Immunocalization of telomerase in cells of lizard tail after amputation suggests cell activation for tail regeneration

Tissue and Cell 48 (2016) 63–71

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Immunocalization of telomerase in cells of lizard tail after amputation suggests cell activation for tail regeneration L. Alibardi Comparative Histolab and Department of Bigea, University of Bologna, via Selmi 3, 40126 Bologna, Italy

a r t i c l e

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Article history: Received 1 September 2015 Received in revised form 25 October 2015 Accepted 25 October 2015 Available online 10 November 2015 Keywords: Lizard Tail Amputation Regeneration Telomerase Immunocytochemistry

a b s t r a c t Tail amputation (autotomy) in most lizards elicits a remarkable regenerative response leading to a new although simplified tail. No information on the trigger mechanism following wounding is known but cells from the stump initiate to proliferate and form a regenerative blastema. The present study shows that telomerases are mainly activated in the nuclei of various connective and muscle satellite cells of the stump, and in other tissues, probably responding to the wound signals. Western blotting detection also indicates that telomerase positive bands increases in the regenerating blastema in comparison to the normal tail. Light and ultrastructural immunocytochemistry localization of telomerase shows that 4–14 days post-amputation in lizards immunopositive nuclei of sparse cells located among the wounded tissues are accumulating into the forming blastema. These cells mainly include fibroblasts and fat cells of the connective tissue and satellite cells of muscles. Also some immature basophilic and polychromatophilic erytroblasts, lymphoblasts and myelocytes present within the Bone Marrow of the vertebrae show telomerase localization in their nuclei, but their contribution to the formation of the regenerative blastema remains undetermined. The study proposes that one of the initial mechanisms triggering cell proliferation for the formation of the blastema in lizards involve gene activation for the production of telomerase that stimulates the following signaling pathways for cell division and migration. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Regeneration of the tail in lizards is a remarkable case of amniote organ regeneration (Bellairs and Bryant, 1985; Alibardi, 2010a, 2014). Ultrastructural studies have shown that after amputation cells of different tissues in the tail stump accumulate on the free surface of the tail and give rise to a mass of mesenchymal-like cells called blastema, covered by a stratified (wound) epidermis, a cone-like structure resembling the developing tail bud formed during embryogenesis (Alibardi and Sala, 1988; Alibardi and Toni, 2005; Alibardi, 2010a,b). However the organogenetic potential of the tail blastema is much limited in comparison to the embryonic tail bud and simplified tissues are regenerated in comparison to the normal tail (Quattrini, 1954; Simpson, 1965; Werner, 1967; Cox, 1969; McLean and Vickaryous, 2011; Fisher et al., 2012; Gilbert et al., 2013; Lozito and Tuan, 2015). The molecular signal that triggers the controlled proliferation, migration, and accumulation of blastema cells remains enigmatic, as well as the initial nuclear and cellular response of the injured tissues to the wound stimulation. Recent studies have indicated

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that the blastema derives from the activation of resident stem cells more than from tissue dedifferentiation (Alibardi, 2014). Other studies have shown that two cell regulator proteins, telomerase and p53/63, are activated during tail regeneration in lizards, addressing future studies on the mechanism allowing a controlled regulation of the process of tissue regeneration (Alibardi, 2015a,b). While p53/63 is mainly involved in the control of epidermal proliferation and differentiation, telomerase appears to be present in sparse connective cells. This enzyme consists in a protein component (TERT, Telomerase Retro Transcriptase) that associates to a special RNA-template (TERC, Telomerase RNA Component) for catalizing the addition of telomere sequences to chromosomal ends during DNA duplication. Since telomerase is believed to be constitutionally active in stem cells, or at least when they give rise to transient amplifying cells, the presence of few telomerase positive cells in the regenerating tail suggests that few stem cells are actually accumulated in regenerating tissues (Alibardi, 2015a,c), an observation also indicated in a recent molecular study on other stem cell markers (Hutchins et al., 2014). This initial observation has suggested searching for the presence of telomerase at earlier stages of wounding and blastema formation in comparison to normal tail tissues using immunological methods. In the present study we have examined, using light and ultrastructural immunocytochemistry, and western blotting, for the

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presence of telomerase in the early events of regeneration, the first 4–14 days post-wounding, when an extensive tissue degeneration, blood cell infiltration, and tissue recovering is taking place, preparing the surface of the tail stump to accumulate mesenchymal-like cells (Alibardi and Sala, 1988; Alibardi, 2010b; McLean and Vickaryous, 2011).

2. Materials and methods 2.1. Western blotting Two normal tails from Podarcis muralis were utilized for protein extraction. Three other samples from P. muralis and two from Anolis carolinensis, consisting of the stump (1–2 mm) and their relative conical blastema (2–3 mm in length) formed at 14 days after amputation were also utilized. Before analysis, the tail stump was separated from the blastema, and the two pieces were analyzed independently. Tissues were homogenized in 8 M urea/50 mM Tris–HCl at pH 7.6 containing 0.1 M 2-mercaptoethanol/1 mM dithiothreithol/ and 1% protease inhibitor (SIGMA, St. Louis, USA). The particulate matter was removed by centrifugation at 10,000 × g for 5 min, and protein concentration was assayed by the Bradford method before electrophoresis. For the electrophoresis analysis, 70 ␮g of proteins were loaded in each lane and separated in 12% SDS-polyacrylamide gels (SDS-PAGE) using the MiniProtean III electrophoresis apparatus (Bio-Rad). For western blotting, the proteins separated in SDS-PAGE were transferred to nitrocellulose paper. After Western blot, membranes were stained with Ponceau red to verify the protein transfer and incubated with primary anti-rabbit antibody. In controls, the primary antibody was omitted. Detection was performed using the enhanced chemiluminescence procedure developed by Amersham (ECL, Plex Western Blotting System, GE Healthcare, UK).

2.2. Fixation and microscopic methods Eight wall lizards (P. muralis) and three anole lizards (A. carolinensis) were utilized in the present microscopic study. The adopted procedures for care and handling the animals followed the Italian guidelines (Art. 5, DL 116/92). After inducing autotomy in P. muralis, a natural process of tail amputation that does not harm the animal, the tail was allowed to repair for 4 (n = 4), 8 (n = 2) and 14 (n = 2) days at room temperature (25–30 ◦ C) when the blastema was formed over the tail stump. Also for A. carolinensis the tail was obtained by induced autotomy (grabbing and twisting the tail to exploit the release of the tail by the animal), and the tail was left to regenerate for 8 days (1 animal) and 14 days (2 animals) until the blastema was formed on the tail stump. The initial, more proximal part of the amputated tail was also fixed or collected for biochemical analysis (see later), and served later as control of the adjacent stump. The stumps or the regenerating tails were collected, halved and the two pieces were fixed for 5 h in cold (0–4 ◦ C) 4% paraformaldehyde in 0.1 M Phosphate buffer at pH 7.4, rinsed in buffer for about 30 min, dehydrated in ethanol (70◦ , 80◦ , 95◦ , 100◦ ), and embedded in Bioacryl Resin (Scala et al., 1992). The tissues were sectioned using an ultramicrotome, and 2–3 ␮m thick sections were collected on glass slides, and stained with 1% Toluidine blue for histology, or were attached to precoated slides for the following immunostaining. Thin sections of 40–90 nm in thickness were collected on Nickel grids for immunogold detection at the transmission electron microscope. The primary antibody, an anti-telomerase-1 component antibody selected on a specific epitope of the lizard A. carolinensis, was produced and affinity purified from immunized rabbits and previously

characterized (Alibardi, 2015a), using a Biotechnology company (Davids Biotechnology, Germany). For light microscopy immunocytochemistry the sections were incubated overnight for 4 h at room temperature with the primary antibody diluted 1:100 in buffer (Tris 0.05 M at pH 7.6 containing 5% BSA). After rinsing in the buffer, the sections were incubated for 60 min at room temperature with a fluorescein-conjugated anti-rabbit antibody (FITC, Sigma, diluted 1: 200), rinsed in buffer, mounted in anti-fading medium (Fluoroshield, Sigma, USA), and observed under a fluorescence microscope using a fluorescein filter. Pictures were taken by a digital camera (Euromex, The Netherlands) and digitalized into a computer using the Adobe Photoshop Program. For electron microscopy immunocytochemistry, sections on Nickel grids were incubated for 4 h at room temperature in the primary antibody diluted in 0.05 M Tris–HCl buffer at pH 7.6 containing 1% Cold Water Fish Gelatin. In negative controls, the primary antibody was omitted. After the incubation period the sections were rinsed in buffer and incubated for 1 h at room temperature with goat anti-rabbit Gold-conjugated secondary antibody (Sigma, 5 or 10 nm gold particles). Some grids went through a silver enhancing treatment, to increase the size of gold particles and make the labeling more easily detectable under the electron microscope. The suggested intensification method followed the manufacturer instructions (British Biocell International, SEKB250). Grids were rinsed in the buffer, dried and stained for 4 min with 2% uranyl acetate, and then observed under an electron microscope (Zeiss C10). 3. Results 3.1. Western blotting The tail extracts from A. carolinensis showed weak bands at 150–160 and weaker at 60 kDa in the tail stump (Fig. 1, first lane). The blastema instead showed more intense bands, mainly at 150–160 kDa, and around 200, 120, and 60–70 kDa (Fig. 1, lane 2). In the control of the regenerating tissues (blastema), no bands were seen (Fig. 1, lane 3). The normal tails and stumps of P. muralis showed similar faint labeling in the expected range, at 100–150 kDa and only a main band around 40–45 kDa was seen (Fig. 1, lane 4).

Fig. 1. Western blot of protein extracts and immunoreacted for telomerase from the normal tail (NT) in comparison to the regenerative blastema (BL, 14 days regeneration) in A. carolinensis and P. muralis. CO, controls (blastema). Numbers on the left indicate the molecular weight in kilo-daltons (see text for description).

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In the regenerating blastema more intense bands were detected at 75–80 and 100 kDa, and a weaker band at 65 kDa (Fig. 1, lane 5). Finally, no labeled bands were present in controls of the regenerating blastema (Fig. 1, lane 6). 3.2. Histology and immunofluorescence The studied stumps at 4 and even more at 8 days postamputation (Fig. 2A) showed the presence of a thick clot covering

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the tail stump, under which a migrating ribbon of elongated keratinocytes (wound epidermis) in the process of covering the stump were present (Fig. 2B). No or occasional labeled cells were observed in the normal tail, aside in the bone marrow (data not shown). Beneath the wound epidermis of the healing stump a loose tissues made of free mesenchymal-like cells and numerous blood cells was present, in continuation with the superficial connective tissues and muscles of the stump (Fig. 2C). This mesenchymalhematogenous loose connective tissue, made of cells accumulating

Fig. 2. Gross view of the tail stump at about 8 days post-amputation (A), with the histological aspect (B, C), and immunofluorescence for telomerase in stump tissues at 4 days post-amputation (D–L) in P. muralis. A, view of a tail stump (arrow on the surface). Bar, 0.5 mm. B, histological section of the surface of the stump featuring the migrating epidermis (arrows) underneath the scab and the loose cells of the pre-blastema granulation tissue present over the injured muscles. Bar, 100 ␮m. C, close-up to the injured muscles surrounded by free cells (arrowheads). Bar, 10 ␮m. D, labeled cells (nuclei) among muscles. The arrowhead indicates a higher labeled nuclear spot. Bar, 10 ␮m. E, inter-muscle blood vessel with a labeled endothelial cell. Bar, 10 ␮m. F, two labeled adipocytes in the fat tissue. Bar, 10 ␮m. G, labeled connective cells on the muscle surface. Bar, 10 ␮m. H, two labeled satellite cells in a spinal ganglion (the arrow shows intensely fluorescent intra-nuclear spots). Bar, 10 ␮m. I, two labeled likely glial cells (arrowheads) on the surface of the white matter of the spinal cord. Bar, 10 ␮m. J, detail on immunolabeled cells within the bone marrow of a vertebra. The arrowheads indicate nuclei containing intensely labeled granules: Bar, 10 ␮m. K, wound epidermis of the forming blastema showing few more intensely labeled cells. The dashes separate the epidermis from the underlying blastema also containing sparse labeled cells. Bar, 10 ␮m. L, control section (CO) of connective on the stump surface where autofluorescent erythrocytes (arrow) are seen but nothing in the surrounding cells. Bar, 10 ␮m. Legends: bl, blastema; c, corneous layer; ga, spinal ganglion; jt, injured muscles; mu, muscles; ns, normal scale; pb, pre-blastematic granulation tissue; sb, scab; v, blood vessel; w, wound epidermis; wsc, white matter of the spinal cord.

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beneath the wound epithelium represented a pre-blastematic granulation tissue (see details in Alibardi and Sala, 1988; Alibardi, 2010b). The observation of immunolabeled sections showed the presence of sparse immunofluorescent cells localized in the loose pre-blastema connective tissue and in cells present among the damaged muscles and the connective tissues of the stump (Fig. 2D–G). The labeling was prevalently nuclear, sometimes even forming 2–4 intranuclear globules more intensely labeled than the remaining nucleoplasm (arrowheads in Fig. 2D, H, J). In slides where also the spinal ganglia and the spinal cord were present, occasional glial cells or ganglion satellite cells appeared labeled (Fig. 2H, I). Sparse labeled cells were particularly and constantly detected in the bone marrow inside the vertebrae of the stump (Fig. 2J). The thickening wound epidermis at the margins of the stump showed few labeled cells together other mesenchymal cells of the forming blastema (Fig. 2K). Immunolabeled cells were not seen in the tissues of the normal tail or in control, negative sections, aside some autofluorescence present in red blood cells (arrow in Fig. 2L).

3.3. Immunogold detection of labeled cells The utilization of the silver enhancement reaction allowed to rapidly detecting immunolabeled cells at 4 days post-amputation, located in the connective tissues or between muscle bundles facing the stump surface. Immunolabeled cells were seen nearby muscle fibers, likely satellite cells (Fig. 3A and B), or were mesenchymallike cells with large nuclei and little cytoplasm (Fig. 3C). No telomerase labeling was seen in the nuclei of muscle fibers. The telomerase labeling was mainly nuclear in these cells and most of them contained euchromatic nuclei, pale or denser, and a simplified cytoplasm where few cell organelles were present among a network of cytoskeletal fibrils (Fig. 3A and B). It appeared that also the nucleolus was labeled for telomerase (arrow in Fig. 3A), as it was later confirmed using immunogold of smaller dimension (5 or 10 nm, see below). Likely hematogenous cells (for their resemblance with those present within the bone marrow of vertebrae, see below) were occasionally seen. The latter showed dense, heterochromatic nuclei and a cytoplasm devoid of organelles, possibly representing polychromatophilic erythroblasts (Fig. 3D). Rare flat

Fig. 3. Four typologies of telomerase positive cells present on the surface of the stump in P. muralis (immunogold and silver enhancement) 4 days post-amputation. A, immunolabeled nucleus and nucleolous (arrow) of satellite cell associated to a muscle fiber (mu). The cell shows vesicles (v) in the cytoplasm. Bar, 0.5 ␮m. B, immunolabeled dense nucleus of connective cell (possibly an adipose cell) on the surface of injured muscles. Arrowheads indicate the fibrous meshwork present among the cytoplasmic vesicles (v). Bar, 0.5 ␮m. C, fibroblast (cl indicates extracellular collagen fibrils) localized among the stump muscles and showing a labeled, euchromatic nucleus (n). Bar, 0.5 ␮m. D, Unidentified cell (likely of haematogenous type) present on the surface of the injured muscles, and featuring a dense and labeled nucleus (n). Bar, 0.5 ␮m.

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cells close to the intervertebral perichondrion of the last vertebra present in the tail stump, also showed labeled nuclei for telomerase (data not shown). In order to better detail the nuclear labeling, we utilized 5 or 10 nm diameter gold particles. The nuclei of the immunopositive mesenchymal-like cells showed that the gold particles tended to form clusters over the chromatin and the nucleolus (Fig. 4A). In other cells the labeling was less intense but tended to localize in roundish regions, possibly identifiable as Cajal bodies, that were observed in the nucleoplasm and were formed by a denser meshwork of 10–12 nm thick coarse filaments (Fig. 4B). In both lizard species here utilized, sparse satellite cells at the periphery of striated myofibres appeared intensely labeled in their nuclei, with a diffuse but mainly clustered labeling over the dense chromatin of these cells, perhaps indicating a process of chromosome condensation before mitosis. Conversely, a lower or absent labeling was detected over the paler euchromatin or in the nucleoplasm (Fig. 5A). The nucleolus, often in contact with a meshwork of coarse filamentous material, appeared immunolabeled (Fig. 5A and B). The examination of control sections showed no labeling in the nuclei of either mesenchymal cells, elongated fibroblasts and in satellite cells (Fig. 5C). Sparse immunolabeled cells for telomerase were also observed within the bone marrow of the proximal vertebra close to the stump surface. The electron microscope allowed to determine that the latter labeled cells were small and roundish, likely lymphocytes with a relatively electron dense but however labeled nucleus, and surrounded by a rim of cytoplasm (Fig. 6A). Rare cells with sparse granules and an irregular surface, likely representing myelocytes of neutrophilic or basophilic types, also showed a prevalent nuclear labeling (Fig. 6B). Finally, also sparse erythropoietic cells, basophilic and polychromatophilic erythroblasts, showed a nuclear labeling (Fig. 6C). Using gold at 5 nm, the labeling appeared mainly localized over the heterochromatin or near the border with the pale euchromatin (arrowheads in Fig. 6D).

4. Discussion 4.1. Telomerase detection after wounding suggests activation of the regenerative response Although qualitative, the present immunological study shows that both the intensity of protein bands and the number of immunopositive cells for telomerase increases from normal to early regenerating tissues during the formation of the regenerative blastema in the tail of lizards. The molecular weight is also in the expected range for the enzyme (120–150 kDa), a better result than those previously obtained from protein extraction during the elongation phase of tail regeneration (Alibardi, 2015a). In the latter case, bands at lower molecular mass were detected like in the present study at 65 and 80 kDa in both species, but whether this result is physiological or derives from protein degradation remains to be clarified. The previous study also showed that sparse telomerase positive cells are present in the elongating blastema and coniform growing tail, while a mesenchymal blastema only remains at the tip of the elongating tail. More frequent telomerase-positive cells were observed in muscles and cartilaginous tube where they are however spread among the prevalent cells not expressing the enzyme, and it is known that these two tissues show the highest rate of cell proliferation in the regenerating tail (Simpson, 1965; Cox, 1969). These same cell types are immunonegative for telomerase in the normal tail, including the satellite cells of un-injured muscle fibers. The above observations suggest that one of the first responses to the wound signal is the activation of telomerase in these cells, that may correspond to silent stem/progenitor cells resting among

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the connective tissues (adipose and inter-muscle fascia) or to the myosatellite cells that a previous study showed to be tritiated thymidine long retaining labeled cells or 5BrdU long retaining labeled cells (Alibardi, 2014, 2015c). We also noted sparse telomerase positive cells in the nervous tissues, glial cells in the spinal cord and satellite cells of the ganglia in the stump. The immunodetection of telomerase in these cells after injury indicates an activation of their proliferative program and the origin of the transient amplifying cells for their accumulation on the stump surface to form a blastema within 7–14 days post-amputation. This pattern indicates that telomerases are present in activated stem/progenitor cells but decrease in transient amplifying cells which multiplication produced the regeneration of the new tissues. This behavior, a signature of a controlled cell proliferation, appears opposite to that of various types of cancer cells where instead telomerase activity tends to increase with cell expansion and the progression of the disease (Counter et al., 1995). The present study indicates that sparse immunolabeled cells for telomerase start to accumulate over the tail stump, coming from the connective and fat tissues of the stump, or accumulating between muscles near the stump surface. The light and ultrastructural detection of the enzyme in sparse clusters located over the condensing chromatin resembles the localization previously observed in the regenerating tail, and in particular over sites of telomerase processing such as the Cajal bodies (Alibardi, 2015a). 4.2. Significance of activated telomerases in tissues of vertebrates The induction of telomerase activity following damaging signals has been reported in various conditions and in different tissues and organs. In the mammalian skin, not only the enzyme increases during cancer but also following diseases such as psoriasis and dermatitis (Taylor et al., 1996). In the stem cells of the hair follicle it appears that telomerase induction determines their activation and the entry of the hair bulb into the phase, another function of telomerase aside the classical mechanism for telomerers elongation (Sarin et al., 2005). A slight increase of telomerase activity was also noted in mice few days after muscle injury, probably in relation to the initial activation of the enzyme in satellite cells (Dell’Aica et al., 2002). The induction of telomerase leading to cell proliferation in an organ has been reported in the lung, a process that determines pulmonary fibrosis (Nozaki et al., 2000). Telomerase activation has also been reported following injuries in the brain and nerve tissues, mainly localized in microglial cells (Flanary and Streit, 2005) or even in some neurons (Kang et al., 2004). The sparse telomerase positive glial cells observed in the spinal cord and satellite cells of the ganglia present in the lizard tail may also indicate their activation toward proliferation. Studies in mice and Xenopus frogs have shown that telomerase, specifically the protein component TERT, modulates gene activity in the Wnt/␤-catenin pathway by interaction with BRG1, a chromatin remodelling protein that regulates Wnt-dependent promoters and eventually determines cell proliferation (Park et al., 2009). While this telomerase activity appears continuous or even follows a positive feedback in cancer cells, it appears regulated, following a negative feedback in cells of the lizard blastema. Telomerase expression, unrelated to longevity, has been demonstrated in regenerating tissues of fish (Lund et al., 2009) and amphibians (Elmore et al., 2008; Alibardi, 2015d). These studies have indicated that telomerase activity is constitutive and is involved in the maintenance of the telomere length in proliferating cells for the regeneration of new tissues. The length of telomeres not shorten or is ever higher in regenerating tissues after numerous cycles of cell divisions (Elmore et al., 2008; Lund et al., 2009). These studies have shown that invertebrate and fish telomerases can recognize pentanucleotide sequences (TTAGG instead or in addition to the

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Fig. 4. Immunogold labeling of mesenchymal-like cells located on the stump surface of A. carolinensis (A) and of P. muralis (B). A, the nuclear labeling is present over condensed regions of chromatin (arrows) and perinuclear chromatin (arrowheads), but also the nucleolous is labeled (nu). Bar, 100 nm. B, nucleus of elongated fibroblast showing a denser region highly labeled (arrow). Bar, 100 nm. The inset (Bar, 50 nm) shows the presence of coarse filaments (arrowheads) in the magnification of this region, probably representing a Cajal body.

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Fig. 5. Immunolabeling of satellite cells in the stump of A. carolinensis (A, C) and P. muralis (B). A, nucleus (n, cy indicates the cytoplasm) showing immunogold particles mainly localized over the perinuclear chromatin and on chromatin material forming roundish bodies (arrows) that may correspond to the immunofluorecent spots observed with the light microscope. Bar, 100 nm. B, detail of the labeled nucleolus and of nearby coarse filaments (arrowhead) that also appeared lightly labeled. Bar, 50 nm. C, immunonegative nucleus (n) in a control section. Bar, 250 nm.

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Fig. 6. Immunolabeled cells present in the bone marrow with silver intensification (A–C) or only using immunogold (D). A, lymphocyte-like cell with dense nucleus. Bar, 0.5 ␮m. B, labeled nucleus (n) of myelocyte (probably basophilic) containing large dense granules (arrowhead). Bar, 1 ␮m. C, labeled nucleus (n; cy indicates the cytoplasm) of polychromatophilic erythroblast. Bar, 0.5 ␮m. D, detail of the nuclear labeling of a polychromatophilic erythrocyte using 5 nm gold particles, seen near the boundary between hetero- and eu-chromatin (arrowheads). Bar, 100 nm.

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classical esanucleotide sequence TTAGGG), suggesting that telomerases can also reconstruct repeats of these pentameric nucleotides at the telomere ends of chromosomes in these species (Elmore et al., 2008). In the lizards studied here and in a study (Alibardi, 2015a) it appears that telomerase is absent or is present in lower level in normal tail tissues than during tail regeneration. The activation of stem/progenitor cells present in the tail tissues (Alibardi, 2014, 2015c) may generate the proliferating cells over many stump tissues, as this was indicated from previous autoradiographic and immunocytochemical studies (Cox, 1964; Simpson, 1965; McLean and Vickaryous, 2011; Delorme et al., 2012). The presence of telomerase positive cells detected not only in the bone marrow of vertebrae of the tail stump and in few cells in normal conditions, but also in similar cells present in the forming blastema, further suggests that immature blood cells are released from the marrow and reach the forming blastema, as indicated by previous studies (Shah et al., 1980; Alibardi, 1994, 2015c). The presence of telomerase in haematogenous cells, especially in fractions enriched with lymphocytes, monocytes and granulocytes, has been reported in normal and especially in malignant human blood diseases (Broccoli et al., 1995; Counter et al., 1995). High levels of telomerases have been reported in the early forms of lymphocytes but also in proliferating B and T lymphocytes, while telomerase activity declines in mature and circulating lymphocytes (Weng et al., 1997). In conclusion, the massive but controlled cell proliferation responsible for the formation of the regenerating blastema in lizard appears somehow related to the activation and successive internal control of the activity of telomerase in the blastema and of p53/63 in the skin (Alibardi, 2015b). In addition to studies on fish and amphibians (Elmore et al., 2008; Lund et al., 2009; Alibardi, 2015d), the elucidation of the mechanisms that control cell proliferation in this non-mammalian amniote model of tissue and organ regeneration may help to understand the loss of proliferation control occurring during cancer in homeothermic amniotes, including humans. Acknowledgments The study was mostly supported by Comparative Histolab, with some contribution from a RFO 2013 from the University of Bologna. References Alibardi, L., 1994. Production of immature erythrocytes in lizard during tail regeneration. Anim. Biol. 3, 139–147. Alibardi, L., 2010a. Morphological and cellular aspects of tail and limb regeneration in lizard: a model system with implications for tissue regeneration in mammals. Adv. Anat. Embryol. Cell. Biol. 207, 1–112. Alibardi, L., 2010b. Ultrastructural features of the process of wound healing after tail and limb amputation in lizard. Acta Zool. 91, 306–318. Alibardi, L., 2014. Histochemical biochemical and cell biological aspects of tail regeneration in lizard, an amniote model for studies on tissue regeneration. Progr. Histoch. Cytoch. 48, 143–244. Alibardi, L., 2015a. Immunolocalization of the telomerase-1 component in cells of the regenerating tail, testis, and intestine of lizards. J. Morphol., http://dx.doi. org/10.1002/jmor.20375. Alibardi, L., 2015b. Immunolocalization of a p53/p63-like protein in the regenerating tail epidermis of the wall lizard (Podarcis muralis) suggests it is involved in the differentiation of the epidermis. Acta Zool., http://dx.doi.org/ 10.1111/azo.12130. Alibardi, L., 2015c. Immunolocalization indicates that both original and regenerated lizard tail tissues contain populations of long retaining cells, putative stem/progenitor cells. Microsc. Res. Techn. Alibardi, L., 2015d. Immunodetection of Telomerase-like immunoreactivity in normal and regenerating tail of amphibians suggests it is related to their regenerative capacity. J. Exp. Zool. A. Alibardi, L., Sala, M., 1988. Fine structure of the blastema in the regenerating tail of the lizard Podarcis sicula. Boll. Zool. 55, 307–313.

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