A common allele increases endometrial Wnt4 expression, with antagonistic implications for pregnancy, reproductive cancers, and endometriosis.

The common human SNP rs3820282 is associated with multiple phenotypes including gestational length and likelihood of endometriosis and cancer, presenting a paradigmatic pleiotropic variant. Deleterious pleiotropic mutations cause the co-occurrence of disorders either within individuals, or across population. When adverse and advantageous effects are combined, pleiotropy can maintain high population frequencies of deleterious alleles. To reveal the causal molecular mechanisms of this pleiotropic SNP, we introduced this substitution into the mouse genome by CRISPR/Cas 9. Previous work showed that rs3820282 introduces a high-affinity estrogen receptor alpha-binding site at the Wnt4 locus. Here, we show that this mutation upregulates Wnt4 transcription in endometrial stroma, following the preovulatory estrogen peak. Effects on uterine transcription include downregulation of epithelial proliferation and induction of progesterone-regulated pro-implantation genes. We propose that these changes increase uterine permissiveness to embryo invasion, whereas they decrease resistance to invasion by cancer and endometriotic foci in other estrogen-responsive tissues.

Wnt signaling regulates ion channel expression to promote smooth muscle and cartilage formation in developing mouse trachea.

Ion channels play critical roles in the physiology and function of the nervous system and contractile tissue; however, their role in noncontractile tissue and embryonic development has yet to be understood. Tracheobronchomalacia (TBM) and complete tracheal rings (CTR) are disorders affecting the muscle and cartilage of the trachea and bronchi, whose etiology remains poorly understood. We demonstrated that trachealis muscle organization and polarity are disrupted after epithelial ablation of Wntless (Wls), a cargo receptor critical for the Wnt signaling pathway, in developing trachea. The phenotype resembles the anomalous trachealis muscle observed after deletion of ion channel encoding genes in developing mouse trachea. We sought to investigate whether and how the deletion of Wls affects ion channels during tracheal development. We hypothesize that Wnt signaling influences the expression of ion channels to promote trachealis muscle cell assembly and patterning. Deleting Wls in developing trachea causes differential regulation of genes mediating actin binding, cytoskeleton organization, and potassium ion channel activity. Wnt signaling regulates the expression of Kcnj13, Kcnd3, Kcnj8, and Abcc9 as demonstrated by in vitro studies and in vivo analysis in Wnt5a and ß-catenin-deficient tracheas. Pharmacological inhibition of potassium ion channels and Wnt signaling impaired contractility of developing trachealis smooth muscle and formation of cartilaginous mesenchymal condensation. Thus, in mice, epithelial-induced Wnt/ß-catenin signaling mediates trachealis muscle and cartilage development via modulation of ion channel expression, promoting trachealis muscle architecture, contractility, and cartilaginous extracellular matrix. In turn, ion channel activity may influence tracheal morphogenesis underlying TBM and CTR.NEW & NOTEWORTHY Ion channels play critical roles in the physiology and function of the nervous system and contractile tissue; however, their role in noncontractile tissue and embryonic development has yet to be understood. In this study, we focused on the role of ion channels in the differentiation and patterning of the large airways of the developing respiratory tract. We identify a mechanism by which Wnt-beta-catenin signaling controls levels of ion channel-encoding genes to promote tracheal differentiation.

Wnt signaling regulates ion channel expression to promote smooth muscle and cartilage formation in developing mouse trachea.

Ion channels play critical roles in the physiology and function of the nervous system and contractile tissue; however, their role in non-contractile tissue and embryonic development has yet to be understood. Tracheobronchomalacia (TBM) and complete tracheal rings (CTR) are disorders affecting the muscle and cartilage of the trachea and bronchi, whose etiology remains poorly understood. We demonstrated that trachealis muscle organization and polarity are disrupted after epithelial ablation of Wls, a cargo receptor critical for the Wnt signaling pathway, in developing trachea. The phenotype resembles the anomalous trachealis muscle observed after deletion of ion channel encoding genes in developing mouse trachea. We sought to investigate whether and how the deletion of Wls affects ion channels during tracheal development. We hypothesize that Wnt signaling influences the expression of ion channels to promote trachealis muscle cell assembly and patterning. Deleting Wls in developing trachea causes differential regulation of genes mediating actin binding, cytoskeleton organization, and potassium ion channel activity. Wnt signaling regulated expression of Kcnj13, Kcnd3, Kcnj8, and Abcc9 as demonstrated by in vitro studies and in vivo analysis in Wnt5a and ß-catenin deficient tracheas. Pharmacological inhibition of potassium ion channels and Wnt signaling impaired contractility of developing trachealis smooth muscle and formation of cartilaginous mesenchymal condensation. Thus, in mice, epithelial-induced Wnt/ß-catenin signaling mediates trachealis muscle and cartilage development via modulation of ion channel expression, promoting trachealis muscle architecture, contractility, and cartilaginous extracellular matrix. In turn, ion channel activity may influence tracheal morphogenesis underlying TBM and CTR.

Airway basal cells show regionally distinct potential to undergo metaplastic differentiation.

Basal cells are multipotent stem cells of a variety of organs, including the respiratory tract, where they are major components of the airway epithelium. However, it remains unclear how diverse basal cells are and how distinct subpopulations respond to airway challenges. Using single cell RNA-sequencing and functional approaches, we report a significant and previously underappreciated degree of heterogeneity in the basal cell pool, leading to identification of six subpopulations in the adult murine trachea. Among these, we found two major subpopulations, collectively comprising the most uncommitted of all the pools, but with distinct gene expression signatures. Notably, these occupy distinct ventral and dorsal tracheal niches and differ in their ability to self-renew and initiate a program of differentiation in response to environmental perturbations in primary cultures and in mouse injury models in vivo. We found that such heterogeneity is acquired prenatally, when the basal cell pool and local niches are still being established, and depends on the integrity of these niches, as supported by the altered basal cell phenotype of tracheal cartilage-deficient mouse mutants. Finally, we show that features that distinguish these progenitor subpopulations in murine airways are conserved in humans. Together, the data provide novel insights into the origin and impact of basal cell heterogeneity on the establishment of regionally distinct responses of the airway epithelium during injury-repair and in disease conditions.

PI3K signaling specifies proximal-distal fate by driving a developmental gene regulatory network in SOX9+ mouse lung progenitors.

The tips of the developing respiratory buds are home to important progenitor cells marked by the expression of SOX9 and ID2. Early in embryonic development (prior to E13.5), SOX9+progenitors are multipotent, generating both airway and alveolar epithelium, but are selective progenitors of alveolar epithelial cells later in development. Transcription factors, including Sox9, Etv5, Irx, Mycn, and Foxp1/2 interact in complex gene regulatory networks to control proliferation and differentiation of SOX9+progenitors. Molecular mechanisms by which these transcription factors and other signaling pathways control chromatin state to establish and maintain cell-type identity are not well-defined. Herein, we analyze paired gene expression (RNA-Seq) and chromatin accessibility (ATAC-Seq) data from SOX9+ epithelial progenitor cells (EPCs) during embryonic development in Mus musculus. Widespread changes in chromatin accessibility were observed between E11.5 and E16.5, particularly at distal cis-regulatory elements (e.g. enhancers). Gene regulatory network (GRN) inference identified a common SOX9+ progenitor GRN, implicating phosphoinositide 3-kinase (PI3K) signaling in the developmental regulation of SOX9+ progenitor cells. Consistent with this model, conditional ablation of PI3K signaling in the developing lung epithelium in mouse resulted in an expansion of the SOX9+ EPC population and impaired airway epithelial cell differentiation. These data demonstrate that PI3K signaling is required for epithelial patterning during lung organogenesis, and emphasize the combinatorial power of paired RNA and ATAC seq in defining regulatory networks in development.

Studying how lungs develop has helped us understand and treat often-devastating lung diseases. This includes diseases like cystic fibrosis which result from spelling mistakes known as mutations in a person's genetic code. However, not all lung diseases involve mutations. Many other diseases, in both adults and children, are caused by genes failing to switch on or off at some point during lung development. DNA is surrounded by various proteins which package it into a compressed structure known as chromatin. Cells can control which genes are turned on or off by modifying how tightly packed parts of the genetic code are within chromatin. Changes in chromatin accessibility, also known as 'epigenetic' changes, are a normal part of development, and guide cells towards specific jobs or identities as an organ matures. However, how this happens in the developing lung is poorly understood. Here, Khattar, Fernandes et al. set out to determine how chromatin accessibility shapes development of the tissue lining the lungs, focusing on a group of progenitor cells which produce the protein SOX9. These cells are initially found at the tips of the early lung, where they go on to develop into the cells that line the whole of the mature organ. Initial experiments used large-scale genetic techniques to measure gene activity and chromatin accessibility simultaneously in progenitor cells extracted from the lungs of mice. Khattar, Fernandes et al. were then able to predict the signaling pathways that shape the lung lining based on which genes were surrounded by unpacked chromatin, and determine the proteins responsible for these epigenetic changes. This included the signaling pathway Phosphatidylinositol 3 kinase (PI3K) which is involved in a number of cellular processes. Additional experiments in mice confirmed that the PI3K pathway became active very early in lung development and remained so until adulthood. In contrast, mice lacking a gene that codes for a key part of the PI3K pathway had defective lungs which failed to develop a proper lining. The data generated in this study will provide an important resource for future studies investigating how epigenetic changes drive normal lung development. Khattar, Fernandes et al. hope that this knowledge will help researchers to better understand the cause of human lung diseases, and identify already available 'epigenetic drugs' which could be repurposed to treat them.

BMP4 and Wnt signaling interact to promote mouse tracheal mesenchyme morphogenesis.

Tracheobronchomalacia and complete tracheal rings are congenital malformations of the trachea associated with morbidity and mortality for which the etiology remains poorly understood. Epithelial expression of Wls (a cargo receptor mediating Wnt ligand secretion) by tracheal cells is essential for patterning the embryonic mouse trachea's cartilage and muscle. RNA sequencing indicated that Wls differentially modulated the expression of BMP signaling molecules. We tested whether BMP signaling, induced by epithelial Wnt ligands, mediates cartilage formation. Deletion of Bmp4 from respiratory tract mesenchyme impaired tracheal cartilage formation that was replaced by ectopic smooth muscle, recapitulating the phenotype observed after epithelial deletion of Wls in the embryonic trachea. Ectopic muscle was caused in part by anomalous differentiation and proliferation of smooth muscle progenitors rather than tracheal cartilage progenitors. Mesenchymal deletion of Bmp4 impaired expression of Wnt/ß-catenin target genes, including targets of WNT signaling: Notum and Axin2. In vitro, recombinant (r)BMP4 rescued the expression of Notum in Bmp4-deficient tracheal mesenchymal cells and induced Notum promoter activity via SMAD1/5. RNA sequencing of Bmp4-deficient tracheas identified genes essential for chondrogenesis and muscle development coregulated by BMP and WNT signaling. During tracheal morphogenesis, WNT signaling induces Bmp4 in mesenchymal progenitors to promote cartilage differentiation and restrict trachealis muscle. In turn, Bmp4 differentially regulates the expression of Wnt/ß-catenin targets to attenuate mesenchymal WNT signaling and to further support chondrogenesis.

Establishing an Endoscopic Chronic Subglottic Stenosis Rabbit Model.

OBJECTIVES/HYPOTHESIS: To develop a reproducible and consistent chronic subglottic stenosis (SGS) in an endoscopic animal model. STUDY DESIGN: Prospective study. METHODS: We conducted a prospective study using New Zealand white rabbits. Chronic SGS was induced endoscopically by Bugbee electrocautery to 50% to 75% of the subglottic area's circumference, followed by 4-hour endotracheal intubation. The rabbit airways were endoscopically assessed and sized with uncuffed endotracheal tubes (ETTs) before the injury, during follow-up, and at the endpoints. There were four endpoints: 2, 4, 6, and 8 weeks post SGS induction. Animals were humanely euthanized for histopathological examination of the subglottic injury site and microscopic measurement of the cricoid lumen. RESULTS: Twenty-two rabbits reached the endpoints, and 18 rabbits developed chronic SGS. ETT size significantly decreased by 0.5 from preinjury to the endpoint in all groups, P < .001. Control median cricoid lumen measurements were 20.48 mm2 , the median cricoid lumen measurement for the 2 weeks endpoint was 14.3 mm2 , 4 weeks 11.69 mm2 , 6 weeks 16.03 mm2 , and 8 weeks endpoint median was 16.33 mm2 . Histopathological examination showed chronic scar tissue and new cartilage formation at the cricoid level, mainly at the posterior subglottic injury site starting from 4 weeks postinjury. Collagen staining revealed substantial amounts of organized collagen and different collagen orientation starting 4 weeks postinjury lasting until 8 weeks postinjury. CONCLUSION: We developed an animal model to study chronic SGS. This model will be utilized to compare different endoscopic treatment interventions in acute SGS versus chronic SGS and further define the molecular basis of SGS. LEVEL OF EVIDENCE: NA Laryngoscope, 132:1909-1915, 2022.

Dysregulated overexpression of Sox9 induces fibroblast activation in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease associated with unremitting fibroblast activation including fibroblast-to-myofibroblast transformation (FMT), migration, resistance to apoptotic clearance, and excessive deposition of extracellular matrix (ECM) proteins in the distal lung parenchyma. Aberrant activation of lung-developmental pathways is associated with severe fibrotic lung disease; however, the mechanisms through which these pathways activate fibroblasts in IPF remain unclear. Sry-box transcription factor 9 (Sox9) is a member of the high-mobility group box family of DNA-binding transcription factors that are selectively expressed by epithelial cell progenitors to modulate branching morphogenesis during lung development. We demonstrate that Sox9 is upregulated via MAPK/PI3K-dependent signaling and by the transcription factor Wilms' tumor 1 in distal lung-resident fibroblasts in IPF. Mechanistically, using fibroblast activation assays, we demonstrate that Sox9 functions as a positive regulator of FMT, migration, survival, and ECM production. Importantly, our in vivo studies demonstrate that fibroblast-specific deletion of Sox9 is sufficient to attenuate collagen deposition and improve lung function during TGF-α-induced pulmonary fibrosis. Using a mouse model of bleomycin-induced pulmonary fibrosis, we show that myofibroblast-specific Sox9 overexpression augments fibroblast activation and pulmonary fibrosis. Thus, Sox9 functions as a profibrotic transcription factor in activating fibroblasts, illustrating the potential utility of targeting Sox9 in IPF treatment.

Mus musculus: biomodelo de expresión de Desyodinasa (DIO3) y Transtiretina (TTR) en el desarrollo placentario / Mus musculus: an experimental model for Deiodinase (DIO3) and Transthyretin (TTR) expression during placental development / Mus musculus: Biomodelo de expressão de Deiodinase (DIO3) e Transtiretina (TTR) no desenvolvimento da placenta

RESUMEN Introducción: El correcto funcionamiento del eje hipotálamo-hipófisis-tiroides es indispensable para el crecimiento y desarrollo embrionario-fetal, al intervenir en la diferenciación de los tejidos, el desarrollo cerebral y somático, la maduración ósea y la regulación del metabolismo. El paso de las hormonas tiroideas maternas al feto a través de la placenta depende de transportadores transmembrana, enzimas desyodinasas (DIO2 y DIO3) y proteínas transportadoras (TTR). Objetivo: Identificar las zonas de expresión de DIO3 y TTR en la placenta de ratón Mus musculus E10.5, E12.5, E14.5. Métodos: La estructura placentaria y expresión de DIO3 y TTR fueron evaluadas con técnicas histoquímicas e inmunofluorescencia. Resultados: Desde E10.5 se encontraron las tres zonas placentarias, laberinto, zona de unión y decidua. En E12.5 se observó la conformación placentaria definitiva. DIO3 y TTR fueron detectadas en los tres estadios, con predominio en la zona del laberinto. Conclusión: DIO3 y TTR se expresan a lo largo del establecimiento y maduración de la placenta de ratón. El biomodelo murino es una herramienta útil para el estudio del transporte placentario de hormonas tiroideas desde la circulación materna a la fetal.

ABSTRACT Introduction: Correct functioning of hypothalamic-pituitary-thyroid axis is essential for embryonic-fetal growth and development, as it is involved in tissue differentiation, brain and somatic development, bone maturation and metabolic regulation. Maternal thyroid hormones passage to the fetus through the placenta depends on transmembrane transporters, deiodinase enzymes (DIO2 and DIO3) and carrier proteins (TTR). Objective: Identify DIO3 and TTR expression within placental layers of Mus musculus E10.5, E12.5 and E14.5. Methods: Placental structure, DIO3 and TTR expression were evaluated using histochemistry and immunofluorescence techniques. Results: We found that the three placental layers, labyrinth zone, junctional zone, and decidua were present since E10.5. At E12.5 placental final conformation was observed. DIO3 and TTR were detected in the three stages with a predominance in the labyrinth. Conclusion: DIO3 and TTR are expressed throughout the establishment and maturation of mouse placenta. Mice are a useful tool for studying how thyroid hormones are transported from maternal t° fetal circulation at the placenta.

RESUMO Introdução: O correto funcionamento do eixo hipotálamo-hipófise-tireoide é essencial para o crescimento e desenvolvimento embrionário-fetal, pois intervém na diferenciação dos tecidos, desenvolvimento cerebral e somático, maturação óssea e regulaçãodo metabolismo. A passagem dos hormônios tireoidianos maternos para o feto através da placenta depende de transportadores transmembranas, enzimas deiodinase (DIO2 e DIO3) e proteínas transportadoras (TTR). Objetivo: Identificar as zonas de expressão de DIO3 e TTR na placenta de rato Mus musculus E10.5, E12.5, E14.5. Métodos: A estrutura placentária e a expressão de DIO3 e TTR foram avaliadas com técnicas histoquímicas e imunofluorescência. Resultados: De E10.5 as três zonas placentárias, labirinto, zona de união e decídua foram encontradas. Em E12.5 a conformação definitiva da placenta foi observada. O DIO3 e o TTR foram detectados nas três fases, com predomínio na área do labirinto. Conclusão: DIO3 e TTR são expressos ao longo do estabelecimento e maturação da placenta de rato O biomodelo murino é uma ferramenta útil para o estudo do transporte placentário dos hormônios tireoidianos da circulação materna para a fetal.

Stearoyl-CoA desaturase 5 (SCD5), a Δ-9 fatty acyl desaturase in search of a function.

A large body of research has demonstrated that human stearoyl-CoA desaturase 1 (SCD1), a universally expressed fatty acid Δ9-desaturase that converts saturated fatty acids (SFA) into monounsaturated fatty acids (MUFA), is a central regulator of metabolic and signaling pathways involved in cell proliferation, differentiation, and survival. Unlike SCD1, stearoyl-CoA desaturase 5 (SCD5), a second SCD isoform found in a variety of vertebrates, including humans, has received considerably less attention but new information on the catalytic properties, regulation and biological functions of this enzyme has begun to emerge. This review will examine the new evidence that supports key metabolic and biological roles for SCD5, as well as the potential implication of this desaturase in the mechanisms of human diseases.

Disruption of a hedgehog-foxf1-rspo2 signaling axis leads to tracheomalacia and a loss of sox9+ tracheal chondrocytes.

Congenital tracheomalacia, resulting from incomplete tracheal cartilage development, is a relatively common birth defect that severely impairs breathing in neonates. Mutations in the Hedgehog (HH) pathway and downstream Gli transcription factors are associated with tracheomalacia in patients and mouse models; however, the underlying molecular mechanisms are unclear. Using multiple HH/Gli mouse mutants including one that mimics Pallister-Hall Syndrome, we show that excessive Gli repressor activity prevents specification of tracheal chondrocytes. Lineage tracing experiments show that Sox9+ chondrocytes arise from HH-responsive splanchnic mesoderm in the fetal foregut that expresses the transcription factor Foxf1. Disrupted HH/Gli signaling results in 1) loss of Foxf1 which in turn is required to support Sox9+ chondrocyte progenitors and 2) a dramatic reduction in Rspo2, a secreted ligand that potentiates Wnt signaling known to be required for chondrogenesis. These results reveal a HH-Foxf1-Rspo2 signaling axis that governs tracheal cartilage development and informs the etiology of tracheomalacia.

Complete Tracheal Ring Deformity. A Translational Genomics Approach to Pathogenesis.

Rationale: Complete tracheal ring deformity (CTRD) is a rare congenital abnormality of unknown etiology characterized by circumferentially continuous or nearly continuous cartilaginous tracheal rings, variable degrees of tracheal stenosis and/or shortening, and/or pulmonary arterial sling anomaly.Objectives: To test the hypothesis that CTRD is caused by inherited or de novo mutations in genes required for normal tracheal development.Methods: CTRD and normal tracheal tissues were examined microscopically to define the tracheal abnormalities present in CTRD. Whole-exome sequencing was performed in children with CTRD and their biological parents ("trio analysis") to identify gene variants in patients with CTRD. Mutations were confirmed by Sanger sequencing, and their potential impact on structure and/or function of encoded proteins was examined using human gene mutation databases. Relevance was further examined by comparison with the effects of targeted deletion of murine homologs important to tracheal development in mice.Measurements and Main Results: The trachealis muscle was absent in all of five patients with CTRD. Exome analysis identified six de novo, three recessive, and multiple compound-heterozygous or rare hemizygous variants in children with CTRD. De novo variants were identified in SHH (Sonic Hedgehog), and inherited variants were identified in HSPG2 (perlecan), ROR2 (receptor tyrosine kinase-like orphan receptor 2), and WLS (Wntless), genes involved in morphogenetic pathways known to mediate tracheoesophageal development in mice.Conclusions: The results of the present study demonstrate that absence of the trachealis muscle is associated with CTRD. Variants predicted to cause disease were identified in genes encoding Hedgehog and Wnt signaling pathway molecules, which are critical to cartilage formation and normal upper airway development in mice.

Fibrillin-2 is a key mediator of smooth muscle extracellular matrix homeostasis during mouse tracheal tubulogenesis.

Epithelial tubes, comprised of polarised epithelial cells around a lumen, are crucial for organ function. However, the molecular mechanisms underlying tube formation remain largely unknown. Here, we report on the function of fibrillin (FBN)2, an extracellular matrix (ECM) glycoprotein, as a critical regulator of tracheal tube formation.We performed a large-scale forward genetic screen in mouse to identify regulators of respiratory organ development and disease. We identified Fbn2 mutants which exhibit shorter and narrowed tracheas as well as defects in tracheal smooth muscle cell alignment and polarity.We found that FBN2 is essential for elastic fibre formation and Fibronectin accumulation around tracheal smooth muscle cells. These processes appear to be regulated at least in part through inhibition of p38-mediated upregulation of matrix metalloproteinases (MMPs), as pharmacological decrease of p38 phosphorylation or MMP activity partially attenuated the Fbn2 mutant tracheal phenotypes. Analysis of human tracheal tissues indicates that a decrease in ECM proteins, including FBN2 and Fibronectin, is associated with tracheomalacia.Our findings provide novel insights into the role of ECM homeostasis in mesenchymal cell polarisation during tracheal tubulogenesis.

Wilms' tumor 1 drives fibroproliferation and myofibroblast transformation in severe fibrotic lung disease.

Wilms' tumor 1 (WT1) is a critical transcriptional regulator of mesothelial cells during lung development but is downregulated in postnatal stages and adult lungs. We recently showed that WT1 is upregulated in both mesothelial cells and mesenchymal cells in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a fatal fibrotic lung disease. Although WT1-positive cell accumulation leading to severe fibrotic lung disease has been studied, the role of WT1 in fibroblast activation and pulmonary fibrosis remains elusive. Here, we show that WT1 functions as a positive regulator of fibroblast activation, including fibroproliferation, myofibroblast transformation, and extracellular matrix (ECM) production. Chromatin immunoprecipitation experiments indicate that WT1 binds directly to the promoter DNA sequence of α-smooth muscle actin (αSMA) to induce myofibroblast transformation. In support, the genetic lineage tracing identifies WT1 as a key driver of mesothelial-to-myofibroblast and fibroblast-to-myofibroblast transformation. Importantly, the partial loss of WT1 was sufficient to attenuate myofibroblast accumulation and pulmonary fibrosis in vivo. Further, our coculture studies show that WT1 upregulation leads to non-cell autonomous effects on neighboring cells. Thus, our data uncovered a pathogenic role of WT1 in IPF by promoting fibroblast activation in the peripheral areas of the lung and as a target for therapeutic intervention.

Notum attenuates Wnt/ß-catenin signaling to promote tracheal cartilage patterning.

Tracheobronchomalacia (TBM) is a common congenital disorder in which the cartilaginous rings of the trachea are weakened or missing. Despite the high prevalence and clinical issues associated with TBM, the etiology is largely unknown. Our previous studies demonstrated that Wntless (Wls) and its associated Wnt pathways are critical for patterning of the upper airways. Deletion of Wls in respiratory endoderm caused TBM and ectopic trachealis muscle. To understand mechanisms by which Wls mediates tracheal patterning, we performed RNA sequencing in prechondrogenic tracheal tissue of Wlsf/f;ShhCre/wt embryos. Chondrogenic Bmp4, and Sox9 were decreased, while expression of myogenic genes was increased. We identified Notum, a deacylase that inactivates Wnt ligands, as a target of Wls induced Wnt signaling. Notum's mesenchymal ventral expression in prechondrogenic trachea overlaps with expression of Axin2, a Wnt/ß-catenin target and inhibitor. Notum is induced by Wnt/ß-catenin in developing trachea. Deletion of Notum activated mesenchymal Wnt/ß-catenin and caused tracheal mispatterning of trachealis muscle and cartilage as well as tracheal stenosis. Notum is required for tracheal morphogenesis, influencing mesenchymal condensations critical for patterning of tracheal cartilage and muscle. We propose that Notum influences mesenchymal cell differentiation by generating a barrier for Wnt ligands produced and secreted by airway epithelial cells to attenuate Wnt signaling.

Hsp90 regulation of fibroblast activation in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a severe fibrotic lung disease associated with fibroblast activation that includes excessive proliferation, tissue invasiveness, myofibroblast transformation, and extracellular matrix (ECM) production. To identify inhibitors that can attenuate fibroblast activation, we queried IPF gene signatures against a library of small-molecule-induced gene-expression profiles and identified Hsp90 inhibitors as potential therapeutic agents that can suppress fibroblast activation in IPF. Although Hsp90 is a molecular chaperone that regulates multiple processes involved in fibroblast activation, it has not been previously proposed as a molecular target in IPF. Here, we found elevated Hsp90 staining in lung biopsies of patients with IPF. Notably, fibroblasts isolated from fibrotic lesions showed heightened Hsp90 ATPase activity compared with normal fibroblasts. 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), a small-molecule inhibitor of Hsp90 ATPase activity, attenuated fibroblast activation and also TGF-ß-driven effects on fibroblast to myofibroblast transformation. The loss of the Hsp90AB, but not the Hsp90AA isoform, resulted in reduced fibroblast proliferation, myofibroblast transformation, and ECM production. Finally, in vivo therapy with 17-AAG attenuated progression of established and ongoing fibrosis in a mouse model of pulmonary fibrosis, suggesting that targeting Hsp90 represents an effective strategy for the treatment of fibrotic lung disease.

Studying Wnt Signaling During Patterning of Conducting Airways.

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis controlled by Wnt signaling is required to understand how tissues are patterned during normal development. This knowledge is also critical to determine the etiology of birth defects such as lung hypoplasia and tracheobronchomalacia. Analysis of earliest stages of development of respiratory tract imposes challenges, as the limited amount of tissue prevents the performance of standard protocols better suited for postnatal studies. In this paper, we discuss methodologies to study cell differentiation and proliferation in the respiratory tract. We describe techniques such as whole mount staining, processing of the tissue for confocal microscopy and immunofluorescence in paraffin sections applied to developing tracheal lung. We also discuss methodologies for the study of tracheal mesenchyme differentiation, in particular cartilage formation. Approaches and techniques discussed in the current paper circumvent the limitation of material while working with embryonic tissue, allowing for a better understanding of the patterning process of developing conducting airways.

Endodermal Wnt signaling is required for tracheal cartilage formation.

Tracheobronchomalacia is a common congenital defect in which the walls of the trachea and bronchi lack of adequate cartilage required for support of the airways. Deletion of Wls, a cargo receptor mediating Wnt ligand secretion, in the embryonic endoderm using ShhCre mice inhibited formation of tracheal-bronchial cartilaginous rings. The normal dorsal-ventral patterning of tracheal mesenchyme was lost. Smooth muscle cells, identified by Acta2 staining, were aberrantly located in ventral mesenchyme of the trachea, normally the region of Sox9 expression in cartilage progenitors. Wnt/ß-catenin activity, indicated by Axin2 LacZ reporter, was decreased in tracheal mesenchyme of Wls(f/f);Shh(Cre/+) embryos. Proliferation of chondroblasts was decreased and reciprocally, proliferation of smooth muscle cells was increased in Wls(f/f);Shh(Cre/+) tracheal tissue. Expression of Tbx4, Tbx5, Msx1 and Msx2, known to mediate cartilage and muscle patterning, were decreased in tracheal mesenchyme of Wls(f/f);Shh(Cre/+) embryos. Ex vivo studies demonstrated that Wnt7b and Wnt5a, expressed by the epithelium of developing trachea, and active Wnt/ß-catenin signaling are required for tracheal chondrogenesis before formation of mesenchymal condensations. In conclusion, Wnt ligands produced by the tracheal epithelium pattern the tracheal mesenchyme via modulation of gene expression and cell proliferation required for proper tracheal cartilage and smooth muscle differentiation.

Mesenchymal Wnt signaling promotes formation of sternum and thoracic body wall.

Midline defects account for approximately 5% of congenital abnormalities observed at birth. However, the molecular mechanisms underlying the formation of the ventral body wall are not well understood. Recent studies linked mutations in Porcupine-an O-acetyl transferase mediating Wnt ligand acylation-with defects in the thoracic body wall. We hypothesized that anomalous Wnt signaling is involved in the pathogenesis of defective closure of the thoracic body wall. We generated a mouse model wherein Wntless (Wls), which encodes a cargo receptor mediating secretion of Wnt ligands, was conditionally deleted from the developing mesenchyme using Dermo1Cre mice. Wls(f/f);Dermo1(Cre/+) embryos died during mid-gestation. At E13.5, skeletal defects were observed in the forelimbs, jaw, and rib cage. At E14.5, midline defects in the thoracic body wall began to emerge: the sternum failed to fuse and the heart protruded through the body wall at the midline (ectopia cordis). To determine the molecular mechanism underlying the phenotype observed in Wls(f/f);Dermo1(Cre/+) embryos, we tested whether Wnt/ß-catenin signaling was operative in developing the embryonic ventral body wall using Axin2(LacZ) and BatGal reporter mice. While Wnt/ß-catenin signaling activity was observed at the midline of the ventral body wall before sternal fusion, this pattern of activity was altered and scattered throughout the body wall after mesenchymal deletion of Wls. Mesenchymal cell migration was disrupted in Wls(f/f);Dermo1(Cre/+) thoracic body wall partially due to anomalous ß-catenin independent Wnt signaling as determined by in vitro assays. Deletion of Lrp5 and Lrp6 receptors, which mediate Wnt/ß-catenin signaling in the mesenchyme, partially recapitulated the phenotype observed in the chest midline of Wls(f/f);Dermo1(Cre/+) embryos supporting a role for Wnt/ß-catenin signaling activity in the normal formation of the ventral body wall mesenchyme. We conclude that Wls-mediated secretion of Wnt ligands from the developing ventral body wall mesenchyme plays a critical role in fusion of the sternum and closure of the secondary body wall. Thus, impaired Wls activity in the ventral body wall mesenchyme is a mechanism underlying ectopia cordis and unfused sternum.