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.     

FGF signaling is required for initiation of feather placode development

Morphogenesis of hairs and feathers is initiated by an as yet unknown dermal signal that induces placode formation in the overlying ectoderm. To determine whether FGF signals are required for this process we over-expressed soluble versions of FGFR1 or FGFR2 in the skin of chicken embryos. This produced a complete failure of feather formation prior to any morphological or molecular signs of placode development. We further show that Fgf10 is expressed in the dermis of nascent feather primordia, and that anti-FGF10 antibodies block feather placode development in skin explants. In addition we show that FGF10 can induce expression of positive and negative regulators of feather development and can induce its own expression under conditions of low BMP signaling. Together these results demonstrate that FGF signaling is required for the initiation of feather placode development and implicate FGF10 as an early dermal signal involved in this process.     

A balance of FGF, BMP and WNT signalling positions the future placode territory in the head

The sensory nervous system in the vertebrate head arises from two different cell populations: neural crest and placodal cells. By contrast, in the trunk it originates from neural crest only. How do placode precursors become restricted exclusively to the head and how do multipotent ectodermal cells make the decision to become placodes or neural crest? At neural plate stages, future placode cells are confined to a narrow band in the head ectoderm, the pre-placodal region (PPR). Here, we identify the head mesoderm as the source of PPR inducing signals, reinforced by factors from the neural plate. We show that several independent signals are needed: attenuation of BMP and WNT is required for PPR formation. Together with activation of the FGF pathway, BMP and WNT antagonists can induce the PPR in na?ve ectoderm. We also show that WNT signalling plays a crucial role in restricting placode formation to the head. Finally, we demonstrate that the decision of multipotent cells to become placode or neural crest precursors is mediated by WNT proteins: activation of the WNT pathway promotes the generation of neural crest at the expense of placodes. This mechanism explains how the placode territory becomes confined to the head, and how neural crest and placode fates diversify.     

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.     

Dermal β-catenin activity in response to epidermal Wnt ligands is required for fibroblast proliferation and hair follicle initiation

Dermal fibroblasts are required for structural integrity of the skin and for hair follicle development. Uniform Wnt signaling activity is present in dermal fibroblast precursors preceding hair follicle initiation, but the functional requirement of dermal Wnt signaling at early stages of skin differentiation and patterning remains largely uncharacterized. We show in mice that epidermal Wnt ligands are required for uniform dermal Wnt signaling/β-catenin activity and regulate fibroblast cell proliferation and initiation of hair follicle placodes. In the absence of dermal Wnt signaling/β-catenin activity, patterned upregulation of epidermal β-catenin activity and Edar expression are absent. Conversely, forced activation of β-catenin signaling leads to the formation of thickened dermis, enlarged epidermal placodes and dermal condensates that result in prematurely differentiated enlarged hair follicles. These data reveal functional roles for dermal Wnt signaling/β-catenin in fibroblast proliferation and in the epidermal hair follicle initiation program.     

Ectodysplasin A1 promotes placodal cell fate during early morphogenesis of ectodermal appendages

Organs developing as appendages of the ectoderm are initiated from epithelial thickenings called placodes. Their formation is regulated by interactions between the ectoderm and underlying mesenchyme, and several signalling molecules have been implicated as activators or inhibitors of placode formation. Ectodysplasin (Eda) is a unique signalling molecule in the tumour necrosis factor family that, together with its receptor Edar, is necessary for normal development of ectodermal organs both in humans and mice. We have shown previously that overexpression of the Eda-A1 isoform in transgenic mice stimulates the formation of several ectodermal organs. In the present study, we have analysed the formation and morphology of placodes using in vivo and in vitro models in which both the timing and amount of Eda-A1 applied could be varied. The hair and tooth placodes of K14-Eda-A1 transgenic embryos were enlarged, and extra placodes developed from the dental lamina and mammary line. Exposure of embryonic skin to Eda-A1 recombinant protein in vitro stimulated the growth and fusion of placodes. However, it did not accelerate the initiation of the first wave of hair follicles giving rise to the guard hairs. Hence, the function of Eda-A1 appears to be downstream of the primary inductive signal required for placode initiation during skin patterning. Analysis of BrdU incorporation indicated that the formation of the epithelial thickening in early placodes does not involve increased cell proliferation and also that the positive effect of Eda-A1 on placode expansion is not a result of increased cell proliferation. Taken together, our results suggest that Eda-A1 signalling promotes placodal cell fate during early development of ectodermal organs.     

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 over-expression, thereby further supporting our model of hair follicle spacing. Furthermore, we suggest future experiments to challenge the RD model of spatial follicle arrangement.     

Involvement of Hex in the initiation of feather morphogenesis

In a previous study, we showed that the proline-rich divergent homeobox gene Hex/Prh is expressed in dorsal skin of the chick embryo before and during feather bud development and that the pattern of Hex mRNA expression in the epidermis is similar to that of Wnt7a mRNA. In order to study the function of Hex and the relationship between Hex and Wnt7a in feather bud development, sense and/or antisense sequences of Hex or Wnt7a were ectopically and transiently expressed in the dorsal skin with the epidermal side toward the cathode by electroporation at the placode stage and then the skin was cultured. Increased expression of Wnt7a and beta-catenin mRNA was observed in the same region where Hex-EGFP fusion protein was expressed 2 days after culture, which was followed by extra bud formation a few days later as a result of the stimulation of cell proliferation. Concomitantly, expression of Notch1 mRNA, which is expressed in normal bud development, increased in Hex-overexpressing skin. However, ectopic Wnt7a expression induced neither Hex expression nor extra bud formation in normal skin. Antisense Wnt7a specifically inhibited bud initiation in Hex-overexpressing skin but did not in normal skin. Taken together, these results suggest that Hex is upstream of Wnt7a and beta-catenin and regulates the Wnt signaling pathway in feather bud initiation and that some other Wnt signals in addition to Wnt7a may be required for bud initiation.     

The role of a reaction-diffusion system in the initiation of primary hair follicles

A mechanism based on a reaction-diffusion system is proposed for the initiation of hair follicles in the epidermis during fetal development. It is demonstrated that initiation of primary follicles in a series of waves, within the proposed mechanism, is a consequence of the size and shape dependent properties of the reaction-diffusion system without the need for the propagation of signals through the skin. The observed trio grouping of follicles and variation of primary follicle density per unit skin area during development are also correctly predicted. An explanation, based on the reaction-diffusion system and the variation of its characteristic spatial wavelength with time during development, is suggested for the termination of both primary and secondary follicle initiation as well as follicle neogenesis. The proposed initiation mechanism is basically the same as that used to explain various spatial patterns observed in hair fibre formation (Nagorcka & Mooney, 1982).     

Gli3-mediated somitic Fgf10 expression gradients are required for the induction and patterning of mammary epithelium along the embryonic axes

Little is known about the regulation of cell fate decisions that lead to the formation of five pairs of mammary placodes in the surface ectoderm of the mouse embryo. We have previously shown that fibroblast growth factor 10 (FGF10) is required for the formation of mammary placodes 1, 2, 3 and 5. Here, we have found that Fgf10 is expressed only in the somites underlying placodes 2 and 3, in gradients across and within these somites. To test whether somitic FGF10 is required for the formation of these two placodes, we analyzed a number of mutants with different perturbations of somitic Fgf10 gradients for the presence of WNT signals and ectodermal multilayering, markers for mammary line and placode formation. The mammary line is displaced dorsally, and formation of placode 3 is impaired in Pax3ILZ/ILZ mutants, which do not form ventral somitic buds. Mammary line formation is impaired and placode 3 is absent in Gli3Xt-J/Xt-J and hypomorphic Fgf10 mutants, in which the somitic Fgf10 gradient is shortened dorsally and less overall Fgf10 is expressed, respectively. Recombinant FGF10 rescued mammogenesis in Fgf10(-/-) and Gli3Xt-J/Xt-J flanks. We correlate increasing levels of somitic FGF10 with progressive maturation of the surface ectoderm, and show that full expression of somitic Fgf10, co-regulated by GLI3, is required for the anteroposterior pattern in which the flank ectoderm acquires a mammary epithelial identity. We propose that the intra-somitic Fgf10 gradient, together with ventral elongation of the somites, determines the correct dorsoventral position of mammary epithelium along the flank.     

The ectodysplasin pathway in feather tract development

The ectodysplasin pathway, comprising the ligand ectodysplasin, its receptor Edar and a dedicated death domain adaptor protein Edaradd, plays an important role in epidermal organ formation in mammals. Mutations in the genes encoding these proteins cause dysplasia or absence of teeth, sweat glands and hair follicles. However, the relative position of this pathway in the regulatory hierarchy directing follicle formation remains unclear. In this work, the chicken orthologs of Eda, Edar and Edaradd were cloned to exploit the temporal precision of the feather tract system in order to study the role of the ectodysplasin pathway. We find that these genes are expressed in a similar pattern during feather and hair development, with the notable difference that the ligand Eda, which is expressed in the epidermis of the mouse, is expressed in the dermis of the feather tract. Contrary to conclusions reached from the analysis of mutant mice, we find that localization of Edar expression to the nascent placode is coincident or subsequent to the local expression of other markers of placodal differentiation, and not an upstream event in tract patterning. Furthermore, forced expression of BMP and activated beta-catenin demonstrate that local expression of Edar is dictated by the interaction between these two pathways. These results suggest that activation of the ectodysplasin pathway may be permissive for activating signals to overcome signals that inhibit placode formation, but the function of this pathway in the specification of follicle initiation lies downstream of other patterning events.     

Expression of Hex during feather bud development

We studied proline-rich divergent homeobox gene Hex/Prh expression in the dorsal skin of chick embryo during feather bud development. Hex mRNA expression was first observed in the dorsolateral ectoderm and mesenchyme at 5 days, then in the epithelium and the dermis of the dorsal skin before placode (primordium of feather bud) formation and then was restricted to the placode and the dermis under the placode. Afterward, Hex expression was seen in the epidermis and the dermis of the posterior region of short bud. In accordance with Hex mRNA expression in the placode, Hex protein was observed in the epidermis as well as in the dermis of the placode. Immunoelectron microscopic study indicated that the protein located both in the nuclei and cytoplasm of the epidermis and the dermis at the short bud stage. The Wnt signaling pathway plays an essential role in the early inductive events in hair (Wnt3a and 7a) and feather (Wnt7a) follicles. The pattern of Hex expression in the epidermis was similar to that of Wnt7a, while little, if any, expression of Wnt7a was detected in the dermis under the placode or the dermis of the short bud compared with that of Hex, suggesting that Hex plays an important role in the initiation of feather morphogenesis.     

A crucial role for Fgfr2-IIIb signalling in epidermal development and hair follicle patterning

To understand the role Fgf signalling in skin and hair follicle development, we analysed the phenotype of mice deficient for Fgfr2-IIIb and its main ligand Fgf10. These studies showed that the severe epidermal hypoplasia found in mice null for Fgfr2-IIIb is caused by a lack of the basal cell proliferation that normally results in a stratified epidermis. Although at term the epidermis of Fgfr2-IIIb null mice is only two to three cells thick, it expresses the classical markers of epidermal differentiation and establishes a functional barrier. Mice deficient for Fgf10 display a similar but less severe epidermal hypoplasia. By contrast, Fgfr2-IIIb-/-, but not Fgf10-/-, mice produced significantly fewer hair follicles, and their follicles were developmentally retarded. Following transplantation onto nude mice, grafts of Fgfr2-IIIb-/- skin showed impaired hair formation, with a decrease in hair density and the production of abnormal pelage hairs. Expression of Lef1, Shh and Bmp4 in the developing hair follicles of Fgfr2-IIIb-/- mice was similar to wild type. These results suggest that Fgf signalling positively regulates the number of keratinocytes needed to form a normal stratified epidermis and to initiate hair placode formation. In addition, Fgf signals are required for the growth and patterning of pelage hairs.     

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.     

Mechanisms of ectodermal organogenesis

All ectodermal organs, e.g. hair, teeth, and many exocrine glands, originate from two adjacent tissue layers: the epithelium and the mesenchyme. Similar sequential and reciprocal interactions between the epithelium and mesenchyme regulate the early steps of development in all ectodermal organs. Generally, the mesenchyme provides the first instructive signal, which is followed by the formation of the epithelial placode, an early signaling center. The placode buds into or out of the mesenchyme, and subsequent proliferation, cell movements, and differentiation of the epithelium and mesenchyme contribute to morphogenesis. The molecular signals regulating organogenesis, such as molecules in the FGF, TGFbeta, Wnt, and hedgehog families, regulate the development of all ectodermal appendages repeatedly during advancing morphogenesis and differentiation. In addition, signaling by ectodysplasin, a recently identified member of the TNF family, and its receptor Edar is required for ectodermal organ development across vertebrate species. Here the current knowledge on the molecular regulation of the initiation, placode formation, and morphogenesis of ectodermal organs is discussed with emphasis on feathers, hair, and teeth.     

Differential requirements for FGF3, FGF8 and FGF10 during inner ear development

FGF signaling is required during multiple stages of inner ear development in many different vertebrates, where it is involved in induction of the otic placode, in formation and morphogenesis of the otic vesicle as well as for cellular differentiation within the sensory epithelia. In this study we have looked to define the redundant and conserved roles of FGF3, FGF8 and FGF10 during the development of the murine and avian inner ear. In the mouse, hindbrain-derived FGF10 ectopically induces FGF8 and rescues otic vesicle formation in Fgf3 and Fgf10 homozygous double mutants. Conditional inactivation of Fgf8 after induction of the placode does not interfere with otic vesicle formation and morphogenesis but affects cellular differentiation in the inner ear. In contrast, inactivation of Fgf8 during induction of the placode in a homozygous Fgf3 null background leads to a reduced size otic vesicle or the complete absence of otic tissue. This latter phenotype is more severe than the one observed in mutants carrying null mutations for both Fgf3 and Fgf10 that develop microvesicles. However, FGF3 and FGF10 are redundantly required for morphogenesis of the otic vesicle and the formation of semicircular ducts. In the chicken embryo, misexpression of Fgf3 in the hindbrain induces ectopic otic vesicles in vivo. On the other hand, Fgf3 expression in the hindbrain or pharyngeal endoderm is required for formation of the otic vesicle from the otic placode. Together these results provide important insights into how the spatial and temporal expression of various FGFs controls different steps of inner ear formation during vertebrate development.