Regeneration among animals: An evolutionary hypothesis related to aquatic versus terrestrial environment.

The present hypothesis tries to explain animal regeneration in relation to the life cycles and environment of different animals. Regeneration is a basic phenomenon present since the origin of life in the sea, as testimonial in lower or more complex extant marine animals. Aquatic animals that evolved an indirect development, forming larvae and transiting into the adult stage through metamorphosis, use gene networks present in their genome for these transformations. In case of injury or organ loss as adults, they can re-utilize most or part of the gene networks previously activated during larval growth and metamorphosis. In contrast, terrestrial animals that evolved life cycles with the elimination of larvae and metamorphosis for the adaptation to land conditions lost some of the genes implicated in these post-developmental processes and consequently also the ability to regenerate. Few arthropods and lizards are capable to form hydrated regenerative blastemas with a similar consistence of embryonic tissues. The present hypothesis submits that regeneration cannot be activated in the dry land environment and consequently was largely or completely abolished in terrestrial animals. After injury or organ loss, nematodes, most arthropods and terrestrial vertebrates can only form scars or a limited healing or regengrow in juveniles. This is a process where somatic growth is superimposed to wound healing so that the apparent regeneration derives from the combination from both processes. When full growth is terminated these terrestrial animals can only heal by scarring.

General aspects on skin development in vertebrates with emphasis on sauropsids epidermis.

General cellular aspects of skin development in vertebrates are presented with emphasis on the epidermis of sauropsids. Anamniote skin develops into a multilayered mucogenic and soft keratinized epidermis made of Intermediate Filament Keratins (IFKs) that is reinforced in most fish and few anurans by dermal bony and fibrous scales. In amniotes, the developing epidermis in contact with the amniotic fluid initially transits through a mucogenic phase recalling that of their anamniotes progenitors. A new gene cluster termed EDC (Epidermal Differentiation Complex) evolved in amniotes contributing to the origin of the stratum corneum. The EDC contains numerous genes coding for over 100 types of corneous proteins (CPs). In sauropsids 2-8 layers of embryonic epidermis accumulate soft keratins (IFKs) but do not form a compact corneous layer. The embryonic epidermis of reptiles and birds produces small amount of other, poorly known proteins in addition to IFKs and mucins. In the following development, a resistant corneous layer is formed underneath the embryonic epidermis that is shed before hatching. The definitive corneous epidermis of sauropsids is mainly composed of CBPs (Corneous beta proteins, formerly indicated as beta-keratins) derived from the EDC. CBPs belong to a gene sub-family of CPs unique for sauropsids, contain an inner amino acid region formed by beta-sheets, are rich in cysteine and glycine, and make most of the protein composition of scales, claws, beaks and feathers. In mammalian epidermis CPs missing the beta-sheet region are instead produced, and include loricrin, involucrin, filaggrin and various cornulins. Small amount of CPs accumulate in the 2-3 layers of mammalian embryonic epidermis and their appendages, that is replaced with the definitive corneous layers before birth. Differently from sauropsids, mammals utilize KAPs (keratin associated proteins) rich in cysteine and glycine for making the hard corneous material of hairs, claws, hooves, horns, and occasionally also scales.

Activation of cell adhesion molecules and Snail during epithelial to mesenchymal transition prior to formation of the regenerative tail blastema in lizards.

After few days from tail amputation in lizards the stump is covered with mesenchymal cells accumulated underneath a wound epidermis and forms a regenerative blastema. During migration, some keratinocytes transit from a compact epidermis into relatively free keratinocytes in a process of "epithelial to mesenchymal transition" (EMT). EMT is also induced after damaging the regenerating epidermis by cauterization, whereas keratinocytes detach and migrate as mesenchymal-like cells among the superficial blastema cells and reconstruct a wound epidermis after about a week from the damage. In normal amputation or after cauterization, no malignant transformation is observed during the transition and migration of keratinocytes. Immunolabeling for markers of EMT confirms the histological description and shows a unique pattern of expression for l-CAM (E-cadherin), N-CAM, and SNAIL-1 and -2 (SLUG). These proteins are present in the cytoplasm and nuclei of migrating keratinocytes. It is hypothesized that the nuclear labeling for E-cadherin coupled to cytoplasmic SNAIL-labeling is somehow related to an initially regulated EMT. After the migrating keratinocytes have reached confluence over the stump, they reverse into a "mesenchymal to epithelial transition" (MET) forming the wound epidermis. The basal layers of the apical wound epidermis of the blastema show some nuclear E-cadherin labeling, while the tail regenerates. It is hypothesized that, together with other tumor suppressors proteins, the apical epidermis and mesenchyme are kept under a tight proliferative control, while in proximal regions the prevalent effect of tumor suppressors determine the differentiation of the new tail tissues.

Regeneration in anamniotes was replaced by regengrow and scarring in amniotes after land colonization and the evolution of terrestrial biological cycles.

An evolutionary hypothesis explaining failure of regeneration among vertebrates is presented. Regeneration derives from postembryonic processes present during the life cycles of fish and amphibians that include larval and metamorphic phases with broad organ reorganizations. Developmental programs imprinted in their genomes are re-utilized with variations also in adults for regeneration. When vertebrates colonized land adopting the amniotic egg, some genes driving larval changes, and metamorphosis were lost and new genes evolved, further limiting regeneration. These included neural inhibitors for maintaining complex nervous systems, behavior and various levels of intelligence, and adaptive immune cells. The latter, that in anamniotes are executioners of metamorphic reorganization, became intolerant to embryonic-oncofetal-antigens impeding organ regeneration, a process that requires de-differentiation of adult cells and/or expansion of stem cells where these early antigens are formed. The evolution of terrestrial lifecycles produced vertebrates with complex bodies but no longer capable to regenerate their organs, mainly repaired by regengrow. Efforts of regenerative medicine to improve healing in humans should determine the diverse developmental pathways evolved between anamniotes and amniotes before attempting genetic manipulations such as the introduction of "anamniote regenerative genes" in amniotes. This operation may determine alteration in amniote developmental programs leading to teratomes, cancer, or death.

Keratinization and Cornification are not equivalent processes but keratinization in fish and amphibians evolved into cornification in terrestrial vertebrates.

The present account offers a generalized view of the evolution of process of terminal differentiation in keratinocytes of the epidermis in anamniotes, indicated as keratinization, into a further differentiating process of cornification in the skin and appendages of terrestrial vertebrates. Keratinization indicates the prevalent accumulation of intermediate filaments of keratins (IFKs) and is present in most fish and amphibian epidermis and inner epithelia of all vertebrates. During land adaptation, terrestrial vertebrates evolved a process of cornification and keratinocytes became dead corneocytes by the addition of numerous others proteins to the IFKs framework, represented by keratin-associated proteins (KAPs) and corneous proteins (CPs). Most of genes coding for these types of proteins are localized in chromosomal loci different and un-related from those of IFKs, and CPs originated from a gene cluster indicated as epidermal differentiation complex. During the evolution of reptiles and birds, the epidermis and corneous derivatives such as scales, claws, beaks and feathers mainly accumulate a type of CPs that overcome IFKs and containing a 34 amino acid beta-sheet core indicated as corneous beta proteins, formerly known as beta-keratins. Mammals did not evolve a beta-sheet core in their CPs and KAPs but instead produced numerous cysteine-rich IFKs in their epidermis and specialized KAPs in hairs, claws, nails, hooves and horns.

Tail regeneration in Lepidosauria as an exception to the generalized lack of organ regeneration in amniotes.

The present review hypothesizes that during the transition from water to land, amniotes lost part of the genetic program for metamorphosis utilized in larvae of their amphibian ancestors, a program that in extant fish and amphibians allows organ regeneration. The direct development of amniotes, with their growth from embryos to adults, occurred with the elimination of larval stages, increases the efficiency of immune responses and the complexity of nervous circuits. In amniotes, T-cells and macrophages likely eliminate embryonic-larval antigens that are replaced with the definitive antigens of adult organs. Among lepidosaurians numerous lizard families during the Permian and Triassic evolved the process of tail autotomy to escape predation, followed by tail regeneration. Autotomy limits inflammation allowing the formation of a regenerative blastema rich in the immunosuppressant and hygroscopic hyaluronic acid. Expression loss of developmental genes for metamorphosis and segmentation in addition to an effective immune system, determined an imperfect regeneration of the tail. Genes involved in somitogenesis were likely lost or are inactivated and the axial skeleton and muscles of the original tail are replaced with a nonsegmented cartilaginous tube and segmental myotomes. Lack of neural genes, negative influence of immune system, and isolation of the regenerating spinal cord within the cartilaginous tube impede the production of nerve and glial cells, and a stratified spinal cord with ganglia. Tissue and organ regeneration in other body regions of lizards and other reptiles is relatively limited, like in the other amniotes, although the cartilage shows a higher regenerative capability than in mammals.

Adhesive pads of gecko and anoline lizards utilize corneous and cytoskeletal proteins for setae development and renewal.

The formation of the complex pattern of setae in adhesive pads of geckos and anoline lizards has been analyzed by ultrastructural, autoradiographic, and immunohistochemical methods. Setae terminate with spatulated ends responsible for adhesion that allow these lizards to climb vertical substrates and conquer arboreal niches. Setae derive from a complex interfaced molding between two specialized epidermal layers of the shedding complex that determines the cyclical skin molting, Oberhautchen and clear layers. Setae result from the action of setae cytoskeleton and the surrounding cytoplasm of clear cells. An intense protein synthesis, indicated by histidine and proline autoradiography, takes place during setae formation. Corneous and cytoskeletal proteins such as beta-proteins (CBPs), intermediate filament keratins (IFKs), actin, RhoV, tubulin, plakophilin-1, are produced during setae formation. Microfilaments of actin and microtubules of tubulin grow inside the elongating setae. Microtubules associated with filaments of unknown IKFs are produced in the cytoplasm of clear cells, forming a helical cytoskeleton that surrounds the growing setae. Oberhautchen and clear cells are tightly joined by numerous desmosomes and plakophilin-1 is mainly localized along the perimeter of these cells. These observations suggest that actin and tubulin are present in a dynamic form in the forming setae and in the surrounding cytoplasm of clear cells. Aside the localized micro-deformations of the cell membranes leading to setae formation the cytoskeleton determines the accumulation of CBPs inside the growing setae and the spatula. How the genome determines the specific pattern of cytoskeletal organization with the resulting species-specific setae branching remains unknown.

Vitamin A administration in lizards during tail regeneration determines epithelial mucogenesis and delays muscle and cartilage differentiation.

Regenerating epidermis and spinal cord is essential to maintain tail regeneration in lizards. The effects of vitamin A, an inhibitor of epithelial cornification, have been studied in lizards during tail regeneration. The injection of high doses of vitamin A induces regeneration of a thinner tail with gummy consistency and suppression of the formation of a normal cartilaginous axial skeleton. Microscopic analysis reveals that all epithelia increase the secretion of glycoprotein-mucus. During the analyzed period the epidermis does not form scales and keratinocytes limit or stop the production of bundles of intermediate filament keratins and packets of corneous beta-proteins (ß-keratins). Differentiation of oberhautchen and ß-layers is much reduced or inhibited while α-keratinization and the formation of a corneous layer are affected as well. The effects of vitamin A are dramatic also on mesoderm cells since the treatment stimulates an invasion of blood cells likely due to the disruption of the wall of blood vessels, mesenchymal cell death (pycnosis), and diffuse phagocytosis by immune cells. A delay of cartilage differentiation and cartilage degradation due to an increase of lysosomes in these cells or released by white blood cells explains the lack of stiffness of the regenerating tail after vitamin A treatment. Regenerating muscles are variably affected, ranging from a variable necrotic effect with partial degradation of internal organelles and myofilaments to a massive or complete loss of myofibrils that do not organize in sarcomeres. In general hypervitaminosis A appears to delay epithelial but also mesodermal cell differentiation and maintains the regenerating tail in an immature condition.

Molecular structure of sauropsid ß-keratins from tuatara (Sphenodon punctatus).

The birds and reptiles, collectively known as the sauropsids, can be subdivided phylogenetically into the archosaurs (birds, crocodiles), the testudines (turtles), the squamates (lizards, snakes) and the rhynchocephalia (tuatara). The structural framework of the epidermal appendages from the sauropsids, which include feathers, claws and scales, has previously been characterised by electron microscopy, infrared spectroscopy and X-ray diffraction analyses, as well as by studies of the amino acid sequences of the constituent ß-keratin proteins (also referred to as the corneous ß-proteins). An important omission in this work, however, was the lack of sequence and structural data relating to the epidermal appendages of the rhynchocephalia (tuatara), one of the two branches of the lepidosaurs. Considerable effort has gone into sequencing the tuatara genome and while this is not yet complete, there are now sufficient sequence data for conclusions to be drawn on the similarity of the ß-keratins from the tuatara to those of other members of the sauropsids. These results, together with a comparison of the X-ray diffraction pattern of tuatara claw with those from seagull feather and goanna claw, confirm that there is a common structural plan in the ß-keratins of all of the sauropsids, and not just those that comprise the archosaurs (birds and crocodiles), the testudines (turtles) and the squamates (lizards and snakes).

Perspective: Appendage regeneration in amphibians and some reptiles derived from specific evolutionary histories.

Some hypotheses on the evolution of regeneration in amphibians and reptiles are presented. Amphibian regeneration is derived from metamorphosis present in sarcopterygian fish and amphibians of the Devonian-Carboniferous. The genetic ability to rebuild organs during metamorphosis was maintained in form of "regeneration" in urodele and anuran tadpoles. Amphibian regeneration may be a consequence of the transition from an aquatic to a terrestrial environment through the evolution of a developmental program for the tadpole stage and replacements of adult organs controlled by the endocrine and immune system. Following metamorphosis, the regeneration program for terrestrial anurans and amniotes was lost or modified, whereas the immune system involved in self-integrity and microbial protection became in charge of regeneration that was replaced by scarring. Among amniotes only lizards regenerate an organ as large and complex as the tail. It is hypothesized that in Permian captorhinids and in Triassic lizards (eosuchians) a regenerative blastema evolved in relation to autotomy, a unique phenomenon present in these reptiles that enhanced survival against the larger predators of the Permian-Mesozoic. Appendage regeneration in amphibians and lizards occurs after the migration of activated mesenchymal and epidermal cells in the wounded areas to form soft and hyaluronate-rich blastemas. Autotomy and production of high hyaluronate levels allows high hydration and immunosuppression, favoring regeneration. It is suggested that a way for regenerative medicine to induce limb regeneration in humans is to develop medical procedures to recreate soft blastemas that can grow, a long and difficult process because it counteracts mammalian evolution toward scarring.

Review: Evolution and diversification of corneous beta-proteins, the characteristic epidermal proteins of reptiles and birds.

In all amniotes specialized intermediate filament keratins (IF-keratins), in addition to keratin-associated and corneous proteins form the outermost cornified layer of the epidermis. Only in reptiles and birds (sauropsids) the epidermis of scales, claws, beaks, and feathers, largely comprises small proteins formerly indicated as "beta-keratins" but here identified as corneous beta-proteins (CBPs) to avoid confusion with true keratins. Genes coding for CBPs have evolved within the epidermal differentiation complex (EDC), a locus with no relationship with those of IF-keratins. CBP genes have the same exon-intron structure as EDC genes encoding other corneous proteins of sauropsids and mammals, but they are unique by encoding a peculiar internal amino acid sequence motif beta-sheet region that allows formation of CBP filaments in the epidermis and epidermal appendages of reptiles and birds. In contrast, skin appendages of mammals, like hairs, claws, horns and nails, contain keratin-associated proteins that, like IF-keratin genes, are encoded by genes in loci different from the EDC. Phylogenetic analysis shows that lepidosaurian (lizards and snakes) and nonlepidosaurian (crocodilians, birds, and turtles) CBPs form two separate clades that likely originated after the divergence of these groups of sauropsids in the Permian Period. Clade-specific CBPs evolved to make most of the corneous material of feathers in birds and of the shell in turtles. Based on the recent identification of the complete sets of CBPs in all major phylogenetic clades of sauropsids, this review provides a comprehensive overview of the molecular evolution of CBPs.

Transcriptome analysis of the regenerating tail vs. the scarring limb in lizard reveals pathways leading to successful vs. unsuccessful organ regeneration in amniotes.

BACKGROUND: Lizards are amniotes regenerating the tail but not the limb, and no information on their different gene expression is available. RESULTS: Transcriptomes of regenerating tail and limb blastemas show differences in gene expression between the two organs. In tail blastemal, snoRNAs and Wnt signals appear up-regulated probably in association with the apical epidermal peg (AEP), an epithelial region that sustains tail regeneration but is absent in the limb. A balance between pro-oncogenes and tumor suppressors is likely present in tail blastema allowing a regulated proliferation. Small collagens, protease inhibitors, embryonic keratins are up-regulated in the regenerating tail blastema but not in the limb where Wnt inhibitors, inflammation-immune and extracellular matrix proteins depress cell growth. CONCLUSIONS: The AEP and the spinal cord in the tail maintains Wnt and fibroblast growth signaling that stimulate blastema cell proliferation and growth while these signals are absent in the limb as a consequence of the intense inflammation. Regeneration of amniote appendages requires a control of cell proliferation and inflammatory-immune reactions to form an apical epidermal cap. Genes that control cell proliferation and inflammation, addressing regeneration and not tumor formation in the tail and scarring in the limb are discussed for future studies. Developmental Dynamics 246:116-134, 2017. © 2016 Wiley Periodicals, Inc.

Comparative Genomics Identifies Epidermal Proteins Associated with the Evolution of the Turtle Shell.

The evolution of reptiles, birds, and mammals was associated with the origin of unique integumentary structures. Studies on lizards, chicken, and humans have suggested that the evolution of major structural proteins of the outermost, cornified layers of the epidermis was driven by the diversification of a gene cluster called Epidermal Differentiation Complex (EDC). Turtles have evolved unique defense mechanisms that depend on mechanically resilient modifications of the epidermis. To investigate whether the evolution of the integument in these reptiles was associated with specific adaptations of the sequences and expression patterns of EDC-related genes, we utilized newly available genome sequences to determine the epidermal differentiation gene complement of turtles. The EDC of the western painted turtle (Chrysemys picta bellii) comprises more than 100 genes, including at least 48 genes that encode proteins referred to as beta-keratins or corneous beta-proteins. Several EDC proteins have evolved cysteine/proline contents beyond 50% of total amino acid residues. Comparative genomics suggests that distinct subfamilies of EDC genes have been expanded and partly translocated to loci outside of the EDC in turtles. Gene expression analysis in the European pond turtle (Emys orbicularis) showed that EDC genes are differentially expressed in the skin of the various body sites and that a subset of beta-keratin genes within the EDC as well as those located outside of the EDC are expressed predominantly in the shell. Our findings give strong support to the hypothesis that the evolutionary innovation of the turtle shell involved specific molecular adaptations of epidermal differentiation.

Hyaluronic acid in the tail and limb of amphibians and lizards recreates permissive embryonic conditions for regeneration due to its hygroscopic and immunosuppressive properties.

The present review focuses on the role of hyaluronate (hyaluronic acid; HA) during limb and tail regeneration in amphibians and lizards mainly in relation to cells of the immune system. This non-sulfated glycosaminoglycan (GAG) increases in early stages of wound healing and blastema formation, like during limb or tail embryogenesis, when the immune system is still immature. The formation of a regenerating blastema occurs by the accumulation of mesenchymal cells displaying embryonic-like antigens and HA. This GAG adsorbs large amount of water and generates a soft tissue over 80% hydrated where mesenchymal and epithelial cells can move and interact, an obligatory passage for organ regeneration. GAGs and HA in particular rise to a high amount and coat plasma membranes of blastema cells forming a shield that likely impedes to the circulating immune cells to elicit an immune reaction against the embryonic-like antigens present on blastema cells. The evolution of limb-tail regeneration in amphibians dates back to the Devonian-Carboniferous, while tail regeneration in lizards is a more recent evolution process, possibly occurred since the Jurassic, which is unique among amniotes. Both processes are associated with the reactivation of proliferating embryonic programs that involve the upregulation of genes for Wnt, non-coding RNAs, and HA synthesis in an immune-suppress organ, the regenerative blastema. Failure of maintaining a lasting HA synthesis for the formation of a highly hydrated blastema leads to scarring, the common healing process of amniotes equipped with an efficient immune system. The study of amphibian and lizard regeneration indicates that attempts to stimulate organ regeneration in other vertebrates require the induction of a highly hydrated and immune-depressed, HA-rich environment, similar to the extracellular environment present during development.

Review: Biological and Molecular Differences between Tail Regeneration and Limb Scarring in Lizard: An Inspiring Model Addressing Limb Regeneration in Amniotes.

Tissue regeneration in lizards represents a unique model of regeneration and scarring in amniotes. The tail and limb contain putative stem cells but also dedifferentiating cells contribute to regeneration. Following tail amputation, inflammation is low and cell proliferation high, leading to regeneration while the intense inflammation in the limb leads to low proliferation and scarring. FGFs stimulate tail and limb regeneration and are present in the wound epidermis and blastema while they disappear in the limb wound epidermis 2-3 weeks postamputation in the scarring outgrowth. FGFs localize in the tail blastema and the apical epidermal peg (AEP), an epidermal microregion that allows tail growth but is absent in the limb. Inflammatory cells invade the limb blastema and wound epidermis, impeding the formation of an AEP. An embryonic program of growth is activated in the tail, dominated by Wnt-positive and -negative regulators of cell proliferation and noncoding RNAs, that represent the key regenerative genes. The balanced actions of these regulators likely impede the formation of a tumor in the tail tip. Genes for FACIT and fibrillar collagens, protease inhibitors, and embryonic keratins are upregulated in the regenerating tail blastema. A strong downregulation of genes for both B and T-lymphocyte activation suggests the regenerating tail blastema is a temporal immune-tolerated organ, whereas a scarring program is activated in the limb. Wnt inhibitors, pro-inflammatory genes, negative regulators of cell proliferation, downregulation of myogenic genes, proteases, and oxidases favoring scarring are upregulated. The evolution of an efficient immune system may be the main limiting barrier for organ regeneration in amniotes, and the poor regeneration of mammals and birds is associated with the efficiency of their mature immune system. This does not tolerate embryonic antigens formed in reprogrammed embryonic cells (as for neoplastic cells) that are consequently eliminated impeding the regeneration of lost organs.

The molecular organization of the beta-sheet region in Corneous beta-proteins (beta-keratins) of sauropsids explains its stability and polymerization into filaments.

The hard corneous material of avian and reptilian scales, claws, beak and feathers is mainly derived from the presence of proteins formerly known as beta-keratins but now termed Corneous beta-proteins of sauropsids to distinguish them from keratins, which are members of the intermediate filament protein family. The modeling of the conserved 34 amino acid residues long central beta-sheet region of Corneous beta-proteins using an ab initio protein folding and structure prediction algorithm indicates that this region is formed by four antiparallel beta-sheets. Molecular dynamic simulations and Molecular Mechanics/Poisson Boltzmann Surface Area (MM-PBSA) analysis showed that the disposition of polar and apolar amino acids within the beta-region gives rise to an amphipathic core whose stability is further increased, especially in an aqueous environment, by the association into a dimer due to apolar interactions and specific amino-acid interactions. The dimers in turn polymerize into a 3nm thick linear beta-filament due to van der Waals and hydrogen-bond interactions. It is suggested that once this nuclear core of anti-parallel sheets evolved in the genome of a reptilian ancestor of the extant reptiles and birds about 300 millions years ago, new properties emerged in the corneous material forming scales, claws, beaks and feathers in these amniotes based on the tendency of these unique corneous proteins to form stable filaments different from keratin intermediate filaments or sterical structures formed by other corneous proteins so far known.

Sauropsids Cornification is Based on Corneous Beta-Proteins, a Special Type of Keratin-Associated Corneous Proteins of the Epidermis.

The evolution of the process of cornification in amniote epidermis from the general process of keratinization present in simple epithelia of anamniotes took place through the evolution of specialized intermediate filament (α) keratins, keratin-associated proteins (KAPs) and corneous proteins (CPs). The scanty information on the three-dimensional conformation of known KAPs and CPs indicate these proteins contain α-helix, random coiled, or beta sheets with different lengths and organizations. CP genes originated in a chromosome locus indicated as epidermal differentiation complex (EDC), and transformed the epidermal keratinization of anamniotes into the cornified epidermis and skin appendages of amniotes (claws, beaks, and feathers). In particular, peculiar genes encoding for small proteins with a central region of 34 amino acids conformed as beta sheets were originated in the EDC of sauropsids (reptiles and birds). These proteins were traditionally indicated as beta-keratins because they form filaments of 3-4 nm in diameter and show an X-ray beta pattern. Different from other proteins of the EDC, dimers of these corneous beta-proteins associate into long polymers of filamentous proteins utilized in sauropsids skin appendages, such as scales and feathers. Future challenges in this area of research will be the study on gene regulation and expression for these proteins, their origin and evolution in different lineages of sauropsids, and their role in determining the material properties of sauropsid scales and other skin appendages.

Evolutionary origin and diversification of epidermal barrier proteins in amniotes.

The evolution of amniotes has involved major molecular innovations in the epidermis. In particular, distinct structural proteins that undergo covalent cross-linking during cornification of keratinocytes facilitate the formation of mechanically resilient superficial cell layers and help to limit water loss to the environment. Special modes of cornification generate amniote-specific skin appendages such as claws, feathers, and hair. In mammals, many protein substrates of cornification are encoded by a cluster of genes, termed the epidermal differentiation complex (EDC). To provide a basis for hypotheses about the evolution of cornification proteins, we screened for homologs of the EDC in non-mammalian vertebrates. By comparative genomics, de novo gene prediction and gene expression analyses, we show that, in contrast to fish and amphibians, the chicken and the green anole lizard have EDC homologs comprising genes that are specifically expressed in the epidermis and in skin appendages. Our data suggest that an important component of the cornified protein envelope of mammalian keratinocytes, that is, loricrin, has originated in a common ancestor of modern amniotes, perhaps during the acquisition of a fully terrestrial lifestyle. Moreover, we provide evidence that the sauropsid-specific beta-keratins have evolved as a subclass of EDC genes. Based on the comprehensive characterization of the arrangement, exon-intron structures and conserved sequence elements of EDC genes, we propose new scenarios for the evolutionary origin of epidermal barrier proteins via fusion of neighboring S100A and peptidoglycan recognition protein genes, subsequent loss of exons and highly divergent sequence evolution.

Immunolocalization of loricrin in the maturing α-layer of normal and regenerating epidermis of the lizard Anolis carolinensis.

Numerous corneous proteins are produced during the differentiation of the complex lizard epidermis, comprising hard ß-layers and softer α-layers. In the present ultrastructural and immunocytochemical study, we have localized a homolog of the mammalian skin barrier protein loricrin in the skin of the green anole lizard (Anolis carolinensis). We used an antibody specific to the carboxyterminus of loricrin 1, a gene of the epidermal differentiation complex (EDC) of A. carolinensis. Lizard loricrin is present in the maturing α-layer (lacunar cells) of normal scale epidermis and in the accumulating corneocytes of the wound epidermis (lacunar cells) of the regenerating epidermis. The protein appears as a component of the α-layer but not of the ß-layer. Lizard loricrin is diffused in the cytoplasm of pre-corneous α-keratinocytes but eventually concentrates in the packing corneous material of the maturing corneocytes of the α-layer (lacunar) in normal epidermis or in the wound epidermis of regenerating epidermis. The protein likely contributes to the composition and pliability of the corneous material but is not specifically accumulated on the corneous cell envelope (marginal layer) that is scarcely differentiated in these cells. The study contributes to the knowledge on the distribution of specific corneous proteins that give rise to the different material properties of α-layers versus ß-layers in lizard epidermis.