RESUMO
Research into mechanisms underlying lung injury and subsequent repair responses is currently of paramount importance. There is a paucity of models that bridge the gap between in vitro and in vivo research. Such intermediate models are critical for researchers to decipher the mechanisms that drive repair and to test potential new treatments for lung repair and regeneration. Here we report the establishment of a new tool, the Acid Injury and Repair (AIR) model, that will facilitate studies of lung tissue repair. In this model, injury is applied to a restricted area of a precision-cut lung slice using hydrochloric acid, a clinically relevant driver. The surrounding area remains uninjured, thus mimicking the heterogeneous pattern of injury frequently observed in lung diseases. We show that in response to injury, the percentage of progenitor cells (pro surfactant protein C, proSP-C and TM4SF1 positive) significantly increases in the injured region. Whereas in the uninjured area, the percentage of proSP-C/TM4SF1 cells remains unchanged but proliferating cells (Ki67 positive) increase. These effects are modified in the presence of inhibitors of proliferation (Cytochalasin D) and Wnt secretion (C59) demonstrating that the AIR model is an important new tool for research into lung disease pathogenesis and potential regenerative medicine strategies.
Assuntos
Pneumopatias , Lesão Pulmonar , Humanos , Pulmão , Células-TroncoRESUMO
Recent advances in cell culture models like air-liquid interface culture and ex vivo models such as organoids have advanced studies of lung biology; however, gaps exist between these models and tools that represent the complexity of the three-dimensional environment of the lung. Precision-cut lung slices (PCLS) mimic the in vivo environment and bridge the gap between in vitro and in vivo models. We have established the acid injury and repair (AIR) model where a spatially restricted area of tissue is injured using drops of HCl combined with Pluronic gel. Injury and repair are assessed by immunofluorescence using robust markers, including Ki67 for cell proliferation and prosurfactant protein C for alveolar type 2/progenitor cells. Importantly, the AIR model enables the study of injury and repair in mouse lung tissue without the need for an initial in vivo injury, and the results are highly reproducible. Here, we present detailed protocols for the generation of PCLS and the AIR model. We also describe methods to analyze and quantify injury in AIR-PCLS by immunostaining with established early repair markers and fluorescence imaging. This novel ex vivo model is a versatile tool for studying lung cell biology in acute lung injury and for semi-high-throughput screening of potential therapeutics. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of precision-cut lung slices Basic Protocol 2: The acid injury and repair model Basic Protocol 3: Analysis of AIR-PCLS: Immunostaining and imaging.
Assuntos
Modelos Animais de Doenças , Pneumopatias/terapia , Lesão Pulmonar/terapia , Animais , Técnicas de Cultura de Células , Humanos , Pneumopatias/etiologia , Lesão Pulmonar/etiologia , CamundongosRESUMO
Congenital pulmonary airway malformations (CPAMs) are rare lung abnormalities that result in cyst formation and are associated with respiratory distress in infants and malignant potential in adults. The pathogenesis of CPAMs remains unknown but data suggest disruption of the normal proximo-distal programme of airway branching and differentiation. Here, we demonstrate that adult human CPAM are lined with epithelium that retains SOX-2 and thyroid transcription factor-1 immunohistochemical markers, characteristic of the developing lung. However, RALDH-1, another key marker, is absent. This suggests a more complex aetiology for CPAM than complete focal arrest of lung development and may provide insight to the associated risk of malignancy.
Assuntos
Pulmão/embriologia , Mucosa Respiratória/metabolismo , Anormalidades do Sistema Respiratório/metabolismo , Anormalidades do Sistema Respiratório/patologia , Adulto , Família Aldeído Desidrogenase 1/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Humanos , Técnicas In Vitro , Retinal Desidrogenase/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/metabolismoRESUMO
During development, intestinal epithelia undergo dramatic morphogenesis mediated by mesenchymal signaling to form villi, which are required for efficient nutrient absorption and host defense. Although both smooth-muscle-induced physical forces and mesenchymal cell clustering beneath emerging villi are implicated in epithelial folding, the underlying cellular mechanisms are unclear. Hedgehog (Hh) signaling can mediate both processes. We therefore analyzed its direct targetome and revealed GLI2 transcriptional activation of atypical cadherin and planar cell polarity (PCP) genes. By examining Fat4 and Dchs1 knockout mice, we demonstrate their critical roles in villus formation. Analyses of PCP-mutant mice and genetic interaction studies show that the Fat4-Dchs1 axis acts in parallel to the core-Vangl2 PCP axis to control mesenchymal cell clustering. Moreover, live light-sheet fluorescence microscopy and cultured PDGFRα+ cells reveal a requirement for PCP in their oriented cell migration guided by WNT5A. Therefore, mesenchymal PCP induced by Hh signaling drives cell clustering and subsequent epithelial remodeling.
Assuntos
Caderinas/metabolismo , Polaridade Celular , Proteínas Hedgehog/metabolismo , Mucosa Intestinal/crescimento & desenvolvimento , Células-Tronco Mesenquimais/metabolismo , Microvilosidades/metabolismo , Animais , Caderinas/genética , Diferenciação Celular , Movimento Celular , Células Cultivadas , Feminino , Proteínas Hedgehog/genética , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Morfogênese , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo , Proteína Gli2 com Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/metabolismoRESUMO
Damage to alveoli, the gas-exchanging region of the lungs, is a component of many chronic and acute lung diseases. In addition, insufficient generation of alveoli results in bronchopulmonary dysplasia, a disease of prematurity. Therefore visualising the process of alveolar development (alveologenesis) is critical for our understanding of lung homeostasis and for the development of treatments to repair and regenerate lung tissue. Here we show live alveologenesis, using long-term, time-lapse imaging of precision-cut lung slices. We reveal that during this process, epithelial cells are highly mobile and we identify specific cell behaviours that contribute to alveologenesis: cell clustering, hollowing and cell extension. Using the cytoskeleton inhibitors blebbistatin and cytochalasin D, we show that cell migration is a key driver of alveologenesis. This study reveals important novel information about lung biology and provides a new system in which to manipulate alveologenesis genetically and pharmacologically.
Assuntos
Movimento Celular/fisiologia , Células Epiteliais/fisiologia , Organogênese/fisiologia , Alvéolos Pulmonares/embriologia , Actomiosina/antagonistas & inibidores , Actomiosina/fisiologia , Animais , Animais Recém-Nascidos , Movimento Celular/efeitos dos fármacos , Citocalasina D/farmacologia , Células Epiteliais/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Microscopia Intravital , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Modelos Animais , Organogênese/efeitos dos fármacos , Alvéolos Pulmonares/efeitos dos fármacos , Imagem com Lapso de TempoRESUMO
Alveoli are the gas-exchange units of lung. The process of alveolar development, alveologenesis, is regulated by a complex network of signaling pathways that act on various cell types including alveolar type I and II epithelial cells, fibroblasts and the vascular endothelium. Dysregulated alveologenesis results in bronchopulmonary dysplasia in neonates and in adults, disrupted alveolar regeneration is associated with chronic lung diseases including COPD and pulmonary fibrosis. Therefore, visualizing alveologenesis is critical to understand lung homeostasis and for the development of effective therapies for incurable lung diseases. We have developed a technique to visualize alveologenesis in real-time using a combination of widefield microscopy and image deconvolution of precision-cut lung slices. Here, we describe this live imaging technique in step-by-step detail. This time-lapse imaging technique can be used to capture the dynamics of individual cells within tissue slices over a long time period (up to 16 h), with minimal loss of fluorescence or cell toxicity.
RESUMO
We previously identified dipeptidylpeptidase 10 (DPP10) on chromosome 2 as a human asthma susceptibility gene, through positional cloning. Initial association results were confirmed in many subsequent association studies but the functional role of DPP10 in asthma remains unclear. Using the MRC Harwell N-ethyl-N-nitrosourea (ENU) DNA archive, we identified a point mutation in Dpp10 that caused an amino acid change from valine to aspartic acid in the ß-propeller region of the protein. Mice carrying this point mutation were recovered and a congenic line was established (Dpp10145D ). Macroscopic examination and lung histology revealed no significant differences between wild-type and Dpp10145D/145D mice. However, after house dust mite (HDM) treatment, Dpp10 mutant mice showed significantly increased airway resistance in response to 100â mg/ml methacholine. Total serum IgE levels and bronchoalveolar lavage (BAL) eosinophil counts were significantly higher in homozygotes than in control mice after HDM treatment. DPP10 protein is present in airway epithelial cells and altered expression is observed in both tissue from asthmatic patients and in mice following HDM challenge. Moreover, knockdown of DPP10 in human airway epithelial cells results in altered cytokine responses. These results show that a Dpp10 point mutation leads to increased airway responsiveness following allergen challenge and provide biological evidence to support previous findings from human genetic studies. This article has an associated First Person interview with the first author of the paper.
Assuntos
Asma/enzimologia , Asma/prevenção & controle , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Sequência de Aminoácidos , Animais , Asma/complicações , Asma/patologia , Sequência de Bases , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Etilnitrosoureia , Genótipo , Homozigoto , Humanos , Hipersensibilidade/complicações , Hipersensibilidade/patologia , Inflamação/complicações , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Pulmão/parasitologia , Pulmão/patologia , Camundongos , Camundongos Mutantes , Mutação/genética , Pyroglyphidae , Reprodutibilidade dos TestesRESUMO
Otitis media (OM), inflammation of the middle ear (ME), is a common cause of conductive hearing impairment. Despite the importance of the disease, the aetiology of chronic and recurrent forms of middle ear inflammatory disease remains poorly understood. Studies of the human population suggest that there is a significant genetic component predisposing to the development of chronic OM, although the underlying genes are largely unknown. Using N-ethyl-N-nitrosourea mutagenesis we identified a recessive mouse mutant, edison, that spontaneously develops a conductive hearing loss due to chronic OM. The causal mutation was identified as a missense change, L972P, in the Nischarin (NISCH) gene. edison mice develop a serous or granulocytic effusion, increasingly macrophage and neutrophil rich with age, along with a thickened, inflamed mucoperiosteum. We also identified a second hypomorphic allele, V33A, with only modest increases in auditory thresholds and reduced incidence of OM. NISCH interacts with several proteins, including ITGA5 that is thought to have a role in modulating VEGF-induced angiogenesis and vascularization. We identified a significant genetic interaction between Nisch and Itga5; mice heterozygous for Itga5-null and homozygous for edison mutations display a significantly increased penetrance and severity of chronic OM. In order to understand the pathological mechanisms underlying the OM phenotype, we studied interacting partners to NISCH along with downstream signalling molecules in the middle ear epithelia of edison mouse. Our analysis implicates PAK1 and RAC1, and downstream signalling in LIMK1 and NF-κB pathways in the development of chronic OM.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Quinases Lim/metabolismo , Mutação de Sentido Incorreto , NF-kappa B/metabolismo , Otite Média/genética , Alelos , Animais , Mapeamento Cromossômico , Doença Crônica , Modelos Animais de Doenças , Orelha Média/metabolismo , Etilnitrosoureia/toxicidade , Feminino , Técnicas de Genotipagem , Heterozigoto , Homozigoto , Humanos , Receptores de Imidazolinas , Inflamação/genética , Integrina alfa6/genética , Integrina alfa6/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Quinases Lim/genética , Masculino , Camundongos , Camundongos Knockout , NF-kappa B/genética , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Otite Média/metabolismo , Penetrância , Análise de Sequência de DNA , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Lung diseases impose a huge economic and health burden worldwide. A key aspect of several adult lung diseases, such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD), including emphysema, is aberrant tissue repair, which leads to an accumulation of damage and impaired respiratory function. Currently, there are few effective treatments available for these diseases and their incidence is rising. The planar cell polarity (PCP) pathway is critical for the embryonic development of many organs, including kidney and lung. We have previously shown that perturbation of the PCP pathway impairs tissue morphogenesis, which disrupts the number and shape of epithelial tubes formed within these organs during embryogenesis. However, very little is known about the role of the PCP pathway beyond birth, partly because of the perinatal lethality of many PCP mouse mutant lines. Here, we investigate heterozygous Looptail (Lp) mice, in which a single copy of the core PCP gene, Vangl2, is disrupted. We show that these mice are viable but display severe airspace enlargement and impaired adult lung function. Underlying these defects, we find that Vangl2Lp/+ lungs exhibit altered distribution of actin microfilaments and abnormal regulation of the actin-modifying protein cofilin. In addition, we show that Vangl2Lp/+ lungs exhibit many of the hallmarks of tissue damage, including an altered macrophage population, abnormal elastin deposition and elevated levels of the elastin-modifying enzyme, Mmp12, all of which are observed in emphysema. In vitro, disruption of VANGL2 impairs directed cell migration and reduces the rate of repair following scratch wounding of human alveolar epithelial cells. Moreover, using population data from a birth cohort of young adults, all aged 31, we found evidence of an interactive effect between VANGL2 and smoking on lung function. Finally, we show that PCP genes VANGL2 and SCRIB are significantly downregulated in lung tissue from patients with emphysema. Our data reveal an important novel role for the PCP pathway in adult lung homeostasis and repair and shed new light on the genetic factors which may modify destructive lung diseases such as emphysema.
Assuntos
Envelhecimento/patologia , Polaridade Celular , Homeostase , Pulmão/patologia , Proteínas do Tecido Nervoso/genética , Cicatrização , Células A549 , Citoesqueleto de Actina/metabolismo , Animais , Movimento Celular , Regulação para Baixo/genética , Elastina/metabolismo , Embrião de Mamíferos/patologia , Técnicas de Silenciamento de Genes , Heterozigoto , Humanos , Pulmão/embriologia , Pulmão/fisiopatologia , Macrófagos/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Modelos Biológicos , Mutação/genética , Fenótipo , Polimorfismo Genético , Doença Pulmonar Obstrutiva Crônica/genética , Fumar/efeitos adversos , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND: Molecular pathways that regulate alveolar development and adult repair represent potential therapeutic targets for emphysema. Signalling via retinoic acid (RA), derived from vitamin A, is required for mammalian alveologenesis, and exogenous RA can induce alveolar regeneration in rodents. Little is known about RA signalling in the human lung and its potential role in lung disease. OBJECTIVES: To examine regulation of human alveolar epithelial and endothelial repair by RA, and characterise RA signalling in human emphysema. METHODS: The role of RA signalling in alveolar epithelial repair was investigated with a scratch assay using an alveolar cell line (A549) and primary human alveolar type 2 (AT2) cells from resected lung, and the role in angiogenesis using a tube formation assay with human lung microvascular endothelial cells (HLMVEC). Localisation of RA synthetic (RALDH-1) and degrading (cytochrome P450 subfamily 26 A1 (CYP26A1)) enzymes in human lung was determined by immunofluorescence. Regulation of RA pathway components was investigated in emphysematous and control human lung tissue by quantitative real-time PCR and Western analysis. RESULTS: RA stimulated HLMVEC angiogenesis in vitro; this was partially reproduced with a RAR-α agonist. RA induced mRNA expression of vascular endothelial growth factor A (VEGFA) and VEGFR2. RA did not modulate AT2 repair. CYP26A1 protein was identified in human lung microvasculature, whereas RALDH-1 partially co-localised with vimentin-positive fibroblasts. CYP26A1 mRNA and protein were increased in emphysema. CONCLUSIONS: RA regulates lung microvascular angiogenesis; the endothelium produces CYP26A1 which is increased in emphysema, possibly leading to reduced RA availability. These data highlight a role for RA in maintenance of the human pulmonary microvascular endothelium.
Assuntos
Pulmão/fisiologia , Neovascularização Fisiológica/efeitos dos fármacos , Enfisema Pulmonar/fisiopatologia , Regeneração/fisiologia , Tretinoína/farmacologia , Idoso , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/fisiologia , Linhagem Celular , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Pulmão/metabolismo , Masculino , Pessoa de Meia-Idade , Neovascularização Fisiológica/fisiologia , Alvéolos Pulmonares/patologia , Enfisema Pulmonar/patologia , RNA Mensageiro/genética , Receptores do Ácido Retinoico/metabolismo , Regeneração/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
BACKGROUND: Genome-wide association studies have identified the ORM (yeast)-like protein isoform 3 (ORMDL3) gene locus on human chromosome 17q to be a highly significant risk factor for childhood-onset asthma. OBJECTIVE: We sought to investigate in vivo the functional role of ORMDL3 in disease inception. METHODS: An Ormdl3-deficient mouse was generated and the role of ORMDL3 in the generation of allergic airways disease to the fungal aeroallergen Alternaria alternata was determined. An adeno-associated viral vector was also used to reconstitute ORMDL3 expression in airway epithelial cells of Ormdl3 knockout mice. RESULTS: Ormdl3 knockout mice were found to be protected from developing allergic airways disease and showed a marked decrease in pathophysiology, including lung function and airway eosinophilia induced by Alternaria. Alternaria is a potent inducer of cellular stress and the unfolded protein response, and ORMDL3 was found to play a critical role in driving the activating transcription factor 6-mediated arm of this response through Xbp1 and downstream activation of the endoplasmic reticulum-associated degradation pathway. In addition, ORMDL3 mediated uric acid release, another marker of cellular stress. In the knockout mice, reconstitution of Ormdl3 transcript levels specifically in the bronchial epithelium resulted in reinstatement of susceptibility to fungal allergen-induced allergic airways disease. CONCLUSIONS: This study demonstrates that ORMDL3, an asthma susceptibility gene identified by genome-wide association studies, contributes to key pathways that promote changes in airway physiology during allergic immune responses.
Assuntos
Asma/imunologia , Proteínas de Membrana/imunologia , Alérgenos/imunologia , Alternaria/imunologia , Animais , Asma/fisiopatologia , Modelos Animais de Doenças , Pulmão/imunologia , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucosa Respiratória/imunologia , Ácido Úrico/metabolismoRESUMO
Otitis media with effusion (OME) is the most common cause of hearing loss in children, and tympanostomy (ear tube insertion) to alleviate the condition remains the commonest surgical intervention in children in the developed world. Chronic and recurrent forms of otitis media (OM) are known to have a very substantial genetic component; however, until recently, little was known of the underlying genes involved. The Jeff mouse mutant carries a mutation in the Fbxo11 gene, a member of the F-box family, and develops deafness due to a chronic proliferative OM. We previously reported that Fbxo11 is involved in the regulation of transforming growth factor beta (TGF-ß) signalling by regulating the levels of phospho-Smad2 in the epithelial cells of palatal shelves, eyelids and airways of the lungs. It has been proposed that FBXO11 regulates the cell's response to TGF-ß through the ubiquitination of CDT2. Additional substrates for FBXO11 have been identified, including p53. Here, we have studied both the genetic and biochemical interactions between FBXO11 and p53 in order to better understand the function of FBXO11 in epithelial development and its potential role in OM. In mice, we show that p53 (also known as Tp53) homozygous mutants and double heterozygous mutants (Jf/+ p53/+) exhibit similar epithelial developmental defects to Fbxo11 homozygotes. FBXO11 and p53 interact in the embryonic lung, and mutation in Fbxo11 prevents the interaction with p53. Both p53 and double mutants show raised levels of pSMAD2, recapitulating that seen in Fbxo11 homozygotes. Overall, our results support the conclusion that FBXO11 regulates the TGF-ß pathway in the embryonic lung via cross-talk with p53.
Assuntos
Embrião de Mamíferos/metabolismo , Epistasia Genética , Proteínas F-Box/genética , Pulmão/embriologia , Otite Média/genética , Proteína Supressora de Tumor p53/genética , Animais , Proteínas Culina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Modelos Animais de Doenças , Desenvolvimento Embrionário , Proteínas F-Box/química , Proteínas F-Box/metabolismo , Heterozigoto , Homozigoto , Pulmão/patologia , Camundongos Knockout , Modelos Moleculares , Mutação , Otite Média/embriologia , Otite Média/patologia , Fenótipo , Fosforilação , Inativadores de Plasminogênio/metabolismo , Proteína Smad2/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismoRESUMO
The DNA damage protein and transcription factor Atmin (Asciz) is required for both lung tubulogenesis and ciliogenesis. Like the lungs, kidneys contain a tubular network that is critical for their function and in addition, renal ciliary dysfunction has been implicated in the pathogenesis of cystic kidney disease. Using the Atmin mouse mutant Gasping6 (Gpg6), we investigated kidney development and found it severely disrupted with reduced branching morphogenesis, resulting in fewer epithelial structures being formed. Unexpectedly, transcriptional levels of key cilia associated genes were not altered in Atmin(Gpg6/Gpg6) kidneys. Instead, Gpg6 homozygous kidneys exhibited altered cytoskeletal organization and modulation of Wnt signaling pathway molecules, including ß-catenin and non-canonical Wnt/planar cell polarity (PCP) pathway factors, such as Daam2 and Vangl2. Wnt signaling is important for kidney development and perturbation of Wnt signaling pathways can result in cystic, and other, renal abnormalities. In common with other PCP pathway mutants, Atmin(Gpg6/Gpg6) mice displayed a shortened rostral-caudal axis and mis-oriented cell division. Moreover, intercrosses between Atmin(Gpg6/+) and Vangl2(Lp/+) mice revealed a genetic interaction between Atmin and Vangl2. Thus we show for the first time that Atmin is critical for normal kidney development and we present evidence that mechanistically, Atmin modifies Wnt signaling pathways, specifically placing it as a novel effector molecule in the non-canonical Wnt/PCP pathway. The identification of a novel modulator of Wnt signaling has important implications for understanding the pathobiology of renal disease.
Assuntos
Nefropatias/embriologia , Rim/crescimento & desenvolvimento , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt , Animais , Cílios/genética , Cílios/metabolismo , Citoesqueleto/metabolismo , Embrião de Mamíferos/patologia , Regulação da Expressão Gênica no Desenvolvimento , Rim/patologia , Nefropatias/patologia , Camundongos , Camundongos Transgênicos , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismoRESUMO
During lung development, proper epithelial cell arrangements are critical for the formation of an arborized network of tubes. Each tube requires a lumen, the diameter of which must be tightly regulated to enable optimal lung function. Lung branching and lumen morphogenesis require close epithelial cell-cell contacts that are maintained as a result of adherens junctions, tight junctions and by intact apical-basal (A/B) polarity. However, the molecular mechanisms that maintain epithelial cohesion and lumen diameter in the mammalian lung are unknown. Here we show that Scribble, a protein implicated in planar cell polarity (PCP) signalling, is necessary for normal lung morphogenesis. Lungs of the Scrib mouse mutant Circletail (Crc) are abnormally shaped with fewer airways, and these airways often lack a visible, 'open' lumen. Mechanistically we show that Scrib genetically interacts with the core PCP gene Vangl2 in the developing lung and that the distribution of PCP pathway proteins and Rho mediated cytoskeletal modification is perturbed in Scrib(Crc/Crc) lungs. However A/B polarity, which is disrupted in Drosophila Scrib mutants, is largely unaffected. Notably, we find that Scrib mediates functions not attributed to other PCP proteins in the lung. Specifically, Scrib localises to both adherens and tight junctions of lung epithelia and knockdown of Scrib in lung explants and organotypic cultures leads to reduced cohesion of lung epithelial cells. Live imaging of Scrib knockdown lungs shows that Scrib does not affect bud bifurcation, as previously shown for the PCP protein Celsr1, but is required to maintain epithelial cohesion. To understand the mechanism leading to reduced cell-cell association, we show that Scrib associates with ß-catenin in embryonic lung and the sub-cellular distribution of adherens and tight junction proteins is perturbed in mutant lung epithelia. Our data reveal that Scrib is required for normal lung epithelial organisation and lumen morphogenesis by maintaining cell-cell contacts. Thus we reveal novel and important roles for Scrib in lung development operating via the PCP pathway, and in regulating junctional complexes and cell cohesion.
Assuntos
Comunicação Celular , Células Epiteliais/citologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pulmão/citologia , Pulmão/embriologia , Mamíferos/embriologia , Morfogênese , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/metabolismo , Animais , Comunicação Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Polaridade Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Epitélio/efeitos dos fármacos , Epitélio/embriologia , Epitélio/metabolismo , Técnicas de Silenciamento de Genes , Imageamento Tridimensional , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Modelos Biológicos , Morfogênese/efeitos dos fármacos , Morfolinos/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-2/metabolismo , beta Catenina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The planar cell polarity (PCP) pathway, incorporating non-canonical Wnt signalling, controls embryonic convergent (CE) extension, polarized cell division and ciliary orientation. It also limits diameters of differentiating renal tubules, with mutation of certain components of the pathway causing cystic kidneys. Mutations in mouse Vangl genes encoding core PCP proteins cause neural tube defects (NTDs) and Vangl2 mutations also impair branching of embryonic mouse lung airways. Embryonic metanephric kidneys also undergo branching morphogenesis and Vangl2 is known to be expressed in ureteric bud/collecting duct and metanephric mesenchymal/nephron lineages. These observations led us to investigate metanephroi in Vangl2 mutant mice, Loop-tail (Lp). Although ureteric bud formation is normal in Vangl2(Lp/Lp) embryos, subsequent in vivo and in vitro branching morphogenesis is impaired. Null mutant kidneys are short, consistent with a CE defect. Differentiating glomerular epithelia express several PCP genes (Vangl1/2, Celsr1, Scrib, Mpk1/2 and Fat4) and glomeruli in Vangl2(Lp/Lp) fetuses are smaller and contain less prominent capillary loops than wild-type littermates. Furthermore, Vangl2(Lp/+) kidneys had modest reduction in glomerular numbers postnatally. Vangl2(Lp/Lp) metanephroi contained occasional dilated tubules but no overt cystic phenotype. These data show for the first time that a PCP gene is required for normal morphogenesis of both the ureteric bud and metanephric mesenchyme-derived structures. It has long been recognized that certain individuals with NTDs are born with malformed kidneys, and recent studies have discovered VANGL mutations in some NTD patients. On the basis of our mutant mouse study, we suggest that PCP pathway mutations should be sought when NTD and renal malformation co-exist.
Assuntos
Glomérulos Renais/embriologia , Rim/embriologia , Proteínas do Tecido Nervoso/genética , Organogênese/genética , Animais , Polaridade Celular/genética , Imunofluorescência , Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Rim/metabolismo , Rim/patologia , Glomérulos Renais/patologia , Camundongos , Camundongos Mutantes , Defeitos do Tubo Neural/embriologia , Defeitos do Tubo Neural/genética , Defeitos do Tubo Neural/metabolismo , Reação em Cadeia da Polimerase , Transdução de Sinais , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
BACKGROUND: The planar cell polarity (PCP) signalling pathway is fundamental to a number of key developmental events, including initiation of neural tube closure. Disruption of the PCP pathway causes the severe neural tube defect of craniorachischisis, in which almost the entire brain and spinal cord fails to close. Identification of mouse mutants with craniorachischisis has proven a powerful way of identifying molecules that are components or regulators of the PCP pathway. In addition, identification of an allelic series of mutants, including hypomorphs and neomorphs in addition to complete nulls, can provide novel genetic tools to help elucidate the function of the PCP proteins. RESULTS: We report the identification of a new N-ethyl-N-nitrosourea (ENU)-induced mutant with craniorachischisis, which we have named chuzhoi (chz). We demonstrate that chuzhoi mutant embryos fail to undergo initiation of neural tube closure, and have characteristics consistent with defective convergent extension. These characteristics include a broadened midline and reduced rate of increase of their length-to-width ratio. In addition, we demonstrate disruption in the orientation of outer hair cells in the inner ear, and defects in heart and lung development in chuzhoi mutants. We demonstrate a genetic interaction between chuzhoi mutants and both Vangl2Lp and Celsr1Crsh mutants, strengthening the hypothesis that chuzhoi is involved in regulating the PCP pathway. We demonstrate that chuzhoi maps to Chromosome 17 and carries a splice site mutation in Ptk7. This mutation results in the insertion of three amino acids into the Ptk7 protein and causes disruption of Ptk7 protein expression in chuzhoi mutants. CONCLUSIONS: The chuzhoi mutant provides an additional genetic resource to help investigate the developmental basis of several congenital abnormalities including neural tube, heart and lung defects and their relationship to disruption of PCP. The chuzhoi mutation differentially affects the expression levels of the two Ptk7 protein isoforms and, while some Ptk7 protein can still be detected at the membrane, chuzhoi mutants demonstrate a significant reduction in membrane localization of Ptk7 protein. This mutant provides a useful tool to allow future studies aimed at understanding the molecular function of Ptk7.
Assuntos
Orelha Interna/anormalidades , Cardiopatias Congênitas/patologia , Pulmão/anormalidades , Mutagênese Insercional , Defeitos do Tubo Neural/patologia , Receptores Proteína Tirosina Quinases/genética , Animais , Polaridade Celular , Modelos Animais de Doenças , Orelha Interna/embriologia , Orelha Interna/patologia , Etilnitrosoureia , Cardiopatias Congênitas/genética , Humanos , Pulmão/embriologia , Camundongos , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/genética , Crista Neural/citologia , Defeitos do Tubo Neural/genética , Receptores Acoplados a Proteínas G/análise , Receptores Acoplados a Proteínas G/genética , Transdução de SinaisRESUMO
The lungs are generated by branching morphogenesis as a result of reciprocal signalling interactions between the epithelium and mesenchyme during development. Mutations that disrupt formation of either the correct number or shape of epithelial branches affect lung function. This, in turn, can lead to congenital abnormalities such as cystadenomatoid malformations, pulmonary hypertension or lung hypoplasia. Defects in lung architecture are also associated with adult lung disease, particularly in cases of idiopathic lung fibrosis. Identifying the signalling pathways which drive epithelial tube formation will likely shed light on both congenital and adult lung disease. Here we show that mutations in the planar cell polarity (PCP) genes Celsr1 and Vangl2 lead to disrupted lung development and defects in lung architecture. Lungs from Celsr1(Crsh) and Vangl2(Lp) mouse mutants are small and misshapen with fewer branches, and by late gestation exhibit thickened interstitial mesenchyme and defective saccular formation. We observe a recapitulation of these branching defects following inhibition of Rho kinase, an important downstream effector of the PCP signalling pathway. Moreover, epithelial integrity is disrupted, cytoskeletal remodelling perturbed and mutant endoderm does not branch normally in response to the chemoattractant FGF10. We further show that Celsr1 and Vangl2 proteins are present in restricted spatial domains within lung epithelium. Our data show that the PCP genes Celsr1 and Vangl2 are required for foetal lung development thereby revealing a novel signalling pathway critical for this process that will enhance our understanding of congenital and adult lung diseases and may in future lead to novel therapeutic strategies.
Assuntos
Pulmão/embriologia , Morfogênese/genética , Morfogênese/fisiologia , Proteínas do Tecido Nervoso/genética , Receptores Acoplados a Proteínas G/genética , Mucosa Respiratória/metabolismo , Transdução de Sinais/genética , Animais , Polaridade Celular/genética , Polaridade Celular/fisiologia , Immunoblotting , Imuno-Histoquímica , Camundongos , Modelos Biológicos , Mutação/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Oligonucleotídeos/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Mucosa Respiratória/embriologiaRESUMO
To isolate new zinc finger genes expressed at early stages of peripheral nerve development, we have used PCR to amplify conserved zinc finger sequences. RNA from rat embryonic day 12 and 13 sciatic nerves, a stage when nerves contain Schwann cell precursors, was used to identify several genes not previously described in Schwann cells. One of them, zinc finger protein (ZFP)-57, proved to be the homologue of a mouse gene found in F9 teratocarcinoma cells. Its mRNA expression profile within embryonic and adult normal and transected peripheral nerves, and its distribution in the rest of the nervous system is described. High levels of expression are seen in embryonic nerves and spinal cord. These drop rapidly during the first few weeks after birth, a pattern mirrored in other parts of the nervous system. ZFP-57 localizes to the nucleus of Schwann and other cells. The sequence contains an N-terminal Krüppel-associated box (KRAB) domain and ZFP-57 constructs containing green fluorescent protein reveal that the protein colocalizes with heterochromatin protein 1alpha to centromeric heterochromatin in a characteristic speckled pattern in NIH3T3 cells. The KRAB domain is required for this localization, because constructs lacking it target the protein to the nucleus but not to the centromeric heterochromatin. When fused to a heterologous DNA binding domain, the KRAB domain of ZFP-57 represses transcription, and full-length ZFP-57 represses Schwann cell transcription from myelin basic protein and P(0) promoters in co-transfection assays. Zfp-57 mRNA is up-regulated in Schwann cells in response to leukemia inhibitory factor and fibroblast growth factor 2.