Topographical changes in extracellular matrix during skin fibrosis and recovery can be evaluated using automated image analysis algorithms.

Skin fibrosis is characterized by fibroblast activation and intradermal fat loss, resulting in excess deposition and remodeling of dermal extracellular matrix (ECM). The topography of the dominant ECM proteins, such as collagens, can indicate skin stiffness and remains understudied in evaluating fibrotic skin. Here, we adapted two different unbiased image analysis algorithms to define collagen topography and alignment in a genetically inducible and reversible Wnt activation fibrosis model. We demonstrated that Wnt-activated fibrotic skin has altered collagen fiber characteristics and a loss of collagen alignment, which were restored in the reversible model. This study highlights how unbiased algorithms can be used to analyze ECM topography, providing novel avenues to evaluate fibrotic skin onset, recovery, and treatment.

Mesenchymal Wnts are required for morphogenetic movements of calvarial osteoblasts during apical expansion.

Apical expansion of calvarial osteoblast progenitors from the cranial mesenchyme (CM) above the eye is integral to calvarial growth and enclosure of the brain. The cellular behaviors and signals underlying the morphogenetic process of calvarial expansion are unknown. Time-lapse light-sheet imaging of mouse embryos revealed calvarial progenitors intercalate in 3D in the CM above the eye, and exhibit protrusive and crawling activity more apically. CM cells express non-canonical Wnt/planar cell polarity (PCP) core components and calvarial osteoblasts are bidirectionally polarized. We found non-canonical ligand Wnt5a-/- mutants have less dynamic cell rearrangements and protrusive activity. Loss of CM-restricted Wntless (CM-Wls), a gene required for secretion of all Wnt ligands, led to diminished apical expansion of Osx+ calvarial osteoblasts in the frontal bone primordia in a non-cell autonomous manner without perturbing proliferation or survival. Calvarial osteoblast polarization, progressive cell elongation and enrichment for actin along the baso-apical axis were dependent on CM-Wnts. Thus, CM-Wnts regulate cellular behaviors during calvarial morphogenesis for efficient apical expansion of calvarial osteoblasts. These findings also offer potential insights into the etiologies of calvarial dysplasias.

Apical expansion of calvarial osteoblasts and suture patency is dependent on fibronectin cues.

The skull roof, or calvaria, is comprised of interlocking plates of bones that encase the brain. Separating these bones are fibrous sutures that permit growth. Currently, we do not understand the instructions for directional growth of the calvaria, a process which is error-prone and can lead to skeletal deficiencies or premature suture fusion (craniosynostosis, CS). Here, we identify graded expression of fibronectin (FN1) in the mouse embryonic cranial mesenchyme (CM) that precedes the apical expansion of calvaria. Conditional deletion of Fn1 or Wasl leads to diminished frontal bone expansion by altering cell shape and focal actin enrichment, respectively, suggesting defective migration of calvarial progenitors. Interestingly, Fn1 mutants have premature fusion of coronal sutures. Consistently, syndromic forms of CS in humans exhibit dysregulated FN1 expression, and we also find FN1 expression altered in a mouse CS model of Apert syndrome. These data support a model of FN1 as a directional substrate for calvarial osteoblast migration that may be a common mechanism underlying many cranial disorders of disparate genetic etiologies.

Tissue fibrosis associated depletion of lipid-filled cells.

Fibrosis is primarily described as the deposition of excessive extracellular matrix, but in many tissues it also involves a loss of lipid or lipid-filled cells. Lipid-filled cells are critical to tissue function and integrity in many tissues including the skin and lungs. Thus, loss or depletion of lipid-filled cells during fibrogenesis, has implications for tissue function. In some contexts, lipid-filled cells can impact ECM composition and stability, highlighting their importance in fibrotic transformation. Recent papers in fibrosis address this newly recognized fibrotic lipodystrophy phenomenon. Even in disparate tissues, common mechanisms are emerging to explain fibrotic lipodystrophy. These findings have implications for fibrosis in tissues composed of fibroblast and lipid-filled cell populations such as skin, lung, and liver. In this review, we will discuss the roles of lipid-containing cells, their reduction/loss during fibrotic transformation, and the mechanisms of that loss in the skin and lungs.

Mesenchymal Wnts are required for morphogenetic movements of calvarial osteoblasts during apical expansion.

Apical expansion of calvarial osteoblast progenitors from the cranial mesenchyme (CM) above the eye is integral for calvarial growth and enclosure of the brain. The cellular behaviors and signals underlying the morphogenetic process of calvarial expansion are unknown. During apical expansion, we found that mouse calvarial primordia have consistent cellular proliferation, density, and survival with complex tissue scale deformations, raising the possibility that morphogenetic movements underlie expansion. Time lapse light sheet imaging of mouse embryos revealed that calvarial progenitors intercalate in 3D to converge supraorbital arch mesenchyme mediolaterally and extend it apically. In contrast, progenitors located further apically exhibited protrusive and crawling activity. CM cells express non-canonical Wnt/Planar Cell Polarity (PCP) core components and calvarial osteoblasts are bidirectionally polarized. We found non-canonical ligand, Wnt5a-/- mutants have less dynamic cell rearrangements, protrusive activity, and a flattened head shape. Loss of cranial mesenchyme-restricted Wntless (CM-Wls), a gene required for secretion of all Wnt ligands, led to diminished apical expansion of OSX+ calvarial osteoblasts in the frontal bone primordia in a non-cell autonomous manner without perturbing proliferation or survival. Calvarial osteoblast polarization, progressive cell elongation and enrichment for actin cytoskeleton protein along the baso-apical axis were dependent on CM-Wnts. Thus, CM-Wnts regulate cellular behaviors during calvarial morphogenesis and provide tissue level cues for efficient apical expansion of calvarial osteoblasts. These findings also offer potential insights into the etiologies of calvarial dysplasias.

Apical expansion of calvarial osteoblasts and suture patency is dependent on graded fibronectin cues.

The skull roof, or calvaria, is comprised of interlocking plates of bone. Premature suture fusion (craniosynostosis, CS) or persistent fontanelles are common defects in calvarial development. Although some of the genetic causes of these disorders are known, we lack an understanding of the instructions directing the growth and migration of progenitors of these bones, which may affect the suture patency. Here, we identify graded expression of Fibronectin (FN1) protein in the mouse embryonic cranial mesenchyme (CM) that precedes the apical expansion of calvarial osteoblasts. Syndromic forms of CS exhibit dysregulated FN1 expression, and we find FN1 expression is altered in a mouse CS model as well. Conditional deletion of Fn1 in CM causes diminished frontal bone expansion by altering cell polarity and shape. To address how osteoprogenitors interact with the observed FN1 prepattern, we conditionally ablate Wasl/N-Wasp to disrupt F-actin junctions in migrating cells, impacting lamellipodia and cell-matrix interaction. Neural crest-targeted deletion of Wasl results in a diminished actin network and reduced expansion of frontal bone primordia similar to conditional Fn1 mutants. Interestingly, defective calvaria formation in both the Fn1 and Wasl mutants occurs without a significant change in proliferation, survival, or osteogenesis. Finally, we find that CM-restricted Fn1 deletion leads to premature fusion of coronal sutures. These data support a model of FN1 as a directional substrate for calvarial osteoblast migration that may be a common mechanism underlying many cranial disorders of disparate genetic etiologies.

The origins of skin diversity: lessons from dermal fibroblasts.

Skin is largely composed of an epidermis that overlies a supporting dermis. Recent advancements in our understanding of how diverse groups of dermal fibroblasts regulate epidermal and hair follicle growth and differentiation have been fueled by tools capable of resolving molecular heterogeneity at a single-cell level. Fibroblast heterogeneity can be traced back to their developmental origin before their segregation into spatially distinct fibroblast subtypes. The mechanisms that drive this lineage diversification during development are being unraveled, with studies showing that both large- and small-scale positional signals play important roles during dermal development. Here, we first delineate what is known about the origins of the dermis and the central role of Wnt/ß-catenin signaling in its specification across anatomical locations. We then discuss how one of the first morphologically recognizable fibroblast subtypes, the hair follicle dermal condensate lineage, emerges. Leveraging the natural variation of skin and its appendages between species and between different anatomical locations, these collective studies have identified shared and divergent factors that contribute to the extraordinary diversity of skin.

EZH2 modulates retinoic acid signaling to ensure myotube formation during development.

Sequential differentiation of presomitic progenitors into myocytes and subsequently into myotubes and myofibers is essential for the myogenic differentiation program (MDP) crucial for muscle development. Signaling factors involved in MDP are polycomb repressive complex 2 (PRC2) targets in various developmental contexts. PRC2 is active in the developing myotomes during MDP, but how it regulates MDP is unclear. Here, we found that myocyte differentiation to myotubes requires Enhancer of Zeste 2 (EZH2), the catalytic component of PRC2. We observed elevated retinoic acid (RA) signaling in the prospective myocytes in the Ezh2 mutants (E8.5-MusEzh2 ), and its inhibition can partially rescue the myocyte differentiation defect. Together, our data demonstrate a new role for PRC2-EZH2 during myocyte differentiation into myotubes by modulating RA signaling.

Skin Fibrosis and Recovery Is Dependent on Wnt Activation via DPP4.

Fibrosis is the life-threatening, excessive accumulation of the extracellular matrix and is sometimes associated with a loss of lipid-filled cells in the skin and other organs. Understanding the mechanisms of fibrosis and associated lipodystrophy and their reversal may reveal new targets for therapeutic intervention. In vivo genetic models are needed to identify key targets that induce recovery from established fibrosis. Wnt signaling is activated in animal and human fibrotic diseases across organs. Here, we developed a genetically inducible and reversible Wnt activation model and showed that it is sufficient to cause fibrotic dermal remodeling, including extracellular matrix expansion and shrinking of dermal adipocytes. Upon withdrawal from Wnt activation, Wnt-induced fibrotic remodeling was reversed in mouse skin-fully restoring skin architecture. Next, we demonstrated CD26/ DPP4 is a Wnt/ß-catenin-responsive gene and a functional mediator of fibrotic transformation. We provide genetic evidence that the Wnt/DPP4 axis is required to drive fibrotic dermal remodeling and is associated with human skin fibrosis severity. Remarkably, DPP4 inhibitors can be repurposed to accelerate recovery from established Wnt-induced fibrosis. Collectively, this study identifies Wnt/DPP4 axis as a key driver of extracellular matrix homeostasis and dermal fat loss, providing therapeutic avenues to manipulate the onset and reversal of tissue fibrosis.

Wnt-Dependent Activation of ERK Mediates Repression of Chondrocyte Fate during Calvarial Development.

Wnt signaling regulates cell fate decisions in diverse contexts during development, and loss of Wnt signaling in the cranial mesenchyme results in a robust and binary cell fate switch from cranial bone to ectopic cartilage. The Extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) and Wnt signaling pathways are activated during calvarial osteoblast cell fate selection. Here, we test the hypothesis that ERK signaling is a mediator of Wnt-dependent cell fate decisions in the cranial mesenchyme. First, we show that loss of Erk1/2 in the cranial mesenchyme results in a diminished domain of osteoblast marker expression and increased expression of cartilage fate markers and ectopic cartilage formation in the frontal bone primordia. Second, we show that mesenchyme Wnt/ß-catenin signaling and Wntless are required for ERK activation in calvarial osteoblasts. Third, we demonstrate that Wnt and ERK signaling pathways function together to repress SOX9 expression in mouse cranial mesenchyme. Our results demonstrate an interaction between the Wnt and ERK signaling pathways in regulating lineage selection in a subset of calvarial cells and provide new insights into Wnt-dependent cell fate decisions.

Dermal EZH2 orchestrates dermal differentiation and epidermal proliferation during murine skin development.

Skin development and patterning is dependent on factors that regulate the stepwise differentiation of dermal fibroblasts concomitant with dermal-epidermal reciprocal signaling, two processes that are poorly understood. Here we show that dermal EZH2, the methyltransferase enzyme of the epigenetic Polycomb Repressive Complex 2 (PRC2), is a new coordinator of both these processes. Dermal EZH2 activity is present during dermal fibroblast differentiation and is required for spatially restricting Wnt/ß-catenin signaling to reinforce dermal fibroblast cell fate. Later in development, dermal EZH2 regulates the expression of reticular dermal markers and initiation of secondary hair follicles. Embryos lacking dermal Ezh2 have elevated epidermal proliferation and differentiation that can be rescued by small molecule inhibition of retinoic acid (RA) signaling. Together, our study reveals that dermal EZH2 is acting like a rheostat to control the levels of Wnt/ß-catenin and RA signaling to impact fibroblast differentiation cell autonomously and epidermal keratinocyte development non-cell autonomously, respectively.

Polycomb Repressive Complex 2: a Dimmer Switch of Gene Regulation in Calvarial Bone Development.

PURPOSE OF REVIEW: Epigenetic regulation is a distinct mechanism of gene regulation that functions by modulating chromatin structure and accessibility. Polycomb Repressive Complex 2 (PRC2) is a conserved chromatin regulator that is required in the developing embryo to control the expression of key developmental genes. An emerging feature of PRC2 is that it not only allows for binary ON/OFF states of gene expression but can also modulate gene expression in feed-forward loops to change the outcome of gene regulatory networks. This striking feature of epigenetic modulation has improved our understanding of musculoskeletal development. RECENT FINDINGS: Recent advances in mouse embryos unravel a range of phenotypes that demonstrate the tissue-specific, temporal, and spatial role of PRC2 during organogenesis and cell fate decisions in vivo. Here, we take a detailed view of how PRC2 functions during the development of calvarial bone and skin. Based on the emerging evidence, we propose that PRC2 serves as a "dimmer switch" to modulate gene expression of target genes by altering the expression of activators and inhibitors. This review highlights the findings from contemporary research that allow us to investigate the unique developmental potential of intramembranous calvarial bones.

What Do Animal Models Teach Us About Congenital Craniofacial Defects?

The formation of the head and face is a complex process which involves many different signaling cues regulating the migration, differentiation, and proliferation of the neural crest. This highly complex process is very error-prone, resulting in craniofacial defects in nearly 10,000 births in the United States annually. Due to the highly conserved mechanisms of craniofacial development, animal models are widely used to understand the pathogenesis of various human diseases and assist in the diagnosis and generation of preventative therapies and treatments. Here, we provide a brief background of craniofacial development and discuss several rare diseases affecting craniofacial bone development. We focus on rare congenital diseases of the cranial bone, facial jaw bones, and two classes of diseases, ciliopathies and RASopathies. Studying the animal models of these rare diseases sheds light not only on the etiology and pathology of each disease, but also provides meaningful insights towards the mechanisms which regulate normal development of the head and face.

A novel mouse model demonstrates that oncogenic melanocyte stem cells engender melanoma resembling human disease.

Melanoma, the deadliest skin cancer, remains largely incurable at advanced stages. Currently, there is a lack of animal models that resemble human melanoma initiation and progression. Recent studies using a Tyr-CreER driven mouse model have drawn contradictory conclusions about the potential of melanocyte stem cells (McSCs) to form melanoma. Here, we employ a c-Kit-CreER-driven model that specifically targets McSCs to show that oncogenic McSCs are a bona fide source of melanoma that expand in the niche, and then establish epidermal melanomas that invade into the underlying dermis. Further, normal Wnt and Endothelin niche signals during hair anagen onset are hijacked to promote McSC malignant transformation during melanoma induction. Finally, molecular profiling reveals strong resemblance of murine McSC-derived melanoma to human melanoma in heterogeneity and gene signatures. These findings provide experimental validation of the human melanoma progression model and key insights into the transformation and heterogeneity of McSC-derived melanoma.

Oscillatory cortical forces promote three dimensional cell intercalations that shape the murine mandibular arch.

Multiple vertebrate embryonic structures such as organ primordia are composed of confluent cells. Although mechanisms that shape tissue sheets are increasingly understood, those which shape a volume of cells remain obscure. Here we show that 3D mesenchymal cell intercalations are essential to shape the mandibular arch of the mouse embryo. Using a genetically encoded vinculin tension sensor that we knock-in to the mouse genome, we show that cortical force oscillations promote these intercalations. Genetic loss- and gain-of-function approaches show that Wnt5a functions as a spatial cue to coordinate cell polarity and cytoskeletal oscillation. These processes diminish tissue rigidity and help cells to overcome the energy barrier to intercalation. YAP/TAZ and PIEZO1 serve as downstream effectors of Wnt5a-mediated actomyosin polarity and cytosolic calcium transients that orient and drive mesenchymal cell intercalations. These findings advance our understanding of how developmental pathways regulate biophysical properties and forces to shape a solid organ primordium.

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.

A tale of two cities: The genetic mechanisms governing calvarial bone development.

The skull bones must grow in a coordinated, three-dimensional manner to coalesce and form the head and face. Mammalian skull bones have a dual embryonic origin from cranial neural crest cells (CNCC) and paraxial mesoderm (PM) and ossify through intramembranous ossification. The calvarial bones, the bones of the cranium which cover the brain, are derived from the supraorbital arch (SOA) region mesenchyme. The SOA is the site of frontal and parietal bone morphogenesis and primary center of ossification. The objective of this review is to frame our current in vivo understanding of the morphogenesis of the calvarial bones and the gene networks regulating calvarial bone initiation in the SOA mesenchyme.

Stage-specific roles of Ezh2 and Retinoic acid signaling ensure calvarial bone lineage commitment.

Development of the skull bones requires the coordination of two stem progenitor populations, the cranial neural crest cells (CNCC) and head paraxial mesoderm (PM), to ensure cell fate selection and morphogenesis. The epigenetic methyltransferase, Ezh2, plays a role in skull bone formation, but the spatiotemporal function of Ezh2 between the CNCC- and PM-derived bone formation in vivo remains undefined. Here, using a temporally-inducible conditional deletion of Ezh2 in both the CNCC- and PM- derived cranial mesenchyme between E8.5 and E9.5, we find a reduction of the CNCC-derived calvarial bones and a near complete loss of the PM-derived calvarial bones due to an arrest in calvarial bone fate commitment. In contrast, deletion of Ezh2 after E9.5 permits PM-derived skull bone development, suggesting that Ezh2 is required early to guide calvarial bone progenitor commitment. Furthermore, exposure to all-trans Retinoic acid at E10.0 can mimic the Ezh2 mutant calvarial phenotype, and administration of the pan retinoic acid receptor (RAR) antagonist, BMS-453, to Ezh2 mutants partially restores the commitment to the calvarial bone lineage and PM-derived bone development in vivo. Exogenous RA signaling activation in the Ezh2 mutants leads to synergistic activation of the anti-osteogenic factors in the cranial mesenchyme in vivo. Thus, RA signaling and EZH2 can function in parallel to guide calvarial bone progenitor commitment by balancing the suppression of anti-osteogenic factors.

Dermal fibroblast in cutaneous development and healing.

The skin is the largest organ of the body and is composed of two layers: the overlying epidermis and the underlying dermis. The dermal fibroblasts originate from distinct locations of the embryo and contain the positional identity and patterning information in the skin. The dermal fibroblast progenitors differentiate into various cell types that are fated to perform specific functions such as hair follicle initiation and scar formation during wound healing. Recent studies have revealed the heterogeneity and plasticity of dermal fibroblasts within skin, which has implications for skin disease and tissue engineering. The objective of this review is to frame our current understanding and provide new insights on the origin and differentiation of dermal fibroblasts and their function during cutaneous development and healing. WIREs Dev Biol 2018, 7:e307. doi: 10.1002/wdev.307 This article is categorized under: Birth Defects > Organ Anomalies Signaling Pathways > Cell Fate Signaling Adult Stem Cells, Tissue Renewal, and Regeneration > Regeneration Nervous System Development > Vertebrates: Regional Development.

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.