In a common theme of organogenesis, certain cells within a multipotent epithelial sheet exchange signals with their neighbors and develop into a bud structure. Using hair bud morphogenesis as a paradigm, we employed mutant mouse models and cultured keratinocytes to dissect the contributions of multiple extracellular cues in orchestrating adhesion dynamics and proliferation to shape the cluster of cells involved. We found that transforming growth factor β2 signaling is necessary to transiently induce the transcription factor Snail and activate the Ras-mitogen-activated protein kinase (MAPK) pathway in the bud. In the epidermis, Snail misexpression leads to hyperproliferation and a reduction in intercellular adhesion. When E-cadherin is transcriptionally down-regulated, associated adhesion proteins with dual functions in signaling are released from cell-cell contacts, a process which we demonstrate leads to Ras-MAPK activation. These studies provide insights into how multipotent cells within a sheet are stimulated to undergo transcriptional changes that result in proliferation, junctional remodeling, and bud formation. This novel signaling pathway further weaves together the web of different morphogens and downstream transcriptional events that guide hair bud formation within the developing skin.

Skin-derived precursors (SKPs) are multipotent dermal stem cells that reside within a hair follicle niche and that share properties with embryonic neural crest precursors. Here, we have asked whether SKPs and their endogenous dermal precursors originate from the neural crest or whether, like the dermis itself, they originate from multiple developmental origins. To do this, we used two different mouse Cre lines that allow us to perform lineage tracing: Wnt1-cre, which targets cells deriving from the neural crest, and Myf5-cre, which targets cells of a somite origin. By crossing these Cre lines to reporter mice, we show that the endogenous follicle-associated dermal precursors in the face derive from the neural crest, and those in the dorsal trunk derive from the somites, as do the SKPs they generate. Despite these different developmental origins, SKPs from these two locations are functionally similar, even with regard to their ability to differentiate into Schwann cells, a cell type only thought to be generated from the neural crest. Analysis of global gene expression using microarrays confirmed that facial and dorsal SKPs exhibit a very high degree of similarity, and that they are also very similar to SKPs derived from ventral dermis, which has a lateral plate origin. However, these developmentally distinct SKPs also retain differential expression of a small number of genes that reflect their developmental origins. Thus, an adult neural crest-like dermal precursor can be generated from a non-neural crest origin, a finding with broad implications for the many neuroendocrine cells in the body.

Skeletal muscles are formed from two cell lineages, myogenic and fibroblastic. Mesoderm-derived myogenic progenitors form muscle cells whereas fibroblastic cells give rise to the supportive connective tissue of skeletal muscles, such as the tendons and perimysium. It remains unknown how myogenic and fibroblastic cell-cell interactions affect cell fate determination and the organization of skeletal muscle. In the present study, we investigated the functional significance of cell-cell interactions in regulating skeletal muscle development. Our study shows that cranial neural crest (CNC) cells give rise to the fibroblastic cells of the tongue skeletal muscle in mice. Loss of Tgfbr2 in CNC cells (Wnt1-Cre;Tgfbr2flox/flox) results in microglossia with reduced Scleraxis and Fgf10 expression as well as decreased myogenic cell proliferation, reduced cell number and disorganized tongue muscles. Furthermore, TGF-β2 beads induced the expression of Scleraxis in tongue explant cultures. The addition of FGF10 rescued the muscle cell number in Wnt1-Cre;Tgfbr2flox/flox mice. Thus, TGF-β induced FGF10 signaling has a critical function in regulating tissue-tissue interaction during tongue skeletal muscle development.

Transforming growth factor-␤ (Tgf-␤) signaling is crucial for regulating craniofacial development. Loss of Tgf-␤ signaling results in defects in cranial neural crest cells (CNCC), but the mechanism by which Tgf-␤ signaling regulates bone formation in CNCC-derived osteogenic cells remains largely unknown. In this study, we discovered that Tgf-␤ regulates the basal transcriptional regulatory machinery to control intramembranous bone development. Specifically, basal transcription factor Taf4b is down-regulated in the CNCC-derived intramembranous bone in Craniofacial skeletal elements are mainly formed by intramembranous ossification through a mechanism that remains relatively uncharacterized. The majority of osteoblasts and chondrocytes in the craniofacial region are derived from cranial neural crest cells (CNCC), 2 which produce the facial skeleton (1, 2). Tgf-␤ signaling plays a crucial role in craniofacial development, and loss of Tgf-␤ signaling in CNCC results in craniofacial skeletal malformations (3, 4). Tgf-␤ transmits signals through a membrane receptor serine/threonine kinase complex that phosphorylates Smad2 and Smad3, and activated Smads form transcriptional complexes with Smad4 and translocate into the nucleus (5). These Tgf-␤ signaling complexes contain other transcription factors and target a variety of genes in an embryonic stage-dependent and cell type-specific manner, but the factors involved in this transcriptional regulatory machinery have yet to be identified. During development, the expression of many genes is associated with changes accompanied by dynamic restructuring of chromatin (6, 7). Recent studies demonstrate that basal transcriptional factors have cell-and promoter-specific functions during embryogenesis (8 -12).RNA polymerase II requires the assembly of a multiprotein complex around the transcriptional start site (13). The general transcriptional factor IID (TFIID) is a large multiprotein transcriptional factor, consisting of the TATA-binding protein and a set of 13-14 TATA-binding protein-associated factors (TAFs), that is responsible for specific binding to the TATA element found in many polymerase II promoters and also demonstrates a coactivator function during transcriptional initiation (14). TAFs are able to regulate gene transcription at multiple steps, with functions in promoter recognition, selective binding to core promoter elements, as well as direct interactions with transcriptional activators (15-17). Mutation and loss of TAFs in yeast and mammalian cells lead to cell cycle arrest and gene-specific transcriptional effects (16). The function of TAFs in gene regulation during embryogenesis has yet to be determined. Here, we show that the interaction between Tgf-␤ signaling and TAFs has a crucial role in regulating CNCC-derived osteogenesis during craniofacial morphogenesis. EXPERIMENTAL PROCEDURES Animals-Mating Tgfbr2fl/ϩ ;Wnt1-Cre with Tgfbr2 fl/fl mice generated Tgfbr2 fl/fl ;Wnt1-Cre conditional null alleles that were genotyped using PCR primers as described previously ...

Regulation of the lymphoid enhancer factor 1 (Lef-1) transcription factor is important for the inductive formation of many epithelial-derived appendages including airway submucosal glands (SMGs). Although Wnts have been linked to developmental processes involving transcriptional activation of the Lef-1 protein, there is little in vivo information directly linking Wnts with the transcriptional regulation of the Lef-1 promoter. In the present study, we hypothesized that Wnt3a directly regulates Lef-1 gene expression required for SMG morphogenesis in mice. In support of this hypothesis, TOPGAL reporter mice demonstrated activation of beta-catenin/Tcf complexes during early phases of SMG development and immunolocalization studies confirmed abundant expression of Tcf4, but not Tcf1 or Tcf3, at this stage. ChIP analysis in primary airway epithelial cells revealed that Tcf4 associates with a known Wnt Responsive Region in the Lef-1 promoter and transfection of Cos-1 cells with dominant active beta-catenin and Tcf4 synergistically activated the Lef-1 promoter. Using Wnt3a deficient and Lef-1 promoter-GFP reporter mice, we also demonstrate that Wnt3a induces Lef-1 gene expression in newly forming SMG buds of mice and is required for the maintenance of gland bud growth. These findings provide the first in vivo evidence that Wnt3a can transcriptionally regulate the Lef-1 gene.