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Fig. 1. Establishment of the Xenopus tropicalis tp53 Δ7 mutant line. (A) gRNA design. TAD, transactivation domain; TD, tetramerization domain. (B) Sanger DNA sequencing data show mutations induced by targeting X. tropicalis tp53. The wild‐type (wt) sequence is shown at the top with the target site in red. Red dashes indicate deletions and lowercase letters indicate insertions. (C) The tp53 Δ7 genotype in the F1 offspring. E, exon. Seven out of 29 F1 adults showed this Δ7‐heterozygous genotype. (D) Histograms of statistical data (mean values ± SD) from three independent X‐ray irradiation (14 Gy) experiments show distribution of abnormal and dead embryos among three genotypes. WT, wild‐type. graphpad prism 9 (GraphPad Software, San Diego, CA, USA) was used for the t‐test (***P < 0.001, ****P < 0.0001). (E) Representative images from one experiment show that P53‐dependent radiosensitivity in WT X. tropicalis embryos was proportionally decreased in tp53
+/Δ7
and tp53
Δ7/Δ7
mutants. Identical results were obtained in three independent experiments, with 9216 embryos used in total. All the embryos were irradiated at stage 10 and evaluated at stage 41. In a dose‐dependent manner, no wild‐type embryos can survive to stage 41 at 8 Gy irradiation. In contrast, tp53
+/Δ7
heterozygotes and tp53
Δ7/Δ7
homozygotes can survive to stage 41 even at 14 and 18 Gy irradiation, respectively (for statistics, see Table S2). Scale bar, 0.5 mm.
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Fig. 2. P53 haploinsufficiency in Xenopus tropicalis tp53
+/Δ7
mutants. (A) RT‐PCR analysis of tp53 and ccng1 expression in samples indicated in the image. odc was used as a RNA loading control. Identical results were obtained in three independent experiments. (B) Western blot analysis shows P53 levels in samples as indicated. Beta tubulin was used as a protein loading control. h, hour; WT, wild‐type. This experiment was carried out once.
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Fig. 3. Nevus and melanoma formation in tp53
Δ7/Δ7
adult frogs. (A) Ventral view of a 14‐month‐old wild‐type (WT) frog shows no nevi. H&E staining indicates representative histological structure of the ventral skin. Similar structure was observed in all 50 serial sections. (B) Higher magnification view of the dashed box area in A shows that the majority of the melanophores are located just beneath the basement membrane and a small portion at the bottom of a mucous gland (red dashed arrows). Ep, epidermis; Me, melanophores; Mu, mucous glands; Gr, granular glands; SSp, stratum spongiosum. Similar structure was observed in all 50 serial sections. (C) Ventral view of a 14‐month‐old tp53
Δ7/Δ7
mutant shows a benign nevus (red dashed arrow and the higher magnification view in the blue dashed box). A representative H&E staining reveled local hyperproliferation of melanophores. Similar phenotype was observed in all 10 serial sections. (D) Ventral view of another 14‐month‐old tp53
Δ7/Δ7
mutant shows a dysplastic nevus (red dashed arrow and the higher magnification view in the blue dashed box). A representative H&E staining reveled aberrant growth of melanophores. Similar phenotype was observed in all 10 serial sections. (E) Ventral view of a 14‐month‐old tp53
Δ7/Δ7
mutant shows a melanoma in situ (red dashed arrow and the higher magnification view in the blue dashed box). A representative H&E staining revealed apparent vertical growth of melanophores across the stratum spongiosum. Similar phenotype was observed in all 10 serial sections. (F) Schematic adapted from [28] shows the histological steps in the progression of human melanoma. B, benign; BM, basement membrane; D, dysplastic; Der, dermis; Epi, epidermis; M, months; M‐melanoma, metastatic melanoma; RGP, radial growth phase; VGP, vertical growth phase; scale bars in frog images are 5 mm. Scale bars in H&E staining images are 50 μm.
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Fig. 4. Invasive melanoma (about 5 mm) developed in a 20‐month‐old tp53
Δ7/Δ7
frog. (A) Ventral view of the 20‐month‐old tp53
Δ7/Δ7
frog shows an invasive melanoma (red dashed arrow and the higher magnification view in the blue dashed box). Scale bar, 5 mm. (B–D) H&E staining of representative sections from the area, as indicated by the dashed lines 1, 2, and 3 in A, respectively. For areas indicated by each line, similar structures were observed in at least 10 neighboring serial sections. Ep, epidermis; M, months; Hd, hypodermis; Sco, stratum corneum; SSp, stratum spongiosum. Scale bars, 50 μm.
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Fig. 5. Histology‐based nevus and melanoma classification is confirmed by the expression levels of molecular marker genes. (A–F) Representative morphology of dissected skin samples under stereo microscope. For each category, two frogs were biopsied and similar morphology was observed. (A) Ventral skin sample from a 19‐month‐old wild‐type frog; (B) ventral skin sample from a 19‐month‐old tp53
Δ7/Δ7
frog without nevus/melanoma lesions; (C–E) ventral skin samples from 19‐month‐old tp53
Δ7/Δ7
frogs with benign nevus, dysplastic nevus, and melanoma in situ, respectively; (F) ventral skin sample from a 25‐month‐old tp53
Δ7/Δ7
frog with an invasive melanoma. Scale bars, 100 μm. (G–L) Representative histological structures (H&E staining) of samples shown in A–F, respectively. Similar structures were observed in 10 serial sections from each sample. Scale bars, 50 μm. (M) RT‐PCR analyses revealed the transcriptional expression levels of genes indicated on the right side in different samples listed on the top. gapdh was used as an RNA loading control. Identical results were obtained in two independent RT‐PCR experiments.
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Fig. 6. Loss of heterozygosity occurred in dysplastic nevus of a tp53
+/Δ7
mutant. (A) A 20 μm thick cryosection of the dysplastic nevus from a tp53
+/Δ7
mutant shows areas for laser capture microdissection‐mediated collection of lesion cells (a) and non‐lesion cells (b). Scale bar, 50 μm. (B) Histograms of Sanger DNA sequencing data (mean values ± SD) from three independent PCR amplifications show the occurrence of LOH in lesion cells. graphpad prism 9 (GraphPad Software) was used for the t‐test (**P < 0.01).
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Fig. 7. Round cell sarcomas and pancreatic ductal adenocarcinoma were developed in a tp53
Δ7/Δ7
adult frogs. (A) Dorsal view of the 14‐month‐old tp53
Δ7/Δ7
frog shows the location of the sarcoma (red dashed arrow). Dissection of the overlying skin revealed that the sarcoma grew from the hypodermis (bottom‐left corner). Scale bar, 5 mm. (B) Representative H&E staining of the sarcoma sections revealed histopathologic characteristics resembling human round cell sarcoma [38]. Similar structure was observed in all 50 serial sections. Scale bar, 50 μm. (C) Representative morphology of a dissected fresh pancreas from a 34‐month‐old wild‐type Xenopus tropicalis frog. Two frogs were dissected and similar pancreas morphology was observed. (D) Representative histology of the pancreas shown in C. Dashed arrow points to a normal pancreatic duct. Similar structure was observed in all 50 serial sections. (E) Morphology of a dissected pancreas from a 34‐month‐old tp53
Δ7/Δ7
frog. Dashed blue arrows highlight the light deposits that are not seen in wild‐type healthy pancreas. (F) Representative histopathology of the lesions seen in E shows neoplastic ducts (dashed blue arrow) embedded in fibrous tissue resulted from severe desmoplastic reaction, thus recapitulating the histopathologic characteristics of human pancreatic ductal adenocarcinoma [39]. Similar structure was observed in all 50 serial sections. Scale bars in C and E, 1 mm. Scale bars in D and F, 50 μm. M, months; WT, wild‐type.
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