Dermal ß-Catenin Is Required for Hedgehog-Driven Hair Follicle Neogenesis.

Hair follicle neogenesis (HFN) occurs following large skin excisions in mice, serving as a rare regenerative model in mammalian wound healing. Wound healing typically results in fibrosis in mice and humans. We previously showed small skin excisions in mice result in scarring devoid of HFN, displaying features of non-regenerative healing, and Hedgehog (Hh) activation in the dermis of such wounds can induce HFN. In this study, we sought to verify the role of dermal Wnt/ß-catenin signaling in HFN, as this pathway is essential for HF development, but is also paradoxically well-characterized in fibrosis of adult wounds. By deletion of ß-catenin in large wound myofibroblasts, we show Wnt/ß-catenin signaling is required for endogenous mechanisms of HFN. Through utilizing a combined mouse model that simultaneously induces deletion of ß-catenin and constitutive activation of Smoothened (Smo) in myofibroblasts, we also found ß-catenin is required for Hh-driven DP formation. Transcriptome analysis confirms Wnt/ß-catenin and Hh pathways are activated in dermal papilla (DP) cells. Our results indicate that Wnt-active fibrotic status may also create a permissive state for the regenerative function of Hh, suggesting that activation of both Wnt and Hh pathways in skin wound fibroblasts must be ensured in future strategies to promote HFN.

The development of hair follicles and nail.

The hair follicle and nail unit develop and regenerate through epithelial-mesenchymal interactions. Here, we review some of the key signals and molecular interactions that regulate mammalian hair follicle and nail formation during embryonic development and how these interactions are reutilized to promote their regeneration during adult homeostasis and in response to skin wounding. Finally, we highlight the role of some of these signals in mediating human hair follicle and nail conditions.

Dedifferentiation maintains melanocyte stem cells in a dynamic niche.

For unknow reasons, the melanocyte stem cell (McSC) system fails earlier than other adult stem cell populations1, which leads to hair greying in most humans and mice2,3. Current dogma states that McSCs are reserved in an undifferentiated state in the hair follicle niche, physically segregated from differentiated progeny that migrate away following cues of regenerative stimuli4-8. Here we show that most McSCs toggle between transit-amplifying and stem cell states for both self-renewal and generation of mature progeny, a mechanism fundamentally distinct from those of other self-renewing systems. Live imaging and single-cell RNA sequencing revealed that McSCs are mobile, translocating between hair follicle stem cell and transit-amplifying compartments where they reversibly enter distinct differentiation states governed by local microenvironmental cues (for example, WNT). Long-term lineage tracing demonstrated that the McSC system is maintained by reverted McSCs rather than by reserved stem cells inherently exempt from reversible changes. During ageing, there is accumulation of stranded McSCs that do not contribute to the regeneration of melanocyte progeny. These results identify a new model whereby dedifferentiation is integral to homeostatic stem cell maintenance and suggest that modulating McSC mobility may represent a new approach for the prevention of hair greying.

Wound-Induced Hair Neogenesis Model.

Skin wounds in adult mammals typically heal with a fibrotic scar and fail to restore ectodermal appendages, such as hair follicles or adipose tissue. Intriguingly, new hair follicles regenerate in the center of large full-thickness wounds of mice in a process called wound-induced hair neogenesis (WIHN). WIHN is followed by neogenesis of dermal adipose tissue. Both neogenic events reactivate embryonic-like cellular and molecular programs. The WIHN model provides a platform for studying mammalian regeneration, and findings from this model could instruct future regenerative medicine interventions for treating wounds and alopecia. Since Ito et al. rediscovered WIHN 15 years ago, numerous investigators have worked on the WIHN model using varying wounding protocols and model interpretations. Because a variety of factors, including environmental variables and choice of mouse strains, can affect the outcomes of a WIHN study, the purpose of this article is to provide an overview of the experimental variables that impact WIHN so that experiments between laboratories can be compared in a meaningful manner.

Phagocytosis of Wnt inhibitor SFRP4 by late wound macrophages drives chronic Wnt activity for fibrotic skin healing.

Human and murine skin wounding commonly results in fibrotic scarring, but the murine wounding model wound-induced hair neogenesis (WIHN) can frequently result in a regenerative repair response. Here, we show in single-cell RNA sequencing comparisons of semi-regenerative and fibrotic WIHN wounds, increased expression of phagocytic/lysosomal genes in macrophages associated with predominance of fibrotic myofibroblasts in fibrotic wounds. Investigation revealed that macrophages in the late wound drive fibrosis by phagocytizing dermal Wnt inhibitor SFRP4 to establish persistent Wnt activity. In accordance, phagocytosis abrogation resulted in transient Wnt activity and a more regenerative healing. Phagocytosis of SFRP4 was integrin-mediated and dependent on the interaction of SFRP4 with the EDA splice variant of fibronectin. In the human skin condition hidradenitis suppurativa, phagocytosis of SFRP4 by macrophages correlated with fibrotic wound repair. These results reveal that macrophages can modulate a key signaling pathway via phagocytosis to alter the skin wound healing fate.

Effects of early detection of peritoneal catheter migration on clinical outcomes: 15-years experiences from a single centre.

AIM: Catheter migration is an important cause of catheter malfunction in peritoneal dialysis (PD). The purpose of this study was to investigate the effect of early detection of catheter migration on clinical outcomes. METHODS: A retrospective review of 135 consecutive patients initiating PD immediately following catheter insertion from 2002 to 2017 was undertaken. In order to detect catheter migration without malfunction early, serial abdominal-pelvic radiographic examinations were performed according to a predefined protocol. Conservative management with rigorous catharsis was undertaken to correct catheter migration. A Kaplan-Meier method was used to calculate survival rate. RESULTS: Mean follow-up period was 42.8 ± 34.9 months. Catheter migration occurred in 62.4%. Among them, 85.9% occurred within the first 2 weeks after catheter insertion. There were no significant associations between catheter migration and variables such as gender, obesity, DM and type of catheter. Success rate of conservative management with rigorous catharsis was 91.1%. Catheter survival at 1 and 5 years were 91.5% and 64.6% in the migration group and 81.2% and 69.9% in the non-migration group, respectively (Log-rank test, P = 0.915). Patient survival at 1 and 5 years were 96.8% and 85.8% in the migration group and 91.9% and 82.3% in the non-migration group, respectively (P = 0.792). CONCLUSION: Early detection of PD catheter migration allowed the migrated tip to be easily corrected with conservative management. Once the migrated catheter tip was restored, catheter migration itself did not affect catheter survival. These findings suggest that early detection and correction of catheter migration is important for improving clinical outcomes.

A novel mouse model demonstrates that oncogenic melanocyte stem cells engender melanoma resembling human disease.

Melanoma, the deadliest skin cancer, remains largely incurable at advanced stages. Currently, there is a lack of animal models that resemble human melanoma initiation and progression. Recent studies using a Tyr-CreER driven mouse model have drawn contradictory conclusions about the potential of melanocyte stem cells (McSCs) to form melanoma. Here, we employ a c-Kit-CreER-driven model that specifically targets McSCs to show that oncogenic McSCs are a bona fide source of melanoma that expand in the niche, and then establish epidermal melanomas that invade into the underlying dermis. Further, normal Wnt and Endothelin niche signals during hair anagen onset are hijacked to promote McSC malignant transformation during melanoma induction. Finally, molecular profiling reveals strong resemblance of murine McSC-derived melanoma to human melanoma in heterogeneity and gene signatures. These findings provide experimental validation of the human melanoma progression model and key insights into the transformation and heterogeneity of McSC-derived melanoma.

Hedgehog stimulates hair follicle neogenesis by creating inductive dermis during murine skin wound healing.

Mammalian wounds typically heal by fibrotic repair without hair follicle (HF) regeneration. Fibrosis and regeneration are currently considered the opposite end of wound healing. This study sought to determine if scar could be remodeled to promote healing with HF regeneration. Here, we identify that activation of the Sonic hedgehog (Shh) pathway reinstalls a regenerative dermal niche, called dermal papilla, which is required and sufficient for HF neogenesis (HFN). Epidermal Shh overexpression or constitutive Smoothened dermal activation results in extensive HFN in wounds that otherwise end in scarring. While long-term Wnt activation is associated with fibrosis, Shh signal activation in Wnt active cells promotes the dermal papilla fate in scarring wounds. These studies demonstrate that mechanisms of scarring and regeneration are not distant from one another and that wound repair can be redirected to promote regeneration following injury by modifying a key dermal signal.

Dissecting Wnt Signaling for Melanocyte Regulation during Wound Healing.

Abnormal pigmentation is commonly seen in the wound scar. Despite advancements in the research of wound healing, little is known about the repopulation of melanocytes in the healed skin. Previous studies have shown the capacity of melanocyte stem cells in the hair follicle to contribute skin epidermal melanocytes after injury in mice and humans. Here, we focused on the Wnt pathway, known to be a vital regulator of melanocyte stem cells in efforts to better understand the regulation of follicle-derived epidermal melanocytes during wound healing. We showed that transgenic expression of Wnt inhibitor Dkk1 in melanocytes reduced epidermal melanocytes in the wound scar. Conversely, forced activation of Wnt signaling by genetically stabilizing ß-catenin in melanocytes increases epidermal melanocytes. Furthermore, we show that deletion of Wntless (Wls), a gene required for Wnt ligand secretion, within epithelial cells results in failure in activating Wnt signaling in adjacent epidermal melanocytes. These results show the essential function of extrinsic Wnt ligands in initiating Wnt signaling in follicle-derived epidermal melanocytes during wound healing. Collectively, our results suggest the potential for Wnt signal regulation to promote melanocyte regeneration and provide a potential molecular window to promote proper melanocyte regeneration after wounding and in conditions such as vitiligo.

Wound Regeneration Deficit in Rats Correlates with Low Morphogenetic Potential and Distinct Transcriptome Profile of Epidermis.

Large excisional wounds in mice prominently regenerate new hair follicles (HFs) and fat, yet humans are deficient for this regenerative behavior. Currently, wound-induced regeneration remains a clinically desirable, but only partially understood phenomenon. We show that large excisional wounds in rats across seven strains fail to regenerate new HFs. We compared wound transcriptomes between mice and rats at the time of scab detachment, which coincides with the onset of HF regeneration in mice. In both species, wound dermis and epidermis share core dermal and epidermal transcriptional programs, respectively, yet prominent interspecies differences exist. Compared with mice, rat epidermis expresses distinct transcriptional and epigenetic factors, markers of epidermal repair, hyperplasia, and inflammation, and lower levels of WNT signaling effectors and regulators. When recombined on the surface of excisional wounds with vibrissa dermal papillae, partial-thickness skin grafts containing distal pelage HF segments, but not interfollicular epidermis, readily regenerated new vibrissa-like HFs. Together, our findings establish rats as a nonregenerating rodent model for excisional wound healing and suggest that low epidermal competence and associated transcriptional profile may contribute to its regenerative deficiency. Future comparison between rat and mouse may lend further insight into the mechanism of wounding-induced regeneration and causes for its deficit.

Regeneration of fat cells from myofibroblasts during wound healing.

Although regeneration through the reprogramming of one cell lineage to another occurs in fish and amphibians, it has not been observed in mammals. We discovered in the mouse that during wound healing, adipocytes regenerate from myofibroblasts, a cell type thought to be differentiated and nonadipogenic. Myofibroblast reprogramming required neogenic hair follicles, which triggered bone morphogenetic protein (BMP) signaling and then activation of adipocyte transcription factors expressed during development. Overexpression of the BMP antagonist Noggin in hair follicles or deletion of the BMP receptor in myofibroblasts prevented adipocyte formation. Adipocytes formed from human keloid fibroblasts either when treated with BMP or when placed with human hair follicles in vitro. Thus, we identify the myofibroblast as a plastic cell type that may be manipulated to treat scars in humans.

EdnrB Governs Regenerative Response of Melanocyte Stem Cells by Crosstalk with Wnt Signaling.

Delineating the crosstalk between distinct signaling pathways is key to understanding the diverse and dynamic responses of adult stem cells during tissue regeneration. Here, we demonstrate that the Edn/EdnrB signaling pathway can interact with other signaling pathways to elicit distinct stem cell functions during tissue regeneration. EdnrB signaling promotes proliferation and differentiation of melanocyte stem cells (McSCs), dramatically enhancing the regeneration of hair and epidermal melanocytes. This effect is dependent upon active Wnt signaling that is initiated by Wnt ligand secretion from the hair follicle epithelial niche. Further, this Wnt-dependent EdnrB signaling can rescue the defects in melanocyte regeneration caused by Mc1R loss. This suggests that targeting Edn/EdnrB signaling in McSCs can be a therapeutic approach to promote photoprotective-melanocyte regeneration, which may be useful for those with increased risk of skin cancers due to Mc1R variants.

Adenine nucleotide translocase 4 is expressed within embryonic ovaries and dispensable during oogenesis.

Adenine nucleotide translocase (Ant) facilitates the exchange of adenosine triphosphate across the mitochondrial inner membrane and plays a critical role for bioenergetics in eukaryotes. Mice have 3 Ant paralogs, Ant1 (Slc25a4), Ant2 (Slc25a5), and Ant4 (Slc25a31), which are expressed in a tissue-dependent manner. We previously identified that Ant4 was expressed exclusively in testicular germ cells in adult mice and essential for spermatogenesis and subsequently male fertility. Further investigation into the process of spermatogenesis revealed that Ant4 was particularly highly expressed during meiotic prophase I and indispensable for normal progression of leptotene spermatocytes to the stages thereafter. In contrast, the expression and roles of Ant4 in female germ cells have not previously been elucidated. Here, we demonstrate that the Ant4 gene is expressed during embryonic ovarian development during which meiotic prophase I occurs. We confirmed embryonic ovary-specific Ant4 expression using a bacterial artificial chromosome transgene. In contrast to male, however, Ant4 null female mice were fertile although the litter size was slightly decreased. They showed apparently normal ovarian development which was morphologically indistinguishable from the control animals. These data indicate that Ant4 is a meiosis-specific gene expressed during both male and female gametogenesis however indispensable only during spermatogenesis and not oogenesis. The differential effects of Ant4 depletion within the processes of male and female gametogenesis may be explained by meiosis-specific inactivation of the X-linked Ant2 gene in male, a somatic paralog of the Ant4 gene.

Evolutionary genomics implies a specific function of Ant4 in mammalian and anole lizard male germ cells.

Most vertebrates have three paralogous genes with identical intron-exon structures and a high degree of sequence identity that encode mitochondrial adenine nucleotide translocase (Ant) proteins, Ant1 (Slc25a4), Ant2 (Slc25a5) and Ant3 (Slc25a6). Recently, we and others identified a fourth mammalian Ant paralog, Ant4 (Slc25a31), with a distinct intron-exon structure and a lower degree of sequence identity. Ant4 was expressed selectively in testis and sperm in adult mammals and was indeed essential for mouse spermatogenesis, but it was absent in birds, fish and frogs. Since Ant2 is X-linked in mammalian genomes, we hypothesized that the autosomal Ant4 gene may compensate for the loss of Ant2 gene expression during male meiosis in mammals. Here we report that the Ant4 ortholog is conserved in green anole lizard (Anolis carolinensis) and demonstrate that it is expressed in the anole testis. Further, a degenerate DNA fragment of putative Ant4 gene was identified in syntenic regions of avian genomes, indicating that Ant4 was present in the common amniote ancestor. Phylogenetic analyses suggest an even more ancient origin of the Ant4 gene. Although anole lizards are presumed male (XY) heterogametic, like mammals, copy numbers of the Ant2 as well as its neighboring gene were similar between male and female anole genomes, indicating that the anole Ant2 gene is either autosomal or located in the pseudoautosomal region of the sex chromosomes, in contrast to the case to mammals. These results imply the conservation of Ant4 is not likely simply driven by the sex chromosomal localization of the Ant2 gene and its subsequent inactivation during male meiosis. Taken together with the fact that Ant4 protein has a uniquely conserved structure when compared to other somatic Ant1, 2 and 3, there may be a specific advantage for mammals and lizards to express Ant4 in their male germ cells.

Adenine nucleotide translocase 4 deficiency leads to early meiotic arrest of murine male germ cells.

Male fertility relies on the highly specialized process of spermatogenesis to continually renew the supply of spermatozoa necessary for reproduction. Central to this unique process is meiosis that is responsible for the production of haploid spermatozoa as well as for generating genetic diversity. During meiosis I, there is a dramatic increase in the number of mitochondria present within the developing spermatocytes, suggesting an increased necessity for ATP production and utilization. Essential for the utilization of ATP is the translocation of ADP and ATP across the inner mitochondrial membrane, which is mediated by the adenine nucleotide translocases (Ant). We recently identified and characterized a novel testis specific Ant, ANT4 (also known as SLC25A31 and Aac4). The generation of Ant4-deficient animals resulted in the severe disruption of the seminiferous epithelium with an apparent spermatocytic arrest of the germ cell population. In the present study utilizing a chromosomal spread technique, we determined that Ant4-deficiency results in an accumulation of leptotene spermatocytes, a decrease in pachytene spermatocytes, and an absence of diplotene spermatocytes, indicating early meiotic arrest. Furthermore, the chromosomes of Ant4-deficient pachytene spermatocyte occasionally demonstrated sustained gammaH2AX association as well as synaptonemal complex protein 1 (SYCP1)/SYCP3 dissociation beyond the sex body. Large ATP supplies from mitochondria may be critical for normal progression of spermatogenesis during early stages of meiotic prophase I, including DNA double-strand break repair and chromosomal synapsis.

Differential CpG island methylation of murine adenine nucleotide translocase genes.

Adenine nucleotide translocase (Ant) mediates the exchange of ADP and ATP across the inner mitochondrial membrane in eukaryotes. Mice possess three distinct but highly homologous Ant isoforms, encoded by independent genes, whose transcription depends upon tissue type. Ant1 is expressed selectively in heart and skeletal muscles, Ant2 is ubiquitously expressed in most tissues but lower in skeletal muscle and testis, while Ant4 is exclusively expressed in the testis. Of interest, each of these Ant genes contains CpG islands in their proximal promoter regions. We investigated the methylation status of the three Ant genes in various tissues with active and inactive transcription. In contrast to the Ant4 gene in which CpG island methylation is essential for gene repression, the CpG islands of Ant1 and Ant2 are hypomethylated regardless of the gene expression status throughout the tissues of male mice. Despite the tissue specific expression profile of Ant1, CpG methylation is unlikely involved in the regulation of the gene. Consistent with these findings, addition of a CpG-demethylating agent, 5-aza-2'-deoxycitidine, to fibroblasts increased the expression of Ant4 but not Ant1 or Ant2 genes. This study provides insight regarding the differential regulation of Ant isoforms in mammals, whereby both the Ant1 and Ant2 genes are capable of expression, but the Ant4 gene is completely repressed throughout somatic tissues. To the best of our knowledge, this is a first example to clearly demonstrate a differential usage of CpG island methylation within a family of genes.

The side population cells in the rabbit limbus sensitively increased in response to the central cornea wounding.

PURPOSE: Side population (SP) cells are known to reside in the limbus as putative corneal epithelial stem cells. This study was performed to demonstrate the presence and the characteristics of SP cells in the rabbit limbal epithelium and explore their sensitivity in response to the central cornea wounding. METHODS: To sort out the SP cells, freshly isolated rabbit limbal and central corneal epithelial cells were subjected to Hoechst 33342 dye efflux assay. For characterization of the sorted SP cells, RT-PCR analysis, semi-dry three-dimensional (3-D) cell culture, and transplantation in nude mice were performed. To explore wound sensitivity of the limbal SP cells, the rabbit central cornea was wounded by direct contact of a 6-mm paper disk soaked with 1 N NaOH, and changes in the population size of the SP cells and the colony-forming efficiency (CFE) was monitored on days 1, 2, and 5 after wounding. RESULTS: The SP cells were present in the rabbit limbal epithelium with an incidence of 0.73% +/- 0.14% (n = 8) and were smaller in cell size than the major population (MP) cells, quiescent in the cell cycle, and in the undifferentiated state. The SP cells were able to regenerate the cornea-like structure with basal enrichment of p63-positive cells by in vitro 3-D culture and in vivo transplantation, all of which were best achieved by the whole population (WP) of cells comprising SP and MP cells. After central cornea wounding, this rare population of the limbal SP cells increased in size fivefold on day 1 and then decreased on day 2. The transient increase in the SP cells was subsequently followed by the propagation of an increase in CFE in the limbal MP cells on day 2 and then in the corneal MP cells on day 5. In the hematopoietic colony-forming assay, the limbal SP cells gave approximately eightfold higher CFU than the limbal MP cells. CONCLUSIONS: The SP cells identified in the rabbit limbus are an undifferentiated and noncycling rare epithelial cell population, which sensitively respond to the central cornea wounding by their transient increase in the population size.