Heterogeneous fates and dynamic rearrangement of regenerative epidermis-derived cells during zebrafish fin regeneration.

The regenerative epidermis (RE) is a specialized tissue that plays an essential role in tissue regeneration. However, the fate of the RE during and after regeneration is unknown. In this study, we performed Cre-loxP-mediated cell fate tracking and revealed the fates of a major population of the RE cells that express fibronectin 1b (fn1b) during zebrafish fin regeneration. Our study showed that these RE cells are mainly recruited from the inter-ray epidermis, and that they follow heterogeneous cell fates. Early recruited cells contribute to initial wound healing and soon disappear by apoptosis, while the later recruited cells contribute to the regenerated epidermis. Intriguingly, many of these cells are also expelled from the regenerated tissue by a dynamic caudal movement of the epidermis over time, and in turn the loss of epidermal cells is replenished by a global self-replication of basal and suprabasal cells in fin. De-differentiation of non-basal epidermal cells into the basal epidermal cells did not occur during regeneration. Overall, our study reveals the heterogeneous fates of RE cells and a dynamic rearrangement of the epidermis during and after regeneration.

Nerve-mediated expression of histone deacetylases regulates limb regeneration in axolotls.

Axolotls have amazing abilities to regenerate their lost limbs. Nerve and wound epidermis have great impacts on this regeneration. Histone deacetylases (HDACs) have been shown to play roles in the regeneration of amphibian tails and limbs. In this study, a bi-phasic up-regulation of HDAC1 was noted before early differentiation stage of axolotl limb regeneration. Limb regeneration was delayed in larvae incubated with an HDAC inhibitor MS-275. Local injection of MS-275 or TSA, another HDAC inhibitor, into amputation sites of the juveniles did not interfere with wound healing but more profoundly inhibited local HDAC activities and blastema formation/limb regeneration. Elevation of HDAC1 expression was more apparent in wound epidermis than in mesenchyme. Prior denervation prohibited this elevation and limb regeneration. Supplementation of nerve factors BMP7, FGF2, and FGF8 in the stump ends after amputation on denervated limbs not only enabled HDAC1 up-regulation but also led to more extent of limb regeneration. In conclusion, nerve-mediated HDAC1 expression is required for blastema formation and limb regeneration.

Regeneration in the ctenophore Mnemiopsis leidyi occurs in the absence of a blastema, requires cell division, and is temporally separable from wound healing.

BACKGROUND: The ability to regenerate is a widely distributed but highly variable trait among metazoans. A variety of modes of regeneration has been described for different organisms; however, many questions regarding the origin and evolution of these strategies remain unanswered. Most species of ctenophore (or "comb jellies"), a clade of marine animals that branch off at the base of the animal tree of life, possess an outstanding capacity to regenerate. However, the cellular and molecular mechanisms underlying this ability are unknown. We have used the ctenophore Mnemiopsis leidyi as a system to study wound healing and adult regeneration and provide some first-time insights of the cellular mechanisms involved in the regeneration of one of the most ancient extant group of multicellular animals. RESULTS: We show that cell proliferation is activated at the wound site and is indispensable for whole-body regeneration. Wound healing occurs normally in the absence of cell proliferation forming a scar-less wound epithelium. No blastema-like structure is generated at the cut site, and pulse-chase experiments and surgical intervention show that cells originating in the main regions of cell proliferation (the tentacle bulbs) do not seem to contribute to the formation of new structures after surgical challenge, suggesting a local source of cells during regeneration. While exposure to cell-proliferation blocking treatment inhibits regeneration, the ability to regenerate is recovered when the treatment ends (days after the original cut), suggesting that ctenophore regenerative capabilities are constantly ready to be triggered and they are somehow separable of the wound healing process. CONCLUSIONS: Ctenophore regeneration takes place through a process of cell proliferation-dependent non-blastemal-like regeneration and is temporally separable of the wound healing process. We propose that undifferentiated cells assume the correct location of missing structures and differentiate in place. The remarkable ability to replace missing tissue, the many favorable experimental features (e.g., optical clarity, high fecundity, rapid regenerative performance, stereotyped cell lineage, sequenced genome), and the early branching phylogenetic position in the animal tree, all point to the emergence of ctenophores as a new model system to study the evolution of animal regeneration.

Fgf signalling controls diverse aspects of fin regeneration.

Studies have shown that fibroblast growth factor (Fgf) signalling is necessary for appendage regeneration, but its exact function and the ligands involved during regeneration have not yet been elucidated. Here, we performed comprehensive expression analyses and identified fgf20a and fgf3/10a as major Fgf ligands in the wound epidermis and blastema, respectively. To reveal the target cells and processes of Fgf signalling, we performed a transplantation experiment of mesenchymal cells that express the dominant-negative Fgf receptor 1 (dnfgfr1) under control of the heat-shock promoter. This mosaic knockdown analysis suggested that Fgf signalling is directly required for fin ray mesenchyme to form the blastema at the early pre-blastema stage and to activate the regenerative cell proliferation at a later post-blastema stage. These results raised the possibility that the early epidermal Fgf20a and the later blastemal Fgf3/10a could be responsible for these respective processes. We demonstrated by gain-of-function analyses that Fgf20a induces the expression of distal blastema marker junbl, and that Fgf3 promotes blastema cell proliferation. Our study highlights that Fgfs in the wound epidermis and blastema have distinct functions to regulate fin regeneration cooperatively.

Tissue regeneration during lower jaw restoration in zebrafish shows some features of epimorphic regeneration.

Zebrafish have the ability to regenerate skeletal structures, including the fin, skull roof, and jaw. Although fin regeneration proceeds by epimorphic regeneration, it remains unclear whether this process is involved in other skeletal regeneration in zebrafish. Initially in epimorphic regeneration, the wound epidermis covers the wound surface. Subsequently, the blastema, an undifferentiated mesenchymal mass, forms beneath the epidermis. In the present study, we re-examined the regeneration of the zebrafish lower jaw in detail, and investigated whether epimorphic regeneration is involved in this process. We performed amputation of the lower jaw at two different positions; the proximal level (presence of Meckel's cartilage) and the distal level (absence of Meckel's cartilage). In both manipulations, a blastema-like cellular mass was initially formed. Subsequently, cartilaginous aggregates were formed in this mass. In the proximal amputation, the cartilaginous aggregates were then fused with Meckel's cartilage and remained as a skeletal component of the regenerated jaw, whereas in the distal amputation, the cartilaginous aggregates disappeared as regeneration progressed. Two molecules that were observed during epimorphic regeneration, Laminin and msxb, were expressed in the regenerating lower jaw, although the domain of msxb expression was out of the main plain of the aggregate formation. Administration of an inhibitor of Wnt/ß-catenin signaling, a pathway associated with epimorphic regeneration, showed few effects on lower jaw regeneration. Our finding suggests that skeletal regeneration of the lower jaw mainly progresses through tissue regeneration that is dependent on the position in the jaw, and epimorphic regeneration plays an adjunctive role in this regeneration.

Cell type-specific transcriptome analysis unveils secreted signaling molecule genes expressed in apical epithelial cap during appendage regeneration.

Wound epidermis (WE) and the apical epithelial cap (AEC) are believed to trigger regeneration of amputated appendages such as limb and tail in amphibians by producing certain secreted signaling molecules. To date, however, only limited information about the molecular signatures of these epidermal structures is available. Here we used a transgenic Xenopus laevis line harboring the enhanced green fluorescent protein (egfp) gene under control of an es1 gene regulatory sequence to isolate WE/AEC cells by performing fluorescence-activated cell sorting during the time course of tail regeneration (day 1, day 2, day 3 and day 4 after amputation). Time-course transcriptome analysis of these isolated WE/AEC cells revealed that more than 8,000 genes, including genes involved in signaling pathways such as those of reactive oxygen species, fibroblast growth factor (FGF), canonical and non-canonical Wnt, transforming growth factor ß (TGF ß) and Notch, displayed dynamic changes of their expression during tail regeneration. Notably, this approach enabled us to newly identify seven secreted signaling molecule genes (mdk, fstl, slit1, tgfß1, bmp7.1, angptl2 and egfl6) that are highly expressed in tail AEC cells. Among these genes, five (mdk, fstl, slit1, tgfß1 and bmp7.1) were also highly expressed in limb AEC cells but the other two (angptl2 and egfl6) are specifically expressed in tail AEC cells. Interestingly, there was no expression of fgf8 in tail WE/AEC cells, whose expression and pivotal role in limb AEC cells have been reported previously. Thus, we identified common and different properties between tail and limb AEC cells.

Regeneration versus scarring in vertebrate appendages and heart.

Injuries to complex human organs, such as the limbs and the heart, result in pathological conditions, for which we often lack adequate treatments. While modern regenerative approaches are based on the transplantation of stem cell-derived cells, natural regeneration in lower vertebrates, such as zebrafish and newts, relies predominantly on the intrinsic plasticity of mature tissues. This property involves local activation of the remaining material at the site of injury to promote cell division, cell migration and complete reproduction of the missing structure. It remains an unresolved question why adult mammals are not equally competent to reactivate morphogenetic programmes. Although organ regeneration depends strongly on the proliferative properties of cells in the injured tissue, it is apparent that various organismic factors, such as innervation, vascularization, hormones, metabolism and the immune system, can affect this process. Here, we focus on a correlation between the regenerative capacity and cellular specialization in the context of functional demands, as illustrated by appendages and heart in diverse vertebrates. Elucidation of the differences between homologous regenerative and non-regenerative tissues from various animal models is essential for understanding the applicability of lessons learned from the study of regenerative biology to clinical strategies for the treatment of injured human organs.

Tissues and Cell Types of Appendage Regeneration: A Detailed Look at the Wound Epidermis and Its Specialized Forms.

Therapeutic implementation of human limb regeneration is a daring aim. Studying species that can regrow their lost appendages provides clues on how such a feat can be achieved in mammals. One of the unique features of regeneration-competent species lies in their ability to seal the amputation plane with a scar-free wound epithelium. Subsequently, this wound epithelium advances and becomes a specialized wound epidermis (WE) which is hypothesized to be the essential component of regenerative success. Recently, the WE and specialized WE terminologies have been used interchangeably. However, these tissues were historically separated, and contemporary limb regeneration studies have provided critical new information which allows us to distinguish them. Here, I will summarize tissue-level observations and recently identified cell types of WE and their specialized forms in different regeneration models.

Timing Does Matter: Nerve-Mediated HDAC1 Paces the Temporal Expression of Morphogenic Genes During Axolotl Limb Regeneration.

Sophisticated axolotl limb regeneration is a highly orchestrated process that requires highly regulated gene expression and epigenetic modification patterns at precise positions and timings. We previously demonstrated two waves of post-amputation expression of a nerve-mediated repressive epigenetic modulator, histone deacetylase 1 (HDAC1), at the wound healing (3 days post-amputation; 3 dpa) and blastema formation (8 dpa onward) stages in juvenile axolotls. Limb regeneration was profoundly inhibited by local injection of an HDAC inhibitor, MS-275, at the amputation sites. To explore the transcriptional response of post-amputation axolotl limb regeneration in a tissue-specific and time course-dependent manner after MS-275 treatment, we performed transcriptome sequencing of the epidermis and soft tissue (ST) at 0, 3, and 8 dpa with and without MS-275 treatment. Gene Ontology (GO) enrichment analysis of each coregulated gene cluster revealed a complex array of functional pathways in both the epidermis and ST. In particular, HDAC activities were required to inhibit the premature elevation of genes related to tissue development, differentiation, and morphogenesis. Further validation by Q-PCR in independent animals demonstrated that the expression of 5 out of 6 development- and regeneration-relevant genes that should only be elevated at the blastema stage was indeed prematurely upregulated at the wound healing stage when HDAC1 activity was inhibited. WNT pathway-associated genes were also prematurely activated under HDAC1 inhibition. Applying a WNT inhibitor to MS-275-treated amputated limbs partially rescued HDAC1 inhibition, resulting in blastema formation defects. We propose that post-amputation HDAC1 expression is at least partially responsible for pacing the expression timing of morphogenic genes to facilitate proper limb regeneration.

Midkine is a dual regulator of wound epidermis development and inflammation during the initiation of limb regeneration.

Formation of a specialized wound epidermis is required to initiate salamander limb regeneration. Yet little is known about the roles of the early wound epidermis during the initiation of regeneration and the mechanisms governing its development into the apical epithelial cap (AEC), a signaling structure necessary for outgrowth and patterning of the regenerate. Here, we elucidate the functions of the early wound epidermis, and further reveal midkine (mk) as a dual regulator of both AEC development and inflammation during the initiation of axolotl limb regeneration. Through loss- and gain-of-function experiments, we demonstrate that mk acts as both a critical survival signal to control the expansion and function of the early wound epidermis and an anti-inflammatory cytokine to resolve early injury-induced inflammation. Altogether, these findings unveil one of the first identified regulators of AEC development and provide fundamental insights into early wound epidermis function, development, and the initiation of limb regeneration.