Hair Placode Research Articles

Radially patterned morphogenesis of murine hair follicle placodes ensures robust epithelial budding.

The bending of simple cellular sheets into complex three-dimensional (3D) forms requires developmental patterning cues to specify where deformations occur, but how positional information directs morphological change is poorly understood. Here, we investigate how morphogen signaling and cell fate diversification contribute to the morphogenesis of murine hair placodes, in which collective cell movements transform radially symmetric primordia into bilaterally symmetric tubes. Through live imaging and 3D volumetric reconstructions, we demonstrate that Wnt and Shh establish radial patterns of cell fate, cell morphology, and movement within developing placodes. Cell fate diversity at different radial positions provides unique and essential contributions to placode morphogenesis. Further, we show that downstream of radial patterning, gradients of classical cadherin expression are required for efficient epithelial rearrangements. Given that the transformation of epithelial discs into 3D tubes is a common morphological motif used to shape diverseorgan primordia, mechanisms of radially patterned morphogenesis are likely highly conserved across evolution.

ARHGEF3 Regulates Hair Follicle Morphogenesis.

During embryogenesis, cells arrange into precise patterns that enable tissues and organs to develop specialized functions. Despite its critical importance, the molecular choreography behind these collective cellular behaviors remains elusive, posing a major challenge in developmental biology and limiting advances in regenerative medicine. By using the mouse hair follicle as a mini-organ system to study the formation of bud-like structures during embryonic development, our work uncovers a crucial role for the Rho GTPase regulator ARHGEF3 in hair follicle morphogenesis. We demonstrate that Arhgef3 expression is upregulated at the onset of hair follicle placode formation. In Arhgef3 knockout animals, we observed defects in placode compaction, leading to impaired hair follicle downgrowth. Through cell culture models, we show that ARHGEF3 promotes F-actin accumulation at the cell cortex and P-cadherin enrichment at cell-cell junctions. Collectively, our study identifies ARHGEF3 as a new regulator of cell shape rearrangements during hair placode morphogenesis, warranting further exploration of its role in other epithelial appendages that arise from similar developmental processes.

Integrating Single‐Cell and Spatial Transcriptomics Reveals Heterogeneity of Early Pig Skin Development and a Subpopulation with Hair Placode Formation (Adv. Sci. 20/2024)

Integrating Single‐Cell and Spatial Transcriptomics Reveals Heterogeneity of Early Pig Skin Development and a Subpopulation with Hair Placode Formation (Adv. Sci. 20/2024)

Integrating Single-Cell and Spatial Transcriptomics Reveals Heterogeneity of Early Pig Skin Development and a Subpopulation with Hair Placode Formation.

The dermis and epidermis, crucial structural layers of the skin, encompass appendages, hair follicles (HFs), and intricate cellular heterogeneity. However, an integrated spatiotemporal transcriptomic atlas of embryonic skin has not yet been described and would be invaluable for studying skin-related diseases in humans. Here, single-cell and spatial transcriptomic analyses are performed on skin samples of normal and hairless fetal pigs across four developmental periods. The cross-species comparison of skin cells illustrated that the pig epidermis is more representative of the human epidermis than mice epidermis. Moreover, Phenome-wide association study analysis revealed that the conserved genes between pigs and humans are strongly associated with human skin-related diseases. In the epidermis, two lineage differentiation trajectories describe hair follicle (HF) morphogenesis and epidermal development. By comparing normal and hairless fetal pigs, it is found that the hair placode (Pc), the most characteristic initial structure in HFs, arises from progenitor-like OGN+/UCHL1+ cells. These progenitors appear earlier in development than the previously described early Pc cells and exhibit abnormal proliferation and migration during differentiation in hairless pigs. The study provides a valuable resource for in-depth insights into HF development, which may serve as a key reference atlas for studying human skin disease etiology using porcine models.

FGF20

Fibroblast growth factor 20 (FGF20) is a neurotrophic factor and a member of the FGF9 subfamily. It was first identified in Xenopus embryos and was isolated shortly thereafter from the adult rat brain. Its receptors include FGFR4, FGFR3b, FGFR2b and the FGFRc splice forms. In adults it is highly expressed in the brain, while it is expressed in a variety of regions during embryonic development, including the inner ear, heart, hair placodes, mammary buds, dental epithelium and limbs. As a result of its wide-spread expression, FGF20 mouse mutants exhibit a variety of phenotypes including congenital deafness, lack of hair, small kidneys and delayed mammary ductal outgrowth. FGF20 is also associated with human diseases including Parkinson's Disease, cancer and hereditary deafness.

Foxi3GFP and Foxi3CreER mice allow identification and lineage labeling of pharyngeal arch ectoderm and endoderm, and tooth and hair placodes.

FOXI3 is a forkhead family transcription factor that is expressed in the progenitors of craniofacial placodes, epidermal placodes, and the ectoderm and endoderm of the pharyngeal arch region. Loss of Foxi3 in mice and pathogenic Foxi3 variants in dogs and humans cause a variety of craniofacial defects including absence of the inner ear, severe truncations of the jaw, loss or reduction in external and middle ear structures, and defects in teeth and hair. To allow for the identification, isolation, and lineage tracing of Foxi3-expressing cells in developing mice, we targeted the Foxi3 locus to create Foxi3GFP and Foxi3CreER mice. We show that Foxi3GFP mice faithfully recapitulate the expression pattern of Foxi3 mRNA at all ages examined, and Foxi3CreER mice can trace the derivatives of pharyngeal arch ectoderm and endoderm, the pharyngeal pouches and clefts that separate each arch, and the derivatives of hair and tooth placodes. Foxi3GFP and Foxi3CreER mice are new tools that will be of use in identifying and manipulating pharyngeal arch ectoderm and endoderm and hair and tooth placodes.

Transcriptomic landscape of early hair follicle and epidermal development

Morphogenesis of ectodermal organs, such as hair, tooth, and mammary gland, starts with the formation of local epithelial thickenings, or placodes, but it remains to be determined how distinct cell types and differentiation programs are established during ontogeny. Here, we use bulk and single-cell transcriptomics andpseudotime modeling to address these questions in developing hair follicles and epidermis and produce a comprehensive transcriptomic profile of cellular populations in the hair placode and interplacodal epithelium. We report previously unknown cell populations and marker genes, including early suprabasal and genuine interfollicular basal markers, and propose the identity of suprabasal progenitors. By uncovering four different hair placode cell populations organized in three spatially distinct areas, with fine gene expression gradients between them, we posit early biases in cell fate establishment. This work is accompanied by a readily accessible online tool to stimulate further research on skin appendages and their progenitors.

Two mutations at KRT74 and EDAR synergistically drive the fine-wool production in Chinese sheep

IntroductionFine-wool sheep are the most common breed used by the wool industry worldwide. Fine-wool sheep have over a three-fold higher follicle density and a 50% smaller fiber diameter than coarse-wool sheep. ObjectivesThis study aims to clarify the underlying genetic basis for the denser and finer wool phenotype in fine-wool breeds. MethodWhole-genome sequences of 140 samples, Ovine HD630K SNP array data of 385 samples, including fine, semi-fine, and coarse wool sheep, as well as skin transcriptomes of nine samples were integrated for genomic selection signature analysis. ResultsTwo loci at keratin 74 (KRT74) and ectodysplasin receptor (EDAR) were revealed. Fine-scale analysis in 250 fine/semi-fine and 198 coarse wool sheep narrowed this association to one C/A missense variant of KRT74 (OAR3:133,486,008, P = 1.02E-67) and one T/C SNP in the regulatory region upstream of EDAR (OAR3:61,927,840, P = 2.50E-43). Cellular over-expression and ovine skin section staining assays confirmed that C-KRT74 activated the KRT74 protein and specifically enlarged cell size at the Huxley’s layer of the inner root sheath (P < 0.01). This structure enhancement shapes the growing hair shaft into the finer wool than the wild type. Luciferase assays validated that the C-to-T mutation upregulated EDAR mRNA expression via a newly created SOX2 binding site and potentially led to the formation of more hair placodes. ConclusionsTwo functional mutations driving finer and denser wool production were characterized and offered new targets for genetic breeding during wool sheep selection. This study not only provides a theoretical basis for future selection of fine wool sheep breeds but also contributes to improving the value of wool commodities.

MiR-29a-5p Inhibits Prenatal Hair Placode Formation Through Targeting EDAR by ceRNA Regulatory Network.

Hair placode formation is an important stage of hair follicle morphogenesis and it is a complex process facilitated by non-coding RNAs. In this study, we conducted whole transcriptome sequencing analysis of skin, heart, liver, lung, and kidney tissues of day 41 (E41) normal and hairless pig embryos, and respectively detected 15, 8, and 515 skin-specific differentially expressed (DE) lncRNAs, miRNAs, and mRNAs. Furthermore, 18 competing endogenous RNA (ceRNA) networks were constructed. Following weighted gene co-expression network analysis (WGCNA) of stages E39, E41, E45, E52, and E60, between normal and hairless pig embryos, only two ceRNAs (lncRNA2162.1/miR-29a-5p/BMPR1b and lncRNA627.1/miR-29a-5p/EDAR) that showed period-specific differential expression in E41 skin were retained. Dual-luciferase reporter assays further indicated that EDAR was a direct, functioning target of miR-29a-5p and that no binding site was found in BMPR1b. Moreover, miR-29a-5p overexpression inhibited the mRNA and protein expression of EDAR while no significant differential expression of BMPR1b was detected. In addition, over-expressed lncRNA627.1 reduces the expression of miR-29a-5p and increase EDAR expression while inhibits lncRNA627.1 resulted in a opposite expression trend. Cell proliferation result demonstrated that lower expression of EDAR and lncRNA627.1 inhibited hair placode precursor cells (HPPCs) proliferation in a manner similar to that shown by over-expressed miR-29a-5p. This study identified that miR-29a-5p inhibited HPPCs proliferation via the suppression of EDAR expression in the EDA/EDAR signaling pathway, while lncRNA627.1 rescues EDAR expression. Our study provides a basis for a better understanding of the mechanisms underlying the ceRNA complex, miR29a-5p/EDAR/lncRNA627.1, that could regulate hair placode formation, which may help decipher diseases affecting human hair.

The combination of hair follicle-specific marker LHX2 and co-expressed marker can distinguish between sweat gland placodes and hair placodes in rat.

Eccrine sweat gland (ESG) and hair follicle (HF) are different skin appendages but share many common development characteristics. Although the morphology of adult ESG and HF is obviously different, it is difficult to distinguish ESG placodes from HFs placodes morphologically. To study the fate determination of ESG and HF, specific antigen markers for ESG placodes and HF placodes must be found first to distinguish them. In the study, we detected the expression of commonly used keratins 4, 5, 7-10, 12, 14, 15, 17-20, 27 and 73, and the reported ESG and HF specific markers, P-cadherin, Lymphoid enhancer factor 1 (LEF1), LIM Homeobox gene 2(LHX2), Na+/K+-ATPase (NKA) and Na+-K+-2Cl- cotransporter 1 (NKCC1) in ESG and HF placodes by single-immunofluorescence staining and double-immunofluorescence staining. To further verify the results of immunofluorescence staining, Western blot (WB) was used to detect the differential antigen and some co-expressed antigens of ESG and HF placodes. The results showed that both ESG and HF placodes expressed K4/5/14/1517/18, P-cadherin and LEF1, neither expressed K7/8/9/10/12/19/20/27/73, NKA or NKCC1. HF placodes specifically expressed LHX2. Combination of LHX2 and co-expressed antigen K14, can distinguish ESG placodes from HF placodes. We conclude that LHX2 is a specific marker for HF placodes, and ESG placodes and HF placodes can be distinguished by double immunofluorescence staining of the specific marker LHX2 and the co-expressed markers, such as K4, K5, K14, K15, K17, K18, P-cadherin and LEF1.

Tracing the origin of hair follicle stem cells.

Tissue stem cells are generated from a population of embryonic progenitors through organ-specific morphogenetic events1,2. Although tissue stem cells are central to organ homeostasis and regeneration, it remains unclear how they are induced during development, mainly because of the lack of markers that exclusively label prospective stem cells. Here we combine marker-independent long-term 3D live imaging and single-cell transcriptomics to capture a dynamic lineage progression and transcriptome changes in the entire epithelium of the mouse hair follicle as it develops. We found that the precursors of different epithelial lineages were aligned in a 2D concentric manner in the basal layer of the hair placode. Each concentric ring acquired unique transcriptomes and extended to form longitudinally aligned, 3D cylindrical compartments. Prospective bulge stem cells were derived from the peripheral ring of the placodebasal layer, but not from suprabasal cells (as was previously suggested3). The fate of placode cells is determined by the cell position, rather than by the orientation of cell division.We also identified 13gene clusters: the ensemble expression dynamics of these clusters drew the entire transcriptional landscape of epithelial lineage diversification, consistent with cell lineage data. Combining these findings with previous work on the development of appendages in insects4,5, we describe the 'telescope model', a generalized model for the development of ectodermal organs in which 2D concentric zones in the placode telescope out to form 3D longitudinally aligned cylindrical compartments.

Differentiated Daughter Cells Regulate Stem Cell Proliferation and Fate through Intra-tissue Tension.

Basal stem cells fuel development, homeostasis, and regeneration of the epidermis. The proliferation and fate decisions of these cells are highly regulated by their microenvironment, including the basement membrane and underlying mesenchymal cells. Basal progenitors give rise to differentiated progeny that generate the epidermal barrier. Here, we present data that differentiated progeny also regulate the proliferation, differentiation, and migration of basal progenitor cells. Using two distinct mouse lines, we found that increasing contractility of differentiated cells resulted in non-cell-autonomous hyperproliferation of stem cells and prevented their commitment to a hair follicle lineage. This increased contractility also impaired movement of basal progenitors during hair placode morphogenesis and diminished migration of melanoblasts. These data suggest that intra-tissue tension regulates stem cell proliferation, fate decisions, and migration and that differentiated epidermal keratinocytes are a component of the stem cell niche that regulates development and homeostasis of the skin.

869 The specification of Merkel cell in the back skin and glabrous paw skin is controlled by FGFR2-meditated signaling

Merkel cells are mechanosensory cells responsible for light touch sensations. In murine hairy skin, Merkel cells are located in touch domes in back skin and in whisker pads. Merkel cells are also present in the glabrous paw skin. While it has been shown that Merkel cells are of epidermal lineage, their progenitor and the regulators that are critical for their specification have not been extensively study. Here, we show that during embryogenesis, SOX9 positive (+) cells located in hair placodes, which were previously known to give rise to hair follicle stem cells, also give rise to Merkel cells in the back skin. In contrast, in the glabrous paw skin, SOX9(+) cells do not give rise to Merkel cells suggesting that hairy and glabrous skins have different Merkel cell precursor populations. We performed RNA-seq transcriptional profiling of Merkel cells on the back and glabrous paw skin and showed that their transcriptomes are similar suggesting that their formation is controlled by similar regulators. Among these regulators is FGFR2-mediated signaling that is essential for Merkel cell formation in both the back skin and glabrous paw skin. Taken together, our results demonstrate that although the precursor cells of Merkel cells in the murine back skin and glabrous paw skin might differ, their formation is controlled by a common genetic program and that FGFR2 plays a critical role in this process.

Dermal Condensate Niche Fate Specification Occurs Prior to Formation and Is Placode Progenitor Dependent

Cell fate transitions are essential for specification of stem cells and their niches, but the precise timing and sequence of molecular events during embryonic development are largely unknown. Here, we identify, with 3D and 4D microscopy, unclustered precursors of dermal condensates (DC), signaling niches for epithelial progenitors in hair placodes. With population-based and single-cell transcriptomics, we define a molecular time-lapse from pre-DC fate specification through DC niche formation and establish the developmental trajectory as the DC lineage emerges from fibroblasts. Co-expression of downregulated fibroblast and upregulated DC genes in niche precursors reveals a transitory molecular state following a proliferation shutdown. Waves of transcription factor and signaling molecule expression then coincide with DC formation. Finally, ablation of epidermal Wnt signaling and placode-derived FGF20 demonstrates their requirement for pre-DC specification. These findings uncover a progenitor-dependent niche precursor fate and the transitory molecular events controlling niche formation and function.

Fate Before Function: Specification of the Hair Follicle Niche Occurs Prior to Its Formation and is Progenitor Dependent

Cell fate transitions are essential for specialization of stem cells and their niches, but the precise timing and sequence of molecular events during embryonic development are largely unknown. Here, we show that dermal condensates (DC), signaling niches for epithelial progenitors in hair placodes, are specified before niche formation and function. With 3D/4D microscopy we identify unclustered DC precursors. With population-based and single-cell transcriptomics we define a molecular time-lapse of dynamic niche signatures and the developmental trajectory as the DC lineage emerges from fibroblasts. Co-expression of downregulated fibroblast and upregulated DC genes in niche precursors reveals a transitory molecular state following a proliferation shutdown. Waves of transcription factor and signaling molecule expression then consolidate DC niche formation. Finally, ablation of epidermal Wnt signaling and placode-derived FGF20 demonstrates their requirement for DC-precursor specification. These findings uncover a progenitor-dependent niche precursor fate and the transitory molecular events controlling niche formation and function.

Counter-rotational cell flows drive morphological and cell fate asymmetries in mammalian hair follicles.

Organ morphogenesis is a complex process coordinated by cell specification, epithelial-mesenchymal interactions, and tissue polarity. A striking example is the pattern of regularly spaced, globally aligned mammalian hair follicles, which emerges through epidermal-dermal signaling and planar polarized morphogenesis. Here, using live-imaging, we discover that developing hair follicles polarize through dramatic cell rearrangements organized in a counter-rotational pattern of cell flows. Upon hair placode induction, Shh signaling specifies a radial pattern of progenitor fates that, together with planar cell polarity (PCP), induce counter-rotational rearrangements through myosin and ROCK-dependent polarized neighbor exchanges. Importantly, these cell rearrangements also establish cell fate asymmetry by repositioning radial progenitors along the anterior-posterior axis. These movements concurrently displace associated mesenchymal cells, which then signal asymmetrically to maintain polarized cell fates. Our results demonstrate how spatial patterning and tissue polarity generate an unexpected collective cell behavior that in turn, establishes both morphological and cell fate asymmetry.

A Cascade of Wnt, Eda, and Shh Signaling Is Essential for Touch Dome Merkel Cell Development.

The Sonic hedgehog (Shh) signaling pathway regulates developmental, homeostatic, and repair processes throughout the body. In the skin, touch domes develop in tandem with primary hair follicles and contain sensory Merkel cells. The developmental signaling requirements for touch dome specification are largely unknown. We found dermal Wnt signaling and subsequent epidermal Eda/Edar signaling promoted Merkel cell morphogenesis by inducing Shh expression in early follicles. Lineage-specific gene deletions revealed intraepithelial Shh signaling was necessary for Merkel cell specification. Additionally, a Shh signaling agonist was sufficient to rescue Merkel cell differentiation in Edar-deficient skin. Moreover, Merkel cells formed in Fgf20 mutant skin where primary hair formation was defective but Shh production was preserved. Although developmentally associated with hair follicles, fate mapping demonstrated Merkel cells primarily originated outside the hair follicle lineage. These findings suggest that touch dome development requires Wnt-dependent mesenchymal signals to establish reciprocal signaling within the developing ectoderm, including Eda signaling to primary hair placodes and ultimately Shh signaling from primary follicles to extrafollicular Merkel cell progenitors. Shh signaling often demonstrates pleiotropic effects within a structure over time. In postnatal skin, Shh is known to regulate the self-renewal, but not the differentiation, of touch dome stem cells. Our findings relate the varied effects of Shh in the touch dome to the ligand source, with locally produced Shh acting as a morphogen essential for lineage specification during development and neural Shh regulating postnatal touch dome stem cell maintenance.

Signaling in tooth, hair, and mammary placodes.

Skin appendages develop from placodes involving reciprocal interactions between the surface ectoderm and the underlying mesenchyme during embryogenesis. Despite their distinct shapes and functions, during early development similar morphological changes are observed among different skin appendages. Previous analyses of genetically modified mice have shown that these skin placodes share many aspects of molecular and cellular programs controlled by a relatively small number of signaling pathways during induction, morphogenesis, and transition to bud stage and beyond. This chapter focuses on the major signaling pathways that are reiteratively utilized to control the early developmental processes of placodes for teeth, hair follicles, and mammary glands. I update knowledge on the roles played by individual pathways and cross talk among them in these placodes and discuss similarities as well as differences among the skin appendages.

CD133 Expression Correlates with Membrane Beta-Catenin and E-Cadherin Loss from Human Hair Follicle Placodes during Morphogenesis

Genetic studies suggest that the major events of human hair follicle development are similar to those in mice, but detailed analyses of this process are lacking. In mice, hair follicle placode ‘budding’ is initiated by invagination of Wnt-induced epithelium into the underlying mesenchyme. Modification of adherens junctions is clearly required for budding. Snail-mediated downregulation of adherens junction component E-cadherin is important for placode budding in mice. Beta-catenin, another adherens junction component, has been more difficult to study due to its essential functions in Wnt signaling, a prerequisite for hair follicle placode induction. Here, we show that a subset of human invaginating hair placode cells expresses the stem cell marker CD133 during early morphogenesis. CD133 associates with membrane beta-catenin in early placodes and its continued expression correlates with loss of beta-catenin and E-cadherin from the cell membrane at a time when E-cadherin transcriptional repressors Snail and Slug are not implicated. Stabilization of CD133 via anti-CD133 antibody treatment of human fetal scalp explants depresses beta-catenin and E-cadherin membrane localization. We discuss this unique correlation and suggest a hypothetical model whereby CD133 promotes morphogenesis in early hair follicle placodes through the localized removal of membrane beta-catenin proteins and subsequent adherens junction dissolution.

Directional Cell Migration, but Not Proliferation, Drives Hair Placode Morphogenesis

Epithelial reorganization involves coordinated changes in cell shapes and movements. This restructuring occurs during formation of placodes, ectodermal thickenings that initiate the morphogenesis of epithelial organs including hair, mammary gland, and tooth. Signaling pathways in ectodermal placode formation are well known, but the cellular mechanisms have remained ill defined. We established imaging methodology for live visualization ofembryonic skin explants during the first wave of hair placode formation. We found that the vast majority of placodal cells were nonproliferative throughout morphogenesis. We show that cell compaction and centripetal migration are the main cellular mechanisms associated with hair placode morphogenesis and that inhibition of actin remodeling suppresses placode formation. Stimulation of both ectodysplasin/NF-κB and Wnt/β-catenin signaling increased cell motility and the number of cells committed to placodal fate. Thus, cell fate choices and morphogenetic events are controlled by the same molecular pathways, providing the framework for coordination of these two processes.