Original Tail Research Articles

Immunolocalization of Wnts in the lizard blastema supports a key role of these signaling proteins for tail regeneration.

A highly upregulated gene during tail regeneration in lizards is Wnt2b, a gene broadly expressed during development. The present study examines the distribution of Wnt proteins, most likely wnt2b, by western blotting and immunofluorescence in the blastema-cone of lizards using a specific antibody produced against a lizard Wnt2b protein. Immunopositive bands at 48-50 and 18 kDa are present in the regenerative blastema, the latter likely as a degradation product. Immunofluorescence is mainly observed in the wound epidermis, including in the Apical Epidermal Peg where the protein appears localized in intermediate and differentiating keratinocytes. Labeling is more intense along the perimeter of keratinocytes, possibly as a secretory product, and indicates that the high epidermal proliferation of the regenerating epidermis is sustained by Wnt proteins. The regenerating spinal cord forms an ependymal tube within the blastema and shows immunolabeling especially in the cytoplasm of ependymal cells contacting the central canal where some secretion might occur. Also, regenerating nerves and proximal spinal ganglia innervating the regenerating blastema contain this signaling protein. In contrast, the blastema mesenchyme, muscles and cartilage show weak immunolabeling that tends to disappear in tissues located in more proximal regions, close to the original tail. However, a distal to proximal gradient of Wnt proteins was not detected. The present study supports the hypothesis that Wnt proteins, in particular Wnt2b, are secreted by the apical epidermis covering the blastema and released into the mesenchyme where they stimulate cell multiplication.

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.

Regeneration in the Podarcis bocagei model organism: a comprehensive immune-/histochemical analysis of the tail

Regeneration is the process of regrowth of an injured/missing tissue/organ from the residual tissue. One outstanding example is the regeneration of tail in lizards in which they can completely regenerate without the formation of scaring or fibrotic tissue. This study presents an analysis of the main differences between a mature regenerated tail and the original tail of the lizard Podarcis bocagei by comparing histological biomarkers for support structures, morphology and pigmentation, vasculature and energy storage. Matured regenerated and original tails were studied with different histological staining’s and immunohistochemistry to highlight the various tissue components. We found differences in the morphological features scalation pattern and pigmentation, as well as in the support structures: muscle organization, collagen and calcification. Vasculature was remodeled. Energy storage was restored with regeneration maintaining therefore the main metabolic function. We demonstrate that the regenerated tail does not lose its main functions like locomotion and energy metabolism. The present findings may open a new window of research with implications in regenerative medicine.

The effect of predator kairomones on caudal regeneration by Allegheny Mountain Dusky Salamanders (Desmognathus ochrophaeus)

Many prey use autotomy as an antipredator mechanism. Rapid regeneration of autotomized appendages is beneficial because forfeited tissues may serve as organs for energy storage, accessories for locomotion, or indicators of social status. We monitored levels of caudal regeneration by Allegheny Mountain Dusky Salamanders (Desmognathus ochrophaeus Cope, 1859) exposed to kairomones from predatory Eastern Garter Snakes (Thamnophis sirtalis (Linnaeus, 1758)). After the induction of autotomy, salamanders were exposed to one of three treatment regimens: blank (water), or acute (30 min per week) or chronic (constant) exposure to predator kairomones during a 12-week study period. Overall, the mean volume of regenerated tissue, as a percentage of the original tail volume, was highest for individuals exposed to the blank versus predator kairomones. When the combined effects of time elapsed since the induction of caudal autotomy and the different treatment regimens were considered, we found that the mean volume of regenerated tissue was significantly greater for control salamanders beginning 8 weeks after autotomy. The mechanism contributing to the differential rates of regeneration among individuals in our treatment groups is unknown, but previous work suggests that elevated stress related to predation threat can have detrimental effects on wound healing and growth in amphibians.

Calcification of Axial Skeleton and Segmentation Muscular Regeneration of Gecko’s Tail (Gekko Gecko Linnaeus, 1758)

Gecko is an animal that can carry out autotomy. Research on gecko tail autotomy has been carried out, but there are still few who research the axial skeleton that focuses on vertebrae caudales and segmentation muscular arranged, this is the background of this research. This research is expected to be data base further research and as a comparison between animals that can induce further autotomy. This study aims to determine the macrostructure and microstructure of axial axial gecko tail regenerates and determine the anatomical microstructure of muscular regeneration of gecko tail. The methods used were X-Ray, Alizarin Red S and Alcian Blue, Paraffin method with hematoxylin-eosin staining, and Mallory Triple Stain. The results showed that the color of the gecko tail regenerate was paler compared to the original tail. On observations using radiological rays and alizarin staining showed that the original tail wo uld look segmented and have a process. The original gecko tail is composed of bones, because it is red which shows perfectly calcified bone. While the gecko tail regenerate is composed of cartilage that is shaped like a long pipe glazed red because it has calcified. At the end of the tail there is also a blue color, this indicates that the gecko tail regenerate has not been completely calcified. Segmentation Muscular of the original gecko tail, when viewed longitudinally, indicates a segment that extends from one process to the skin and when viewed from the cross has only four muscle segments separated by the septum. While the gecko tail regenerate when viewed in a longitudinal manner there is no segment and when seen transversely there are 12 muscle segments seen. Muscles are composed of a collection of myotubes that form myotomes, each myotomes limited by myoseptum.

Macro and Microanatomical Structures of Lizard’s Regenerate Tail (Mabouya multifasciata Kuhl, 1820)

Lizards are animals that can do autotomy. Research on the autotomy of lizard’s tail has been carried out, but few have examined about axial skeletons that focus on vertebrae caudales or arranged muscles. That is the background of this research. This research is expected to be a database for further research and as a comparison between animals that can perform other autotomies. This research intend to determine the macro and micro structures of the anatomical axial skeleton lizard’s regenerate tail and to know the micro structures of anatomy muscular segmentation lizard’s regenerate tail. The used methods are X-Ray, Alizarin Red S and Alcian Blue, Paraffin method with Hematoxylin-Eosin and Mallory Triple Strain staining. The result showed that the lizard's regenerate tail was darker than the original. On observations using X-Ray and Alizarin staining showed that the original tail is seen broad and have a Processus. The original lizard's tail is composed of bones, because it is red which shows perfectly calcified bones. While the regenerate of the lizard's tail is composed of bones in the form of long pipes which are red-colored because they have calcified from outside. Muscular segmentation of the original lizard's tail when viewed transversely has irregular segments. While the lizard's regenerate tail has 15 muscle segments. Muscles are composed of a collection of myotubes that become myotomes, each of myotomes confined by the myoseptum.

Morphology of setae in regenerating caudal adhesive pads of the gecko Lygodactylus capensis (Smith, 1849)

After tail loss in the African gecko Lygodactylus capensis (Smith, 1949) a new tail is regenerated, including caudal adhesive pads. The axial skeleton of the regenerating tail consists in an elastic cartilaginous tube replacing the original vertebrae that allows interacting with the substrate like in the original tail. The formation of adhesive setae has been analyzed using transmission and scanning electron microscopy coupled to immunolabeling for Corneous Beta Proteins. During progressive stages of epidermal differentiation new setae are developed at stage 4 of the shedding cycle and contain Corneous Beta Proteins. These structural proteins are faintly localized in the Oberhäutchen but are abundant in the beta-layer, indicating that the two epidermal layers have a different protein composition. The setae originate from the growth of Oberhäutchen spinulae into the cytoplasm of clear cells and the latter produce a thick fibrous meshwork of keratin and other unknown proteins localized around the growing setae. This cytoskeleton likely allows molding tail setae like for digital setae. A graded development of setae is observed from the base to the tip of regenerated pads and from the periphery to more central areas. The terminal end of the setae is subdivided into numerous filamentous spatulae that increase the adhesion contact. Sensory boutons are frequently detected at the margin of tail scales and adhesive pads, likely improving compliance with the substrate. The present study indicates that tail regeneration is a convenient experimental model to analyze adhesive setae formation, microstructures that allow to these lizards climbing vertical and arboreal substrates.

Microscopical observations on the regenerating tail in the tuatara Sphenodon punctatus indicate a tendency to scarring, but also influence from somatic growth.

The process of tail regeneration in the tuatara (Sphenodon punctatus) is not entirely known. Similarity to and differences from lizard tail regenerations are indicated in the present histological and ultrastructural study. Regeneration is influenced by the animal's age and ambient temperature, but in comparison to that of lizards it is very slow and tends to produce outgrowths that do not reach the length of the original tail. Although microscopically similar to lizard blastemas, the mesenchyme rapidly gives rise to a dense connective tissue that contains few muscle bundles, nerves, and fat cells. The unsegmented cartilaginous tube forming the axial skeleton is not calcified after 5 months of regeneration, but calcification in the inner region of the cartilage, present after 10 months, increases thereafter. Amyelinic and myelinic peripheral nerves are seen within the regenerating tails of 2-3 mm in length and the spinal cord forms an ependymal tube inside a cartilaginous casing. Tissues of the original tail, like muscles, vertebrae and the adipose mass, are largely replaced by dense connective tissue that occupies most of the volume of the new tail at 5 and 10 months of regeneration. It is unknown whether the differentiation of the dense connective tissue is caused by the relatively low temperature that this species lives under or stems from a genetic predisposition toward scarring as with most other amniotes. Increases of muscle and adipose tissues seen in older regenerated tails derive from somatic growth of the new tail in the years following tail loss and not from a rapid regeneration process like that in lizards.

Review: The Regenerating Tail Blastema of Lizards as a Model to Study Organ Regeneration and Tumor Growth Regulation in Amniotes.

Tail regeneration in lizards is a unique case of organ regeneration among amniotes. The Review summarizes past and recent studies indicating that tail regeneration utilizes numerous signaling pathways typical for tumor growth. The regenerative blastema-cone contains sparse proliferating cells that utilize coding and noncoding RNAs in an environment rich in water and hyaluronate, as typical for tumor outgrowth. Differently from tumors, the blastema appears as a polarized outgrowth where the distal region contains proliferating cells mainly driven by the up-regulation of Wnt, snoRNAs, and associated onco-genes. The down-regulation of immune-genes coupled with the high production of hyaluronate coating blastema cells likely protect them from attach by immune cells. Immunoevasion of blastema cells allows the proliferation and migration necessary for the morphogenesis of a new tail. Transcriptome and immunolabeling data suggest that gradients for wnts, shh, msx, and signaling receptors are present in the tail blastema. It is hypothesized that cells along these gradients activate different genes, including tumor suppressors that are expressed in more proximal regions where cells stop proliferating and differentiate into tissues of the new tail. The continuous proliferation at the apex of the blastema is turned into a regulated growth in more proximal regions near the original tail. In contrast, it is hypothesized that no or nonresponding gradients of signaling proteins are present in tumor outgrowths so that cell proliferation but no differentiation occurs in expanding tumors. Considering signaling gradients, the lizard model of regeneration can help in understanding the lack of regulation of tumor growth. Anat Rec, 302:1469-1490, 2019. © 2018 American Association for Anatomy.

Alizarin Red S-Alcian Blue Staining for Regenerated tail of Common House Gecko (Hemidactylus frenatus)

Common House Gecko (Hemidactylus frenatus) is one of reptiles that have ability to autotomy their tails. Tail autotomy is a mechanism to protect it self from predators. After the tail broke, there will be wound healing on the tail which is then followed by a tail regeneration event. Original tail and regenerate tail is very different morphologically and anatomically. The original tail is composed of bones while the tail of the regenerate is composed of cartilage. Histochemical staining using Alizarin Red-S Alcian Blue was done to differentiate bone and cartilage. This method will stained bones red while the cartilage will stained blue.

Differences in neural stem cell identity and differentiation capacity drive divergent regenerative outcomes in lizards and salamanders

While lizards and salamanders both exhibit the ability to regenerate amputated tails, the outcomes achieved by each are markedly different. Salamanders, such as Ambystoma mexicanum, regenerate nearly identical copies of original tails. Regenerated lizard tails, however, exhibit important morphological differences compared with originals. Some of these differences concern dorsoventral patterning of regenerated skeletal and spinal cord tissues; regenerated salamander tail tissues exhibit dorsoventral patterning, while regrown lizard tissues do not. Additionally, regenerated lizard tails lack characteristically roof plate-associated structures, such as dorsal root ganglia. We hypothesized that differences in neural stem cells (NSCs) found in the ependyma of regenerated spinal cords account for these divergent regenerative outcomes. Through a combination of immunofluorescent staining, RT-PCR, hedgehog regulation, and transcriptome analysis, we analyzed NSC-dependent tail regeneration. Both salamander and lizard Sox2+ NSCs form neurospheres in culture. While salamander neurospheres exhibit default roof plate identity, lizard neurospheres exhibit default floor plate. Hedgehog signaling regulates dorsalization/ventralization of salamander, but not lizard, NSCs. Examination of NSC differentiation potential in vitro showed that salamander NSCs are capable of neural differentiation into multiple lineages, whereas lizard NSCs are not, which was confirmed by in vivo spinal cord transplantations. Finally, salamander NSCs xenogeneically transplanted into regenerating lizard tail spinal cords were influenced by native lizard NSC hedgehog signals, which favored salamander NSC floor plate differentiation. These findings suggest that NSCs in regenerated lizard and salamander spinal cords are distinct cell populations, and these differences contribute to the vastly different outcomes observed in tail regeneration.

Establishment and characterization of rough-tailed gecko original tail cells.

Some of lizard species have the ability to lose their tail in order to defend against predators and regenerate the new tail. Lizard's regenerated tail has attracted scientists' attention for unraveling the regeneration process, but less information is known about the cellular characterization and cell growth properties of original tail. This research aimed to report cell culture and banking process of rough-tailed gecko or Cyrtopodion scabrum's original tail cell sample from inner tissue without skin using tissue explant technique. For banking reports, it is essential to analyze this cells' potential to proliferate, to investigate biological aspects such as cell culture features, differentiation and chromosome number and to report its species identification and quality control. To achieve optimal growth conditions, three different temperatures for incubation including 18, 23 and 37°C and two different media including DMEM and L-15 were applied. The expanded cells were studied for their potential to adipose and osteoblast differentiation. Results indicated that lizard's original tail cells could be successfully obtained by explant technique. The cells demonstrated fibroblast like morphology with population doubling times of approximately 24 ± 0.5h. Karyotyping analysis showed a distribution of 2n = 40 chromosome number for this cell line. The comparison of different incubation media and temperatures showed that cell growth is equally optimal in all mentioned conditions according to growth curves. Adipose and osteoblast differentiation was obviously observed in these cells which confirms the hint of stem-ness in the produced mixed cells. According to cell banking policies, produced cells were also checked for bacterial, fungal, yeast and mycoplasma contaminations and no contamination was observed. Multiplex PCR for identification of species confirmed the species of lizard with no cross-contamination with other cells in the cell bank. Establishment of authenticated and well-characterized lizard's original tail cell line will provide a valuable source for subsequent in vitro regenerative research and molecular studies which are not feasible in in vivo methods. This finding will allow us to get an opportunity to create and preserve a new collection of lizard cell lines in the future.

A new species of large-bodied, tuberculate Hemidactylus Oken(Squamata: Gekkonidae) from the Eastern Ghats, India.

A distinct new gecko of the genus Hemidactylus is described from Andhra Pradesh, India. This large-sized (snout to vent length up to at least 105 mm), scansorial Hemidactylus is characterized by dorsal scalation of small granules intermixed with large, pointed, trihedral tubercles that form 16-17 fairly regularly arranged longitudinal rows at midbody; 9-11 subdigital lamellae below the first and 11-13 below the fourth digit; 6-8 strongly pointed and keeled enlarged tubercles on the original tail; 20-23 femoral pores separated by 4 poreless scales in males; 11-13 supralabials and 9-11 infralabials. This is the third vertebrate endemic to the Mahendragiri Range, highlighting the significance of this topographically complex region.

Development and clinical application of a new type of intramedullary nail tail cap

To develop a new type of tail cap for closing the tail of the central hole of the interlocking intramedullary nail so as to reduce difficulty in implantation of the tail cap into intramedullary nail, and ensure the accuracy of implantation, as well as reduce unnecessary operation time. In 2014, the new type of intramedullary nail tail cap ("new tail cap" for short) was successfully developed, and has been granted national utility model patent. A randomized controlled clinical trial was conducted in 34 patients with non pathological femoral shaft fractures treated between August 2014 and March 2016. The patients were randomly divided into groups A and B, 17 patients each group. There was no significant difference in gender, age, side, body mass index, cause of injury, injury to operation time, and fracture AO classification between groups ( P>0.05), with comparability. All patients underwent fixation with interlocking intramedullary nail, and the new tail cap and original tail cap were used in groups A and B respectively. The blood loss and operation time during tail cap implantation, fracture healing time, and complications were recorded and compared between 2 groups; the limb function was evaluated by Klemm function evaluation standard. The blood loss and operation time during tail cap implantation of group A [(3.5±0.8) mL and (10.57±3.15) s] were significantly less than those of group B [(5.5±1.7) mL and (21.99±6.90) s] ( t=-8.281, P=0.010; t=-10.743, P=0.009). All new tail caps were successfully implanted by one-time in group A, and the one-time success rate was 100%; the original tail cap exfoliation occurred in 3 cases of group B, and the one-time success rate was 82.4%. All the patients were followed up 6-16 months (mean, 9.7 months); there was no deep infection, loosening or breakage of internal fixation, and other serious complications. The time of fracture healing was (16.4±3.2) weeks in group A and (15.8±3.5) weeks in group B, showing no significant difference ( t=0.514, P=0.611). At last follow-up, according to the Klemm function evaluation standard, the results were excellent in 14 cases and good in 3 cases in group A, and were excellent in 11 cases and good in 6 cases in group B, showing no significant difference between 2 groups ( χ2=0.142, P=0.707). The new type of femoral intramedullary nail tail cap can simplify operation, shorten operation time, and reduce blood loss, and it has satisfactory effectiveness.

Wnt-1 immunodetection in the regenerating tail of lizard suggests it is involved in the proliferation and distal growth of the blastema

Lizard tail regeneration depends from growth of the apical tip and autonomous regeneration of a new spinal cord, cartilaginous tube and muscles. The presence of embryonic signaling pathways is likely involved and we have focused on immunolocalization of Wnt1 protein in regenerating tissues, a protein promoting proliferation and tumorigenesis. Western blot indicates some immunoreactive bands in the expected range at 46 and 33kDa in the regenerating tail. Immunolocalization indicates that Wnt1 is prevalently detected in the apical wound epidermis, blastema, and ependyma ampulla of the regenerating tail while it lowers in other tissues of more proximal regions close to the original tail stump. Although a gradient for Wnt1 was not detected, the higher immunofluorescence present in the apical region of the blastema and around the regenerating spinal cord indicates that the protein could be secreted from the apical wound epidermis and the ependyma and might influence cell proliferation in the blastema, the distal-most growing center of the new tail. The present observations suggest the involvement of the Wnt pathway to direct the process of tail regeneration in lizard. The stimulation of proliferation of epidermal and mesenchymal cells in the apical blastema by Wnt proteins remains to be experimentally validated.

Regeneration of adhesive tail pad scales in the New Zealand gecko (Hoplodactylus maculatus)(Reptilia;Squamata;Lacertilia) can serve as an experimental model to analyze setal formation in lizards generally.

During the regeneration of the tail in the arboreal New Zealand gecko (Hoplodactylus maculatus) a new set of tail scales, modified into pads bearing setae 5-20 μm long, is also regenerated. Stages of the formation of these specialized scales from epidermal pegs that invaginate the dermis of the regenerating tail are described on the basis of light and electron microscopic images. Within the pegs a differentiating clear layer interfaces with the spinulae and setae of the Oberhäutchen according to a process similar to that described for the digital pads. A layer of clear cytoplasm surrounds the growing tiny setae and eventually cornifies around them and their spatular ends, later leaving the new setae freestanding on the epidermal surface. The fresh adhesive pads help the gecko to maintain the prehensile function of its regenerated tail as together with the axial skeleton (made of a cylinder of elastic cartilage) the pads allow the regenerated tail to curl around twigs and small branches just like the original tail. The regeneration of caudal adhesive pads represents an ideal system to study the cellular processes that determine setal formation under normal or experimental manipulation as the progressive phases of the formation of the setae can be sequentially analyzed.

Histological Study of Common House Gecko (Hemidactylus frenatus) Regenerated Tail

Common house gecko<strong> </strong>(<em>Hemidactylus frenatus</em>) belongs to suborder Lacertilia that has capacity to shed its tail (autotomy) as a self-defense mechanism. After autotomy, tail regeneration occurs. Axiale skeleton of an original tail is composed of bony vertebrae, whereas the the regenerate one is comprised of cartilaginous tube. The purpose of this study was to determine the histological difference between axial skeleton of the original tail and the regenerate one of the common house gecko. Twenty four individuals consist of twelve common house gecko with original tail and twelve with regenerate tail were used. Microanatomical observations were carried on histological slide of original and regenerated tail stained with Hematoxylin-Eosin and Mallory Acid Fuchsin. The results showed that the original tail comprised of bony vertebrae whereas regenerated tail supported by cartilaginous tube.

Bacteriophage LM33_P1, a fast-acting weapon against the pandemic ST131-O25b:H4 Escherichia coli clonal complex.

Amongst the highly diverse Escherichia coli population, the ST131-O25b:H4 clonal complex is particularly worrisome as it is associated with a high level of antibiotic resistance. The lack of new antibiotics, the worldwide continuous increase of infections caused by MDR bacteria and the need for narrow-spectrum antimicrobial agents have revived interest in phage therapy. In this article, we describe a virulent bacteriophage, LM33_P1, which specifically infects O25b strains, and provide data related to its therapeutic potential. A large panel of E. coli strains (n = 283) was used to assess both the specificity and the activity of bacteriophage LM33_P1. Immunology, biochemistry and genetics-based methods confirmed this specificity. Virology methods and sequencing were used to characterize this bacteriophage in vitro, while three relevant mouse models were employed to show its in vivo efficacy. Bacteriophage LM33_P1 exclusively infects O25b E. coli strains with a 70% coverage on sequence types associated with high antibiotic resistance (ST131 and ST69). This specificity is due to an interaction with the LPS mediated by an original tail fibre. LM33_P1 also has exceptional intrinsic properties with a high adsorption constant and produces over 300 virions per cell in <10 min. Using animal pneumonia, septicaemia and urinary tract infection models, we showed the in vivo efficacy of LM33_P1 to reduce the bacterial load in several organs. Bacteriophage LM33_P1 represents the first weapon that specifically and quickly kills O25b E. coli strains. Therapeutic approaches derived from this bacteriophage could be developed to stop or slow down the spread of the ST131-O25b:H4 drug-resistant clonal complex in humans.

Ependymal Cells are a Heterogeneous Population in the Spinal Cord of the Leopard Gecko

The leopard gecko (Eublepharis macularius) is an emerging model for the study of spinal cord regeneration. As for many lizards, leopard geckos are able to self‐detach their tails (to escape predation) and then spontaneously regenerate a replacement. The regenerate tail is characterized by a centrally positioned spinal cord with a relatively simple morphology: a layer of ependymal cells surrounded by nerve tracts. We hypothesize that ependymal cells in the spinal cord are a heterogenous population. Prior to injury, virtually all ependymal cells of the original (uninjured) spinal cord express the neural stem/progenitor cell (NSPC) marker Sox2, and a subset of these cells also express the astrocyte marker glial fibrillary acid protein (GFAP). Neurons of the grey matter, but not ependymal cells, co‐express the neuronal markers HuCD and NeuN. During regeneration, ependymal cells remain Sox2+ and begin to express additional NSPC markers including Musashi‐1, βIII‐Tubulin and Sox9. Interestingly, a subset of ependymal cells in both the original tail stump and the newly formed regenerate spinal cord begin expressing HuCD, a protein otherwise characteristic of neuronal differentiation. These cells are negative for NeuN (a marker of neuronal nuclei) and are positionally restricted to locations bordering the central canal. A second subset of ependymal cells within the regenerating spinal cord begin to express GFAP and Vimentin but not HuCD. Our results suggest that there are at least three distinct populations of ependymal cells present during regeneration. The majority of ependymal cells are HuCD/GFAP− and display a dynamic NSPC profile following injury (Musashi‐1+/ βIII‐Tubulin+/ Sox9+). Two other populations are less widespread. HuCD+/GFAP−/NeuN− cells may represent a population of neuronal‐like central canal contacting cells, while HuCD−/GFAP+/Vimentin+ cells appear to be ependymo‐radial glia. This complex assemblage of cells may explain the remarkable regenerative abilities of the spinal cord in this species.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada, Discovery Grant (400358)

New species of Blaesodactylus (Squamata: Gekkonidae) from Tsingy karstic outcrops in Ankarana National Park, northern Madagascar.

We describe a new gecko of the genus Blaesodactylus from a karstic outcrop in deciduous dry forest of Ankarana National Park, northern Madagascar. Blaesodactylus microtuberculatus sp. nov., the fifth recognized species of Blaesodactylus, is distinguished from all other congeners, B. ambonihazo, B. antongilensis, B. boivini and B. sakalava by a combination of small, homogeneous gular granules, unspotted venter and lack of tubercles on distal part of original tail. Mitochondrial (ND2 and ND4) and nuclear (RAG-1) DNA identify a consistent divergence between B. microtuberculatus and its allotopic sister species B. boivini. We highlight habitat partitioning in these allotopic congeners where Blaesodactylus microtuberculatus inhabits karstic outcrops in Tsingy massif, and B. boivini dwells on tree trunks in deciduous dry forest.