The global objective of this research is to elucidate the molecular mechanisms underlying the balance between proliferation and differentiation in the developing mammalian epidermis and its appendages, and to understand how this process goes awry in human skin disorders, including cancers. Central to achieving this goal is to understand how embryonic cells choose between an epidermal vs. hair cell fate, and how adult skin drives new growth during the hair cycle and repairs the epidermis on injury. Two key signaling pathways, Wnt and Bmp, are involved in transcriptionally orchestrating this process, but the underlying mechanisms and critical genes they govern remain unknown. The parent R01 of this revision application uses gene targeting, microarray and chromatin IP analyses to identify the molecular changes that are directly downstream from Wnt and BMP transcriptional complexes in regulating embryonic and postnatal epidermis. While we have made substantial progress in discerning the functional significance of downstream transcriptional target genes, progress has been impeded by cumbersome and time-consuming approaches of mouse genetics. In this revision application, we propose to use analyze our array data develop a systems biology approach in vitro and in vivo which will greatly enhance the efficiency at which the functional significance of changes in these gene expression patterns can be revealed. Specifically, we propose to exploit our embryonic microarray data in which we transcriptionally profile the genes upregulated in the Wnt activated hair placode cells vs Wnt restricted epidermal cells and 1) establish a sufficiently faithful hair placode cell culture system that will enable us to test the efficiency of action of ShRNA lentiviral vectors in vitro;2) subject cultured hair follicle stem cells from our Wnt reporter mice to lentiviral ShRNA knockdown for these array signature genes; and evaluate the degree to which the ShRNA affects the ability of follicle stem cells to respond to exogenous Wnt signaling and to proliferate and differentiate; and 3) subject the most promising candidates to functional ShRNA knockdown in vivo and evaluate the ability of these Wnt signature genes to affect hair follicle specification and down growth. This new methodology stands the potential to greatly enhance the rate at which we can dissect how Wnt and BMP signaling pathways operate in transcriptionally balancing stem cell activation, proliferation and differentiation in the embryonic and adult hair follicle. This interdisciplinary strategy also holds promise for progressing these applications to genetic skin disorders and medicine.
Our skin epithelium provides an essential barrier to keep harmful microbes out and body fluids in. Skin begins as a single layered epithelium. In response to external cues involving Wnt signaling, the skin responds by changing its program of gene expression to generate hair follicles and an interfollicular stratified epidermis. This research develops new technologies aimed at elucidating the functional importance of these changes. This study is a fundamental prerequisite to deciphering the complex transcriptional mechanisms that balance growth, differentiation and development and to elucidating how aberrations in these basic pathways go awry in skin cancers.
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