DOI: 10.5061/dryad.wpzgmsc0t
title: Data from: Evolutionary adaptations of TRPA1 thermosensitivity and skin thermoregulation in vertebrates

description: Altering skin color and reflectance is crucial for temperature regulation
 in poikilothermic vertebrates, while less so in homeotherms like birds and
 mammals, which evolved feathers, fur, and other insulation for endothermy.
 Heat-sensing in vertebrates relies primarily on Transient Receptor
 Potential (TRP) channels, with certain channels (TRPA1) shifting
 thermosensitivity over evolution and others retaining heat sensitivity
 (TRPV1). Exploration of a role for TRP channels in skin physiology has
 largely focused on human pigmentation and overlooked the evolution of
 different thermoregulatory structures in the integument of distinct
 vertebrates. For instance, colour/reflector pigment cells in ectotherms,
 fur and feathers in endotherms, hairless skin in hominids, and blubber in
 marine mammals. Therefore, we investigated whether a TRP channel mediates
 skin darkening induced by heat in the ectotherm Xenopus laevis and then
 explored the evolution of TRPA1 thermal sensitivity and its link with skin
 physiology. We find Trpa1 mediates heat-induced melanosome dispersion,
 darkening skin under warmer conditions. In contrast, TRPA1 is known to
 mediate cold sensation in rodents and UV-induced tanning in humans,
 leading us to investigate the co-evolution of TRPA1 and skin
 thermoregulation. Our findings reveal that TRPA1 is a heat sensor in
 ectotherms with uncovered integuments. In mammals, we suggest that TRPA1
 was thermally insensitive in Euarchontoglires but became cold-sensitive in
 several rodent lineages. TRPA1 shows reduced selection pressure for
 thermosensitivity in aquatic mammals (manatees, whales) that depend on
 blubber for insulation as compared to their terrestrial relatives. These
 findings emphasize adaptive evolution of TRPA1 in vertebrates, linking
 thermal sensitivity to the evolution of skin physiology.
description: <strong>Embryos, drug treatment, and warm
 treatment</strong> The Animal Care and Use
 Committee, University of Calgary, approved procedures involving frogs and
 embryos (AC21-0148; signed by Dr. Derrick Rancourt). Embryos were obtained
 by induced egg production from chorionic gonadotrophin (Intervet Canada
 Ltd.) injected females and in vitro fertilization according to the
 standard procedures (see protocols at Xenbase (http://www.xenbase.org)). Embryos were maintained at 16 °C in Marc’s modified Ringer’s (MMR) solution (100mM NaCl, 2mM KC1, 2mM CaCl<sub>2</sub>, 1mM MgCl<sub>2</sub>, 5mM HEPES, pH 7.4) until stage 43/44 (approximately 1 week) and staged according to Nieuwkoop and Faber on Xenbase (http://www.xenbase.org). The embryos were reared under light cycles of 12 h ON/ 12 h OFF (light =1000 lux or approximately 1.5 × 10–4W/cm<sup>2</sup>) on a white background. For experiments, the embryos were set in 35 mm dishes with 4 ml of MMR in a 16 °C or 32 °C incubator for the indicated times. A non-noxious warm temperature was chosen (32 °C) based on the average temperatures recorded over the last 30 years for three national parks in southern Africa (Etosha/Namibia; Kruger/South Africa, and Hawange/Zimbabwe) obtained from the Meteoblue website (https://www.meteoblue.com/en/weather/historyclimate/). For immunohistochemistry, embryos at 48 h post-fertilization were treated with 0.02% 1-phenyl-2-thiourea (PTU), an inhibitor of eumelanin pigment formation (Bertolesi et al., 2015). Pharmacological studies were performed with the following TRP agonists added to the MMR rearing solution containing tadpoles or MEX cells at 16 °C to determine their effect on pigmentation: 1) Piperine, a TRPV1 agonist [Abcam; (Ab142933)]; 2) GSK1016790A, a TRPV4 agonist [Millipore Sigma (G0798)] and 3) Allyl isothiocyanate (AITC), a TRPA1 agonist (Millipore Sigma; W203408). The following antagonists were added at 16 °C to MMR solution containing tadpoles or melanophores cells in growth medium, which were then switched immediately to 32°C: 1) Capsazepine, a TRPV1 antagonist [Abcam (Ab120025)]; 2) GSK2193874, a TRPV4 antagonist (Tocris Bioscience; #5106); and 3) A-967079, a TRPA1 antagonist (Millipore Sigma; SML0085). Of note, the maximum dose tested varied depending on either solubility in aqueous solution or drug toxicity. The agonists and antagonists used are organic compounds with low solubility in aqueous solutions; therefore were initially dissolved in dimethyl sulfoxide (DMSO) before dilution (1/1000) in MMR or growth medium. For example, the highest piperine dose tested was 100 µM (diluted from a 100 mM DMSO stock solution) as its maximum solubility in water is 140 µM. Toxicity causing death of tadpoles was identified by stasis after one hour, which failed to reappear upon switching to a drug-free condition. Note that in general, toxicity and death were followed by skin lightening. <strong>Determination of pigmentation index</strong> We quantified changes in skin pigmentation by measuring skin pigmentation indices as described previously (Bertolesi et al., 2015). Briefly, pictures of the dorsal head of tadpoles were taken using a stereoscope (Stemi SV11; Carl Zeiss Canada, Ltd., Toronto, Canada) and a camera (Zeiss; Axiocam HRC), with identical conditions of light, exposure time, and diaphragm aperture. Pictures were converted to binary white/black images using NIH ImageJ (U. S. National Institutes of Health, Bethesda, MD) public domain software. <em><strong>Xenopus laevis</strong></em> <strong>melanophore (MEX) cell culture</strong> To test the pigmentation response to temperature, we employed the melanophore (MEX) cell line originally generated from stage 35 <em>Xenopus laevis</em> embryos (Kashina et al., 2004). Cells were maintained in growth medium (70% Leibovitz’s L15 medium with 25% added water and supplemented with 5% fetal bovine serum (Invitrogen) without antibiotics). To mimic the conditions observed <em>in vivo</em>, cells were maintained in medium without phenol red to maximize light penetration. For the pigmentation heat response, cultures at 16 °C were switched to 32 °C. During the warming paradigm, light from above (1000 lux) was maintained, as described previously for tadpoles. Cells were fixed with 4% paraformaldehyde and stained with DAPI (1 µg/µl) before imaging. <strong>Identification and expression of</strong> <em><strong>Xenopus</strong></em> <strong>TRP channels</strong> The screening and identification of <em>Xenopus laevis</em> <em>TRPV</em> and <em>trpa</em> members was described recently (Malik et al., 2023). The expression of <em>trp</em> mRNAs in whole stage 43/44 embryos and melanophores was assessed by RT-PCR. Total RNA was obtained from whole embryos, surgically isolated tails, and MEX cells using TRIzol (Invitrogen) according to the manufacturer’s protocol. Single-strand cDNA was produced from RNA samples (5 µg) by priming with oligo(dT) primers using SuperScriptTM IV reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. All PCR amplifications were carried out in a total volume of 20 µL with 1 µL of cDNA, 2 µL of primers, 7 µL of water, and 10 µL of 2X PCR master mix (Thermo Scientific, IL). PCR amplifications were carried out between 40 and 45 cycles and with an annealing temperature of 55 °C. PCR products obtained from cDNA were cloned into TOPO-pCRII (Invitrogen) vectors and sequenced to confirm identity. Specific primers were designed to amplify both homologous variants (on L and S chromosomes) of the different Trp channels (Supplementary Table 1). The sequence of the different <em>trp</em> channels can be obtained from the GenBank-NCBI database with the gene name accession numbers provided in Fig. 3A. <strong>Immunohistochemistry for Trpv1 and Trpa1</strong> Immunohistochemistry against Trpv1 and Trpa1 was performed as described recently (Malik et al., 2023) using the following antibodies: anti-crocodile Trpv1 (rabbit polyclonal; 1/200 dilution; Thermo-Fisher Scientific; #OST00058W) and anti-human TRPA1 (rabbit polyclonal; 1/200 dilution; Novus Biologicals; NB110-4076SS). The identification of skin melanophores was performed with a rabbit polyclonal antibody that recognizes the Tyrp-1 (1:200 dilution; Thermo-Fisher Scientific, IL; PA5-81909), a specific enzyme involved in melanin synthesis. Since antibodies against Trp channels and the melanophore marker (Tyrp-1) were all generated in rabbits, the analysis of co-expression was performed in consecutive 12 µm transverse frozen cryostat sections obtained from stage 43/44 embryos. Following detection with primary antibodies, slides were treated with a secondary antibody (1:1000 dilution of Alexa Fluor 488) and DAPI (1 µg/µl) to stain nuclei. <strong>Grouping in clades and analysis of TRPA1 thermal sensitivity from the literature.</strong> Vertebrates were divided into 26 groups based on taxon clades [(Integrated Taxonomic Information System (ITIS) (https://www.itis.gov/)] with demonstrated evolutionary relationships. Group 1 contains species in the infraphylum agnathan (cyclostomes), while Groups 2 to 26 correspond to some, but not all, extant gnathostomes. We initially grouped based on the taxonomic clade “Class”. The Group 2 contains the cartilaginous fish (sharks; taxonomic class, Chondrichthyes), while the ray‐finned fish (Actinopterygii) were divided into 4 groups also representing taxonomic class; The Cladistei (Group 3, bichir), Chondrostei (Group 4; sturgeons and paddlefishes) Holostei (Group 5; gars and bowfins) and Teleostei (Group 6; the largest clade of bony fish with several examples of TRPA1 cloned and characterized). The following class groups are Coelacanths and Dipnoi (Group 7; Sarcopterygii, lung fish), the Amphibians (Group 8; frogs, salamanders, and caecilians), reptiles (Group 9; lizards, snakes, turtl,es and crocodiles), Aves (Group 10 and 11), and mammals (Group 12 to 26). The Avian Sauropods were divided into two groups representing the ‘Inferior class’ Paleognathae (Group 10), where most primitive and mainly flightless birds reside, and Neognathae (Group 11), which include almost all living flight bird species. The three mammalian infraclasses correspond to monotremes (Group 12; egg-laying mammals), marsupials (Group 13), and eutherians (Groups 14 to 26). Groups 14 to 23 were generated to compare three marine mammals with their terrestrial relatives: i) In the superorder Afrotheria, the Sirenians (Group 14; manatees and dugongs) were compared with elephants (Group 15; Proboscidea) and African insectivores (Group 16; aardvarks and others), ii) The cetaceans (Group 17; whales) were compared to hippopotamuses (Group 18), ruminants (Group 19), and suids (Group 20; pigs), and iii) Seals and sea lions (Group 21; Pinnipeds), marine mammals, which were compared with other carnivores including minks, otters, and ferrets (Group 22; Musteloidea), and bears (Group 23; Ursidae). The superorder Euarchontoglires (Group 24 to 26) contains most of the species where TRPA1 is well characterized, including the lagomorphs (Group 24; rabbits, hares, and pikas), rodents (Group 25; mice and rats), and primates (Group 26; humans and monkeys). <strong>Alignment and phylogenetic analysis</strong> Validated sequences (NCBI numbers in Supplementary Table 2) were aligned using MUSCLE (multiple sequence alignment) to build a hidden Markov model (HMM) by using a maximum‐likelihood architecture construction algorithm. All phylogenetic analyses and alignments were performed using the public domain MEGA X software (www.megasoftware.net) (Kumar et al., 2018). <strong>Microscopy</strong> Images of embryos were taken with an Axio-Cam HRc (Carl Zeiss) on the Stemi SVII stereomicroscope (Carl Zeiss). Section images were processed for brightness and contrast with Adobe Photoshop. <strong>Statistics analysis and reproducibility</strong> GraphPad Prism 10.0 software was used for statistical analysis of data and graphic preparation. Statistical analysis is ANOVA followed by Bonferroni’s test. Significance was considered at p &lt; 0.05. Experiments were performed three independent times (N=3) unless otherwise indicated. Since pigmentation index varies between hatches, a representative experiment is shown in each figure. The independent experiments showed similar trends. Each experimental treatment contained a minimum of 9 tadpoles (n; generally, ≥9), which are represented in the figures as individual data points. Additionally, figures show a box plot (25th to 75th percentile) of the mean and 95% confidence interval. Immunohistochemical analyses were performed at less than three times (N 3), with 4 independent tadpoles (n = 4) in each experiment. CorelDRAW 10.0 was used to compile multipaneled figures.
description: # Data from: Evolutionary adaptations of TRPA1 thermosensitivity and skin
 thermoregulation in vertebrates Dataset DOI:
 [10.5061/dryad.wpzgmsc0t](10.5061/dryad.wpzgmsc0t) Name: Gabriel E.
 Bertolesi Institution: University of Calgary Email:
 [gbertole@ucalgary.ca](mailto:gbertole@ucalgary.ca) ## Description of the
 data and file structure We analyzed the effect of temperature on the
 pigmentation of tadpoles (Fig 1; variable: pigmentation index in  File
 Figure 1 A, C and D). The effect on melanosome dispersion is analyzed on
 melanophores (MEX cells) in-vitro (Fig 2; variable: percentage of cells
 with dispersed melanosomes; File Figure 2). Those studies are analyzed
 again in response to TRP agonist and antagonist (Figure 4; File   Figure 4
 in vivo and Figure 4 in vitro) as well as after transfection with siRNA
 for TRPA1 (Figure 5; File Figure 5). An evolutionary analysis of TRPA1 is
 performed in the second part of the manuscript. Figure_1_D.txt: Kinetic
 from 16 to the indicated temperatures Variable: Pigmentation Index
 Figure_1A_1C.txt:  Figure 1A: Kinetic from 16 to 32 degrees and
 reversibility Variable: Pigmentation Index Figure 1C:  Pigmentation Index
 melanophores different body parts Figure_2_B_2_C.txt:  Figure 2B: MEX
 cells Kinetic from 16 to 32 degrees and reversibility Variable: Number of
 cells with melanin disperse Figure 2C: MEX cells Kinetic from 16 to
 indicated temperature Figure_4_in_vitro.txt: Temperature of agonist
 measured at 16 Celsius degrees antagonist, added at 16 Celcius degrees and
 switched to 32 Celsius degrees Figure_4_in_vivo.txt: Concentration of
 agonists and antagonists of TRP channels; Temperature agonist measured at
 16 Celsius degrees and antagonist, added at 16 Celsius degrees and
 switched to 32 Celsius degrees. Figure_5.txt: MEX cells transfected with
 GFP alone or GFP plus AS-L/S or GPF plus Sense