Cheng-Ming Chuong - US grants

limbs; cartilage development; cell adhesion; gene expression; cartilage disorder; developmental genetics; cell differentiation; cell cell interaction; intercellular connection; cell cycle; molecular biology; histogenesis; tissue /cell culture; autoradiography; chick embryo; chickens; laboratory rabbit; laboratory mouse; microelectrodes; immunofluorescence technique; enzyme linked immunosorbent assay; density gradient ultracentrifugation; gel electrophoresis; cinematography; fluorescence microscopy; nucleic acid hybridization;

9317397 Chuong Pattern formation is a crucial event in embryonic development. Identification of molecules and mechanisms involved in pattern formation have been the central interest of developmental biologists. Dr. Chuong is using feather morphogenesis as a model to study pattern formation and has focused on adhesion molecules and homeobox (Hox) genes. Dr. Chuong has shown that there are two kinds of Hox gradients in the integument: a "macro gradient" across the feather tract and a "micro gradient" across a single feather bud. The asynchronous alignment of different Hox macro gradient leads to a unique repertoire of position specific Hox expression patterns named Hox codes of skin appendages, which he hypothesizes determine the phenotype of skin appendages. Retinoic acid (RA) can transform feather phenotypes and alter the anterior-posterior axis of skin appendages, with concomitant changes in Hox expression. Epithelial-mesenchymal recombination revealed that the Hox micro gradient may be regulated by a signal from the epithelium. By the use of RA responsive element-LacZ- containing F9 cells it has been shown that a wave of endogenous RA activity propagates bilaterally from the midline. Dr. Chuong now suggests that 1) Hox codes of skin appendages determine the phenotype of skin appendages while the micro gradient determines the A-P axis of skin appendages, 2) The two gradients are regulated independently: the macro gradient may originate from the mesoderm, but the micro gradient is determined later and may be determined by the epithelium, and 3) RA is an endogenous morphogen in skin morphogenesis, with a dynamic distribution pattern during feather development similar to that of morphogenetic furrow in Drosophila eye development. To test these hypotheses, Dr. Chuong will ectopically express Hox genes on skin appendages in ovo using retroviral gene delivery. The ontogeny of Hox codes will be traced back to dermatome stage using DiI tracing and somite transpos ition. To search for the signals that regulate Hox gene expression, a model using epithelial-mesenchymal rotation/recombination of skin explants was developed in which the inducing signal can be analyzed. To test whether endogenous RA is involved in pattern formation, RARE-LacZ containing cell monolayers will be used to detect the dynamic RA distribution in developing skin. Aided by the distinct morphology of feathers, these novel approaches will add new dimensions to our understanding of pattern formation. ***

The formation of the integument requires the interaction and integration of epithelial and mesenchymal cells to form skin and specialized skin appendages with unique functions such as hair and glands. Elucidation of this process is of central importance to the management of impaired skin conditions resulting from trauma, burn, grafts, auto-immune responses, genetics, chemotherapy, UV irradiation, and other causes. We have been using cultured embryonic skin explants as a model to analyze the roles of adhesion molecules in skin appendage morphogenesis. The general hypothesis is that adhesion molecules play specific roles in different developmental stages of skin appendages morphogenesis, and that they are downstream molecules of growth factors involved in epithelial-mesenchymal interaction. Previously we demonstrated unique temporal and spatial roles of NCAM and tenascin during skin appendage development. Here we propose to further analyze their roles through ectopically and untimely expression of these genes using the newly developed retroviral technology to produce "transgenic skin" and "chimeric skin explant". To search for signals that initiate skin appendage and induce adhesion molecules, a new model using epithelial-mesenchymal rotation/recombination was developed in which new induction occurs. This provide a sensitive model on which the roles of various growth factors and intracellular signaling modulators on epithelial-mesenchymal interaction can be tested. Furthermore, we have recently identified a new epithelial adhesion molecule, cDCC (chicken homologue to a gene deleted in human colo-rectal carcinoma), which is enriched in the proliferating basal layer and feather collar. We will characterize cDCC in skin development and analyze its function in skin appendage formation with antibodies and retroviral technology. The results will further our understanding in the mechanism of skin appendage formation and help to advance gene therapy which eventually may push the management of damaged skin or skin appendages to a new frontier. These findings will be applied to benefit those who suffer from abnormal skin conditions.

Chuong 9723889 The long term objective of this laboratory is to understand the molecular mechanism of pattern formation in embryonic development. Along these lines, Dr. Chuong has been using feather morphogenesis as a model and studying its molecular mechanism. In this pilot proposal, he will focus on signals involved in periodic patterning of feather primordia. He suggests that initial cell aggregates form randomly and are unstable. Through a reaction diffusion mechanism, only some aggregates survive to become feather primordia. The final number, size, and inter primordia space reaches an equilibrium regulated by positive and negative signals. Preliminary data suggest that FGFs may be candidates of positive signals, while BMPs may be candidates of negative signals. Candidate molecules for these events will be tested using novel feather reconstitution procedures and retroviral infection. The role of cell density on feather bud density, size and interbud spacing will be analyzed. The corollary of the reaction diffusion mechanism is that the primary row and sequential propagation of feather primordia is not essential. This will be tested using DiI labeling of feather primordia followed by dissociation and reconstitution. The work will shed new light on the molecular basis and modeling of periodic pattern formation for feathers and other organs.

The long term objective of the Chuong laboratory is to understand the molecular basis of pattern formation in embryonic development. Along this line, the PI has been using feather morphogenesis as a model and studying its molecular mechanisms. In this proposal, the PI will focus on signals involved in periodic patterning of feather primordia.

The formation of periodic patterns is fundamental in biology. Theoretical models describing these phenomena have been proposed for feather patterning. However, no molecular candidates have been identified. Based on results and preliminary data, the Chuong lab hypothesizes that the mechanism of periodic patterning in feathers has a global and a local component. 1 ) Local activators (candidates: FGFs, SHH) and inhibitors (candidates: BMPs) triggered from initial unstable small cell aggregates act in a reaction-diffusion mechanism to form individual primordia through competition for survival. 2) A global wave of competence (candidates: beta-catenin, signaling molecule receptors) propagating from one end of the primary row, imposing additional activator activities, thus organizing competent cells in the tract field progressively and effectively into a functional feather tract.

The general approach is to map and analyze, with in situ hybridization and immunochemical localization, the expression sequences and modes of candidate molecules in normal and experimental conditions. Signaling molecules coated on beads and candidate genes and their mutated forms carried in retroviral vectors will be used to perturb feather formation.

Induction is the key event in organ formation. Many researchers have focused on the pursuit of the inducer. Previously, the PI showed that SHH is one of the earliest molecules in feather initiation, but what molecules are upstream of SHH? If it is X, what is upstream of X? This type of pursuit will have no ending. Somewhere, there should be a forming process resulted from intrinsic cell properties, rather than from a specific molecular marker. This proposal represents a new approach to answer the fundamental "induction" question. The Chuong lab will test alternative ideas, molecules and to revise the hypotheses depending on the results obtained from the laboratory and the field. The works will contribute to the understanding of the cellular and molecular mechanisms of organogenesis in general.

The long-term objective of this laboratory is to understand the molecular basis of epithelial appendage morphogenesis. Epithelial appendages including hair, nail, feather, teeth, etc. are integral parts of the integument. Diseases and injuries involving these appendages can be detrimental to the survival of the animal. However, research on the development and regeneration of epithelial appendages has lacked. We hope this research will form the basis to promote novel approaches and ideas to manage pathological conditions involving epithelial appendages. We use the chicken feather as an experimental model. In the last funding period, we studied the roles of adhesion molecules and searched for their regulators. We found heterogenous molecular distribution in the epithelial placode develop from an initially homogeneous feather primordia. Molecules like Sonic hedgehog and FGF4 are radically symmetrically distributed, while others become anterior-posterior asymmetrically compartmentalized, with BMP2, 4 and Msx1, 2 in the anterior side, Serrate 1, Wnt7a, BrdU labeling in the posterior side, and Notch 1 forming a mid-stripe. Preliminary perturbation of the molecular balance arrested placode outgrowth in different ways. We hypothesize that the interactions between the anterior and posterior compartments are essential for the generation of a continuous growth zone required to drive the formation of the proximal-distal axis. This growth zone later becomes the feather collar (=hair matrix). Failure to form the growth zone leads to limited growth as seen in scale, but can be rescued to form feather buds by retinoic acid which may induce an ectopic compartment. This process is analogous to the formation of Drosophila appendages from epithelial imaginal discs and the limb bud apical ectodermal ridge, resulting from interactions between dorsal and ventral compartment signals. To test the hypothesis, we will use in situ, BrdU labeling and DiI labeling to establish molecular expression pattern and cell behavior. Differences among scales, feathers and feathery scales will be compared. For molecular candidates, we will focus on Notch and Wnt 7a pathways, and their interactions with other molecules. Retroviral gene transduction together with embryology techniques will be used to generate several experimental conditions including gene over-expression, dominant negative receptors, virus susceptible/resistant chimeric explants, and juxtaposition of anterior/posterior domains. Results and techniques developed here will advance our understanding in epithelial appendage morphogenesis and contribute to the development of gene therapy on hair matrix cells or dissociated keratinocytes for the regeneration of fully functional skin.

DESCRIPTION (provided by applicant): The goal of cancer genomics is to determine the gene expression profile of normal, precancerous, and cancerous cells, with the hope of improving detection, diagnosis, and treatment for patients. The completion of the human genome project has accelerated this progression, and gene microarrays have set the stage for genomic pathology analysis. However, current technology to obtain RNA from specimens is inefficient and requires at least 2 million cells. This is especially problematic with tumors where little starting material is available, or where tumors are heterogeneous and require microdissection or similar methods that drastically limit the amount of starting material. To this end, invention of aRNA (amplified antisense) was a major step ahead but is not ideal for serial amplification or preparation for microarray analysis from a scant amount of sample. Based on the recently developed RNA-PCR Technology, we propose a format that can amplify mRNA from less than 100 cells. We postulate that further development and validation of this technology will lead to a routine procedure to obtain gene expression profiles (GEPs) from just a few or even single cells. In the R21 phase, we will use an NCI RNA standard and cultured sarcoma cell lines to compare amplified and non-amplified RNA with rigorous statistical analyses using Genetrix, a software tool developed here to analyze microarray data. Conditions will be optimized and worked out for pathological specimens. When the fidelity and efficacy of RNA-PCR amplification are validated, we will progress to the R33 phase to field test in cancer using childhood sarcoma as a model. We hope to achieve high resolution GEP using Laser Capture Micro-dissection microscopy to dissect histopathology sections. We will verify whether GEPs from a few cells are as predictive of biologic behavior as conventional GEPs, using the same clinical material used in Triche?s Director?s Challenge grant. We will also apply microarray analysis to skinny needle biopsies and cytological preparations, which were previously not possible. Bioinformatic analysis with Genetrix, which allows correlation of expression on a gene-by-gene basis with patients? clinical data (e.g., age, sex, pathological diagnosis, therapeutic protocol, etc.), may identify new markers of high predictive value that are obscured due to tissue heterogeneity, or unavailable due to limited amount of materials (e.g., those in tissue banks). This protocol is simple, fast, and reliable and uses minimal samples widely obtainable. The method can be applied to all cancers.

DESCRIPTION (provided by the applicant): The regulation of organ size is a fundamental mechanism in development. The skin appendage is the best model to study size regulation because of the large number of appendages and the extensive size variation found within one individual organism. Many clinical conditions such as alopecia, hirsutism and even cancer are basically problems of size. The chicken feather is an excellent model because of the large spectrum of graded feather sizes on one individual and the possible variations in special breeds. We hypothesize that the size of feathers is determined through multiple stages of epithelial-mesenchymal interactions, from initiation to growth to resting phases, each modulated by combinatorial positive and negative molecular regulators. We will do molecular mapping of different phases of feather follicles with in situ hybridization and immuno-staining. Preliminary data showed the presence of members of major signaling pathways (e.g. SHH, BMP, Wnt) in different regions of the follicle. We also have identified a novel feather follicle bulge zone that shows long term BrdU label retention that may contain feather stem cells. To search for cellular determinants of feather size, we will engineer chimeric feather follicles made with components from different-sized follicles. To identify molecular determinants, we have developed a feather plucking/regeneration model and can introduce genes to the regenerating feathers using gene therapy technology. Candidate molecules will be chosen by comparative molecular mapping of different-sized follicles, and library subtraction. Preliminary data suggest that the SHH pathway is one of the positive regulators and the BMP pathway is one of the negative regulators for feather size. Alterations of cell proliferation, differentiation (judged by keratin markers) and other signaling molecules will be analyzed to identify crosstalk among molecular pathways. This work will lead to a new understanding of the molecular basis of the size regulation of skin appendages.

DESCRIPTION (provided by applicant): Our long term objective is to study how ectodermal organ size is determined. In the last funding period, we studied the regulation of organ size and shape in feather, hair, beak, tooth, etc. We developed the topobiological concept of localized growth zone (LoGZ), suggesting the number, size, position and activity of clustered transient amplifying (TA) cells can determine an organ's size and shape. In this renewal, we ask what unique characteristics in hairs and feathers enable them to increase in size and regenerate continuously throughout adult life. We hypothesize it is made possible by forming a sustaining growth unit, composed of epidermal stem / TA / differentiated cells and a dermal signaling center, topologically arranged in a follicle design that allows the continuous flow of growth and regeneration without structural constraints. Using the feather model, we postulate that FGF / MARK is the major driving force of cell proliferation for size increases throughout bud and follicle stages. Organized growth is essential in organ building and tissue engineering. In the upward feather bud outgrowth, the Notch pathway may specify a growth - maturation gradient to the feather filament growth zone, conferring orientation to the elongating feather buds (Aim 1). In the downward follicle wall invagination, the dermal papilla may work as a signaling center to direct the epithelial tongue extension via FGF 10 (Aim 2). In the growing follicle, FGF activity is modulated by sprouty and antagonized by BMP to generate different structures along the feather axis (Aim 3). Preliminary microarray data are consistent with these candidate pathways. Efforts are made to alter dermal papilla properties so we can convert small feathers into large ones via modulation of the stem cell microenvironment, rather than stem cells themselves (Aim 4). RCAS retroviral vectors, siRNAs, electroporation, protein coated beads, chicken/quail chimeric explant, follicle dermal papilla transplantations, Oil tracing, time lapse movies, microarrays, bioinformatics analyses, etc. are the techniques we will apply to these studies. Using this excellent animal model, we will identify gene pathways critical for follicle morphogenesis and essential topobiological principles for this process. We should learn the developmental origin of appendage stem cells and dermal papilla. The knowledge acquired can be directed to make short hairs grow longer, and to engineer ectodermal stem cells into follicles with sustaining growth ability.

[unreadable] DESCRIPTION (provided by applicant): Regenerative medicine and tissue engineering have emerged as some of the most fascinating fields with potentially high clinical applications. If an adult human unfortunately loses a digit or limb, the wounds heal without signs of regeneration. Among amniotes, some lizards show a remarkable ability to regenerate tails following amputation. Here we propose to develop this unique in vivo model of regeneration to understand the unique molecular/ cellular mechanisms underlying repair and regeneration. The long term goal is to learn how animals do this process in Nature and to be able to apply these principles to benefit regeneration and bio-engineering following human amputation (e.g., limb, digit). Examples of ideal animal models that helped to improve our understanding of physiological and pathological phenomena include the use of Drosophila, C. elegans, etc. A unique phenomenon in reptiles is "autotomy" in which the stressed lizards automatically break their tails at specific fracture planes. Regeneration then initiates from these planes. Since this becomes part of the "physiological" process, we hypothesize that stem cells may reside in niches located near the facture plane, similar to bulge stem cells in hair molting cycles. Alternatively, cells in wounds may undergo de- differentiation to generate pluri-potential blastema. In either case, the regeneration is initiated and tissue morphogenesis progresses beyond simple repair. To test for these possibilities, we will pursue the following aims. 1) Characterize the regenerative events following wounding of reptile tails, including cell proliferation, apoptosis and migration. 2) Locate the origin of precursor cells in regenerating reptile tails, including the use of long-term label retention and chromatin status markers. 3) What molecular events are involved during reptile tail regeneration? Development related signaling molecule pathways (e.g., Msx, beta catenin, Shh, BMP, FGF, etc.) will be prioritized for mapping studies. Likely candidates will be perturbed using electroporation of siRNA. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The objective of this proposal is to develop reconstituted skin that can form new hairs from dissociated stem cells. Progress has been made in producing hair follicles using dissociated multi-potential dermal and epidermal stem cells. However, couple gaps need to be filled before significant progress in clinical applications can be made. Recently, we have improved significantly the previous assays by Lichti and Stenn into a simpler hair forming assay with clinically proper appearance and potential for a high throughput assay. Our general hypothesis is that multi-potent skin stem cells can be generated by molecular reprogramming, and that arrangement and patterning of hair primordia can be regulated by environmental conditions. With the newly developed planar hair forming procedure, we are able to apply our expertise in feather pattern formation to regulate the number and size of hair primordia. We will also apply our recent experience on macro-environment regulation of hair stem cells to improve the efficiency of hair formation. Recent success of molecular re-programmig in other organs has inspired excitement. This simple assay will allow us to do high throughput analyses to identify molecules or combinations of molecules important to reprogram adult skin cells into hair forming dermal and epidermal cells. The development of this technology will bring new hope to those who suffer from severe wounds and alopecia. PUBLIC HEALTH RELEVANCE: Currently, over 100,000 patients are treated for burn injury in the United States per year, and burns can cause lasting cosmetic and functional defects to visible areas on the skin. Here we propose to develop a novel planar hair forming protocol which integrates progress from our parent grant and others hair research and may bring new hope to those who suffer from severe wounds, and alopecia.

DESCRIPTION (Provided by Applicant): The University of Southern California (USC) Training Program in Developmental Biology, Stem Cells, and Regeneration (DSCR) provides graduate students with unified training in mechanistic studies of fundamental developmental processes combined with training in the biology and application of stem cell technology. A distinguishing theme of this program is the incorporation of clinical and translational science into the curriculum of students in the DSCR track, based on the premise that strong basic science graduate training can and should be coupled with an appropriately structured exposure to clinical and translational science in a way that will better train the next generation of scientists to be able to realize the clinical and therapeutic potential of developmental biology discoveries. The faculty of this program includes a balanced mix of full, associate, and assistant professors from several USC schools and campuses. These faculty members have proven records of graduate student training, a long history of collaborative research, and approximately half hold clinical degrees and are active clinical translational scientists. The DSCR Program draws students primarily from a highly successful USC interdepartmental graduate student recruitment and first year of studies program (PIBBS; Programs in Biological and Biomedical Sciences); this outstanding cohort of students comes from diverse backgrounds, and includes a prominent representation of training-grant-eligible underrepresented minority students. Predoctoral students are supported by the PIBBS program in their first year of graduate studies, during which they take core classes and do research laboratory rotations. Three new students per year enter the training program at the beginning of their second year, and are supported for two years. Specialized courses and additional program functions provide a rigorous and diverse training experience for students to realize the full potential of the convergent fields of developmental biology, stem cell biology, and regenerative medicine.

DESCRIPTION (provided by applicant): The multi-faceted functions of skin are conferred by its unique three dimensional architecture made up of multiple modules interwoven into an integral organ. To function normally, the size, number, ratio and relative positions of each component have to be precisely regulated. Few studies have focused on how the complex pattern of the skin is built. Current management of severe skin injury has achieved the goal of saving patients' lives by growing a flat layer of epidermis and dermis over the wound. Life-saving as it may be, the replaced skin is composed of relatively simple flat epidermis and dermis and does not function in full due to the lack of skin appendages and key functional modules. Toward the long term objectives of regenerative medicine, we aspire to learn fundamental principles of skin organogenesis that we can apply to better wound healing/regeneration and tissue engineering. In the previous funding period, we focused on the morphogenesis of a single appendage. We now want to study the multi-faceted skin as a whole and to reveal the unifying framework of skin morphogenesis at multiple spatio-temporal scales. We propose to study how the key functional modules of skin are built and patterned. Based on our preliminary data, we postulate that the construction of skin structures occurs through a series of tissue interactions, each with distinct patterning behaviors, built layer by layer, module by module, leading to the integration of the skin as a whole. We choose to focus on three components critical to avian skin function: the feathers, muscles that connect feathers for functionality, and pigment that decorates feathers for communication. Each component represents a different category of patterning behavior in a hierarchical framework. We will study how the boundary between appendage primordia and surrounding dermis is consolidated and hypothesize that periodic patterning, the most fundamental process of skin organogenesis, is established via competition and stabilization of cell adhesion / motility. We will study how the dermal muscle network is established and hypothesize that this adaptive patterning is achieved using appendage primordia as anchor points. We will analyze how the final patterns are pleomorphic and how the process is modulated by environment factors. We will study how skin pigment patterns are painted by the regulatory patterning process. We hypothesize that it is achieved through combinatorial regulation of the migration, proliferation, survival, and / or differentiation of melanocyte progenitors. Similar principles may be used in the patterning of other tissue components. Understanding these patterning behaviors will allow us to apply the principles and initiate self-organizing regenerative processes in various skin disease conditions. PUBLIC HEALTH RELEVANCE: Here we study the patterning mechanisms involved in the development of the skin organ. They include periodic patterning to set up periodically arranged appendage primordia, adaptive patterning to add more components onto extant architecture and regulatory patterning to modulate cell migration or differentiation under physiological or pathological conditions. The study should provide new insights into how the different components are integrated to build a complex organ, and will be useful toward the tissue engineering of a fully functional skin.

? DESCRIPTION (provided by applicant): Our long term objectives are to study the development and regeneration of skin ectodermal organs and to apply the principles toward regenerative medicine. Over the last two decades, my laboratory has made a long term commitment to this goal; we have produced several basic scientific discoveries and made conceptual progress toward the advancement of skin regenerative biology. In the next phase, we will consolidate new findings from two recently completed RO1 grants to focus on deciphering the mechanism of phenotypic specification in skin organogenesis, a major challenge in the next phase of cutaneous regenerative medicine. Through progress in stem cell biology and reprogramming technology, scientists are now able to generate epidermal progenitors, in the form of dissociated cells or a keratinocyte monolayer. How to guide these progenitors to form skin with different architecture remains unknown. Understanding these processes is crucial to ensure proper regeneration of a wound graft. A fundamental feature of the skin is the striking regional variation, where distinct skin types (e.g., facial skin and scalp glabrous skin, etc) develop at different body regions to serve different purposes.2,3 We postulate that competent, dissociated, epidermal progenitors interact with dermal cells to form reconstituted skin, a process which is regulated by environmental signals which change over developmental time to generate specific skin appendage phenotypes. We will focus on three fundamental aspects, using the most appropriate animal models for each process. Mouse and human cells are used in Aim 1 whereas chicken skin is used for Aims 2 and 3 because it features remarkable regional differences ideal for experimental analyses. In Aim 1, we will take a multi-disciplinary approach to study how the reconstituted skin layer is generated from dissociated cells. Time-lapse imaging, transcriptome analyses and molecular perturbation will be used to study how the dissociated cells can self-assemble into a layered configuration via a series of multicellular morphological transitions. We will focus on the roles of MMPs in this morphogenetic transition and will attempt to reactivate the morphogenetic ability in adult mouse and human cells by modifying the Wnt-MMP-ECM module. In Aim 2, we will study how skin progenitors are specified during regionalization. Our preliminary data suggests that the conversion of scales to feathers proceeds in a stepwise manner via a hierarchy of signaling molecules. Based on preliminary studies, we will focus on the role of Sox genes in establishing this hierarchy. In Aim 3, we will study the morphogenetic principles of organ shaping. We will focus on the regulation of appendage length using feathers as a model. We will evaluate the hypothesis that complex organ shapes are specified by a core circuit that defines a prototypic phenotype qualitatively and modulator circuits that modify specific dimensional parameters quantitatively in a temporal-spatial manner. We hypothesize that FGF and Zic signaling, respectively, may be examples of such molecular circuits.

Summary The objective is to understand the organizing principles of the skin for future use in regenerative medicine. Progress has been made toward generating skin progenitor cells. However, before significant progress in clinical applications can be made, we must know how the skin exhibits region-specific characteristics with unique functions and architectures. While human skins show regional specificity (scalp, face, palm, etc), mice do not. Chicken skin does show dramatic regional specificity and is accessible to experimentation, making it an ideal model for this type of study. Tissue recombination studies showed dermis controls the phenotypes, and epidermal progenitors respond by making appropriate appendage phenotypes. In this proposal, we will focus on how regional specific keratinocyte differentiation is controlled by epigenetic processes. Our recent work showed that chicken ?-keratin clusters on Chr25 are organized in five sub-clusters, each enriched in a globally different skin region (feather, scale, claw, ?macro-regional specificity?); whereas the ?-keratin clusters on Chr27 are differentially expressed in different within-feather regions (rachis vs barb) or feather generations (downy vs adult contour feathers) made from stem cells in the same follicle (?micro- regional specificity?). Thus, we hypothesize there is a hierarchical correspondence between the skin regional topographic map and genomic organization of b-keratin clusters. Preliminary data of Chr25 keratins show typical enhancers in front of keratin sub-clusters, while Chr27 keratins surprisingly show 38 peaks of CTCF/KLF4 binding motifs, strongly suggesting intra-cluster chromatin looping and complex combination potential. In Aim 1, we will use histone ChIP-Seq to identify how typical enhancer(s) differentially regulate the b-keratin cluster within the EDC in different skin regions. We will also analyze how intra-cluster higher-order chromatin looping in the Chr27 keratin cluster may lead to differential keratin expression in within-feather differences. Feather and scales at different competent stages, tissue recombinant explants with reprogrammed epithelial fate, and experimental conditions in which scales are converted toward feathers, and adult contour feathers converted toward downy feathers will be used to monitor the epigenetic changes. In Aim 2, we will analyze how the genome organizers CTCF, KLF4, and SATB1/2 bind DNA and configure chromatin conformation at the keratin loci in the above conditions. Appendage phenotypes caused by suppression of these genome organizers will be analyzed.

Summary Our long term objective is to heal skin wounds with full functional restoration (regenerative wound healing) instead of scarring (reparative wound healing). We aspire to learn what factors control regeneration as supposed to repair during wound healing under the newly established paradigm of ?Wound-Induced Hair Neogenesis? (WIHN). In this model, new hair follicles emerge from the wound center when a large (>1cm) skin wound is made on the mice (e.g., C57BL/6). Our new finding in Spiny mice (Acomys) shows, however, that WIHN starts to form from the periphery of wounds toward the center. Our preliminary data further shows a distinct spatial distribution of mechanical stiffness across the wound field, and that perturbation of mechanotransduction in the wound bed alters the outcome of WIHN. These new findings prompted us to hypothesize that tissue mechanics modulate tissue regeneration and WIHN. WIHN is easily accessible and is a good model to evaluate this hypothesis. While epithelial placode formation can be initiated by different chemical morphogens present during embryonic development (which converge to induce beta-catenin signaling), in WIHN of both C57BL/6 and spiny mouse, the mechanical environment of the wound can modulate, in parallel or independently, the threshold of successful placode formation. This acts to alter the status of epithelia activation, basement membrane remodeling, and dermal condensation. In Aim 1A, we will compare the different cellular and molecular events leading to WIHN, contrasting spiny and C57BL/6 mice. Supported by the bioinformatic analyses, Twist1 is proposed as a master regulator for placode formation in the spiny mouse. Therefore, the role of Twist1 and its downstream Msx2 in WIHN will be examined in Aim1B. In Aim 2A, we will map the stiffness in different parts of the wound field employing atomic force microscopy accompanied by cell shape analyses and FRET-biosensors to indirectly ?visualize? the consequence of cell forces within the wound site. The role of tissue mechanics in WIHN will be further tested by perturbation studies. Aim 2B will investigate and define the molecular circuits which are functioning in the conductive mechanical environment for placode regeneration. Overall, the proposal aims to explore novel epidermal-dermal networks during regenerative wound healing from the perspective of epigenetic, molecular, and mechanical inputs that construct the grand theme of elements necessary for future progression of regenerative medicine. !

Our long-term objective is to study the organizing principles that establish complex skin architectures required for optimal skin functions. We have been using developing skin as experimental models because of their distinct patterns which we use as a readout and their accessibility to experimentation. In the past, we have taken an analytical approach to study the disruption of tissue patterns by altering expression levels of different diffusible morphogens. Recent work from us and others have enlightened us that biophysical processes also play integral roles in tissue patterning, and these roles have been under appreciated. We now explore the ramifications of this novel understanding. Here we formulate a general hypothesis that tissue patterning occurs by integrating molecular signaling and biophysical events. Newly expressed molecules on cells lead to changes in the physical properties of cells and their surrounding matrix, causing disequilibrium that drives biophysical processes to the next stage. Mechano-chemical coupling leads to new molecular expression and so on, leading to the building of complex architectures. We will evaluate three key morphogenetic events. First, the periodic patterning process that converts the skin from one morphogenetic field to multiple skin appendage units. We suggest there are multi-layered controls. Tissue mechanics may facilitate Turing patterning and help the propagation of the bud forming wave, while gap junctions may alter the diffusion of intra-cellular second messengers to modulate the results of Turing patterns. Second, the formation of stem cell-based skin appendage follicles in each unit. The margin of the feather bud shows planar cell polarity, Tenascin C, and MMP14-FRET activity that drives bud margin epithelium to invaginate into the dermis. Fate maps of stem cells and dermal papillae will be generated using singe cell RNA sequencing and lineage tracing, and the results will be compared with that of hair follicles for key similarities. Third, adaptive patterning in which adnexal structures such as blood vessels, nerves, dermal muscles, etc. are integrated back to the skin to form one functional unit. We will use the assembly of the dermal muscle network as a paradigm to explore these principles. The competence, specification and plasticity of dermal cell fates during this process will be analyzed epigenetically using a SMA mechanosensor in living explants. By exploring the roles of tissue mechanics in during tissue patterning of the skin, we can obtain a more holistic understanding of the self- organizing processes in the development and regeneration of the skin. This knowledge will have significant applications toward regenerative medicine in the future.

Our long-term objective is to understand the principles that orchestrate skin morphogenesis in development and wound regeneration. The understanding of biochemical signaling is well advanced. Yet, research into the roles of non-neural bioelectricity lags behind, although evidence for a role of bioelectricity in development, regeneration (McLaughlin and Levin 2018 16; Li et al., 2020 5) and wound healing (Zhao et al. 2012 32) is growing. Our research objective is to study the mechanisms underlying the development and regeneration of skin appendages. In two of our recent research papers, we were inspired to see bioelectricity in action in two tissue patterning processes. First, the orientation of elongating feather buds is regulated by synchronization of oscillating calcium channel activities in bud dermal cells, which is controlled by epidermal Shh signaling (Li et al., 2018 11). Second, the skin frequently shows pigment stripes along the body. The size and spacing of longitudinal pigmentation stripes in Japanese quail was recently shown to be controlled autonomously within melanocyte progenitor populations in a gap junction-dependent manner (Inaba et al., 2019 12). At the time these periodic black/yellow stripes form in embryos, the spacing is in millimeters, a large-scale patterning process that cannot be explained by the classical Turing reaction-diffusion mechanism (patterning in micrometer range). The results led us to think hard about how large-scale tissue architecture is built. While localized signaling centers involving morphogens (e.g., WNT, BMP, FGF) were shown to initiate periodic patterning of feather/hair buds, some unidentified mechanism capable of spanning large distances dynamically must work together to transduce the information over the long-distance scale (Inaba and Chuong, 2019 15). Bioelectricity work here provides a clue. Thus, we organized a multi-disciplinary team to analyze the mechanisms on how biochemical and bioelectric signals integrate to achieve the large-scale tissue patterning. We hypothesize, among other possibilities, transient bioelectrical signaling across gap-junction-coupled cell collectives may allow rapid, long-distance signaling with minimal decrement. Electropotential gradients are harnessed to propagate signals rapidly over the long distance (millimeters in milliseconds) to regulate intracellular messengers and pattern the much larger morphogenetic field. The developing avian skin explants provide an excellent model because of the quantifiable distinct patterns, planar topology for easier channel activity visualization, electric current perturbation and optogenetic gene activation ? not easy in the mouse model. Experimentally, we will first gauge the endogenous bioelectric landscape and evaluate the importance of bioelectricity in these two tissue patterning processes (Aim 1A, 2A). Then we will study how ion channels / gap junctions cross-talk with biochemical signals to achieve tissue patterns (Aim 1B, 2B). The work is likely to produce new findings and insights for future applications to use bioelectricity to benefit wound regeneration.