Mouse Digit Tip Regeneration Research Articles

En1 and Lmx1b do not recapitulate embryonic dorsal-ventral limb patterning functions during mouse digit tip regeneration.

The mouse digit tip regenerates following amputation. How the regenerate is patterned is unknown, but a long-standing hypothesis proposes developmental patterning mechanisms are re-used during regeneration. The digit tip bone exhibits dorsal-ventral (DV) polarity, so we focus on En1 and Lmx1b, two factors necessary for DV patterning during limb development. We investigate whether they are re-expressed during regeneration in a developmental-like pattern and whether they direct DV morphology of the regenerate. We find that both En1 and Lmx1b are expressed in the regenerating digit tip epithelium and mesenchyme, respectively, but without DV polarity. Conditional genetics and quantitative analysis of digit tip bone morphology determine that genetic deletion of En1 or Lmx1b in adult digit tip regeneration modestly reduces bone regeneration but does not affect DV patterning. Collectively, our data suggest that, while En1 and Lmx1b are re-expressed during mouse digit tip regeneration, they do not define the DV axis during regeneration.

Digit specific denervation does not inhibit mouse digit tip regeneration

It is long-established that innervation-dependent production of neurotrophic factors is required for blastema formation and epimorphic regeneration of appendages in fish and amphibians. The regenerating mouse digit tip and the human fingertip are mammalian models for epimorphic regeneration, and limb denervation in mice inhibits this response. A complicating issue of limb denervation studies in terrestrial vertebrates is that the experimental models also cause severe paralysis therefore impairing appendage use and diminishing mechanical loading of the denervated tissues. Thus, it is unclear whether the limb denervation impairs regeneration via loss of neurotrophic signaling or loss of mechanical load, or both. Herein, we developed a novel surgical procedure in which individual digits were specifically denervated without impairing ambulation and mechanical loading. We demonstrate that digit specific denervation does not inhibit but attenuates digit tip regeneration, in part due to a delay in wound healing. However, treating denervated digits with a wound dressing that enhances closure results in a partial rescue of the regeneration response. Contrary to the current understanding of mammalian epimorphic regeneration, these studies demonstrate that mouse digit tip regeneration is not peripheral nerve dependent, an observation that should inform continued mammalian regenerative medicine approaches.

Tissue origin of cytotoxic natural killer cells dictates their differential roles in mouse digit tip regeneration and progenitor cell survival.

SummaryRegeneration of amputated digit tips relies on mesenchymal progenitor cells and their differentiation into replacement bone and tissue stroma. Natural killer (NK) cells have well-characterized roles in antigen-independent killing of virally infected, pre-tumorous, or stressed cells; however, the potential for cytotoxic activity against regenerative progenitor cells is unclear. We identified NK cell recruitment to the regenerating digit tip, and NK cytotoxicity was observed against osteoclast and osteoblast progenitors. Adoptive cell transplants of spleen NK (SpNK) or thymus NK (ThNK) donor cells into immunodeficient mice demonstrated ThNK cell-induced apoptosis with a reduction in osteoclasts, osteoblasts, and proliferative cells, resulting in inhibition of regeneration. Adoptive transfer of NK cells deficient in NK cell activation genes identified that promotion of regeneration by SpNK cells requires Ncr1, whereas inhibition by ThNK cells is mediated via Klrk1 and perforin. Successful future therapies aimed at enhancing regeneration will require a deeper understanding of progenitor cell protection from NK cell cytotoxicity.

Activation of the Melanocortin-4 receptor signaling by \u03b1-MSH stimulates nerve-dependent mouse digit regeneration

BackgroundExpression of Mc4r in peripheral organs indicates it has broader roles in organ homeostasis and regeneration. However, the expression and function of Mc4r in the mouse limb and digit has not been fully investigated. Our previous work showed that Mc4r−/− mice fail to regenerate the digit, but whether activation of MC4R signaling could rescue digit regeneration, or stimulate proximal digit regeneration is not clear.ResultsWe analyzed the expression dynamics of Mc4r in the embryonic and postnatal mouse limb and digit using the Mc4r-gfp mice. We found that Mc4r-GFP is mainly expressed in the limb nerves, and in the limb muscles that are undergoing secondary myogenesis. Expression of Mc4r-GFP in the adult mouse digit is restricted to the nail matrix. We also examined the effect of α-MSH on mouse digit regeneration. We found that administration of α-MSH in the Mc4r+/− mice rescue the delayed regeneration of distal digit tip. α-MSH could rescue distal digit regeneration in denervated hindlimbs. In addition, α-MSH could stimulate regeneration of the proximally amputated digit, which is non-regenerative.ConclusionsMc4r expression in the mouse limb and digit is closely related to nerve tissues, and α-MSH/MC4R signaling has a neurotrophic role in mouse digit tip regeneration.

Cellular Heterogeneity and Lineage Restriction during Mouse Digit Tip Regeneration at Single-Cell Resolution.

Innate regeneration following digit tip amputation is one of the few examples of epimorphic regeneration in mammals. Digit tip regeneration is mediated by the blastema, the same structure invoked during limb regeneration in some lower vertebrates. By genetic lineage analyses, the digit tip blastema has been defined as a population of heterogeneous, lineage-restricted progenitor cells. These previous studies, however, do not comprehensively evaluate blastemaheterogeneity or address lineage restriction of closely related cell types. In this report, we present single-cell RNA sequencing of over 38,000 cells from mouse digit tip blastemas and unamputated control digit tips and generate an atlas of the cell types participating in digit tip regeneration. We computationally define differentiation trajectories of vascular, monocytic, and fibroblastic lineages over regeneration, and while our data confirm broad lineage restriction of progenitors, our analysis reveals 67 genes enriched in blastema fibroblasts including a novel regeneration-specific gene, Mest.

Bone growth as the main determinant of mouse digit tip regeneration after amputation

Regeneration is classically demonstrated in mammals using mice digit tip. In this study, we compared different amputation plans and show that distally amputated digits regrow with morphology close to normal but fail to regrow the fat pad. Proximally amputated digits do not regrow the phalangeal bone, but the remaining structures (nail, skin and connective tissue), all with intrinsic regenerative capacity, re-establishing integrity indistinguishably in distally and proximally amputated digits. Thus, we suggest that the bone growth promoted by signals and progenitor cells not removed by distal amputations is responsible for the re-establishment of a drastically different final morphology after distal or proximal digit tip amputations. Despite challenging the use of mouse digit tip as a model system for limb regeneration in mammals, these findings evidence a main role of bone growth in digit tip regeneration and suggest that mechanisms that promote joint structures formation should be the main goal of regenerative medicine for limb and digit regrowth.

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.

Level‐specific amputations and resulting regenerative outcomes in the mouse distal phalanx

Mouse digit tip regeneration involves an intricate coordinated regrowth of the terminal phalanx, nail, dermis and epidermis. During this time, regenerating digits undergo wound healing, blastema formation, and differentiation. However, the regenerative response of the digit is dependent on the level of the amputation. Amputation of <30% of the distal phalanx (P3), with part of the base nail remaining, results in extensive digit regeneration. In contrast, >60% P3 removal results in no regeneration. This level-dependent regenerative ability of the mouse digit provides a comparative model between regeneration and non-regeneration that may enable identification of specific factors critical to regeneration. Although the ability to create regenerating and non-regenerating conditions has been well established, the regenerative response between these regions ("intermediate" zone) has received less scrutiny, and may add insight to the regenerative processes, including the degree of histolysis, and the level of blastema formation. The objective of this study is then to compare the regeneration capacity between amputation levels within the regenerating (<30%), intermediate (40-59%), and non-regenerating (>60%) regions. Results indicated that regenerative and intermediate amputations led to significant histolysis and blastema formation of the distal phalanx 14 days post-amputation. Unlike the regenerating digits, intermediate amputations led to incomplete regeneration whereby regrowth of the digits were not to the levels of the intact or regenerating digits. Non-regenerating amputations did not exhibit significant histolysis or blastema formation. Remarkably, the histolytic process resulted in day 14 P3 lengths that were similar regardless of the initial amputation over 19%. The differences in histolysis, blastema formation and injury outcomes were also marked by changes in the number of proliferating cells and osteoclasts. Altogether, these results indicate that although intermediate amputations result in histolysis and blastema formation similar to regenerating digits, the resulting cellular composition of the blastema differs, contributing to incomplete regeneration.

Macrophages are required to coordinate mouse digit tip regeneration.

In mammals, macrophages are known to play a major role in tissue regeneration. They contribute to inflammation, histolysis, re-epithelialization, revascularization and cell proliferation. Macrophages have been shown to be essential for regeneration in salamanders and fish, but their role has not been elucidated in mammalian epimorphic regeneration. Here, using the regenerating mouse digit tip as a mammalian model, we demonstrate that macrophages are essential for the regeneration process. Using cell-depletion strategies, we show that regeneration is completely inhibited; bone histolysis does not occur, wound re-epithelialization is inhibited and the blastema does not form. Although rescue of epidermal wound closure in the absence of macrophages promotes blastema accumulation, it does not rescue cell differentiation, indicating that macrophages play a key role in the redifferentiation of the blastema. We provide additional evidence that although bone degradation is a component, it is not essential to the overall regenerative process. These findings show that macrophages play an essential role in coordinating the epimorphic regenerative response in mammals.

Principles and mechanisms of regeneration in the mouse model for wound-induced hair follicle neogenesis.

Wound‐induced hair follicle neogenesis (WIHN) describes a regenerative phenomenon in adult mammalian skin wherein fully functional hair follicles regenerate de novo in the center of large excisional wounds. Originally described in rats, rabbits, sheep, and humans in 1940−1960, the WIHN phenomenon was reinvestigated in mice only recently. The process of de novo hair regeneration largely duplicates the morphological and signaling features of normal embryonic hair development. Similar to hair development, WIHN critically depends on the activation of canonical WNT signaling. However, unlike hair development, WNT activation in WIHN is dependent on fibroblast growth factor 9 signaling generated by the immune system's γδ T cells. The cellular bases of WIHN remain to be fully characterized; however, the available evidence leaves open the possibility for a blastema‐like mechanism wherein epidermal and/or dermal wound cells undergo epigenetic reprogramming toward a more plastic, embryonic‐like state. De novo hair follicles do not regenerate from preexisting hair‐fated bulge stem cells. This suggests that hair neogenesis is not driven by preexisting lineage‐restricted progenitors, as is the case for amputation‐induced mouse digit tip regeneration, but rather may require a blastema‐like mechanism. The WIHN model is characterized by several intriguing features, which await further explanation. These include (1) the minimum wound size requirement for activating neogenesis, (2) the restriction of hair neogenesis to the wound's center, and (3) imperfect patterning outcomes, both in terms of neogenic hair positioning within the wound and in terms of their orientation. Future enquiries into the WIHN process, made possible by a wide array of available skin‐specific genetic tools, will undoubtedly expand our understanding of the regeneration mechanisms in adult mammals.

Connective tissue fibroblast properties are position-dependent during mouse digit tip regeneration.

A key factor that contributes to the regenerative ability of regeneration-competent animals such as the salamander is their use of innate positional cues that guide the regeneration process. The limbs of mammals has severe regenerative limitations, however the distal most portion of the terminal phalange is regeneration competent. This regenerative ability of the adult mouse digit is level dependent: amputation through the distal half of the terminal phalanx (P3) leads to successful regeneration, whereas amputation through a more proximal location, e.g. the subterminal phalangeal element (P2), fails to regenerate. Do the connective tissue cells of the mammalian digit play a role similar to that of the salamander limb in controlling the regenerative response? To begin to address this question, we isolated and cultured cells of the connective tissue surrounding the phalangeal bones of regeneration competent (P3) and incompetent (P2) levels. Despite their close proximity and localization, these cells show very distinctive profiles when characterized in vitro and in vivo. In vitro studies comparing their proliferation and position-specific interactions reveal that cells isolated from the P3 and P2 are both capable of organizing and differentiating epithelial progenitors, but with different outcomes. The difference in interactions are further characterized with three-dimension cultures, in which P3 regenerative cells are shown to lack a contractile response that is seen in other fibroblast cultures, including the P2 cultures. In in vivo engraftment studies, the difference between these two cell lines is made more apparent. While both P2 and P3 cells participated in the regeneration of the terminal phalanx, their survival and proliferative indices were distinct, thus suggesting a key difference in their ability to interact within a regeneration permissive environment. These studies are the first to demonstrate distinct positional characteristics of connective tissue cells that are associated with their regenerative capabilities.

The “Stars and Stripes” Metaphor for Animal Regeneration-Elucidating Two Fundamental Strategies along a Continuum

A number of challenges have hindered the development of a unified theory for metazoan regeneration. To describe the full range of complex regeneration phenomena in Animalia, we suggest that metazoans that regenerate missing body parts exhibit biological attributes that are tailored along a morpho-spatial regeneration continuum, illustrated in its polar scenarios by the USA “stars and stripes” flag. Type 1 organisms (“T1, ‘stars’”) are typical colonial organisms (but contain unitary taxa) that are able to regenerate “whole new stars”, namely, whole bodies and colonial modules, through systemic induction and sometimes multiple regeneration foci (hollow regeneration spheres, resembling the blastula) that compete for dominance. They regenerate soma and germ constituents with pluripotent adult stem cells and exhibit somatic-embryogenesis mode of ontogeny. Type 2 organisms (“T2, ‘stripes’”) are capable of limited regeneration of somatic constituents via fate-restricted stem cells, and regenerate through centralized inductions that lead to a single regeneration front. T2 organisms are unitary and use preformistic mode of ontogeny. T1 and T2 organisms also differ in interpretation of what constitutes positional information. T2 organisms also execute alternative, less effective, regeneration designs (i.e., scar formation). We assigned 15 characteristics that distinguish between T1/T2 strategies: those involving specific regeneration features and those operating on biological features at the whole-organism level. Two model organisms are discussed, representing the two strategies of T1/T2 along the regeneration continuum, the Botrylloides whole body regeneration (T1) and the mouse digit-tip regeneration (T2) phenomena. The above working hypothesis also postulates that regeneration is a primeval attribute of metazoans. As specified, the “stars and stripes” paradigm allows various combinations of the biological features assigned to T1 and T2 regeneration strategies. It does not consider any concentration gradient or thresholds and does not refer to the “epimorphosis” and “morphallaxis” terms, regeneration types across phyla or across body plans. The “stars and stripes” paradigm also ignores, at this stage of analysis, cases of regeneration loss that may obscure biological trajectories. The main advantage of the “stars and stripes” paradigm is that it allows us to compare T1/T2 regeneration, as well as other modes of regeneration, through critical determining characteristics.

Mouse digit tip regeneration is mediated by fate-restricted progenitor cells

Regeneration of appendages is frequent among invertebrates as well as some vertebrates. However, in mammals this has been largely relegated to digit tip regeneration, as found in mice and humans. The regenerated structures are formed from a mound of undifferentiated cells called a blastema, found just below the site of amputation. The blastema ultimately gives rise to all of the tissues in the regenerate, excluding the epidermis, and has classically been thought of as a homogenous pool of pluripotent stem cells derived by dedifferentiation of stump tissue, although this has never been directly tested in the context of mammalian digit tip regeneration. Successful digit tip regeneration requires that the level of amputation be within the nail bed and depends on expression of Msx1. Because Msx1 is strongly expressed in the nail bed mesenchyme, it has been proposed that the Msx1-expressing cells represent a pluripotent cell population for the regenerating digit. In this report, we show that Msx1 is dynamically expressed during digit tip regeneration, and it does not mark a pluripotent stem cell population. Moreover, we show that both the ectoderm and mesoderm contain fate-restricted progenitor populations that work in concert to regenerate their own lineages within the digit tip, supporting the hypothesis that the blastema is a heterogeneous pool of progenitor cells.

19-P039 The fate of Msx1 positive nail bed cells in mouse digit tip regeneration

19-P039 The fate of Msx1 positive nail bed cells in mouse digit tip regeneration

Mammalian regeneration and regenerative medicine

Mammals are generally considered to be poor regenerators, yet there are a handful of mammalian models that display a robust ability to regenerate. One such system is the regenerating tips of digits in both humans and mice. In vitro studies of regenerating fetal human and mouse digit tips display both anatomical and molecular similarities, indicating that the mouse digit is a clinically relevant model. At the same time, genetic studies on mouse digit tip regeneration have identified signaling pathways required for the regeneration response that parallel those known to be important for regeneration in lower vertebrates. In addition, recent studies establish that digit tip regeneration involves the formation of a blastema that shares similarities with the amphibian blastema, thus establishing a conceptual bridge between clinical application and basic research in regeneration. In this review we discuss how the study of endogenous regenerating mammalian systems is enhancing our understanding of regenerative mechanisms and helping to shed light on the development of therapeutic strategies in regenerative medicine.