Serial block-face scanning electron microscopy of the tail tip of post-metamorphic amphioxus finds novel myomeres with odd shapes and unusually prominent sclerocoels.

Serial block-face scanning electron microscopy of the tail tip of post-metamorphic amphioxus (Branchiostoma floridae) revealed some terminal myomeres never been seen before with other techniques. The morphology of these myomeres differed markedly from the chevron shapes of their more anterior counterparts. Histologically, these odd-shaped myomeres ranged from empty vesicles bordered by undifferentiated cells to ventral sacs composed of well-developed myotome, dermatome, and sclerotome. Strikingly, several of these ventral sacs gave rise to a nipple-like dorsal projection composed either entirely of sclerotome or a mixture of sclerotome and myotome. Considered as a whole, from posterior to anterior, these odd-shaped posterior myomeres suggested that their more substantial ventral part may represent the ventral limb of a chevron, while the delicate projection represents a nascent dorsal limb. This scenario contrasts with formation of chevron-shaped myomeres along most of the antero-posterior axis. Although typical chevron formation in amphioxus is surprisingly poorly studied, it seems to be attained by a dorso-ventral extension of the myomere accompanied by the assumption of a V-shape; this is similar to what happens (at least superficially) in developing fishes. Another unusual feature of the odd-shaped posterior myomeres of amphioxus is their especially distended sclerocoels. One possible function for these might be to protect the posterior end of the central nervous system from trauma when the animals burrow into the substratum.

Wnt1 and Wnt2b immunodetection of the regenerating tail and comparative ultrastructure of tail spinal cord in the Scincella tsinlingensis / Inmunodetección Wnt1 y Wnt2b de la cola en regeneración y ultraestructura comparativa de la médula espinal de la cola en Scincella tsinlingensis

SUMMARY: The Wnt pathway is essential for the initiation of lizard tail regeneration. The regenerated lizard tails exhibit obvious morphological differences compared to the original ones. The expression of Wnt1 and Wnt2b proteins in the regenerating tail of Scincella tsinlingensis was detected by immunohistochemistry and then comparatively analyzed for ultrastructural changes in the original and regenerated spinal cord. The ependymal layer of the original spinal cord was pseudostratified with multiciliated cells and primary monociliated cells, while the cells of the ependymal layer of the regenerated spinal cord were organized in a monolayer with a few biciliated cells. Immunolocalization indicated that Wnt1 and Wnt2b were mainly distributed in the dermis near the original tail stump, spinal cord, and clot-positive migratory cells during Stage I, 0-1 days post-amputation (dpa). Wnt1 and Wnt2b were predominantly detected in the epaxial and hypaxial musculature near the original tail stump, wound epithelium, and spinal cord in the original tail during Stage II, 1-7 dpa. Mesenchymal cells and wound epithelium showed immunostaining during Stage III and IV, 7-15 dpa. The ependymal tubes contained these signaling proteins during Stage V and VI, 20- 30 dpa. Labeling was mainly observed in nearby regenerative blood vessels, ependymal cells, epaxial and hypaxial musculature in the apical epithelial layer (AEC) after 45-160 dpa. These findings indicated that Wnt1 and Wnt2b proteins presented primarily in regenerating epidermis and nerve tissues were a critical signal for tail regeneration in S. tsinlingensis.

RESUMEN: La vía Wnt es esencial para el inicio de la regeneración de la cola del lagarto. Las colas de lagarto regeneradas exhiben diferencias morfológicas obvias en comparación con las originales. La expresión de las proteínas Wnt1 y Wnt2b en la cola en regeneración de Scincella tsinlingensis se detectó mediante inmunohistoquímica y luego se analizaron comparativamente los cambios ultraestructurales en la médula espinal original y regenerada. La capa ependimaria de la médula espinal original se pseudoestratificó con células multiciliadas y células monociliadas primarias, mientras que las células de la capa ependimaria de la médula espinal regenerada se organizaron en monocapa con algunas células bicilicadas. La inmunolocalización indicó que Wnt1 y Wnt2b se distribuyeron principalmente en la dermis cerca del muñón de la cola original, la médula espinal y las células migratorias positivas en el coágulo durante la Etapa I, 0-1 días después de la amputación (dpa). Wnt1 y Wnt2b se detectaron predominantemente en la musculatura epaxial e hipaxial cerca del muñón de la cola original, el epitelio de la herida y la médula espinal en la cola original durante la Etapa II, 1-7 dpa. Las células mesenquimales y el epitelio de la herida mostraron inmunomarcaje durante la Etapa III y IV, 7- 15 dpa. Los tubos ependimarios contenían estas proteínas de señalización durante la Etapa V y VI, 20-30 dpa. El marcaje se observó principalmente en vasos sanguíneos regenerativos cercanos, células ependimarias, musculatura epaxial e hipaxial en la capa epitelial apical (AEC) después de 45-160 dpa. Estos hallazgos indicaron que las proteínas Wnt1 y Wnt2b están presentes principalmente en la epidermis en regeneración y en los tejidos nerviosos y eran una señal crítica para la regeneración de la cola en S. tsinlingensis.

Tail regeneration in a cephalochordate, the Bahamas lancelet, Asymmetron lucayanum.

Lancelets (Phylum Chordata, subphylum Cephalochordata) readily regenerate a lost tail. Here, we use light microscopy and serial blockface scanning electron microscopy (SBSEM) to describe tail replacement in the Bahamas lancelet, Asymmetron lucayanum. One day after amputation, the monolayered epidermis has migrated over the wound surface. At 4 days, the regenerate is about 3% as long as the tail length removed. The re-growing nerve cord is a tubular outgrowth of ependymal cells, and the new part of the notochord consists of several degenerating lamellar cells anterior to numerous small vacuolated cells. The cut edges of the mesothelium project into the regenerate as tubular extensions. These tubes anastomose with each other and with midline mesodermal canals beneath the regenerating edges of the dorsal and ventral fins. SBSEM did not reveal a blastema-like aggregation of undifferentiated cells anywhere in the regenerate. At 6 days, the regenerate (10% of the amputated tail length) includes a notochord in which the small vacuolated cells mentioned above are differentiating into lamellar cells. At 10 days, the regenerate is 22% of the amputated tail length: myocytes have appeared in the walls of the myomeres, and sclerocoels have formed. By 14 days, the regenerate is 35% the length of the amputated tail, and the new tissues resemble smaller versions of those originally lost. The present results for A. lucayanum, a species regenerating quickly and with little inter-specimen variability, provide the morphological background for future cell-tracer, molecular genetic, and genomic studies of cephalochordate regeneration.

Appendage regeneration in anamniotes utilizes genes active during larval-metamorphic stages that have been lost or altered in amniotes: The case for studying lizard tail regeneration.

This review elaborates the idea that organ regeneration derives from specific evolutionary histories of vertebrates. Regenerative ability depends on genomic regulation of genes specific to the life-cycles that have differentially evolved in anamniotes and amniotes. In aquatic environments, where fish and amphibians live, one or multiple metamorphic transitions occur before the adult stage is reached. Each transition involves the destruction and remodeling of larval organs that are replaced with adult organs. After organ injury or loss in adult anamniotes, regeneration uses similar genes and developmental process than those operating during larval growth and metamorphosis. Therefore, the broad presence of regenerative capability across anamniotes is possible because generating new organs is included in their life history at metamorphic stages. Soft hyaluronate-rich regenerative blastemas grow in submersed or in hydrated environments, that is, essential conditions for regeneration, like during development. In adult anamniotes, the ability to regenerate different organs decreases in comparison to larval stages and becomes limited during aging. Comparisons of genes activated during metamorphosis and regeneration in anamniotes identify key genes unique to these processes, and include thyroid, wnt and non-coding RNAs developmental pathways. In the terrestrial environment, some genes or developmental pathways for metamorphic transitions were lost during amniote evolution, determining loss of regeneration. Among amniotes, the formation of soft and hydrated blastemas only occurs in lizards, a morphogenetic process that evolved favoring their survival through tail autotomy, leading to a massive although imperfect regeneration of the tail. Deciphering genes activity during lizard tail regeneration would address future attempts to recreate in other amniotes regenerative blastemas that grow into variably completed organs.

The denticle surface of thresher shark tails: Three-dimensional structure and comparison to other pelagic species.

Shark skin denticles (scales) are diverse in morphology both among species and across the body of single individuals, although the function of this diversity is poorly understood. The extremely elongate and highly flexible tail of thresher sharks provides an opportunity to characterize gradients in denticle surface characteristics along the length of the tail and assess correlations between denticle morphology and tail kinematics. We measured denticle morphology on the caudal fin of three mature and two embryo common thresher sharks (Alopias vulpinus), and we compared thresher tail denticles to those of eleven other shark species. Using surface profilometry, we quantified 3D-denticle patterning and texture along the tail of threshers (27 regions in adults, and 16 regions in embryos). We report that tails of thresher embryos have a membrane that covers the denticles and reduces surface roughness. In mature thresher tails, surfaces have an average roughness of 5.6 µm which is smoother than some other pelagic shark species, but similar in roughness to blacktip, porbeagle, and bonnethead shark tails. There is no gradient down the tail in roughness for the middle or trailing edge regions and hence no correlation with kinematic amplitude or inferred magnitude of flow separation along the tail during locomotion. Along the length of the tail there is a leading-to-trailing-edge gradient with larger leading edge denticles that lack ridges (average roughness = 9.6 µm), and smaller trailing edge denticles with 5 ridges (average roughness = 5.7 µm). Thresher shark tails have many missing denticles visible as gaps in the surface, and we present evidence that these denticles are being replaced by new denticles that emerge from the skin below.

The sensory peripheral nervous system in the tail of a cephalochordate studied by serial blockface scanning electron microscopy.

Serial blockface scanning electron microscopy (SBSEM) is used to describe the sensory peripheral nervous system (PNS) in the tail of a cephalochordate, Asymmetron lucayanum. The reconstructed region extends from the tail tip to the origin of the most posterior peripheral nerves from the dorsal nerve cord. As peripheral nerves ramify within the dermis, all the nuclei along their course belong to glial cells. Invaginations in the glial cell cytoplasm house the neurites, an association reminiscent of the nonmyelinated Schwann cells of vertebrates. Peripheral nerves pass from the dermis to the epidermis via small fenestrae in the sub-epidermal collagen fibril layer; most nerves exit abruptly, but a few run obliquely within the collagen fibril layer for many micrometers before exiting. Within the epidermis, each nerve begins ramifying repeatedly, but the branches are too small to be followed to their tips with SBSEM at low magnification (previous studies on other cephalochordates indicate that the branches end freely or in association with epidermal sensory cells). In Asymmetron, two morphological kinds of sensory cells are scattered in the epidermis, usually singly, but sometimes in pairs, evidently the recent progeny of a single precursor cell. The discussion considers the evolution of the sensory PNS in the phylum Chordata. In cephalochordates, Retzius bipolar neurons with intramedullary perikarya likely correspond to the Rohon-Beard cells of vertebrates. However, extramedullary neurons originating from ventral epidermis in cephalochordates (and presumably in ancestral chordates) contrast with vertebrate sensory neurons, which arise from placodes and neural crest.

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

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

Ultrastructural analysis of early regenerating lizard tail suggests that a process of dedifferentiation is involved in the formation of the regenerative blastema.

The formation of the regenerating tail blastema of lizards occurs by the multiplication of stem cells but also some dedifferentiation from adult cells may take place after tail loss by autotomy, as it is suggested in the present study. Using 5BrdU-immunocytochemistry and transmission electron microscopy it is shown that part of the damaged tissues undergo progressive cytological de-differentiation (cell reprogramming). This occurs for muscle, fibrocytes, chondrocytes, adipocytes, and cells derived from the spinal cord during the initial 3-8 days post-autotomy of the tail in the wall lizard Podarcis muralis. Dedifferentiating cells loose most endoplasmic reticulum, sarcomeres in myocells, lipid droplets in adipocytes, extracellular matrix in chondrocytes. Numerous cytoplasmic vesicles are formed, perhaps reflecting an initial sufferance of dedifferentiating cells. These cells are not dying because they incorporate 5BrdU and proliferate. Nuclei of small fibrocytes present in the dermis and inter-muscle connective tissues, initially heterochromatic, become euchromatic and their cytoplasm increases in volume although the endoplasmic reticulum remains limited, as it is typical for mesenchymal cells. The present study, supported by previous transcriptome and 5BrdU-labeling data, and from recent tracing studies, suggests that aside stem cells present in different tissues of the tail, also cell dedifferentiation occurs in the injured tail of lizards. The relative contribution between de-differentiation and stem cells for the formation of the regenerating lizard blastema likely depends from the extension of the trauma.

Ultrastructural immunolocalization of hyaluronate in regenerating tail of lizards and amphibians supports an immune-suppressive role to favor regeneration.

Hyaluronate is produced in high amount during the initial stages of regeneration of the tail and limbs of lizards, newts, and frog tadpoles. The fine distribution of hyaluronate in the regenerating tail blastemas has been assessed by ultrastructural immunolocalization of the Hyaluronate Binding Protein (HABP), a protein that indirectly reveals the presence of hyaluronate in tissues. The present electron microscopic study shows that HABP is detected in the cytoplasm but this proteins is mainly localized on the surfaces of cells in the wound epidermis and mesenchymal cells of the blastema. HABP appears, therefore, accumulated along the cell surface, indicating that hyaluronate coats these embryonic-like cells and their antigens. The high level of hyaluronate in the blastema, aside favoring tissue hydration, cell movements, and remodeling for blastema formation and growth, likely elicits a protection from the possible immune-reaction of lymphocytes and macrophages to embryonic-fetal-like antigens present on the surface of blastema and epidermal cells. Their survival, therefore, allows the continuous multiplication of these cells in regions rich in hyaluronate, promoting the regeneration of a new tail or limbs. The study suggests that organ regeneration in vertebrates is only possible in the presence of high hyaluronate content and hydration. These two conditions facilitate cell movement, immune-protection, and activate the Wnt signaling pathway, like during development.

Neural stem/progenitor cells are activated during tail regeneration in the leopard gecko (Eublepharis macularius).

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.

Imaging the Nonlinear Susceptibility Tensor of Collagen by Nonlinear Optical Stokes Ellipsometry.

Nonlinear optical Stokes ellipsometric (NOSE) microscopy was demonstrated for the analysis of collagen-rich biological tissues. NOSE is based on polarization-dependent second harmonic generation imaging. NOSE was used to access the molecular-level distribution of collagen fibril orientation relative to the local fiber axis at every position within the field of view. Fibril tilt-angle distribution was investigated by combining the NOSE measurements with ab initio calculations of the predicted molecular nonlinear optical response of a single collagen triple helix. The results were compared with results obtained previously by scanning electron microscopy, nuclear magnetic resonance imaging, and electron tomography. These results were enabled by first measuring the laboratory-frame Jones nonlinear susceptibility tensor, then extending to the local-frame tensor through pixel-by-pixel corrections based on local orientation.

Open and closed evolutionary paths for drastic morphological changes, involving serial gene duplication, sub-functionalization, and selection.

Twin-tail goldfish strains are examples of drastic morphological alterations that emerged through domestication. Although this mutation is known to be caused by deficiency of one of two duplicated chordin genes, it is unknown why equivalent mutations have not been observed in other domesticated fish species. Here, we compared the chordin gene morphant phenotypes of single-tail goldfish and common carp (close relatives, both of which underwent chordin gene duplication and domestication). Morpholino-induced knockdown depleted chordin gene expression in both species; however, while knockdown reproduced twin-tail morphology in single-tail goldfish, it had no effect on common carp morphology. This difference can be explained by the observation that expression patterns of the duplicated chordin genes overlap completely in common carp, but are sub-functionalized in goldfish. Our finding implies that goldfish drastic morphological changes might be enhanced by the subsequent occurrence of three different types of evolutionary event (duplication, sub-functionalization, and selection) in a certain order.

A metameric origin for the annelid pygidium?

BACKGROUND: Segmented body organizations are widely represented in the animal kingdom. Whether the last common bilaterian ancestor was already segmented is intensely debated. Annelids display broad morphological diversity but many species are among the most homonomous metameric animals. The front end (prostomium) and tail piece (pygidium) of annelids are classically described as non-segmental. However, the pygidium structure and development remain poorly studied. RESULTS: Using different methods of microscopy, immunolabelling and a number of molecular markers, we describe the neural and mesodermal structures of the pygidium of Platynereis dumerilii. We establish that the pygidium possesses a complicated nervous system with a nerve ring and a pair of sensory ganglia, a complex intrinsic musculature, a large terminal circular blood sinus and an unusual unpaired torus-shaped coelomic cavity. We also describe some earlier steps of pygidial development and pygidial structure of mature animals after epitokous transformation. CONCLUSIONS: We describe a much more complex organization of the pygidium of P. dumerilii than previously suggested. Many of the characteristics are strikingly similar to those found in the trunk segments, opening the debate on whether the pygidium and trunk segments derive from the same ancestral metameric unit. We analyze these scenarios in the context of two classical theories on the origin of segmentation: the cyclomeric/archicoelomate concept and the colonial theory. Both theories provide possible explanations for the partial or complete homology of trunk segments and pygidium.

Structural origin of the drastic modification of second harmonic generation intensity pattern occurring in tail muscles of climax stages xenopus tadpoles.

Second harmonic generation (SHG) microscopy is a powerful tool for studying submicron architecture of muscles tissues. Using this technique, we show that the canonical single frequency sarcomeric SHG intensity pattern (SHG-IP) of premetamorphic xenopus tadpole tail muscles is converted to double frequency (2f) sarcomeric SHG-IP in metamorphic climax stages due to massive physiological muscle proteolysis. This conversion was found to rise from 7% in premetamorphic muscles to about 97% in fragmented muscular apoptotic bodies. Moreover a 66% conversion was also found in non-fragmented metamorphic tail muscles. Also, a strong correlation between predominant 2f sarcomeric SHG-IPs and myofibrillar misalignment is established with electron microscopy. Experimental and theoretical results demonstrate the higher sensitivity and the supra resolution power of SHG microscopy over TPEF to reveal 3D myofibrillar misalignment. From this study, we suggest that 2f sarcomeric SHG-IP could be used as signature of triad defect and disruption of excitation-contraction coupling. As the mechanism of muscle proteolysis is similar to that found in mdx mouse muscles, we further suggest that xenopus tadpole tail resorption at climax stages could be used as an alternative or complementary model of Duchene muscular dystrophy.

Ultrastructural immunolocalization of chatelicidin-like peptides in granulocytes of normal and regenerating lizard tissues.

The presence and localization of cathelicidin anti-microbial peptides in the lizard, Anolis carolinensis, were investigated by immunocytochemistry. The study showed that immunoreactivity for cathelicidins 1 and 2 was only present in large granules of heterophilic-basophilic granulocytes, rarely found in the dermis and sub-dermal muscle in normal and more frequently in wound and regenerating skin tissues or in the blood. Some immunopositive granulocytes were also observed among the keratinocytes of the wound epithelium covering the tail stump and occasionally in the regenerating epidermis of the tail. Immunolabeling for cathelicidins was also seen in low electrondense amorphous material present on the surface of the wound epidermis and on the plasma membrane of bacteria present on the surface of corneocytes of the epidermis. Immunolabeling for cathelicidins was absent in the other cell types and in control sections. The study suggests that cathelicidins are normally stored in granulocytes in the blood or in connective tissues, while keratinocytes can be stimulated to produce and possibly release these molecules only after injury or microbial invasion.

A histological comparison of the original and regenerated tail in the green anole, Anolis carolinensis.

This study provides a histological comparison of the mature regenerated and original tail of the lizard Anolis carolinensis. These data will provide a framework for future studies of this emerging model organism whose genome was recently published. This study demonstrated that the cartilage skeleton of the regenerated tail enclosed a spinal cord with an ependymal core, but there was no evidence that dorsal root ganglia or peripheral nerves are regenerated. The cartilage tube contained foramina that allowed the vasculature to cross, but was otherwise a rigid structure. The original tail has muscle groups arranged in quadrants in a regular pattern that attach to the vertebral column. The regenerated tail has irregular muscle bundles of variable number that form unusual attachments to each other and to the cartilage tube. Furthermore, the data show that there was increased connective tissue within the muscle bundles. Implications for functionality of the regenerated tail and for future biomechanical studies are discussed.

Scar-free wound healing and regeneration following tail loss in the leopard gecko, Eublepharis macularius.

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-ß3 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.

Elastic and viscoelastic properties of a type I collagen fiber.

A new mathematical model is presented to describe the elastic and viscoelastic properties of a single collagen fiber. The model is formulated by accounting for the mechanical contribution of the collagen fiber's main constituents: the microfibrils, the interfibrillar matrix and crosslinks. The collagen fiber is modeled as a linear elastic spring, which represents the mechanical contribution of the microfibrils, and an arrangement in parallel of elastic springs and viscous dashpots, which represent the mechanical contributions of the crosslinks and interfibrillar matrix, respectively. The linear elastic spring and the arrangement in parallel of elastic springs and viscous dashpots are then connected in series. The crosslinks are assumed to gradually break under strain and, consequently, the interfibrillar is assumed to change its viscous properties. Incremental stress relaxation tests are conducted on dry collagen fibers reconstituted from rat tail tendons to determine their elastic and viscoelastic properties. The elastic and total stress-strain curves and the stress relaxation at different levels of strain collected by performing these tests are then used to estimate the parameters of the model and evaluate its predictive capabilities.

Cell culture from lizard skin: a tool for the study of epidermal differentiation.

An in vitro system of isolated skin cells has been developed in order to address the understanding on the factors that control the shedding cycle and differentiation of lizard epidermis. The skin from the regenerating lizard tail has been separated in epidermis and dermis, cells have been dissociated, cultivated in vitro, and studied ultrastructurally after 1-30 days of culture condition. Dissociated keratinocytes after 12 days in culture show numerous cell elongations and contain bundles of keratin or sparse keratin filaments. These cells often contain one to three 0.5-3 µm large and dense "keratinaceous bodies", an organelle representing tonofilament disassembling. Most keratinocytes have sparse tonofilaments in the cytoplasm and form shorter bundles of keratin in the cell periphery. The dissociated dermis mainly consists of mesenchymal cells containing sparse bundles of intermediate filaments. These cells proliferate and form multi-stratified layers and a dermal pellicle in about 2-3 weeks in vitro in our basic medium. Conversely, cultures of keratinocytes do not expand but eventually reduce to few viable cells within 2-3 weeks of in vitro condition. It is suggested that dermal cells sustain themselves through the production of growth factors but that epidermal cells requires specific growth factors still to be identified before setting-up an in vitro system that allows analyzing the control of the shedding cycle in lizards.

Monitoring micrometer-scale collagen organization in rat-tail tendon upon mechanical strain using second harmonic microscopy.

We continuously monitored the microstructure of a rat-tail tendon during stretch/relaxation cycles. To that purpose, we implemented a new biomechanical device that combined SHG imaging and mechanical testing modalities. This multi-scale experimental device enabled simultaneous visualization of the collagen crimp morphology at the micrometer scale and measurement of macroscopic strain-stress response. We gradually increased the ultimate strain of the cycles and showed that preconditioning mostly occurs in the first stretching. This is accompanied by an increase of the crimp period in the SHG image. Our results indicate that preconditioning is due to a sliding of microstructures at the scale of a few fibrils and smaller, that changes the resting length of the fascicle. This sliding can reverse on long time scales. These results provide a proof of concept that continuous SHG imaging performed simultaneously with mechanical assay allows analysis of the relationship between macroscopic response and microscopic structure of tissues.