RESUMEN
Members of the Sp family of transcription factors regulate gene expression via binding GC boxes within promoter regions. Unlike Sp1, which stimulates transcription, the closely related Sp3 can either repress or activate gene expression and is required for perinatal survival in mice. Here, we use RNA-seq and cellular phenotyping to show how Sp3 regulates murine fetal cell differentiation and proliferation. Homozygous Sp3-/- mice were smaller than wild-type and Sp+/- littermates, died soon after birth and had abnormal lung morphogenesis. RNA-seq of Sp3-/- fetal lung mesenchymal cells identified alterations in extracellular matrix production, developmental signaling pathways and myofibroblast/lipofibroblast differentiation. The lungs of Sp3-/- mice contained multiple structural defects, with abnormal endothelial cell morphology, lack of elastic fiber formation, and accumulation of lipid droplets within mesenchymal lipofibroblasts. Sp3-/- cells and mice also displayed cell cycle arrest, with accumulation in G0/G1 and reduced expression of numerous cell cycle regulators including Ccne1. These data detail the global impact of Sp3 on in vivo mouse gene expression and development.
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Desarrollo Embrionario , Factores de Transcripción , Animales , Ratones , División Celular , Pulmón , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/metabolismoRESUMEN
A number of inflammatory lung diseases, including chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and pneumonia, are modulated by WNT/ß-catenin signaling. However, the underlying molecular mechanisms remain unclear. Here, starting with a forward genetic screen in mouse, we identify the WNT coreceptor Related to receptor tyrosine kinase (RYK) acting in mesenchymal tissues as a cell survival and antiinflammatory modulator. Ryk mutant mice exhibit lung hypoplasia and inflammation as well as alveolar simplification due to defective secondary septation, and deletion of Ryk specifically in mesenchymal cells also leads to these phenotypes. By analyzing the transcriptome of wild-type and mutant lungs, we observed the up-regulation of proapoptotic and inflammatory genes whose expression can be repressed by WNT/RYK signaling in vitro. Moreover, mesenchymal Ryk deletion at postnatal and adult stages can also lead to lung inflammation, thus indicating a continued role for WNT/RYK signaling in homeostasis. Our results indicate that RYK signaling through ß-catenin and Nuclear Factor kappa B (NF-κB) is part of a safeguard mechanism against mesenchymal cell death, excessive inflammatory cytokine production, and inflammatory cell recruitment and accumulation. Notably, RYK expression is down-regulated in the stromal cells of pneumonitis patient lungs. Altogether, our data reveal that RYK signaling plays critical roles as an antiinflammatory modulator during lung development and homeostasis and provide an animal model to further investigate the etiology of, and therapeutic approaches to, inflammatory lung diseases.
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Neumonía , Proteínas Tirosina Quinasas Receptoras , Vía de Señalización Wnt , beta Catenina , Animales , Humanos , Pulmón/enzimología , Pulmón/crecimiento & desarrollo , Mesodermo/metabolismo , Ratones , FN-kappa B/metabolismo , Neumonía/enzimología , Neumonía/genética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Células del Estroma/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
A cell is a dynamic system in which various processes occur simultaneously. In particular, intra- and intercellular signaling pathway crosstalk has a significant impact on a cell's life cycle, differentiation, proliferation, growth, regeneration, and, consequently, on the normal functioning of an entire organ. Hippo signaling and YAP/TAZ nucleocytoplasmic shuttling play a pivotal role in normal development, homeostasis, and tissue regeneration, particularly in lung cells. Intersignaling communication has a significant impact on the core components of the Hippo pathway and on YAP/TAZ localization. This review describes the crosstalk between Hippo signaling and key lung signaling pathways (WNT, SHH, TGFß, Notch, Rho, and mTOR) using lung cells as an example and highlights the remaining unanswered questions.
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Pulmón , Transducción de Señal , Factores de Transcripción , Humanos , Pulmón/metabolismo , Pulmón/citología , Animales , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Señalizadoras YAP/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Vía de Señalización Hippo , Espacio Intracelular/metabolismoRESUMEN
BACKGROUND: Hox genes encode transcription factors that are important for establishing the body plan. Hoxa5 is a member of the mammalian Hox5 paralogous group that regulates the patterning and morphology of the cervical-thoracic region of the axial skeleton. Hoxa5 also plays crucial functions in lung morphogenesis. RESULTS: We generated a Hoxa5eGFP reporter mouse line using CRISPR technology, allowing real-time visualization of Hoxa5 expression. Hoxa5eGFP recapitulates reported embryonic Hoxa5 mRNA expression patterns. Specifically, Hoxa5eGFP can be visualized in the developing mouse neural tube, somites, lung, diaphragm, foregut, and midgut, among other organs. In the stomach, posteriorly biased Hoxa5eGFP expression correlates with a drastic morphological reduction of the corpus in Hox5 paralogous mutants. Expression of Hoxa5eGFP in the lung continues in all lung fibroblast populations through postnatal and adult stages. CONCLUSIONS: We identified cell types that express Hoxa5 in postnatal and adult mouse lungs, including various fibroblasts and vascular endothelial cells. This reporter line will be a powerful tool for studies of the function of Hoxa5 during mouse development, homeostasis, and disease processes.
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Células Endoteliales , Proteínas de Homeodominio , Ratones , Animales , Células Endoteliales/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/metabolismo , Pulmón , Genes Homeobox , Organogénesis/genética , Fosfoproteínas/genética , Mamíferos/metabolismoRESUMEN
Most lung development occurs in the context of cyclic stretch. Alteration of the mechanical microenvironment is a common feature of many pulmonary diseases, with congenital diaphragmatic hernia (CDH) and fetal tracheal occlusion (FETO, a therapy for CDH) being extreme examples with changes in lung structure, cell differentiation, and function. To address limitations in cell culture and in vivo mechanotransductive models, we developed two mouse lung organoid (mLO) mechanotransductive models using postnatal day 5 (PND5) mouse lung CD326-positive cells and fibroblasts subjected to increased, decreased, and cyclic strain. In the first model, mLOs were exposed to forskolin (FSK) and/or disrupted (DIS) and evaluated at 20 h. mLO cross-sectional area changed by +59%, +24%, and -68% in FSK, control, and DIS mLOs, respectively. FSK-treated organoids had twice as many proliferating cells as other organoids. In the second model, 20 h of 10.25% biaxial cyclic strain increased the mRNAs of lung mesenchymal cell lineages compared with static stretch and no stretch. Cyclic stretch increased TGF-ß and integrin-mediated signaling, with upstream analysis indicating roles for histone deacetylases, microRNAs, and long noncoding RNAs. Cyclic stretch mLOs increased αSMA-positive and αSMA-PDGFRα-double-positive cells compared with no stretch and static stretch mLOs. In this PND5 mLO mechanotransductive model, cell proliferation is increased by static stretch, and cyclic stretch induces mesenchymal gene expression changes important in postnatal lung development.
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Pulmón/patología , Organoides/patología , Estrés Mecánico , Animales , Colforsina/farmacología , Fibroblastos/patología , Regulación de la Expresión Génica/efectos de los fármacos , Mecanotransducción Celular/efectos de los fármacos , Mecanotransducción Celular/genética , Mesodermo/patología , Ratones Endogámicos C57BL , Modelos BiológicosRESUMEN
The TGF-ß signaling pathway plays a pivotal role in controlling organogenesis during fetal development. Although the role of TGF-ß signaling in promoting lung alveolar epithelial growth has been determined, mesenchymal TGF-ß signaling in regulating lung development has not been studied in vivo due to a lack of genetic tools for specifically manipulating gene expression in lung mesenchymal cells. Therefore, the integral roles of TGF-ß signaling in regulating lung development and congenital lung diseases are not completely understood. Using a Tbx4 lung enhancer-driven Tet-On inducible Cre transgenic mouse system, we have developed a mouse model in which lung mesenchyme-specific deletion of TGF-ß receptor 2 gene (Tgfbr2) is achieved. Reduced airway branching accompanied by defective airway smooth muscle growth and later peripheral cystic lesions occurred when lung mesenchymal Tgfbr2 was deleted from embryonic day 13.5 to 15.5, resulting in postnatal death due to respiratory insufficiency. Although cell proliferation in both lung epithelium and mesenchyme was reduced, epithelial differentiation was not significantly affected. Tgfbr2 downstream Smad-independent ERK1/2 may mediate these mesenchymal effects of TGF-ß signaling through the GSK3ß-ß-catenin-Wnt canonical pathway in fetal mouse lung. Our study suggests that Tgfbr2-mediated TGF-ß signaling in prenatal lung mesenchyme is essential for lung development and maturation, and defective TGF-ß signaling in lung mesenchyme may be related to abnormal airway branching morphogenesis and congenital airway cystic lesions.
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Quistes/metabolismo , Enfermedades Pulmonares/patología , Mesodermo/citología , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Animales , Quistes/patología , Células Epiteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Pulmón/metabolismo , Pulmón/patología , Enfermedades Pulmonares/metabolismo , Ratones , Ratones Transgénicos , Morfogénesis/efectos de los fármacos , Morfogénesis/fisiología , Organogénesis/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The lung mesenchyme consists of a widely heterogeneous population of cells that play crucial roles during development and homeostasis after birth. These cells belong to myogenic, adipogenic, chondrogenic, neuronal and other lineages. Yet, no clear hierarchy for these lineages has been established. We have previously generated a novel Fgf10(iCre) knock-in mouse line that allows lineage tracing of Fgf10-positive cells during development and postnatally. Using these mice, we hereby demonstrate the presence of two waves of Fgf10 expression during embryonic lung development: the first wave, comprising Fgf10-positive cells residing in the submesothelial mesenchyme at early pseudoglandular stage (as well as their descendants); and the second wave, comprising Fgf10-positive cells from late pseudoglandular stage (as well as their descendants). Our lineage-tracing data reveal that the first wave contributes to the formation of parabronchial and vascular smooth muscle cells as well as lipofibroblasts at later developmental stages, whereas the second wave does not give rise to smooth muscle cells but to lipofibroblasts as well as an Nkx2.1(-) E-Cad(-) Epcam(+) Pro-Spc(+) lineage that requires further in-depth analysis. During alveologenesis, Fgf10-positive cells give rise to lipofibroblasts rather than alveolar myofibroblasts, and during adult life, a subpopulation of Fgf10-expressing cells represents a pool of resident mesenchymal stromal (stem) cells (MSCs) (Cd45(-) Cd31(-) Sca-1(+)). Taken together, we show for the first time that Fgf10-expressing cells represent a pool of mesenchymal progenitors in the embryonic and postnatal lung. Our findings suggest that Fgf10-positive cells could be useful for developing stem cell-based therapies for treating interstitial lung diseases.
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Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Pulmón/embriología , Pulmón/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Animales , Linaje de la Célula , Movimiento Celular , Femenino , Factor 10 de Crecimiento de Fibroblastos/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Pulmón/crecimiento & desarrollo , Ratones , Ratones Transgénicos , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Embarazo , Alveolos Pulmonares/embriología , Alveolos Pulmonares/crecimiento & desarrollo , Alveolos Pulmonares/metabolismoRESUMEN
Recent studies have shown that mesothelial progenitors contribute to mesenchymal lineages of developing organs. To what extent the overlying mesothelium contributes to lung development remains unknown. To rigorously address this question, we employed Wt1(CreERT2/+) mice for high-fidelity lineage tracing after confirming that Cre recombinase was mesothelial specific and faithfully recapitulated endogenous Wilms' tumor 1 (Wt1) gene expression. We visualized WT1(+) mesothelial cell entry into the lung by live imaging and identified their progenies in subpopulations of bronchial smooth muscle cells, vascular smooth muscle cells and desmin(+) fibroblasts by lineage tagging. Derivation of these lineages was only observed with Cre recombinase activation during early lung development. Using loss-of-function assays in organ cultures, and targeted mesothelial-restricted hedgehog loss-of-function mice, we demonstrated that mesothelial cell movement into the lung requires the direct action of hedgehog signaling. By contrast, hedgehog signaling was not required for fetal mesothelial heart entry. These findings further support a paradigm wherein the mesothelium is a source of progenitors for mesenchymal lineages during organogenesis and indicate that signals controlling mesothelial cell entry are organ specific.
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Linaje de la Célula/fisiología , Feto/citología , Proteínas Hedgehog/metabolismo , Pulmón/embriología , Mesodermo/embriología , Transducción de Señal/fisiología , Animales , Cartilla de ADN/genética , Inmunohistoquímica , Hibridación in Situ , Integrasas/metabolismo , Pulmón/citología , Mesodermo/citología , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Imagen de Lapso de Tiempo , Proteínas WT1/genética , Proteínas WT1/metabolismo , beta-GalactosidasaRESUMEN
Mutations in the T-box transcription factor TBX4 gene have been reported in patients with Ischiocoxopodopatellar syndrome (MIM# 147891) and childhood-onset pulmonary arterial hypertension. Whole exome sequencing of DNA from a 1 day old deceased newborn, with severe diffuse developmental lung disorder exhibiting features of acinar dysplasia, and her unaffected parents identified a de novo TBX4 missense mutation p.E86Q (c.256G>C) in the DNA-binding T-box domain. We propose phenotypic expansion of the TBX4-related clinical disease spectrum to include acinar dysplasia of the lungs. The reported mutation is the first identified genetic variant causative for acinar dysplasia. © 2016 Wiley Periodicals, Inc.
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Estudios de Asociación Genética , Pulmón/anomalías , Mutación , Fenotipo , Proteínas de Dominio T Box/genética , Alelos , Autopsia , Cromosomas Humanos Par 16 , Variaciones en el Número de Copia de ADN , Análisis Mutacional de ADN , Resultado Fatal , Femenino , Genotipo , Heterocigoto , Humanos , Recién Nacido , Cariotipo , Pulmón/patología , Linaje , Radiografía TorácicaRESUMEN
Sonic hedgehog (Shh) is expressed and secreted from the embryonic lung epithelium and acts on the adjacent mesenchymal cells via its receptor Patched (Ptch)/Smoothened (Smo) and transcriptional effectors Gli proteins. Genetic studies showed that the Shh pathway plays critical roles in mouse lung development. However, little is known about microRNAs (miRNAs) downstream of Shh in embryonic lungs. Here we profiled miRNAs in embryonic lung cultures treated with cyclopamine, a specific Smo antagonist or with Smo agonist by next-generation of sequencing. We then performed functional screening to examine whether some of these miRNAs can modulate the induction of Gli-responsive luciferase by Shh treatment. These analyses revealed that expression of miR-326 and its host gene, Arrestin ß1, is selectively enriched in embryonic lung mesenchymal cells and is specifically influenced by Shh activity. Furthermore, functional analyses showed that miR-326 acts as a negative modulator for Shh signaling by directly targeting Smo and Gli2. Together, these findings suggest a novel miR-326-negative feedback loop in regulating the activity of Shh signaling.
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Proteínas Hedgehog/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Pulmón/metabolismo , MicroARNs/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Arrestinas/genética , Arrestinas/metabolismo , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Proteínas Hedgehog/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Factores de Transcripción de Tipo Kruppel/genética , Pulmón/efectos de los fármacos , Pulmón/embriología , Ratones , MicroARNs/genética , Receptores Acoplados a Proteínas G/genética , Transducción de Señal , Receptor Smoothened , Transfección , Alcaloides de Veratrum/farmacología , Proteína Gli2 con Dedos de Zinc , beta-ArrestinasRESUMEN
During all the stages of lung development, the lung mesoderm (or mesenchyme) is closely related to the endoderm, and their cross-regulation promotes, controls, and drives all lung developmental processes. Generation of 3D organoids in vitro, RNA assays, and mitochondrial respiration studies are used to analyze lung development and regeneration to better understand the interactions between epithelium and mesenchyme, as well as for the study of redox alterations and the metabolic status of the cells. Moreover, to avoid using immortalized cell lines in these in vitro approaches, standardized murine neonatal primary lung fibroblast isolation techniques are essential. Here, we present an optimized method to isolate, culture, and freeze primary lung fibroblasts from neonatal lungs. Our current method includes step-by-step instructions accompanied by graphical explanations and critical steps.
RESUMEN
Millions suffer from incurable lung diseases, and the donor lung shortage hampers organ transplants. Generating the whole organ in conjunction with the thymus is a significant milestone for organ transplantation because the thymus is the central organ to educate immune cells. Using lineage-tracing mice and human pluripotent stem cell (PSC)-derived lung-directed differentiation, we revealed that gastrulating Foxa2 lineage contributed to both lung mesenchyme and epithelium formation. Interestingly, Foxa2 lineage-derived cells in the lung mesenchyme progressively increased and occupied more than half of the mesenchyme niche, including endothelial cells, during lung development. Foxa2 promoter-driven, conditional Fgfr2 gene depletion caused the lung and thymus agenesis phenotype in mice. Wild-type donor mouse PSCs injected into their blastocysts rescued this phenotype by complementing the Fgfr2-defective niche in the lung epithelium and mesenchyme and thymic epithelium. Donor cell is shown to replace the entire lung epithelial and robust mesenchymal niche during lung development, efficiently complementing the nearly entire lung niche. Importantly, those mice survived until adulthood with normal lung function. These results suggest that our Foxa2 lineage-based model is unique for the progressive mobilization of donor cells into both epithelial and mesenchymal lung niches and thymus generation, which can provide critical insights into studying lung transplantation post-transplantation shortly.
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Células Endoteliales , Células Madre Pluripotentes , Ratones , Humanos , Animales , Adulto , Células Madre Pluripotentes/metabolismo , Diferenciación Celular , Pulmón , Blastocisto/metabolismo , Factor Nuclear 3-beta del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/metabolismoRESUMEN
The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These "bud tip progenitors" are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2+ mesenchyme show that RSPO2 is a critical niche cue that potentiates WNT signaling in bud tip progenitors to support their maintenance and multipotency.
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Células Madre Mesenquimatosas , Organogénesis , Humanos , Pulmón , Organoides , Vía de Señalización WntRESUMEN
We performed tissue recombination experiments to discover the mesenchymal influences on differentiation of epithelia in chicken digestive organs. Epithelia and mesenchymes were taken from the lung, esophagus, proventriculus, gizzard, small intestine and large intestine of 6-day chicken embryos and recombined in various associations and cultivated in vitro for 6 days. Rather unexpectedly, embryonic chicken pepsinogen (ECPg) gene, a marker of the proventricular epithelium, was induced in the gizzard epithelium, which does not express ECPg in normal development, by the proventricular and lung mesenchymes. In the second half of this study, we investigated the mode of action of mesenchymal cells on ECPg expression in gizzard epithelial cells more precisely using the cell aggregate culture system, in which gizzard epithelial cells were mixed with proventricular, gizzard or lung mesenchymal cells. We found that supporting action of lung mesenchymal cells on ECPg expression was even stronger than that of proventricular mesenchymal cells, and suggest that the action of lung mesenchyme may be due partly to the enhancement of epithelial cell proliferation. According to the results of this study, together with many facts obtained so far, we will discuss a new model for restricted expression of ECPg in the proventricular epithelium in normal development.