Foxi3 Deficiency Compromises Hair Follicle Stem Cell Specification and Activation

Abstract

The hair follicle is an ideal system to study stem cell specification and homeostasis due to its well characterized morphogenesis and stereotypic cycles of stem cell activation upon each hair cycle to produce a new hair shaft. The adult hair follicle stem cell niche consists of two distinct populations, the bulge and the more activation-prone secondary hair germ (HG). Hair follicle stem cells are set aside during early stages of morphogenesis. This process is known to depend on the Sox9 transcription factor, but otherwise the establishment of the hair follicle stem cell niche is poorly understood. Here, we show that that mutation of Foxi3, a Forkhead family transcription factor mutated in several hairless dog breeds, compromises stem cell specification. Further, loss of Foxi3 impedes hair follicle downgrowth and progression of the hair cycle. Genome-wide profiling revealed a number of downstream effectors of Foxi3 including transcription factors with a recognized function in hair follicle stem cells such as Lhx2, Runx1, and Nfatc1, suggesting that the Foxi3 mutant phenotype results from simultaneous downregulation of several stem cell signature genes. We show that Foxi3 displays a highly dynamic expression pattern during hair morphogenesis and cycling, and identify Foxi3 as a novel secondary HG marker. Absence of Foxi3 results in poor hair regeneration upon hair plucking, and a sparse fur phenotype in unperturbed mice that exacerbates with age, caused by impaired secondary HG activation leading to progressive depletion of stem cells. Thus, Foxi3 regulates multiple aspects of hair follicle development and homeostasis. STEM CELLS 2016;34:1896-1908 SIGNIFICANCE STATEMENTAdult tissue-specific stem cells are intensively studied, but how they are established is poorly understood for most tissues, including the hair follicle. A profound knowledge of stem cell origins is an essential step not only for understanding tissue homeostasis, but also for diseases where stem cell behavior is misregulated such as cancer, as well as for regenerative biology. Here, we identify a critical role for transcription factor Foxi3 in hair follicle stem cell niche establishment. We show that Foxi3 integrates cues that govern hair follicle morphogenesis and stem cell specification during embryogenesis, and morphogenesis and stem cell activation during regeneration.

Functional anatomy of the hair follicle: The Secondary Hair Germ

“…Gene expression profiling of embryonic skin in these mice has revealed downregulation of a number of targets expressed concurrently in both the bulge and the SHG of the telogen HF, for example Sox9, Lhx2 and K15 (Table ; Figure E). Downregulation of markers differentially labelling these two HF cell populations (Runx1 and hr for the SHG and Nfatc1 for the bulge) was also observed . Given the exclusive expression of Foxi3 in the SHG of the telogen follicles, downregulation of bulge‐specific Nfatc1 suggests a possibility of SHG influence upon bulge cells.…”

“…Most recently, Foxi3, a transcription factor of the forkhead family and a target of ectodysplasin A (Eda) pathway in hair placodes, was identified as a specific marker of the telogen SHG . Foxi3‐deficiency in mice, generated using K14‐driven Cre recombination, results in abrogation of HF anagen downgrowth and impediment of the hair cycle.…”

“…All in all, the currently prevailing view is that during telogen, the HF stem cell pool is composed of two compartments—the bulge and the SHG . Consequently, the SHG is simply considered as a “second stem cell population” [eg] or, more specifically, as a population of “primed stem cells” . At the same time, the functional rationale for the existence of two stem cell populations next to each other is usually not discussed ( see sections 2.1 and 5 on SHG morphology and origin ).…”

“…Sfrp1 is a Hedgehog target which restrains canonical Wnt signalling in stem or progenitor cells . It is noteworthy that another general Wnt inhibitor, Dkk4 , is among the primary negative targets of Foxi3, which is specifically expressed in the SHG and not found in the bulge . Presumptive deficiency of Wnt‐inhibiting activity in the telogen SHG may provide further evidence that this HF cell population does not fit the classical gene expression signature of “stemness” ( see also Supplementary text ).…”

“…Thus, the SHG was brought back into relevance, especially in light of growing interest in HF stem cell kinetics. During recent years, a number of studies have been published addressing the role of SHG in anagen induction, in maintenance of the HF stem cell niche and in restoration of HF structure after its damage [eg]. Nevertheless, so far, there has been only one systemic attempt to elucidate the structural, functional and expressional nature of this cell population .…”