Canonical WNT Signalling Controls Hair Follicle Spacing

Canonical WNT signals play an important role in hair follicle development. In addition to being crucial for epidermal appendage initiation, they control the interfollicular spacing pattern and contribute to the spatial orientation and largely parallel alignment of hair follicles. However, owing to the complexity of canonical WNT signalling and its interconnections with other pathways, many details of hair follicle formation await further clarification. Here, we discuss the recently suggested reaction-diffusion (RD) mechanism of spatial hair follicle arrangement in the light of yet unpublished data and conclusions. They clearly demonstrate that the observed hair follicle clustering in dickkopf (DKK) transgenic mice cannot be explained by any trivial process caused by protein overexpression, thereby further supporting our model of hair follicle spacing. Furthermore, we suggest future experiments to challenge the RD model of spatial follicle arrangement.Key Words: hair follicle, pattern formation, WNT, DKK, KRM, LRPIn order to stimulate the canonical WNT signalling pathway, members of the WNT protein family have to bind to their cognate Frizzled receptors as well as to a co-receptor encoded by Lrp5 and 6, respectively.^1–3 Pathway activation is competitively inhibited by soluble WNT binding proteins such as secreted frizzled related proteins (SFRPs).^4,5 Moreover, members of the DKK family bind non-competitively to LRPs;^6,7 simultaneous interaction with Kremen (KRM) 1 or 2 causes depletion of WNT co-receptors from the cell surface, thereby inhibiting canonical WNT signals.⁸Based on previous findings concerning the importance of WNT signalling in hair follicle initiation and orientation,^9,10 we recently hypothesised that the pathway may also have an essential role in the spatial arrangement of follicles. Using a combined experimental and computational modelling approach, we provided evidence for WNTs and DKKs controlling interfollicular spacing through a reaction-diffusion mechanism (Fig. 1).¹¹ By confirming the prediction of hair follicle clustering in the presence of moderate DKK overexpression, we demonstrated the biological implementation of a fundamental principle of pattern formation the mathematical basis of which has been described by Alan Turing in the 1950s.¹²Open in a separate windowFigure 1Schematic of early hair follicle development in mouse and the hypothesised distribution of WNTs and DKKs as the critical regulators of interfollicular spacing. According to the RD hypothesis of Alan Turing, patterning starts with an almost even distribution of activator and inhibitor (solid and dashed lines, respectively) (A, bottom). Transferring the model to murine hair follicle morphogenesis, this molecular pattern is associated with a morphologically unstructured epidermis (A, top); (the underlying dermis is indicated by black spots). Of note, WNTs and DKKs were recently suggested to represent an activator/inhibitor pair in follicle development. In the RD model, small fluctuations in the initially even protein distribution are enhanced and, eventually, give rise to a distinct and stable pattern of activator and inhibitor distribution (B, bottom). This is mainly achieved by an activator controlling expression of its own as well of the inhibitor, an inhibitor antagonising the activator''s action, and an increased mobility of the inhibitor as compared to the activator. Although protein distribution in the developing skin is still hypothetical, the predicted pattern could control hair follicle morphogenesis, the first sign of which are epithelial thickenings (B, top). They stimulate the formation of dermal condensates which become dermal papillae later on. Of note, interfollicular spacing is solely determined by the parameters of the underlying RD mechanism. Hence, upon embryo growth, areas in between previously formed follicles again become capable of hair follicle formation owing to local protein levels (C). While the Turing model cannot describe this transitional state, it clearly predicts the formation of new follicles after enlargement of the interfollicular space (D); without changing the underlying parameters, the RD mechanism generates a fixed spacing pattern. Indeed, hair follicle development in mouse does occur by consecutive inductive waves.Unexpectedly, DKK2 was capable of stimulating the WNT pathway in the absence of Krm2 expression.¹³ As discussed by Stark et al., this finding raises the possibility that transgenic overexpression of Dkk2 in our Foxn1::Dkk2 mice may directly activate the canonical WNT pathway.¹⁴ Thus, since stabilised β-catenin is sufficient for follicle formation,¹⁵ new appendages may be initiated adjacent to previously formed, Dkk2 expressing follicles, if their neighborhood lacks KRM protein.Indeed, during early hair follicle development, interfollicular epidermis shows only weak Krm2 expression as compared to follicle buds (Fig. 2). Moreover, at more advanced stages, the distal part of emerging follicles may even lack any Krm2 gene activity. However, although Krm1 is also predominantly expressed in the developing hair bulb, moderate gene activity is found throughout the epidermis and the distal part of hair follicles (Fig. 3). Hence, developing follicles and their neighborhood do not represent a KRM-negative compartment. As a consequence, hair follicle induction by DKK2-mediated stimulation of the canonical WNT pathway is very unlikely.Open in a separate windowFigure 2Expression of Krm2 during early hair follicle development, demonstrated by non-radioactive in situ hybridisation. Bars, 100 µm.Open in a separate windowFigure 3Expression of Krm1 during early hair follicle development, demonstrated by non-radioactive in situ hybridisation. Bars, 100 µm.In contrast to DKK2, DKK1 is unable to stimulate the canonical WNT pathway.^16,17 This difference could be attributed to the amino-terminal domain. To investigate whether transgenic DKK2 may cause hair follicle clustering just by pathway activation, we generated Foxn1::Dkk1 transgenic mice. However, they showed essentially the same phenotype as Foxn1::Dkk2 animals.¹¹ Moreover, transgenic mice expressing amino-terminally truncated DKK1 protein were largely indistinguishable from Foxn1::Dkk1 animals instead of showing an enhanced patterning abnormality (data not shown).If direct stimulation of the canonical WNT signalling pathway by transgenic DKKs would be responsible for the severely altered spatial arrangement of hair follicles, increasing transgene expression should at least preserve or even enhance hair follicle clustering, while the distances between clusters may increase. However, mice with particularly strong Dkk2 transgene expression did not show hair follicle clusters but single, well-developed follicles with large interfollicular distances.¹¹In summary, our data do strongly argue against hair follicle clustering in transgenic mice by DKK-mediated activation of the WNT pathway. By contrast, all data are in line with the recently suggested RD model of hair follicle spacing.Nevertheless, several questions remain to be answered. First, the identity of the WNT protein(s) involved in interfollicular patterning is unknown. Second, the contribution of the inhibitors DKK1 and DKK4 both of which are expressed during hair follicle initiation is still a matter of debate. In the light of multiple WNTs being expressed during early hair follicle morphogenesis,¹⁸ some redundancy appears to be likely. Hence, the effects of single gene knockouts may be limited and transgenic approaches with their intrinsic capability of dramatically changing overall WNT levels may be favourable to challenge the RD model and to identify the WNT family members that are involved in the patterning process. Likewise, gene inactivation of either Dkk1 or Dkk4 would be insufficient to provide further support for our model. In principle, the inhibitor(s) may be crucial for follicle formation while they are not involved in the interfollicular patterning process. By contrast, experimentally lowering the inhibitors'' mobility should unequivocally support or disprove the proposed mechanism of hair follicle spacing. According to the underlying mathematical model, it should dramatically affect patterning in the presence of normal levels of functional protein.     

Dietary vitamin A regulates wingless-related MMTV integration site signaling to alter the hair cycle

Alopecia areata (AA) is an autoimmune hair loss disease caused by a cell-mediated immune attack of the lower portion of the cycling hair follicle. Feeding mice 3–7 times the recommended level of dietary vitamin A accelerated the progression of AA in the graft-induced C3H/HeJ mouse model of AA. In this study, we also found that dietary vitamin A, in a dose dependent manner, activated the hair follicle stem cells (SCs) to induce the development and growth phase of the hair cycle (anagen), which may have made the hair follicle more susceptible to autoimmune attack. Our purpose here is to determine the mechanism by which dietary vitamin A regulates the hair cycle. We found that vitamin A in a dose-dependent manner increased nuclear localized beta-catenin (CTNNB1; a marker of canonical wingless-type Mouse Mammary Tumor Virus integration site family (WNT) signaling) and levels of WNT7A within the hair follicle bulge in these C3H/HeJ mice. These findings suggest that feeding mice high levels of dietary vitamin A increases WNT signaling to activate hair follicle SCs.     

Spatial patterns produced by a reaction-diffusion system in primary hair follicles

This paper is the third in a series examining the role of a reaction-diffusion (RD) system as the principal mechanism providing spatial information for cell differentiation during hair follicle initiation and development and hair fibre formation. A theoretical mechanism is described by which the RD system supplies positional information during hair follicle development. Solutions of the RD system within the primordial follicle are described as well as the sequence of spatial patterns provides the follicle/epidermis boundary conditions required to account for the density and grouping of follicles during initiation. At the same time the spatial patterns are also shown to be capable of providing the positional information which determines various geometrical aspects of follicle development; in particular the development of follicles at an angle to the skin surface and the initiation and location of sweat glands and sebaceous glands on the follicle.     

Characterization of Wnt gene expression in developing and postnatal hair follicles and identification of Wnt5a as a target of Sonic hedgehog in hair follicle morphogenesis

Mutations in WNT effector genes perturb hair follicle morphogenesis, suggesting key roles for WNT proteins in this process. We show that expression of Wnts 10b and 10a is upregulated in placodes at the onset of follicle morphogenesis and in postnatal hair follicles beginning a new cycle of hair growth. The expression of additional Wnt genes is observed in follicles at later stages of differentiation. Among these, we find that Wnt5a is expressed in the developing dermal condensate of wild type but not Sonic hedgehog (Shh)-null embryos, indicating that Wnt5a is a target of SHH in hair follicle morphogenesis. These results identify candidates for several key follicular signals and suggest that WNT and SHH signaling pathways interact to regulate hair follicle morphogenesis.     

Localization of Shh expression by Wnt and Eda affects axial polarity and shape of hairs

Axial patterning is a recurrent theme during embryonic development. To elucidate its fundamental principles, the hair follicle is an attractive model due to its easy accessibility and dispensability. Hair follicle asymmetry is evident from its angling and the localization of associated structures. However, axial patterning is not restricted to the follicle itself but also generates rotational hair shaft asymmetry which, for zigzag hairs, generates 3-4 bends that alternately point into opposite directions. Here we show by analyzing mutant and transgenic mice that WNT and ectodysplasin signaling are involved in the control of the molecular and morphological asymmetry of the follicle and the associated hair shaft, respectively. Asymmetry is affected by polarized WNT and ectodysplasin signaling in mature hair follicles. When endogenous signaling is impaired, molecular asymmetry is lost and mice no longer form zigzag hairs. Both signaling pathways affect the polarized expression of Shh which likely functions as a directional reference for hair shaft production in all follicles. We propose that this regulatory pathway also establishes follicular asymmetry during morphogenesis. Moreover, the identified molecular hierarchy offers a model for the periodic patterning of zigzag hairs mechanistically similar to mesodermal segmentation.     

Comparative study on seasonal hair follicle cycling by analysis of the transcriptomes from cashmere and milk goats

Guard hair and cashmere undercoat are developed from primary and secondary hair follicle, respectively. Little is known about the gene expression differences between primary and secondary hair follicle cycling. In this study, we obtained RNA-seq data from cashmere and milk goats grown at four different seasons. We studied the differentially expressed genes (DEGs) during the yearly hair follicle cycling, and between cashmere and milk goats. WNT, NOTCH, MAPK, BMP, TGFβ and Hedgehog signaling pathways were involved in hair follicle cycling in both cashmere and milk goat. However, Milk goat DEGs between different months were significantly more than cashmere goat DEGs, with the largest difference being identified in December. Some expression dynamics were confirmed by quantitative PCR and western blot, and immunohistochemistry. This study offers new information sources related to hair follicle cycling in milk and cashmere goats, which could be applicable to improve the wool production and quality.     

WNT signals are required for the initiation of hair follicle development

Hair follicle morphogenesis is initiated by a dermal signal that induces the development of placodes in the overlying epithelium. To determine whether WNT signals are required for initiation of follicular development, we ectopically expressed Dickkopf 1, a potent diffusible inhibitor of WNT action, in the skin of transgenic mice. This produced a complete failure of placode formation prior to morphological or molecular signs of differentiation, and blocked tooth and mammary gland development before the bud stage. This phenotype indicates that activation of WNT signaling in the skin precedes, and is required for, localized expression of regulatory genes and initiation of hair follicle placode formation.     

Canonical WNT signaling promotes mammary placode development and is essential for initiation of mammary gland morphogenesis

Mammary glands, like other skin appendages such as hair follicles and teeth, develop from the surface epithelium and underlying mesenchyme; however, the molecular controls of embryonic mammary development are largely unknown. We find that activation of the canonical WNT/beta-catenin signaling pathway in the embryonic mouse mammary region coincides with initiation of mammary morphogenesis, and that WNT pathway activity subsequently localizes to mammary placodes and buds. Several Wnt genes are broadly expressed in the surface epithelium at the time of mammary initiation, and expression of additional Wnt and WNT pathway genes localizes to the mammary lines and placodes as they develop. Embryos cultured in medium containing WNT3A or the WNT pathway activator lithium chloride (LiCl) display accelerated formation of expanded placodes, and LiCl induces the formation of ectopic placode-like structures that show elevated expression of the placode marker Wnt10b. Conversely, expression of the secreted WNT inhibitor Dickkopf 1 in transgenic embryo surface epithelium in vivo completely blocks mammary placode formation and prevents localized expression of all mammary placode markers tested. These data indicate that WNT signaling promotes placode development and is required for initiation of mammary gland morphogenesis. WNT signals play similar roles in hair follicle formation and thus may be broadly required for induction of skin appendage morphogenesis.     

Modelling Hair Follicle Growth Dynamics as an Excitable Medium

The hair follicle system represents a tractable model for the study of stem cell behaviour in regenerative adult epithelial tissue. However, although there are numerous spatial scales of observation (molecular, cellular, follicle and multi follicle), it is not yet clear what mechanisms underpin the follicle growth cycle. In this study we seek to address this problem by describing how the growth dynamics of a large population of follicles can be treated as a classical excitable medium. Defining caricature interactions at the molecular scale and treating a single follicle as a functional unit, a minimal model is proposed in which the follicle growth cycle is an emergent phenomenon. Expressions are derived, in terms of parameters representing molecular regulation, for the time spent in the different functional phases of the cycle, a formalism that allows the model to be directly compared with a previous cellular automaton model and experimental measurements made at the single follicle scale. A multi follicle model is constructed and numerical simulations are used to demonstrate excellent qualitative agreement with a range of experimental observations. Notably, the excitable medium equations exhibit a wider family of solutions than the previous work and we demonstrate how parameter changes representing altered molecular regulation can explain perturbed patterns in Wnt over-expression and BMP down-regulation mouse models. Further experimental scenarios that could be used to test the fundamental premise of the model are suggested. The key conclusion from our work is that positive and negative regulatory interactions between activators and inhibitors can give rise to a range of experimentally observed phenomena at the follicle and multi follicle spatial scales and, as such, could represent a core mechanism underlying hair follicle growth.     

Multiple roles for elastic fibers in the skin.

Dermal elastic fibers are believed to have a primary role in providing elastic stretch and recoil to the skin. Here we compare the structural arrangement of dermal elastic fibers of chick skin and different animal species. Most elastic fibers in chick skin are derived from cells that line the feather follicle and/or smooth muscle that connects the pterial and apterial muscle bundles to feather follicles. Elastic fibers in the dermis of animals with single, primary hair follicles are derived from cells lining the hair follicle or from the ends of the pili muscle, which anchors the muscle to the matrix or to the hair follicle. Each follicle is interconnected with elastic fibers. Follicles of animals with primary and secondary (wool) hair follicles are also interconnected by elastic fibers, yet only the elastic fibers derived from the primary follicle are connected to each primary follicle. Only the primary hair follicles are connected to the pili muscle. Human skin, but not the skin of other primates, is significantly different from other animals with respect to elastic fiber organization and probably cell of origin. The data suggest that the primary role for elastic fibers in animals, with the possible exception of humans, is movement and/or placement of feathers or hair.     

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.     

Melatonin promotes Cashmere goat (Capra hircus) secondary hair follicle growth: a view from integrated analysis of long non-coding and coding RNAs

The role of melatonin in promoting the yield of Cashmere goat wool has been demonstrated for decades though there remains a lack of knowledge regarding melatonin mediated hair follicle growth. Recent studies have demonstrated that long non-coding RNAs (lncRNAs) are widely transcribed in the genome and play ubiquitous roles in regulating biological processes. However, the role of lncRNAs in regulating melatonin mediated hair follicle growth remains unclear. In this study, we established an in vitro Cashmere goat secondary hair follicle culture system, and demonstrated that 500 ng/L melatonin exposure promoted hair follicle fiber growth. Based on long intergenic RNA sequencing, we demonstrated that melatonin promoted hair follicle elongation via regulating genes involved in focal adhesion and extracellular matrix receptor pathways and further cis predicting of lncRNAs targeted genes indicated that melatonin mediated lncRNAs mainly targeted vascular smooth muscle contraction and signaling pathways regulating the pluripotency of stem cells. We proposed that melatonin exposure not only perturbed key signals secreted from hair follicle stem cells to regulate hair follicle development, but also mediated lncRNAs mainly targeted to pathways involved in the microvascular system and extracellular matrix, which constitute the highly orchestrated microenvironment for hair follicle stem cell. Taken together, our findings here provide a profound view of lncRNAs in regulating Cashmere goat hair follicle circadian rhythms and broaden our knowledge on melatonin mediated hair follicle morphological changes.     

Spatial and temporal expression of PORCN is highly dynamic in the developing mouse cochlea

The mammalian organ of Corti is a highly specialized sensory organ of the cochlea with a fine-grained pattern that is essential for auditory function. The sensory epithelium, the organ of Corti consists of a single row of inner hair cells and three rows of outer hair cells that are intercalated by support cells in a mosaic pattern. Previous studies show that the Wnt pathway regulates proliferation, promotes medial compartment formation in the cochlea, differentiation of the mechanosensory hair cells and axon guidance of Type II afferent neurons. WNT ligand expressions are highly dynamic throughout development but are insufficient to explain the roles of the Wnt pathway. We address a potential way for how WNTs specify the medial compartment by characterizing the expression of Porcupine (PORCN), an O-acyltransferase that is required for WNT secretion. We show PORCN expression across embryonic ages (E)12.5 - E14.5, E16.5, and postnatal day (P)1. Our results showed enriched PORCN in the medial domains during early stages of development, indicating that WNTs have a stronger influence on patterning of the medial compartment. PORCN was rapidly downregulated after E14.5, following the onset of sensory cell differentiation; residual expression remained in some hair cells and supporting cells. On E14.5 and E16.5, we also examined the spatial expression of Gsk3β, an inhibitor of canonical Wnt signaling to determine its potential role in radial patterning of the cochlea. Gsk3β was broadly expressed across the radial axis of the epithelium; therefore, unlikely to control WNT-mediated medial specification. In conclusion, the spatial expression of PORCN enriches WNT secretion from the medial domains of the cochlea to influence the specification of cell fates in the medial sensory domain.     

The TGF-beta2 isoform is both a required and sufficient inducer of murine hair follicle morphogenesis.

Hair follicle development serves as an excellent model to study control of organ morphogenesis. Three specific isoforms of TGF-beta exist which exhibit a distinct pattern of expression during hair follicle morphogenesis. To clarify the still elusive role of these factors in hair follicle development, we have used a combined genetic and functional approach: analysis of hair follicle development in mice with disruptions of the TGF-beta1, 2, and 3 genes was coupled with a direct functional test of the effect of added purified factors on fetal hair follicle development in skin organ cultures. TGF-beta2 null mice exhibited a profound delay of hair follicle morphogenesis, with a 50% reduced number of hair follicles. In contrast to hair follicle development, growth and differentiation of interfollicular keratinocytes proceeded unimpaired. Unlike TGF-beta2-/- mice, mice with a disruption of the TGF-beta1 gene showed slightly advanced hair follicle formation, while lack of the TGF-beta3 gene did not have any effects. Treatment of wild-type, embryonic skin explants (E14.5 or E15.5) with TGF-beta2 protein in either soluble form or slow release beads induced hair follicle development and epidermal hyperplasia, while similar TGF-beta1 treatment exerted suppressive effects. Thus, the TGF-beta2 isoform plays a specific role, not shared by the other TGF-beta isoforms, as an inducer of hair follicle morphogenesis and is both required and sufficient to promote this process.     

Molecular principles of hair follicle induction and morphogenesis

Hair follicle (HF) development is the result of neuroectodermal-mesodermal interactions, and can be divided into morphologically distinguishable stages (induction, organogenesis and cytodifferentiation). The spacing, polarity and differentiation patterns of HFs are driven by interacting, self-assembling gradients of inhibitors and activators, which are established jointly by the skin epithelium and mesenchyme. For HF development to occur, the dominant-negative influence of inhibitors of the HF differentiation pathway must be locally counteracted by specific antagonists and/or overriden by stimulators of hair placode formation. Once a mesenchymal condensate of inductive fibroblasts has formed, it takes over control of most subsequent steps of HF organogenesis and of epithelial stem cell differentiation into distinct lineages. In this review we introduce the morphological characteristics, major underlying principles and molecular key players that control HF development. The focus is on recent insights into the molecular interactions leading to hair follicle induction, and we close with synthesizing a corresponding working hypothesis.     

Multipotent nestin-expressing stem cells capable of forming neurons are located in the upper,middle and lower part of the vibrissa hair follicle

We have previously demonstrated that the neural stem-cell marker nestin is expressed in hair follicle stem cells. Nestin-expressing cells were initially identified in the hair follicle bulge area (BA) using a transgenic mouse model in which the nestin promoter drives the green fluorescent protein (ND-GFP). The hair-follicle ND-GFP-expressing cells are keratin 15-negative and CD34-positive and could differentiate to neurons, glia, keratinocytes, smooth muscle cells and melanocytes in vitro. Subsequently, we showed that the nestin-expressing stem cells could affect nerve and spinal cord regeneration after injection in mouse models. In the present study, we separated the mouse vibrissa hair follicle into three parts (upper, middle and lower). Each part of the follicle was cultured separately in DMEM-F12 containing B-27 and 1% methylcellulose supplemented with basic FGF. After 2 mo, the nestin-expressing cells from each of the separated parts of the hair follicle proliferated and formed spheres. Upon transfer of the spheres to RPMI 1640 medium containing 10% FBS, the nestin-expressing cells in the spheres differentiated to neurons, as well as glia, keratinocytes, smooth muscle cells and melanocytes. The differentiated cells were produced by spheres which formed from nestin-expressing cells from all segments of the hair follicle. However, the differentiation potential is greatest in the upper part of the follicle. This result is consistent with trafficking of nestin-expressing cells throughout the hair follicle from the bulge area to the dermal papilla that we previously observed. The nestin-expressing cells from the upper part of the follicle produced spheres in very large amounts, which in turn differentiated to neurons and other cell types. The results of the present study demonstrate that multipotent, nestin-expressing stem cells are present throughout the hair follicle and that the upper part of the follicle can produce the stem cells in large amounts that could be used for nerve and spinal cord repair.     

Cultured dermal papilla cells induce follicle formation and hair growth by transdifferentiation of an adult epidermis.

Adult rat pelage follicle dermal papilla cells induced follicle neogenesis and external hair growth when associated with adult footpad skin epidermis. They thus demonstrated a capacity to completely change the structural arrangement and gene expression of adult epidermis--an ability previously undocumented for cultured adult cells. Isolation chambers ensured that de novo follicle formation must have occurred by eliminating the possibility of cellular contributions, and/or inductive influences, from local skin follicles. These findings argue against previous suggestions of vibrissa follicle specificity, and imply that the potential for hair follicle induction may be common to all adult papilla cells.