The Making of a Flight Feather: Bio-architectural Principles and Adaptation.

The evolution of flight in feathered dinosaurs and early birds over millions of years required flight feathers whose architecture features hierarchical branches. While barb-based feather forms were investigated, feather shafts and vanes are understudied. Here, we take a multi-disciplinary approach to study their molecular control and bio-architectural organizations. In rachidial ridges, epidermal progenitors generate cortex and medullary keratinocytes, guided by Bmp and transforming growth factor ß (TGF-ß) signaling that convert rachides into adaptable bilayer composite beams. In barb ridges, epidermal progenitors generate cylindrical, plate-, or hooklet-shaped barbule cells that form fluffy branches or pennaceous vanes, mediated by asymmetric cell junction and keratin expression. Transcriptome analyses and functional studies show anterior-posterior Wnt2b signaling within the dermal papilla controls barbule cell fates with spatiotemporal collinearity. Quantitative bio-physical analyses of feathers from birds with different flight characteristics and feathers in Burmese amber reveal how multi-dimensional functionality can be achieved and may inspire future composite material designs. VIDEO ABSTRACT.

Genetic Mapping and Biochemical Basis of Yellow Feather Pigmentation in Budgerigars.

Parrot feathers contain red, orange, and yellow polyene pigments called psittacofulvins. Budgerigars are parrots that have been extensively bred for plumage traits during the last century, but the underlying genes are unknown. Here we use genome-wide association mapping and gene-expression analysis to map the Mendelian blue locus, which abolishes yellow pigmentation in the budgerigar. We find that the blue trait maps to a single amino acid substitution (R644W) in an uncharacterized polyketide synthase (MuPKS). When we expressed MuPKS heterologously in yeast, yellow pigments accumulated. Mass spectrometry confirmed that these yellow pigments match those found in feathers. The R644W substitution abolished MuPKS activity. Furthermore, gene-expression data from feathers of different bird species suggest that parrots acquired their colors through regulatory changes that drive high expression of MuPKS in feather epithelia. Our data also help formulate biochemical models that may explain natural color variation in parrots. VIDEO ABSTRACT.

Organ-level quorum sensing directs regeneration in hair stem cell populations.

Coordinated organ behavior is crucial for an effective response to environmental stimuli. By studying regeneration of hair follicles in response to patterned hair plucking, we demonstrate that organ-level quorum sensing allows coordinated responses to skin injury. Plucking hair at different densities leads to a regeneration of up to five times more neighboring, unplucked resting hairs, indicating activation of a collective decision-making process. Through data modeling, the range of the quorum signal was estimated to be on the order of 1 mm, greater than expected for a diffusible molecular cue. Molecular and genetic analysis uncovered a two-step mechanism, where release of CCL2 from injured hairs leads to recruitment of TNF-α-secreting macrophages, which accumulate and signal to both plucked and unplucked follicles. By coupling immune response with regeneration, this mechanism allows skin to respond predictively to distress, disregarding mild injury, while meeting stronger injury with full-scale cooperative activation of stem cells.

Gap junctions in Turing-type periodic feather pattern formation.

Periodic patterning requires coordinated cell-cell interactions at the tissue level. Turing showed, using mathematical modeling, how spatial patterns could arise from the reactions of a diffusive activator-inhibitor pair in an initially homogeneous 2D field. Most activators and inhibitors studied in biological systems are proteins, and the roles of cell-cell interaction, ions, bioelectricity, etc. are only now being identified. Gap junctions (GJs) mediate direct exchanges of ions or small molecules between cells, enabling rapid long-distance communications in a cell collective. They are therefore good candidates for propagating nonprotein-based patterning signals that may act according to the Turing principles. Here, we explore the possible roles of GJs in Turing-type patterning using feather pattern formation as a model. We found 7 of the 12 investigated GJ isoforms are highly dynamically expressed in the developing chicken skin. In ovo functional perturbations of the GJ isoform, connexin 30, by siRNA and the dominant-negative mutant applied before placode development led to disrupted primary feather bud formation. Interestingly, inhibition of gap junctional intercellular communication (GJIC) in the ex vivo skin explant culture allowed the sequential emergence of new feather buds at specific spatial locations relative to the existing primary buds. The results suggest that GJIC may facilitate the propagation of long-distance inhibitory signals. Thus, inhibition of GJs may stimulate Turing-type periodic feather pattern formation during chick skin development, and the removal of GJ activity would enable the emergence of new feather buds if the local environment were competent and the threshold to form buds was reached. We further propose Turing-based computational simulations that can predict the sequential appearance of these ectopic buds. Our models demonstrate how a Turing activator-inhibitor system can continue to generate patterns in the competent morphogenetic field when the level of intercellular communication at the tissue scale is modulated.

Cyclic growth of dermal papilla and regeneration of follicular mesenchymal components during feather cycling.

How dermis maintains tissue homeostasis in cyclic growth and wounding is a fundamental unsolved question. Here, we study how dermal components of feather follicles undergo physiological (molting) and plucking injury-induced regeneration in chickens. Proliferation analyses reveal quiescent, transient-amplifying (TA) and long-term label-retaining dermal cell (LRDC) states. During the growth phase, LRDCs are activated to make new dermal components with distinct cellular flows. Dermal TA cells, enriched in the proximal follicle, generate both peripheral pulp, which extends distally to expand the epithelial-mesenchymal interactive interface for barb patterning, and central pulp, which provides nutrition. Entering the resting phase, LRDCs, accompanying collar bulge epidermal label-retaining cells, descend to the apical dermal papilla. In the next cycle, these apical dermal papilla LRDCs are re-activated to become new pulp progenitor TA cells. In the growth phase, lower dermal sheath can generate dermal papilla and pulp. Transcriptome analyses identify marker genes and highlight molecular signaling associated with dermal specification. We compare the cyclic topological changes with those of the hair follicle, a convergently evolved follicle configuration. This work presents a model for analyzing homeostasis and tissue remodeling of mesenchymal progenitors.

Cis-acting mutation affecting GJA5 transcription is underlying the Melanotic within-feather pigmentation pattern in chickens.

Melanotic (Ml) is a mutation in chickens that extends black (eumelanin) pigmentation in normally brown or red (pheomelanin) areas, thus affecting multiple within-feather patterns [J. W. Moore, J. R. Smyth Jr, J. Hered. 62, 215-219 (1971)]. In the present study, linkage mapping using a back-cross between Dark Cornish (Ml/Ml) and Partridge Plymouth Rock (ml+/ml+ ) chickens assigned Ml to an 820-kb region on chromosome 1. Identity-by-descent mapping, via whole-genome sequencing and diagnostic tests using a diverse set of chickens, refined the localization to the genomic region harboring GJA5 encoding gap-junction protein 5 (alias connexin 40) previously associated with pigmentation patterns in zebrafish. An insertion/deletion polymorphism located in the vicinity of the GJA5 promoter region was identified as the candidate causal mutation. Four different GJA5 transcripts were found to be expressed in feather follicles and at least two showed differential expression between genotypes. The results showed that Melanotic constitutes a cis-acting regulatory mutation affecting GJA5 expression. A recent study established the melanocortin-1 receptor (MC1R) locus and the interaction between the MC1R receptor and its antagonist agouti-signaling protein as the primary mechanism underlying variation in within-feather pigmentation patterns in chickens. The present study advances understanding the mechanisms underlying variation in plumage color in birds because it demonstrates that the activity of connexin 40/GJA5 can modulate the periodic pigmentation patterns within individual feathers.

Regional specific differentiation of integumentary organs: SATB2 is involved in α- and ß-keratin gene cluster switching in the chicken.

BACKGROUND: Animals develop skin regional specificities to best adapt to their environments. Birds are excellent models in which to study the epigenetic mechanisms that facilitate these adaptions. Patients suffering from SATB2 mutations exhibit multiple defects including ectodermal dysplasia-like changes. The preferential expression of SATB2, a chromatin regulator, in feather-forming compared to scale-forming regions, suggests it functions in regional specification of chicken skin appendages by acting on either differentiation or morphogenesis. RESULTS: Retrovirus mediated SATB2 misexpression in developing feathers, beaks, and claws causes epidermal differentiation abnormalities (e.g. knobs, plaques) with few organ morphology alterations. Chicken ß-keratins are encoded in 5 sub-clusters (Claw, Feather, Feather-like, Scale, and Keratinocyte) on Chromosome 25 and a large Feather keratin cluster on Chromosome 27. Type I and II α-keratin clusters are located on Chromosomes 27 and 33, respectively. Transcriptome analyses showed these keratins (1) are often tuned up or down collectively as a sub-cluster, and (2) these changes occur in a temporo-spatial specific manner. CONCLUSIONS: These results suggest an organizing role of SATB2 in cluster-level gene co-regulation during skin regional specification.

Instructive role of melanocytes during pigment pattern formation of the avian skin.

Animal skin pigment patterns are excellent models to study the mechanism of biological self-organization. Theoretical approaches developed mathematical models of pigment patterning and molecular genetics have brought progress; however, the responsible cellular mechanism is not fully understood. One long unsolved controversy is whether the patterning information is autonomously determined by melanocytes or nonautonomously determined from the environment. Here, we transplanted purified melanocytes and demonstrated that melanocytes could form periodic pigment patterns cell autonomously. Results of heterospecific transplantation among quail strains are consistent with this finding. Further, we observe that developing melanocytes directly connect with each other via filopodia to form a network in culture and in vivo. This melanocyte network is reminiscent of zebrafish pigment cell networks, where connexin is implicated in stripe formation via genetic studies. Indeed, we found connexin40 (cx40) present on developing melanocytes in birds. Stripe patterns can form in quail skin explant cultures. Several calcium channel modulators can enhance or suppress pigmentation globally, but a gap junction inhibitor can change stripe patterning. Most interestingly, in ovo, misexpression of dominant negative cx40 expands the black region, while overexpression of cx40 expands the yellow region. Subsequently, melanocytes instruct adjacent dermal cells to express agouti signaling protein (ASIP), the regulatory factor for pigment switching, which promotes pheomelanin production. Thus, we demonstrate Japanese quail melanocytes have an autonomous periodic patterning role during body pigment stripe formation. We also show dermal agouti stripes and how the coupling of melanocytes with dermal cells may confer stable and distinct pigment stripe patterns.

Self-organization process in newborn skin organoid formation inspires strategy to restore hair regeneration of adult cells.

Organoids made from dissociated progenitor cells undergo tissue-like organization. This in vitro self-organization process is not identical to embryonic organ formation, but it achieves a similar phenotype in vivo. This implies genetic codes do not specify morphology directly; instead, complex tissue architectures may be achieved through several intermediate layers of cross talk between genetic information and biophysical processes. Here we use newborn and adult skin organoids for analyses. Dissociated cells from newborn mouse skin form hair primordia-bearing organoids that grow hairs robustly in vivo after transplantation to nude mice. Detailed time-lapse imaging of 3D cultures revealed unexpected morphological transitions between six distinct phases: dissociated cells, cell aggregates, polarized cysts, cyst coalescence, planar skin, and hair-bearing skin. Transcriptome profiling reveals the sequential expression of adhesion molecules, growth factors, Wnts, and matrix metalloproteinases (MMPs). Functional perturbations at different times discern their roles in regulating the switch from one phase to another. In contrast, adult cells form small aggregates, but then development stalls in vitro. Comparative transcriptome analyses suggest suppressing epidermal differentiation in adult cells is critical. These results inspire a strategy that can restore morphological transitions and rescue the hair-forming ability of adult organoids: (i) continuous PKC inhibition and (ii) timely supply of growth factors (IGF, VEGF), Wnts, and MMPs. This comprehensive study demonstrates that alternating molecular events and physical processes are in action during organoid morphogenesis and that the self-organizing processes can be restored via environmental reprogramming. This tissue-level phase transition could drive self-organization behavior in organoid morphogenies beyond the skin.

Morpho-regulation in diverse chicken feather formation: Integrating branching modules and sex hormone-dependent morpho-regulatory modules.

Many animals can change the size, shape, texture and color of their regenerated coats in response to different ages, sexes, or seasonal environmental changes. Here, we propose that the feather core branching morphogenesis module can be regulated by sex hormones or other environmental factors to change feather forms, textures or colors, thus generating a large spectrum of complexity for adaptation. We use sexual dimorphisms of the chicken to explore the role of hormones. A long-standing question is whether the sex-dependent feather morphologies are autonomously controlled by the male or female cell types, or extrinsically controlled and reversible. We have recently identified core feather branching molecular modules which control the anterior-posterior (bone morphogenetic orotein [BMP], Wnt gradient), medio-lateral (Retinoic signaling, Gremlin), and proximo-distal (Sprouty, BMP) patterning of feathers. We hypothesize that morpho-regulation, through quantitative modulation of existing parameters, can act on core branching modules to topologically tune the dimension of each parameter during morphogenesis and regeneration. Here, we explore the involvement of hormones in generating sexual dimorphisms using exogenously delivered hormones. Our strategy is to mimic male androgen levels by applying exogenous dihydrotestosterone and aromatase inhibitors to adult females and to mimic female estradiol levels by injecting exogenous estradiol to adult males. We also examine differentially expressed genes in the feathers of wildtype male and female chickens to identify potential downstream modifiers of feather morphogenesis. The data show male and female feather morphology and their color patterns can be modified extrinsically through molting and resetting the stem cell niche during regeneration.

Comparative regenerative biology of spiny (Acomys cahirinus) and laboratory (Mus musculus) mouse skin.

Wound-induced hair follicle neogenesis (WIHN) has been demonstrated in laboratory mice (Mus musculus) after large (>1.5 × 1.5 cm2 ) full-thickness wounds. WIHN occurs more robustly in African spiny mice (Acomys cahirinus), which undergo autotomy to escape predation. Yet, the non-WIHN regenerative ability of the spiny mouse skin has not been explored. To understand the regenerative ability of the spiny mouse, we characterized skin features such as hair types, hair cycling, and the response to small and large wounds. We found that spiny mouse skin contains a large portion of adipose tissue. The spiny mouse hair bulge is larger and shows high expression of stem cell markers, K15 and CD34. All hair types cycle synchronously. To our surprise, the hair cycle is longer and less frequent than in laboratory mice. Newborn hair follicles in anagen are more mature than C57Bl/6 and demonstrate molecular features similar to C57Bl/6 adult hairs. The second hair cycling wave begins at week 4 and lasts for 5 weeks, then telogen lasts for 30 weeks. The third wave has a 6-week anagen, and even longer telogen. After plucking, spiny mouse hairs regenerate in about 5 days, similar to that of C57Bl/6. After large full-thickness excisional wounding, there is more de novo hair formation than C57Bl/6. Also, all hair types are present and pigmented, in contrast to the unpigmented zigzag hairs in C57Bl/6 WIHN. These findings shed new light on the regenerative biology of WIHN and may help us understand the control of skin repair vs regeneration.

Transcriptome analyses of reprogrammed feather / scale chimeric explants revealed co-expressed epithelial gene networks during organ specification.

BACKGROUND: The molecular mechanism controlling regional specific skin appendage phenotypes is a fundamental question that remains unresolved. We recently identified feather and scale primordium associated genes and with functional studies, proposed five major modules are involved in scale-to-feather conversion and their integration is essential to form today's feathers. Yet, how the molecular networks are wired and integrated at the genomic level is still unknown. RESULTS: Here, we combine classical recombination experiments and systems biology technology to explore the molecular mechanism controlling cell fate specification. In the chimeric explant, dermal fate is more stable, while epidermal fate is reprogrammed to be similar to the original appendage type of the mesenchyme. We analyze transcriptome changes in both scale-to-feather and feather-to-scale transition in the epidermis. We found a highly interconnected regulatory gene network controlling skin appendage types. These gene networks are organized around two molecular hubs, ß-catenin and retinoic acid (RA), which can bind to regulatory elements controlling downstream gene expression, leading to scale or feather fates. ATAC sequencing analyses revealed about 1000 altered widely distributed chromatin open sites. We find that perturbation of a key gene alters the expression of many other co-expressed genes in the same module. CONCLUSIONS: Our findings suggest that these feather / scale fate specification genes form an interconnected network and rewiring of the gene network can lead to changes of appendage phenotypes, acting similarly to endogenous reprogramming at the tissue level. This work shows that key hub molecules, ß-catenin and retinoic acid, regulate scale / feather fate specification gene networks, opening up new possibilities to understand the switches controlling organ phenotypes in a two component (epithelial and mesenchyme) system.

Shaping organs by a wingless-int/Notch/nonmuscle myosin module which orients feather bud elongation.

How organs are shaped to specific forms is a fundamental issue in developmental biology. To address this question, we used the repetitive, periodic pattern of feather morphogenesis on chicken skin as a model. Avian feathers within a single tract extend from dome-shaped primordia to thin conical structures with a common axis of orientation. From a systems biology perspective, the process is precise and robust. Using tissue transplantation assays, we demonstrate that a "zone of polarizing activity," localized in the posterior feather bud, is necessary and sufficient to mediate the directional elongation. This region contains a spatially well-defined nuclear ß-catenin zone, which is induced by wingless-int (Wnt)7a protein diffusing in from posterior bud epithelium. Misexpressing nuclear ß-catenin randomizes feather polarity. This dermal nuclear ß-catenin zone, surrounded by Notch1 positive dermal cells, induces Jagged1. Inhibition of Notch signaling disrupts the spatial configuration of the nuclear ß-catenin zone and leads to randomized feather polarity. Mathematical modeling predicts that lateral inhibition, mediated by Notch signaling, functions to reduce Wnt7a gradient variations and fluctuations to form the sharp boundary observed for the dermal ß-catenin zone. This zone is also enriched for nonmuscle myosin IIB. Suppressing nonmuscle myosin IIB disrupts directional cell rearrangements and abolishes feather bud elongation. These data suggest that a unique molecular module involving chemical-mechanical coupling converts a pliable chemical gradient to a precise domain, ready for subsequent mechanical action, thus defining the position, boundary, and duration of localized morphogenetic activity that molds the shape of growing organs.

Specialized stem cell niche enables repetitive renewal of alligator teeth.

Reptiles and fish have robust regenerative powers for tooth renewal. However, extant mammals can either renew their teeth one time (diphyodont dentition) or not at all (monophyodont dentition). Humans replace their milk teeth with permanent teeth and then lose their ability for tooth renewal. Here, we study tooth renewal in a crocodilian model, the American alligator, which has well-organized teeth similar to mammals but can still undergo life-long renewal. Each alligator tooth is a complex family unit composed of the functional tooth, successional tooth, and dental lamina. Using multiple mitotic labeling, we map putative stem cells to the distal enlarged bulge of the dental lamina that contains quiescent odontogenic progenitors that can be activated during physiological exfoliation or artificial extraction. Tooth cycle initiation correlates with ß-catenin activation and soluble frizzled-related protein 1 disappearance in the bulge. The dermal niche adjacent to the dermal lamina dynamically expresses neural cell adhesion molecule, tenascin-C, and other molecules. Furthermore, in development, asymmetric ß-catenin localization leads to the formation of a heterochronous and complex tooth family unit configuration. Understanding how these signaling molecules interact in tooth development in this model may help us to learn how to stimulate growth of adult teeth in mammals.

Emergence of differentially regulated pathways associated with the development of regional specificity in chicken skin.

BACKGROUND: Regional specificity allows different skin regions to exhibit different characteristics, enabling complementary functions to make effective use of the integumentary surface. Chickens exhibit a high degree of regional specificity in the skin and can serve as a good model for when and how these regional differences begin to emerge. RESULTS: We used developing feather and scale regions in embryonic chickens as a model to gauge the differences in their molecular pathways. We employed cosine similarity analysis to identify the differentially regulated and co-regulated genes. We applied low cell techniques for expression validation and chromatin immunoprecipitation (ChIP)-based enhancer identification to overcome limited cell availabilities from embryonic chicken skin. We identified a specific set of genes demonstrating a high correlation as being differentially expressed during feather and scale development and maturation. Some members of the WNT, TGF-beta/BMP, and Notch family known to be involved in feathering skin differentiation were found to be differentially regulated. Interestingly, we also found genes along calcium channel pathways that are differentially regulated. From the analysis of differentially regulated pathways, we used calcium signaling pathways as an example for further verification. Some voltage-gated calcium channel subunits, particularly CACNA1D, are expressed spatio-temporally in the skin epithelium. These calcium signaling pathway members may be involved in developmental decisions, morphogenesis, or epithelial maturation. We further characterized enhancers associated with histone modifications, including H3K4me1, H3K27ac, and H3K27me3, near calcium channel-related genes and identified signature intensive hotspots that may be correlated with certain voltage-gated calcium channel genes. CONCLUSION: We demonstrated the applicability of cosine similarity analysis for identifying novel regulatory pathways that are differentially regulated during development. Our study concerning the effects of signaling pathways and histone signatures on enhancers suggests that voltage-gated calcium signaling may be involved in early skin development. This work lays the foundation for studying the roles of these gene pathways and their genomic regulation during the establishment of skin regional specificity.

Proper BMP Signaling Levels Are Essential for 3D Assembly of Hepatic Cords from Hepatoblasts and Mesenchymal Cells.

BACKGROUND: Because the molecular mechanisms of morphogenesis of the hepatic cord and sinus are unclear, we investigated the involvement of bone morphogenetic protein (BMP4) in hepatic sinusoid morphogenesis. METHODS: We used embryonic chicken livers, which develop rapidly, as our model, and investigated expression of BMP-related genes. BMP4 activity was manipulated by overexpressing BMP4 and its antagonist, noggin. RESULTS: During hepatic cord morphogenesis, BMP4 and its receptors are expressed in both peri-sinusoidal cells and hepatoblasts as the sinusoids form, whereas noggin is expressed transiently in peri-sinusoidal cells at early stages. Suppression of BMP activity with noggin overexpression disrupted normal hepatic sinusoid structure, leading to liver congestion, failure of fibronectin deposition, and markedly reduced numbers of peri-sinusoidal cells. However, overexpression of BMP did not change sinusoidal morphology but increased endothelial cell number. Noggin overexpression resulted in disrupted cord organization, and dilated sinusoidal space, eventually leading to increased apoptosis and failed hepatocyte differentiation. CONCLUSIONS: Our results show that proper BMP signaling mediates peri-sinusoidal cell-hepatoblast interactions during development; this is essential for hepatic cord organization among hepatoblasts, endothelium, and presumptive hepatic stellate cells.

Adaptive patterning of vascular network during avian skin development: Mesenchymal plasticity and dermal vasculogenesis.

A vasculature network supplies blood to feather buds in the developing skin. Does the vasculature network during early skin development form by sequential sprouting from the central vasculature or does local vasculogenesis occur first that then connect with the central vascular tree? Using transgenic Japanese quail Tg(TIE1p.H2B-eYFP), we observe that vascular progenitor cells appear after feather primordia formation. The vasculature then radiates out from each bud and connects with primordial vessels from neighboring buds. Later they connect with the central vasculature. Epithelial-mesenchymal recombination shows local vasculature is patterned by the epithelium, which expresses FGF2 and VEGF. Perturbing noggin expression leads to abnormal vascularization. To study endothelial origin, we compare transcriptomes of TIE1p.H2B-eYFP+ cells collected from the skin and aorta. Endothelial cells from the skin more closely resemble skin dermal cells than those from the aorta. The results show developing chicken skin vasculature is assembled by (1) physiological vasculogenesis from the peripheral tissue, and (2) subsequently connects with the central vasculature. The work implies mesenchymal plasticity and convergent differentiation play significant roles in development, and such processes may be re-activated during adult regeneration. SUMMARY STATEMENT: We show the vasculature network in the chicken skin is assembled using existing feather buds as the template, and endothelia are derived from local bud dermis and central vasculature.

Conserved regulatory switches for the transition from natal down to juvenile feather in birds.

The transition from natal downs for heat conservation to juvenile feathers for simple flight is a remarkable environmental adaptation process in avian evolution. However, the underlying epigenetic mechanism for this primary feather transition is mostly unknown. Here we conducted time-ordered gene co-expression network construction, epigenetic analysis, and functional perturbations in developing feather follicles to elucidate four downy-juvenile feather transition events. We report that extracellular matrix reorganization leads to peripheral pulp formation, which mediates epithelial-mesenchymal interactions for branching morphogenesis. α-SMA (ACTA2) compartmentalizes dermal papilla stem cells for feather renewal cycling. LEF1 works as a key hub of Wnt signaling to build rachis and converts radial downy to bilateral symmetry. Novel usage of scale keratins strengthens feather sheath with SOX14 as the epigenetic regulator. We show that this primary feather transition is largely conserved in chicken (precocial) and zebra finch (altricial) and discuss the possibility that this evolutionary adaptation process started in feathered dinosaurs.

Sprouty/FGF signaling regulates the proximal-distal feather morphology and the size of dermal papillae.

In a feather, there are distinct morphologies along the proximal-distal axis. The proximal part is a cylindrical stalk (calamus), whereas the distal part has barb and barbule branches. Here we focus on what molecular signaling activity can modulate feather stem cells to generate these distinct morphologies. We demonstrate the drastic tissue remodeling during feather cycling which includes initiation, growth and resting phases. In the growth phase, epithelial components undergo progressive changes from the collar growth zone to the ramogenic zone, to maturing barb branches along the proximal-distal axis. Mesenchymal components also undergo progressive changes from the dermal papilla, to the collar mesenchyme, to the pulp along the proximal-distal axis. Over-expression of Spry4, a negative regulator of receptor tyrosine kinases, promotes barb branch formation at the expense of the epidermal collar. It even induces barb branches from the follicle sheath (equivalent to the outer root sheath in hair follicles). The results are feathers with expanded feather vane regions and small or missing proximal feather shafts (the calamus). Spry4 also expands the pulp region while reducing the size of dermal papillae, leading to a failure to regenerate. In contrast, over-expressing Fgf10 increases the size of the dermal papillae, expands collar epithelia and mesenchyme, but also prevents feather branch formation and feather keratin differentiation. These results suggest that coordinated Sprouty/FGF pathway activity at different stages is important to modulate feather epidermal stem cells to form distinct feather morphologies along the proximal-distal feather axis.

Physiological regeneration of skin appendages and implications for regenerative medicine.

The concept of regenerative medicine is relatively new, but animals are well known to remake their hair and feathers regularly by normal regenerative physiological processes. Here, we focus on 1) how extrafollicular environments can regulate hair and feather stem cell activities and 2) how different configurations of stem cells can shape organ forms in different body regions to fulfill changing physiological needs.