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/β-catenin target genes, including targets of WNTsignaling: Notum, and Axin2. In vitro, rBMP4 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 co-regulated 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/β-catenin targets to attenuate mesenchymal WNT signaling and to further support chondrogenesis.

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Amygdala-Based Altered miRNome and Epigenetic Contribution of miR-128-3p in Conferring Susceptibility to Depression-Like Behavior via Wnt Signaling

The International Journal of Neuropsychopharmacology ◽

10.1093/ijnp/pyz071 ◽

2020 ◽

Vol 23 (3) ◽

pp. 165-177 ◽

Cited By ~ 5

Author(s):

Bhaskar Roy ◽

Michael Dunbar ◽

Juhee Agrawal ◽

Lauren Allen ◽

Yogesh Dwivedi

Keyword(s):

Wnt Signaling ◽

Target Genes ◽

Rna Binding ◽

Statistical Significance ◽

Neuroblastoma Cell ◽

Wnt Signaling Pathway ◽

Target Prediction ◽

In Vivo Model

Abstract Background Recent studies suggest that microRNAs (miRNAs) can participate in depression pathogenesis by altering a host of genes that are critical in corticolimbic functioning. The present study focuses on examining whether alterations in the miRNA network in the amygdala are associated with susceptibility or resiliency to develop depression-like behavior in rats. Methods Amygdala-specific altered miRNA transcriptomics were determined in a rat depression model following next-generation sequencing method. Target prediction analyses (cis- and trans) and qPCR-based assays were performed to decipher the functional role of altered miRNAs. miRNA-specific target interaction was determined using in vitro transfection assay in neuroblastoma cell line. miRNA-specific findings from the rat in vivo model were further replicated in postmortem amygdala of major depressive disorder (MDD) subjects. Results Changes in miRNome identified 17 significantly upregulated and 8 significantly downregulated miRNAs in amygdala of learned helpless (LH) compared with nonlearned helpless rats. Prediction analysis showed that the majority of the upregulated miRNAs had target genes enriched for the Wnt signaling pathway. Among altered miRNAs, upregulated miR-128-3p was identified as a top hit based on statistical significance and magnitude of change in LH rats. Target validation showed significant downregulation of Wnt signaling genes in amygdala of LH rats. A discernable increase in expression of amygdalar miR-128-3p along with significant downregulation of key target genes from Wnt signaling (WNT5B, DVL, and LEF1) was noted in MDD subjects. Overexpression of miR-128-3p in a cellular model lead to a marked decrease in the expression of Dvl1 and Lef1 genes, confirming them as validated targets of miR-128-3p. Additional evidence suggested that the amygdala-specific diminished expression of transcriptional repressor Snai1 could be potentially linked to induced miR-128-2 expression in LH rats. Furthermore, an amygdala-specific posttranscriptional switching mechanism could be active between miR-128-3p and RNA binding protein Arpp21 to gain control over their target genes such as Lef1. Conclusion Our study suggests that in amygdala a specific set of miRNAs may play an important role in depression susceptibility, which could potentially be mediated through Wnt signaling.

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Endotoxin stimulated cytokine production in rat vascular smooth muscle cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.281.2.h661 ◽

2001 ◽

Vol 281 (2) ◽

pp. H661-H668 ◽

Cited By ~ 16

Author(s):

Kristina Detmer ◽

Zhongbiao Wang ◽

Debra Warejcka ◽

Sandra K. Leeper-Woodford ◽

Walter H. Newman

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Target Genes ◽

Interleukin 1 ◽

Muscle Cells ◽

Nuclear Factor Κb ◽

Tnf Α

Because inflammatory processes may promote the development of atherosclerosis, we examined the activation of cytokine genes in rat vascular smooth muscle cells in vitro after treatment with bacterial lipopolysaccharide (LPS). Interleukin-1 (IL-1), IL-6 and tumor necrosis factor-α (TNF-α) mRNA increased in response to LPS. Activation of nuclear factor-κB (NF-κB) presumably results in NF-κB binding to regulatory regions of target genes and activating transcription. We therefore compared the kinetics of NF-κB activation, cytokine message production, and TNF-α secretion. Maximum active NF-κB was found at 30 min after the addition of LPS and decreased thereafter. Increased IL-6 mRNA was detected at 30 min, increased TNF-α mRNA at 60 min, and increased IL-1 mRNA at 120 min. Secretion of TNF-α was dependent on LPS concentration and was first detected 120 min after LPS addition. Aspirin, which has been shown to inhibit NF-κB activation and cytokine secretion in other cell types, did not inhibit NF-κB activation or TNF-α secretion. However, aspirin reduced the amount of both TNF-α and IL-6 mRNA present 30 min after LPS addition by half ( P < 0.05).

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In vivo loads on airway smooth muscle: the role of noncontractile airway structures

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y92-077 ◽

1992 ◽

Vol 70 (4) ◽

pp. 602-606 ◽

Cited By ~ 5

Author(s):

Philip Robinson ◽

Mitsushi Okazawa ◽

Tony Bai ◽

Peter Paré

Keyword(s):

Smooth Muscle ◽

Airway Smooth Muscle ◽

Muscle Activation ◽

Muscle Length ◽

Tracheal Cartilage ◽

Elastic Load ◽

Muscle Shortening ◽

Trachealis Muscle

The degree of airway smooth muscle contraction and shortening that occurs in vivo is modified by many factors, including those that influence the degree of muscle activation, the resting muscle length, and the loads against which the muscle contracts. Canine trachealis muscle will shorten up to 70% of starting length from optimal length in vitro but will only shorten by around 30% in vivo. This limitation of shortening may be a result of the muscle shortening against an elastic load such as could be applied by tracheal cartilage. Limitation of airway smooth muscle shortening in smaller airways may be the result of contraction against an elastic load, such as could be applied by lung parenchymal recoil. Measurement of the elastic loads applied by the tracheal cartilage to the trachealis muscle and by lung parenchymal recoil to smooth muscle of smaller airways were performed in canine preparations. In both experiments the calculated elastic loads applied by the cartilage and the parenchymal recoil explained in part the limitation of maximal active shortening and airway narrowing observed. We conclude that the elastic loads provided by surrounding structures are important in determining the degree of airway smooth muscle shortening and the resultant airway narrowing.Key words: elastic loads, tracheal cartilage, airway smooth muscle shortening.

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The LIM-only protein FHL2 interacts with β-catenin and promotes differentiation of mouse myoblasts

The Journal of Cell Biology ◽

10.1083/jcb.200202075 ◽

2002 ◽

Vol 159 (1) ◽

pp. 113-122 ◽

Cited By ~ 98

Author(s):

Bernd Martin ◽

Richard Schneider ◽

Stefanie Janetzky ◽

Zoe Waibler ◽

Petra Pandur ◽

...

Keyword(s):

Mammalian Cells ◽

Muscle Development ◽

Target Genes ◽

Myogenic Differentiation ◽

Myoblast Differentiation ◽

Lim Domains ◽

Vitro Binding ◽

Specific Proteins

FHL2 is a LIM-domain protein expressed in myoblasts but down-regulated in malignant rhabdomyosarcoma cells, suggesting an important role of FHL2 in muscle development. To investigate the importance of FHL2 during myoblast differentiation, we performed a yeast two-hybrid screen using a cDNA library derived from myoblasts induced for differentiation. We identified β-catenin as a novel interaction partner of FHL2 and confirmed the specificity of association by direct in vitro binding tests and coimmunoprecipitation assays from cell lysates. Deletion analysis of both proteins revealed that the NH2-terminal part of β-catenin is sufficient for binding in yeast, but addition of the first armadillo repeat is necessary for binding FHL2 in mammalian cells, whereas the presence of all four LIM domains of FHL2 is needed for the interaction. Expression of FHL2 counteracts β-catenin–mediated activation of a TCF/LEF-dependent reporter gene in a dose-dependent and muscle cell–specific manner. After injection into Xenopus embryos, FHL2 inhibited the β-catenin–induced axis duplication. C2C12 mouse myoblasts stably expressing FHL2 show increased myogenic differentiation reflected by accelerated myotube formation and expression of muscle-specific proteins. These data imply that FHL2 is a muscle-specific repressor of LEF/TCF target genes and promotes myogenic differentiation by interacting with β-catenin.

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Bidirectional Wnt signaling between endoderm and mesoderm confer tracheal identity in mouse and human

10.1101/758235 ◽

2019 ◽

Cited By ~ 1

Author(s):

Keishi Kishimoto ◽

Kana T. Furukawa ◽

Agustin Luz Madrigal ◽

Akira Yamaoka ◽

Chisa Matsuoka ◽

...

Keyword(s):

Smooth Muscle ◽

Wnt Signaling ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cell ◽

Bmp Signaling ◽

Lateral Plate ◽

Tracheal Tissue ◽

Canonical Wnt ◽

Human And Mouse

AbstractThe periodic cartilage and smooth muscle structures in mammalian trachea are derived from tracheal mesoderm, and tracheal malformations result in serious respiratory defects in neonates. Here we show that canonical Wnt signaling in mesoderm is critical to confer trachea mesenchymal identity in human and mouse. Loss of β-catenin in fetal mouse mesoderm caused loss of Tbx4+ tracheal mesoderm and tracheal cartilage agenesis. The Tbx4 expression relied on endodermal Wnt activity and its downstream Wnt ligand but independent of known Nkx2.1-mediated respiratory development, suggesting that bidirectional Wnt signaling between endoderm and mesoderm promotes trachea development. Repopulating in vivo model, activating Wnt, Bmp signaling in mouse embryonic stem cell (ESC)-derived lateral plate mesoderm (LPM) generated tracheal mesoderm containing chondrocytes and smooth muscle cells. For human ESC-derived LPM, SHH activation was required along with Wnt to generate proper tracheal mesoderm. Together, these findings may contribute to developing applications for human tracheal tissue repair.

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Interaction of interleukin-6 and the BMP pathway in pulmonary smooth muscle

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00197.2006 ◽

2007 ◽

Vol 292 (6) ◽

pp. L1473-L1479 ◽

Cited By ~ 104

Author(s):

Moira Hagen ◽

Karen Fagan ◽

Wolfgang Steudel ◽

Michelle Carr ◽

Kirk Lane ◽

...

Keyword(s):

Smooth Muscle ◽

Transgenic Mice ◽

Bmp Signaling ◽

Dominant Negative ◽

Luciferase Reporter ◽

Second Hit ◽

Protein Receptor ◽

Bmp Pathway

The majority of familial pulmonary arterial hypertension (PAH) cases are caused by mutations in the type 2 bone morphogenetic protein receptor (BMPR2). However, less than one-half of BMPR2 mutation carriers develop PAH, suggesting that the most important function of BMPR2 mutation is to cause susceptibility to a “second hit.” There is substantial evidence from the literature implicating dysregulated inflammation, in particular the cytokine IL-6, in the development of PAH. We thus hypothesized that the BMP pathway regulates IL-6 in pulmonary tissues and conversely that IL-6 regulates the BMP pathway. We tested this in vivo using transgenic mice expressing an inducible dominant negative BMPR2 in smooth muscle, using mice injected with an IL-6-expressing virus, and in vitro using small interfering RNA (siRNA) to BMPR2 in human pulmonary artery smooth muscle cells (PA SMC). Consistent with our hypothesis, we found upregulation of IL-6 in both the transgenic mice and in cultured PA SMC with siRNA to BMPR2; this could be abolished with p38MAPK inhibitors. We also found that IL-6 in vivo caused a twofold increase in expression of the BMP signaling target Id1 and caused increased BMP activity in a luciferase-reporter assay in PA SMC. Thus we have shown both in vitro and in vivo a complete negative feedback loop between IL-6 and BMP, suggesting that an important consequence of BMPR2 mutations may be poor regulation of cytokines and thus vulnerability to an inflammatory second hit.

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Autophagy Is Involved in Mesenchymal Stem Cell Death in Coculture with Chondrocytes

Cartilage ◽

10.1177/1947603520941227 ◽

2020 ◽

pp. 194760352094122

Author(s):

Carlo Alberto Paggi ◽

Amel Dudakovic ◽

Yao Fu ◽

Catalina Galeano Garces ◽

Mario Hevesi ◽

...

Keyword(s):

Cell Death ◽

Rna Sequencing ◽

Human Mscs ◽

Chondrocyte Proliferation ◽

Apoptotic Pathway ◽

Primary Chondrocytes ◽

Cartilage Formation ◽

Cell Death Pathways

Objective Cartilage formation is stimulated in mixtures of chondrocytes and human adipose–derived mesenchymal stromal cells (MSCs) both in vitro and in vivo. During coculture, human MSCs perish. The goal of this study is to elucidate the mechanism by which adipose tissue–derived MSC cell death occurs in the presence of chondrocytes. Methods Human primary chondrocytes were cocultured with human MSCs derived from 3 donors. The cells were cultured in monoculture or coculture (20% chondrocytes and 80% MSCs) in pellets (200,000 cells/pellet) for 7 days in chondrocyte proliferation media in hypoxia (2% O2). RNA sequencing was performed to assess for differences in gene expression between monocultures or coculture. Immune fluorescence assays were performed to determine the presence of caspase-3, LC3B, and P62. Results RNA sequencing revealed significant upregulation of >90 genes in the 3 cocultures when compared with monocultures. STRING analysis showed interconnections between >50 of these genes. Remarkably, 75% of these genes play a role in cell death pathways such as apoptosis and autophagy. Immunofluorescence shows a clear upregulation of the autophagic machinery with no substantial activation of the apoptotic pathway. Conclusion In cocultures of human MSCs with primary chondrocytes, autophagy is involved in the disappearance of MSCs. We propose that this sacrificial cell death may contribute to the trophic effects of MSCs on cartilage formation.

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Transcriptional profiling of bovine muscle-derived satellite cells during differentiation in vitro by high throughput RNA sequencing

Cellular & Molecular Biology Letters ◽

10.1515/cmble-2015-0019 ◽

2015 ◽

Vol 20 (3) ◽

Cited By ~ 12

Author(s):

Hui Li Tong ◽

Hong Yan Yin ◽

Wei Wei Zhang ◽

Qian Hu ◽

Shu Feng Li ◽

...

Keyword(s):

Gene Expression ◽

Rna Sequencing ◽

Gene Expression Profile ◽

Expression Profile ◽

High Throughput ◽

Satellite Cells ◽

Muscle Development ◽

Future Research ◽

Pathway Enrichment Analysis

AbstractIn this study, we utilized high throughput RNA sequencing to obtain a comprehensive gene expression profile of muscle-derived satellite cells (MDSCs) upon induction of differentiation. MDSCs were cultured in vitro and RNA was extracted for sequencing prior to differentiation (MDSC-P), and again during the early and late differentiation (MDSC-D1, and MDSC-D3, respectively) stages. Sequence tags were assembled and analyzed by digital gene expression profile to screen for differentially expressed genes, Gene Ontology annotation, and pathway enrichment analysis. Quantitative real-time PCR was used to confirm the results of RNA sequencing. Our results indicate that certain of genes were changed during skeletal muscle cell development, cell cycle progression, and cell metabolism during differentiation of bovine MDSCs. Furthermore, we identified certain genes that could be used as novel candidates for future research of muscle development. Additionally, the sequencing results indicated that lipid metabolism might be the predominant cellular process that occurs during MDSC differentiation.

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Muscle LIM Proteins Are Associated with Muscle Sarcomeres and Require dMEF2 for Their Expression during DrosophilaMyogenesis

Molecular Biology of the Cell ◽

10.1091/mbc.10.7.2329 ◽

1999 ◽

Vol 10 (7) ◽

pp. 2329-2342 ◽

Cited By ~ 38

Author(s):

Beth E. Stronach ◽

Patricia J. Renfranz ◽

Brenda Lilly ◽

Mary C. Beckerle

Keyword(s):

Muscle Development ◽

Target Genes ◽

Striated Muscle ◽

Myogenic Differentiation ◽

Muscle Differentiation ◽

Epistasis Analysis ◽

Lim Proteins ◽

Muscle Lim Proteins ◽

Genetic Hierarchy

A genetic hierarchy of interactions, involving myogenic regulatory factors of the MyoD and myocyte enhancer-binding 2 (MEF2) families, serves to elaborate and maintain the differentiated muscle phenotype through transcriptional regulation of muscle-specific target genes. Much work suggests that members of the cysteine-rich protein (CRP) family of LIM domain proteins also play a role in muscle differentiation; however, the specific functions of CRPs in this process remain undefined. Previously, we characterized two members of the Drosophila CRP family, the muscle LIM proteins Mlp60A and Mlp84B, which show restricted expression in differentiating muscle lineages. To extend our analysis ofDrosophila Mlps, we characterized the expression of Mlps in mutant backgrounds that disrupt specific aspects of muscle development. We show a genetic requirement for the transcription factor dMEF2 in regulating Mlp expression and an ability of dMEF2 to bind, in vitro, to consensus MEF2 sites derived from those present inMlp genomic sequences. These data suggest that theMlp genes may be direct targets of dMEF2 within the genetic hierarchy controlling muscle differentiation. Mutations that disrupt myoblast fusion fail to affect Mlp expression. In later stages of myogenic differentiation, which are dedicated primarily to assembly of the contractile apparatus, we analyzed the subcellular distribution of Mlp84B in detail. Immunofluorescent studies revealed the localization of Mlp84B to muscle attachment sites and the periphery of Z-bands of striated muscle. Analysis of mutations that affect expression of integrins and α-actinin, key components of these structures, also failed to perturb Mlp84B distribution. In conclusion, we have used molecular epistasis analysis to position Mlp function downstream of events involving mesoderm specification and patterning and concomitant with terminal muscle differentiation. Furthermore, our results are consistent with a structural role for Mlps as components of muscle cytoarchitecture.

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Patterning the embryonic pulmonary mesenchyme

10.1101/2020.08.20.259101 ◽

2020 ◽

Author(s):

Katharine Goodwin ◽

Jacob M. Jaslove ◽

Hirotaka Tao ◽

Min Zhu ◽

Sevan Hopyan ◽

...

Keyword(s):

Smooth Muscle ◽

Wnt Signaling ◽

Single Cell ◽

Airway Smooth Muscle ◽

Branching Morphogenesis ◽

Muscle Differentiation ◽

Embryonic Mouse ◽

Mesenchymal Progenitors ◽

Mammalian Lung ◽

Smooth Muscle Differentiation

AbstractSmooth muscle guides morphogenesis of epithelia during development of several organs, including the mammalian lung. However, it remains unclear how airway smooth-muscle differentiation is spatiotemporally patterned and whether it originates from distinct mesenchymal progenitors. Using single-cell RNA-sequencing of embryonic mouse lungs, we show that the pulmonary mesenchyme contains a continuum of cell identities, but no distinct progenitors. Transcriptional variability correlates with sub-epithelial and sub-mesothelial mesenchymal compartments that are regulated by Wnt signaling. Live-imaging and tension sensors reveal patterned migratory behaviors and cortical forces in each compartment, and show that sub-epithelial mesenchyme gives rise to airway smooth muscle. Differentiation trajectory reconstruction reveals that cytoskeleton, adhesion, and Wnt signaling pathways are activated early in differentiation. Finally, we show that Wnt activation stimulates the earliest stages of differentiation and induces local accumulation of mesenchymal F-actin, which influences epithelial morphology. Our work provides the first single-cell view of pulmonary mesenchymal patterning during branching morphogenesis.

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