Epithelial Wnt Ligand Secretion Is Required for Adult Hair Follicle Growth and Regeneration

β-Catenin, a key transducer molecule of Wnt signaling, is required for adult hair follicle growth and regeneration. However, the cellular source of Wnt ligands required for Wnt/β-catenin activation during anagen induction is unknown. In this study, we genetically deleted Wntless (Wls), a gene required for Wnt ligand secretion by Wnt-producing cells, specifically in the hair follicle epithelium during telogen phase. We show that epithelial Wnt ligands are required for anagen, as loss of Wls in the follicular epithelium resulted in a profound hair cycle arrest. Both the follicular epithelium and dermal papilla showed markedly decreased Wnt/β-catenin signaling during anagen induction compared with control hair follicles. Surprisingly, hair follicle stem cells that are responsible for hair regeneration maintained expression of stem cell markers but exhibited significantly reduced proliferation. Finally, we demonstrate that epidermal Wnt ligands are critical for adult wound-induced de novo hair formation. Collectively, these data show that Wnt ligands secreted by the hair follicle epithelium are required for adult hair follicle regeneration and provide new insight into potential cellular targets for the treatment of hair disorders such as alopecia

Wnt/β-catenin Signaling Pathway Regulates Specific lncRNAs That Impact Dermal Fibroblasts and Skin Fibrosis

Wnt/β-catenin signaling is required for embryonic dermal fibroblast cell fate, and dysregulation of this pathway is sufficient to promote fibrosis in adult tissue. The downstream modulators of Wnt/β-catenin signaling required for controlling cell fate and dermal fibrosis remain poorly understood. The discovery of regulatory long non-coding RNAs (lncRNAs) and their pivotal roles as key modulators of gene expression downstream of signaling cascades in various contexts prompted us to investigate their roles in Wnt/β-catenin signaling. Here, we have identified lncRNAs and protein-coding RNAs that are induced by β-catenin activity in mouse dermal fibroblasts using next generation RNA-sequencing. The differentially expressed protein-coding mRNAs are enriched for extracellular matrix proteins, glycoproteins, and cell adhesion, and many are also dysregulated in human fibrotic tissues. We identified 111 lncRNAs that are differentially expressed in response to activation of Wnt/β-catenin signaling. To further characterize the role of mouse lncRNAs in this pathway, we validated two novel Wnt signaling- Induced Non-Coding RNA (Wincr) transcripts referred to as Wincr1 and Wincr2. These two lncRNAs are highly expressed in mouse embryonic skin and perinatal dermal fibroblasts. Furthermore, we found that Wincr1 expression levels in perinatal dermal fibroblasts affects the expression of key markers of fibrosis (e.g., Col1a1 and Mmp10), enhances collagen contraction, and attenuates collective cell migration. Our results show that β-catenin signaling-responsive lncRNAs may modulate dermal fibroblast behavior and collagen accumulation in dermal fibrosis, providing new mechanistic insights and nodes for therapeutic intervention

PRC2 Is Dispensable in Vivo for β-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/β-catenin signaling, and loss of β-catenin leads to an ectopic chondrogenic cell fate switch. The mechanism by which Wnt/β-catenin activity suppresses the cartilage fate is unclear. Upon conditional deletion of β-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/β-catenin-mediated repression of chondrogenesis in the embryonic CM. We find that β-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 β-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/β-catenin signaling

Distinct Requirements for Cranial Ectoderm and Mesenchyme-Derived Wnts in Specification and Differentiation of Osteoblast and Dermal Progenitors

<div><p>The cranial bones and dermis differentiate from mesenchyme beneath the surface ectoderm. Fate selection in cranial mesenchyme requires the canonical Wnt effector molecule β-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.</p></div

Distinct requirements for <i>Wntless</i> in the cranial ectoderm and mesenchyme.

<p>(A, B, C, D, C′, D′) Von Kossa staining, or (E–H) alcian blue staining was performed on coronal mouse embryonic head sections and counterstained with eosin. Br, brain, fb, frontal bone, vhf, supraorbital vibrissae hair follicle, mn, meningeal progenitors. Black arrowheads indicate guard hair follicles (hf), red arrowheads indicate dorsal extent of ossified frontal bone, and open black arrows indicate ectopic cartilage. (C′, D′ C″, D″) Black dotted line demarcates the lower limit of the dermal layer and the black bracket shows dermal thickness. Diagrams inset (B) figure depicts lateral view of E15.5 embryonic head with plane of section and region of interest. Red regions in diagram represent bone primordia. Scale bars (A,E) represent 100 µm.</p

Ectoderm deletion of <i>Wntless</i> leads to loss of cranial bone and dermal lineage markers in the mesenchyme.

<p>Indirect immunofluorescence with DAPI-stained (blue) nuclei was performed on coronal mouse embryonic head sections at E12.5 or as indicated (A,B, F, G, H, I, M, N, P, R, T, V). Alkaline Phosphatase staining (C, J), in situ hybridization (D, E, K, L, O, S), or β-galactosidase staining with eosin counterstain (Q, U) was performed on coronal tissue sections. Diagram in (A) demonstrates plane of section and region of interest for E12.5-E13.5 (A–T). Box and dashed lines in (Q, U) demonstrate the region of high magnification, and β-galactosidase stained sections were included for perspective for (R, V). Diagram inset in high magnification photograph from (Q) shows plane of section and region of interest for E15.5. Red arrows indicate changes in marker expression and black arrows in (U) high magnification indicate ectopic cartilage. Scale bars represent 100 µm.</p

Primer sequences for RT-PCR of mouse Wnt genes.

<p>Primer sequences for RT-PCR of mouse Wnt genes.</p

Expression of Wnt ligands, Wntless, and Wnt signaling response in cranial ectoderm and mesenchyme.

<p>(A, B) RT-PCR for individual Wnt ligands was performed on cDNA from purified mouse embryonic cranial mesenchyme and surface ectoderm. (C, D G, H) Indirect immunofluorescence with DAPI counterstained nuclei (blue), (E) <i>in situ</i> hybridization, or immunohistochemistry (F, I) was performed on coronal mouse embryonic head sections. (G, H, I) Boxes indicate region in insets at higher magnification. White arrowheads indicate co-expression of (G) Wls/Runx2 or (D,H) Lef1/Runx2, (I) red arrowheads indicate osteoblast progenitors, and blue arrowheads indicate dermal progenitors. (F–I) White hatched lines demarcate ectoderm from mesenchyme. (J) Summary scheme of E12.5 supraorbital cranial mesenchyme. (J) Embryonic axes, figure depicts lateral view of embryonic head, region of interest in sections used in figures are shown. Scale bars represent 100 µm.</p

Mesenchyme deletion of <i>Wntless</i> leads to diminished differentiation and Wnt responsiveness in the bone lineage.

<p>Indirect immunofluorescence with DAPI-stained (blue) nuclei (A, B, D, F, G, H, J, L, P, T) and immunohistochemistry (M,Q) was performed on coronal mouse embryonic head sections In situ hybridization (C, I, N, O, R, S) or eosin counterstain (E, K), was performed on coronal tissue sections of embryonic murine heads at the indicated stages. Diagram in (A) demonstrates plane of section and region of interest for E11.5-E12.5. Box in (D, J) demonstrate the region of high magnification. (I, S, T) Red arrows highlight changes in marker expression in osteoprogenitor domain. (E,K) vhf: subraorbital vibrissae hair follicle and black bracket indicates the dermal layer. (A,G) Scale bars represent 100 µm.</p