Dab2 (Disabled-2), an adaptor protein, regulates self-renewal of hair follicle stem cells.

Disabled 2 (Dab2), an adaptor protein, is up regulated in the hair follicle stem cells (HFSCs); however, its role in any tissue stem cells has not been studied. In the present study, we have reported that Dab2 conditional knockout (Dab2-cKO) mice exhibited a delay in the HF cycle due to perturbed activation of HFSCs. Further, Dab2-cKO mice showed a reduction in the number of HFSCs and reduced colony forming ability of HFSCs. Dab2-cKO mice showed extended quiescence of HFSCs concomitant with an increased expression of Nfatc1. Dab2-cKO mice showed a decreased expression of anti-aging genes such as Col17a1, decorin, Sirt2 and Sirt7. Dab2-cKO mice did not show full hair coat recovery in aged mice thereby suggesting an accelerated aging process. Overall, we unveil for the first time, the role of Dab2 that regulate activation and self-renewal of HFSCs.

Alk1 acts in non-endothelial VE-cadherin+ perineurial cells to maintain nerve branching during hair homeostasis.

Vascular endothelial (VE)-cadherin is a well-recognized endothelial cell marker. One of its interacting partners, the TGF-ß receptor Alk1, is essential in endothelial cells for adult skin vasculature remodeling during hair homeostasis. Using single-cell transcriptomics, lineage tracing and gene targeting in mice, we characterize the cellular and molecular dynamics of skin VE-cadherin+ cells during hair homeostasis. We describe dynamic changes of VE-cadherin+ endothelial cells specific to blood and lymphatic vessels and uncover an atypical VE-cadherin+ cell population. The latter is not a predicted adult endovascular progenitor, but rather a non-endothelial mesenchymal perineurial cell type, which forms nerve encapsulating tubular structures that undergo remodeling during hair homeostasis. Alk1 acts in the VE-cadherin+ perineurial cells to maintain proper homeostatic nerve branching by enforcing basement membrane and extracellular matrix molecular signatures. Our work implicates the VE-cadherin/Alk1 duo, classically known as endothelial-vascular specific, in perineurial-nerve homeostasis. This has broad implications in vascular and nerve disease.

Single-cell transcriptomics of adult skin VE-cadherin expressing lineages during hair cycle.

Adult skin homeostasis involves global reorganization of dermal lineages at different stages of the mouse hair growth cycle. Vascular endothelial cadherin (VE-cadherin encoded by Cdh5 ) expressing cells from blood and lymphatic vasculature structures are known to remodel during the adult hair cycle. Here we employ single-cell RNA-sequencing (scRNA-seq) 10x-genomics analysis of FACS-sorted VE-cadherin expressing cells marked via Cdh5-CreER genetic labeling at resting (telogen) and growth (anagen) stage of hair cycle. Our comparative analysis between the two stages uncovers a persistent Ki67 + proliferative EC population and documents changes in EC population distribution and gene expression. Global gene expression changes in all the analyzed populations revealed bioenergetic metabolic changes that may drive vascular remodeling during HF growth phase, alongside a few highly restricted cluster-specific gene expression differences. This study uncovers active cellular and molecular dynamics of adult skin endothelial lineages during hair cycle that may have broad implications in adult tissue regeneration and for understanding vascular disease.

Blood endothelial ALK1-BMP4 signaling axis regulates adult hair follicle stem cell activation.

Blood vessels can play dual roles in tissue growth by transporting gases and nutrients and by regulating tissue stem cell activity via signaling. Correlative evidence implicates skin endothelial cells (ECs) as signaling niches of hair follicle stem cells (HFSCs), but functional demonstration from gene depletion of signaling molecules in ECs is missing to date. Here, we show that depletion of the vasculature-factor Alk1 increases BMP4 secretion from ECs, which delays HFSC activation. Furthermore, while previous evidence suggests a lymphatic vessel role in adult HFSC activation possibly through tissue drainage, a blood vessel role has not yet been addressed. Genetic perturbation of the ALK1-BMP4 axis in all ECs or the lymphatic ECs specifically unveils inhibition of HFSC activation by blood vessels. Our work suggests a broader relevance of blood vessels, adding adult HFSCs to the EC functional repertoire as signaling niches for the adult stem cells.

Binary organization of epidermal basal domains highlights robustness to environmental exposure.

Adulte interfollicular epidermis (IFE) renewal is likely orchestrated by physiological demands of its complex tissue architecture comprising spatial and cellular heterogeneity. Mouse tail and back skin display two kinds of basal IFE spatial domains that regenerate at different rates. Here, we elucidate the molecular and cellular states of basal IFE domains by marker expression and single-cell transcriptomics in mouse and human skin. We uncover two paths of basal cell differentiation that in part reflect the IFE spatial domain organization. We unravel previously unrecognized similarities between mouse tail IFE basal domains defined as scales and interscales versus human rete ridges and inter-ridges, respectively. Furthermore, our basal IFE transcriptomics and gene targeting in mice provide evidence supporting a physiological role of IFE domains in adaptation to differential UV exposure. We identify Sox6 as a novel UV-induced and interscale/inter-ridge preferred basal IFE-domain transcription factor, important for IFE proliferation and survival. The spatial, cellular, and molecular organization of IFE basal domains underscores skin adaptation to environmental exposure and its unusual robustness in adult homeostasis.

High-resolution single-cell transcriptomics reveals heterogeneity of self-renewing hair follicle stem cells.

Multipotent bulge stem cells (SCs) fuel the hair follicle (HF) cyclic growth during adult skin homeostasis, but their intrinsic molecular heterogeneity is not well understood. These hair follicle stem cells (HFSCs) engage in bouts of self-renewal, migration and differentiation during the hair cycle. Here, we perform high-resolution single-cell RNA sequencing (scRNA-seq) of HFSCs sorted as CD34+ /K14-H2BGFP+ from mouse skin at mid-anagen, the self-renewal stage. We dissect the transcriptomic profiles and unravel population-specific transcriptional heterogeneity. Unsupervised clustering reveals five major HFSC populations, which distinguished by known markers associated with both the bulge and the outer root sheath (ORS) underneath. These populations include quiescent bulge, ORS cellular states and proliferative cells. Lineage trajectory analysis predicted the prospective differentiation path of these cellular states and their corresponding self-renewing subpopulations. The bulge population itself can be further sub-divided into distinct subpopulations that can be mapped to the upper, mid and lower bulge regions, and present a decreasing quiescence score. Gene set enrichment analysis (GSEA) revealed new markers and suggested potentially distinct functions of the ORS and bulge subpopulations. This included communications between the upper bulge subpopulation and sensory nerves and between the upper ORS and skin vasculature, as well as enrichment of a bulge subset in cell migratory functions. The lower ORS enriched genes may potentially enable nutrients passing from the surrounding fat and vasculature cells towards the proliferating hair matrix cells. Thus, we provide a comprehensive account of HFSC molecular heterogeneity during their self-renewing stage, which enables future HF functional studies.

Publisher Correction: Secretory phospholipase A2-IIA overexpressing mice exhibit cyclic alopecia mediated through aberrant hair shaft differentiation and impaired wound healing response.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Context-dependent effect of sPLA2-IIA induced proliferation on murine hair follicle stem cells and human epithelial cancer.

BACKGROUND: Tissue stem cells (SCs) and cancer cells proliferation is regulated by many common signalling mechanisms. These mechanisms temporally balance proliferation and differentiation events during normal tissue homeostasis and repair. However, the effect of these aberrant signalling mechanisms on the ultimate fate of SCs and cancer cells remains obscure. METHODS: To evaluate the functional effects of Secretory Phospholipase A2-IIA (sPLA2-IIA) induced abnormal signalling on normal SCs and cancer cells, we have used K14-sPLA2-IIA transgenic mice hair follicle stem cells (HFSCs), DMBA/TPA induced mouse skin tumour tissues, human oral squamous cell carcinoma (OSCC) and skin squamous cell carcinoma (SCC) derived cell lines. FINDINGS: Our study demonstrates that sPLA2-IIA induces rapid proliferation of HFSCs, thereby altering the proliferation dynamics leading to a complete loss of the slow cycling H2BGFP positive HFSCs. Interestingly, in vivo reversion study by JNK inhibition exhibited a significant delay in post depilation hair growth, confirming that sPLA2-IIA promotes HFSCs proliferation through JNK/c-Jun signalling. In a different cellular context, we showed increased expression of sPLA2-IIA in human OSCC and mouse skin cancer tissues. Importantly, a xenograft of sPLA2-IIA knockdown cells of OSCC and SCC cell lines showed a concomitant reduction of tumour volume in NOD-SCID mice and decreased JNK/c-Jun signalling. INTERPRETATION: This study unravels how an increased proliferation induced by a common proliferation inducer (sPLA2-IIA) alters the fate of normal SCs and cancer cells distinctively through common JNK/c-Jun signalling. Thus, sPLA2-IIA can be a potential target for various diseases including cancer. FUND: This work was partly supported by the Indian Council of Medical Research (ICMR-3097) and ACTREC (42) grants.

Establishment and characterization of novel human oral squamous cell carcinoma cell lines from advanced­stage tumors of buccal mucosa.

Oral squamous cell carcinoma (OSCC) is a leading cause of mortality in India owing to the high percentage of tobacco chewers, smokers and alcohol consumption. OSCC is highly heterogeneous in nature; therefore poses a challenge in the treatment of the patient. To better understand the heterogeneity of the tumors, an in vitro cell line model is required. However, the efficiency of establishing cell lines from the oral tumors is low. In the present study, three novel cell lines, namely ACOSC3, ACOSC4, and ACOSC16, were isolated and characterized from advanced­stage treatment­naive OSCCs originating from the buccal mucosa. The three cell lines exhibited polygonal morphology, which is typical of epithelial cells. Furthermore, immunofluorescence revealed the expression of keratins 8 and 14, thereby confirming the epithelial origin of the cells. DNA content analysis of the three OSCC cell lines revealed aneuploidy. Furthermore, an in vitro orosphere assay revealed the formation of primary orospheres. Notably, the OSCC cell lines were able to give rise to tumors when administered subcutaneously into non­obese diabetic/severe combined immune deficiency mice. The novelty of the cell lines was also validated by performing short tandem repeat profiling; the STR profiles of the present cell lines did not significantly match with any known established OSCC cell lines present in the DSMZ database, thereby confirming the unique identity of these lines. These cell lines established from tumor samples derived from Indian OSCC patients provide a valuable resource to understand the molecular mechanism involved in tumor resistance and recurrence.

Epigenetic control in skin development, homeostasis and injury repair.

Cell-type- and cell-state-specific patterns of covalent modifications on DNA and histone tails form global epigenetic profiles that enable spatiotemporal regulation of gene expression. These epigenetic profiles arise from coordinated activities of transcription factors and epigenetic modifiers, which result in cell-type-specific outputs in response to dynamic environmental conditions and signalling pathways. Recent mouse genetic and functional studies have highlighted the physiological significance of global DNA and histone epigenetic modifications in skin. Importantly, specific epigenetic profiles are emerging for adult skin stem cells that are associated with their cell fate plasticity and proper activity in tissue regeneration. We can now begin to draw a more comprehensive picture of how epigenetic modifiers orchestrate their cell-intrinsic role with microenvironmental cues for proper skin development, homeostasis and wound repair. The field is ripe to begin to implement these findings from the laboratory into skin therapies.

Secretory phospholipase A2-IIA overexpressing mice exhibit cyclic alopecia mediated through aberrant hair shaft differentiation and impaired wound healing response.

Secretory phospholipase A2 Group-IIA (sPLA2-IIA) is involved in lipid catabolism and growth promoting activity. sPLA2-IIA is deregulated in many pathological conditions including various cancers. Here, we have studied the role of sPLA2-IIA in the development of cyclic alopecia and wound healing response in relation to complete loss of hair follicle stem cells (HFSCs). Our data showed that overexpression of sPLA2-IIA in homozygous mice results in hyperproliferation and terminal epidermal differentiation followed by hair follicle cycle being halted at anagen like stage. In addition, sPLA2-IIA induced hyperproliferation leads to compl pathological conditions including various cancers. Here ete exhaustion of hair follicle stem cell pool at PD28 (Postnatal day). Importantly, sPLA2-IIA overexpression affects the hair shaft differentiation leading to development of cyclic alopecia. Molecular investigation study showed aberrant expression of Sox21, Msx2 and signalling modulators necessary for proper differentiation of inner root sheath (IRS) and hair shaft formation. Further, full-thickness skin wounding on dorsal skin of K14-sPLA2-IIA homozygous mice displayed impaired initial healing response. Our results showed the involvement of sPLA2-IIA in regulation of matrix cells differentiation, hair shaft formation and complete loss of HFSCs mediated impaired wound healing response. These novel functions of sPLA2-IIA may have clinical implications in alopecia, cancer development and ageing.

sPLA2 -IIA Overexpression in Mice Epidermis Depletes Hair Follicle Stem Cells and Induces Differentiation Mediated Through Enhanced JNK/c-Jun Activation.

Secretory phospholipase A2 Group-IIA (sPLA2 -IIA) catalyzes the hydrolysis of the sn-2 position of glycerophospholipids to yield fatty acids and lysophospholipids. sPLA2 -IIA is deregulated in various cancers; however, its role in hair follicle stem cell (HFSC) regulation is obscure. Here we report a transgenic mice overexpressing sPLA2 -IIA (K14-sPLA2 -IIA) showed depletion of HFSC pool. This was accompanied with increased differentiation, loss of ortho-parakeratotic organization and enlargement of sebaceous gland, infundibulum and junctional zone. The colony forming efficiency of keratinocytes was significantly reduced. Microarray profiling of HFSCs revealed enhanced level of epithelial mitogens and transcription factors, c-Jun and FosB that may be involved in proliferation and differentiation. Moreover, K14-sPLA2 -IIA keratinocytes showed enhanced activation of EGFR and JNK1/2 that led to c-Jun activation, which co-related with enhanced differentiation. Further, depletion of stem cells in bulge is associated with high levels of chromatin silencing mark, H3K27me3 and low levels of an activator mark, H3K9ac suggestive of alteration in gene expression contributing toward stem cells differentiation. Our results, first time uncovered that overexpression of sPLA2 -IIA lead to depletion of HFSCs and differentiation associated with altered histone modification. Thus involvement of sPLA2 -IIA in stem cells regulation and disease pathogenesis suggest its prospective clinical implications. Stem Cells 2016;34:2407-2417.