Evolutionary origin of Hoxc13-dependent skin appendages in amphibians

Abstract

Cornified skin appendages, such as hair and nails, are major evolutionary innovations of terrestrial vertebrates. Human hair and nails consist largely of special intermediate filament proteins, known as hair keratins, which are expressed under the control of the transcription factor Hoxc13. Here, we show that the cornified claws of Xenopus frogs contain homologs of hair keratins and the genes encoding these keratins are flanked by promoters in which binding sites of Hoxc13 are conserved. Furthermore, these keratins and Hoxc13 are co-expressed in the claw-forming epithelium of frog toe tips. Upon deletion of hoxc13, the expression of hair keratin homologs is abolished and the development of cornified claws is abrogated in X. tropicalis. These results indicate that Hoxc13-dependent expression of hair keratin homologs evolved already in stem tetrapods, presumably as a mechanism for protecting toe tips, and that this ancestral genetic program was coopted to the growth of hair in mammals.

Fig. 1. Synteny analysis and molecular phylogenetics identify hair keratin homologs in amphibians.

a, b Schematic representation of the keratin type I (a) and type II (b) gene clusters of selected vertebrates: human, Homo sapiens; frog, Xenopus tropicalis; axolotl, Ambystoma mexicanum; caecilian, Rhinatrema bivittatum; lungfish, Protopterus annectens. Genes are shown as triangles pointing in the direction of transcription. Homologs of hair keratin genes are highlighted by blue shading. Note that KRT18 is the only type I keratin gene located in the type II cluster. Slanted double lines indicate sites where genes are omitted for clarity. c, d Phylogenetic analysis of type I (c) and type II (d) keratins. Keratin orthologs in amphibians and human or lungfish with bootstrap values > 50 (indicated in c, d) are shown with matching colors in panels (a) and (c) (type I) and (b) and (d) (type II). Nodes without numbers have bootstrap values < 50. Note the strong support (bootstrap >90) for orthology of amphibian krt34 (c) and krt59 (d) with human hair keratins (highlighted in blue) of the respective keratin type. Only fully sequenced keratin genes were included in this analysis. e Model for the evolution of hair keratin homologs in relation to the evolution of amphibians. mya, million years ago; +, present; -, absent.

Fig. 2. Expression of hair keratin homologs of Xenopus tropicalis is associated with cornified claws.

a Schematic depiction of a clawed frog (X. tropicalis) bearing cornified claws on the hindlimb inner (HI) toes (toes I, II, III) and no claws on the hindlimb outer (HO) toes (toes IV, V) and on toes of the forelimbs. b Schematic of a frog clawed toe tip. The term “claw” refers to the cornified claw that is formed by the differentiation of the epithelium on the tip of the toe. c–e Quantitative RT-PCR analysis of mRNA expression of krt34 (c), krt59 (d), krt53 (e) relative to the housekeeping gene, eef1a1. Mass-spectrometric quantification of Krt34 (f), Krt59 (g) and Krt53 (h). a.u., arbitrary units. RNA and protein were sampled at an age of 7 months. Statistics was calculated by one-way ANOVA for n = 4 (c–e) and three (f–h) biological replicates, respectively, in each of 3 groups. Bars and error bars indicate means and standard deviations, respectively.

Fig. 3. Co-expression with krt34 in clawed toes and promoter assays suggest Hoxc13 as regulator of hair keratin expression.

a–f mRNA in situ hybridization using hoxc13 (a), krt34 (b), and krt59 (c) antisense (as) probes on sections of HI toes of X. tropicalis sampled at an age of 4 months (3 replicates each). Hybridizations with sense (s) probes (d–f) served as negative controls. Artefacts such as the detachment of the cornified claw during tissue sectioning (*) are indicated. cl, claw; ep, epidermis. Scale bars: 100 µm. g Consensus Hoxc13 binding sites in the proximal promoters of human and frog hair keratin homologs. The distance to the TATA box (number of nucleotides) is indicated on the right. The consensus sequence of Hoxc13 binding sites in human hair keratin promoters is shown below the sequence alignment. h, i Luciferase activity of cells co-transfected with a Hoxc13 expression vector and a luciferase reporter under the control of the krt34 (h) or krt59 (i) promoter (prom) containing wildtype (wt) or mutated (mut) Hoxc13 binding sites. Statistics was calculated by one-way ANOVA for n = 4 biological replicates in each of 4 groups. Bars and error bars indicate means and standard deviations, respectively. RLU relative light units.

Fig. 4. hoxc13 knockout frogs lack claws and do not express hair keratins homologs.

a Schematic depiction of hoxc13 knockout (KO) in X. tropicalis frogs. The number of animals bearing claws is shown on the right. Macroscopic appearance of a wildtype (WT) (b) and a KO (c) frog. Arrows point to claws. d–g Hindlimb phenotypes (d, e) and histology (f, g) of hindlimb inner (HI) toes of WT (d, f) and KO (e, g) frogs. Images in b-g are representative for at least 3 biological replicates. cl claw, ep epidermis. Scale bars: 2 mm (d, e), 200 µm (f, g). Quantitative RT-PCR analysis of krt34 (h) and krt59 (i) in HI toes of WT and KO. Statistics was calculated by unpaired two-tailed t-test. n = 4 biological replicates for WT and KO. Age of the frogs: 7 months. a.u., arbitrary units. Bars and error bars indicate means and standard deviations, respectively.

Fig. 5. Hoxc13 and hair keratin homologs are expressed at the toe tips of the axolotl.

a Macroscopic view of an albino axolotl. b View of the pigmented toe tips. Sections of toe tips were stained with hematoxylin and eosin (c) or were left unstained (d). e RT-PCR analysis of hoxc13, hair keratin homologs (krt34 and krt59), epithelial keratins (krt-T1-3 and krt78L2) and the housekeeping gene eef1a1 in toes and belly skin. f In situ hybridization of krt34 mRNA using an anti-sense (as) probe. g Negative control experiment using a krt34 sense (s) probe. Images are representative for n = 3 adult individuals of albino axolotl. Artefacts such as the detachment of tissue during tissue sectioning (*) are indicated. cl cornified layer, ep epidermis. Dashed lines indicate the junction of epidermis and dermis. Scale bars: 5 mm (b), 100 µm (c, d, f, g).

Fig. 6. Model for the evolutionary origin of Hoxc13 and hair keratin-dependent cornified skin appendages.

The evolutionary changes in the expression pattern of Hoxc13 and the appearance of the regulatory link between Hoxc13 and “hair keratins” were inferred from data obtained in extant vertebrates and their known phylogeny. “Hair keratins” refers to human keratins KRT31-KRT40 and KRT81-KRT86 and their orthologs in other species. Red and blue shading mark sites of expression of Hoxc13 and hair keratin homologs, respectively.