Hair follicle patterning

Individual mammals can produce different types of hairs that share basic architectural principles. In the dorsal skin of mice, HF development occurs in three successive waves: (1) large, straight, primary guard or tylotrich hairs, which comprise 1–3% of total hairs and have two sebaceous glands, form on embryonic day 14.5; (2) secondary awl (straight, ~30% of the coat) and auchene (one bend, ~0.1% of the coat) hairs form on embryonic day 16.5; and (3) tertiary zigzag hairs, which have several bends and account for ~70% of the coat, form on embryonic day 18.5. The mouse coat therefore contains four distinct hair types, in addition to specialized hairs such as vibrissae (whiskers) and tail hairs . EDAR is necessary for placode development in primary HFs but not for induction of secondary HFs, which utilize signaling pathways that involve Noggin and SRY-box 18 (SOX18) expression within the dermal papillae .

In humans, hypohidrotic ectodermal dysplasia (HED) with abnormal morphogenesis of scalp hair, sweat glands, and teeth results from mutations in the genes encoding components of the EDAR/NF-κB signaling pathway, including EDAR, its ligand ectodysplasin A, and the downstream effector EDAR-associated death domain . In addition, a non-synonymous single nucleotide polymorphism (SNP) within EDAR is associated with increased hair thickness in Asian populations . Deep deformations in the hair shaft were identified in Swedish patients with an autosomal dominant form of HED due to an EDAR mutation . These clinical observations underscore the importance of the EDAR/NF-κB signaling pathway in human appendage development. Interestingly, mutations in human SOX18 cause lymphedema and telangiectasias as well as hypotrichosis .

The hairs of the mouse dorsal coat have anterior–posterior polarity along the body axis, which contributes to formation of a sleek coat. SHH and neural cell adhesion molecule (NCAM) within the HF have an asymmetric distribution pattern, which is established by a conserved planar cell polarity (PCP) pathway during hair germ development . PCP proteins Van Gogh-like 2 (VANGL2) and cadherin epidermal growth factor laminin A seven-pass G-type receptor 1 (CELSR1) respond to directional cues and are necessary for the correct polarization of follicles within the skin . Mutations in Vangl2 or the frizzled family receptor 6 ( Fzd6 ) affect the early global alignment of HFs, resulting in germs that first grow vertically. Over time the follicles reorient themselves in competing patterns, producing characteristic whorls on the back of the mouse . In Fzd6 ^−/− mice, an additional mutation in Astrotactin2 ( Astn2 ), which has roles in neuronal migration, leads to a 180-degree reorientation of the dorsal coat to a posterior–anterior polarity . Hair whorls are frequently observed in mammals, such as on the occipital scalp in humans, between the eyes in horses and cattle, and on the upper chest in dogs .

Under physiologic conditions, human HF morphogenesis occurs only once. Lanugo, vellus, and terminal hairs ( Table 68.1 ) have the same basic architecture. The first “coat” is composed of fine, long, variably pigmented lanugo hair, which is shed in an anterior-to-posterior wave during the third trimester. A second coat of fine, shorter, unpigmented lanugo hair then grows in all areas except the scalp and is shed 3–4 months after birth; pigmented scalp hairs, which have variable lengths and diameters, are also shed postnatally in an anterior-to-posterior wave. Thereafter, the hair grows in an asynchronous “mosaic” pattern rather than waves. In prepubertal children, hair is terminal on the scalp, eyelashes and eyebrows, while it is vellus on the face, trunk, and extremities. During puberty, vellus hairs in some sites become terminal hairs under the influence of androgens. Paradoxically, terminal hairs on the scalp exposed to the same androgens revert to miniaturized vellus-like hairs in androgenetic alopecia.

Congenital generalized hypertrichosis (Ambras syndrome) is characterized by a disruption in the patterning of hairs, with terminal hairs growing at sites that normally have vellus follicles . A position effect that down-regulates the expression of the trichorhinophalangeal syndrome I gene ( TRPS1 ) was found to underlie this hypertrichosis phenotype in both humans and mice . In addition, X-linked congenital generalized hypertrichosis in a Mexican family was associated with a position effect that decreased the expression of FGF13 .

In most mammals, the hair pattern differs depending on the body site. While the ectoderm involved in skin and hair development is non-neural and relatively homogeneous, the mesenchyme has various origins which may explain this regionalization. The dorsal dermis is derived from the somitic dermomyotome, the ventral dermis has a somatopleural origin, and the craniofacial dermis arises from the neural crest . The dermal progenitors at each of these sites are specified by different signaling pathways. Mice display regional differences in skin thickness as well as hair color and type. The postnatal identity of their skin is acquired by embryonic day 12.5, with the T-box 15 (TBX15) transcription factor appearing within dorsal skin to demarcate the dorsal–ventral boundary .

In humans, the effects of androgens on HFs depend upon the body site. Follicles in the frontal scalp are normally androgen-dependent, whereas occipital scalp follicles are hormonally insensitive and therefore less susceptible to androgenetic alopecia. When HFs from the occipital scalp are transplanted to the frontal scalp, they typically retain the pigmentary characteristics, androgen sensitivity, and growth rate of the donor site . However, in other instances, transplanted hairs take on characteristics of the recipient site, such as scalp hairs moved into the eyebrow region. It remains to be established exactly which characteristics are more influenced by the recipient site versus the donor site following transplantation.

Postnatal hair follicle development

Although HF formation in general occurs prior to birth, adult mammalian skin retains the capacity for HF morphogenesis. In adult mouse and rabbit skin, WNT-dependent de novo HF formation occurs after wounding ; this is mediated by FGF-9 secreted by infiltrating γδ-T cells and wound fibroblasts . It is therefore reasonable to consider possible therapeutic interventions that would initiate de novo HF formation on the human scalp. Trans-species end-bulb formation and hair fiber growth has been demonstrated after implantation of intact human dermal papillae onto transected mouse whisker follicles . In humans, allogeneic transplantation of tissue from the dermal sheath that surrounds the dermal papilla can induce new follicle formation in the recipient, with immune privilege providing protection from rejection . In addition, cultured tooth mesenchymal papilla cells from humans and rats have been shown to induce hair fiber formation in an amputated follicle, highlighting the conservation of inductive signals among different adnexa .

Dermal papilla cells in rodents show much more inductive potency than their human counterparts. Rodent HF papilla cells can reprogram differentiated glabrous epidermis to produce a de novo HF, and they are also capable of initiating trans-differentiation of amnion or even adult corneal epithelium into epidermis complete with HFs . Human dermal papilla cells lose their inductive properties quickly in a standard monolayer culture, which hampers the utilization of these cells for hair regeneration. However, dermal papilla cells grown in a three-dimensional spheroid culture have been shown to retain their ability to induce hair growth in glabrous skin, suggesting that cellular interactions are critical to maintenance of dermal papilla identity .

Anagen

Anagen is the period of active hair fiber production, and its duration varies depending upon factors such as species and body site ( Table 68.3 ). Anagen is divided into six substages, anagen I–VI; it lasts 1–3 weeks in mice and up to several years on the human scalp , explaining the longer lengths of human scalp hair. The start of anagen (anagen I), as observed in mouse skin, is associated with an increased size of the dermal papilla, due mainly to an influx of cells from the surrounding dermal sheath. Dermal sheath cells at the base of the follicle also contribute to dermal papilla regeneration after removal of the lower follicle . The influx of cells at the start of anagen is accompanied by an increase in ECM material. This doubles the size of the dermal papilla , the width of which is directly proportional to the thickness of the hair shaft produced by the follicle. The degree of axial symmetry within the hair bulb determines the curvature of the final hair structure .

Table 68.3

Basic information on human hair follicles (HFs).

 

The dermal papilla provides signals to the surrounding matrix cells, whose position with respect to the dermal papilla specifies their fate, e.g. central cells become the medulla . As the matrix cells begin to differentiate, they form precursor cells of the different follicle lineages. Migrating upwards, these cells undergo specialized programs of differentiation, expressing defined sets of keratins to form seven concentric epithelial lineages of the follicle, from the medulla to the companion layer ( Figs 68.5 & 68.6 ). In contrast, the outer root sheath (ORS), which is the outermost layer of the epidermal portion of the follicle, does not originate from upward growth of matrix cells, but rather directly from the bulge (see below) . The growing HF ends up nestled in the subcutaneous fat by anagen VI. In the mouse, dermal adipocyte precursors secrete platelet-derived growth factor-A (PDGF-A), which acts on the dermal papilla to support anagen .

Terminal human hair follicle in anagen VI.

The expression of keratins in the hair follicle.

Hair keratins are in red font. KRT, keratin.

In rodents and rabbits, plucking telogen hairs can accelerate onset of the next anagen phase, and this has been utilized to study the mammalian hair cycle in a controlled manner. The trauma of plucking causes release of chemokine C-C motif ligand 2 (CCL2) by the follicle, leading to recruitment of macrophages that secrete tumor necrosis factor (TNF), which promotes local follicles to enter anagen . Other microenvironmental cues and “quorum-sensing” among HFs also likely play a role in hair cycle coordination.

Catagen

At the end of anagen, the follicle enters the regressive catagen phase, which is divided into eight substages (catagen I–VIII) . Catagen lasts ~2 weeks in humans, regardless of the site and follicle type . The initiation of catagen is characterized by a nearly 50% reduction in the volume of the dermal papilla due to loss of ECM and migration of cells into the surrounding dermal sheath, which acts as a repository that replenishes the dermal papilla in the subsequent anagen . Melanocytes in the hair bulb also down-regulate melanogenesis. Later, there is cessation of mitotic activity within the matrix cells of the bulb, and the remaining hair precursor cells at the base of the shaft differentiate to form a “club fiber”.

The club fiber migrates up the follicle, and a germ capsule termed the trichilemmal sac forms around its base. This club fiber lacks pigmentation and is formed from cortical and cuticle cells only. The electron-dense, non-nucleated layer of trichilemmal keratin around its base has a characteristic “brush” structure that protrudes outwards and between surrounding ORS cells . Abundant desmosomes between the trichilemmal keratin and surrounding tissue are thought to physically anchor the club fiber within its trichilemmal sac until it receives the signal for release .

As the club fiber moves up the follicle, the dermal papilla, which is initially still in the subcutaneous fat, becomes separated from the club by an epithelial strand that is surrounded by basement membrane (the glassy membrane) . Apoptosis in the epithelial strand creates an “apoptotic force” that draws the dermal papilla up to just beneath the club fiber in the dermis, leaving behind a trailing dermal sheath called the fibrous streamer or stele . FGF-5 produced by macrophage-like cells facilitates regression of human HFs , and FGF5 mutations underlie familial trichomegaly characterized by abnormally long eyelashes . Macrophages also participate in the clearance of cellular debris during catagen .

Loss-of-function mutations in the hairless gene ( HR ) can result in atrichia with papular lesions (APL) , a rare autosomal recessive disease characterized by complete hair loss early in life and later eruption of papular lesions. Hairless is a transcription factor that is required for apoptosis in the epithelial strand, which draws the dermal papilla up beneath the club fiber and secondary hair germ in a position to activate the next anagen phase. Thus, individuals with APL are born with hair, which never grows back after it is shed. Activating mutations in the upstream open reading frame of HR lead to another form of sparse hair, Marie Unna hypotrichosis . Hairless interacts with the vitamin D receptor (VDR), which has ligand-independent functions in hair cycle control . Patients with hereditary vitamin D-resistant rickets caused by VDR mutations present with alopecia that is not corrected by vitamin D supplementation .

Telogen

Telogen begins when the dermal papilla reaches the base of the permanent portion of the HF. The duration of telogen can be as short as 2 days in mice and as long as 3 months for human scalp hairs. The hair germ is derived from the bulge during the catagen-to-telogen transition and lies inferior to the bulge, separating the latter from the dermal papilla . As in HF morphogenesis, epithelial–mesenchymal cross-talk is required to activate the new anagen phase. Anagen progression recapitulates the middle and later stages of HF development, and many factors that drive follicle morphogenesis (e.g. WNT, SHH, noggin) also play important roles in telogen-to-anagen transition ( Fig. 68.4B ). The bulge is relatively quiescent during telogen, traditionally referred to as the “resting” phase, due to inhibitory signals (e.g. FGF-18, BMP-6) from keratin 6-positive inner bulge cells . The apparent dormancy of the HF during telogen may be an energy-efficient adaptation allowing for the maintenance of stem cells in preparation for the next anagen phase .

The macro-environment surrounding the HF also takes part in regulating cycle transitions . BMPs in the subcutaneous fat maintain HFs in a “refractory” telogen, and cessation of this inhibitory signal enables the follicle to progress to a “competent” telogen with a hair germ (biochemically distinct from the bulge) that is responsive to anagen-initiation signals and capable of entering a new anagen phase . Fluctuations of microRNA levels (e.g. microRNA-31) in the follicle and its macro-environment may also silence certain gene expression patterns, promoting a switch from telogen to anagen . After secondary hair germ activation, the germ cells begin to contribute to the hair matrix and concentric layers of the follicle via an upward movement, while bulge cells generate the ORS through a downward movement . In follicles with a longer anagen duration, the proliferative capacity of the matrix is not sufficient to sustain growth. The “stem cell trafficking theory” proposes that a continuous migration of precursor cells from the bulge down the ORS prevents exhaustion of the germinative matrix during anagen .

Exogen

The stimulus for entry into catagen, and hence the formation of a club hair, typically occurs several months before the hair is shed in humans . Shedding of overhair is loosely coupled with anagen in mouse pelage follicles , while club fiber release is a very predictable event in rat whisker follicles. In the latter instance, the club fiber is retained through the following anagen and then shed prior to the onset of catagen in a regulated manner . In humans, the club fiber is not usually retained through subsequent anagens; instead, it is believed to be shed in the telogen to anagen transition. The term “exogen” was introduced into the literature in 1998 to represent the shedding of the club fiber (teloptosis) as well as the preceding signals and structural changes in the follicle . Exogen is considered to be an active proteolytic event rather than a passive process where the club fiber is dislodged due solely to mechanical forces from the new growing fiber. A gradual decrease in the adhesion of the club fiber within its epithelial silo has been observed prior to its programmed release from the follicle .

Structure of the hair fiber

In response to signals from the dermal papilla, the matrix cells at the base of the follicle proliferate and differentiate during anagen, forming the layers of the follicle. The three central lineages – medulla, cortex, and hair cuticle – form the hair fiber, which fulfills the primary hair-producing function of the anagen follicle. Surrounding the hair fiber are the three concentric layers that compose the inner root sheath (IRS): the IRS cuticle, Huxley’s layer, and Henle’s layer. The IRS undergoes a complex program of differentiation during anagen, with Henle’s layer terminally differentiating shortly after formation. The IRS cuticle differentiates further up the follicle, followed by Huxley’s layer; all three layers undergo desmosomal remodeling during terminal differentiation . The IRS forms a mechanically supportive tube around the hair fiber and is degraded by proteolytic enzymes at the level of the isthmus , allowing the hair shaft to enter the hair canal and emerge from the skin surface. Heparanase expressed in the IRS during anagen acts on heparin sulfate proteoglycans and controls IRS differentiation by influencing growth factor sequestration and diffusion .

The seventh concentric layer that originates from the matrix in line with the hair fiber and IRS is the companion layer, which is a single cell thick. Regarded as the “slippage plane” between the IRS and the surrounding ORS during anagen, the companion layer enables directional hair growth. It is attached to the IRS through “Flugelzellen” structures that extend from Huxley’s layer through Henle’s layer and link to the companion layer via numerous desmosomes . A “barrel” of transversely oriented intermediate filaments on the inner side of the companion layer are thought to provide support for the inner root sheath, protecting it from the compressive forces of the expanding ORS .