Novel roles of cardiac-derived erythropoietin in cardiac development and function.

The role of erythropoietin (EPO) has extended beyond hematopoiesis to include cytoprotection, inotropy, and neurogenesis. Extra-renal EPO has been reported for multiple tissue/cell types, but the physiological relevance remains unknown. Although the EPO receptor is expressed by multiple cardiac cell types and human recombinant EPO increases contractility and confers cytoprotection against injury, whether the heart produces physiologically meaningful amounts of EPO in vivo is unclear. We show a distinct circadian rhythm of cardiac EPO mRNA expression in adult mice and increased mRNA expression during embryogenesis, suggesting physiological relevance to cardiac EPO production throughout life. We then generated constitutive, cardiomyocyte-specific EPO knockout mice driven by the Mlc2v promoter (EPOfl/fl:Mlc2v-cre+/-; EPOΔ/Δ-CM). During cardiogenesis, cardiac EPO mRNA expression and cellular proliferation were reduced in EPOΔ/Δ-CM hearts. However, in adult EPOΔ/Δ- CM mice, total heart weight was preserved through increased cardiomyocyte cross-sectional area, indicating the reduced cellular proliferation was compensated for by cellular hypertrophy. Echocardiography revealed no changes in cardiac dimensions, with modest reductions in ejection fraction, stroke volume, and tachycardia, whereas invasive hemodynamics showed increased cardiac contractility and lusitropy. Paradoxically, EPO mRNA expression in the heart was elevated in adult EPOΔ/Δ-CM, along with increased serum EPO protein content and hematocrit. Using RNA fluorescent in situ hybridization, we found that Epo RNA colocalized with endothelial cells in the hearts of adult EPOΔ/Δ-CM mice, identifying the endothelial cells as a cell responsible for the EPO hyper-expression. Collectively, these data identify the first physiological roles for cardiomyocyte-derived EPO. We have established cardiac EPO mRNA expression is a complex interplay of multiple cell types, where loss of embryonic cardiomyocyte EPO production results in hyper-expression from other cells within the adult heart.

Comparative analysis of osteoderms across the lizard body.

Osteoderms (ODs) are mineralized tissue embedded within the skin and are particularly common in reptiles. They are generally thought to form a protective layer between the soft tissues of the animal and potential external threats, although other functions have been proposed. The aim of this study was to characterize OD variation across the lizard body. Adults of three lizard species were chosen for this study. After whole body CT scanning of each lizard, single ODs were extracted from 10 different anatomical regions, CT scanned, and characterized using sectioning and nanoindentation. Morphological analysis and material characterization revealed considerable diversity in OD structure across the species investigated. The scincid Tiliqua gigas was the only studied species in which ODs had a similar external morphology across the head and body. Greater osteoderm diversity was found in the gerrhosaurid Broadleysaurus major and the scincid Tribolonotus novaeguineae. Dense capping tissue, like that reported for Heloderma, was found in only one of the three species examined, B. major. Osteoderm structure can be surprisingly complex and variable, both among related taxa, and across the body of individual animals. This raises many questions about OD function but also about the genetic and developmental factors controlling OD shape.

The role of cranial osteoderms on the mechanics of the skull in scincid lizards.

Osteoderms (ODs) are calcified organs formed directly within the skin of most major extant tetrapod lineages. Lizards possibly show the greatest diversity in ODs morphology and distribution. ODs are commonly hypothesized to function as a defensive armor. Here we tested the hypothesis that cranial osteoderms also contribute to the mechanics of the skull during biting. A series of in vivo experiments were carried out on three specimens of Tiliqua gigas. Animals were induced to bite a force plate while a single cranial OD was strain gauged. A finite element (FE) model of a related species, Tiliqua scincoides, was developed and used to estimate the level of strain across the same OD as instrumented in the in vivo experiments. FE results were compared to the in vivo data and the FE model was modified to test two hypothetical scenarios in which all ODs were (i) removed from, and (ii) fused to, the skull. In vivo data demonstrated that the ODs were carrying load during biting. The hypothetical FE models showed that when cranial ODs were fused to the skull, the overall strain across the skull arising from biting was reduced. Removing the ODs showed an opposite effect. In summary, our findings suggest that cranial ODs contribute to the mechanics of the skull, even when they are loosely attached.

Spontaneous neuronal regeneration in the forebrain of the leopard gecko (Eublepharis macularius) following neurochemical lesioning.

BACKGROUND: Neurogenesis is the ability to generate new neurons from resident stem/progenitor populations. Although often understood as a homeostatic process, several species of teleost fish, salamanders, and lacertid lizards are also capable of reactive neurogenesis, spontaneously replacing lost or damaged neurons. Here, we demonstrate that reactive neurogenesis also occurs in a distantly related lizard species, Eublepharis macularius, the leopard gecko. RESULTS: To initiate reactive neurogenesis, the antimetabolite 3-acetylpyridine (3-AP) was administered. Four days following 3-AP administration there is a surge in neuronal cell death within a region of the forebrain known as the medial cortex (homolog of the mammalian hippocampal formation). Neuronal cell death is accompanied by a shift in resident microglial morphology and an increase neural stem/progenitor cell proliferation. By 30 days following 3-AP administration, the medial cortex was entirely repopulated by NeuN+ neurons. At the same time, local microglia have reverted to a resting state and cell proliferation by neural stem/progenitors has returned to levels comparable with uninjured controls. CONCLUSIONS: Together, these data provide compelling evidence of reactive neurogenesis in leopard geckos, and indicate that the ability of lizards to spontaneously replace lost or damaged forebrain neurons is more taxonomically widespread and evolutionarily conserved than previously considered.

Biomechanical behaviour of lizard osteoderms and skin under external loading.

Many species of lizards are partially enveloped by a dermal armour made of ossified units called osteoderms. Lizard osteoderms demonstrate considerable species-specific variation in morphology and histology. Although a physical/protective role (against predators, prey, conspecifics and impact loading during falls) is frequently advanced, empirical data on the biomechanics of lizard osteoderms are scarce, limiting our understanding of form-function relationships. Here, we report deformation recorded at the surface of temporal osteoderms during controlled external loading of preserved specimens of 11 lizard species (Tiliqua rugosa, Tiliqua scincoides, Corucia zebrata, Pseudopus apodus, Timon lepidus, Matobosaurus validus, Broadleysaurus major, Tribolonotus gracilis, Tribolonotus novaeguineae, Heloderma horridum and Heloderma suspectum). Based on the strain recorded in situ and from isolated osteoderms, the skin of the species investigated can be ranked along a marked stiffness gradient that mostly reflects the features of the osteoderms. Some species such as T. rugosa and the two Heloderma species had very stiff osteoderms and skin while others such as T. lepidus and P. apodus were at the other end of the spectrum. Histological sections of the osteoderms suggest that fused (versus compound) osteoderms with a thick layer of capping tissue are found in species with a stiff skin. In most cases, loading neighbouring osteoderms induced a large strain in the instrumented osteoderm, attesting that, in most species, lizard osteoderms are tightly interconnected. These data empirically confirm that the morphological diversity observed in lizard osteoderms is matched by variability in biomechanical properties.

Radial Glia and Neuronal-like Ependymal Cells Are Present within the Spinal Cord of the Trunk (Body) in the Leopard Gecko (Eublepharis macularius).

As is the case for many lizards, leopard geckos (Eublepharis macularius) can self-detach a portion of their tail to escape predation, and then regenerate a replacement complete with a spinal cord. Previous research has shown that endogenous populations of neural stem/progenitor cells (NSPCs) reside within the spinal cord of the original tail. In response to tail loss, these NSPCs are activated and contribute to regeneration. Here, we investigate whether similar populations of NSPCs are found within the spinal cord of the trunk (body). Using a long-duration 5-bromo-2'-deoxyuridine pulse-chase experiment, we determined that a population of cells within the ependymal layer are label-retaining following a 20-week chase. Tail loss does not significantly alter rates of ependymal cell proliferation within the trunk spinal cord. Ependymal cells of the trunk spinal cord express SOX2 and represent at least two distinct cell populations: radial glial-like (glial fibrillary acidic protein- and Vimentin-expressing) cells; and neuronal-like (HuCD-expressing) cells. Taken together, these data demonstrate that NSPCs of the trunk spinal cord closely resemble those of the tail and support the use of the tail spinal cord as a less invasive proxy for body spinal cord injury investigations.

Unravelling the structural variation of lizard osteoderms.

Vertebrate skin is a remarkable organ that supports and protects the body. It consists of two layers, the epidermis and the underlying dermis. In some tetrapods, the dermis includes mineralised organs known as osteoderms (OD). Lizards, with over 7,000 species, show the greatest diversity in OD morphology and distribution, yet we barely understand what drives this diversity. This multiscale analysis of five species of lizards, whose lineages diverged ∼100-150 million years ago, compared the micro- and macrostructure, material properties, and bending rigidity of their ODs, and examined the underlying bones of the skull roof and jaw (including teeth when possible). Unsurprisingly, OD shape, taken alone, impacts bending rigidity, with the ODs of Corucia zebrata being most flexible and those of Timon lepidus being most rigid. Macroscopic variation is also reflected in microstructural diversity, with differences in tissue composition and arrangement. However, the properties of the core bony tissues, in both ODs and cranial bones, were found to be similar across taxa, although the hard, capping tissue on the ODs of Heloderma and Pseudopus had material properties similar to those of tooth enamel. The results offer evidence on the functional adaptations of cranial ODs, but questions remain regarding the factors driving their diversity. STATEMENT OF SIGNIFICANCE: Understanding nature has always been a significant source of inspiration for various areas of the physical and biological sciences. Here we unravelled a novel biomineralization, i.e. calcified tissue, OD, forming within the skin of lizards which show significant diversity across the group. A range of techniques were used to provide an insight into these exceptionally diverse natural structures, in an integrated, whole system fashion. Our results offer some suggestions into the functional and biomechanical adaptations of OD and their hierarchical structure. This knowledge can provide a potential source of inspiration for biomimetic and bioinspired designs, applicable to the manufacturing of light-weight, damage-tolerant and multifunctional materials for areas such as tissue engineering.

Lizard osteoderms - Morphological characterisation, biomimetic design and manufacturing based on three species.

Osteoderms (OD) are mineralised dermal structures consisting mainly of calcium phosphate and collagen. The sheer diversity of OD morphologies and their distribution within the skin of lizards makes these reptiles an ideal group in which to study ODs. Nonetheless, our understanding of the structure, development, and function of lizard ODs remains limited. The specific aims of this study were: (1) to carry out a detailed morphological characterisation of ODs in three lizard species; (2) to design and manufacture biomimetic sheets of ODs corresponding to the OD arrangement in each species; and (3) to evaluate the impact resistance of the manufactured biomimetic sheets under a drop weight test. Skin samples of the anguimorphsH. suspectumandO. ventralis, and the skinkC. zebratawere obtained from frozen lab specimens. Following a series of imaging and image characterisations, 3D biomimetic models of the ODs were developed. 3D models were then printed using additive manufacturing techniques and subjected to drop weight impact tests. The results suggest that a 3D printed compound of overlapping ODs as observed inCoruciacan potentially offers a higher energy absorption by comparison with the overlapping ODs ofOphisaurusand the non-overlapping ODs ofHeloderma.Compound overlapping ODs need to be further tested and explored as a biomimetic concept to increase the shock absorption capabilities of devices and structures.

The Dendrite Arbor of Purkinje Cells Is Altered Following to Tail Regeneration in the Leopard Gecko.

Purkinje cells of the cerebellum have a complex arborized arrangement of dendrites and are among the most distinctive cell types of the nervous system. Although the neuromorphology of Purkinje cells has been well described for some mammals and teleost fish, for most vertebrates less is known. Here we used a modified Golgi-Cox method to investigate the neuromorphology of Purkinje cells from the lizard Eublepharis macularius, the leopard gecko. Using Sholl and Branch Structure Analyses, we sought to investigate whether the neuromorphology of gecko Purkinje cells was altered in response to tail loss and regeneration. Tail loss is an evolved mechanism commonly used by geckos to escape predation. Loss of the tail represents a significant and sudden change in body length and mass, which is only partially recovered as the tail is regenerated. We predicted that tail loss and regeneration would induce a quantifiable change in Purkinje cell dendrite arborization. Post hoc comparisons of Sholl analyses data showed that geckos with regenerated tails have significant changes in dendrite diameter and the number of dendrite intersections in regions corresponding to the position of parallel fiber synapses. We propose that the neuromorphological alterations observed in gecko Purkinje cells represent a compensatory response to tail regrowth, and perhaps a role in motor learning.

Cranial ornamentation in the Late Cretaceous nodosaurid ankylosaur Hungarosaurus.

Bony cranial ornamentation is developed by many groups of vertebrates, including ankylosaur dinosaurs. To date, the morphology and ontogenetic origin of ankylosaurian cranial ornamentation has primarily focused on a limited number of species from only one of the two major lineages, Ankylosauridae. For members of the sister group Nodosauridae, less is known. Here, we provide new details of the cranial anatomy of the nodosaurid Hungarosaurus from the Santonian of Europe. Based on a number of previously described and newly identified fragmentary skulls and skull elements, we recognize three different size classes of Hungarosaurus. We interpret these size classes as representing different stages of ontogeny. Cranial ornamentation is already well-developed in the earliest ontogenetic stage represented herein, suggesting that the presence of outgrowths may have played a role in intra- and interspecific recognition. We find no evidence that cranial ornamentation in Hungarosaurus involves the contribution of coossified osteoderms. Instead, available evidence indicates that cranial ornamentation forms as a result of the elaboration of individual elements. Although individual differences and sexual dimorphism cannot be excluded, the observed variation in Hungarosaurus cranial ornamentation appears to be associated with ontogeny.

Cutaneous tactile sensitivity before and after tail loss and regeneration in the leopard gecko (Eublepharis macularius).

Amongst tetrapods, mechanoreceptors on the feet establish a sense of body placement and help to facilitate posture and biomechanics. Mechanoreceptors are necessary for stabilizing the body while navigating through changing terrains or responding to a sudden change in body mass and orientation. Lizards such as the leopard gecko (Eublepharis macularius) employ autotomy - a voluntary detachment of a portion of the tail - to escape predation. Tail autotomy represents a natural form of significant (and localized) mass loss. Semmes-Weinstein monofilaments were used to investigate the effect of tail autotomy (and subsequent tail regeneration) on tactile sensitivity of each appendage of the leopard gecko. Prior to autotomy, we identified site-specific differences in tactile sensitivity across the ventral surfaces of the hindlimbs, forelimbs and tail. Repeated monofilament testing of both control (tail-intact) and tail-loss geckos had a significant sensitization effect (i.e. decrease in tactile threshold, maintained over time) in all regions of interest except the palmar surfaces of the forelimbs in post-autotomy geckos, compared with baseline testing. Although the regenerated tail is not an exact replica of the original, tactile sensitivity is shown to be effectively restored at this site. Re-establishment of tactile sensitivity on the ventral surface of the regenerate tail points towards a (continued) role in predator detection.

A comparative histological study of the osteoderms in the lizards Heloderma suspectum (Squamata: Helodermatidae) and Varanus komodoensis (Squamata: Varanidae).

We describe the histological appearance of the osteoderms (ODs) of Heloderma suspectum and Varanus komodoensis using multiple staining and microscopy techniques to yield information about their morphology and development. Histological analysis showed that the ODs of H. suspectum are composed of three main tissue types, a superficial layer, herein identified as osteodermine, capping a base composed of Sharpey-fibre bone and lamellar bone rich in secondary osteons (Haversian bone tissue). In contrast, ODs in V. komodoensis are composed of a core of woven bone surrounded by parallel-fibred bone without a capping tissue. Thus, in these two species, ODs differ both in terms of their structural composition and in details of their skeletogenesis. The histology of the mineralised tissues observed in these two reptile taxa provides insights into the mechanism of formation of lizard ODs and presents a direct comparison of the histological properties between the ODs of the two species. These data allow greater understanding of the comparative histological appearance of the dermal bones of lizards and highlight their structural diversity.

Anatomy and Ontogeny of the Mandibular Symphysis in Alligator mississippiensis.

Crocodylians evolved some of the most characteristic skulls of the animal kingdom with specializations for semiaquatic and ambush lifestyles, resulting in a feeding apparatus capable of tolerating high biomechanical loads and bite forces and a head with a derived sense of trigeminal-nerve-mediated touch. The mandibular symphysis accommodates these specializations being both at the end of a biomechanical lever and an antenna for sensation. Little is known about the anatomy of the crocodylian mandibular symphysis, hampering our understanding of form, function, and evolution of the joint in extant and extinct lineages. We explore mandibular symphysis anatomy of an ontogenetic series of Alligator mississippiensis using imaging, histology, and whole mount methods. Complex sutural ligaments emanating about a midline-fused Meckel's cartilage bridge the symphysis. These tissues organize during days 37-42 of in ovo development. However, interdigitations do not manifest until after hatching. These soft tissues leave a hub and spoke-like bony morphology of the symphyseal plate, which never fuses. Interdigitation morphology varies within the symphysis suggesting differential loading about the joint. Neurovascular canals extend throughout the mandibles to alveoli, integument, and bone adjacent to the symphysis. These features suggest the Alligator mandibular symphysis offers compliance in an otherwise rigid skull. We hypothesize a fused Meckel's cartilage offers stiffness in hatchling mandibles prior to the development of organized sutural ligaments and mineralized bone while offering a scaffold for somatic growth. The porosity of the dentaries due to neurovascular tissues likely allows transmission of sensory and proprioceptive information from the surroundings and the loaded symphysis. Anat Rec, 302:1696-1708, 2019. © 2019 American Association for Anatomy.

Reptile Embryology and Regeneration.

Reptiles (lizards, snakes, turtles, and crocodilians) are becoming increasingly popular as models for developmental investigations. In this review the leopard gecko, Eublepharis macularius, is presented as a reptilian model for embryonic and tissue regeneration studies. We provide details of husbandry and breeding and discuss aspects of embryonic nutrition, egg anatomy, and sex determination. We provide comprehensive protocols for transcardial perfusion, short-term anesthesia using the injectable anesthetic Alfaxan, and full-thickness cutaneous biopsy punches, used in geckos for the study of scar-free wound healing. We also provide modifications to three popular histological techniques (whole-mount histochemistry, immunohistochemistry, and double-label immunofluorescence) and provide details on bromodeoxyuridine (BrdU) labeling and immuno-detection.

Constitutive cardiomyocyte proliferation in the leopard gecko (Eublepharis macularius).

Although the contractile function of the heart is universally conserved, the organ itself varies in structure across species. This variation includes the number of ventricular chambers (one, two, or an incompletely divided chamber), the structure of the myocardial wall (compact or trabeculated), and the proliferative capacity of the resident cardiomyocytes. Whereas zebrafish are capable of comparatively high rates of constitutive cardiomyocyte proliferation, humans and rodents are not. However, for most species, the capacity to generate new cardiomyocytes under homeostatic conditions remains unclear. Here, we investigate cardiomyocyte proliferation in the lizard Eublepharis macularius, the leopard gecko. As for other lizards, the leopard gecko heart has a partially septated ventricular lumen with a trabeculated myocardial wall. To test our hypothesis that leopard gecko cardiomyocytes routinely proliferate, we performed 5-bromo-2'-deoxyuridine incorporation and immunostained for the mitotic marker phosphorylated histone H3 (pHH3) and the DNA synthesis phase (S phase) marker proliferating cell nuclear antigen (PCNA). Using double immunofluorescence, we co-localized pHH3 or PCNA with the cardiomyocyte marker myosin heavy chain (MHC). We found that ~0.5% of cardiomyocytes were mitotically active (pHH3+/MHC+), while ~10% were in S phase (PCNA+/MHC+). We also determined that cell cycling by gecko cardiomyocytes is not impacted by caudal autotomy (tail loss), a dramatic form of self-amputation. Finally, we show that populations of cardiac cells are slow cycling. Overall, our findings provide predictive evidence that geckos may be capable of spontaneous cardiac self-repair and regeneration following a direct injury.

Evidence for neurogenesis in the medial cortex of the leopard gecko, Eublepharis macularius.

Although lizards are often described as having robust neurogenic abilities, only a handful of the more than 6300 species have been explored. Here, we provide the first evidence of homeostatic neurogenesis in the leopard gecko (Eublepharis macularius). We focused our study on the medial cortex, homologue of the mammalian hippocampal formation. Using immunostaining, we identified proliferating pools of neural stem/progenitor cells within the sulcus septomedialis, the pseudostratified ventricular zone adjacent to the medial cortex. Consistent with their identification as radial glia, these cells expressed SOX2, glial fibrillary acidic protein, and Vimentin, and demonstrated a radial morphology. Using a 5-bromo-2'-deoxyuridine cell tracking strategy, we determined that neuroblast migration from the ventricular zone to the medial cortex takes ~30-days, and that newly generated neuronal cells survived for at least 140-days. We also found that cell proliferation within the medial cortex was not significantly altered following rupture of the tail spinal cord (as a result of the naturally evolved process of caudal autotomy). We conclude that the sulcus septomedialis of the leopard gecko demonstrates all the hallmarks of a neurogenic niche.

VEGF, FGF-2 and TGFß expression in the normal and regenerating epidermis of geckos: implications for epidermal homeostasis and wound healing in reptiles.

The skin is a bilayered organ that serves as a key barrier between an organism and its environment. In addition to protecting against microbial invasion, physical trauma and environmental damage, skin participates in maintaining homeostasis. Skin is also capable of spontaneous self-repair following injury. These functions are mediated by numerous pleiotrophic growth factors, including members of the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and transforming growth factor ß (TGFß) families. Although growth factor expression has been well documented in mammals, particularly during wound healing, for groups such as reptiles less is known. Here, we investigate the spatio-temporal pattern of expression of multiple growth factors in normal skin and following a full-thickness cutaneous injury in the representative lizard Eublepharis macularius, the leopard gecko. Unlike mammals, leopard geckos can heal cutaneous wounds without scarring. We demonstrate that before, during and after injury, keratinocytes of the epidermis express a diverse panel of growth factor ligands and receptors, including: VEGF, VEGFR1, VEGFR2, and phosphorylated VEGFR2; FGF-2 and FGFR1; and phosphorylated SMAD2, TGFß1, and activin ßA. Unexpectedly, only the tyrosine kinase receptors VEGFR1 and FGFR1 were dynamically expressed, and only during the earliest phases of re-epithelization; otherwise all the proteins of interest were constitutively present. We propose that the ubiquitous pattern of growth factor expression by keratinocytes is associated with various roles during tissue homeostasis, including protection against ultraviolet photodamage and coordinated body-wide skin shedding.

Tail regeneration and other phenomena of wound healing and tissue restoration in lizards.

Wound healing is a fundamental evolutionary adaptation with two possible outcomes: scar formation or reparative regeneration. Scars participate in re-forming the barrier with the external environment and restoring homeostasis to injured tissues, but are well understood to represent dysfunctional replacements. In contrast, reparative regeneration is a tissue-specific program that near-perfectly replicates that which was lost or damaged. Although regeneration is best known from salamanders (including newts and axolotls) and zebrafish, it is unexpectedly widespread among vertebrates. For example, mice and humans can replace their digit tips, while many lizards can spontaneously regenerate almost their entire tail. Whereas the phenomenon of lizard tail regeneration has long been recognized, many details of this process remain poorly understood. All of this is beginning to change. This Review provides a comparative perspective on mechanisms of wound healing and regeneration, with a focus on lizards as an emerging model. Not only are lizards able to regrow cartilage and the spinal cord following tail loss, some species can also regenerate tissues after full-thickness skin wounds to the body, transections of the optic nerve and even lesions to parts of the brain. Current investigations are advancing our understanding of the biological requirements for successful tissue and organ repair, with obvious implications for biomedical sciences and regenerative medicine.

Blood vessel formation during tail regeneration in the leopard gecko (Eublepharis macularius): The blastema is not avascular.

Unique among amniotes, many lizards are able to self-detach (autotomize) their tail and then regenerate a replacement. Tail regeneration involves the formation of a blastema, an accumulation of proliferating cells at the site of autotomy. Over time, cells of the blastema give rise to most of the tissues in the replacement tail. In non-amniotes capable of regenerating (such as urodeles and some teleost fish), the blastema is reported to be essentially avascular until tissue differentiation takes place. For tail regenerating lizards less is known. Here, we investigate neovascularization during tail regeneration in the leopard gecko (Eublepharis macularius). We demonstrate that the gecko tail blastema is not an avascular structure. Beginning with the onset of regenerative outgrowth, structurally mature (mural cell supported) blood vessels are found within the blastema. Although the pattern of blood vessel distribution in the regenerate tail differs from that of the original, a hierarchical network is established, with vessels of varying luminal diameters and wall thicknesses. Using immunostaining, we determine that blastema outgrowth and tissue differentiation is characterized by a dynamic interplay between the pro-angiogenic protein vascular endothelial growth factor (VEGF) and the anti-angiogenic protein thrombospondin-1 (TSP-1). VEGF-expression is initially widespread, but diminishes as tissues differentiate. In contrast, TSP-1 expression is initially restricted but becomes more abundant as VEGF-expression wanes. We predict that variation in the neovascular response observed between different regeneration-competent species likely relates to the volume of the blastema. J. Morphol. 278:380-389, 2017. © 2017 Wiley Periodicals, Inc.

The phylogenetic distribution, anatomy and histology of the post-cloacal bones and adnexa of geckos.

Post-cloacal bones of gekkotans may be present as a single (medial) pair, two pairs (medial and lateral), or may be lacking. We, herein, demonstrate that the presence of a single medial pair is the ancestral condition for the Gekkota, that the lateral pair is of sporadic occurrence within and between families, except for the Eublepharidae where it is universal, and that absence is also of sporadic occurrence except for the Sphaerodactylidae where it is the ancestral condition. Adult male Tokay geckos (Gekko gecko) possess only the medial pair of bones, and these exhibit a regionally-specific expression of woven, fibrolamellar, and lamellar bone, and an enclosed medullary cavity. Females and small juvenile males lack bony elements but exhibit a conspicuous band of dense connective tissue located about the anterior and lateral margins of the cloacal sacs. As males grow and attain sexual maturity, the medial post-cloacal bones condense in this band of dense connective tissue, and are thus shown to be dermal ossifications, similar to osteoderms but with muscular associations (although this is also known for crocodylians). Based upon ontogenetic data we set forth a scenario to explain the loss of the medial post-cloacal bones in various lineages. Differential staining of the cloacal sacs failed to reveal any specialized glandular structures. Investigation of the post-cloacal spurs shows them to be associated with cellular connective tissue of a type similar to that found in the vicinity of the medial post-cloacal bones. This suggests that the lateral post-cloacal bones may also be dermal bones, but histological evidence is needed to corroborate this.