Complex tissue architecture is achieved through multiple rounds of morphological transitions. Here, we analyzed cellular flows and tissue mechanics during avian skin development by employing chicken and transgenic quail skin explant models. We demonstrate how novel cellular flows initiate chemo-mechanical circuits that guide epithelial protrusion, folding, invagination, and spatial cell fate specification. During initial feather bud formation, stiff dermal condensates protrude vertically from the locally softened epithelial sheet. As the bud elongates, it stretches the epithelial cells at the base, thus mechanically activating YAP, which causes the epithelial sheet to fold downward and form a stiff cylindrical wall that invaginates into the skin. This stiff epithelial tongue is essential for the compaction and formation of the tightly packed dermal papillae. These topological transformational events are mechanically interconnected, and the completion of one circuit initiates the next. In contrast, during scale development, the rigid epithelial sheet restricts dermal cell flows, preventing further topological transformation. Based on these findings, we developed a topological transformation model describing how this process enabled the evolution of feather follicles from scales.

Developmental competence allows tissues to respond to inductive cues before committing to specialized forms, but how this potential is encoded at clustered gene-family loci is poorly understood. We use vertebrate skin to address this problem. Epidermis responds to regional dermal signals before committing to feather, scale, or differentiated programs, and α-keratin loci provide a stringent genomic test: separated type-I/type-II clusters show coordinated transcriptional pairing, yet individual keratin genes are selectively deployed across appendage, differentiation, and disease states. Using chicken developmental genomics with comparative mouse and human epidermal datasets, we show that α-keratin clusters are organized before commitment as scaffolded chromatin domains. Within these domains, regulatory elements remain broadly accessible but acquire state-specific activity during commitment and differentiation. Inter-cluster contacts and chromatin-factor perturbation link this architecture to keratin output and morphology. These findings reveal a locus-level chromatin basis for developmental competence, enabling domain-level coordination with gene-level selectivity during epidermal diversification.

ABSTRACT Humans are tight-skinned mammals who typically fail to regenerate large full-thickness skin wounds, instead healing with substantial scarring and concomitant loss of function. Mechanical context is a major determinant of this outcome: elevated tissue tension or stiffness promotes fibrotic repair associated with hypertrophic or keloid scarring. Accordingly, regenerative medicine research has relied on diverse animal models to understand scar development and skin regeneration. Loose-skinned mammals exhibit greater regeneration ability. Furthermore, spiny mouse skin is significantly less stiff and associated with enhanced regenerative ability. Interestingly, this skin wound stiffness can be modulated to shift healing toward more regenerative or more fibrotic trajectories. Despite of this progress, the restoration of normal skin architecture after large-full thickness injury has not been elucidated in tight-skinned mammals. Can large full-thickness wounds regenerate with minimal scarring in tight-skinned mammals? Here we show the tight-skinned mammal Fraser’s Dolphin regenerates de novo a complex rete ridge architecture with associated vasculature and minimal scar following large full-thickness wound healing. Counterintuitively, this skin regeneration occurs in an aqueous, high-shear stress and high-tension environment. Complete rete ridge regeneration in tight-skinned mammals has not been documented and not observed in humans except in utero . This unique ability to rebuild elaborate rete ridges under tension is an opportunity to uncover molecular, cellular, and tissue-level mechanisms that enable regenerative wound healing in a mechanical regime typically associated with fibrosis.

Tissue patterning is integral to development and regeneration, yet the factors that initiate morphogenetic patterning remain to be explored. Here, using embryonic chicken skin as a model, we show that perturbation of calcium signaling induces de novo feather bud formation in regions that normally do not form feather buds. This is achieved through coordinated changes in calcium dynamics, endogenous bioelectric currents, transcriptional regulation of calcium and potassium channel genes, and morphogen signaling. Different combinations of channel perturbations altered the number, distribution, size, and shape of induced feather buds. Live calcium imaging and extracellular electrophysiological recordings revealed homeostatic regulation, in which initially depressed calcium activity is followed by elevated calcium activity. Inward bioelectric currents emerge as de novo feather buds appear. Potassium channel blockade suppressed calcium dynamics, abolished endogenous currents, and inhibited new bud formation. Canonical feather morphogenesis pathways including Shh and β-catenin are induced in these new buds. Our findings support a model in which developmental bioelectricity contributes to regulating the threshold of feather bud formation. These results identify developmental bioelectricity as an unrecognized regulatory layer of tissue patterning that warrants further study.

Reaction-diffusion circuits generate self-organized spatial patterns through local activation and long-range inhibition, but synthetic implementations in mammalian cells have been limited by the differential-diffusion requirement. Here, we introduce a novel architecture, juxtacrine activation with paracrine inhibition (JAPI), where the activator propagates through cell-cell contacts rather than diffusion. We demonstrate mathematically and numerically that JAPI accesses the same patterning regimes as classical diffusion-based circuits with one fewer free parameter. We then engineer compact synNotch-based JAPI circuits in mammalian fibroblasts and demonstrate their sufficiency for self-organized patterning through tunable, size-limited signal propagation. Functionalized to spatially control morphogen secretion, these circuits perturb feather bud formation on adjacent embryonic chicken epidermis. Finally, we develop a library-based approach to explore coupled, dual-JAPI circuits with tunable cross-inhibition, enabling programmable interactions between patterns and access to a broad morphospace of spatial states. Together, JAPI provides a compact, modular platform for programming self-organized multicellular patterning.

Bioelectricity plays a key role in shaping tissues during early development. We previously demonstrated that elongating chicken feather buds establish a transient standing electrical current loop, with calcium channel-mediated inward current at the bud tip driving collective distal dermal cell movement that orients feather bud growth. Here, we evaluate the hypothesis that potassium channels carry the outward current at the bud base. We found potassium channel inhibition converts periodic feather primordia into horizontal stripes and alters bud aspect ratios by disrupting the bud elongation process. Bioelectric measurements show disruption of the entire current loop, affecting both outward current at the base and inward current at the feather bud tip. Hexagonally arrayed bud patterns become horizontal stripes and buds with irregular contours. In situ hybridization shows a thinner dermal condensation layer and failure to form distinct primordia. Despite disorganized morphology, dermal cells still express feather markers (NCAM, TnC, DKK1 and BMP4), and epidermis exhibits aberrant β-catenin, Shh, EDA and EDAR expression patterns. These findings show that potassium channel activity is required to couple cell fate specification with morphogenesis and highlight that ion channels are essential for cell-cell communication during periodic feather patterning and bud shaping.

Self-organization in organoid morphogenesis involves the coordinated arrangement of interacting cells into higher-order structures, yet the underlying principles remain elusive. Here, we investigate how epidermal and dermal cells respond distinctively to elevated levels of hypoxia during skin organoid morphogenesis that largely resembles the skin development during embryogenesis. We unveil that autonomously generated hypoxic environment-induced metabolic adaptation drives the transition from coalesced spheroids to a planarized structure in skin organoids through the following three levels. Hif1a-mediated anaerobic metabolism positions epidermal cells in the liquid phase of the cultures under lower oxygen levels, facilitating tissue phase separation of the epidermal layer from the dermal layer. Hypoxia-driven activation of lysosomal hydrolases eliminates suprabasal keratin debris during planar epidermis formation. Fibroblasts adjacent to the basal epidermis have differential metabolic adaptation to hypoxia, which exhibit enhanced retinoid metabolism and become putative papillary dermis. Together, these hypoxia-induced metabolic adaptations contribute to reconstructing skin architecture similar to physiological development. Our findings highlight the ability of hypoxia-induced metabolic alteration to trigger varied cellular responses, leading to self-organizing coalesced spheroids-to-planar topological transformations and the restoration of tissue homeostasis.

The integument serves as the interface between an organism and its environment. It primarily comprises ectoderm-derived epithelium and mesenchyme derived from various embryonic sources. These integumentary organs serve as a barrier defining the physiological boundary between the internal and exterior environments and fulfill diverse functions. How does the integument generate such a large diversity? Here, we attempt to decipher the organizational principles. We focus on amniotes and use appendage follicles as the primary examples. The integument begins as a simple planar sheet of coupled epithelial and mesenchymal cells, then becomes more complex through the following patterning processes. 1) De novo Turing periodic patterning process: This process converts the integument into multiple skin appendage units. 2) Adaptive patterning process: Dermal muscle, blood vessels, adipose tissue, and other components are assembled and organized around appendage follicles when present. 3) Cyclic renewal: Skin appendage follicles contain stem cells and their niches, enabling physiological molting and regeneration in the adult animal. 4) Spatial variations: Multiple appendage units allow modulation of shape, size, keratin types, and color patterns of feathers and hairs across the animal's surface. 5) Temporal phenotypic plasticity: Cyclic renewal permits temporal transition of appendage phenotypes, i.e. regulatory patterning or integumentary metamorphosis, throughout an animal's lifetime. The diversities in (4) and (5) can be generated epigenetically within the same animal. Over the evolutionary timescale, different species can modulate the number, size, and distributions of existing ectodermal organs in the context of micro-evolution, allowing effective adaptation to new climates as seen in the variation of hair length among mammals. Novel ectodermal organs can also emerge in the context of macro-evolution, enabling animals to explore new ecological niches, as seen in the emergence of feathers on dinosaurs. These principles demonstrate how multi-scale organ adaption in the amniotes can maximize diverse and flexible integumentary organ phenotypes, producing a vast repertoire for natural selection and thereby providing effective adaptation and evolutionary advantages.

The color patterns of mammalian fur are determined by melanocytes' ability to respond to and adapt to microenvironmental cues. However, these patterns can be lost following injury or under pathological conditions, and the underlying biological mechanisms remain poorly understood. In this study, reconstituted hair-bearing skin is generated using skin organoids derived from dissociated epidermal cells, dermal cells, and melanocyte progenitors. The reconstituted skin exhibited pigmented hair patterns. By investigating the molecular cues involved in re-establishing pigment patterns, it is demonstrated that this process is regulated through a two-step mechanism. First, during skin organoid culture, signaling from dermal fibroblasts to melanocytes via the COL6A3-CD44 pathway promotes the early maintenance of organotypic melanocytes. Subsequently, during hair follicle morphogenesis after skin organoid transplantation, signaling from the bulge to melanocytes via the SEMA3C-NRP1 pathway regulates microtubule stability. This regulation guides melanocytes to migrate to their bulge stem cell niche, thereby enhancing hair pigmentation by promoting the adaptive patterning of melanocytes within the hair follicle. The study reveals two novel signaling mechanisms that shape melanocyte adaptive patterning and highlight the hair follicle as a regulatory hub for melanocyte physiological behaviors. These findings may inspire new clinical strategies for preventing hair greying.

Ectodermal appendages display a range of renewal mechanisms, with some undergoing continuous growth and others experiencing cyclic regeneration. The latter requires sustainable epithelial stem cells and mesenchymal niche essential for interacting with these stem cells. Furthermore, certain appendages dynamically adjust their mesenchymal niche in response to environmental factors, such as hormonal fluctuations, sex, and seasonal changes, enabling them to cyclically renew with different appendages phenotypes to adapt to different environments and to different life stages. Here we focus on amniotes, including reptiles, birds, and mammals, which exhibit integumentary adaptations that enable their survival across various ecological environments, from aquatic habitats and terrestrial landscapes to aerial domains. We highlight three representative integument appendage follicles: teeth, feathers, and hairs. Despite independent evolutionary origins, these structures share a fundamental architectural design characterized by the presence of stem cells and mesenchymal niches. They differ in the spatial arrangement and topology of these components. By examining the distinct architectural features of these follicles, we demonstrate the different strategies they use to orchestrate the physiological regenerative cycling, from growth initiation to cessation and molting, and regeneration after wounding. We delve into known molecular controls that govern these processes and unravel the evolutionary insights. We also identify new cell interactions that underlie the emergence of evolutionary novel follicle components. Various amniote scales have evolved independently with different configurations, but all lack follicle architecture and maintain homeostasis using a strategy similar to that of skin. The convergently evolved follicles in hairs, feathers, and teeth utilize different designs to achieve cyclic renewability, allowing them to produce spatially and temporally specific appendage phenotypes, thus enhancing the adaptability of the integumentary interface to external environmental pressures. This, in turn, enriches our understanding of evolutionary developmental biology (Evo-Devo) of the integument, shedding light on the intricate interplay between form and function across diverse taxa.

The integument plays a critical role in functional adaptation, with macro-regional specification forming structures like beaks, combs, feathers, and scales, while micro-regional specification modifies skin appendage shapes. However, the molecular mechanisms remain largely unknown. Craniofacial integument displays dramatic diversity, exemplified by the Polish chicken (PC) with a homeotic transformation of comb-to-crest feathers, caused by a 195-base pair (bp) duplication in HoxC10 intron. Micro-C analyses show that HoxC-containing topologically associating domain (TAD) is normally closed in the scalp but open in the dorsal and tail regions, allowing multiple long-distance contacts. In the PC scalp, the TAD is open, resulting in high HoxC expression. CRISPR-Cas9 deletion of the 195-bp duplication reduces crest feather formation, and HoxC misexpression alters feather shapes. The 195-bp sequence is found only in Archelosauria (crocodilians and birds) and not in mammals. These findings suggest that higher-order regulation of the HoxC cluster modulates gene expression, driving the evolution of adaptive integumentary appendages in birds.

Introduction The exogen phase of the hair follicle cycle, during which club hairs are shed and new hairs emerge, represents a critical transitional step involving distinct structural remodeling and adhesive changes. Despite its fundamental importance, the molecular mechanisms orchestrating exogen remain largely unknown. Objective To investigate the role of Msx2 in regulating hair anchoring, hair follicle stem cell (HFSC) lineage commitment, and extracellular matrix (ECM) remodeling during exogen. Methods We employed Msx2-knockout (KO) mice, bulk RNA sequencing, and late exogen transcriptome comparisons to identify differentially expressed genes (DEGs) and disrupted pathways. Additionally, protein interaction assays were conducted for functional studies. Results Msx2 deficiency mice still form new bulges containing HFSCs for subsequent hair cycles, but this resulted in the failed anchoring of older bulges and club hairs, leading to aberrant hair retention and accelerated hair shedding. Transcriptomic analysis revealed widespread transcriptional reprogramming, particularly in ECM-related genes, with significant overlap between Msx2-KO HFSCs and late exogen DEGs. Msx2-KO mice exhibited downregulated adhesion molecules, disrupted HFSC niche integrity, increased keratinization, and aberrant differentiation in HFSC and hair germ cells. Mechanistically, MSX2 directly interacts with SMAD proteins to regulate TGF-β signaling, thereby modulating ECM gene expression and suppressing HFSC trans-epidermal differentiation. Conclusions Our findings establish Msx2 as a master regulator of exogen control, orchestrating hair shedding, HFSC niche stability, and ECM remodeling. This study provides new insights into hair follicle cycling dynamics and identifies Msx2 as a potential therapeutic target for promoting hair regeneration and mitigating alopecia phenotypes.

Germline-competent embryonic stem (ES) cells have been successfully derived from mice and rats, but not from other species. Here we report the development of culture conditions for deriving ES cells from chickens and seven other avian species. Chicken ES cells express core pluripotency markers and can differentiate into cells of all embryonic germ layers, as well as extra-embryonic lineages. Notably, chicken ES cells contribute to high rates of chimerism when injected into chicken embryos and give rise to germ cells both in vitro and in ovo, confirming their germline competence. In addition, we demonstrated that ES cell self-renewal pathways are conserved among avian species, allowing ES cells from multiple avian species to be established using optimized chicken ES cell culture conditions. The establishment of authentic avian ES cells lays the groundwork for future applications in genetic engineering and the conservation of avian biodiversity. Embryonic stem cells for chicken and seven other avian species are derived.

Introduction: Aging causes striking changes in the extracellular matrix (ECM) in hair follicles, which has a profound influence on hair growth. How the ECM of dermal papilla (DP), the master regulator of hair growth, changes during aging remains largely unknown. Methods: Herovici staining, Western Blotting and immunofluorescence were used to assess DP ECM and protein expression in hair follicles. Bulk and single cell RNA-sequencing were used to analyze gene expression and predict upstream and downstream regulators of target genes. Skin organoid and mouse models were used for functional validation of molecular mechanisms. Results: Aged follicle DP shows drastic depletion of ECM in which Thrombospondin Type 1 Domain Containing 4 (Thsd4) is highly downregulated. THSD4 is specifically expressed in the interface between DP and hair matrix (HM). It promotes hair growth by enhancing the interaction between dermal (DP) and epithelial cells (HM) through the SDC4-THSD4-CXCL1 signaling axis in both skin organoids and mouse models. Murine dorsal hair follicles show upregulated THSD4, enhanced DP-HM interaction, and hair growth following exposure to low temperature. Conclusions: THSD4 is a key micro- and macro-environmental mediator to promote hair growth by facilitating epidermal-mesenchymal interactions during aging. These findings demonstrate the therapeutic potential of low-temperature treatment for treating unwanted hair loss.

The evolutionary adaptation of skin repair drives sequential regenerative phases: epidermal proliferation rapidly restores barrier function, followed by dermal reconstruction through extracellular matrix remodeling to establish structural support, yet the molecular coordination of this spatiotemporal program remains unclear. While the endocrine system is crucial in modulating wound repair, the critical hormone receptors orchestrating tissue-layer-specific responses are unidentified. Here, bulk and single-cell RNA sequencing, spatial transcriptomics, and in vivo/in vitro analyses in mouse models of hyperthyroidism and hypothyroidism, as well as wound and skin organoid models, are employed to identify the thyroid hormone receptor Thra as a key regulator of phase-coupled regeneration through two distinct yet coordinated mechanisms. In the initial phase, epidermal Thra activates glutathione metabolism via Gamma-Glutamylcyclotransferase (GGCT), driving keratin filament assembly to accelerate reepithelialization. In the subsequent phase, dermal Thra mediates the Serum Amyloid A3 (SAA3)-Fibronectin 1 (FN1) interaction, establishing angiogenic niches essential for matrix maturation. Using the self-assembled epidermis-dermis dynamic skin organoid model, Thra's role in simulating the wound healing process is further confirmed. This study highlights the essential role of spatiotemporal adaptability in wound repair using Thra as a paradigm and provides insights for developing clinical strategies to enhance skin wound healing.

Feathers, the primary skin appendage covering the avian body, undergo dynamic phenotypic changes throughout a bird's life. Males and females of the same species can exhibit sexually dichromatic plumage colors which play a critical role in mating choice, survival, and ecological interactions. In this study, we investigate the molecular mechanisms underlying the changes of color that occur during the transition from juvenile to adult feathers, known as the secondary transition. We focus on sexual dichromatism of craniofacial plumage and use the male cheek domain of the zebra finch (Taeniopygia guttata) as the major model. The transcriptome of the cheek and scalp (crown) domains in males and females of wild-type and genetic color variants were compared. We found that (1) Craniofacial color patterning operates through two regulatory layers. The first layer involves transcription factor (TF) genes that define the cheek domain such as PITX1, PAX1, PAX6. The second layer comprises pigment-related genes responsible for specific colors, including male-biased TFs (SOX10 and DMRT1) and transporters associated with red pigment synthesis. (2) Surprisingly, ASIP, which controls pheomelanin production in other species, was expressed in both male (red) and female (gray) cheeks. Instead, PAX1 in cheek dermal fibroblasts may serve as an upstream regulator, potentially triggering the male-biased color pattern through PAX6 and SOX10. PAX6 and SOX10 in melanocytes potentially enhance the expression of GPR143, SLC45A2, and TMEM163, driving increased pheomelanin production in males. (3) Sexual dichromatism is associated with sex-linked genes on the Z chromosome, notably SLC45A2. In addition, motif analysis comparing the binding strength between regional transcription factors and melanogenesis genes suggests that craniofacial pigmentation may have evolved convergently in passerine birds. These findings provide novel insights into the molecular control of color patterning and lay the groundwork for further studies on avian sexual dichromatism and secondary feather transition.

Mammalian genomes are organized by multi-level folding; yet how this organization contributes to cell-type-specific transcription remains unclear. SATB1 forms a nuclear substructure that resists high-salt extraction. SATB1 binds base-unpairing regions (BURs), genomic elements with high unwinding propensities. In mouse thymocytes, we found that SATB1 establishes a two-tiered chromatin organization: one through indirect binding to transcriptionally active DNase 1-accessible chromatin and another by direct binding to BURs in the DNase 1-inaccessible nuclear substructure. Recently published ChIP-seq datasets show SATB1 binding to accessible chromatin at enhancers and CTCF sites, but not to BURs. By employing urea ChIP-seq, which retains only directly bound protein:DNA complexes, we found that BURs, but not CTCF sites, are direct SATB1 binding targets genome-wide. BURs bound to the SATB1 nuclear substructure interact with accessible chromatin, crossing multiple topologically associated domains (TADs). SATB1 is required for these megabase-scale interactions linked to cell-type-specific gene expression. BURs are highly enriched within transcriptionally repressive lamina-associated domains (LADs). Besides these BURs, SATB1 anchors some BURs (18%) outside LADs near genes in otherwise accessible chromatin to the SATB1 nuclear substructure. Only a subset of total BURs is bound to SATB1, depending on cell type. Notably, despite the mutually exclusive SATB1-binding profiles uncovered by the two ChIP-seq methods, we found most peaks in both profiles are valid and require SATB1. Based on these and previous data, we propose that the SATB1 protein network forms a chromatin scaffold, providing an interface that connects accessible chromatin to a subnuclear architectural structure, thereby facilitating the three-dimensional organization linked to cell-type-specific gene expression.