A transgenic reporter under control of an es1 promoter/enhancer marks wound epidermis and apical epithelial cap during tail regeneration in Xenopus laevis tadpole

Rapid wound healing and subsequent formation of the apical epithelial cap (AEC) are believed to be required for successful appendage regeneration in amphibians. Despite the significant role of AEC in limb regeneration, its role in tail regeneration and the mechanisms that regulate the wound healing and AEC formation are not well understood. We previously identified Xenopus laevis es1, which is preferentially expressed in wounded regions, including the AEC after tail regeneration. In this study we established and characterized transgenic Xenopus laevis lines harboring the enhanced green fluorescent protein (EGFP) gene under control of an es1 gene regulatory sequence (es1:egfp).The EGFP reporter expression was clearly seen in several regions of the embryo and then declined to an undetectable level in larvae, recapitulating the endogenous es1 expression. After amputation of the tadpole tail, EGFP expression was re-activated at the edge of the stump epidermis and then increased in the wound epidermis (WE) covering the amputation surface. As the stump started to regenerate, the EGFP expression became restricted to the most distal epidermal region, including the AEC. EGFP was preferentially expressed in the basal or deep cells but not in the superficial cells of the WE and AEC.We performed a small-scale pharmacological screening for chemicals that affected the expression of EGFP in the stump epidermis after tail amputation. The EGFP expression was attenuated by treatment with an inhibitor for ERK, TGF-β or reactive oxygen species (ROS) signaling. These treatments also impaired wound closure of the amputation surface, suggesting that the three signaling activities are required for es1 expression in the WE and successful wound healing after tail amputation.These findings showed that es1:egfp Xenopus laevis should be a useful tool to analyze molecular mechanisms regulating wound healing and appendage regeneration.     

Transforming growth factor-β signaling confers hepatic stellate cells progenitor features after partial hepatectomy

Liver regeneration involves not only hepatocyte replication but progenitor aggregation and scarring. Partial hepatectomy (PH), an established model for liver regeneration, reactivates transforming growth factor-β (TGF-β) signaling. Hepatic stellate cells (HSCs) are primarily responding cells for TGF-β and resident in stem cell niche. In the current study, PH mice were treated with SB-431542, an inhibitor of TGF-β Type I receptor, aiming to address the role of TGF-β signaling on the fate determination of HSCs during liver regeneration. After PH, control mice exhibited HSCs activation, progenitor cells accumulation, and a fraction of HSCs acquired the phenotype of hepatocyte or cholangiocyte. Blocking TGF-β signaling delayed proliferation, impaired progenitor response, and scarring repair. In SB-431542 group, merely no HSCs were found coexpressed progenitor makers, such as SOX9 and AFP. Inhibition of TGF-β pathway disturbed the epithelial-mesenchymal transitions and diminished the nuclear accumulation of β-catenin as well as the expression of cytochrome P450 2E1 in HSC during liver regeneration. We identify a key role of TGF-β signaling on promoting HSC transition, which subsequently becomes progenitor for generating liver epithelial cells after PH. This process might interact with an acknowledged stem cell function signaling, Wnt/β-catenin.     

Neurotrophic regulation of fibroblast dedifferentiation during limb skeletal regeneration in the axolotl (Ambystoma mexicanum)

The ability of animals to repair tissue damage is widespread and impressive. Among tissues, the repair and remodeling of bone occurs during growth and in response to injury; however, loss of bone above a threshold amount is not regenerated, resulting in a “critical-size defect” (CSD). The development of therapies to replace or regenerate a CSD is a major focus of research in regenerative medicine and tissue engineering. Adult urodeles (salamanders) are unique in their ability to regenerate complex tissues perfectly, yet like mammals do not regenerate a CSD. We report on an experimental model for the regeneration of a CSD in the axolotl (the Excisional Regeneration Model) that allows for the identification of signals to induce fibroblast dedifferentiation and skeletal regeneration. This regenerative response is mediated in part by BMP signaling, as is the case in mammals; however, a complete regenerative response requires the induction of a population of undifferentiated, regeneration-competent cells. These cells can be induced by signaling from limb amputation to generate blastema cells that can be grafted to the wound, as well as by signaling from a nerve and a wound epithelium to induce blastema cells from fibroblasts within the wound environment.     

Plasticity of photoreceptor-generating retinal progenitors revealed by prolonged retinoic acid exposure

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.     

Apoptosis is required during early stages of tail regeneration in Xenopus laevis

The Xenopus tadpole is able to regenerate its tail, including skin, muscle, notochord, spinal cord and neurons and blood vessels. This process requires rapid tissue growth and morphogenesis. Here we show that a focus of apoptotic cells appears in the regeneration bud within 12 h of amputation. Surprisingly, when caspase-3 activity is specifically inhibited, regeneration is abolished. This is true of tails both before and after the refractory period. Programmed cell death is only required during the first 24 h after amputation, as later inhibition has no effect on regeneration. Inhibition of caspase-dependent apoptosis results in a failure to induce proliferation in the growth zone, a mispatterning of axons in the regenerate, and the appearance of ectopic otoliths in the neural tube, in the context of otherwise normal continued development of the larva. Larvae amputated during the refractory stage exhibit a much broader domain of caspase-3-positive cells, suggesting a window for the amount of apoptosis that is compatible with normal regeneration. These data reveal novel roles for apoptosis in development and indicate that a degree of apoptosis is an early and obligate component of normal tail regeneration, suggesting the possibility of the existence of endogenous inhibitory cells that must be destroyed by programmed cell death for regeneration to occur.     

Initiation of limb regeneration: the critical steps for regenerative capacity

While urodele amphibians (newts and salamanders) can regenerate limbs as adults, other tetrapods (reptiles, birds and mammals) cannot and just undergo wound healing. In adult mammals such as mice and humans, the wound heals and a scar is formed after injury, while wound healing is completed without scarring in an embryonic mouse. Completion of regeneration and wound healing takes a long time in regenerative and non-regenerative limbs, respectively. However, it is the early steps that are critical for determining the extent of regenerative response after limb amputation, ranging from wound healing with scar formation, scar-free wound healing, hypomorphic limb regeneration to complete limb regeneration. In addition to the accumulation of information on gene expression during limb regeneration, functional analysis of signaling molecules has recently shown important roles of fibroblast growth factor (FGF), Wnt/beta-catenin and bone morphogenic protein (BMP)/Msx signaling. Here, the routine steps of wound healing/limb regeneration and signaling molecules specifically involved in limb regeneration are summarized. Regeneration of embryonic mouse digit tips and anuran amphibian (Xenopus) limbs shows intermediate regenerative responses between the two extremes, those of adult mammals (least regenerative) and urodele amphibians (more regenerative), providing a range of models to study the various abilities of limbs to regenerate.     

The regulation of TGF-β/SMAD signaling by protein deubiquitination

Transforming growth factor-β (TGF-β) members are key cytokines that control embryogenesis and tissue homeostasis via transmembrane TGF-β type II (TβR II) and type I (TβRI) and serine/threonine kinases receptors. Aberrant activation of TGF-β signaling leads to diseases, including cancer. In advanced cancer, the TGF-β/SMAD pathway can act as an oncogenic factor driving tumor cell invasion and metastasis, and thus is considered to be a therapeutic target. The activity of TGF-β/SMAD pathway is known to be regulated by ubiquitination at multiple levels. As ubiquitination is reversible, emerging studies have uncovered key roles for ubiquitin-removals on TGF-β signaling components by deubiquitinating enzymes (DUBs). In this paper, we summarize the latest findings on the DUBs that control the activity of the TGF-β signaling pathway. The regulatory roles of these DUBs as a driving force for cancer progression as well as their underlying working mechanisms are also discussed.     

SDF-1α/CXCR4 signaling mediates digit tip regeneration promoted by BMP-2

Previously we demonstrated that BMP signaling is required for endogenous digit tip regeneration, and that treatment with BMP-2 or -7 induces a regenerative response following amputation at regeneration-incompetent levels (Yu et al., 2010 and Yu et al., 2012). Both endogenous regeneration and BMP-induced regeneration are associated with the transient formation of a blastema, however the formation of a regeneration blastema in mammals is poorly understood. In this study, we focus on how blastema cells respond to BMP signaling during neonatal digit regeneration in mice. First, we show that blastema cells retain regenerative properties after expansion in vitro, and when re-introduced into the amputated digit, these cells display directed migration in response to BMP-2. However, in vitro studies demonstrate that BMP-2 alone does not influence blastema cell migration, suggesting a requirement of another pivotal downstream factor for cell recruitment. We show that blastema cell migration is stimulated by the cytokine, SDF-1α, and that SDF-1α is expressed by the wound epidermis as well as endothelial cells of the blastema. Blastema cells express both SDF-1α receptors, CXCR4 and CXCR7, although the migration response is inhibited by the CXCR4-specific antagonist, AMD3100. Mice treated with AMD3100 display a partial inhibition of skeletal regrowth associated with the regeneration response. We provide evidence that BMP-2 regulates Sdf-1α expression in endothelial cells but not cells of the wound epidermis. Finally, we show that SDF-1α-expressing COS1 cells engrafted into a regeneration-incompetent digit amputation wound resulted in a locally enhanced population of CXCR4 positive cells, and induced a partial regenerative response. Taken together, this study provides evidence that one downstream mechanism of BMP signaling during mammalian digit regeneration involves activation of SDF-1α/CXCR4 signaling by endothelial cells to recruit blastema cells.     

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.     

Cellular and molecular mechanisms of regeneration in Xenopus

We have employed transgenic methods combined with embryonic grafting to analyse the mechanisms of regeneration in Xenopus tadpoles. The Xenopus tadpole tail contains a spinal cord, notochord and segmented muscles, and all tissues are replaced when the tail regenerates after amputation. We show that there is a refractory period of very low regenerative ability in the early tadpole stage. Tracing of cell lineage with the use of single tissue transgenic grafts labelled with green fluorescent protein (GFP) shows that there is no de-differentiation and no metaplasia during regeneration. The spinal cord, notochord and muscle all regenerate from the corresponding tissue in the stump; in the case of the muscle the satellite cells provide the material for regeneration. By using constitutive or dominant negative gene products, induced under the control of a heat shock promoter, we show that the bone morphogenetic protein (BMP) and Notch signalling pathways are both essential for regeneration. BMP is upstream of Notch and has an independent effect on regeneration of muscle. The Xenopus limb bud will regenerate completely at the early stages but regenerative ability falls during digit differentiation. We have developed a procedure for making tadpoles in which one hindlimb is transgenic and the remainder wild-type. This has been used to introduce various gene products expected to prolong the period of regenerative capacity, but none has so far been successful.     

The regenerative capacity of the zebrafish heart is dependent on TGFβ signaling

Mammals respond to a myocardial infarction by irreversible scar formation. By contrast, zebrafish are able to resolve the scar and to regenerate functional cardiac muscle. It is not known how opposing cellular responses of fibrosis and new myocardium formation are spatially and temporally coordinated during heart regeneration in zebrafish. Here, we report that the balance between the reparative and regenerative processes is achieved through Smad3-dependent TGFβ signaling. The type I receptor alk5b (tgfbr1b) is expressed in both fibrotic and cardiac cells of the injured heart. TGFβ ligands are locally induced following cryoinjury and activate the signaling pathway both in the infarct area and in cardiomyocytes in the vicinity of the trauma zone. Inhibition of the relevant type I receptors with the specific chemical inhibitor SB431542 qualitatively altered the infarct tissue and completely abolished heart regeneration. We show that transient scar formation is an essential step to maintain robustness of the damaged ventricular wall prior to cardiomyocyte replacement. Taking advantage of the reversible action of the inhibitor, we dissected the multifunctional role of TGFβ signaling into three crucial processes: collagen-rich scar deposition, Tenascin C-associated tissue remodeling at the infarct-myocardium interface, and cardiomyocyte proliferation. Thus, TGFβ signaling orchestrates the beneficial interplay between scar-based repair and cardiomyocyte-based regeneration to achieve complete heart regeneration.     

Wnt3a induces myofibroblast differentiation by upregulating TGF-β signaling through SMAD2 in a β-catenin-dependent manner

Growing evidence suggests the Wnt family of secreted glycoproteins and their associated signaling pathways, linked to development, are recapitulated during wound repair and regeneration events. However, the role of the Wnt pathway in such settings remains unclear. In the current study, we treated mouse fibroblasts with 250 ng/mL of recombinant Wnt3a for 72 hours and examined its affect on cell morphology and function. Wnt3a induced a spindle-like morphology in fibroblasts characterized by the increased formation of stress fibres. Wnt3a decreased the proliferation of fibroblasts, but significantly increased cell migration as well as fibroblast-mediated contraction of a collagen lattice. Wnt3a significantly increased the expression of TGF-β and its associated signaling through SMAD2. Consistent with this, we observed significantly increased smooth muscle α-actin expression and incorporation of this contractile protein into stress fibres following Wnt3a treatment. Knockdown of β-catenin using siRNA reversed the Wnt3a-induced smooth muscle α-actin expression, suggesting these changes were dependent on canonical Wnt signaling through β-catenin. Neutralization of TGF-β with a blocking antibody significantly inhibited the Wnt3a-induced smooth muscle α-actin expression, indicating these changes were dependent on the increased TGF-β signaling. Collectively, this data strongly suggests Wnt3a promotes the formation of a myofibroblast-like phenotype in cultured fibroblasts, in part, by upregulating TGF-β signaling through SMAD2 in a β-catenin-dependent mechanism. As myofibroblasts are critical regulators of wound healing responses, these findings may have important implications for our understanding of normal and aberrant injury and repair events.