RESUMEN
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.
RESUMEN
Visceral organs, such as the lungs, stomach and liver, are derived from the fetal foregut through a series of inductive interactions between the definitive endoderm (DE) and the surrounding splanchnic mesoderm (SM). While DE patterning is fairly well studied, the paracrine signaling controlling SM regionalization and how this is coordinated with epithelial identity is obscure. Here, we use single cell transcriptomics to generate a high-resolution cell state map of the embryonic mouse foregut. This identifies a diversity of SM cell types that develop in close register with the organ-specific epithelium. We infer a spatiotemporal signaling network of endoderm-mesoderm interactions that orchestrate foregut organogenesis. We validate key predictions with mouse genetics, showing the importance of endoderm-derived signals in mesoderm patterning. Finally, leveraging these signaling interactions, we generate different SM subtypes from human pluripotent stem cells (hPSCs), which previously have been elusive. The single cell data can be explored at: https://research.cchmc.org/ZornLab-singlecell .
Asunto(s)
Sistema Digestivo/metabolismo , Endodermo/metabolismo , Redes Reguladoras de Genes , Mesodermo/metabolismo , Organogénesis/genética , Transducción de Señal/genética , Animales , Linaje de la Célula/genética , Sistema Digestivo/citología , Sistema Digestivo/embriología , Endodermo/citología , Endodermo/embriología , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Humanos , Internet , Mesodermo/citología , Mesodermo/embriología , Ratones Endogámicos C57BL , Análisis de la Célula Individual/métodos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Multipotent Nkx2-1-positive lung epithelial primordial progenitors of the foregut endoderm are thought to be the developmental precursors to all adult lung epithelial lineages. However, little is known about the global transcriptomic programs or gene networks that regulate these gateway progenitors in vivo. Here we use bulk RNA-sequencing to describe the unique genetic program of in vivo murine lung primordial progenitors and computationally identify signaling pathways, such as Wnt and Tgf-ß superfamily pathways, that are involved in their cell-fate determination from pre-specified embryonic foregut. We integrate this information in computational models to generate in vitro engineered lung primordial progenitors from mouse pluripotent stem cells, improving the fidelity of the resulting cells through unbiased, easy-to-interpret similarity scores and modulation of cell culture conditions, including substratum elastic modulus and extracellular matrix composition. The methodology proposed here can have wide applicability to the in vitro derivation of bona fide tissue progenitors of all germ layers.
Asunto(s)
Células Epiteliales/citología , Pulmón/citología , Ratones/genética , Células Madre Pluripotentes/citología , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Epiteliales/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Femenino , Estratos Germinativos/embriología , Estratos Germinativos/metabolismo , Pulmón/embriología , Pulmón/metabolismo , Masculino , Ratones/embriología , Ratones/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Madre Pluripotentes/metabolismo , Transducción de Señal , Factor Nuclear Tiroideo 1/genética , Factor Nuclear Tiroideo 1/metabolismo , Transcriptoma , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Organogenesis is regulated by mesenchymal-epithelial signaling events that induce expression of cell-type specific transcription factors critical for cellular proliferation, differentiation and appropriate tissue patterning. While mesenchymal transcription factors play a key role in mesenchymal-epithelial interactions, transcriptional networks in septum transversum and splanchnic mesenchyme remain poorly characterized. Forkhead Box F1 (FOXF1) transcription factor is expressed in mesenchymal cell lineages; however, its role in organogenesis remains uncharacterized due to early embryonic lethality of Foxf1-/- mice. In the present study, we generated mesenchyme-specific Foxf1 knockout mice (Dermo1-Cre Foxf1-/-) and demonstrated that FOXF1 is required for development of respiratory, cardiovascular and gastrointestinal organ systems. Deletion of Foxf1 from mesenchyme caused embryonic lethality in the middle of gestation due to multiple developmental defects in the heart, lung, liver and esophagus. Deletion of Foxf1 inhibited mesenchyme proliferation and delayed branching lung morphogenesis. Gene expression profiling of micro-dissected distal lung mesenchyme and ChIP sequencing of fetal lung tissue identified multiple target genes activated by FOXF1, including Wnt2, Wnt11, Wnt5A and Hoxb7. FOXF1 decreased expression of the Wnt inhibitor Wif1 through direct transcriptional repression. Furthermore, using a global Foxf1 knockout mouse line (Foxf1-/-) we demonstrated that FOXF1-deficiency disrupts the formation of the lung bud in foregut tissue explants. Finally, deletion of Foxf1 from smooth muscle cell lineage (smMHC-Cre Foxf1-/-) caused hyper-extension of esophagus and trachea, loss of tracheal and esophageal muscle, mispatterning of esophageal epithelium and decreased proliferation of smooth muscle cells. Altogether, FOXF1 promotes lung morphogenesis by regulating mesenchymal-epithelial signaling and stimulating cellular proliferation in fetal lung mesenchyme.
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Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Pulmón/embriología , Animales , Proliferación Celular , Factores de Transcripción Forkhead/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Pulmón/citología , Pulmón/metabolismo , Mesodermo/metabolismo , Ratones/embriología , Ratones Endogámicos C57BL , Ratones Noqueados , Organogénesis/fisiología , Factores de Transcripción/metabolismo , Transcriptoma/genéticaRESUMEN
A small number of signaling pathways are used repeatedly during organogenesis, and they can have drastically different effects on the same population of cells depending on the embryonic stage. How cellular competence changes over developmental time is not well understood. Here we used Xenopus, mouse, and human pluripotent stem cells to investigate how the temporal sequence of Wnt, BMP, and retinoic acid (RA) signals regulates endoderm developmental competence and organ induction, focusing on respiratory fate. While Nkx2-1+ lung fate is not induced until late somitogenesis stages, here we show that lung competence is restricted by the gastrula stage as a result of Wnt and BMP-dependent anterior-posterior (A-P) patterning. These early Wnt and BMP signals make posterior endoderm refractory to subsequent RA/Wnt/BMP-dependent lung induction. We further mapped how RA modulates the response to Wnt and BMP in a temporal specific manner. In the gastrula RA promotes posterior identity, however in early somite stages of development RA regulates respiratory versus pharyngeal potential in anterior endoderm and midgut versus hindgut potential in posterior endoderm. Together our data suggest a dynamic and conserved response of vertebrate endoderm during organogenesis, wherein early Wnt/BMP/RA impacts how cells respond to later Wnt/BMP/RA signals, illustrating how reiterative combinatorial signaling can regulate both developmental competence and subsequent fate specification.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Endodermo/embriología , Organogénesis/efectos de los fármacos , Tretinoina/farmacología , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Endodermo/citología , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Organogénesis/fisiología , Somitos/citología , Somitos/embriología , Especificidad de la Especie , Xenopus laevisRESUMEN
The in vitro-directed differentiation of pluripotent stem cells (PSCs) through stimulation of developmental signaling pathways can generate mature somatic cell types for basic laboratory studies or regenerative therapies. However, there has been significant uncertainty regarding a method to separately derive lung versus thyroid epithelial lineages, as these two cell types each originate from Nkx2-1+ foregut progenitors and the minimal pathways claimed to regulate their distinct lineage specification in vivo or in vitro have varied in previous reports. Here, we employ PSCs to identify the key minimal signaling pathways (Wnt+BMP versus BMP+FGF) that regulate distinct lung- versus thyroid-lineage specification, respectively, from foregut endoderm. In contrast to most previous reports, these minimal pathways appear to be evolutionarily conserved between mice and humans, and FGF signaling, although required for thyroid specification, unexpectedly appears to be dispensable for lung specification. Once specified, distinct Nkx2-1+ lung or thyroid progenitor pools can now be independently derived for functional 3D culture maturation, basic developmental studies or future regenerative therapies.
Asunto(s)
Tipificación del Cuerpo , Diferenciación Celular , Pulmón/citología , Pulmón/embriología , Células Madre Pluripotentes/citología , Transducción de Señal , Glándula Tiroides/citología , Animales , Biomarcadores/metabolismo , Tipificación del Cuerpo/genética , Proteínas Morfogenéticas Óseas/metabolismo , Linaje de la Célula , Embrión de Mamíferos/citología , Desarrollo Embrionario , Endodermo/citología , Endodermo/metabolismo , Células Epiteliales/citología , Factores de Crecimiento de Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Reproducibilidad de los Resultados , Esferoides Celulares/citología , Esferoides Celulares/metabolismo , Glándula Tiroides/embriología , Transcriptoma/genética , Proteínas Wnt/metabolismoRESUMEN
After influenza infection, lineage-negative epithelial progenitors (LNEPs) exhibit a binary response to reconstitute epithelial barriers: activating a Notch-dependent ΔNp63/cytokeratin 5 (Krt5) remodelling program or differentiating into alveolar type II cells (AEC2s). Here we show that local lung hypoxia, through hypoxia-inducible factor (HIF1α), drives Notch signalling and Krt5pos basal-like cell expansion. Single-cell transcriptional profiling of human AEC2s from fibrotic lungs revealed a hypoxic subpopulation with activated Notch, suppressed surfactant protein C (SPC), and transdifferentiation toward a Krt5pos basal-like state. Activated murine Krt5pos LNEPs and diseased human AEC2s upregulate strikingly similar core pathways underlying migration and squamous metaplasia. While robust, HIF1α-driven metaplasia is ultimately inferior to AEC2 reconstitution in restoring normal lung function. HIF1α deletion or enhanced Wnt/ß-catenin activity in Sox2pos LNEPs blocks Notch and Krt5 activation, instead promoting rapid AEC2 differentiation and migration and improving the quality of alveolar repair.
Asunto(s)
Linaje de la Célula , Proliferación Celular , Transdiferenciación Celular , Células Epiteliales/metabolismo , Hipoxia/metabolismo , Gripe Humana/metabolismo , Infecciones por Orthomyxoviridae/metabolismo , Oxígeno/metabolismo , Alveolos Pulmonares/metabolismo , Regeneración , Animales , Movimiento Celular , Células Cultivadas , Modelos Animales de Enfermedad , Células Epiteliales/patología , Células Epiteliales/virología , Femenino , Perfilación de la Expresión Génica , Genotipo , Humanos , Hipoxia/genética , Hipoxia/patología , Hipoxia/virología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Gripe Humana/genética , Gripe Humana/patología , Gripe Humana/virología , Queratina-5/genética , Queratina-5/metabolismo , Masculino , Ratones Transgénicos , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , Fenotipo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Alveolos Pulmonares/patología , Alveolos Pulmonares/virología , Receptores Notch/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Análisis de la Célula Individual , Factores de Tiempo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Vía de Señalización WntRESUMEN
Gastric and small intestinal organoids differentiated from human pluripotent stem cells (hPSCs) have revolutionized the study of gastrointestinal development and disease. Distal gut tissues such as cecum and colon, however, have proved considerably more challenging to derive in vitro. Here we report the differentiation of human colonic organoids (HCOs) from hPSCs. We found that BMP signaling is required to establish a posterior SATB2+ domain in developing and postnatal intestinal epithelium. Brief activation of BMP signaling is sufficient to activate a posterior HOX code and direct hPSC-derived gut tube cultures into HCOs. In vitro, HCOs express colonic markers and contained colon-specific cell populations. Following transplantation into mice, HCOs undergo morphogenesis and maturation to form tissue that exhibits molecular, cellular, and morphologic properties of human colon. Together these data show BMP-dependent patterning of human hindgut into HCOs, which will be valuable for studying diseases including colitis and colon cancer.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Colon/metabolismo , Organoides/metabolismo , Células Madre Pluripotentes/metabolismo , Transducción de Señal , Animales , Colon/citología , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos NOD , Organoides/citología , Organoides/trasplante , Células Madre Pluripotentes/citologíaRESUMEN
It has been postulated that during human fetal development, all cells of the lung epithelium derive from embryonic, endodermal, NK2 homeobox 1-expressing (NKX2-1+) precursor cells. However, this hypothesis has not been formally tested owing to an inability to purify or track these progenitors for detailed characterization. Here we have engineered and developmentally differentiated NKX2-1GFP reporter pluripotent stem cells (PSCs) in vitro to generate and isolate human primordial lung progenitors that express NKX2-1 but are initially devoid of differentiated lung lineage markers. After sorting to purity, these primordial lung progenitors exhibited lung epithelial maturation. In the absence of mesenchymal coculture support, this NKX2-1+ population was able to generate epithelial-only spheroids in defined 3D cultures. Alternatively, when recombined with fetal mouse lung mesenchyme, the cells recapitulated epithelial-mesenchymal developing lung interactions. We imaged these progenitors in real time and performed time-series global transcriptomic profiling and single-cell RNA sequencing as they moved through the earliest moments of lung lineage specification. The profiles indicated that evolutionarily conserved, stage-dependent gene signatures of early lung development are expressed in primordial human lung progenitors and revealed a CD47hiCD26lo cell surface phenotype that allows their prospective isolation from untargeted, patient-specific PSCs for further in vitro differentiation and future applications in regenerative medicine.
Asunto(s)
Células Madre Pluripotentes Inducidas/fisiología , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular , Separación Celular , Células Cultivadas , Citometría de Flujo , Regulación Enzimológica de la Expresión Génica , Humanos , Ratones , Factor Nuclear Tiroideo 1 , TranscriptomaRESUMEN
Crosstalk between mesenchymal and epithelial cells influences organogenesis in multiple tissues, such as lung, pancreas, liver, and the nervous system. Lung mesenchyme comprises multiple cell types, however, and precise identification of the mesenchymal cell type(s) that drives early events in lung development remains unknown. Endothelial cells have been shown to be required for some aspects of lung epithelial patterning, lung stem cell differentiation, and regeneration after injury. Furthermore, endothelial cells are involved in early liver and pancreas development. From these observations we hypothesized that endothelial cells might also be required for early specification of the respiratory field and subsequent lung bud initiation. We first blocked VEGF signaling in E8.5 cultured foreguts with small molecule VEGFR inhibitors and found that lung specification and bud formation were unaltered. However, when we examined E9.5 mouse embryos carrying a mutation in the VEGFR Flk-1, which do not develop endothelial cells, we found that respiratory progenitor specification was impeded. Because the E9.5 embryos were substantially smaller than control littermates, suggesting the possibility of developmental delay, we isolated and cultured foreguts from mutant and control embryos on E8.5, when no size differences were apparent. We found that both specification of the respiratory field and lung bud formation occurred in mutant and control explants. These observations were unaffected by the presence or absence of serum. We also observed that hepatic specification and initiation occurred in the absence of endothelial cells, and that expansion of the liver epithelium in culture did not differ between mutant and control explants. Consistent with previously published results, we also found that pancreatic buds were not maintained in cultured foreguts when endothelial cells were absent. Our observations support the conclusion that endothelial cells are not required for early specification of lung progenitors and bud initiation, and that the diminished lung specification seen in E9.5 Flk-/- embryos is likely due to developmental delay resulting from the insufficient delivery of oxygen, nutrients, and other factors in the absence of a vasculature.
Asunto(s)
Células Endoteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Pulmón/metabolismo , Organogénesis/genética , Animales , Movimiento Celular/genética , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Células Endoteliales/citología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Inmunohistoquímica , Hibridación in Situ , Pulmón/citología , Pulmón/embriología , Ratones , Ratones Noqueados , Ratones Transgénicos , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor Nuclear Tiroideo 1 , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Despite the global prevalence of gastric disease, there are few adequate models in which to study the fundus epithelium of the human stomach. We differentiated human pluripotent stem cells (hPSCs) into gastric organoids containing fundic epithelium by first identifying and then recapitulating key events in embryonic fundus development. We found that disruption of Wnt/ß-catenin signalling in mouse embryos led to conversion of fundic to antral epithelium, and that ß-catenin activation in hPSC-derived foregut progenitors promoted the development of human fundic-type gastric organoids (hFGOs). We then used hFGOs to identify temporally distinct roles for multiple signalling pathways in epithelial morphogenesis and differentiation of fundic cell types, including chief cells and functional parietal cells. hFGOs are a powerful model for studying the development of the human fundus and the molecular bases of human gastric physiology and pathophysiology, and also represent a new platform for drug discovery.
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Fundus Gástrico/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , Animales , Tipificación del Cuerpo , Diferenciación Celular , Linaje de la Célula , Descubrimiento de Drogas/métodos , Células Epiteliales/citología , Células Epiteliales/metabolismo , Epitelio/embriología , Epitelio/metabolismo , Femenino , Fundus Gástrico/citología , Fundus Gástrico/embriología , Proteínas de Homeodominio/metabolismo , Humanos , Masculino , Ratones , Organoides/citología , Organoides/embriología , Organoides/metabolismo , Células Parietales Gástricas/citología , Células Parietales Gástricas/metabolismo , Células Madre Pluripotentes/citología , Factores de Transcripción SOXB1/metabolismo , Esferoides Celulares/citología , Esferoides Celulares/metabolismo , Transactivadores/metabolismo , Vía de Señalización Wnt/genética , beta Catenina/agonistasRESUMEN
Organogenesis of the trachea and lungs requires a complex series of mesoderm-endoderm interactions mediated by WNT, BMP, retinoic acid (RA), and hedgehog (Hh), but how these pathways interact in a gene regulatory network is less clear. Using Xenopus embryology, mouse genetics, and human ES cell cultures, we identified a conserved signaling cascade that initiates respiratory lineage specification. We show that RA has multiple roles; first RA pre-patterns the lateral plate mesoderm and then it promotes Hh ligand expression in the foregut endoderm. Hh subsequently signals back to the pre-patterned mesoderm to promote expression of the lung-inducing ligands Wnt2/2b and Bmp4. Finally, RA regulates the competence of the endoderm to activate the Nkx2-1+ respiratory program in response to these mesodermal WNT and BMP signals. These data provide insights into early lung development and a paradigm for how mesenchymal signals are coordinated with epithelial competence during organogenesis.
Asunto(s)
Tipificación del Cuerpo , Endodermo/embriología , Proteínas Hedgehog/metabolismo , Pulmón/embriología , Mesodermo/embriología , Transducción de Señal , Tretinoina/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Embrión de Mamíferos/metabolismo , Embrión no Mamífero/metabolismo , Endodermo/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Intestinos/embriología , Ligandos , Pulmón/metabolismo , Mesodermo/metabolismo , Ratones , Respiración , Células Madre/metabolismo , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
Differentiation of functional thyroid epithelia from pluripotent stem cells (PSCs) holds the potential for application in regenerative medicine. However, progress toward this goal is hampered by incomplete understanding of the signaling pathways needed for directed differentiation without forced overexpression of exogenous transgenes. Here we use mouse PSCs to identify key conserved roles for BMP and FGF signaling in regulating thyroid lineage specification from foregut endoderm in mouse and Xenopus. Thyroid progenitors derived from mouse PSCs can be matured into thyroid follicular organoids that provide functional secretion of thyroid hormones in vivo and rescue hypothyroid mice after transplantation. Moreover, by stimulating the same pathways, we were also able to derive human thyroid progenitors from normal and disease-specific iPSCs generated from patients with hypothyroidism resulting from NKX2-1 haploinsufficiency. Our studies have therefore uncovered the regulatory mechanisms that underlie early thyroid organogenesis and provide a significant step toward cell-based regenerative therapy for hypothyroidism.
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Diferenciación Celular , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/trasplante , Regeneración , Glándula Tiroides/citología , Glándula Tiroides/fisiología , Animales , Línea Celular , Humanos , Ratones , Ratones Transgénicos , XenopusRESUMEN
Pulmonary surfactant, a mixture of proteins and phospholipids, plays an important role in facilitating gas exchange by maintaining alveolar stability. Saturated phosphatidylcholine (SatPC), the major component of surfactant, is synthesized both de novo and by the remodeling of unsaturated phosphatidylcholine (PC) by lyso-PC acyltransferase 1 (LPCAT1). After synthesis in the endoplasmic reticulum, SatPC is routed to lamellar bodies (LBs) for storage prior to secretion. The mechanism by which SatPC is transported to LB is not understood. The specificity of LPCAT1 for lyso-PC as an acyl acceptor suggests that formation of SatPC via LPCAT1 reacylation is a final step in SatPC synthesis prior to transport. We hypothesized that LPCAT1 forms a transient complex with SatPC and specific phospholipid transport protein(s) to initiate trafficking of SatPC from the endoplasmic reticulum to the LB. Herein we have assessed the ability of different StarD proteins to interact with LPCAT1. We found that LPCAT1 interacts with StarD10, that this interaction is direct, and that amino acids 79-271 of LPCAT1 and the steroidogenic acute regulatory protein-related lipid transfer (START) domain of START domain-containing protein 10 (StarD10) are sufficient for this interaction. The role of StarD10 in trafficking of phospholipid to LB was confirmed by the observation that knockdown of StarD10 significantly reduced transport of phospholipid to LB. LPCAT1 also interacted with one isoform of StarD7 but showed no interaction with StarD2/PC transfer protein.
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1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Metabolismo de los Lípidos , Fosfolípidos/biosíntesis , Fosfoproteínas/metabolismo , Alveolos Pulmonares/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferasa/genética , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , Humanos , Ratones , Fosfatidilcolinas/biosíntesis , Fosfatidilcolinas/metabolismo , Fosfolípidos/metabolismo , Fosfoproteínas/genética , Mapas de Interacción de Proteínas/genética , Alveolos Pulmonares/citología , Surfactantes Pulmonares/metabolismoRESUMEN
BACKGROUND: Early lung morphogenesis is driven by tissue interactions. Signals from the lung mesenchyme drive epithelial morphogenesis, but which individual mesenchymal cell types are influencing early epithelial branching and differentiation remains unclear. It has been shown that endothelial cells are involved in epithelial repair and regeneration in the adult lung, and they may also play a role in driving early lung epithelial branching. These data, in combination with evidence that endothelial cells influence early morphogenetic events in the liver and pancreas, led us to hypothesize that endothelial cells are necessary for early lung epithelial branching. RESULTS: We blocked vascular endothelial growth factor (VEGF) signaling in embryonic day (E) 12.5 lung explants with three different VEGF receptor inhibitors (SU5416, Ki8751, and KRN633) and found that in all cases the epithelium was able to branch despite the loss of endothelial cells. Furthermore, we found that distal lung mesenchyme depleted of endothelial cells retained its ability to induce terminal branching when recombined with isolated distal lung epithelium (LgE). Additionally, isolated E12.5 primary mouse lung endothelial cells, or human lung microvascular endothelial cells (HMVEC-L), were not able to induce branching when recombined with LgE. CONCLUSIONS: Our observations support the conclusion that endothelial cells are not required for early lung branching.
Asunto(s)
Células Endoteliales/citología , Células Epiteliales/citología , Epitelio/embriología , Pulmón/embriología , Pulmón/metabolismo , Animales , Diferenciación Celular , Humanos , Pulmón/patología , Mesodermo/metabolismo , Ratones , Morfogénesis , Mutación , Miocitos del Músculo Liso/citología , Pericitos/citología , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Inherited syndromes provide unique opportunities to identify key regulatory mechanisms governing human disease. We previously identified germline loss-of-function DICER1 mutations in a human syndrome defined by the childhood lung neoplasm pleuropulmonary blastoma (PPB), which arises during lung development. DICER1 regulates many biological processes critical in development and disease pathogenesis. Significant challenges in defining the role of DICER1 in human disease are identifying cause-effect relationships and generating manipulatable systems that model the complexity of organ development and disease pathogenesis. Here we report the generation of a murine model for PPB and demonstrate that precise temporal and cell type-specific Dicer1 ablation is necessary and sufficient for the development of cystic lungs that histologically and phenotypically model PPB. Dicer1 ablation in the distal airway epithelium during early stages of lung development resulted in a cystic lung phenotype indistinguishable from PPB, whereas DICER1 function was not required for development of the proximal airway epithelium or during later stages of organogenesis. Mechanistic studies demonstrate that Dicer1 loss results in epithelial cell death, followed by cystic airway dilatation accompanied by epithelial and mesenchymal proliferation. These studies define precise temporal and epithelial cell type-specific DICER1 functions in the developing lung and demonstrate that loss of these DICER1 functions is sufficient for the development of cystic PPB. These results also provide evidence that PPB arise through a novel mechanism of non-cell-autonomous tumour initiation, in which the genetic abnormality initiating the neoplasm does not occur in the cells that ultimately transform, but rather occurs in a benign-appearing epithelial cell component that predisposes underlying mesenchymal cells to malignant transformation.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Mutación de Línea Germinal/genética , Neoplasias Pulmonares/metabolismo , Blastoma Pulmonar/metabolismo , Ribonucleasa III/metabolismo , Animales , ARN Helicasas DEAD-box/genética , Modelos Animales de Enfermedad , Epitelio/metabolismo , Epitelio/patología , Humanos , Neoplasias Pulmonares/patología , Ratones , Blastoma Pulmonar/patología , Ribonucleasa III/genéticaRESUMEN
RATIONALE: Inactivating mutations in the Forkhead Box transcription factor F1 (FOXF1) gene locus are frequently found in patients with alveolar capillary dysplasia with misalignment of pulmonary veins, a lethal congenital disorder, which is characterized by severe abnormalities in the respiratory, cardiovascular, and gastrointestinal systems. In mice, haploinsufficiency of the Foxf1 gene causes alveolar capillary dysplasia and developmental defects in lung, intestinal, and gall bladder morphogenesis. OBJECTIVE: Although FOXF1 is expressed in multiple mesenchyme-derived cell types, cellular origins and molecular mechanisms of developmental abnormalities in FOXF1-deficient mice and patients with alveolar capillary dysplasia with misalignment of pulmonary veins remain uncharacterized because of lack of mouse models with cell-restricted inactivation of the Foxf1 gene. In the present study, the role of FOXF1 in endothelial cells was examined using a conditional knockout approach. METHODS AND RESULTS: A novel mouse line harboring Foxf1-floxed alleles was generated by homologous recombination. Tie2-Cre and Pdgfb-CreER transgenes were used to delete Foxf1 from endothelial cells. FOXF1-deficient embryos exhibited embryonic lethality, growth retardation, polyhydramnios, cardiac ventricular hypoplasia, and vascular abnormalities in the lung, placenta, yolk sac, and retina. Deletion of FOXF1 from endothelial cells reduced endothelial proliferation, increased apoptosis, inhibited vascular endothelial growth factor signaling, and decreased expression of endothelial genes critical for vascular development, including vascular endothelial growth factor receptors Flt1 and Flk1, Pdgfb, Pecam1, CD34, integrin ß3, ephrin B2, Tie2, and the noncoding RNA Fendrr. Chromatin immunoprecipitation assay demonstrated that Flt1, Flk1, Pdgfb, Pecam1, and Tie2 genes are direct transcriptional targets of FOXF1. CONCLUSIONS: FOXF1 is required for the formation of embryonic vasculature by regulating endothelial genes critical for vascular development and vascular endothelial growth factor signaling.
Asunto(s)
Vasos Sanguíneos/metabolismo , Células Endoteliales/metabolismo , Factores de Transcripción Forkhead/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Apoptosis/genética , Secuencia de Bases , Vasos Sanguíneos/embriología , Western Blotting , Línea Celular , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Factores de Transcripción Forkhead/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Inmunohistoquímica , Pulmón/irrigación sanguínea , Pulmón/embriología , Pulmón/metabolismo , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido NucleicoRESUMEN
Hypoxia inducible factor (HIF) 1a, EPAS1 and NEPAS are expressed in the embryonic mouse lung and each isoform exhibits distinct spatiotemporal expression patterns throughout morphogenesis. To further assess the role of the HIF1a isoform in lung epithelial cell differentiation and homeostasis, we created transgenic mice that express a constitutively active isoform of human HIF-1a (HIF-1a three point mutant (TPM)), in a doxycycline-dependent manner. Expression of HIF1a TPM in the developing pulmonary epithelium resulted in lung hypoplasia characterized by defective branching morphogenesis, altered cellular energetics and impaired epithelial maturation, culminating in neonatal lethality at birth from severe respiratory distress. Histological and biochemical analyses revealed expanded glycogen pools in the pulmonary epithelial cells at E18.5, concomitant with decreased pulmonary surfactant, suggesting a delay or an arrest in maturation. Importantly, these defects occurred in the absence of apoptosis or necrosis. In addition, sub-pleural hemorrhaging was evident as early as E14.5 in HIF1a TPM lungs, despite normal patterning of the blood vasculature, consistent with defects in endothelial barrier function. Epithelial expression of HIF1a TPM also resulted in increased VEGFA and VEGFC production, an increase in the number of lymphatic vessels and indirect activation of the multiple Notch pathway components in endothelial precursor cells. Collectively, these data indicate that HIF-1a protein levels in the pulmonary epithelium must be tightly controlled for proper development of the epithelial and mesenchymal compartments.