Immunolabeling for filaggrin and acidic keratins in the granular layer of mammalian epidermis indicates that an acidic-basic interaction is involved in cornification.

The soft epidermis of mammals derives from the accumulation of keratohyaline granules in the granular layer, before maturing into corneocytes. Main proteins accumulated in the granular layer are pro-filaggrin and filaggrin that determine keratin clumping and later moisturization of the stratum corneum that remains flexible. This soft epidermis allows the high sensitivity of mammalian skin. Presence and thickness of the stratum granulosum varies among different species of mammals and even between different body regions of the same animal, from discontinuous to multilayered. These variations are evident using antibodies for filaggrin, a large protein that share common epitopes among placentals. Here we have utilized filaggrin antibodies (8959 and 466) and an acidic keratin antibody (AK2) for labeling placental, marsupial and monotreme epidermis. AK2 labeling appears mainly to detect K24 keratin, and less likely other acidic keratins. Immunoreactivity for filaggrin is absent in platypus, discontinuous in Echidna and in the tested marsupials. In placentals, it is inconstantly or hardly detected in the thin epidermis of bat, rodents, and lagomorphs with a narrow, mono-stratified and/or discontinuous granular layer. In contrast, where the granular layer is continuous or even stratified, both filaggrin and AK2 antibodies decorate granular cells. The ultrastructural analysis using the AK2 antibody on human epidermis reveals that a weak labeling is associated with keratohyalin granules and filamentous keratins of transitional keratinocytes and corneocytes. This observation suggests that basophilic filaggrin interacts with acidic keratins like K24 and determines keratin condensation into corneocytes of the stratum corneum.

Immunolocalization of telomerases in human hairs identifies proliferating cells in the bulb matrix and outer root sheath.

Telomerase is present in cells with numerous or even un-limited replicative cycles, and some studies suggest it is a stemness marker. In order to determine whether this is the case for the human hair bulbs, an immunohistochemical and ultrastructural study has been carried out using antibodies against telomerase and PCNA (a cell proliferation marker). The observed labeling is similar for the two antibodies here utilized and is mainly nuclear. More frequent telomerase-positive cells are seen in the matrix epithelium of anagen hair bulbs but sparse labeled cells are also seen in the outer root sheath. In late catagen and also in telogen hair follicles only sparse labeled cells are present in the outer root sheath and few cells also in the secondary germinal epithelium formed at the base of the hair bulb in telogen. Electron microscopic immunogold shows a prevalent nuclear distribution and a lower cytoplasmic distribution in sparse cells of anagen bulb matrix that contain few keratin bundles. The nuclear localization is generally seen over the euchromatin or in areas occupied by more compact chromatin that may indicate an activity of telomerase in chromatin assemblage or dis-assemblage. The study concludes that the localization of telomerase is present in cells undergoing proliferation, namely transit amplifying cells of the outer root sheath that are sparsely detected in the lowermost secondary germinal hair bulb also in telogen.

Regeneration Abilities among Extant Animals Depend on Their Evolutionary History and Life Cycles.

The present brief manuscript summarizes the main points supporting recently proposed hypotheses explaining the different distributions of regenerative capacity among invertebrates and vertebrates. The new hypotheses are based on the evolution of regeneration from marine animals to the terrestrial animals derived from them. These speculations suggest that animals that were initially capable of broad regeneration in the sea underwent epigenetic modifications during terrestrial adaptation that determined the loss of their regenerative abilities in sub-aerial conditions. These changes derived from the requirements of life on land that include variable dry and UV-exposed conditions. Terrestrial conditions do not allow for organ regeneration, especially in arthropods and amniotes. Nematodes, the other main metazoan group unable of regeneration, instead evolved eutely (a fixed number of body cells), a process which is incompatible with regeneration. All these changes involved gene loss, modification and new gene interactions within the genomes of terrestrial adapting animals that gave rise to sophisticated invertebrates and vertebrates adapted to living on land but with low cellular plasticity.

Comparative immunohistochemical analysis suggests a conserved role of EPS8L1 in epidermal and hair follicle barriers of mammals.

The mammalian skin and its appendages depend on tightly coordinated differentiation of epithelial cells. Epidermal growth factor receptor (EGFR) pathway substrate 8 (EPS8) like 1 (EPS8L1) is enriched in the epidermis among human tissues and has also been detected in the epidermis of lizards. Here, we show by the analysis of single-cell RNA-sequencing data that EPS8L1 mRNA is co-expressed with filaggrin and loricrin in terminally differentiated human epidermal keratinocytes. Comparative genomics indicated that EPS8L1 is conserved in all main clades of mammals, whereas the orthologous gene has been lost in birds. Using a polyclonal antibody against EPS8L1, we performed an immunohistochemical screening of skin from diverse mammalian species and immuno-electron microscopy of human skin. EPS8L1 was detected predominantly in the granular layer of the epidermis in monotremes, marsupial, and placental mammals. The labeling was partly associated with cell membranes, and it was evident along the perimeter of keratinocytes at the transition with the cornified layer of the epidermis, similar to involucrin distribution. Basal, spinous, and the fully mature cornified layers lacked immunolabeling of EPS8L1. In addition to the epidermis, the hair follicle inner root sheath (IRS) was immunolabeled. Both epidermal granular layer and IRS contribute to the barrier function of the skin, suggesting that EPS8L1 is involved in the regulation of these barriers.

Actin-templated Structures: Nature's Way to Hierarchical Surface Patterns (Gecko's Setae as Case Study).

The hierarchical design of the toe pad surface in geckos and its reversible adhesiveness have inspired material scientists for many years. Micro- and nano-patterned surfaces with impressive adhesive performance have been developed to mimic gecko's properties. While the adhesive performance achieved in some examples has surpassed living counterparts, the durability of the fabricated surfaces is limited and the capability to self-renew and restore function-inherent to biological systems-is unimaginable. Here the morphogenesis of gecko setae using skin samples from the Bibron´s gecko (Chondrodactylus bibronii) is studied. Gecko setae develop as specialized apical differentiation structures at a distinct cell-cell layer interface within the skin epidermis. A primary role for F-actin and microtubules as templating structural elements is necessary for the development of setae's hierarchical morphology, and a stabilization role of keratins and corneus beta proteins is identified. Setae grow from single cells in a bottom layer protruding into four neighboring cells in the upper layer. The resulting multicellular junction can play a role during shedding by facilitating fracture of the cell-cell interface and release of the high aspect ratio setae. The results contribute to the understanding of setae regeneration and may inspire future concepts to bioengineer self-renewable patterned adhesive surfaces.

Immunolocalization of lix1 in the regenerating tail of lizard indicates that the protein is mainly present in the nervous tissue.

PURPOSE: Lizard regeneration derives from the re-activation of a number of developmental genes after tail amputation. Among genes with the highest expression, as indicated from the transcriptome, is lix1 which functional role is not known. METHOD: An antibody that cross-reacts with the lizard Podarcis muralis lix1 has been utilized to detect by immunofluorescence the sites of localization of the protein in the regenerating tail. RESULTS: Lix1-protein is almost exclusively localized in the regenerating spinal cord (ependyma) and nerves growing into the blastema, in sparse blastema cells but is undetectable in other tissues. CONCLUSIONS: Since the spinal cord is essential to stimulate tail regeneration it is hypothesized that the lix1 protein is part of the signaling or growing factors produced from the regenerating spinal cord that are needed for tail regeneration of the lizard tail.

Scales of non-avian reptiles and their derivatives contain corneous beta proteins coded from genes localized in the Epidermal Differentiation Complex.

The evolution of modern reptiles from basic reptilian ancestors gave rise to scaled vertebrates. Scales are of different types, and their corneous layer can shed frequently during the year in lepidosaurians (lizards, snakes), 1-2 times per year in the tuatara and in some freshwater turtle, irregularly in different parts of the body in crocodilians, or simply wore superficially in marine and terrestrial turtles. Lepidosaurians possess tuberculate, non-overlapped or variably overlapped scales with inter-scale (hinge) regions. The latter are hidden underneath the outer scale surface or may be more exposed in specific body areas. Hinge regions allow stretching during growth and movement so that the skin remains mechanically functional. Crocodilian and turtles feature flat and shield scales (scutes) with narrow inter-scale regions for stretching and growth. The epidermis of non-avian reptilian hinge regions is much thinner than the exposed outer surface of scales and is less cornified. Despite the thickness of the epidermis, scales are mainly composed of variably amount of Corneous Beta Proteins (CBPs) that are coded in a gene cluster known as EDC (Epidermal Differentiation Complex). These are small proteins, 100-200 amino acid long of 8-25 kDa, rich in glycine and cysteine but also in serine, proline and valine that participate to the formation of beta-sheets in the internal part of the protein, the beta-region. This region determines the further polymerization of CBPs in filamentous proteins that, together a network of Intermediate Filament Keratins (IFKs) and other minor epidermal proteins from the EDC make the variable pliable or inflexible corneous material of reptilian scales, claws and of turtle beak. The acquisition of scales and skin derivatives with different mechanical and material properties, mainly due to the evolution of reptile CBPs, is essential for the life and different adaptations of these vertebrates.

Immunolocalization of Some Epidermal Proteins and Glycoproteins in the Growing Skin of the Australian Lungfish (Neoceratodus forsteri).

Here we report the immunolocalization of mucin, nestin, elastin and three glycoproteins involved in tissue mineralization in small and large juveniles of Neoceratodus forsteri. Both small and larger juvenile epidermis are mucogenic and contain a diffuse immunolabeling for nestin. Sparse PCNA-labeled cells, indicating proliferation, are found in basal and suprabasal epidermal layers. No scales are formed in small juveniles but are present in a 5 cm long juvenile and in larger juveniles. Elastin and a mineralizing matrix are localized underneath the basement membrane of the tail epidermis where lepidotriches are forming. The latter appears as "circular bodies" in cross sections and are made of elongated cells surrounding a central amorphous area containing collagen and elastin-like proteins that undergo calcification as evidenced using the von Kossa staining. However, the first calcification sites are the coniform teeth of the small juveniles of 2-3 cm in length. In the superficial dermis of juveniles (16-26 cm in length) where scales are formed, the spinulated outer bony layer (squamulin) of the elasmoid scales contains osteonectin, alkaline phosphatase, osteopontin, and calcium deposits that are instead absent in the underlying layer of elasmodin. In particular, these glycoproteins are localized along the scale margin in juveniles where scales grow, as indicated by the presence of PCNA-labeled cells (proliferating). These observations suggest a continuous deposition of new bone during the growth of the scales, possibly under the action of these mineralizing glycoproteins, like in the endoskeleton of terrestrial vertebrates.

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.

Protein extracts from regenerating lizard tail show an inhibitory effect on human cancer cells cultivated in-vitro.

BACKGROUND: accumulating evidence indicates that during tail regeneration in lizards the initial stage of regenerative blastema is a tumor-like proliferative outgrowth that rapidly elongates into a new tail composed of fully differentiated tissues. Both oncogenes and tumor-suppressors are expressed during regeneration, and it has been hypothesized that an efficient control of cell proliferation avoids that the blastema is turned into a tumor outgrowth. METHODS: in order to determine whether functional tumor-suppressors are present in the growing blastema we have utilized protein extracts collected from early regenerating tails of 3-5 mm that have been tested for a potential anti-tumor effect on in-vitro culture by using cancer cell lines from human mammary gland (MDA-MB-231) and prostate cancer (DU145). RESULTS: at specific dilutions, the extract determines a reduction of viability in cancer cells after 2-4 days of culture, as supported by statistical and morphological analyses. While control cells appear viable, treated cells result damaged and produce an intense cytoplasmic granulation and degeneration. CONCLUSIONS: this negative effect on cell viability and proliferation is absent using tissues from the original tail supporting the hypothesis that only regenerating tissues synthesize tumor-suppressor molecules. The study suggests that the regenerating tail of lizard at the stages here selected contains some molecules that determine inhibition of cell viability on the cancer cells analyzed.

Regeneration or Scarring Derive from Specific Evolutionary Environmental Adaptations of the Life Cycles in Different Animals.

The ability to heal or even regenerate large injuries in different animals derives from the evolution of their specific life cycles during geological times. The present, new hypothesis tries to explain the distribution of organ regeneration among animals. Only invertebrates and vertebrates that include larval and intense metamorphic transformations can broadly regenerate as adults. Basically, regeneration competent animals are aquatic while terrestrial species have largely or completely lost most of the regeneration ability. Although genomes of terrestrial species still contain numerous genes that in aquatic species allow a broad regeneration ("regenerative genes"), the evolution of terrestrial species has variably modified the genetic networks linking these genes to the others that evolved during land adaptation, resulting in the inhibition of regeneration. Loss of regeneration took place by the elimination of intermediate larval phases and metamorphic transformations in the life cycles of land invertebrates and vertebrates. Once the evolution along a specific lineage generated species that could no longer regenerate, this outcome could not change anymore. It is therefore likely that what we learn from regenerative species will explain their mechanisms of regeneration but cannot or only partly be applied to non-regenerative species. Attempts to introduce "regenerative genes" in non-regenerative species most likely would disorder the entire genetic networks of the latter, determining death, teratomas and cancer. This awareness indicates the difficulty to introduce regenerative genes and their activation pathways in species that evolved genetic networks suppressing organ regeneration. Organ regeneration in non-regenerating animals such as humans should move to bio-engineering interventions in addition to "localized regenerative gene therapies" in order to replace lost tissues or organs.

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.

Micro-ornamentation patterns in different areas of the epidermis in the gecko Tarentola mauritanica reflect variations in the accumulation of corneous material in Oberhautchen cells.

Micro-ornamentations characterize the surface of scales in lepidosaurians and are summarized in four main patterns, i.e., spinulated, lamellated, lamellate-dentate, and honeycomb, although variations of these patterns are present in different species. Although geckos are known to possess a spinulated pattern derived from the Oberhautchen layer, also other pattern variations of the spinulated micro-ornamentation are present such as those indicated as dendritic ramification, corneous belts, and small bare patches. The present study mainly describes the variation of micro-ornamentations present in scales of different skin regions in the Mediterranean gecko Tarentula mauritanica using scannig and transmission electron microscopy. The study reports that the accumulation of corneous material in Oberhautchen cells is not homogenous in different areas of body scales and, when mature, this process gives rise to different sculpturing on the epidermal surface generating not only spinulae but also transitional zones leading to the other main patterns. It is hypothesized that spinulae formation derives from the vertical and lateral symmetric growth of tubercolate, non-overlapped scales of geckos. Sparse areas also result smooth or with serpentine-ridges likely revealing the beta-layer located underneath and merged with the Oberhautchen. The eco-functional role of this variable micro-ornamentation in the skin of lizards however remains largely speculative.

Immunolocalization of Pglyrp3 and Eps8l1 proteins in the regenerating lizard epidermis indicates they contribute to epidermal barrier formation.

During tail regeneration in lizards the new corneous layer formed in the regenerating epidermis includes antimicrobial peptides, cystatin and serpins, likely forming an anti-microbial barrier. The present study aims to reveal other proteins potentially contributing to this protective barrier of the epidermis. Using immunohistochemistry we have detected a peptidoglycan-like recognition protein-3 (pglyrp3), an antimicrobial molecule, and an epidermal growth factor receptor kinase 8 l (eps8l), a receptor of EGF (Epidermal Growth Factor) that stimulates epidermal formation. The study shows that the two proteins are mostly accumulated in the forming wound epidermis and in the shedding layer of the regenerating scales. The shedding layer is the intra-epidermal layer that allows the separation of the initial corneous layer from the regenerating epidermis. While presence of pglyrp3 is likely related to the formation of the anti-microbial barrier, the function of the eps8l protein in epidermal regeneration remains unknown. Whether the latter protein is involved in keratinocyte movement within the regenerating epidermis has to be specifically determined in future studies. Together with the antimicrobial peptides cystatin and serpins, previously detected in the wound epidermis and shedding layer, the present study indicates that pglyp3, and potentially eps8l, contribute to protect the new skin and underlying regenerated tissues from the potential microbe invasion.

Introduction to the Development of Skin in Vertebrates.

The integument of vertebrates is a complex and large organ positioned at the interface with the aquatic or terrestrial environment, and is derived from the embryonic ectoderm (epidermis) and mesoderm (dermis and hypodermis) [...].

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.

Electron microscopic analysis in the gecko Lygodactylus reveals variations in micro-ornamentation and sensory organs distribution in the epidermis that indicate regional functions.

Possible pattern variations of micro-ornamentation in different areas of the skin in the gecko Lygodactylus have been analyzed by scanning and transmission electron microscopy. A map of micro-ornamentation present in various areas of the skin has been obtained. Differences in micro-ornamentation pattern and sensory organ distribution were detected. The "spinulated pattern" consists of shorter spinulae in dorsal versus ventral scales, and spinules are shorter in inner scale surface and hinge regions with respect to the outer scale surface. The spines derive from the accumulation of struts of corneous material mainly composed of corneous beta proteins (CBPs, formerly indicated as beta-keratins) that merge into pointed micro-ornamentation. The 3D-accumulation of CBPs within Oberhautchen cells can vary in some regions of different scales during Oberhautchen-beta cell differentiation, perhaps also under physical tensile forces derived from continuous scale growth. Three other main patterns of micro-ornamentation were detected and indicated as "corneous belts," "corneous dendritic ramification," and "serpentine-pit and groove." These variations from the typical spinulated pattern present in gecko epidermis are interpreted as transitional regions where the accumulation of corneous material in Oberhautchen cells that merges with underlying beta-cells gives rise to nonspinulated surfaces. Spinulated sensory organs with bristles and lenticular-shaped or knob-like tactile corpuscles are more numerous in ventral scales of the tail tip close to adhesive pads and near the digital pads. These regions are likely those most involved in the fine control of movements and response to vibrational stimuli derived from air and objects movements, including potential preys or predators.

Immunolocalization of tumor suppressors arhgap28 and retinoblastoma in the lizard Podarcis muralis suggests that they contribute to the regulated regeneration of the tail.

Tail regeneration in lizards is an outstanding and unique postembryonic morphogenetic process. This developmental process is regulated by poorly known factors, but recent studies have suggested that it derives from a balanced activity between oncoproteins and tumor suppressors. Transcriptome and expression data have indicated that arhgap28 and retinoblastoma proteins are among the main tumor suppressors activated during tail regeneration. However, their cellular localization is not known. Therefore, in the present immunohistochemical study, two proteins have been detected in various tissues at the beginning of their differentiation. Both proteins are present especially in the new scales, axial cartilage, and muscle bundles of the regenerating tail, the main tissues forming the new tail. Sparse or occasionally labeled cells are observed in the blastema, but intense labeling is seen in the basal layers of the wound (regenerating) epidermis and in external differentiating epidermal layers. Numerous keratinocytes also show a nuclear localization for both proteins, suggesting that the latter may activate a gene program for tissue differentiation after the inhibition of cell multiplication. Based on microscopic, molecular, experimental, and in vitro studies, a hypothesis on the "inhibition of contact" among the apical cells of the blastema and those of proximal differentiating tissues is proposed to explain the permanence of an active blastema only at the apex of the regenerating tail without tail growth can degenerate into a tumorigenic outgrowth.

Immunohistochemistry indicates that persistent inflammation determines failure of tail, limb and finger regeneration in the Lizard Podarcis muralis.

BACKGROUND: The presence of white blood inflammatory cells in injured tissues and their effect on the process of organ regeneration in lizards has been assessed on tail, limb and digits. METHODS: The present immunohistochemical survey analyzes the occurrence of CD68-labeled cells in lizard organs uncapable of regenerating tissues that exhibit strong inflammatory activity. RESULTS: This marker mainly identifies macrophages and mast cells present in large number within tissues of injured limbs and digits. Also a high inflammation is associated with amputated tails that do not regenerate, derived from cauterization or infection of tissues of the tail stump. In the healing limbs and fingers at 12-20 days post-amputation, numerous CD68-labeled cells, most likely macrophages, are seen among superficial connective tissues and injured muscles and bones. These cells likely stimulate and give rise to scarring tissues and no regeneration of limb and fingers occurs. In the cauterized or in the infected tail stump a strong accumulation of CD68-positive mast cells and macrophages is observed, where they likely evoke epidermal coagulation, formation of scarring connective tissue, and loss of regeneration. CONCLUSIONS: The present observations provide further cytological evidence that support the notion that a strong and lasting inflammatory condition impedes organ regeneration in specifically lizards and, more generally other vertebrates as well.

Immunolocalization of adenomatous polyposis coli protein (apc) in the regenerating lizard tail suggests involvement in tissue differentiation and regulation of growth.

Lizard tail regeneration is likely regulated by the balanced activity of oncogenes and tumor suppressors that control cell proliferation avoiding tumorigenic degeneration. One of the main tumor suppressor genes present in the regenerating tail is the "adenomatous polyposis coli (apc)" but the localization of its coded protein (apc) is not known. This protein may be involved in regulation of apical-basal tail regeneration in lizards. The present immunohistochemical study shows that apc is localized in apical wound epidermis and regenerating ependyme, two tissues that proliferate and also express onco-genes. Apc is not present in blastema cells but localizes in differentiating cells of regenerating scales, muscles and less intensely in the non-apical ependymal epithelium and cartilage. This suggests that apc is involved in the induction of their differentiation. The apc immunolabeling is mainly nuclear in the basal epidermal layer of the apical wound epidermis where it may be involved in modulating keratinocytes proliferation, like in the forming scales. In regenerating muscle and cartilage apc is mainly cytoplasmic while sparse labeled nuclei are seen in proliferative areas of these tissues. In the regenerating spinal cord, the nuclear and cytoplasmic apc labeling is present in ependymal cells of the distal-most ependymal ampulla but the labeling fades in more proximal regions and mainly remains in the cytoplasm facing the central canal and in sparse nuclei. It is suggested that the pattern of immunolabeling for apc indicates that this tumor suppressor may contribute to tissue differentiation within the regenerating tail.