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Dev Growth Differ
2023 May 01;654:194-202. doi: 10.1111/dgd.12848.
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Xenopus: An in vivo model for studying skin response to ultraviolet B irradiation.
El Mir J
,
Fedou S
,
Thézé N
,
Morice-Picard F
,
Cario M
,
Fayyad-Kazan H
,
Thiébaud P
,
Rezvani HR
.
Abstract
Ultraviolet B (UVB) in sunlight cause skin damage, ranging from wrinkles to photoaging and skin cancer. UVB can affect genomic DNA by creating cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidine (6-4) photoproducts (6-4PPs). These lesions are mainly repaired by the nucleotide excision repair (NER) system and by photolyase enzymes that are activated by blue light. Our main goal was to validate the use of Xenopus laevis as an in vivo model system for investigating the impact of UVB on skin physiology. The mRNA expression levels of xpc and six other genes of the NER system and CPD/6-4PP photolyases were found at all stages of embryonic development and in all adult tissues tested. When examining Xenopus embryos at different time points after UVB irradiation, we observed a gradual decrease in CPD levels and an increased number of apoptotic cells, together with an epidermal thickening and an increased dendricity of melanocytes. We observed a quick removal of CPDs when embryos are exposed to blue light versus in the dark, confirming the efficient activation of photolyases. A decrease in the number of apoptotic cells and an accelerated return to normal proliferation rate was noted in blue light-exposed embryos compared with their control counterparts. Overall, a gradual decrease in CPD levels, detection of apoptotic cells, thickening of epidermis, and increased dendricity of melanocytes, emulate human skin responses to UVB and support Xenopus as an appropriate and alternative model for such studies.
FIGURE 1. Gene expression pattern of NER proteins, CPD photolyase (phr), and 6–4PP photolyase (6–4phr) in Xenopus laevis. (a) Conserved syntenic regions between human (Hsa), mouse (Mmu), and Xenopus laevis (Xl) chromosome regions containing xpc, ercc2 to ercc6 and ddb1 loci. (b) Comparison of syntenic regions around Xenopus phr and 6–4 phr genes showing the absence of the genes in the mammalian genomes. The chromosome number for each species is indicated. Boxes with the same color correspond to ortholog genes. The versions of the genomes used to establish are as follows: Human, GRCH38; p7; mouse, GRCm38; Xenopus laevis version 9.1; the drawing is not on scale to avoid complexity. (c–d) RT–PCR analysis of RNA expression of xpc, phr, and 6–4phr in embryos at stage 2 to stage 42 (c) and in adult tissues (d). (e–f) RT–PCR analysis of RNA expression of ercc3 to 5 and ddb1 in embryos at stage 2 to stage 42 (e) and in adult tissues (f). Br, brain; H, heart; Li, liver; Lu, lung; M, skeletal muscle; Ov, ovary; Sk, skin; Sp, spleen; St, stomach; T, testis. Odc and rpl8 gene expression was used as control. -RT, control without reverse transcription.
FIGURE 2. Removal kinetics of CPDs from genome following exposure of Xenopus embryos to UVB irradiation and blue light. (a) UVB irradiation device. Embryos were placed on agarose dish (upper panel) and exposed to UVB on their left side (lower panel). (b) Immunostaining analysis of a histological section at the level of the head of a stage 42 irradiated embryo. CPDs (red arrow) are found on the irradiated side (Ir) but absent from the non-irradiated side (nIr). Br, brain; Scale bar is 150 μm. The nuclei were stained with DAPI (blue color). (c) Photoreactivation and NER systems cooperate together in Xenopus embryos to remove UVB-induced DNA damage. Following their activation with blue light, photolyases break up directly pyrimidine dimers. The NER system is a multistep process, in which several proteins are involved in recognition of DNA damage, excision of the lesion and surrounding nucleotides and finally filling the gap by de novo DNA synthesis. (d) CPD quantities (DNA repair capacity) were assessed on embryos genomic DNA by immune dot blot analysis at indicated time points after irradiation. Embryos were kept after irradiation either in dark (Dark) or under blue light (Blue). SYBR green was used as loading control. (e) Quantification of the dot bot results showed in d. n = 4 per condition, *p < 0.05, **p < 0.01, and ***p < 0.0005 for irradiated embryos at the indicated time points versus the initial levels. §p < 0.01 for embryos kept in dark versus embryos exposed to blue light.
FIGURE 3. Skin homeostasis following exposure of Xenopus embryos to UVB irradiation. (a) Experimental design. Embryos were UVB irradiated and in toto analyzed 3 hr postirradiation for pHH3 and dendricity and 24 hr postirradiation for apoptosis and skin histological analysis. (b) Histological section of a stage 42 embryo stained by hematoxylin–eosin (Left panel) showing epidermal thickness of the irradiated (Ir) side compared with non-irradiated (nIr) side. Scale bar is 250 μm. Upper right panel, enlargement of the framed regions displayed on the left (solid line for nIr side and dotted line for Ir side). Lower right panel, immunostaining of the framed regions with anti-keratin 2 antibody. Scale bar is 20 μm. (c) Quantification of epidermal thickness. Values represent the mean ± SD of epidermal thickness measured in nine randomly selected fields of view per embryos (n = 6). (d) Detection of apoptosis by TUNEL assay of irradiated embryos that has been kept in the dark (Dark) or under blue light (Blue) after irradiation. The irradiated (Ir) versus non-irradiated (nIr) sides of embryos are shown (Scale bar is 1 mm). An enlarged region is shown on the right (Scale bar is 200 μm). (e) Quantification of TUNEL-positive cells. Cells that showed intense TUNEL staining were scored positive (n = 6). The numbers of TUNEL-positive cells (i.e., apoptotic cells) is remarkably reduced in blue light-exposed embryos (Blue) compared with embryos kept in the dark (Dark). (f) Measurement of caspase-3 activity indicates that apoptotic cell death induction is reduced in blue light-exposed embryos compared with embryos kept in the dark (n = 5 embryos per condition). (g) Proliferation rate analysis by pHH3 assay of irradiated embryos that have been kept in the dark (Dark) or under blue light (Blue) after irradiation. The irradiated (Ir) versus non-irradiated (nIr) sides of embryos are shown (Scale bar is 1 mm). An enlarged region is shown on the right (Scale bar is 200 μm). (H) The number of pHH3-positive cells was reduced in irradiated embryos in the dark compared with blue light-exposed embryos where proliferation rate has been restored (n = 5 embryos per condition). (i–j) Ventral region of a stage 42 embryo showing melanocytes at 9 hr after irradiation (Ir). The proportion of the skin surface covered by melanocytes was significantly increased after irradiation, suggesting an increased number of dendrites (n = 5 embryos per condition). Scale bar is 500 μm. *p < 0.05 for Ir versus nIr embryos. § < 0.05 for embryos exposed to blue light versus embryos kept in dark.
FIGURE S1. Protein sequence alignment of Xenopus and mammalian XPC. The primary structures of XPC from X. laevis (XP_018114551.1), human (NP_004619.3), and mouse (NP_033557.2) are shown. Identical amino acids are denoted by an asterisk (*), highly similar residues by a colon (:), and weakly similar residues by a period (.). Gaps are indicated as dashes (−). The DNA-binding domain of the proteins is highlighted in yellow. Sequence alignment was performed with Clustal Omega.
FIGURE S2. Protein sequence alignment of photolyases and 6–4 photolyases. The primary structures of photolyases and 6–4 photolyases from different species are shown. (a) Photolyase (phr) (NP_001089127.1) and 6–4 photolyase (6–4) (NP_001081421.1) from Xenopus laevis have been aligned. (b) Photolyases from Xenopus laevis (Xenopus) (NP_001089127.1), Danio rerio (Danio) (NP_957358.1), Orysias latipes (Orizias) (NP_001098271.1), and Drosophila melanogaster (Drosophila) (NP_523653.2) have been aligned. (c) 6–4 photolyases from Xenopus laevis (Xenopus) (NP_001081421.1), Danio rerio (Danio) (BAA96852.1), Drosophila melanogaster (Drosophila) (NP_724274.1), and Arabidopsis thaliana (Arabidopsis) (NP_566520.1) have been aligned. Identical amino acids are denoted by an asterisk (*), highly similar residues by a colon (:), and weakly similar residues by a period (.). Gaps are indicated as dashes (−). Sequence alignment was performed with Clustal Omega.
