsies, there is aberrant Notch expression and dual K14/K10 expression (Ota et al., 341 2014). Dysregulated epidermal calcium gradients can also produce dual expression of K14 and 342 K10, such as in the skin blistering disorders Hailey-Hailey disease and Darier disease (Mikkelsen 343 et al., 2018; Robia and Young, 2018), and in TRPV4 KO mice (Moore et al., 2013) which suffer 344 defective tight junction formation (Sokabe et al., 2010). Interestingly, TRPV4 is also a nociceptor 345 and those mice had altered pain perception and inflammatory responses after sunburn (Moore et 346 al., 2013). Aberrant cell cycle regulation can also cause dual K14/K10 expression as mice lacking 347 epidermal C/EBPβ and C/EBPα exhibit defective cornified envelope formation (Lopez et al., 348 2009). In contrast to these pathological examples of K14/K10 co-expression, this suprabasal layer 349 formed after UVB in A. cahirinus appears to be a physiological skin damage response. Notably, 350 A. cahirinus executes this distinctive program of epidermal differentiation without marked changes 351 in inflammatory signaling factors, which is similar to the blunted inflammatory response reported 352 during wound healing (Brant et al., 2015). 353 The study of organisms with long lifespans such as the naked mole rat (H. glaber) has 354 revealed several unique mechanisms of stress resistance (Pérez et al., 2009; Seluanov et al., 2009). 355 Long-lived model organisms often also exhibit altered inflammatory and cellular damage 356 responses distinct from humans or laboratory mice which provide cancer resistance or augmented 357 tissue repair (Eming et al., 2009; Kowalczyk et al., 2020). In H. glaber, cellular senescence is rare, 358 yet cancer rates are very low due to sensitive cellular growth inhibition, modified tumor suppressor 359 pathways, altered extracellular matrix composition, and a stable epigenome (Seluanov et al., 2018). 360 The evolutionary path to these adaptive differences was likely guided by evolutionary adaptation 361 to the hypoxic subterranean environment of H. glaber and the fructose biased metabolism resulting 362 from hypoxia (Seluanov et al., 2018). We reasoned that A. cahirinus might have also evolved bioRxiv preprint doi: https://doi.org/10.1101/2020.05.07.083287; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

363 distinct cellular stress mechanisms to enable extensive tissue regeneration, which parallels the 364 unique survival mechanisms recently found in A. cahirinus fibroblasts in vitro (Saxena et al., 2019). 365 We found that A. cahirinus epidermis was remarkably resilient to chronological aging stress with 366 minimal changes in epidermal thickness or biomarkers of aging and senescence. It is interesting to 367 note that the MRL/MpJ healer strains of mice also exhibit reduced inflammatory responses to 368 injury and improved recovery outcomes in the corneal epithelium (Ueno et al., 2005) and ear punch 369 wounds (Kench et al., 1999). Thus, an attenuated inflammatory response may enable the unique 370 growth and repair patterns of A. cahirinus epidermis compared to M. musculus and may also 371 explain the differences in biological aging between the species. 372 Our study of the regenerative African spiny mouse, A. cahirinus, has revealed a unique 373 skin response to acute UVB-irradiation which consists of rapid differentiation and apoptosis 374 without prototypical epidermal hyperplasia or inflammation. However, we do not yet understand 375 the precise cellular mechanisms underlying this response. While proliferation and differentiation 376 may be intrinsically regulated, inflammation and clearance of cells is likely related to the altered 377 immunity and inflammation previously reported during skin repair in A. cahirinus (Maden, 2018; 378 Simkin et al., 2017). Moreover, our findings of improved epidermal tissue repair in A. cahirinus 379 extend recent work using models of spinal cord injury (Streeter et al., 2019), musculoskeletal 380 regeneration (Gawriluk et al., 2016), and muscle damage (Maden et al., 2018). Future work should 381 clarify the molecular underpinnings of improved tissue repair in A. cahirinus in order to advance 382 regenerative medicine. 383 384 Acknowledgements 385 The authors wish to thank J. Monaghan for helpful discussions related to the project and E. Crane 386 for assistance with some of the IHC analysis. 387 388 Competing interests 389 The authors declare no competing or financial interests. 390 391 Author contributions 392 Conceptualization: W.W., J.D.C.; Investigation: W.W., A.K.; Formal Analysis: W.W., A.K.; 393 Resources: J.D.C., A.W.S., M.M.; Writing – original draft: W.W., J.D.C.; Writing – review & bioRxiv preprint doi: https://doi.org/10.1101/2020.05.07.083287; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

394 editing: W.W., A.K., J.D.C, A.W.S., M.M.; Visualization: W.W.; Supervision: W.W., J.D.C.; 395 Project administration: J.D.C.; Funding acquisition: J.D.C. 396 397 Funding 398 This work was supported by startup funding from the Northeastern University Provost’s Office (to 399 J. D. C.). Work in A.W.S. lab is supported by grants from NSF (IOS-1353713) and NIH 400 (R01AR070313). 401 402 List of abbreviations 403 K10 Keratin 10 404 K14 Keratin 14 405 Lor Loricrin 406 PBS Phosphate buffered saline 407 BrdU 5-Bromo-2’-deoxyuridine 408 mJ Millijoule 409 TBS Tris-buffered saline 410 DAPI 4’,6-diamidino-2-phenylindole 411 GFP Green fluorescent protein 412 DsRed Red fluorescent protein 413 Cy5 Cyanine 5 414 T-T dimer Thymine dimer 415 CC3 Cleaved caspase-3 416 417 References 418 419 Bald, T., Quast, T., Landsberg, J., Rogava, M., Glodde, N., Lopez-Ramos, D., Kohlmeyer, 420 J., Riesenberg, S., van den Boorn-Konijnenberg, D., Hömig-Hölzel, C., et al. (2014). 421 Ultraviolet-radiation-induced inflammation promotes angiotropism and metastasis in 422 melanoma. 507, 109–113. 423 Barker, J. N., Mitra, R. S., Griffiths, C. E., Dixit, V. M. and Nickoloff, B. J. (1991). 424 Keratinocytes as initiators of inflammation. Lancet 337, 211–214. 425 Brant, J. O., Lopez, M.-C., Baker, H. V., Barbazuk, W. B. and Maden, M. (2015). A 426 Comparative Analysis of Gene Expression Profiles during Skin Regeneration in Mus and 427 Acomys. PLoS One 10, e0142931. bioRxiv preprint doi: https://doi.org/10.1101/2020.05.07.083287; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

428 Brash, D. E., Rudolph, J. A., Simon, J. A., Lin, A., McKenna, G. J., Baden, H. P., Halperin, 429 A. J. and Pontén, J. (1991). A role for sunlight in skin cancer: UV-induced p53 430 mutations in squamous cell carcinoma. Proc. Natl. Acad. Sci. USA 88, 10124–10128. 431 Brown-Borg, H. M. (2011). Role of the somatotropic axis in mammalian aging. In Handbook of 432 the biology of aging, pp. 25–45. Elsevier. 433 Crane, J. D., MacNeil, L. G., Lally, J. S., Ford, R. J., Bujak, A. L., Brar, I. K., Kemp, B. E., 434 Raha, S., Steinberg, G. R. and Tarnopolsky, M. A. (2015). Exercise-stimulated 435 interleukin-15 is controlled by AMPK and regulates skin metabolism and aging. Aging 436 Cell 14, 625–634. 437 Davalos, A. R., Kawahara, M., Malhotra, G. K., Schaum, N., Huang, J., Ved, U., 438 Beausejour, C. M., Coppe, J.-P., Rodier, F. and Campisi, J. (2013). p53-dependent 439 release of Alarmin HMGB1 is a central mediator of senescent phenotypes. J. Cell Biol. 440 201, 613–629. 441 Edrey, Y. H., Casper, D., Huchon, D., Mele, J., Gelfond, J. A., Kristan, D. M., Nevo, E. and 442 Buffenstein, R. (2012). Sustained high levels of neuregulin-1 in the longest-lived 443 ; a key determinant of rodent longevity. Aging Cell 11, 213–222. 444 El-Abaseri, T. B., Putta, S. and Hansen, L. A. (2006). Ultraviolet irradiation induces 445 keratinocyte proliferation and epidermal hyperplasia through the activation of the 446 epidermal growth factor receptor. Carcinogenesis 27, 225–231. 447 Eming, S. A., Hammerschmidt, M., Krieg, T. and Roers, A. (2009). Interrelation of immunity 448 and tissue repair or regeneration. Semin. Cell Dev. Biol. 20, 517–527. 449 Flurkey, K., Papaconstantinou, J. and Harrison, D. E. (2002). The Snell dwarf mutation 450 Pit1(dw) can increase life span in mice. Mech. Ageing Dev. 123, 121–130. 451 Freije, A., Molinuevo, R., Ceballos, L., Cagigas, M., Alonso-Lecue, P., Rodriguez, R., 452 Menendez, P., Aberdam, D., De Diego, E. and Gandarillas, A. (2014). Inactivation of 453 p53 in human keratinocytes leads to squamous differentiation and shedding via 454 replication stress and mitotic slippage. Cell Rep. 9, 1349–1360. 455 Freund, A., Laberge, R.-M., Demaria, M. and Campisi, J. (2012). Lamin B1 loss is a 456 senescence-associated biomarker. Mol. Biol. Cell 23, 2066–2075. 457 Fuchs, E. (2008). Skin stem cells: rising to the surface. J. Cell Biol. 180, 273–284. 458 Gawriluk, T. R., Simkin, J., Thompson, K. L., Biswas, S. K., Clare-Salzler, Z., Kimani, J. 459 M., Kiama, S. G., Smith, J. J., Ezenwa, V. O. and Seifert, A. W. (2016). Comparative 460 analysis of ear-hole closure identifies epimorphic regeneration as a discrete trait in 461 . Nat. Commun. 7, 11164. 462 Gawriluk, T. R., Simkin, J., Hacker, C. K., Kimani, J. M., Kiama, S. G., Ezenwa, V. O. and 463 Seifert, A. W. (2019). Mammalian musculoskeletal regeneration is associated with 464 reduced inflammatory cytokines and an influx of T cells. BioRxiv. 465 Hadi, F., Kulaberoglu, Y., Lazarus, K., Beattie, P., Smith, E. S. J. and Khaled, W. (2018). 466 Naked Mole-Rat Cells are Susceptible to Malignant Transformation by SV40LT and 467 Oncogenic Ras. BioRxiv. 468 Joven, A., Elewa, A. and Simon, A. (2019). Model systems for regeneration: salamanders. 469 Development 146,. 470 Kench, J. A., Russell, D. M., Fadok, V. A., Young, S. K., Worthen, G. S., Jones-Carson, J., 471 Henson, J. E., Henson, P. M. and Nemazee, D. (1999). Aberrant Wound Healing and 472 TGF-β Production in the Autoimmune-Prone MRL/+ Mouse. Clin. Immunol. 92, 300– 473 310. bioRxiv preprint doi: https://doi.org/10.1101/2020.05.07.083287; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

474 Keyes, B. E., Liu, S., Asare, A., Naik, S., Levorse, J., Polak, L., Lu, C. P., Nikolova, M., 475 Pasolli, H. A. and Fuchs, E. (2016). Impaired epidermal to dendritic T cell signaling 476 slows wound repair in aged skin. Cell 167, 1323–1338.e14. 477 Kowalczyk, A., Partha, R., Clark, N. L. and Chikina, M. (2020). Pan-mammalian analysis of 478 molecular constraints underlying extended lifespan. Elife 9,. 479 Liu, N., Matsumura, H., Kato, T., Ichinose, S., Takada, A., Namiki, T., Asakawa, K., 480 Morinaga, H., Mohri, Y., De Arcangelis, A., et al. (2019). Stem cell competition 481 orchestrates skin homeostasis and ageing. Nature 568, 344–350. 482 Lopez, R. G., Garcia-Silva, S., Moore, S. J., Bereshchenko, O., Martinez-Cruz, A. B., 483 Ermakova, O., Kurz, E., Paramio, J. M. and Nerlov, C. (2009). C/EBPalpha and beta 484 couple interfollicular keratinocyte proliferation arrest to commitment and terminal 485 differentiation. Nat. Cell Biol. 11, 1181–1190. 486 Maden, M. (2018). Optimal skin regeneration after full thickness thermal burn injury in the 487 spiny mouse, Acomys cahirinus. Burns 44, 1509–1520. 488 Maden, M., Brant, J. O., Rubiano, A., Sandoval, A. G. W., Simmons, C., Mitchell, R., 489 Collin-Hooper, H., Jacobson, J., Omairi, S. and Patel, K. (2018). Perfect chronic 490 skeletal muscle regeneration in adult spiny mice, Acomys cahirinus. Sci. Rep. 8, 8920. 491 Mikkelsen, S. A., Vangheluwe, P. and Andersen, J. P. (2018). A Darier disease mutation 492 relieves kinetic constraints imposed by the of sarco(endo)plasmic reticulum Ca2+- 493 ATPase 2b. J. Biol. Chem. 293, 3880–3889. 494 Moore, C., Cevikbas, F., Pasolli, H. A., Chen, Y., Kong, W., Kempkes, C., Parekh, P., Lee, 495 S. H., Kontchou, N.-A., Yeh, I., et al. (2013). UVB radiation generates sunburn pain and 496 affects skin by activating epidermal TRPV4 ion channels and triggering endothelin-1 497 signaling. Proc. Natl. Acad. Sci. USA 110, E3225-34. 498 Ota, T., Takekoshi, S., Takagi, T., Kitatani, K., Toriumi, K., Kojima, T., Kato, M., Ikoma, 499 N., Mabuchi, T. and Ozawa, A. (2014). Notch signaling may be involved in the 500 abnormal differentiation of epidermal keratinocytes in psoriasis. Acta Histochem. 501 Cytochem. 47, 175–183. 502 Ouhtit, A., Muller, H. K., Davis, D. W., Ullrich, S. E., McConkey, D. and Ananthaswamy, 503 H. N. (2000). Temporal events in skin injury and the early adaptive responses in 504 ultraviolet-irradiated mouse skin. Am. J. Pathol. 156, 201–207. 505 Pérez, V. I., Buffenstein, R., Masamsetti, V., Leonard, S., Salmon, A. B., Mele, J., Andziak, 506 B., Yang, T., Edrey, Y., Friguet, B., et al. (2009). Protein stability and resistance to 507 oxidative stress are determinants of longevity in the longest-living rodent, the naked 508 mole-rat. Proc. Natl. Acad. Sci. USA 106, 3059–3064. 509 Qiang, L., Sample, A., Shea, C. R., Soltani, K., Macleod, K. F. and He, Y.-Y. (2017). 510 Autophagy gene ATG7 regulates ultraviolet radiation-induced inflammation and skin 511 tumorigenesis. Autophagy 13, 2086–2103. 512 Robia, S. L. and Young, H. S. (2018). Skin cells prefer a slower calcium pump. J. Biol. Chem. 513 293, 3890–3891. 514 Saxena, S., Vekaria, H., Sullivan, P. G. and Seifert, A. W. (2019). Connective tissue 515 fibroblasts from highly regenerative mammals are refractory to ROS-induced cellular 516 senescence. Nat. Commun. 10, 4400. 517 Seifert, A. W., Kiama, S. G., Seifert, M. G., Goheen, J. R., Palmer, T. M. and Maden, M. 518 (2012). Skin shedding and tissue regeneration in African spiny mice (Acomys). Nature 519 489, 561–565. bioRxiv preprint doi: https://doi.org/10.1101/2020.05.07.083287; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

520 Seluanov, A., Hine, C., Azpurua, J., Feigenson, M., Bozzella, M., Mao, Z., Catania, K. C. 521 and Gorbunova, V. (2009). Hypersensitivity to contact inhibition provides a clue to 522 cancer resistance of naked mole-rat. Proc. Natl. Acad. Sci. USA 106, 19352–19357. 523 Seluanov, A., Gladyshev, V. N., Vijg, J. and Gorbunova, V. (2018). Mechanisms of cancer 524 resistance in long-lived mammals. Nat. Rev. Cancer 18, 433–441. 525 Simkin, J., Gawriluk, T. R., Gensel, J. C. and Seifert, A. W. (2017). Macrophages are 526 necessary for epimorphic regeneration in African spiny mice. Elife 6,. 527 Sokabe, T., Fukumi-Tominaga, T., Yonemura, S., Mizuno, A. and Tominaga, M. (2010). 528 The TRPV4 channel contributes to intercellular junction formation in keratinocytes. J. 529 Biol. Chem. 285, 18749–18758. 530 Streeter, K. A., Sunshine, M. D., Brant, J. O., Sandoval, A. G. W., Maden, M. and Fuller, 531 D. D. (2019). Molecular and histologic outcomes following spinal cord injury in spiny 532 mice, Acomys cahirinus. J. Comp. Neurol. 533 Tian, X., Azpurua, J., Ke, Z., Augereau, A., Zhang, Z. D., Vijg, J., Gladyshev, V. N., 534 Gorbunova, V. and Seluanov, A. (2015). INK4 of the tumor-resistant rodent, the 535 naked mole rat, expresses a functional p15/p16 hybrid isoform. Proc. Natl. Acad. Sci. 536 USA 112, 1053–1058. 537 Trevithick, J. R., Xiong, H., Lee, S., Shum, D. T., Sanford, S. E., Karlik, S. J., Norley, C. 538 and Dilworth, G. R. (1992). Topical tocopherol acetate reduces post-UVB, sunburn- 539 associated erythema, edema, and skin sensitivity in hairless mice. Arch. Biochem. 540 Biophys. 296, 575–582. 541 Ueno, M., Lyons, B. L., Burzenski, L. M., Gott, B., Shaffer, D. J., Roopenian, D. C. and 542 Shultz, L. D. (2005). Accelerated wound healing of alkali-burned corneas in MRL mice 543 is associated with a reduced inflammatory signature. Invest. Ophthalmol. Vis. Sci. 46, 544 4097–4106. 545 Wang, M., Qin, X., Mudgett, J. S., Ferguson, T. A., Senior, R. M. and Welgus, H. G. (1999). 546 Matrix metalloproteinase deficiencies affect contact hypersensitivity: stromelysin-1 547 deficiency prevents the response and gelatinase B deficiency prolongs the response. Proc. 548 Natl. Acad. Sci. USA 96, 6885–6889. 549 Wang, A. S., Ong, P. F., Chojnowski, A., Clavel, C. and Dreesen, O. (2017). Loss of lamin 550 B1 is a biomarker to quantify cellular senescence in photoaged skin. Sci. Rep. 7, 15678. 551 Williams, S. E., Beronja, S., Pasolli, H. A. and Fuchs, E. (2011). Asymmetric cell divisions 552 promote Notch-dependent epidermal differentiation. Nature 470, 353–358. 553 Wong, W., Crane, E. D., Kuo, Y., Kim, A. and Crane, J. D. (2019). The exercise cytokine 554 interleukin-15 rescues slow wound healing in aged mice. J. Biol. Chem. 294, 20024– 555 20038. 556 Yoshinaga, K., Obata, H., Jurukovski, V., Mazzieri, R., Chen, Y., Zilberberg, L., Huso, D., 557 Melamed, J., Prijatelj, P., Todorovic, V., et al. (2008). Perturbation of transforming 558 growth factor (TGF)-beta1 association with latent TGF-beta binding protein yields 559 inflammation and tumors. Proc. Natl. Acad. Sci. USA 105, 18758–18763. 560 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.07.083287; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Figure 1. Acute UVB-exposure induces distinctive pattern of skin epidermal differentiation in A. cahirinus. A, representative brightfield microscopy images of hematoxylin and eosin-stained skin from control (sham) and UV-irradiated M. musculus and A. cahirinus, collected 24 and 48 hours after exposure. Scale bar = 100 µm. B, quantification of epidermal thickness. n = sham both speces, 3; 24hr M. musculus, 6; 24hr A. cahirinus, 5; 48hr both species, 5 mice per group. C, representative immunofluorescence images of epidermal BrdU labeling. The epidermal basement membrane is indicated by the yellow dashed line. Positive cells are indicated by the pink arrows. Scale bar = 25 µm. D and E, quantification BrdU labeling in (D) basal epidermis and (E) suprabasal epidermis. n = sham both species, 3; 24hr both species, 5; 48hr M. musculus, 4; 48hr A. cahirinus, 5 mice per group. F, representative immunofluorescence images of epidermal differentiation markers keratin 14 (K14) and keratin 10 (K10) labeling with magnified inset of the basal-suprabasal junction. Scale bar = 50 µm. G, quantification of individual differentiation marker layer thickness. n = 3 mice per group. Loricrin images and individual layer comparisons included in Supplemental Figure 1A-E. Data points are biological replicates. Lines indicate group means. *, significantly different from the indicated group (p<0.05). #, significantly different relative to sham control (p<0.05). †, significantly different (p<0.05) to M. musculus at the same timepoint.

Figure 2. Efficient removal of damaged and dying skin epidermal cells through rapid turnover and apoptosis in acute UVB-exposed A. cahirinus. A, representative immunofluorescence images of epidermal thymine dimer (T-T dimer) labeling of skin from control (sham) and UV-irradiated M. musculus and A. cahirinus, collected 24 and 48 hours after exposure. B and C, quantification of thymine dimer labeling in (B) basal epidermis and (C) suprabasal epidermis. D, representative immunofluorescence images of epidermal γH2AX labeling. E and F, quantification γH2AX labeling in (D) basal epidermis and (E) suprabasal epidermis. G, representative immunofluorescence images of epidermal cleaved caspase-3 (CC3) labeling. Positive cells are indicated by the pink arrows. H and I, quantification of cleaved caspase-3 labeling in (H) basal and (I) suprabasal epidermis. Data points are biological replicates. Lines indicate group means. *, significantly different from the indicated group (p<0.05). #, significantly different relative to sham control (p<0.05). †, significantly different (p<0.05) to M. musculus at the same timepoint. The epidermal basement membrane is indicated by the yellow dashed line. All scale bars = 50 µm. All measurements n = sham both speces, 3; 24hr both species, 5; 48hr both species, 5 mice per group.

Figure 3. Attenuated skin epidermal inflammatory response following UV- irradiation in A. cahirinus. A, representative immunofluorescence images of epidermal HMGB1 labeling of skin from control (sham) and UV-irradiated M. musculus and A. cahirinus, collected 24 and 48 hours after exposure. The epidermal basement membrane is indicated by the yellow dashed line. Scale bar = 50 µm. B and C, quantification of nuclear HMGB1 labeling in (B) basal epidermis and (C) suprabasal epidermis. n = sham both speces, 3; 24hr both species, 5; 48hr both species, 5 mice per group. D, E, F, and G, mRNA expression (ΔCt) of (D) Cxcl1, (E) Il1b, (F) Tgfb1, and (G) Mmp9 in each treatment group as measured by qPCR. n = sham M. musculus, 3; sham A. cahirinus, 3; bioRxiv preprint doi: https://doi.org/10.1101/2020.05.07.083287; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

24hr M. musculus, 6, 24hr A. cahirinus, 5; 48hr M. musculus, 6; 48hr A. cahirinus, 3 mice per group for Cxcl1. n = sham M. musculus, 4; sham A. cahirinus, 3; 24hr M. musculus, 6; 24hr A. cahirinus, 3; 48hr M. musculus, 5; 48hr A. cahirinus, 3 mice per group for Il1b. n = sham M. musculus, 4; sham A. cahirinus, 3; 24hr M. musculus, 5; 24hr A. cahirinus, 5; 48hr M. musculus, 6; 48hr A. cahirinus, 5 mice per group for Tgfb1. n = sham M. musculus, 4; sham A. cahirinus, 3; 24hr M. musculus, 5; 24hr A. cahirinus, 5; 48hr M. musculus, 6; 48hr A. cahirinus, 5 mice per group for Mmp9. Data points are biological replicates. Lines indicate group means. *, significantly different from the indicated group (p<0.05). #, significantly different relative to sham control (p<0.05). †, significantly different (p<0.05) to M. musculus at the same timepoint.

Figure 4. Aging associated epidermal thinning, inflammation, and senescence are absent in A. cahirinus. A, representative brightfield microscopy images of hematoxylin and eosin-stained intact skin from control (Young) and aged (Old) M. musculus and A. cahirinus. Scale bar = 100 µm. B, quantification of epidermal thickness. C, representative immunofluorescence images of epidermal Lamin B1 labeling of skin from each treatment group. The epidermal basement membrane is indicated by the yellow dashed line. Scale bar = 50 µm. n = young M. musculus, 7; young A. cahirinus, 8; old both species, 8 mice per group. D, quantification of epidermal Lamin B1 labeling. n = young M. musculus, 6; young A. cahirinus, 8; old M. musculus, 5, old A. cahirinus, 6 mice per group. E, representative immunofluorescence images of epidermal HMGB1 labeling of skin from each treatment group. The epidermal basement membrane is indicated by the yellow dashed line. Scale bar = 50 µm. F, quantification of epidermal nuclear HMGB1 labeling. n = young M. musculus, 6; young A. cahirinus, 7; old M. musculus, 6, old A. cahirinus, 7 mice per group. Data points are biological replicates. Lines indicate group means. *, significantly different from the indicated group (p<0.05).

Supplemental Figure 1. Acute UVB-exposure induces distinctive pattern of skin epidermal differentiation in A. cahirinus. A, representative immunofluorescence images of epidermal differentiation markers keratin 14 (K14) and loricrin (Lor) labeling with magnified inset of the basal-suprabasal junction. B, C, D, E individual layer thickness quantification of the (B), K14+/K10– single positive basal layer (C), K14+/K10+ double positive middle suprabasal layer (D), K14–/K10– single positive spinous layer and (E), Lor+ single positive cornified envelope. n = 3 mice per group. Data points are biological replicates. Lines indicate group means. *, significantly different from the indicated group (p<0.05).

A G Layer thickness (μm) + E 48 hours 24 hours 20 40 60 80 Suprabasal BrdU cells 0 3 post-UV post-UV Sham .musculus M.

per 1020 μm40 epidermis60 80 sham MM 0 .musculus M. hm2h 48hr 24hr sham bioRxiv preprint 24hr K14 K14 MM 10 was notcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade 0 µm + + /K10 /K10 48hr MM .cahirinus A. – + * sham AC .cahirinus A. # # doi: # † 24hr K14 Lor AC https://doi.org/10.1101/2020.05.07.083287 + – B 48hr /K10 AC Epidermal thickness (μm) 100 20 40 60 80 + 0 .cahirinus A. musculus M. F 48 hours 24 hours 48hr 24hr sham post-UV post-UV Sham .musculus M. available undera * * DAPI K10 K14 Merge 5 # 0 µm .cahirinus A.

48 hours 24 hours C CC-BY-NC-ND 4.0Internationallicense post-UV post-UV

Sham ; .musculus M. this versionpostedMay10,2020. .musculus M. DAPI BrdU 2 5 µm .cahirinus A. K14 .cahirinus A.

Basal BrdU+ cells D The copyrightholderforthispreprint(which 3 . .musculus M. per 10 μm epidermis 100 20 40 60 80 0 hm2h 48hr 24hr sham K10 .cahirinus A. # iue1 Figure * # # D A 48 hours 24 hours G 48 hours 24 hours 48 hours 24 hours post-UV post-UV Sham post-UV post-UV Sham post-UV post-UV Sham .musculus M. .musculus M. .musculus M. 5 0 µm bioRxiv preprint 5 5 0 0 was notcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade µm µm .cahirinus A. .cahirinus A. .cahirinus A. - dimer T-T γH2AX doi: DAPI DAPI DAPI CC3 https://doi.org/10.1101/2020.05.07.083287 B H E Basal CC3+ Basal γH2AX+ cells Basal thymine dimer+ cells per 103 μm epidermis per 103 μm epidermis cells per 103 μm epidermis 10 15 20 40 60 80 20 40 60 0 5 0 0 .cahirinus A. musculus M. hm2h 48hr 24hr sham hm2h 48hr 24hr sham hm2h 48hr 24hr sham p=0.0664 available undera # # # p=0.0700 # † † * * # † C CC-BY-NC-ND 4.0Internationallicense + I + F + Suprabasal CC3 Suprabasal γH2AX cells Suprabasal thymine dimer ; this versionpostedMay10,2020. cells per 103 μm epidermis per 103 μm epidermis cells per 103 μm epidermis 100 80 20 40 60 10 15 20 20 40 60 80 0 0 5 0 hm2h 48hr 24hr sham hm2h 48hr 24hr sham hm2h 48hr 24hr sham NS p=0.0685 p=0.0913 # # # † † * # # # # † # † The copyrightholderforthispreprint(which . iue2 Figure 48 hours 24 hours A post-UV post-UV Sham .msuu .cahirinus A. musculus M. HMGB1 bioRxiv preprint DAPI was notcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade 2 5 µm E Il1b mRNA (ΔCt) 0 1 2 3 4 doi: sham https://doi.org/10.1101/2020.05.07.083287 † + B

4r48hr 24hr Basal Nuclear HMGB1 NS cells per 103 μm epidermis † 100 150 50 0 .cahirinus A. musculus M. hm2h 48hr 24hr sham † available undera F Tgfb1 mRNA (ΔCt) 0.0 0.2 0.4 0.6 sham # * CC-BY-NC-ND 4.0Internationallicense C 4r48hr 24hr + Suprabasal Nuclear HMGB1 ; this versionpostedMay10,2020. † * cells per 103 μm epidermis 100 150 200 50 0 hm2h 48hr 24hr sham G Mmp9 mRNA (ΔCt) 0.0 0.2 0.4 0.6 NS sham * p 4r48hr 24hr 0.0650 = * D The copyrightholderforthispreprint(which Cxcl1 mRNA (ΔCt) . 0.0 0.2 0.4 0.6 sham 4r48hr 24hr * iue3 Figure p 0.054 = bioRxiv preprint doi: https://doi.org/10.1101/2020.05.07.083287; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A B M. musculus A. cahirinus M. musculus A. cahirinus ) 100µm

m 18 * μ 16 Young

14

12 Old

10 Epidermal thickness ( Young Old C M. musculus A. cahirinus D DAPI 150 Lamin B1 * Young cells 100 + epidermis m μ

3 50 Old Lamin B1

50 µm per 10 0 Young Old E M. musculus A. cahirinus F DAPI 150 HMGB1 * cells +

Young 100 m epidermis μ

3 50 Old

50 µm per 10 Nuclear HMGB1 0 Young Old Figure 4 D B A K14-/K10+ K14+/K10- 48 hours 24 hours Layer thickness (μm) Layer thickness (μm) post-UV post-UV Sham .musculus M. 10 15 20 10 20 30 40 0 5 0 bioRxiv preprint sham sham MM MM was notcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade .musculus M. .musculus M. Merge 5 0 µm 24hr 24hr MM MM * .cahirinus A. 48hr 48hr MM MM doi: NS DAPI https://doi.org/10.1101/2020.05.07.083287 K14 Lor sham sham AC AC .cahirinus A. .cahirinus A. .musculus M. 24hr 24hr AC AC 48hr 48hr AC AC K14 available undera .cahirinus A. Lor+ E K14+/K10+ C Layer thickness (μm) Layer thickness (μm) 10 15 20 0 5 0 2 4 6 8 sham sham MM MM .musculus M. CC-BY-NC-ND 4.0Internationallicense .musculus M. .musculus M. ; this versionpostedMay10,2020. 24hr 24hr MM MM Loricrin 48hr 48hr MM MM .cahirinus A. NS sham sham AC AC .cahirinus A. .cahirinus A. 24hr 24hr AC AC * 48hr 48hr AC AC The copyrightholderforthispreprint(which . upeetl1 Supplemental