Peggy S. Myung, Ph.D. - US grants

DESCRIPTION (provided by applicant): Tissue homeostasis and regeneration are mediated by the coordinated growth of multiple cell types to generate tissue that sustains integrity and function of the organism. This ability to durably regenerate tissue relies on adult stem cells that reside in a specialized environment called the niche, which influences their self- renewal, growth and differentiation. Failure to maintain or mobilize stem cells results in tissue loss and dysfunction, while uncontrolled activation of these cells can fuel disorganized growth and cancer. Elucidating how key molecular signals govern stem cell behavior holds tremendous implications for designing targeted therapies to treat human diseases. A major challenge to examining how mammalian stem cells are regulated is the inability of conventional static analysis to follow the fate and behavior of cell populations in vivo over time. This proposal aims to address a major gap in our knowledge of how signals can engage a population of undifferentiated cells to yield robust and organized growth. In particular, is still unclear (1) wht cellular behaviors such as cell divisions and movement are regulated by key morphogenetic signals during regeneration, (2) how these signals are disseminated throughout a field of cells in to ensure robust but compartmentalized growth, and (3) how these mechanisms can contribute to disorganized and uncontrolled growth during tumorigenesis. The hair follicle is the ideal model to address these questions as it is exceptionally accessible and undergoes well-characterized cyclical regeneration in a manner dependent on resident stem cells. By coupling this model to a novel live imaging technique we are now uniquely poised to address these outstanding questions. In this project, we examine how Wnt/¿-catenin signaling, a key molecular pathway required for hair follicle regeneration, is propagated throughout a population of undifferentiated cells to promote synchronous and coordinated growth. By live imaging, we have found that only a subset of cells is required to fuel the non-cell autonomous activation of this signal and growth behaviors throughout surrounding epithelial cells and is associated with upregulation of diffusible Wnt ligands. Our first aim uses both in vitro cell culture and genetic mouse models that activate ¿-catenin in the presence or absence of Wnt secretion to determine the requirement for Wnt ligand secretion in promoting hair follicle regeneration. The second examines if Wnt secretion is required for epithelial Wnt activation in basal cell carcinomas (BCCs), the most common human skin cancer, which is dependent upon Wnt signaling for growth. We will also determine how sustained epithelial Wnt signaling can influence the mesenchyme to regulate BCC growth, using tissue grafting and genetic approaches to modulate epithelial and dermal Wnt activation in a BCC mouse model. Accomplishing these aims will provide novel insight into the principle mechanisms that ensure proper tissue regeneration and how they can also be exploited deleteriously to promote collective growth in cancer.

PROJECT SUMMARY Loss of tissue integrity and function is a unifying basis of disease, and the ability to regenerate lost or damaged tissue to its original form holds broad potential for human health. However, while some vertebrates such as newts can replace lost tissue perfectly, mammals repair injured tissue with scar, resulting in altered form and function. Why mammals have limited regenerative capacity is not known. A major barrier to addressing this limitation is our incomplete understanding of 1) how tissue is originally formed during development and 2) if and how these developmental programs can be resurrected in adults to drive regeneration. Currently, we lack mammalian models to explore these questions in parallel, and we lack a molecular handle to open up groundwork for systematic studies. Moreover, tools to dynamically trace how signals control specific cell behaviors that govern tissue formation are needed. In this grant, we address these challenges by examining mammalian skin, a model that uniquely exhibits regenerative wound healing. Here, full-thickness skin wounds on adult mice regenerate lost hair follicles through epithelial-mesenchymal interactions that chronicle embryonic skin development. This remarkable process is one of the few promising indications that mammals can achieve regeneration. Yet, how embryonic programs are reclaimed during wound healing is still unclear due to our fragmented understanding of how hair follicle formation is originally initiated during development and the lack of tools to trace molecular signals to specific cell behaviors that orchestrate hair follicle formation. In this grant, we examine how temporal activation of dermal Wnt/b-catenin prior to hair follicle initiation, coordinately regulates epithelial and dermal cell behaviors that mediate hair formation. We found that genetic modulation of dermal b-catenin at different time points prior to hair follicle initiation regulates hair follicle size. Based on this novel finding, we hypothesize that the timing of dermal Wnt/b-catenin signaling regulates hair follicle epithelial and dermal cell number in a tunable fashion during development and adult wound-induced hair follicle neogenesis. In Aim 1, we will use two-photon live imaging of mouse embryonic skin explants to coordinately delineate epithelial and dermal cell behaviors during hair follicle initiation and to dynamically track how temporal genetic loss or gain of dermal Wnt activity prior to hair follicle induction influences these cell behaviors. To identify molecular targets of dermal Wnt signaling, we will analyze transcriptomes of epithelial and dermal cell populations following ablation of dermal b-catenin at different time points. In Aim 2, we will begin to determine if dermal Wnt activation similarly regulates adult wound-induced hair follicle neogenesis. Specifically, we will define the spatiotemporal activity of dermal Wnt following wounding and analyze the size of neogenic hair follicles following genetic ablation of dermal Wnt at different times during wound healing. Success of this project will reveal mechanisms that drive hair follicle initiation and will set the stage for future studies that dynamically track the molecular and cellular mechanisms governing adult tissue regeneration.

SUMMARY The hair follicle (HF) is composed of an epithelial and dermal population and is a classic model to study the epithelial-mesenchymal interactions governing appendage development. During HF development, the first morphologically distinct dermal population is the dermal condensate (DC), a dense cluster of specialized cells that matures into the dermal papilla (DP). As the DP holds the revered capacity to induce new HF growth, large efforts have been made to program undifferentiated fibroblasts into differentiated DC/DP cells but met with limited efficacy. The principal challenge has been the inability to assess molecular differences between cells before they are morphologically apparent. As such, we lack a molecular ?roadmap? of the transition states that direct lineage commitment and morphogenesis that could guide faithful methods to recapitulate these events in vitro. To meet this challenge, we recently used an unbiased diffusion map technique to systemize single-cell RNA sequencing (scRNA-seq) data from mouse embryonic skin. Using this technique, we identified a molecular DC differentiation trajectory, an inferred pathway of transcriptional states through which DC cells pass, before and during HF morphogenesis. Guided by this map, we showed that dermal Wnt/?-catenin signaling is required to progress to an intermediate phase of DC cell differentiation and that DC cells are immediate quiescent progeny of a molecularly distinct (Dkk1+), highly proliferative population. Currently, the critical transition steps that Dkk1+ cells pass through and the signals that regulate them remain unknown. Combining innovative computational methods and mouse models, our preliminary data reveal that Dkk1+ progenitors utilize two molecular pathways to generate DC cells that distinguishes DC initiation from DC expansion processes prior to morphogenesis. We hypothesize that DC formation is a dynamic process wherein DC initiation and DC expansion utilize distinct molecular pathways to generate DC cells and that signals that regulate the transition from proliferation to quiescence are essential for DC differentiation by Dkk1+ progenitors. In this grant, we use an integrative approach to build a temporospatial map of DC transition states that govern DC formation. In Aim 1, we will couple transcriptional kinetic scRNA-seq data (RNA velocity) with in vivo lineage tracing to define transition steps that lead to DC initiation and DC expansion, coupled with live imaging and quantitative FISH to spatially locate critical transition steps. Using this same approach, we will define how local epithelial signals regulate key transition states within distinct DC paths. In Aim 2, we will examine the role of local proliferation in DC formation and signals (e.g. YAP/TAZ) that regulate the transition between proliferation and quiescence using genetic mouse models. This complementary approach will overcome major challenges in dissecting the early events that lead to DC cell fate. The overall goal of this project is to build a high-resolution roadmap that delineates how DCs form to accelerate regenerative efforts, while also providing an experimental paradigm to study the formation of other cutaneous appendages.