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Over the past decade the concept that the hair follicle plays an important role in cutaneous wound repair has been established. Several elegant lineage tracing studies have demonstrated that hair follicle derived cells contribute to the long term maintenance of the epidermis following repair, while an absence of hair follicles is known to delay repair. The exact mechanisms surrounding hair follicle derived repair are unknown. Moreover, while multiple stem cell niches are present within the hair follicle, their relative importance during wound repair is still unclear. The hair follicle is also a regenerative mini-organ, undergoing regular cycles of growth and regression throughout life, yet surprisingly this has not been previously investigated with respect to wound repair. Data presented in this thesis reveals an unappreciated, yet fundamental link between the independent processes of hair cycle and wound repair, with a substantial acceleration in the rate of repair (~50%) observed in anagen phase. Importantly, the hair follicle appears to play a global role in repair, with differences in the contribution of multiple cell types to wound repair. In addition, this thesis addresses the early kinetics of hair follicle wound response for the first time. Anagen hair follicles are found predisposed to a more rapid and extensive response to injury, suggesting a higher overall percentage of repair derived from the hair follicle in anagen phase. Surprisingly, the bulge stem cell region, while critical for hair cycle appears to play little role in the events immediately following injury, and is not required for initiation of re-epithelialisation. Gene expression profiling reveals numerous genes associated with anagen accelerated repair, and identifies altered modulation of the immune system as a key mechanism. Further, anagen wounds are associated with an upregulation of developmental transcription factors, which may imply a more regenerative healing phenotype. These data reveal numerous targets with the potential to accelerate repair, which now require validation for their therapeutic potential. These targets could be of importance in promoting the repair of chronic wounds, an area of unmet clinical need. More generally, this thesis has established hair cycle as an important experimental variable, which must be controlled for in all future in vivo murine wounding studies.

Androgens are the main regulators of human hair growth stimulating larger, terminal hair development e.g. beard and causing scalp balding, androgenetic alopecia. Hair disorders cause psychological distress but are poorly controlled. Androgens probably act by altering regulatory paracrine factors produced by the mesenchyme-derived dermal papilla. This study aimed to investigate paracrine factors involved in androgen-regulated alopecia, particularly hepatocyte growth factor (HGF) family members, by investigating their in vivo status. Balding and non-balding scalp hair follicles and their component tissues were isolated and analysed by molecular biological methods (reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative PCR and DNA microarray analysis), cell culture and immunohistochemistry. Scalp follicles expressed a range of paracrine messenger genes. The dermal papilla, cultured dermal papilla cells and dermal sheath expressed several HGF family genes, while matrix cells only produced the receptor RON suggesting autocrine roles for HGF and MSP, but a paracrine route only for MSP. Comparing balding and non-balding follicles from the same individuals revealed the expected reduction in several keratin and keratin-related protein genes supporting this approach's validity. There were also significant differences in paracrine factors previously implicated in androgen action by in vitro studies. Several factors believed to increase during androgen stimulation of larger, darker follicles, e.g. IGF-I and SCF, were lowered in balding follicles, while putative inhibitory factors, e.g. TGFß-1, were increased. HGF and MSP and their receptors, c-Met and RON, were significantly reduced. These results increase our understanding of androgen action in human hair follicles; this could lead to better treatments for hair disorders.

Hair disorders cause significant distress. The main, but limited, treatment for hair loss is minoxidil, an ATP-sensitive potassium (KATP) channel opener whose mechanism of stimulation is unclear. The regulatory component of KATP channels has three forms: SUR1, SUR2A and SUR2B which all respond to different molecules. Minoxidil only opens SUR2B channels, though SUR1 and SUR2B are present in human hair follicles. To expand our understanding, the red deer hair follicle model was used initially. Deer follicles expressed the same KATP channel genes as human follicles when growing (anagen), but no channels were detected in resting follicles. This reinforces the importance of KATP channels in active hair growth and the usefulness of the deer model. To assess whether SUR1 KATP channels are actually involved in human hair growth, the effects of a selective SUR1 channel opener, NNC55-9216, on scalp follicle growth in organ culture was examined. NNC55-9216 stimulated anagen; its effect was augmented by minoxidil. This creates the potential for more effective pharmaceuticals to treat hair loss via SUR1 channels, either alone or in combination with minoxidil. The dermal papilla plays a crucial regulatory role in hair follicle activity determining the type of hair produced. Minoxidil had no effect on dermal papilla cell proliferation, but altered the profile of proteins produced when assessed by proteomics. Further research into the roles of KATP channels and greater understanding of the significance of these protein changes should enhance our knowledge of hair biology and help the development of new, improved therapies for hair pathologies.

Regeneration of hair follicles (HFs) and dermal adipocytes (DAs) occurs in mouse skin wounds upon large excisional wounding. Although HF regeneration is observed in African spiny mice of the genus Acomys and northern elephant seals after apex predator-inflicted wounding, laboratory rats do not display such regenerative phenotype. Such regeneration defect was observed in large excisional wound healing models in several rat strains, which undergo otherwise normal wound re-epithelialization. Inter-species transcriptome analyses between laboratory mouse and rat wound tissues attributed such lack of HF regeneration to differences in expression of inflammation markers, epigenetic remodelers and pleiotropic signaling molecules, including Satb1, Setd1b, Setdb1, and Whsc1l1. In mice, the origin of de novo HF regeneration has been partially elucidated, whereas the origin of DAs, a complex tissue that proceeds HF regeneration, remained elusive. Functional lineage tracing revealed the origin of DAs to be myofibroblastic. Bulk RNA-sequencing of genetically-labeled, FACS-purified myofibroblasts across a wound healing time course identified Zfp423 to be markedly up-regulated at a time-point coincident with initiation of DA regeneration. Pharmacological and genetic ablation/down-modulation of BMP signaling resulted in a significant DA regeneration defect. Because the origin of myofibroblasts appears to be tissue- and injury context-specific, the origin of myofibroblasts that contribute to DA regeneration in skin wounds was interrogated. Droplet-enabled single cell transcriptome analyses on unsorted, viable cells from wound dermal tissues collected prior the onset of HF regeneration was performed. Dimensionality reduction analyses revealed a large degree of cellular heterogeneity in the dermal compartment of early stage wounds. Furthermore, sub-clustering of wound fibroblasts further revealed a large degree of fibroblast heterogeneity. Pseudotime analyses revealed a putative fibroblast-myofibroblast differentiation trajectory and identified genes, including transcription factors, that may be important in myofibroblast differentiation in skin wounds in vivo. A subset of myofibroblasts expressed hematopoetic markers, most notably Lyz2, suggesting a common monocytic-origin. Full-length single cell RNA-sequencing and immunoblotting analyses of genetically labeled myofibroblasts confirmed these in silico observations. Bone marrow transplantation and functional lineage tracing using pan-hematopoetic Cre drivers demonstrated labeling of DA in regenerated skin wounds, suggesting that a population of hematopoetic-derived myofibroblasts contributes to regeneration of mouse skin wounds.