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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+/-; EPO Δ/Δ-CM ). During cardiogenesis, cardiac EPO mRNA expression and cellular proliferation were reduced in EPO Δ/Δ-CM hearts. However, in adult EPO Δ/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 EPO Δ/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 EPO Δ/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., Competing Interests: Declaration of competing interest The authors report no commercial or proprietary interest in any product or concept discussed in this article., (Copyright © 2023. Published by Elsevier Ltd.)

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., (© 2022 American Association for Anatomy.)

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

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.

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

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., (© 2019 American Association for Anatomy.)

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.

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., (© 2018 Wiley Periodicals, Inc.)

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.

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 β (TGFβ) 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, TGFβ1, and activin βA. 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., (© 2018 Anatomical Society.)

As for many lizards, the leopard gecko (Eublepharis macularius) can self-detach its tail to avoid predation and then regenerate a replacement. The replacement tail includes a regenerated spinal cord with a simple morphology: an ependymal layer surrounded by nerve tracts. We hypothesized that cells within the ependymal layer of the original spinal cord include populations of neural stem/progenitor cells (NSPCs) that contribute to the regenerated spinal cord. Prior to tail loss, we performed a bromodeoxyuridine pulse-chase experiment and found that a subset of ependymal layer cells (ELCs) were label-retaining after a 140-day chase period. Next, we conducted a detailed spatiotemporal characterization of these cells before, during, and after tail regeneration. Our findings show that SOX2, a hallmark protein of NSPCs, is constitutively expressed by virtually all ELCs before, during, and after regeneration. We also found that during regeneration, ELCs express an expanded panel of NSPC and lineage-restricted progenitor cell markers, including MSI-1, SOX9, and TUJ1. Using electron microscopy, we determined that multiciliated, uniciliated, and biciliated cells are present, although the latter was only observed in regenerated spinal cords. Our results demonstrate that cells within the ependymal layer of the original, regenerating and fully regenerate spinal cord represent a heterogeneous population. These include radial glia comparable to Type E and Type B cells, and a neuronal-like population of cerebrospinal fluid-contacting cells. We propose that spinal cord regeneration in geckos represents a truncation of the restorative trajectory observed in some urodeles and teleosts, resulting in the formation of a structurally distinct replacement., (© 2017 Wiley Periodicals, Inc.)

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

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., (© 2017 Wiley Periodicals, Inc.)

Transforming growth factor beta (TGFβ) signalling is essential for wound healing, including both non-specific scar formation and tissue-specific regeneration. Specific TGFβ isoforms and downstream mediators of canonical and non-canonical signalling play different roles in each of these processes. Here we review the role of TGFβ signalling during tissue repair, with a particular focus on the prototypic isoforms TGFβ1, TGFβ2, and TGFβ3. We begin by introducing TGFβ signalling and then discuss the role of these growth factors and their key downstream signalling mediators in determining the balance between scar formation and tissue regeneration. Next we discuss examples of the pleiotropic roles of TGFβ ligands during cutaneous wound healing and blastema-mediated regeneration, and how inhibition of the canonical signalling pathway (using small molecule inhibitors) blocks regeneration. Finally, we review various TGFβ-targeting therapeutic strategies that hold promise for enhancing tissue repair.

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., (© 2015 Wiley Periodicals, Inc.)

Osteoderms are bone-rich organs found in the dermis of many scleroglossan lizards sensu lato, but are only known for two genera of gekkotans (geckos): Tarentola and Gekko. Here, we investigate their sequence of appearance, mode of development, structural diversity and ability to regenerate following tail loss. Osteoderms were present in all species of Tarentola sampled (Tarentola annularis, T. mauritanica, T. americana, T. crombei, T. chazaliae) as well as Gekko gecko, but not G. smithii. Gekkotan osteoderms first appear within the integument dorsal to the frontal bone or within the supraocular scales. They then manifest as mineralized structures in other positions across the head. In Tarentola and G. gecko, discontinuous clusters subsequently form dorsal to the pelvis/base of the tail, and then dorsal to the pectoral apparatus. Gekkotan osteoderm formation begins once the dermis is fully formed. Early bone deposition appears to involve populations of fibroblast-like cells, which are gradually replaced by more rounded osteoblasts. In T. annularis and T. mauritanica, an additional skeletal tissue is deposited across the superficial surface of the osteoderm. This tissue is vitreous, avascular, cell-poor, lacks intrinsic collagen, and is herein identified as osteodermine. We also report that following tail loss, both T. annularis and T. mauritanica are capable of regenerating osteoderms, including osteodermine, in the regenerated part of the tail. We propose that osteoderms serve roles in defense against combative prey and intraspecific aggression, along with anti-predation functions., (© 2015 Wiley Periodicals, Inc.)

Cutaneous wounds heal with two possible outcomes: scarification or near-perfect integumentary restoration. Whereas scar formation has been intensively investigated, less is known about the tissue-level events characterising wounds that spontaneously heal scar-free, particularly in non-foetal amniotes. Here, a spatiotemporal investigation of scar-free cutaneous wound healing following full-thickness excisional biopsies to the tail and body of leopard geckos (Eublepharis macularius) is provided. All injuries healed without scarring. Cutaneous repair involves the development of a cell-rich aggregate within the wound bed, similar to scarring wounds. Unlike scar formation, scar-free healing involves a more rapid closure of the wound epithelium, and a delay in blood vessel development and collagen deposition within the wound bed. It was found that, while granulation tissue of scarring wounds is hypervascular, scar-free wound healing conspicuously does not involve a period of exuberant blood vessel formation. In addition, during scar-free wound healing the newly formed blood vessels are typically perivascular cell-supported. Immunohistochemistry revealed widespread expression of both the pro-angiogenic factor vascular endothelial growth factor A and the anti-angiogenic factor thrombospondin-1 within the healing wound. It was found that scar-free wound healing is an intrinsic property of leopard gecko integument, and involves a modulation of the cutaneous scar repair program. This proportional revascularisation is an important factor in scar-free wound healing., (© 2015 Anatomical Society.)

Although amniotes (reptiles, including birds, and mammals) are capable of replacing certain tissues, complete appendage regeneration is rare. Perhaps the most striking example is the lizard tail. Tail loss initiates a spontaneous epimorphic (blastema-mediated) regenerative program, resulting in a fully functional but structurally non-identical replacement. Here we review lizard tail regeneration with a particular focus on the blastema. In many lizards, the original tail has evolved a series of fracture planes, anatomical modifications that permit the tail to be self-detached or autotomized. Following tail loss, the wound site is covered by a specialized wound epithelium under which the blastema develops. An outgrowth of the spinal cord, the ependymal tube, plays a key role in governing growth (and likely patterning) of the regenerate tail. In some species (e.g., geckos), the blastema forms as an apical aggregation of proliferating cells, similar to that of urodeles and teleosts. For other species (e.g., anoles) the identification of a proliferative blastema is less obvious, suggesting an unexpected diversity in regenerative mechanisms among tail-regenerating lizards.

Objective: Ongoing research is aimed at increasing cartilage tissue yield and quality from multipotent mesenchymal stromal cells (MSC) for the purpose of treating cartilage damage in horses. Low oxygen culture has been shown to enhance chondrogenesis, and novel membrane culture has been proposed to increase tissue yield and homogeneity. The objective of this study was to evaluate and compare the effect of reduced oxygen and membrane culture during in vitro chondrogenesis of equine cord blood (CB) MSC., Methods: CB-MSC (n = 5 foals) were expanded at 21% oxygen prior to 3-week differentiation in membrane or pellet culture at 5% and 21% oxygen. Assessment included histological examination (H&E, toluidine Blue, immunohistochemistry (IHC) for collagen type I and II), protein quantification by hydroxyproline assay and dimethylmethylene assay, and mRNA analysis for collagen IA1, collagen IIA1, collagen XA1, HIF1α and Sox9., Results: Among treatment groups, 5% membrane culture produced neocartilage most closely resembling hyaline cartilage. Membrane culture resulted in increased wet mass, homogenous matrix morphology and an increase in total collagen content, while 5% oxygen culture resulted in higher GAG and type II collagen content. No significant differences were observed for mRNA analysis., Conclusion: Membrane culture at 5% oxygen produces a comparatively larger amount of higher quality neocartilage. Matrix homogeneity is attributed to a uniform diffusion gradient and reduced surface tension. Membrane culture holds promise for scale-up for therapeutic purposes, for cellular preconditioning prior to cytotherapeutic applications, and for modeling system for gas-dependent chondrogenic differentiation studies., (Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.)

Abstract Caudal autotomy-the ability to self-detach the tail-is a dramatic adaptation common to many structural-grade lizards. For most species, tail loss is followed by the equally dramatic phenomenon of tail regeneration. Here we review the anatomy and histology of caudal autotomy and regeneration in lizards, drawing heavily from research published over the past 2 decades. The autotomous tail is characterized by various structural adaptations, which act to minimize blood loss and trauma to adjacent tissues. The early phase of wound healing involves a leukocytic response but limited inflammation. Reepithelialization via a specialized wound epithelium is not only critical for scar-free healing but also necessary for subsequent tissue patterning and regenerative outgrowth. Regeneration begins with the formation of the blastema, a mass of proliferating mesenchymal-like cells. As the blastema expands, it is invaded by blood vessels and the spinal cord. Whereas the replacement tail outwardly resembles the original appendage, it differs in several notable respects, including the tissue composition and organization of the skeleton, muscular system, and spinal cord. Increasingly, the lizard tail is being recognized among biomedical scientists as an important model for the study of wound healing and multitissue restoration.

Introduction: The transforming growth factor beta (TGFβ)/activin signaling pathway has a number of documented roles during wound healing and is increasingly appreciated as an essential component of multi-tissue regeneration that occurs in amphibians and fish. Among amniotes (reptiles and mammals), less is known due in part to the lack of an appropriate model organism capable of multi-tissue regeneration. The leopard gecko Eublepharis macularius is able to spontaneously, and repeatedly, regenerate its tail following tail loss. We examined the expression and localization of several key components of the TGFβ/activin signaling pathway during tail regeneration of the leopard gecko., Results: We observed a marked increase in phosphorylated Smad2 expression within the regenerate blastema indicating active TGFβ/activin signaling. Interestingly, during early regeneration, TGFβ1 expression is limited whereas activin-βA is strongly upregulated. We also observe the expression of EMT transcription factors Snail1 and Snail2 in the blastema., Conclusions: Combined, these observations provide strong support for the importance of different TGFβ ligands during multi-tissue regeneration and the potential role of TGFβ/activin-induced EMT programs during this process., (Copyright © 2013 Wiley Periodicals, Inc.)

Statements about morphological variation in extinct taxa often suffer from insufficient sampling that can be remedied by taking advantage of larger sample sizes provided by related, extant taxa. This analysis quantitatively and qualitatively examines histological and morphological variation of osteoderms from extant and extinct alligatoroid specimens. Statistically significant differences were correlated with changes in osteoderm size and shape. These differences are independent of position on the body, taxonomy, or evolution. Histological variation in alligatoroid osteoderms is due to morphological constraints on the elements themselves, and not taxonomic differences. This has implications for the recognition of histological characters in the osteoderms of extinct archosaur groups that lack extant representatives., (Copyright © 2013 Wiley Periodicals, Inc.)

The advantages of the embryonic chick as a model for studying neural development range from the relatively low cost of fertilized eggs to the rapid rate of development. We investigated in ovo cerebellar development in the chick, which has a nearly identical embryonic period as the mouse (19-22 days). We focused on three antigens: Calbindin (CB), Zebrin II (ZII), and Calretinin (CR), and our results demonstrate asynchronous expression patterns during cerebellar development. Presumptive CB+ Purkinje cells are first observed at embryonic day (E)10 in clusters in posterior cerebellum. At E12, corresponding with global expression of CB across the cerebellum, Purkinje cells began to express ZII. By E14-E16, Purkinje cells disperse into a monolayer and develop a pattern of alternating immunopositive and immunonegative ZII stripes. CR is initially expressed by clusters of presumptive Purkinje cells in the nodular zone at E8. However, this expression is transient and at later stages, CR is largely confined to the granule and molecular layers. Before hatch (E18-E20), Purkinje cells adopt a morphologically mature phenotype with complex dendritic arborizations. Comparing this data to that seen in mice, we found that the sequence of Purkinje cell formation, protein expression, and development is similar in both species, but these events consistently begin ∼5-7 days earlier in the precocial chick cerebellum, suggesting an important role for heterochrony in neurodevelopment., (Copyright © 2012 Wiley Periodicals, Inc.)

Unlike most tetrapods, in extant crocodylians the acetabulum is formed by only two of the three skeletal elements that constitute the pelvis, the ilium, and ischium. This peculiar arrangement is further confused by various observations that suggest the crocodylian pelvis initially develops from four skeletal elements: the ilium, ischium, pubis, and a novel element, the prepubis. According to one popular historical hypothesis, in crocodylians (and many extinct archosaurs), the pubis fuses with the ischium during skeletogenesis, leaving the prepubis as a distinct element, albeit one which is excluded from the acetabulum. Whereas the notion of a distinct prepubic element was once a topic of considerable interest, it has never been properly resolved. Here, we combine data gleaned from a developmental series of Alligator mississippiensis embryos, with a revised interpretation of fossil evidence from numerous outgroups to Crocodylia. We demonstrate that the modern crocodylian pelvis is composed of only three elements: the ilium, ischium, and pubis. The reported fourth pelvic element is an unossified portion of the ischium. Interpretations of pelvic skeletal homology have featured prominently in sauropsid systematics, and the unambiguous identification of the crocodylian pubis provides an important contribution to address larger scale evolutionary questions associated with locomotion and respiration., (Copyright © 2012 Wiley Periodicals, Inc.)

Many lizards are able to undergo scar-free wound healing and regeneration following loss of the tail. In most instances, lizard tail loss is facilitated by autotomy, an evolved mechanism that permits the tail to be self-detached at pre-existing fracture planes. However, it has also been reported that the tail can regenerate following surgical amputation outside the fracture plane. In this study, we used the leopard gecko, Eublepharis macularius, to investigate and compare wound healing and regeneration following autotomy at a fracture plane and amputation outside the fracture plane. Both forms of tail loss undergo a nearly identical sequence of events leading to scar-free wound healing and regeneration. Early wound healing is characterized by transient myofibroblasts and the formation of a highly proliferative wound epithelium immunoreactive for the wound keratin marker WE6. The new tail forms from what is commonly referred to as a blastema, a mass of proliferating mesenchymal-like cells. Blastema cells express the protease matrix metalloproteinase-9. Apoptosis (demonstrated by activated caspase 3 immunostaining) is largely restricted to isolated cells of the original and regenerating tail tissues, although cell death also occurs within dermal structures at the original-regenerated tissue interface and among clusters of newly formed myocytes. Furthermore, the autotomized tail is unique in demonstrating apoptosis among cells adjacent to the fracture planes. Unlike mammals, transforming growth factor-β3 is not involved in wound healing. We demonstrate that scar-free wound healing and regeneration are intrinsic properties of the tail, unrelated to the location or mode of tail detachment., (Copyright © 2012 Wiley Periodicals, Inc.)

The persistence of the notochord into the skeletally mature life stage is characteristic of gekkotans, but is otherwise of rare occurrence among amniotes. The taxonomic diversity of Gekkota affords the opportunity to investigate the structure and development of this phylogenetically ancestral component of the skeleton, and to determine its basic characteristics. The gekkotan notochord spans almost the entire postcranial long axis and is characterized by a moniliform morphology with regularly alternating zones of chordoid and chondroid tissue. Chordoid tissue persists in the region of intervertebral articulations and occupies the cavitations that lie between the centra of the amphicoelous vertebrae. Chondroid tissue is restricted to zones in which the diameter of the notochord is reduced, corresponding to mid-vertebral locations. In the tail, these zones of chondroid tissue are associated with the autotomic fracture planes. Chondroid tissue first manifests during late embryogenesis, appears to differentiate from pre-existing chordoid tissue, and has the histological and histochemical characteristics of cartilage. Our observations lend support to the hypothesis that cartilage can be derived directly from notochordal tissue, and suggest that the latter may be an evolutionary and developmental precursor to chordate cartilage. The persistence of chordoid tissue in the intervertebral regions of amphicoelous vertebrae is consistent with a suite of paedomorphic traits exhibited by gekkotans and suggests that the typical hydrostatic nature of notochordal tissue may play a role in mechanically governing patterns of displacement between adjacent amphicoelous vertebrae that lack extensive centrum-to-centrum contact., (Copyright © 2012 Wiley Periodicals, Inc.)

The skeleton is of fundamental importance in research in comparative vertebrate morphology, paleontology, biomechanics, developmental biology, and systematics. Motivated by research questions that require computational access to and comparative reasoning across the diverse skeletal phenotypes of vertebrates, we developed a module of anatomical concepts for the skeletal system, the Vertebrate Skeletal Anatomy Ontology (VSAO), to accommodate and unify the existing skeletal terminologies for the species-specific (mouse, the frog Xenopus, zebrafish) and multispecies (teleost, amphibian) vertebrate anatomy ontologies. Previous differences between these terminologies prevented even simple queries across databases pertaining to vertebrate morphology. This module of upper-level and specific skeletal terms currently includes 223 defined terms and 179 synonyms that integrate skeletal cells, tissues, biological processes, organs (skeletal elements such as bones and cartilages), and subdivisions of the skeletal system. The VSAO is designed to integrate with other ontologies, including the Common Anatomy Reference Ontology (CARO), Gene Ontology (GO), Uberon, and Cell Ontology (CL), and it is freely available to the community to be updated with additional terms required for research. Its structure accommodates anatomical variation among vertebrate species in development, structure, and composition. Annotation of diverse vertebrate phenotypes with this ontology will enable novel inquiries across the full spectrum of phenotypic diversity.

Background: Epimorphic regeneration results in the restoration of lost tissues and structures from an aggregation of proliferating cells known as a blastema. Among amniotes the most striking example of epimorphic regeneration comes from tail regenerating lizards. Although tail regeneration is often studied in the context of ecological costs and benefits, details of the sequence of tissue-level events are lacking. Here we investigate the anatomical and histological events that characterize tail regeneration in the leopard gecko, Eublepharis macularius., Results: Tail structure and tissue composition were examined at multiple days following tail loss, revealing a conserved pattern of regeneration. Removal of the tail results in a consistent series of morphological and histological events. Tail loss is followed by a latent period of wound healing with no visible signs of regenerative outgrowth. During this latent period basal cells of the epidermis proliferate and gradually cover the wound. An additional aggregation of proliferating cells accumulates adjacent to the distal tip of the severed spinal cord marking the first appearance of the blastema. Continued growth of the blastema is matched by the initiation of angiogenesis, followed by the re-development of peripheral axons and the ependymal tube of the spinal cord. Skeletal tissue differentiation, corresponding with the expression of Sox9, and muscle re-development are delayed until tail outgrowth is well underway., Conclusions: We demonstrate that tail regeneration in lizards involves a highly conserved sequence of events permitting the establishment of a staging table. We show that tail loss is followed by a latent period of scar-free healing of the wound site, and that regeneration is blastema-mediated. We conclude that the major events of epimorphic regeneration are highly conserved across vertebrates and that a comparative approach is an invaluable biomedical tool for ongoing regenerative research.

Cranial kinesis is a widespread feature of gekkotan lizards. Previous studies of kinesis in lizards often described the relevant, mobile joints as synovial, thus characterized by the presence of a synovial cavity lined with articular cartilage. To date however, detailed investigations of cranial joint histology are lacking. We examined eight cranial joints (quadrate-articular, quadrate-pterygoid, quadrate-otooccipital, quadrate-squamosal, epipterygoid-prootic, epipterygoid-pterygoid, basisphenoid-pterygoid, and frontal-parietal) in five gekkotan species (Oedura lesueuerii, Eublepharis macularius, Hemitheconyx caudicinctus, Tarentola annularis, and Chondrodactylous bibronii) using microcomputed tomography and serial histology. Particular focus was given to the relationship between the bony and soft-tissue components of the joint. Our results demonstrate that only three of these joints are synovial: the quadrate-articular, epipterygoid-pterygoid, and basisphenoid-pterygoid joints. The frontal-parietal and quadrate-pterygoid joints are syndesmosis (fibrous), the epipterygoid-prootic and quadrate-otooccipital joints are synchondroses (cartilaginous without a synovial cavity) and the quadrate-squamosal joint was not present. Based on previous descriptions, we determine that the structure of some cranial joints is variable among lizard taxa. We caution that osteology does not necessarily predict cranial joint histology. Although the functional implications of these findings remain to be explored we note that the development of synovial joints appears to be associated with a neural crest origin for the elements involved., (Copyright © 2011 Wiley-Liss, Inc.)

Reptiles (lizards, snakes, turtles and crocodylians) are becoming increasing popular as models for developmental investigations. In this review the leopard gecko, Eublepharis macularius, is presented as a reptilian model for embryonic studies. We provide details of husbandry, breeding and modifications to two popular histological techniques (whole-mount histochemistry and immunohistochemistry). In addition, we provide a summary of basic reptilian husbandry requirements and discuss important details of embryonic nutrition, egg anatomy and sex determination.

The pectoral apparatus (shoulder girdle plus sternum) of amniotes plesiomorphically includes an unpaired element of dermal origin. In crocodylians, lepidosaurs, and nontherian synapsids (monotremes and their ancestors) this element is identified as the interclavicle, in Testudines (turtles and tortoises) as the entoplastron, and in Aves as the furcula. We investigated embryonic development of the interclavicle in Alligator mississippiensis (American alligator) and of the furcula in Gallus gallus (domestic chicken). The interclavicle and furcula are among the first skeletal elements to ossify, beginning at Ferguson stage 19 (Alligator) and Hamburger and Hamilton stage 33 (Gallus). Both elements: occupy a similar mid-ventral position within the pectoral apparatus; develop from paired (bilateral) cell condensations; never coexist at anytime during ontogeny or in the adult; and undergo intramembranous (i.e., direct) ossification. For both the interclavicle and the furcula, the initial onset of ossification is concomitant with mineralization of elements of the dermatocranium, and occurs in advance of mineralization of the replacement bones (e.g., scapula, metacoracoid) of the pectoral apparatus. Shortly after the initiation of ossification the paired condensations of both elements fuse. For each of Alligator and Gallus, only one pair of skeletogenic condensations is present during embryonic development. Based on these data and a review of the evolution and development of dermal elements in the pectoral apparatus, we conclude that the interclavicle is equally parsimonious as a homolog of the furcula., (2009 Wiley-Liss, Inc.)

Squamates constitute a major vertebrate radiation, representing almost one-third of all known amniotes. Although speciose and morphologically diverse, they remain poorly represented in developmental studies. Here, we present an embryonic staging table of in ovo development for the basal gekkotan Eublepharis macularius (the leopard gecko) and advocate this species as a laboratory-appropriate developmental model. E. macularius, is a hardy and tractable species of relatively large body size (with concomitantly relatively large eggs and embryos), that is widely available and easy to maintain and propagate. Additionally, E. macularius displays a body plan appropriate to the study of the plesiomorphic quadrupedal condition of early pentadactylous terrestrial amniotes. Although not unexpected, it is worth noting that the morphological events characterizing limb development in E. macularius are comparable with those described for the avian Gallus gallus. Therefore, E. macularius holds great promise as a model for developmental studies focusing on pentadactyly and the formation of digits. Furthermore, it is also attractive as a developmental model because it demonstrates temperature-dependent sex determination. The staging table presented herein is based on an all-female series and represents the entire 52 day in ovo period. Overall, embryogenesis of E. macularius is similar to that of other squamates in terms of developmental stage attained at the time of oviposition, patterns of limb and pharyngeal arch development, and features of the appearance of scalation and pigmentation, indicative of a conserved developmental program., ((c) 2009 Wiley-Liss, Inc.)

Most non-tetrapod vertebrates develop mineralized extra-oral elements within the integument. Known collectively as the integumentary skeleton, these elements represent the structurally diverse skin-bound contribution to the dermal skeleton. In this review we begin by summarizing what is known about the histological diversity of the four main groups of integumentary skeletal tissues: hypermineralized (capping) tissues; dentine; plywood-like tissues; and bone. For most modern taxa, the integumentary skeleton has undergone widespread reduction and modification often rendering the homology and relationships of these elements confused and uncertain. Fundamentally, however, all integumentary skeletal elements are derived (alone or in combination) from only two types of cell condensations: odontogenic and osteogenic condensations. We review the origin and diversification of the integumentary skeleton in aquatic non-tetrapods (including stem gnathostomes), focusing on tissues derived from odontogenic (hypermineralized tissues, dentines and elasmodine) and osteogenic (bone tissues) cell condensations. The novelty of our new scenario of integumentary skeletal evolution resides in the demonstration that elasmodine, the main component of elasmoid scales, is odontogenic in origin. Based on available data we propose that elasmodine is a form of lamellar dentine. Given its widespread distribution in non-tetrapod lineages we further propose that elasmodine is a very ancient tissue in vertebrates and predict that it will be found in ancestral rhombic scales and cosmoid scales.

Although often overlooked, the integument of many tetrapods is reinforced by a morphologically and structurally diverse assemblage of skeletal elements. These elements are widely understood to be derivatives of the once all-encompassing dermal skeleton of stem-gnathostomes but most details of their evolution and development remain confused and uncertain. Herein we re-evaluate the tetrapod integumentary skeleton by integrating comparative developmental and tissue structure data. Three types of tetrapod integumentary elements are recognized: (1) osteoderms, common to representatives of most major taxonomic lineages; (2) dermal scales, unique to gymnophionans; and (3) the lamina calcarea, an enigmatic tissue found only in some anurans. As presently understood, all are derivatives of the ancestral cosmoid scale and all originate from scleroblastic neural crest cells. Osteoderms are plesiomorphic for tetrapods but demonstrate considerable lineage-specific variability in size, shape, and tissue structure and composition. While metaplastic ossification often plays a role in osteoderm development, it is not the exclusive mode of skeletogenesis. All osteoderms share a common origin within the dermis (at or adjacent to the stratum superficiale) and are composed primarily (but not exclusively) of osseous tissue. These data support the notion that all osteoderms are derivatives of a neural crest-derived osteogenic cell population (with possible matrix contributions from the overlying epidermis) and share a deep homology associated with the skeletogenic competence of the dermis. Gymnophionan dermal scales are structurally similar to the elasmoid scales of most teleosts and are not comparable with osteoderms. Whereas details of development are lacking, it is hypothesized that dermal scales are derivatives of an odontogenic neural crest cell population and that skeletogenesis is comparable with the formation of elasmoid scales. Little is known about the lamina calcarea. It is proposed that this tissue layer is also odontogenic in origin, but clearly further study is necessary. Although not homologous as organs, all elements of the integumentary skeleton share a basic and essential relationship with the integument, connecting them with the ancestral rhombic scale.

The dermal skeleton (=exoskeleton) has long been recognized as a major determinant of vertebrate morphology. Until recently however, details of tissue development and diversity, particularly among amniotes, have been lacking. This investigation explores the development of the dermatocranium, gastralia, and osteoderms in the American alligator, Alligator mississippiensis. With the exception of osteoderms, elements of the dermal skeleton develop early during skeletogenesis, with most initiating ossification prior to mineralization of the endoskeleton. Characteristically, circumoral elements of the dermatocranium, including the pterygoid and dentigerous elements, are among the first to form. Unlike other axially arranged bones, gastralia develop in a caudolateral to craniomedial sequence. Osteoderms demonstrate a delayed onset of development compared with the rest of the skeleton, not appearing until well after hatching. Osteoderm development is asynchronous across the body, first forming dorsally adjacent to the cervical vertebrae; the majority of successive elements appear in caudal and lateral positions. Exclusive of osteoderms, the dermal skeleton initiates osteogenesis via intramembranous ossification. Following the establishment of skeletal condensations, some preossified spicules become engorged with many closely packed clusters of chondrocyte-like cells in a bone-like matrix. This combination of features is characteristic of chondroid bone, a tissue otherwise unreported among nonavian reptiles. No secondary cartilage was identified in any of the specimens examined. With continued growth, dermal bone (including chondroid bone) and osteoid are resorbed by multinucleated osteoclasts. However, there is no evidence that these cells contribute to the rugose pattern of bony ornamentation characteristic of the crocodylian dermatocranium. Instead, ornamentation develops as a result of localized concentrations of bone deposited by osteoblasts. Osteoderms develop in the absence of osteoblastic cells, osteoid, and periosteum; bone develops via the direct transformation of the preexisting dense irregular connective tissue. This mode of bone formation is identified as metaplasia. Importantly, it is also demonstrated that osteoderms are not histologically uniform but involve a range of tissues including calcified and uncalcified dense irregular connective tissue. Between taxa, not all osteoderms develop by homologous processes. However, it is concluded that all osteoderms may share a deep homology, connected by the structural and skeletogenic properties of the dermis., (Copyright (c) 2007 Wiley-Liss, Inc.)

Among modern mammals, armadillos (Xenarthra, Cingulata) are the only group that possesses osteoderms, bony inclusions within the integument. Along the body, osteoderms are organized into five discrete assemblages: the head, pectoral, banded, pelvic, and tail shields. The pectoral, banded, and pelvic shields articulate to form the carapace. We examined osteoderm skeletogenesis in the armadillo Dasypus novemcinctus using serial and whole-mount histochemistry. Compared with the rest of the skeleton, osteoderms have a delayed onset of development. Skeletogenesis begins as condensations of osteoblasts secreting osteoid, localized within the papillary layer of the dermis. Osteoderm formation is asynchronous both within each shield and across the body. The first osteoderms to mineralize are situated within the pectoral shield of the carapace, followed by elements within the banded, head, pelvic, and tail shields. In general, within each shield ossification begins craniomedially and proceeds caudally and laterally, except over the head, where the earliest elements form over the frontal and parietal bones. The absence of cartilage precursors indicates that osteoderms are dermal elements, possibly related to the all-encompassing vertebrate dermal skeleton (exoskeleton). The mode of development of D. novemcinctus osteoderms is unlike that described for squamate osteoderms, which arise via bone metaplasia, and instead is comparable with intramembranously derived elements of the skull., (Copyright 2006 Wiley-Liss, Inc.)

Metazoans are composed of a finite number of recognisable cell types. Similar to the relationship between species and ecosystems, knowledge of cell type diversity contributes to studies of complexity and evolution. However, as with other units of evolution, the cell type often resists definition. This review proposes guidelines for characterising cell types and discusses cell homology and the various developmental pathways by which cell types arise, including germ layers, blastemata (secondary development/neurulation), stem cells, and transdifferentiation. An updated list of cell types is presented for a familiar, albeit overlooked model taxon, adult Homo sapiens, with 411 cell types, including 145 types of neurons, recognised. Two methods for organising these cell types are explored. One is the artificial classification technique, clustering cells using commonly accepted criteria of similarity. The second approach, an empirical method modeled after cladistics, resolves the classification in terms of shared features rather than overall similarity. While the results of each scheme differ, both methods address important questions. The artificial classification provides compelling (and independent) support for the neural crest as the fourth germ layer, while the cladistic approach permits the evaluation of cell type evolution. Using the cladistic approach we observe a correlation between the developmental and evolutionary origin of a cell, suggesting that this method is useful for predicting which cell types share common (multipotential) progenitors. Whereas the current effort is restricted by the availability of phenotypic details for most cell types, the present study demonstrates that a comprehensive cladistic classification is practical, attainable, and warranted. The use of cell types and cell type comparative classification schemes has the potential to offer new and alternative models for therapeutic evaluation.

Although poorly appreciated, the vertebrate eye and adnexa are relatively common sites for skeletogenesis. In many taxa, the skeleton contributes to internal reinforcement in addition to the external housing of the eye (e.g., the circumorbital bones and eyelids). Eyeball elements such as scleral cartilage and scleral ossicles are present within a broad diversity of vertebrates, albeit not therian mammals, and have been used as important models for the study of condensations and epithelial-mesenchymal interactions. In contrast, other elements invested within the eye or its close surroundings remain largely unexplored. The onset and mode of development of these skeletal elements are often variable (early versus late; involving chondrogenesis, osteogenesis, or both), and most (if not all) of these elements appear to share a common neural crest origin. This review discusses the development and distribution of the skeletal elements within and associated with the developing eye and comments on homology of the elements where these are questionable., ((c) 2006 Wiley-Liss, Inc.)

As in monotreme mammals, the pectoral apparatus of basal (fossil) amniotes includes two coracoid elements, the procoracoid and metacoracoid. Among extant reptiles the metacoracoid has long been assumed lost; this notion is herein challenged. A comprehensive review of data from numerous sources, including the fossil record, experimental embryology, genetic manipulations and an analysis of morphology at the level cell condensations, supports the conclusion that the metacoracoid gives rise to the majority of the reptilian coracoid. By contrast, the reptilian procoracoid remains as a rudiment that is incorporated as a process of the (meta)coracoid and/or the glenoid region of the scapula early during development, prior to skeletogenesis. Application of this integrated approach corroborates and enhances previous work describing the evolution of the pectoral apparatus in mammals. A revised scenario of amniote coracoid evolution is presented emphasizing the importance of considering cell condensations when evaluating the homology of a skeletal complex.