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.

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.

Single-cell transcriptome analysis reveals the dynamics of human immune cells during early fetal skin development.

The immune system of skin develops in stages in mice. However, the developmental dynamics of immune cells in human skin remains elusive. Here, we perform transcriptome profiling of CD45+ hematopoietic cells in human fetal skin at an estimated gestational age of 10-17 weeks by single-cell RNA sequencing. A total of 13 immune cell types are identified. Skin macrophages show dynamic heterogeneity over the course of skin development. A major shift in lymphoid cell developmental states occurs from the first to the second trimester that implies an in situ differentiation process. Gene expression analysis reveals a typical developmental program in immune cells in accordance with their functional maturation, possibly involving metabolic reprogramming. Finally, we identify transcription factors (TFs) that potentially regulate cellular transitions by comparing TFs and TF target gene networks. These findings provide detailed insight into how the immune system of the human skin is established during development.

Overexpression of Flii during Murine Embryonic Development Increases Symmetrical Division of Epidermal Progenitor Cells.

Epidermal progenitor cells divide symmetrically and asymmetrically to form stratified epidermis and hair follicles during late embryonic development. Flightless I (Flii), an actin remodelling protein, is implicated in Wnt/ß-cat and integrin signalling pathways that govern cell division. This study investigated the effect of altering Flii on the divisional orientation of epidermal progenitor cells (EpSCs) in the basal layer during late murine embryonic development and early adolescence. The effect of altering Flii expression on asymmetric vs. symmetric division was assessed in vitro in adult human primary keratinocytes and in vivo at late embryonic development stages (E16, E17 and E19) as well as adolescence (P21 day-old) in mice with altered Flii expression (Flii knockdown: Flii+/-, wild type: WT, transgenic Flii overexpressing: FliiTg/Tg) using Western blot and immunohistochemistry. Flii+/- embryonic skin showed increased asymmetrical cell division of EpSCs with an increase in epidermal stratification and elevated talin, activated-Itgb1 and Par3 expression. FliiTg/Tg led to increased symmetrical cell division of EpSCs with increased cell proliferation rate, an elevated epidermal SOX9, Flap1 and ß-cat expression, a thinner epidermis, but increased hair follicle number and depth. Flii promotes symmetric division of epidermal progenitor cells during murine embryonic development.

Zebrafish twist2/dermo1 regulates scale shape and scale organization during skin development and regeneration.

Scales are skin appendages in fishes that evolutionarily predate feathers in birds and hair in mammals. Zebrafish scales are dermal in origin and develop during metamorphosis. Understanding regulation of scale development in zebrafish offers an exciting possibility of unraveling how the mechanisms of skin appendage formation evolved in lower vertebrates and whether these mechanisms remained conserved in birds and mammals. Here we have investigated the expression and function of twist 2/dermo1 gene - known for its function in feather and hair formation - in scale development and regeneration. We show that of the four zebrafish twist paralogues, twist2/dermo1 and twist3 are expressed in the scale forming cells during scale development. Their expression is also upregulated during scale regeneration. Our knockout analysis reveals that twist2/dermo1 gene functions in the maintenance of the scale shape and organization during development as well as regeneration. We further show that the expression of twist2/dermo1 and twist3 is regulated by Wnt signaling. Our results demonstrate that the function of twist2/dermo1 in skin appendage formation, presumably under regulation of Wnt signaling, originated during evolution of basal vertebrates.

Histochemical and Morphometrical Analyses of Scarless Wound Healing in Mouse Fetal Model.

OBJECTIVE: Scar formation is an inevitable outcome after craniofacial surgery in the congenital facial anomaly. Scarless healing is the ultimate treatment after the surgery. Therefore, we elucidate the mechanism underlying scarless healing during fetal development. METHODS: A full-thickness back skin excision (1 × 0.5 mm) was made at embryonic day 16.5 (E16.5) and 18.5 (E18.5) in fetal C57BL/6J mice and examined the histochemical and morphometrical findings of wound healing after 48 hours. RESULTS: The wound made at E16.5 showed almost complete re-epithelialization with fine reticular dermal collagen fibers, but not at E18.5. The ratio of CK5 positive area was significantly higher in the wound of E16.5 operation than in the E18.5. The wounds made at E18.5 showed granulation tissue formation which will lead to subsequent scar formation. The collagen fibers tended to be thinner in wound than in normal skin, while the decrease in the number of fibers but the increase in the straightness of fibers were evident in the wound at E18.5. CONCLUSION: Transition point of scarless healing seemed between E16.5 and E18.5 in mice, which may imply that the potential of epithelial regeneration and matrix formation was changed, possibly due to alteration of cell constitution and decrease in stemness, at that time.

Inference and analysis of cell-cell communication using CellChat.

Understanding global communications among cells requires accurate representation of cell-cell signaling links and effective systems-level analyses of those links. We construct a database of interactions among ligands, receptors and their cofactors that accurately represent known heteromeric molecular complexes. We then develop CellChat, a tool that is able to quantitatively infer and analyze intercellular communication networks from single-cell RNA-sequencing (scRNA-seq) data. CellChat predicts major signaling inputs and outputs for cells and how those cells and signals coordinate for functions using network analysis and pattern recognition approaches. Through manifold learning and quantitative contrasts, CellChat classifies signaling pathways and delineates conserved and context-specific pathways across different datasets. Applying CellChat to mouse and human skin datasets shows its ability to extract complex signaling patterns. Our versatile and easy-to-use toolkit CellChat and a web-based Explorer ( http://www.cellchat.org/ ) will help discover novel intercellular communications and build cell-cell communication atlases in diverse tissues.

αßγδ T cells play a vital role in fetal human skin development and immunity.

T cells in human skin play an important role in the immune defense against pathogens and tumors. T cells are present already in fetal skin, where little is known about their cellular phenotype and biological function. Using single-cell analyses, we identified a naive T cell population expressing αß and γδ T cell receptors (TCRs) that was enriched in fetal skin and intestine but not detected in other fetal organs and peripheral blood. TCR sequencing data revealed that double-positive (DP) αßγδ T cells displayed little overlap of CDR3 sequences with single-positive αß T cells. Gene signatures, cytokine profiles and in silico receptor-ligand interaction studies indicate their contribution to early skin development. DP αßγδ T cells were phosphoantigen responsive, suggesting their participation in the protection of the fetus against pathogens in intrauterine infections. Together, our analyses unveil a unique cutaneous T cell type within the native skin microenvironment and point to fundamental differences in the immune surveillance between fetal and adult human skin.

Preterm Infant Skin Structure Is Qualitatively and Quantitatively Different From That of Term Newborns.

BACKGROUND: The immature skin of preterm infants is uniquely vulnerable to pressure and chemical injury. We sought to qualitatively and quantitatively describe the histopathologic patterns of skin development in preterm infants. METHODS: Autopsy skin samples were examined for 48 liveborn preterm infants born at 18+ to 36 weeks, and control groups of term neonates and older infants/children. Quantitative variables included thickness of the stratum corneum, epidermis, and dermis. Qualitative features included stratum corneum, rete ridges, and hair follicles. RESULTS: Patterns of maturation were reproducible. Compact keratin appeared beginning at 21-22 weeks. Basketweave keratin appeared first around hair follicles, and then became more generalized from ∼28 weeks corrected gestational age (CGA) onward. Rete ridges began to appear at ∼30 weeks. Epidemal and dermal thickness increased with age. Infants who survived ≤7 days had thicker dermis than those who survived longer, even adjusted for CGA. CONCLUSIONS: Skin development in preterm infants has reproducible milestones. Significant structural changes occurring around 28-30 weeks may improve barrier function, with implications for use of topical compounds such as chlorhexidine. The findings also highlight challenges in evaluating pressure injuries in preterm infants, and postnatal changes in skin parameters.

Developmental cell programs are co-opted in inflammatory skin disease.

The skin confers biophysical and immunological protection through a complex cellular network established early in embryonic development. We profiled the transcriptomes of more than 500,000 single cells from developing human fetal skin, healthy adult skin, and adult skin with atopic dermatitis and psoriasis. We leveraged these datasets to compare cell states across development, homeostasis, and disease. Our analysis revealed an enrichment of innate immune cells in skin during the first trimester and clonal expansion of disease-associated lymphocytes in atopic dermatitis and psoriasis. We uncovered and validated in situ a reemergence of prenatal vascular endothelial cell and macrophage cellular programs in atopic dermatitis and psoriasis lesional skin. These data illustrate the dynamism of cutaneous immunity and provide opportunities for targeting pathological developmental programs in inflammatory skin diseases.

Parallel evolution of direct development in frogs - Skin and thyroid gland development in African Squeaker Frogs (Anura: Arthroleptidae: Arthroleptis).

BACKGROUND: Cases of parallel evolution offer the possibility to identify adaptive traits and to uncover developmental constraints on the evolutionary trajectories of these traits. The independent evolution of direct development from the ancestral biphasic life history in frogs is such a case of parallel evolution. In frogs, aquatic larvae (tadpoles) differ profoundly from their adult forms and exhibit a stunning diversity regarding their habitats, morphology and feeding behaviors. The transition from the tadpole to the adult is a climactic, thyroid hormone (TH)-dependent process of profound and fast morphological rearrangement called metamorphosis. One of the organ systems that experiences the most comprehensive metamorphic rearrangements is the skin. Direct-developing frogs lack a free-swimming tadpole and hatch from terrestrial eggs as fully formed froglets. In the few species examined, development is characterized by the condensed and transient formation of some tadpole-specific features and the early formation of adult-specific features during a "cryptic" metamorphosis. RESULTS: We show that skin in direct-developing African squeaker frogs (Arthroleptis) is also repatterned from a tadpole-like to an adult-like histology during a cryptic metamorphosis. This repatterning correlates with histological thyroid gland maturation. A comparison with data from the Puerto Rican coqui (Eleutherodactylus coqui) reveals that the evolution of direct development in these frogs is associated with a comparable heterochronic shift of thyroid gland maturation. CONCLUSION: This suggests that the development of many adult features is still dependent on, and possibly constrained by, the ancestral dependency on thyroid hormone signaling.

Advances in scarless foetal wound healing and prospects for scar reduction in adults.

Healing after mammalian skin injury involves the interaction between numerous cellular constituents and regulatory factors, which together form three overlapping phases: an inflammatory response, a proliferation phase and a remodelling phase. Any slight variation in these three stages can substantially alter the healing process and resultant production of scars. Of particular significance are the mechanisms responsible for the scar-free phenomenon observed in the foetus. Uncovering such mechanisms would offer great expectations in the treatment of scars and therefore represents an important area of investigation. In this review, we provide a comprehensive summary of studies on injury-induced skin regeneration within the foetus. The information contained in these studies provides an opportunity for new insights into the treatment of clinical scars based on the cellular and molecular processes involved.

Identification of key genes and pathways associated with duck (Anas platyrhynchos) embryonic skin development using weighted gene co-expression network analysis.

Skin and feather follicle morphogenesis are important processes for duck development; however, the mechanisms underlying morphogenesis at the embryonic stage remain unclear. To improve the understanding of these processes, we used transcriptome and weighted gene co-expression network analyses to identify the critical genes and pathways involved in duck skin development. Five modules were found to be the most related to five key stages in skin development that span from embryonic day 8 (E8) to postnatal day 7 (D7). Using STEM software, 6519 genes from five modules were clustered into 10 profiles to reveal key genes. Above all, we obtained several key module genes including WNT3A, NOTCH1, SHH, BMP2, NOG, SMAD3, and TGFß2. Furthermore, we revealed that several pathways play critical roles throughout the skin development process, including the Wnt pathway and cytoskeletal rearrangement-related pathways, whereas others are involved in specific stages of skin development, such as the Notch, Hedgehog, and TGF-beta signaling pathways. Overall, this study identified the pathways and genes that play critical roles in skin development, which may provide a basis for high-quality down-type meat duck breeding.

Identification of epidermal differentiation genes of the tuatara provides insights into the early evolution of lepidosaurian skin.

The tuatara (Sphenodon punctatus) is the phylogenetically closest relative of squamates (including lizards and snakes) from which it diverged around 250 million years ago. Together, they constitute the clade Lepidosauria. Fully terrestrial vertebrates (amniotes) form their skin barrier to the environment under the control of a gene cluster, termed the epidermal differentiation complex (EDC). Here we identified EDC genes in the genome of the tuatara and compared them to those of other amniotes. The organization of the EDC and proteins encoded by EDC genes are most similar in the tuatara and squamates. A subcluster of lepidosaurian EDC genes encodes corneous beta-proteins (CBPs) of which three different types are conserved in the tuatara. Small proline-rich proteins have undergone independent expansions in the tuatara and some, but not all subgroups of squamates. Two genes encoding S100 filaggrin-type proteins (SFTPs) are expressed during embryonic skin development of the tuatara whereas SFTP numbers vary between 1 and 3 in squamates. Our comparative analysis of the EDC in the tuatara genome suggests that many molecular features of the skin that were previously identified in squamates have evolved prior to their divergence from the lineage leading to the tuatara.

Ontogeny of the Moorish gecko Tarentola mauritanica with emphasis on morphogenesis of the skin and its derivatives.

Studying reptilian embryonic development provides answers to many questions related to the development of tetrapods. Reptilian skin has been recently considered in studies at the evo-devo level. The lizard epidermis has to be shed periodically. At the embryonic level, contention exists regarding the first layers to appear, whether the oberhautchen or the clear layer, and whether the shedding complex develops before hatching. Geckos exhibit diverse morphologies independently evolved multiple times within the clade, such as subdigital pad lamellae. Here we investigate the embryonic development of Tarentola mauritanica and establishing its embryonic table. Primarily we follow the development of the integument. This is a closely related species to Tarentola annularis and it is crucial to investigate whether it has the same derived digital condition of claw regression. Eleven embryonic stages are described according to the external morphological characteristics of the embryos. Interestingly, the oviposition stage appears earlier than its close relative T. annularis, and the total incubation time is less. We also describe skin development, adding clear evidence to the debate on the development of the shedding complex, which we found is developed before hatching. We describe one layer of periderm and the clear layer as the first embryonic epidermal layers. Generally, our results show the genus Tarentola to have the advantage of being a unique taxon, easily breed at the laboratory, with multiple clutches per year, and with an earlier stage at oviposition. That could be a model animal for embryonic development and experimental embryology studies.

Formation, structure, and function of extra-skeletal bones in mammals.

This review describes the formation, structure, and function of bony compartments in antlers, horns, ossicones, osteoderm and the os penis/os clitoris (collectively referred to herein as AHOOO structures) in extant mammals. AHOOOs are extra-skeletal bones that originate from subcutaneous (dermal) tissues in a wide variety of mammals, and this review elaborates on the co-development of the bone and skin in these structures. During foetal stages, primordial cells for the bony compartments arise in subcutaneous tissues. The epithelial-mesenchymal transition is assumed to play a key role in the differentiation of bone, cartilage, skin and other tissues in AHOOO structures. AHOOO ossification takes place after skeletal bone formation, and may depend on sexual maturity. Skin keratinization occurs in tandem with ossification and may be under the control of androgens. Both endochondral and intramembranous ossification participate in bony compartment formation. There is variation in gradients of density in different AHOOO structures. These gradients, which vary according to function and species, primarily reduce mechanical stress. Anchorage of AHOOOs to their surrounding tissues fortifies these structures and is accomplished by bone-bone fusion and Sharpey fibres. The presence of the integument is essential for the protection and function of the bony compartments. Three major functions can be attributed to AHOOOs: mechanical, visual, and thermoregulatory. This review provides the first extensive comparative description of the skeletal and integumentary systems of AHOOOs in a variety of mammals.

The developing immune network in human prenatal skin.

Establishment of a well-functioning immune network in skin is crucial for its barrier function. This begins in utero alongside the structural differentiation and maturation of skin, and continues to expand and diversify across the human lifespan. The microenvironment of the developing human skin supports immune cell differentiation and has an overall anti-inflammatory profile. Immunologically inert and skewed immune populations found in developing human skin promote wound healing, and as such may play a crucial role in the structural changes occurring during skin development.

Cortical contraction drives the 3D patterning of epithelial cell surfaces.

Cellular protrusions create complex cell surface topographies, but biomechanical mechanisms regulating their formation and arrangement are largely unknown. To study how protrusions form, we focused on the morphogenesis of microridges, elongated actin-based structures that are arranged in maze-like patterns on the apical surfaces of zebrafish skin cells. Microridges form by accreting simple finger-like precursors. Live imaging demonstrated that microridge morphogenesis is linked to apical constriction. A nonmuscle myosin II (NMII) reporter revealed pulsatile contractions of the actomyosin cortex, and inhibiting NMII blocked apical constriction and microridge formation. A biomechanical model suggested that contraction reduces surface tension to permit the fusion of precursors into microridges. Indeed, reducing surface tension with hyperosmolar media promoted microridge formation. In anisotropically stretched cells, microridges formed by precursor fusion along the stretch axis, which computational modeling explained as a consequence of stretch-induced cortical flow. Collectively, our results demonstrate how contraction within the 2D plane of the cortex can pattern 3D cell surfaces.

The comprehensive detection of miRNA, lncRNA, and circRNA in regulation of mouse melanocyte and skin development.

BACKGROUND: Pigmentation development, is a complex process regulated by many transcription factors during development. With the development of the RNA sequencing (RNA-seq), non-coding RNAs, such as miRNAs, lncRNAs, and circRNAs, are found to play an important role in the function detection of related regulation factors. In this study, we provided the expression profiles and development of ncRNAs related to melanocyte and skin development in mice with black coat color skin and mice with white coat color skin during embryonic day 15 (E15) and postnatal day 7 (P7). The expression profiles of different ncRNAs were detected via RNA-seq and also confirmed by the quantitative real-time PCR (qRT-PCR) method. GO and KEGG used to analyze the function the related target genes. RESULTS: We identified an extensive catalogue of 206 and 183 differently expressed miRNAs, 600 and 800 differently expressed lncRNAs, and 50 and 54 differently expressed circRNAs, respectively. GO terms and pathway analysis showed the target genes of differentially expressed miRNA and lncRNA. The host genes of circRNA were mainly enriched in cellular process, single organism process. The target genes of miRNAs were mainly enriched in chromatin binding and calcium ion binding in the nucleus. The function of genes related to lncRNAs are post translation modification. The competing endogenous RNA (ceRNA) network of lncRNAs and circRNAs displays a complex interaction between ncRNA and mRNA related to skin development, such as Tcf4, Gnas, and Gpnms related to melanocyte development. CONCLUSIONS: The ceRNA network of lncRNA and circRNA displays a complex interaction between ncRNA and mRNA related to skin development and melanocyte development. The embryonic and postnatal development of skin provide a reference for further studies on the development mechanisms of ncRNA during pigmentation.

Exploration of key regulators driving primary feather follicle induction in goose skin.

The primary feather follicles are universal skin appendages widely distributed in the skin of feathered birds. The morphogenesis and development of the primary feather follicles in goose skin remain largely unknown. Here, the induction of primary feather follicles in goose embryonic skin (pre-induction vs induction) was investigated by de novo transcriptome analyses to reveal 409 differentially expressed genes (DEGs). The DEGs were characterized to potentially regulate the de novo formation of feather follicle primordia consisting of placode (4 genes) and dermal condensate (12 genes), and the thickening of epidermis (5 genes) and dermal fibroblasts (17 genes), respectively. Further analyses enriched DEGs into GO terms represented as cell adhesion and KEGG pathways including Wnt and Hedgehog signaling pathways that are highly correlated with cell communication and molecular regulation. Six selected Wnt pathway genes were detected by qPCR with up-regulation in goose skin during the induction of primary feather follicles. The localization of WNT16, SFRP1 and FRZB by in situ hybridization showed weak expression in the primary feather primordia, whereas FZD1, LEF1 and DKK1 were expressed initially in the inter-follicular skin and feather follicle primordia, then mainly restricted in the feather primordia. The spatial-temporal expression patterns indicate that Wnt pathway genes DKK1, FZD1 and LEF1 are the important regulators functioned in the induction of primary feather follicle in goose skin. The dynamic molecular changes and specific gene expression patterns revealed in this report provide the general knowledge of primary feather follicle and skin development in waterfowl, and contribute to further understand the diversity of hair and feather development beyond the mouse and chicken models.