The Evolution of Transglutaminases Underlies the Origin and Loss of Cornified Skin Appendages in Vertebrates

Abstract Transglutaminases (TGMs) cross-link proteins by introducing covalent bonds between glutamine and lysine residues. These cross-links are essential for epithelial cornification which enables tetrapods to live on land. Here, we investigated which evolutionary adaptations of vertebrates were associated with specific changes in the family of TGM genes. We determined the catalog of TGMs in the main clades of vertebrates, performed a comprehensive phylogenetic analysis of TGMs, and localized the distribution of selected TGMs in tissues. Our data suggest that TGM1 is the phylogenetically oldest epithelial TGM, with orthologs being expressed in the cornified teeth of the lamprey, a basal vertebrate. Gene duplications led to the origin of TGM10 in stem vertebrates, the origin of TGM2 in jawed vertebrates, and an increasing number of epithelium-associated TGM genes in the lineage leading to terrestrial vertebrates. TGM9 is expressed in the epithelial egg tooth, and its evolutionary origin in stem amniotes coincided with the evolution of embryonic development in eggs that are surrounded by a protective shell. Conversely, viviparous mammals have lost both the epithelial egg tooth and TGM9. TGM3 and TGM6 evolved as regulators of cornification in hair follicles and underwent pseudogenization upon the evolutionary loss of hair in cetaceans. Taken together, this study reveals the gain and loss of vertebrate TGM genes in association with the evolution of cornified skin appendages and suggests an important role of TGM9 in the evolution of amniotes.


Introduction
Transglutaminases (TGMs) are calcium-dependent enzymes which introduce covalent bonds (Nϵ-(γ-glutamyl) lysine bridges) between lysine and glutamine residues of proteins (Greenberg et al. 1991).This process, known as transglutamination, allows the formation of large macromolecular structures which have been implicated in blood coagulation, apoptotic cell death, and the cornification of epithelia (Eckert et al. 2014).In addition, TGMs can catalyze the deamidation of glutamine residues and the linkage of amines to glutamine residues of proteins, known as transamidation (Lorand and Iismaa 2019).TGM genes have been identified and partially characterized in a broad range of metazoans ranging from the fruit fly (Shibata and Kawabata 2018) to humans (Greenberg et al. 1991).However, the evolution of TGMs has remained incompletely understood.In the present study, we utilized comparative genomics to infer the evolutionary history of TGMs in vertebrates, focusing on the evolutionary lineage leading to humans.
Humans have nine TGM genes, namely TGM1 through TGM7, F13A1, and EPB42 with different expression patterns (Eckert et al. 2014).TGM2 is expressed in a wide variety of cell types, whereas expression of F13A1 (coagulation factor XIII A chain) is confined to macrophages (Beckers et al. 2017).EPB42 (erythrocyte membrane protein band 4.2) is expressed as a catalytically inactive TGM family protein in erythrocytes (Korsgren et al. 1990), and TGM4 is specific for the prostate (Dubbink et al. 1996).Similarly, the expression of TGM1, TGM3, and TGM5 is confined to stratified epithelia, such as the epidermis of the skin, skin appendages, and the esophageal epithelium.Epithelial cells, known as keratinocytes, of the epidermis, hair, and nails undergo cornification, that is, a conversion into dead, mechanically stable and interconnected cell remnants, known as corneocytes (Candi et al. 2005;Yamane et al. 2010;Surbek et al. 2023).Protein crosslinking is an essential step of cornification (Eckhart et al. 2013), and the importance of TGMs for cornification is highlighted by the effects of TGM gene mutations in knockout mouse models and human patients.Deletion of Tgm1 in mice leads to a skin barrier defect and perinatal death due to excessive transepidermal water loss (Matsuki et al. 1998;Kuramoto et al. 2002).Humans can partially compensate inactivating mutations of TGM1, but develop severe ichthyosis (Rice et al. 2003;Zhang et al. 2019).Mutations of TGM3 and TGM5 cause deformations of hair fibers and acral peeling skin syndrome, respectively (Cassidy et al. 2005;Ü Basmanav et al. 2016).TGM6 and TGM7 are the least characterized human TGMs, with contradictory data on expression being available in the literature (Grenard et al. 2001;Thomas et al. 2013) and in the Genotype-Tissue Expression database (https://www.gtexportal.org/,last accessed on 2024 February 9).In addition to orthologs of human TGMs, a gene termed TGM8 was identified in zebrafish (Deasey et al. 2012).
Cornified integumentary structures are not specific for mammals, but also form in other vertebrates (Wu et al. 2004;Akat et al. 2022;Alibardi 2022).The cornified layer of the epidermis, the stratum corneum, is conserved in all land-dwelling vertebrates (Alibardi 2003;Candi et al. 2005;Akat et al. 2022).The stratum corneum consists of a single layer of dead cells in amphibians and many layers thereof in amniotes (Bereiter-Hahn et al. 1986).Epidermal cornification is diminished or modified at the subcellular level in mammals that have secondarily acquired a fully aquatic lifestyle (cetaceans and sirenians) (Ehrlich et al. 2019;Menon et al. 2022).Hard skin appendages, such as hair, claws, and feathers, are cornified by transglutamination (Sachslehner et al. 2023) and by the formation of disulfide bonds (Strasser et al. 2015;Harland and Plowman 2018;Ehrlich et al. 2020).The cornified epithelial egg tooth, also known as caruncle, is conserved in many but not all amniotes (Mlitz et al. 2021;Fenelon et al. 2023).Various species of teleost fish have cornified breeding tubercles (Fischer et al. 2014), and the most basal vertebrates, the agnathans (lamprey and hagfish), have cornified epithelial teeth (Fig. 1) (Rice et al. 1994;Zaccone et al. 1995;Alibardi and Segalla 2011).The distribution of cornification among vertebrate taxa suggests that TGMs are active in phylogenetically diverse clades.However, it is unknown whether and how the evolution of cornified structures was linked to the evolution of the TGM gene family.
Here, we determined the repertoire of TGM genes in phylogenetically and phenotypically diverse vertebrates and reconstructed the evolutionary history of TGMs in different lineages.Our results point to key roles of TGM gene innovation and degeneration in the evolution of skin appendages that were critical for evolutionary transitions of vertebrates.

Results
The Phylogenetically Basal TGM1 and F13A1 Genes are Conserved in Vertebrates, Whereas TGM4 has been Lost in At Least Four Lineages We identified the complete set of TGM genes in representatives from major groups of vertebrates (Chordata, Olfactores, Vertebrata) and subjected them to a phylogenetic analysis (supplementary table S1 and supplementary fig.S1, Supplementary Material online).TGM genes of a tunicate (Chordata, Olfactores, Tunicata) and lancelet (Chordata, Cephalochordata, Leptocardii) were included for comparison.Besides tunicate TGM genes, which do not cluster together with any TGM of the other species, three distinct clades of TGMs (Fig. 2) were identified: (i) the clade comprising TGM1 and F13A1, (ii) TGM4, and (iii) the clade comprising TGM2 through TGM8, EPB42, and two genes that we tentatively name TGM9 and TGM10.A comparison of the exon-intron structures showed that, in agreement with a  S3, Supplementary Material online) exist in all species investigated, except the tunicates.TGM1 and F13A1-like homologs of the lancelet form the outgroup to the vertebrate TGM1/F13A1 genes (Fig. 2).Sea lamprey, zebrafish, and coelacanth have more than one copy of TGM1, indicating lineage-specific gene duplications (supplementary fig.S4, Supplementary Material online).By mass spectrometry-based proteomics, we detected TGM1-like proteins in the cornified epithelial teeth of the sea lamprey (supplementary table S4, Supplementary Material online), suggesting that orthologs of mammalian TGM1 contribute to epithelial cornification in agnathans.
TGM4 is expressed predominantly in the prostate of humans and mice (Dubbink et al. 1996;Tian et al. 2009).Our comparative genomics and phylogenetic analyses revealed orthologs of TGM4 in sauropsids, amphibians, the coelacanth, and actinopterygians (ray-finned fish), but not in sharks (supplementary fig.S6 and supplementary table S5, Supplementary Material online), indicating that TGM4 has originated in stem Osteichthyes (bony fish including tetrapods) and not at the emergence of land vertebrates, as suggested in a previous study (Tian et al. 2009).TGM4 was independently duplicated in the gray birchir (Polypterus senegalus), the diploid frog Xenopus tropicalis, and the green anole lizard (Anolis carolinensis) and has been lost or pseudogenized in the lungfish (supplementary fig.S6, Supplementary Material online) and at least three groups of mammals, namely marsupials, Paenungulata (a subclade of Afrotheria, comprising elephants and sirenians), and Laurasiatheria (supplementary fig.S6, Supplementary Material online).The origin of TGM4 in fish and its loss in several taxa of mammals argue against a strict association of TGM4 with a function in the prostate, because this organ is confined to mammals and conserved among them.
The Previously Uncharacterized Gene TGM10 is Present in Fish and has been Lost Upon the Water-to-Land Transition of Tetrapods According to molecular phylogenetics (Fig. 2), the largest clade of TGMs is subdivided into two subclades, of which the first one comprises TGM2 and a TGM2-like gene (Lisetto et al. 2023) which we tentatively name TGM10, and the second subclade comprises the orthologs of human TGM3, TGM5, TGM6, TGM7, EPB42, fish TGM8, and another uncharacterized gene, tentatively named TGM9.For further analysis of homology, we performed a synteny analysis of TGM gene loci.

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In most species investigated, the genes TGM2, TGM3, TGM5, TGM6, TGM7, EPB42, and, if present, TGM8 are clustered at one chromosomal locus (Fig. 3a, supplementary tables S6 and S7, Supplementary Material online).Deviations from this organization are likely due to rearrangements of genes or chromosomal segments.TGM9 (see the last section of Results) and TGM10 (Fig. 3b) were found at loci lacking synteny with the aforementioned TGM gene cluster.Importantly, a basal vertebrate, the lamprey, has a TGM10 gene but lacks TGM genes at the loci corresponding to those of TGM2 and TGM9, suggesting that TGM10 originated prior to all other TGMs of this clade.Of note, an additional TGM10-like gene at another locus of the lamprey genome is likely the product of a lamprey-specific TGM10 gene duplication (supplementary fig.S7, Supplementary Material online).TGM10 is conserved in fish including the lungfish but not in tetrapods, indicating that this gene was lost upon the evolutionary transition to life on land.
A cluster of TGM2 and other TGM genes is located next to CCNDBP1 (cyclin D1-binding protein 1) in jawed vertebrates.In zebrafish, TGM8 localizes to this cluster, confirming the association in molecular phylogenetics (Fig. 2).Analysis of tissue transcriptomes (supplementary table S8, Supplementary Material online) showed that TGM8 is predominantly expressed in the skin of the zebrafish, with TGM1, TGM1L1, TGM5-like (TGM5L), and F13A1B being other skin-enhanced TGMs (Fig. 3, c to f, supplementary fig.S8, Supplementary Material online).A monophyletic subcluster of TGM3-like genes (Fig. 2) is present in the frog (Fig. 3a), indicating lineage-specific gene duplications.Mammalian TGM6 is Associated with Hair Follicles and has been Lost in Cetaceans The TGM2-related gene cluster contains at least one gene, namely TGM3, that is strongly expressed in epithelial cells of hair follicles (Thibaut et al. 2009;Eckert et al. 2014;Ü Basmanav et al. 2016;Harland and Plowman 2018).The functions of the TGM3-related gene TGM6 (Fig. 2) are not known.We hypothesized that TGMs with a predominant role in hair follicles may have become dispensable in mammals that have entirely lost their hair coat.Thus, we investigated the TGM genes of cetaceans (whales and dolphins), which are the only clade of mammals in which hair has completely disappeared.We found that cetaceans lack protein-coding TGM3 through TGM7 genes (Fig. 4a).In contrast to TGM4, which is also absent in the closest land-dwelling relatives of cetaceans (Fig. 4a), TGM3, TGM5, TGM6, and TGM7 have been lost specifically in cetaceans.The loss of TGM5 was reported previously (Sharma et al. 2018).Detailed sequence analysis of TGM6 showed that, besides other mutations, a frameshift mutation in exon 5 is shared among all cetaceans investigated (Fig. 4b), indicating that TGM6 underwent pseudogenization in stem cetaceans.By analyzing publicly available gene expression (supplementary tables S9 and S10, Supplementary Material online) and hair proteome data (supplementary tables S11, Supplementary Material online) from mouse studies (Joost et al. 2020;Sukseree et al. 2024), we found evidence for expression of TGM6 in hair keratinocytes (Fig. 4c, supplementary tables S9 and S11, Supplementary Material online).Of note, TGM3 and TGM5 are also expressed in human and mouse tissues other than hair follicles (Fig. 4c, list of "other expression sites"), suggesting that the loss of these genes in cetaceans occurred despite pleiotropic functions of the TGMs.We conclude that the loss of hair follicles, possibly together with modifications of other TGM-associated structures, allowed the pseudogenization of TGM6 and the contraction of the entire TGM gene repertoire in cetaceans.
Origin and Loss of TGM9 Paralleled the Evolution of the Epithelial Egg Tooth in Amniotes and the Evolution of Claws in Squamates Our phylogenetic analysis (Fig. 2) revealed that TGM9 is most closely related to TGM3, TGM5, TGM6, and TGM7.TGM9 is present in the genomes of turtles, crocodilians, birds, and the platypus, a monotreme mammal, where it is flanked by the conserved genes TMX3 (thioredoxin-related transmembrane protein 3) and DSEL (dermatan sulfate epimerase-like) (Fig. 5a).The species distribution of TGM9 orthologs suggests that TGM9 has originated, presumably by duplication of a TGM3/TGM5-like gene (Fig. 2), in stem amniotes and that it has been lost or pseudogenized independently in at least three clades of amniotes, namely therian mammals, snakes, and worm lizards (Fig. 5, a and b, supplementary table S12, Supplementary Material online).
We noticed that all of the TGM9-negative amniotes lack an epithelial egg tooth, which is a cornified structure that evolved in stem amniotes to facilitate hatching from the egg after completion of embryonic development.To test the hypothesis that the epithelial egg tooth is associated with TGM9, we localized TGM9 mRNA in chicken embryos isolated from eggs (Fig. 5c).TGM9 mRNA was prominently detected by in situ hybridization in the epithelial cells that give rise to the egg tooth, but only at minute amounts in the beak and the periderm, a transient epithelium that covers the egg tooth and epidermis during development (Fig. 5c).TGM9 co-localized with TGM activity (Fig. 5d).Reverse transcription polymerase chain reaction (RT-PCR) confirmed the expression of TGM9 in the egg tooth and revealed additional expression of TGM9 in the wings and back skin where feathers were developing (Fig. 5e).To substantiate these data, we determined the proteome of the epithelial egg tooth and detected the TGM9 protein in three biological replicates there of (PRIDE dataset: PXD048875, accession number TGM9: XP_040519905.1).Reanalysis of a publicly available proteomic dataset showed that TGM9 is also present in developing feathers of the pied flycatcher (Ficedula hypoleuca) (protein accession number Ensembl: ENSTGUP00000009578, corresponding to GenBank: XP_030121789.1)(Leskinen et al. 2012).In the anole lizard (A.carolinensis), TGM9 mRNA was detected by RT-PCR in the claws (Fig. 5f), whereas internal organs were TGM9-negative.
These data suggest that TGM9 plays roles of in diverse cornified skin appendages of amniotes and that the loss of the epithelial egg tooth has been a prerequisite for the degeneration of the TGM9 gene.Additional constraints on the evolution of TGM9 appear to arise from its roles in claws, so that claw-bearing squamates have retained the TGM9 gene after the loss the epithelial egg tooth, whereas limbless and therefore also clawless squamates (snakes and worm lizards) have lost TGM9.

Discussion
The results of this study suggest a new model for the evolution of TGMs in vertebrates (Fig. 6a).TGM1 and F13A1 are the phylogenetically basal TGM genes, which evolved from a single ancestral gene prior to the emergence of vertebrates.A subsequent gene duplication in stem vertebrates gave rise to TGM10 from which the ancestor of TGM2 evolved at a new locus.Subsequent gene duplications starting from TGM2 led to the evolution of TGM3 through TGM8 and EPB42 during the diversification of vertebrates.TGM9 arose from the TGM2-like gene cluster by From the presence or absence of individual TGMs in extant vertebrates, we infer a stepwise diversification of TGMs during the evolution of vertebrates and an important role of gene loss in shaping the human TGM repertoire: (i) TGM10 was lost after the divergence of tetrapods from other sarcopterygians (lobe-finned fishes) and (ii) TGM9 was lost after the divergence of therian mammals from prototherians.Moreover, our study indicates that duplications (Fig. 6a) and loss (Fig. 6b) of TGM genes occurred also in other lineages of vertebrates.
By combining the model of gene origin and gene loss (Fig. 6a) with the data on gene expression in tissues of diverse species (Fig. 6b), we conclude that two phylogenetically distinct groups of TGMs (Fig. 2) are expressed predominantly in stratified epithelia and cornified skin appendages: (i) TGM1 and (ii) the group of TGM3, TGM5, TGM6, TGM7, TGM8, and TGM9.

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Other TGMs are expressed either predominantly in nonepithelial tissues or at similar levels in epithelial and nonepithelial tissue.Thus, the role of TGMs in epithelial cornification was either an ancestral trait of vertebrate TGMs with roles in nonepithelial tissues evolving for F13A1, TGM10, TGM2, and possibly TGM4, or TGM-mediated epithelial cornification evolved at least twice during the diversification of TGMs.Importantly, the evolution of epithelial TGMs was constrained by pleiotropic functions of TGMs, correlating with the expression of individual TGMs at more than one epithelial site, and influenced by redundancy of TGMs due to expression of more than one TGM at particular epithelial sites.We propose that, in general, pleiotropy favors conservation of a gene, whereas redundancy favors gene loss.Our findings of TGM gene losses in cetaceans (Fig. 4) and loss of TGM9 in therian mammals and legless squamates (Fig. 5) indicate that phenotypic adaptations can be associated with the degeneration of genes even when the genes have pleiotropic roles in other phylogenetic lineages.These data support the concept that the conservative effects of pleiotropy are limited by gene redundancy and parallel adaptations of multiple body sites (Hecker et al. 2017).TGM1 is the phylogenetically oldest epithelial TGM (Fig. 6).Previous studies have shown expression of TGM1 HH36 HH40 in the epidermis of mammals, birds, and ray-finned fish (Rodriguez Cruz et al. 2017).Here, we added proteomic evidence for the presence of TGM1 orthologs in the cornified epithelial teeth of the sea lamprey, which diverged from the aforementioned TGM1-positive taxa more than 500 million years ago (Kumar et al. 2022).The group of TGM3/TGM5-like genes evolved more recently than TGM1 and was not as strictly conserved as TGM1.Upon the evolutionary transition to life in water and loss of hair in cetaceans, TGM3, TGM5, TGM6, and TGM7 were pseudogenized or lost (Fig. 6b).The loss of functional TGM6 in cetaceans and the evidence for expression of TGM6 in hair follicles, particularly in the medulla of growing hair fibers (supplementary table S9, Supplementary Material online), suggest that TGM6 resembles its presumable parental gene, TGM3, with regard to a primary function in hair Evolution of Transglutaminases in Vertebrates • https://doi.org/10.1093/molbev/msae100MBE (John et al. 2012).It is worth mentioning that a role of TGM6 in the central nervous system, as discussed in the literature (Thomas et al. 2013), is not supported by the distribution of TGM6 mRNA in human tissues, which is limited to the skin (https://www.gtexportal.org/home/gene/TGM6), last accessed on 2024 February 9), and a hypothetical link between mutations of TGM6 and spinocerebellar ataxia (Wang et al. 2010) was refuted by a recent genetic study (Chen et al. 2020).
Our phylogenetic and synteny analyses define TGM9 as a gene that evolved in amniotes prior to the divergence of the lineages leading to sauropsids and mammals.Previous studies of chicken keratinocytes cultured in vitro had detected TGM9 mRNA (Couteaudier et al. 2015;Lachner et al. 2021), which was then named protein-glutamine gamma-glutamyltransferase 5-like according to the GenBank annotation or TG5 (TGM5, ENSGALG00000013762).The data of the current study show that TGM9 is not orthologous to TGM5 and, more importantly, reveal expression of TGM9 in the epithelial egg tooth (Fig. 5, c and e), which is a cornified skin appendage that has played an essential role in the evolutionary transition of tetrapods to fully terrestrial life (Fenelon et al. 2023).The egg tooth must have co-evolved with the egg shell in stem amniotes to facilitate hatching.However, the epithelial egg tooth was lost in specific subgroups of amniotes, namely squamate reptiles, in which the epithelial egg tooth was functionally substituted by a calcified "real" tooth, and in therian mammals, which have replaced embryonic development in ovo by development in utero.Of note, monotremes have both an epithelial egg tooth (caruncle) and a calcified egg tooth, which, however, differs histologically from the egg tooth of squamates (Fenelon et al. 2023).We could confirm the presence of a complete TGM9 gene in the platypus (Fig. 5; supplementary table S1, Supplementary Material online), but we found only one exon of TGM9 in the current genome sequence assembly of the echidna (Tachyglossus aculeatus) (GenBank accession number NC_052070.1,nucleotides 28415425 to 28415602).Thus, the evolution of TGM9 in monotremes requires further investigation.Based on gene expression evidence, we propose that TGM9 contributes to the cornification of the epithelial egg tooth and thereby helps to establish the hardness required for its function in opening the egg shell.The loss of TGM9 in viviparous mammals supports the primary association of TGM9 with the epithelial egg tooth, whereas a role of TGM9 in the claws of squamates is suggested by the conservation of TGM9 in claw-bearing lizards and geckos (Fig. 5a).

Conclusion
In conclusion, the evolution of the TGM gene family was linked to important adaptations of the skin epithelium in vertebrates and, indirectly, to major changes in the embryonic development of tetrapods.Future studies will explore the possible coevolution of TGMs with their substrate proteins and the evolution of the mechanisms that regulate the expression of established and newly identified TGMs in vertebrates.

Comparative Genomics and Sequence Alignments
For the comparative genomic analysis, the genome sequences of human (GCF_000001405.40,International Human Genome Sequencing Consortium), Tasmanian devil (GCF_902635505.1,Stammnitz et al. 2023) TGM sequences were collected from NCBI GenBank.The chicken TGM1 homolog was identified in a previous study (Sachslehner, Surbek, et al. 2021).For several genes, the predictions of exon sequences were corrected according to the results of tBLASTn searches with other TGMs as queries (supplementary table S1, Supplementary Material online).TGM1 and TGM1L4 of the coelacanth (Latimeria chalumnae) lacked large parts of their coding sequences in the annotations available in GenBank.For identification of the complete coding sequences, RNA-seq data of pooled tissues of the coelacanth were downloaded from NCBI GenBank (accession number: SRX112771, run accession: SRR391920) with the prefetch (version 3.0.6)package of SRA Toolkit (https://github.com/ncbi/sra-tools,last accessed on 2024 January 18) and converted to the fastq format with fastq-dump (version 3.0.6)package of SRA Toolkit.A quality check was performed with FastQC (version 0.12.1, https:// www.bioinformatics.babraham.ac.uk/projects/fastqc/, last accessed on 2024 January 18).The transcriptome was assembled with Trinity (Grabherr et al. 2011, version 2.15.1), and coding regions were predicted with TransDecoder (version 5.7.1, https://github.com/TransDecoder/TransDecoder,last accessed on 2024 January 18).The complete TGM1 and TGM1L4 coding sequences were identified via BLASTp (Altschul et al. 1990, version 2.14.0+).The assembled transcriptome is available at https://doi.org/10.5281/zenodo.10619730.

Molecular Phylogenetics
Multiple sequence alignments for phylogeny were created and manually trimmed with aliview (Larsson 2014).After trimming, the amino acid sequences contained only the residues that span from the "Transglut_N superfamily" domain to the second "

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template for in vitro transcription with the DIG RNA labeling mix (Roche).T7 RNA polymerase was used for transcription of antisense and sense probes.RNA probes were precipitated with 4 M LiCl (Sigma-Aldrich) and ethanol overnight at −20 °C, followed by subsequent washing steps with 70% and 100% ethanol and resuspension in RNase-free water.The riboprobes were stored at −80 °C.
mRNA In Situ Hybridization Sections were prepared with a Microm HM 335E microtome at a thickness of 5 µm and collected in a water bath set to 42 °C on super-adhesive slides (Menzel).mRNA in situ hybridization was performed on tissue sections of formalin-fixed paraffin-embedded chicken beaks of stage HH36 (Hamburger and Hamilton 1951) based on published protocols (Mlitz et al. 2021;Sachslehner, Zieger, et al. 2021).Briefly, paraffin sections were melted at 58 °C for 1 h, deparaffinized in xylene (Fisher Chemical) for 20 min, and further hydrated in a descending ethanol series.The samples were treated with 20 µg/ml proteinase K for 10 min at 37 °C.Specimens were subsequently incubated for 10 min in 1% triethanolamine (Sigma-Aldrich) in PBS, for 5 min in 1% triethanolamine with 0.15% acetic anhydride (Prolabo), and for 5 min in 1% triethanolamine with 0.3% acetic anhydride to neutralize charged probe binding.Samples were postfixed with 4% paraformaldehyde in PBS for 40 min followed by dehydration in an ascending ethanol series.Samples were dried at room temperature for 1 h and prehybridized in hybridization buffer at 60 °C for 2 h.Corresponding sense and antisense riboprobes were prepared at a concentration of 2 ng/ µl and preheated in hybridization buffer (4 M urea, VWR; 5x saline sodium citrate (SSC), Roth; 100 μg/ml heparin, Sigma-Aldrich; 5 mM EDTA, Roth; 1x Denhardt's block reagent, Sigma-Aldrich; 100 μg/ml salmon sperm DNA, Agilent; 5% dextran sulfate, Sigma-Aldrich) at 85 °C for 10 min.Hybridization took place overnight at 60 °C.To remove unbound probes, samples were washed thrice with 4 M urea and 4x SSC, once with a solution containing 4 M urea and 2x SSC, and once with 4 M urea and 1x SSC at 58 °C for 15 min each, followed by one wash with 1x SSC at 37 °C for 15 min.Specimens were incubated for 2 h in 0.1 M maleic acid buffer (MAB, Sigma-Aldrich), pH 7.5 containing 2% BSA to prevent nonspecific antidigoxigenin antibody binding.Afterward, specimens were incubated with an antidigoxigenin antibody conjugated to the alkaline phosphatase enzyme (1:5,000, Roche) in 2% MAB block solution overnight at 4 °C.A buffer suitable for alkaline phosphatase enzyme activity (0.5 M Tris pH 9.5; 0.5 M NaCl) was used, and samples were washed twice with alkaline phosphatase buffer for 15 min at room temperature.The staining was developed with alkaline phosphatase buffer supplemented with 0.05 M MgCl 2 , 3.75 μl/ml 5-bromo-4-chloro-3-indolyl phosphate (Roche), and 5 µl/ml nitroblue tetrazolium (Roche).Sections were mounted with Aquatex (Sigma-Aldrich), and photographs were taken with an Olympus UC-90 camera on an Olympus BX63 light microscope.

Mass Spectrometry-Based Proteomics
The beaks of stage HH44 chicken embryos were dissected and dried for some minutes at room temperature.The egg tooth became clear after the drying step and was removed from the beak with a pointy scalpel.The epidermis of the upper beak was removed with forceps.Samples were frozen in liquid nitrogen and stored at −80 °C until lysate preparation.Lamprey teeth were obtained from a museum sample that was stored in ethanol at room temperature.The specimens were placed in 200 µl of lysis buffer, made of 30 mM Tris, 7 M urea (VWR), 2 M thiourea (Sigma-Aldrich), and 4% CHAPSO (Pierce).0.2 M dithiothreitol (DTT) was added to the samples.After incubation at 70 °C for 3 h, the samples were homogenized with a homogenizer (Precellys, VWR) and centrifuged at 18,000 × g for 15 min at 4 °C, and the supernatant was collected.The pellet was sonicated with a sonicator (Hielscher Ultrasound Technology) twice for 30 s at an amplitude of 100, and centrifugation was repeated as before.The supernatant was pooled with the one from the initial homogenization and stored at −80 °C until analysis was started.The proteomic analysis and database search were performed like in a previous study (Cruz-Bustos et al. 2023) with the following modification: After protein reduction with 200 mM DTT (37 °C, 30 min), the proteins were alkylated with 500 mM iodoacetamide (Sigma) at 37 °C for 30 min.The database for the lamprey (NCBI_ Petromyzon_marinus_tx7757_230919.fasta) and the chicken (NCBI_Gallus_gallus_tx9031_230919.fasta) was downloaded from crap.fasta (https://www.thegpm.org/crap/,last accessed on 2024 February 7).

Fig. 1 .Fig. 2 .
Fig. 1.Cornified skin structures evolved in vertebrates and depend on protein crosslinking by transglutamination.Schematic depiction of the cornified integumentary structures (red) in different lineages of vertebrates (left panel).Transglutamination is a mechanism of covalently crosslinking proteins at glutamine (Q) and lysine (K) residues, which is catalyzed by TGMs, including coagulation factor XIIIa (F13A1) (right panel).This study tests the hypothesis that the evolutionary origin and loss of skin appendages, such as the egg tooth, claws, or hair, were associated with specific changes in the TGM gene family.Parts of the figure are modified from Carron et al. 2024.

Fig. 3 .
Fig.3.Synteny analysis of TGM2-related genes in vertebrates.a) Schematic depiction of gene clusters comprised of TGM2 and TGM genes closely related to TGM2 (Fig.2).Genes are shown as triangles pointing in the direction of transcription.Horizontal lines indicate chromosomes or sequence scaffolds.Because of space limitations, not all genes are shown.Vertical double lines indicate gaps in the display where several genes are not shown.An asterisk besides the vertical double lines indicates a complex rearrangement of the left and right segments of the chicken gene locus.b) Locus of the TGM10 gene in different species.Note that TGM10 is not present in tunicates and terrestrial vertebrates.c to f) Expression of zebrafish TGMs in tissues.The colors of bars correspond to the colors of the genes in panel (a).RNA-seq data were analyzed for the mRNA levels of tgm8 (c), tgm5 l, which is orthologous to human TGM5 (d), tgm2b, which is one of two orthologs of human TGM2 (e) and tgm10 (f).P-values were calculated by ANOVA.n.s., not significant.Species: Human (H.sapiens), chicken (Gallus gallus), lizard (A.carolinensis), frog (X.tropicalis), lungfish (P.annectens), coelacanth (L.chalumnae), zebrafish (D. rerio), shark (C.carcharias), lamprey (P.marinus), tunicate (C.intestinalis), and lancelet (B.floridae).

Fig. 4 .
Fig. 4. Cetaceans have lost TGM homologs implicated in epithelial cell differentiation within hair follicles.a) Loss of TGM genes (flash symbol) was mapped onto a phylogenetic tree of mammals.The presence (+) or absence (−) of TGM orthologs is indicated for each species.Lineages of fully aquatic mammals are highlighted by blue lines and fonts.b) Nucleotide sequence alignment of exon 4 of human TGM6 and its homologs in cetaceans.Identical nucleotides in all five species are shown with blue fonts.A nucleotide leading to a frame shift and a premature stop codon are shaded red.Sequences of the flanking introns are shaded gray.Amino acid sequences obtained by translation of the exonic nucleotide sequences are shown for human and minke whale.Note that additional mutations are present in other exons of TGM6 in cetaceans.c) Schematic depiction of the epithelial compartments of a hair follicles and summary of evidence for expression of individual TGM genes.IRS, inner root sheath; ORS, inner root sheath; sc, single cell.

Fig. 5 .
Fig. 5. TGM9 is expressed in the epithelial egg tooth of chicken embryos and in claws of lizards.a) Evolution of TGM9 in relation to the evolution of cornified skin appendages.The locus of TGM9 and its neighboring genes was compared between diverse species of tetrapods (right panel).The relationship of species is shown by a phylogenetic tree (left) on which the inferred origin (arrow) and loss of TGM9 (flash symbol) are indicated.Traits that are correlated with the conservation of TGM9 are shown for each lineage, whereby "+" and "−" indicate the presence or absence of the respective trait.Red circles highlight the evolutionary loss of traits.Species: human (H.sapiens), marsupial (Tasmanian devil, Sarcophilus harrisii), platypus (O.anatinus), chicken (Gallus gallus), alligator (Alligator sinensis), turtle (Mauremys reevesii), worm lizard (Rhineura floridana), wall lizard (Podarcis muralis), snake (Pantherophis guttatus), anole lizard (A.carolinensis), gecko (Gekko japonicus), and frog (X.tropicalis).b) Nucleotide sequence alignment of exon 6 of the TGM9 genes of the wall lizard and the legless worm lizard.Splicing signals (underlined) and further intronic sequences are included in the alignment.Sequence identity is highlighted by blue fonts.A premature stop codon is highlighted by red shading.c) mRNA in situ hybridization of TGM9 in the chicken egg tooth at developmental stage HH36.An arrow points to the strongest signal in the epithelial egg tooth (et).In the negative control reaction (right panel), the antisense probe was replaced by the corresponding sense probe.Scale bars, 200 µm.peri, periderm.d) TGM activity (red) was localized by using a fluorescently labeled (red) substrate on a cryosection of the chicken beak and egg tooth at developmental stage HH36.In the negative control (neg.con.) reaction (right panel), calcium ions were replaced by EDTA.Unspecific signals are indicated by an asterisk.Scale bars, 200 µm.e) RT-PCR analysis of TGM9 expression in chicken embryos at developmental stages HH36 and HH40.f) RT-PCR analysis of TGM9 expression in the adult anole lizard (A.carolinensis).The house-keeping gene EEF1A1 was amplified to confirm the integrity of tissue cDNAs in panels e) and f).

Fig. 6 .
Fig.6.Gene duplications and gene loss have shaped the repertoire of TGMs in humans and other vertebrates.a) The evolution of the TGM gene family is schematically depicted on a phylogenetic tree focusing on the lineage leading to humans.The origin (vertical arrows) and loss (flash symbols) of TGM genes are indicated at positions inferred from the species distribution of TGMs in extant species.The presence of TGM10 and TGM9 is indicated by blue and red colors of branches, respectively.b) Summary of functional diversification of TGMs in vertebrates.The phylogenetic relationships of TGMs are indicated by a simplified tree (left side) in which well-supported nodes (Fig.2) are marked.
Sachslehner et al. • https://doi.org/10.1093/molbev/msae100 Evolution of Transglutaminases in Vertebrates • https://doi.org/10.1093/molbev/msae100MBEEPB42,which is specifically expressed in erythrocytes(Wang  et al. 2020), is present in tetrapods but not in fish, suggesting an evolutionary origin at the time of the water-to-land transition.Residues critical for catalytic TGM activity are conserved in EPB42 orthologs of some but not all amphibians and sauropsids and in none of the mammalian species investigated (supplementary fig.S9, Supplementary Material online), suggesting that EPB42 was originally an active enzyme which lost its activity in distinct lineages, including the one leading to humans.