Axonal regrowth is impaired during digit tip regeneration in mice

Mice are intrinsically capable of regenerating the tips of their digits after amputation. Mouse digit tip regeneration is reported to be a peripheral nerve-dependent event. However, it is presently unknown what types of nerves and Schwann cells innervate the digit tip, and to what extent these cells regenerate in association with the regenerative response. Given the necessity of peripheral nerves for mammalian regeneration, we investigated the neuroanatomy of the unamputated, regenerating, and regenerated mouse digit tip. Using immunohistochemistry for β-III-tubulin (β3T) or neurofilament H (NFH), substance P (SP), tyrosine hydroxylase (TH), myelin protein zero (P0), and glial fibrillary acidic protein (GFAP), we identified peripheral nerve axons (sensory and sympathetic), and myelinating- and non-myelinating-Schwann cells. Our findings show that the digit tip is innervated by two digital nerves that each bifurcate into a bone marrow (BM) and connective tissue (CT) branch. The BM branches are composed of sympathetic axons that are ensheathed by non-myelinating-Schwann cells whereas the CT branches are composed of sensory and sympathetic axons and are ensheathed by myelinating- and non-myelinating-Schwann cells. The regenerated digit neuroanatomy differs from unamputated digit in several key ways. First, there is 7.5 fold decrease in CT branch axons in the regenerated digit compared to the unampuated digit. Second, there is a 5.6 fold decrease in myelinating-Schwann cells in the regenerated digit compared to the unamputated digit that is consistent with the decrease in CT branch axons. Importantly, we also find that the central portion of the regenerating digit blastema is aneural, with axons and Schwann cells restricted to peripheral and distal blastema regions. Finally, we show that even with impaired innervation, digits maintain the ability to regenerate after re-amputation. Taken together, these data indicate that nerve regeneration is impaired in the context of mouse digit tip regeneration.     

Wound healing and blastema formation in regenerating digit tips of adult mice

Amputation of the distal region of the terminal phalanx of mice causes an initial wound healing response followed by blastema formation and the regeneration of the digit tip. Thus far, most regeneration studies have focused in embryonic or neonatal models and few studies have examined adult digit regeneration. Here we report on studies that include morphological, immunohistological, and volumetric analyses of adult digit regeneration stages. The regenerated digit is grossly similar to the original, but is not a perfect replacement. Re-differentiation of the digit tip occurs by intramembranous ossification forming a trabecular bone network that replaces the amputated cortical bone. The digit blastema is comprised of proliferating cells that express vimentin, a general mesenchymal marker, and by comparison to mature tissues, contains fewer endothelial cells indicative of reduced vascularity. The majority of blastemal cells expressing the stem cell marker SCA-1, also co-express the endothelial marker CD31, suggesting the presence of endothelial progenitor cells. Epidermal closure during wound healing is very slow and is characterized by a failure of the wound epidermis to close across amputated bone. Instead, the wound healing phase is associated with an osteoclast response that degrades the stump bone allowing the wound epidermis to undercut the distal bone resulting in a novel re-amputation response. Thus, the regeneration process initiates from a level that is proximal to the original plane of amputation.     

Hedgehog and Wnt coordinate signaling in myogenic progenitors and regulate limb regeneration

Amphibians have a remarkable capacity for limb regeneration. Following a severe injury, there is complete regeneration with restoration of the patterning and cellular architecture of the amputated limb. While studies have focused on the structural anatomical changes during amphibian limb regeneration, the signaling mechanisms that govern cellular dedifferentiation and blastemal progenitors are unknown. Here, we demonstrate the temporal and spatial requirement for hedgehog (Hh) signaling and its hierarchical correlation with respect to Wnt signaling during newt limb regeneration. While the dedifferentiation process of mature lineages does not depend on Hh signaling, the proliferation and the migration of the dedifferentiated cells are dependent on Hh signaling. Temporally controlled chemical inactivation of the Hh pathway indicates that Hh-mediated antero-posterior (AP) specification occurs early during limb regeneration and that Hh is subsequently required for expansion of the blastemal progenitors. Inhibition of Hh signaling results in G0/G1 arrest with a concomitant reduction in S-phase and G2/M population in myogenic progenitors. Furthermore, Hh inhibition leads to reduced Pax7-positive cells and fewer regenerating fibers relative to control tissue. We demonstrate that activation of Wnt signaling rescues the inhibition of Hh pathway mainly by enhancing proliferative signals, possibly mediated through TCF4 activity. Collectively, our results demonstrate coordinated signaling of Hh and Wnt activities in regulating blastemal progenitors and their hierarchical positioning during limb regeneration.     

Morphological,cellular and molecular characterization of posterior regeneration in the marine annelid Platynereis dumerilii

Regeneration, the ability to restore body parts after an injury or an amputation, is a widespread but highly variable and complex phenomenon in animals. While having fascinated scientists for centuries, fundamental questions about the cellular basis of animal regeneration as well as its evolutionary history remain largely unanswered. Here, we present a study of regeneration of the marine annelid Platynereis dumerilii, an emerging comparative developmental biology model, which, like many other annelids, displays important regenerative abilities. When P. dumerilii worms are amputated, they are able to regenerate the posteriormost differentiated part of their body and a stem cell-rich growth zone that allows the production of new segments replacing the amputated ones. We show that posterior regeneration is a rapid process that follows a well reproducible path and timeline, going through specific stages that we thoroughly defined. Wound healing is achieved one day after amputation and a regeneration blastema forms one day later. At this time point, some tissue specification already occurs, and a functional posterior growth zone is re-established as early as three days after amputation. Regeneration timing is only influenced, in a minor manner, by worm size. Comparable regenerative abilities are found for amputations performed at different positions along the antero-posterior axis of the worm, except when amputation planes are very close to the pharynx. Regenerative abilities persist upon repeated amputations without important alterations of the process. We also show that intense cell proliferation occurs during regeneration and that cell divisions are required for regeneration to proceed normally. Finally, 5-ethynyl-2’-deoxyuridine (EdU) pulse and chase experiments suggest that blastemal cells mostly derive from the segment immediately abutting the amputation plane. The detailed characterization of P. dumerilii posterior body regeneration presented in this article provides the foundation for future mechanistic and comparative studies of regeneration in this species.     

Dynamic gene expression profiles during arm regeneration in the brittle star Amphiura filiformis

Echinoderms and in particular brittle stars display a remarkable ability to regenerate lost or damaged tissues. They offer an excellent model in which to study regeneration displaying extensive regenerative ability and close relationship to vertebrates providing the opportunity for comparative studies. Previous studies of gene expression during arm regeneration in brittle stars have focused on single genes commonly associated with the regenerative process. In this study we present the first microarray investigation of gene expression during arm regeneration in the brittle star Amphiura filiformis. We show the large-scale gene expression changes associated with the complex process of regeneration with over 50% of the clones measured showing a significant change at some point during the process when compared to non-regenerating arms. Particular attention is paid to genes associated with Hox gene expression regulation, neuronal development and the bone morphogenic protein BMP-1. Our data give an insight into the molecular control required during the various stages of regeneration from the stem cell rich blastema stage through to the highly differentiated regenerate. This work also forms an important basis for future gene expression investigations in this emerging model of limb regeneration.     

Planarian regeneration involves distinct stem cell responses to wounds and tissue absence

Regeneration requires signaling from a wound site for detection of the wound and a mechanism that determines the nature of the injury to specify the appropriate regenerative response. Wound signals and tissue responses to wounds that elicit regeneration remain poorly understood. Planarians are able to regenerate from essentially any type of injury and present a novel system for the study of wound responses in regeneration initiation. Newly developed molecular and cellular tools now enable study of regeneration initiation using the planarian Schmidtea mediterranea. Planarian regeneration requires adult stem cells called neoblasts and amputation triggers two peaks in neoblast mitoses early in regeneration. We demonstrate that the first mitotic peak is a body-wide response to any injury and that a second, local, neoblast response is induced only when injury results in missing tissue. This second response was characterized by recruitment of neoblasts to wounds, even in areas that lack neoblasts in the intact animal. Subsequently, these neoblasts were induced to divide and differentiate near the wound, leading to formation of new tissue. We conclude that there exist two functionally distinct signaling phases of the stem cell wound response that distinguish between simple injury and situations that require the regeneration of missing tissue.     

Relative digit lengths and testosterone levels in Guinea baboons

A growing body of literature suggests that the ratio of the lengths of the second to fourth digits (2D:4D) on human hands is sexually dimorphic and associated with prenatal exposure to gonadal hormones, circulating serum testosterone, and a number of psychological and behavioral measures. Little research has investigated digit ratios in nonhuman species. In the present study, we investigated sex differences in digit ratios and their possible association with serum testosterone in a captive group of Guinea baboons (Papio papio). Contrary to the sex difference typically reported in humans, male baboons exhibited a substantially larger 2D:4D than did female baboons. Consistent with the human data, however, lower 2D:4D was associated with higher serum testosterone among the males. The present findings suggest that the relationship between digit ratios and male gonadal hormones may be phylogenetically well-conserved, although they also suggest possible species differences in the causal relationships between developmental mechanisms and sex-differentiated digit length patterns.     

Three genes control the timing, the site and the size of blastema formation in Drosophila

Regeneration is a vital process to maintain and repair tissues. Despite the importance of regeneration, the genes responsible for regenerative growth remain largely unknown. In Drosophila, imaginal disc regeneration can be induced either by fragmentation and in vivo culture or in situ by ubiquitous expression of wingless (wg/wnt1). Imaginal discs, like appendages in lower vertebrates, initiate regeneration by wound healing and proliferation at the wound site, forming a regeneration blastema. Most blastema cells maintain their disc-specific identity during regeneration; a few cells however, exhibit stem-cell like properties and switch to a different fate, in a phenomenon known as transdetermination. We identified three genes, regeneration (rgn), augmenter of liver regeneration (alr) and Matrix metalloproteinase-1 (Mmp1) expressed specifically in blastema cells during disc regeneration. Mutations in these genes affect both fragmentation- and wg-induced regeneration by either delaying, reducing or positioning the regeneration blastema. In addition to the modifications of blastema homeostasis, mutations in the three genes alter the rate of regeneration-induced transdetermination. We propose that these genes function in regenerative proliferation, growth and regulate cellular plasticity.     

Positional cloning of a temperature-sensitive mutant emmental reveals a role for sly1 during cell proliferation in zebrafish fin regeneration

Here, we used classical genetics in zebrafish to identify temperature-sensitive mutants in caudal fin regeneration. Gross morphological, histological, and molecular analyses revealed that one of these strains, emmental (emm), failed to form a functional regeneration blastema. Inhibition of emm function by heat treatment during regenerative outgrowth rapidly blocked regeneration. This block was associated with reduced proliferation in the proximal blastema and expansion of the nonproliferative distal blastemal zone. Positional cloning revealed that the emm phenotype is caused by a mutation in the orthologue of yeast sly1, a gene product involved in protein trafficking. sly1 is upregulated in the newly formed blastema as well as during regenerative outgrowth. Thus, sly1 is essential for blastemal organization and proliferation during two stages of fin regeneration.     

Role of RhoC in digit morphogenesis during limb development

Here we report a new role for the small GTPase RhoC in the control of limb chondrogenesis. Expression of rhoC is a precocious marker of the zeugopodial and digit blastemas and is induced by treatments with TGFbetas preceding the formation of ectopic digits. As development progresses, expression of rhoC outlines the growing distal tip of the digits, and marks the regions of interphalangeal joint formation. Functional experiments show that RhoC is a negative regulator of chondrogenesis, which controls digit outgrowth and joint segmentation. These functions appear to be mediated by reorganization of the actin cytoskeleton and modification of the adhesive properties of the mesenchymal cells.     

Development and regeneration of the retinotectal map in goldfish: a computational study

We present a simple computational model to study the interplay of activity-dependent and intrinsic processes thought to be involved in the formation of topographic neural projections. Our model consists of two input layers which project to one target layer. The connections between layers are described by a set of synaptic weights. These weights develop according to three interacting developmental rules: (i) an intrinsic fibre-target interaction which generates chemospecific adhesion between afferent fibres and target cells; (ii) an intrinsic fibre-fibre interaction which generates mutual selective adhesion between the afferent fibres; and (iii) an activity-dependent fibre-fibre interaction which implements Hebbian learning. Additionally, constraints are imposed to keep synaptic weights finite. The model is applied to a set of eleven experiments on the regeneration of the retinotectal projection in goldfish. We find that the model is able to reproduce the outcome of an unprecedented range of experiments with the same set of model parameters, including details of the size of receptive and projective fields. We expect this mathematical framework to be a useful tool for the analysis of developmental processes in general. <br>     

Dynamics of actinotrichia regeneration in the adult zebrafish fin

The skeleton of adult zebrafish fins comprises lepidotrichia, which are dermal bones of the rays, and actinotrichia, which are non-mineralized spicules at the distal margin of the appendage. Little is known about the regenerative dynamics of the actinotrichia-specific structural proteins called Actinodins. Here, we used immunofluorescence analysis to determine the contribution of two paralogous Actinodin proteins, And1/2, in regenerating fins. Both proteins were detected in the secretory organelles in the mesenchymal cells of the blastema, but only And1 was detected in the epithelial cells of the wound epithelium. The analysis of whole mount fins throughout the entire regenerative process and longitudinal sections revealed that And1-positive fibers are complementary to the lepidotrichia. The analysis of another longfin fish, a gain-of-function mutation in the potassium channel kcnk5b, revealed that the long-fin phenotype is associated with an extended size of actinotrichia during homeostasis and regeneration. Finally, we investigated the role of several signaling pathways in actinotrichia formation and maintenance. This revealed that the pulse-inhibition of either TGFβ/Activin-βA or FGF are sufficient to impair deposition of Actinodin during regeneration. Thus, the dynamic turnover of Actinodin during fin regeneration is regulated by multiple factors, including the osteoblasts, growth rate in a potassium channel mutant, and instructive signaling networks between the epithelium and the blastema of the regenerating fin.     

IHH and FGF8 coregulate elongation of digit primordia

In the developing limb bud, digit pattern arises from anterior-posterior (A-P) positional information which is provided by the concentration gradient of SHH. However, the mechanisms of translating early asymmetry into morphological form are still unclear. Here, we examined the ability of IHH and FGF8 signaling to regulate digital chondrogenesis, by implanting protein-loaded beads in the interdigital space singly and in combination. We found that IHH protein induced an elongated digit and that FGF8 protein blocked the terminal phalange formation. Molecular marker analysis showed that IHH expanded Sox9 expression in mesenchymal cells possibly through up-regulated FGF8 expression. Application of both IHH and FGF8 protein induced a large terminal phalange. These results suggest that both enhanced IHH and FGF8 signaling are required for the development of additional cartilage element in limbs. IHH and FGF8 maybe play different roles and act synergistically to promote chondrogenesis during digit primordia elongation.     

Retinoic acid-dependent attraction of adult spinal cord axons towards regenerating newt limb blastemas in vitro

Adult urodele amphibians possess the unique ability to regenerate amputated limbs and to re-innervate these regenerating structures; however, the factors involved in mediating this re-innervation are largely unknown. Here, we investigated the role of retinoic acid (RA) and one of its receptors, RARbeta, in the reciprocal neurotropic interactions between regenerating limb blastemas and spinal cord explants from the adult newt Notophthalmus viridescens. First, we showed that retinoic acid induced directed axonal outgrowth from cultured spinal cord tissue. This RA-induced outgrowth was significantly reduced when spinal cord explants were pre-treated with either the synthetic RAR pan antagonist, LE540, or the specific RARbeta antagonist, LE135. The role of RARbeta was also investigated using co-cultured regenerating limb blastemas and spinal cord explants. Blastemas induced significantly more axonal outgrowth from the near side of co-cultured explants, than from the far side (when cultured less than 1 mm apart). This blastema-induced directed outgrowth from co-cultured spinal cord explants was also abolished in the presence of the RARbeta antagonist, LE135. These data strongly suggest that endogenous retinoic acid is one of the tropic factors produced by the blastema and that it may be capable of guiding re-innervating axons to their targets. Moreover, this interaction is likely mediated by the retinoic acid beta nuclear receptor.     

Evidence for the reversibility of digit loss: a phylogenetic study of limb evolution in Bachia (Gymnophthalmidae: Squamata)

Reevolution of lost characters constitutes evidence that the capacity for producing specific phenotypes may remain latent after a trait is lost and be transmitted over many generations without visible effect. Although some evolutionary changes are easily reversible, it can be argued that the reappearance of complex characters would be nearly impossible. This idea is based on the assumption that, after a structure is lost, the genes related to its development will degenerate. In the present paper we test this idea with respect to digit loss in the gymnophthalmid genus Bachia. We present a molecular phylogeny of the genus Bachia and investigate the evolution of digit number in this taxon. Most members of this South American genus have undergone major reduction in hind limbs without ever losing all the digits in the forelimbs. We apply three statistical methods to test the hypothesis that trait loss is irreversible (Dollo's law). These are tree tests, parsimony-cost curves, and likelihood-ratio tests. Data is also analyzed under a simple probability model. All analyses provided strong evidence for reevolution of digit number in derived Bachia species. The evidence is stronger in toes (hind limb) than in fingers (forelimb). Other published examples of reevolution of complex traits are discussed in the light of the statistical approaches used in this paper. We conclude that there are a limited number of cases with strong evidence for the reevolution of lost morphological structures, raising questions about the mechanisms that retain the genetic information for a latent character.     

Development of regeneration protocols for selected cucurbit cultivars

A shoot regeneration protocol was developed for five cultivars of theCucurbitaceae. The influence of the seed coat, an ethanol pre-treatment,different seed sources, NaOCl concentrations and treatment times ondecontamination were investigated. The effects of combinations of BA, kinetin,iP and TDZ with IAA in the culture medium on shoot regeneration of cotyledonaryexplants were tested. The cultivars Cucurbita maxima cv.A-line, C. maxima cv. Chicago Warted and C.pepo cv. Rolet, did not form shoots on any of the treatments.However, somatic embryos formed on C. maxima cv. ChicagoWarted explants. Cucumis melo cv. Hales Best 36 was highlyregenerable and shoots formed on all the treatments containing cytokinin. Theshoot regeneration response was significantly higher on BA-containing media thanwith the other cytokinins tested. Although Cucumis sativuscv. Ashley responded poorly, shoot development was observed on media containingBA or iP.     

Failure of digit tip regeneration in the absence of Lmx1b suggests Lmx1b functions disparate from dorsoventral polarity

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Achieving stable myocardial regeneration after apical resection in neonatal mice

The neonatal heart completely regenerates after apical resection (AR), providing a desirable research model to study the mechanism of cardiac regeneration and cardiomyocyte proliferation. However, AR-induced neonatal heart regenerative phenomenon is controversial due to the variation of operative details in different laboratories. Here, we provide an optimized AR operation procedure with stable regeneration and high survival rate by achieving heart exposure, normalizing myocardium cut-offs, and reducing operation duration. We also established a whole-heart-slice approach to estimate the myocardial regeneration after the AR operation, which ensures no false-negative/positive results. The combination of the optimized AR operation and the whole-heart-slice analysis provides a stable system to study neonatal heart regeneration and cardiomyocyte proliferation in situ.