De novo hair follicle formation in embryonic skin and new hair growth in adult skin are initiated when specialized mesenchymal dermal papilla (DP) cells send cues to multipotent epithelial stem cells. Subsequently, DP cells are enveloped by epithelial stem cell progeny and other cell types to form a niche orchestrating hair growth. Understanding the general biological principles that govern the mesenchymal–epithelial interactions within the DP niche, however, has been hampered so far by the lack of systematic approaches to dissect the complete molecular make-up of this complex tissue. Here, we take a novel multicolor labeling approach, using cell type–specific transgenic expression of red and green fluorescent proteins in combination with immunolabeling of specific antigens, to isolate pure populations of DP and four of its surrounding cell types: dermal fibroblasts, melanocytes, and two different populations of epithelial progenitors (matrix and outer root sheath cells). By defining their transcriptional profiles, we develop molecular signatures characteristic for the DP and its niche. Validating the functional importance of these signatures is a group of genes linked to hair disorders that have been largely unexplored. Additionally, the DP signature reveals novel signaling and transcription regulators that distinguish them from other cell types. The mesenchymal–epithelial signatures include key factors previously implicated in ectodermal-neural fate determination, as well as a myriad of regulators of bone morphogenetic protein signaling. These findings establish a foundation for future functional analyses of the roles of these genes in hair development. Overall, our strategy illustrates how knowledge of the genes uniquely expressed by each cell type residing in a complex niche can reveal important new insights into the biology of the tissue and its associated disease states.

Wnt signaling has been implicated in stem cell (SC) biology, but little is known about how stabilized ␤-catenin functions within native SC niches. We address this by defining the impact of ␤-catenin stabilization on maintenance, proliferation, and lineage commitment of multipotent follicle SCs when in their native niche and in culture. We employ gain of function mutations and inducible loss of function mutations to demonstrate that ␤-catenin stabilization is essential for promoting the transition between SC quiescence and conversion to proliferating transit amplifying (TA) progeny. We transcriptionally profile purified SCs isolated directly from wild-type and elevated ␤-catenin follicles in both resting and activated states to uncover the discrete set of genes whose expression in native SCs is dependent upon ␤-catenin stabilization. Finally, we address the underlying mechanism and show that in the SC niche, Wnt signaling and ␤-catenin stabilization transiently activate Lef1/Tcf complexes and promote their binding to target genes that promote TA cell conversion and proliferation to form the activated cells of the newly developing hair follicle. We also show that these changes precede subsequent Wnt signals that impact on the TA progeny to specify the differentiation lineages of the follicle.

No abstract

TBX5 is a member of the T-box gene family and encodes a transcription factor that regulates the expression of other gene(s) in the developing heart and limbs. Mutations of TBX5 cause Holt-Oram syndrome (HOS), an autosomal dominant condition characterized by congenital heart defects and limb anomalies. How TBX5 gene expression is regulated is still largely unknown. In order to identify transcription factors regulating TBX5 expression, we examined the 5'-flanking region of the human TBX5 gene. We determined that up to 300 bp of the 5'-flanking region of the TBX5 gene was necessary for promoter activity in mouse cardiomyocyte ECL2 cells. One GC box, three potential T-box-like binding elements (TBE-A, -B, and -C), and one NKX2.5 binding site were identified. Site-directed mutagenesis of the potential binding sites revealed that the GC box, TBE-B, TBE-C, and NKX2.5 are functionally positive for the expression of TBX5. DNA footprint analysis showed that these binding regions are resistant to DNaseI digestion. Electrophoretic mobility shift assays (EMSAs) further demonstrated the protein-DNA interactions at the GC box and the potential TBE-B, TBE-C, and NKX2.5 sites in a sequence-specific manner. The ability of TBX5 to regulate its own promoter was demonstrated by the ability of ectopically expressed human TBX5 to increase reporter expression. We conclude that the GC box, T-box-like binding elements, and NKX2.5 binding site play important roles in the regulation of TBX5 expression, and that TBX5 is likely to be autoregulated as part of the mechanism of its transcription.