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the recently suggested reaction-diffusion (RD) mechanism of spatial hair follicle arrangement in the light of yet unpublished data and conclusions.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.     

经典WNT信号通路与哺乳动物肺器官发育

肺器官发育是上皮和问充质相互作用的过程,由多条信号通路共同调控。已知经典WNT信号通路对细胞的增殖、凋亡和分化起着重要的调控作用,在小鼠等模式生物上研究发现,它也参与了哺乳动物肺器官发育的调控过程。综述近年来经典WNT信号通路成员在哺乳动物肺器官发育过程中的表达变化、作用功能及表达异常可能诱发的肺部疾病,以期为研究经典WNT信号通路调控人类肺器官发育的分子机制及相关肺部疾病的诊治奠定基础。     

毛囊干细胞及其隆突微环境

毛囊隆突(bulge)是毛囊干细胞特定的微环境,它维持并调节干细胞的特性,使干细胞在静息态、自我更新和分化上保持平衡。现重点从毛囊隆突的物理结构上来简要揭示微环境对干细胞的调控。     

Canonical WNT/beta-catenin signaling is required for ureteric branching

WNT/beta-catenin signaling has an established role in nephron formation during kidney development. Yet, the role of beta-catenin during ureteric morphogenesis in vivo is undefined. We generated a murine genetic model of beta-catenin deficiency targeted to the ureteric bud cell lineage. Newborn mutant mice demonstrated bilateral renal aplasia or renal dysplasia. Analysis of the embryologic events leading to this phenotype revealed that abnormal ureteric branching at E12.5 precedes histologic abnormalities at E13.5. Microarray analysis of E12.5 kidney tissue identified decreased Emx2 and Lim1 expression among a small subset of renal patterning genes disrupted at the stage of abnormal branching. These alterations are followed by decreased expression of genes downstream of Emx2, including Lim1, Pax2, and the ureteric tip markers, c-ret and Wnt 11. Together, these data demonstrate that beta-catenin performs essential functions during renal branching morphogenesis via control of a hierarchy of genes that control ureteric branching.     

Characterization of Hair Follicle Development in Engineered Skin Substitutes

Generation of skin appendages in engineered skin substitutes has been limited by lack of trichogenic potency in cultured postnatal cells. To investigate the feasibility and the limitation of hair regeneration, engineered skin substitutes were prepared with chimeric populations of cultured human keratinocytes from neonatal foreskins and cultured murine dermal papilla cells from adult GFP transgenic mice and grafted orthotopically to full-thickness wounds on athymic mice. Non-cultured dissociated neonatal murine-only skin cells, or cultured human-only skin keratinocytes and fibroblasts without dermal papilla cells served as positive and negative controls respectively. In this study, neonatal murine-only skin substitutes formed external hairs and sebaceous glands, chimeric skin substitutes formed pigmented hairs without sebaceous glands, and human-only skin substitutes formed no follicles or glands. Although chimeric hair cannot erupt readily, removal of upper skin layer exposed keratinized hair shafts at the skin surface. Development of incomplete pilosebaceous units in chimeric hair corresponded with upregulation of hair-related genes, LEF1 and WNT10B, and downregulation of a marker of sebaceous glands, Steroyl-CoA desaturase. Transepidermal water loss was normal in all conditions. This study demonstrated that while sebaceous glands may be involved in hair eruption, they are not required for hair development in engineered skin substitutes.     

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.     

Hair Follicle Development in Mouse Pluripotent Stem Cell-Derived Skin Organoids

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iRhom2 Mutation Leads to Aberrant Hair Follicle Differentiation in Mice

iRhom1 and iRhom2 are inactive homologues of rhomboid intramembrane serine proteases lacking essential catalytic residues, which are necessary for the maturation of TNFα-converting enzyme (TACE). In addition, iRhoms regulate epidermal growth factor family secretion. The functional significance of iRhom2 during mammalian development is largely unclear. We have identified a spontaneous single gene deletion mutation of iRhom2 in Uncv mice. The iRhom2^Uncv/Uncv mice exhibit hairless phenotype in a BALB/c genetic background. In this study, we observed dysplasia hair follicles in iRhom2^Uncv/Uncv mice from postnatal day 3. Further examination found decreased hair matrix proliferation and aberrant hair shaft and inner root sheath differentiation in iRhom2^Uncv/Uncv mutant hair follicles. iRhom2 is required for the maturation of TACE. Our data demonstrate that iRhom2^Uncv cannot induce the maturation of TACE in vitro and the level of mature TACE is also significantly reduced in the skin of iRhom2^Uncv/Uncv mice. The activation of Notch1, a substrate of TACE, is disturbed, associated with dramatically down-regulation of Lef1 in iRhom2^Uncv/Uncv hair follicle matrix. This study identifies iRhom2 as a novel regulator of hair shaft and inner root sheath differentiation.     

Genome Array of Hair Follicle Genes in Lambskin with Different Patterns

Hu sheep lambskin comes from a specific breed of sheep of China. Hu sheep are considered a protected breed by the Chinese government. The hair follicles of these sheep have three types of waves, large, medium, and small. There are only few histological reports of Hu sheep lambskin, and there are no modern molecular or biological studies, so the molecular mechanisms underlying the formation of hair follicles with different patterns are not currently known. The aim of this article was to study the molecular mechanism of the formation of these types of hair follicles in Hu sheep. Histological and microscopic analysis indicated that the number of follicles with small waves was not significantly higher than the number of follicles with large waves (P>0.05). The diameters of primary and secondary small-wave follicles were significantly smaller than those of large-wave follicles (P<0.05; P<0.01). The ratio between the number primary follicles and the number of secondary follicles was significantly higher among small-wave follicles than among large-wave follicles (P<0.05). Differentially expressed genes in the skin tissue were screened using an Agilent gene chip and RT-PCR. Differential expression analysis revealed 3 groups of large waves and small waves; 1067, 2071, and 3879 differentially expressed genes; and 137 genes common to all 3 groups. Differentially expressed genes were classified using gene ontology. They were found to be mainly involved in cell differentiation, proliferation, apoptosis, growth, immune response, and ion transport. RT-PCR results of 4 differentially expressed genes were consistent with gene chip results. Combined with related literature, our results suggest that BMP7, MMP2, SNAI1, SFXN1, CDKNIC, MT3, and POU1F1 may have important effects on the formation of large-wave and small-wave hair follicles. This study may enrich knowledge of hair follicle development, and may identify the genes responsible for the formation of hair follicles with different patterns.     

NEDD4L Is Downregulated in Colorectal Cancer and Inhibits Canonical WNT Signaling

The NEDD4 family of E3 ubiquitin ligases includes nine members. Each is a modular protein, containing an N-terminal C2 domain for cell localization, two-to-four central WW domains for substrate recognition, and a C-terminal, catalytic HECT domain, which is responsible for catalyzing the ubiquitylation reaction. Members of this family are known to affect pathways central to the pathogenesis of colorectal cancer, including the WNT, TGFβ, EGFR, and p53 pathways. Recently, NEDD4 mRNA was reported to be overexpressed in colorectal cancer, but tumor stage was not considered in the analysis. Expression of the other family members has not been studied in colorectal cancer. Herein, we determined the expression patterns of all nine NEDD4 family members in 256 patients who presented with disease ranging from premalignant adenoma to stage IV colorectal cancer. NEDD4 mRNA was significantly increased in all stages of colorectal cancer. In contrast, NEDD4L mRNA, the closest homolog to NEDD4, was the most highly downregulated family member, and was significantly downregulated in all tumor stages. We also found NEDD4L protein was significantly decreased by western blotting in colorectal cancer samples compared to adjacent normal mucosa. In addition, NEDD4L, but not catalytically inactive NEDD4L, inhibited canonical WNT signaling at or below the level of β-catenin in vitro. These findings suggest that NEDD4L may play a tumor suppressive role in colorectal cancer, possibly through inhibition of canonical WNT signaling.     

Circadian Clock Genes Contribute to the Regulation of Hair Follicle Cycling

Hair follicles undergo recurrent cycling of controlled growth (anagen), regression (catagen), and relative quiescence (telogen) with a defined periodicity. Taking a genomics approach to study gene expression during synchronized mouse hair follicle cycling, we discovered that, in addition to circadian fluctuation, CLOCK–regulated genes are also modulated in phase with the hair growth cycle. During telogen and early anagen, circadian clock genes are prominently expressed in the secondary hair germ, which contains precursor cells for the growing follicle. Analysis of Clock and Bmal1 mutant mice reveals a delay in anagen progression, and the secondary hair germ cells show decreased levels of phosphorylated Rb and lack mitotic cells, suggesting that circadian clock genes regulate anagen progression via their effect on the cell cycle. Consistent with a block at the G1 phase of the cell cycle, we show a significant upregulation of p21 in Bmal1 mutant skin. While circadian clock mechanisms have been implicated in a variety of diurnal biological processes, our findings indicate that circadian clock genes may be utilized to modulate the progression of non-diurnal cyclic processes.     

Tyrosinase Depletion Prevents the Maturation of Melanosomes in the Mouse Hair Follicle

The mechanisms that lead to variation in human skin and hair color are not fully understood. To better understand the molecular control of skin and hair color variation, we modulated the expression of Tyrosinase (Tyr), which controls the rate-limiting step of melanogenesis, by expressing a single-copy, tetracycline-inducible shRNA against Tyr in mice. Moderate depletion of TYR was sufficient to alter the appearance of the mouse coat in black, agouti, and yellow coat color backgrounds, even though TYR depletion did not significantly inhibit accumulation of melanin within the mouse hair. Ultra-structural studies revealed that the reduction of Tyr inhibited the accumulation of terminal melanosomes, and inhibited the expression of genes that regulate melanogenesis. These results indicate that color in skin and hair is determined not only by the total amount of melanin within the hair, but also by the relative accumulation of mature melanosomes.