Wnt/ß-catenin signaling in the mouse embryonic cranial mesenchyme is required to sustain the emerging differentiated meningeal layers.

Cranial neural crest cells (CNCCs) give rise to cranial mesenchyme (CM) that differentiates into the forebrain meningeal progenitors in the basolateral and apical regions of the head. This occurs in close proximity to the other CNCC-CM-derivatives, such as calvarial bone and dermal progenitors. We found active Wnt signaling transduction in the forebrain meningeal progenitors in basolateral and apical populations and in the non-meningeal CM preceding meningeal differentiation. Here, we dissect the source of Wnt ligand secretion and requirement of Wnt/ß-catenin signaling for the lineage selection and early differentiation of the forebrain meninges. We find persistent canonical Wnt/ß-catenin signal transduction in the meningeal progenitors in the absence of Wnt ligand secretion in the CM or surface ectoderm, suggesting additional sources of Wnts. Conditional mutants for Wntless and ß-catenin in the CM showed that Wnt ligand secretion and Wnt/ß-catenin signaling were dispensable for specification and proliferation of early meningeal progenitors. In the absence of ß-catenin in the CM, we found diminished laminin matrix and meningeal hypoplasia, indicating a structural and trophic role of mesenchymal ß-catenin signaling. This study shows that ß-catenin signaling is required in the CM for maintenance and organization of the differentiated meningeal layers in the basolateral and apical populations of embryonic meninges.

Distinct requirements for cranial ectoderm and mesenchyme-derived wnts in specification and differentiation of osteoblast and dermal progenitors.

The cranial bones and dermis differentiate from mesenchyme beneath the surface ectoderm. Fate selection in cranial mesenchyme requires the canonical Wnt effector molecule ß-catenin, but the relative contribution of Wnt ligand sources in this process remains unknown. Here we show Wnt ligands are expressed in cranial surface ectoderm and underlying supraorbital mesenchyme during dermal and osteoblast fate selection. Using conditional genetics, we eliminate secretion of all Wnt ligands from cranial surface ectoderm or undifferentiated mesenchyme, to uncover distinct roles for ectoderm- and mesenchyme-derived Wnts. Ectoderm Wnt ligands induce osteoblast and dermal fibroblast progenitor specification while initiating expression of a subset of mesenchymal Wnts. Mesenchyme Wnt ligands are subsequently essential during differentiation of dermal and osteoblast progenitors. Finally, ectoderm-derived Wnt ligands provide an inductive cue to the cranial mesenchyme for the fate selection of dermal fibroblast and osteoblast lineages. Thus two sources of Wnt ligands perform distinct functions during osteoblast and dermal fibroblast formation.

Twist1 contributes to cranial bone initiation and dermal condensation by maintaining Wnt signaling responsiveness.

BACKGROUND: Specification of cranial bone and dermal fibroblast progenitors in the supraorbital arch mesenchyme is Wnt/ß-catenin signaling-dependent. The mechanism underlying how these cells interpret instructive signaling cues and differentiate into these two lineages is unclear. Twist1 is a target of the Wnt/ß-catenin signaling pathway and is expressed in cranial bone and dermal lineages. RESULTS: Here, we show that onset of Twist1 expression in the mouse cranial mesenchyme is dependent on ectodermal Wnts and mesenchymal ß-catenin activity. Conditional deletion of Twist1 in the supraorbital arch mesenchyme leads to cranial bone agenesis and hypoplastic dermis, as well as craniofacial malformation of eyes and palate. Twist1 is preferentially required for cranial bone lineage commitment by maintaining Wnt responsiveness. In the conditional absence of Twist1, the cranial dermis fails to condense and expand apically leading to extensive cranial dermal hypoplasia with few and undifferentiated hair follicles. CONCLUSIONS: Thus, Twist1, a target of canonical Wnt/ß-catenin signaling, also functions to maintain Wnt responsiveness and is a key effector for cranial bone fate selection and dermal condensation.

Sustained ß-catenin activity in dermal fibroblasts promotes fibrosis by up-regulating expression of extracellular matrix protein-coding genes.

Fibrosis is an end-stage response to tissue injury that is associated with loss of organ function as a result of excess extracellular matrix (ECM) production by fibroblasts. In skin, pathological fibrosis is evident during keloid scar formation, systemic sclerosis (SSc) and morphea. Dermal fibroblasts in these fibrotic diseases exhibit increased Wnt/ß-catenin signalling, a pathway that is sufficient to cause fibrosis in mice. However, in the context of this complex pathology, the precise pro-fibrotic consequences of Wnt/ß-catenin signalling are not known. We found that expression of stabilized ß-catenin in mouse dermal fibroblasts resulted in spontaneous, progressive skin fibrosis with thickened collagen fibres and altered collagen fibril morphology. The fibrotic phenotype was predominated by resident dermal fibroblasts. Genome-wide profiling of the fibrotic mouse dermis revealed elevated expression of matrix-encoding genes, and the promoter regions of these genes were enriched for Tcf/Lef family transcription factor binding sites. Additionally, we identified 32 ß-catenin-responsive genes in our mouse model that are also over-expressed in human fibrotic tissues and poised for regulation by Tcf/Lef family transcription factors. Therefore, we have uncovered a matrix-regulatory role for stabilized ß-catenin in fibroblasts in vivo and have defined a set of ß-catenin-responsive genes with relevance to fibrotic disease.

PRC2 Is Dispensable in Vivo for ß-Catenin-Mediated Repression of Chondrogenesis in the Mouse Embryonic Cranial Mesenchyme.

A hallmark of craniofacial development is the differentiation of multiple cell lineages in close proximity to one another. The mouse skull bones and overlying dermis are derived from the cranial mesenchyme (CM). Cell fate selection of the embryonic cranial bone and dermis in the CM requires Wnt/ß-catenin signaling, and loss of ß-catenin leads to an ectopic chondrogenic cell fate switch. The mechanism by which Wnt/ß-catenin activity suppresses the cartilage fate is unclear. Upon conditional deletion of ß-catenin in the CM, several key determinants of the cartilage differentiation program, including Sox9, become differentially expressed. Many of these differentially expressed genes are known targets of the Polycomb Repressive Complex 2 (PRC2). Thus, we hypothesized that PRC2 is required for Wnt/ß-catenin-mediated repression of chondrogenesis in the embryonic CM. We find that ß-catenin can physically interact with PRC2 components in the CM in vivo However, upon genetic deletion of Enhancer of Zeste homolog 2 (EZH2), the catalytic component of PRC2, chondrogenesis remains repressed and the bone and dermis cell fate is preserved in the CM. Furthermore, loss of ß-catenin does not alter either the H3K27me3 enrichment levels genome-wide or on cartilage differentiation determinants, including Sox9 Our results indicate that EZH2 is not required to repress chondrogenesis in the CM downstream of Wnt/ß-catenin signaling.