RESUMO
Smooth muscle cells (SMCs) are a crucial component of the mesenchymal wall of the ureter, as they account for the efficient removal of the urine from the renal pelvis to the bladder by means of their contractile activity. Here, we show that the zinc-finger transcription factor gene Gata6 is expressed in mesenchymal precursors of ureteric SMCs under the control of BMP4 signaling. Mice with a conditional loss of Gata6 in these precursors exhibit a delayed onset and reduced level of SMC differentiation and peristaltic activity, as well as dilatation of the ureter and renal pelvis (hydroureternephrosis) at birth and at postnatal stages. Molecular profiling revealed a delayed and reduced expression of the myogenic driver gene Myocd, but the activation of signaling pathways and transcription factors previously implicated in activation of the visceral SMC program in the ureter was unchanged. Additional gain-of-function experiments suggest that GATA6 cooperates with FOXF1 in Myocd activation and SMC differentiation, possibly as pioneer and lineage-determining factors, respectively.
Assuntos
Ureter , Animais , Diferenciação Celular/genética , Camundongos , Desenvolvimento Muscular , Músculo Liso , Miócitos de Músculo Liso/fisiologia , Ureter/metabolismoRESUMO
The TBX18 transcription factor regulates patterning and differentiation programs in the primordia of many organs yet the molecular complexes in which TBX18 resides to exert its crucial transcriptional function in these embryonic contexts have remained elusive. Here, we used 293 and A549 cells as an accessible cell source to search for endogenous protein interaction partners of TBX18 by an unbiased proteomic approach. We tagged endogenous TBX18 by CRISPR/Cas9 targeted genome editing with a triple FLAG peptide, and identified by anti-FLAG affinity purification and subsequent LC-MS analysis the ZMYM2 protein to be statistically enriched together with TBX18 in both 293 and A549 nuclear extracts. Using a variety of assays, we confirmed the binding of TBX18 to ZMYM2, a component of the CoREST transcriptional corepressor complex. Tbx18 is coexpressed with Zmym2 in the mesenchymal compartment of the developing ureter of the mouse, and mutations in TBX18 and in ZMYM2 were recently linked to congenital anomalies in the kidney and urinary tract (CAKUT) in line with a possible in vivo relevance of TBX18-ZMYM2 protein interaction in ureter development.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteômica/métodos , Transdução de Sinais/genética , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/metabolismo , Células A549 , Animais , Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Introdução de Genes , Humanos , Camundongos , Mutação , Gravidez , Ligação Proteica , Proteínas com Domínio T/genética , Fatores de Transcrição/genética , Transfecção , Ureter/embriologia , Ureter/metabolismo , Anormalidades Urogenitais/genética , Anormalidades Urogenitais/metabolismo , Refluxo Vesicoureteral/genética , Refluxo Vesicoureteral/metabolismoRESUMO
The differentiated cell types of the epithelial and mesenchymal tissue compartments of the mature ureter of the mouse arise in a precise temporal and spatial sequence from uncommitted precursor cells of the distal ureteric bud epithelium and its surrounding mesenchyme. Previous genetic efforts identified a member of the Hedgehog (HH) family of secreted proteins, Sonic hedgehog (SHH) as a crucial epithelial signal for growth and differentiation of the ureteric mesenchyme. Here, we used conditional loss- and gain-of-function experiments of the unique HH signal transducer Smoothened (SMO) to further characterize the cellular functions and unravel the effector genes of HH signaling in ureter development. We showed that HH signaling is not only required for proliferation and SMC differentiation of cells of the inner mesenchymal region but also for survival of cells of the outer mesenchymal region, and for epithelial proliferation and differentiation. We identified the Forkhead transcription factor gene Foxf1 as a target of HH signaling in the ureteric mesenchyme. Expression of a repressor version of FOXF1 in this tissue completely recapitulated the mesenchymal and epithelial proliferation and differentiation defects associated with loss of HH signaling while re-expression of a wildtype version of FOXF1 in the inner mesenchymal layer restored these cellular programs when HH signaling was inhibited. We further showed that expression of Bmp4 in the ureteric mesenchyme depends on HH signaling and Foxf1, and that exogenous BMP4 rescued cell proliferation and epithelial differentiation in ureters with abrogated HH signaling or FOXF1 function. We conclude that SHH uses a FOXF1-BMP4 module to coordinate the cellular programs for ureter elongation and differentiation, and suggest that deregulation of this signaling axis occurs in human congenital anomalies of the kidney and urinary tract (CAKUT).
Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/metabolismo , Ureter/embriologia , Animais , Proteína Morfogenética Óssea 4/genética , Diferenciação Celular , Proliferação de Células , Modelos Animais de Doenças , Epitélio/embriologia , Epitélio/metabolismo , Feminino , Fatores de Transcrição Forkhead/genética , Proteínas Hedgehog/genética , Processamento de Imagem Assistida por Computador , Masculino , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Análise em Microsséries , Organogênese/genética , Reprodutibilidade dos Testes , Transdução de Sinais , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Ureter/metabolismoRESUMO
Vertebrate organ development relies on the precise spatiotemporal orchestration of proliferation rates and differentiation patterns in adjacent tissue compartments. The underlying integration of patterning and cell cycle control during organogenesis is insufficiently understood. Here, we have investigated the function of the patterning T-box transcription factor gene Tbx2 in lung development. We show that lungs of Tbx2-deficient mice are markedly hypoplastic and exhibit reduced branching morphogenesis. Mesenchymal proliferation was severely decreased, while mesenchymal differentiation into fibrocytes was prematurely induced. In the epithelial compartment, proliferation was reduced and differentiation of alveolar epithelial cells type 1 was compromised. Prior to the observed cellular changes, canonical Wnt signaling was downregulated, and Cdkn1a (p21) and Cdkn1b (p27) (two members of the Cip/Kip family of cell cycle inhibitors) were strongly induced in the Tbx2-deficient lung mesenchyme. Deletion of both Cdkn1a and Cdkn1b rescued, to a large degree, the growth deficits of Tbx2-deficient lungs. Prolongation of Tbx2 expression into adulthood led to hyperproliferation and maintenance of mesenchymal progenitor cells, with branching morphogenesis remaining unaffected. Expression of Cdkn1a and Cdkn1b was ablated from the lung mesenchyme in this gain-of-function setting. We further show by ChIP experiments that Tbx2 directly binds to Cdkn1a and Cdkn1b loci in vivo, defining these two genes as direct targets of Tbx2 repressive activity in the lung mesenchyme. We conclude that Tbx2-mediated regulation of Cdkn1a and Cdkn1b represents a crucial node in the network integrating patterning information and cell cycle regulation that underlies growth, differentiation, and branching morphogenesis of this organ.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Pulmão , Proteínas com Domínio T , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Pulmão/crescimento & desenvolvimento , Pulmão/metabolismo , Mesoderma , Camundongos , Morfogênese , Transdução de Sinais , Proteínas com Domínio T/deficiência , Proteínas com Domínio T/genéticaRESUMO
Vertebrate limb outgrowth is driven by a positive feedback loop that involves Sonic hedgehog (Shh) and Gremlin1 (Grem1) in the posterior limb bud mesenchyme and Fibroblast growth factors (Fgfs) in the overlying epithelium. Proper spatio-temporal control of these signaling activities is required to avoid limb malformations such as polydactyly. Here we show that, in Tbx2-deficient hindlimbs, Shh/Fgf4 signaling is prolonged, resulting in increased limb bud size and duplication of digit 4. In turn, limb-specific Tbx2 overexpression leads to premature termination of this signaling loop with smaller limbs and reduced digit number as phenotypic manifestation. We show that Tbx2 directly represses Grem1 in distal regions of the posterior limb mesenchyme allowing Bone morphogenetic protein (Bmp) signaling to abrogate Fgf4/9/17 expression in the overlying epithelium. Since Tbx2 itself is a target of Bmp signaling, our data identify a growth-inhibiting positive feedback loop (Bmp/Tbx2/Grem1). We propose that proliferative expansion of Tbx2-expressing cells mediates self-termination of limb bud outgrowth due to their refractoriness to Grem1 induction.
Assuntos
Fatores de Crescimento de Fibroblastos/genética , Proteínas Hedgehog/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Botões de Extremidades/crescimento & desenvolvimento , Proteínas com Domínio T/genética , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Citocinas , Epitélio/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Botões de Extremidades/metabolismo , Mesoderma/crescimento & desenvolvimento , Mesoderma/metabolismo , Camundongos , Fenótipo , Transdução de Sinais , Proteínas com Domínio T/metabolismoRESUMO
BACKGROUND & AIMS: Current hepatic differentiation protocols for human embryonic stem cells (ESCs) require substantial improvements. MicroRNAs (miRNAs) have been reported to regulate hepatocyte cell fate during liver development, but their utility to improve hepatocyte differentiation from ESCs remains to be investigated. Therefore, our aim was to identify and to analyse hepatogenic miRNAs for their potential to improve hepatocyte differentiation from ESCs. METHODS: By miRNA profiling and in vitro screening, we identified miR-199a-5p among several potential hepatogenic miRNAs. Transplantation studies of miR-199a-5p-inhibited hepatocyte-like cells (HLCs) in the liver of immunodeficient fumarylacetoacetate hydrolase knockout mice (Fah(-/-)/Rag2(-/-)/Il2rg(-/-)) were performed to assess their in vivo liver repopulation potential. For target determination, western blot and luciferase reporter assay were carried out. RESULTS: miRNA profiling revealed 20 conserved candidate hepatogenic miRNAs. By miRNA screening, only miR-199a-5p inhibition in HLCs was found to be able to enhance the in vitro hepatic differentiation of mouse as well as human ESCs. miR-199a-5p inhibition in human ESCs-derived HLCs enhanced their engraftment and repopulation capacity in the liver of Fah(-/-)/Rag2(-/-)/Il2rg(-/-) mice. Furthermore, we identified SMARCA4 and MST1 as novel targets of miR-199a-5p that may contribute to the improved hepatocyte generation and in vivo liver repopulation. CONCLUSIONS: Our findings demonstrate that miR-199a-5p inhibition in ES-derived HLCs leads to improved hepatocyte differentiation. Upon transplantation, HLCs were able to engraft and repopulate the liver of Fah(-/-)/Rag2(-/-)/Il2rg(-/-) mice. Thus, our findings suggest that miRNA modulation may serve as a promising approach to generate more mature HLCs from stem cell sources for the treatment of liver diseases.
Assuntos
Regulação da Expressão Gênica , Hepatócitos/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Transplante de Fígado , MicroRNAs/genética , RNA/genética , Animais , Western Blotting , Diferenciação Celular , Células Cultivadas , Hepatócitos/citologia , Células-Tronco Embrionárias Humanas/citologia , Humanos , Camundongos , Camundongos Knockout , MicroRNAs/antagonistas & inibidores , MicroRNAs/biossíntese , Reação em Cadeia da Polimerase em Tempo RealRESUMO
RATIONALE: The embryonic epicardium plays a crucial role in the formation of the coronary vasculature and in myocardial development, yet the exact contribution of epicardium-derived cells (EPDCs) to the vascular and connective tissue of the heart, and the factors that regulate epicardial differentiation, are insufficiently understood. OBJECTIVE: To define the role of Notch signaling in murine epicardial development. METHODS AND RESULTS: Using in situ hybridization and RT-PCR analyses, we detected expression of a number of Notch receptor and ligand genes in early epicardial development, as well as during formation of coronary arteries. Mice with epicardial deletion of Rbpj, the unique intracellular mediator of Notch signaling, survived to adulthood and exhibited enlarged coronary venous and arterial beds. Using a Tbx18-based genetic lineage tracing system, we show that EPDCs give rise to fibroblasts and coronary smooth muscle cells (SMCs) but not to endothelial cells in the wild type, whereas in Rbpj-deficient embryos EPDCs form and surround the developing arteries but fail to differentiate into SMCs. Conditional activation of Notch signaling results in premature SMC differentiation of epicardial cells and prevents coronary angiogenesis. We further show that Notch signaling regulates, and cooperates with transforming growth factor ß signaling in SM differentiation of EPDCs. CONCLUSIONS: Notch signaling is a crucial regulator of SM differentiation of EPDCs, and thus, of formation of a functional coronary system.
Assuntos
Diferenciação Celular/fisiologia , Miócitos de Músculo Liso/citologia , Pericárdio/citologia , Receptores Notch/fisiologia , Transdução de Sinais/fisiologia , Animais , Aterosclerose/fisiopatologia , Células Cultivadas , Vasos Coronários/citologia , Vasos Coronários/fisiopatologia , Feminino , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/deficiência , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/fisiologia , Masculino , Camundongos , Camundongos Knockout , Modelos Animais , Pericárdio/embriologia , Pericárdio/fisiologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/fisiologia , Receptores Notch/genética , Fator de Crescimento Transformador beta/fisiologiaRESUMO
Formation of the mammalian secondary palate is a highly regulated and complex process. Impairment of the underlying cellular and molecular programs often results in cleft palate, a common birth defect in mammals. Here we report that Tbx2 and Tbx3, two closely related genes encoding T-box transcription factors, are expressed in the mesenchyme of the mouse palatal structures during development. Mice homozygous mutant for Tbx2 and mice double heterozygous for Tbx2 and Tbx3 exhibit a cleft palate phenotype arguing for an important contribution of Tbx2 and Tbx3 to palatogenesis. In Tbx2-deficient embryos, the bilateral primordial palatal shelves form but are smaller and retarded in the outgrowth process. They do not make contact but retain the potential to fuse. Development of other craniofacial structures appears normal, suggesting that impaired palate formation in Tbx2-mutant mice is caused by a primary defect in the palatal shelf mesenchyme. This is further supported by increased cell proliferation and apoptosis accompanied by increased expression of Bmp4 and CyclinD1 in Tbx2-deficient palatal shelves. Hence, Tbx2 and Tbx3 function overlappingly to control growth of the palatal shelf mesenchyme.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Palato/embriologia , Proteínas com Domínio T/genética , Animais , Proteína Morfogenética Óssea 4/genética , Ciclina D1/genética , Hibridização In Situ , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas com Domínio T/fisiologiaRESUMO
UNLABELLED: After specification of the hepatic endoderm, mammalian liver organogenesis progresses through a series of morphological stages that culminate in the migration of hepatocytes into the underlying mesenchyme to populate the hepatic lobes. Here, we show that in the mouse the transcriptional repressor Tbx3, a member of the T-box protein family, is required for the transition from a hepatic diverticulum with a pseudo-stratified epithelium to a cell-emergent liver bud. In Tbx3-deficient embryos, proliferation in the hepatic epithelium is severely reduced, hepatoblasts fail to delaminate, and cholangiocyte rather than hepatocyte differentiation occurs. Molecular analyses suggest that the primary function of Tbx3 is to maintain expression of hepatocyte transcription factors, including hepatic nuclear factor 4a (Hnf4a) and CCAAT/enhancer binding protein (C/EBP), alpha (Cebpa), and to repress expression of cholangiocyte transcription factors such as Onecut1 (Hnf6) and Hnf1b. CONCLUSION: Tbx3 controls liver bud expansion by suppressing cholangiocyte and favoring hepatocyte differentiation in the liver bud.
Assuntos
Ductos Biliares Extra-Hepáticos/embriologia , Diferenciação Celular/fisiologia , Células Epiteliais/citologia , Fígado/embriologia , Fígado/metabolismo , Organogênese/fisiologia , Proteínas com Domínio T/metabolismo , Animais , Ductos Biliares Extra-Hepáticos/citologia , Ductos Biliares Extra-Hepáticos/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proliferação de Células , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Células Epiteliais/metabolismo , Feminino , Fator 1-beta Nuclear de Hepatócito/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Fator 6 Nuclear de Hepatócito/metabolismo , Fígado/citologia , Camundongos , Camundongos Knockout , Gravidez , Proteínas com Domínio T/genéticaRESUMO
The embryonic origin of the dermis in vertebrates can be traced back to the dermomyotome of the somites, the lateral plate mesoderm and the neural crest. The dermal precursors directly overlying the neural tube display a unique dense arrangement and are the first to induce skin appendage formation in vertebrate embryos. These dermal precursor cells have been shown to derive from the dorsomedial lip of the dermomyotome (DML). Based on its expression pattern in the DML, Wnt11 is a candidate regulator of dorsal dermis formation. Using EGFP-based cell labelling and time-lapse imaging, we show that the Wnt11 expressing DML is the source of the dense dorsal dermis. Loss-of-function studies in chicken embryos show that Wnt11 is indeed essential for the formation of dense dermis competent to support cutaneous appendage formation. Our findings show that dermogenic progenitors cannot leave the DML to form dense dorsal dermis following Wnt11 silencing. No alterations were noticeable in the patterning or in the epithelial state of the dermomyotome including the DML. Furthermore, we show that Wnt11 expression is regulated in a manner similar to the previously described early dermal marker cDermo-1. The analysis of Wnt11 mutant mice exhibits an underdeveloped dorsal dermis and strongly supports our gene silencing data in chicken embryos. We conclude that Wnt11 is required for dense dermis and subsequent cutaneous appendage formation, by influencing the cell fate decision of the cells in the DML.
Assuntos
Movimento Celular , Galinhas/metabolismo , Derme/citologia , Derme/embriologia , Células-Tronco/citologia , Proteínas Wnt/metabolismo , Animais , Biomarcadores/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Embrião de Galinha , Regulação da Expressão Gênica no Desenvolvimento , Folículo Piloso/citologia , Camundongos Knockout , Interferência de RNA , Transdução de Sinais , Células-Tronco/metabolismoRESUMO
BACKGROUND: The atypical protein kinases C (PKC) isoforms ι/λ and ζ play crucial roles in many cellular processes including development, cell proliferation, differentiation and cell survival. Possible redundancy between the two isoforms has always been an issue since most biochemical tools do not differentiate between the two proteins. Thus, much effort has been made during the last decades to characterize the functions of aPKCs using gene targeting approaches and depletion studies. However, little is known about the specific roles of each isoform in mouse development. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the importance of PKCι in mouse development we designed PKCι deletion mutants using the gene targeting approach. We show that the deletion of PKCι, results in a reduced size of the amniotic cavity at E7.5 and impaired growth of the embryo at E8.5 with subsequent absorption of the embryo. Our data also indicate an impaired localization of ZO-1 and disorganized structure of the epithelial tissue in the embryo. Importantly, using electron microscopy, embryoid body formation and immunofluorescence analysis, we found, that in the absence of PKCι, tight junctions and apico-basal polarity were still established. Finally, our study points to a non-redundant PKCι function at E9.5, since expression of PKCζ is able to rescue the E7.5 phenotype, but could not prevent embryonic lethality at a later time-point (E9.5). CONCLUSION: Our data show that PKCι is crucial for mouse embryogenesis but is dispensable for the establishment of polarity and tight junction formation. We present a compensatory function of PKCζ at E7.5, rescuing the phenotype. Furthermore, this study indicates at least one specific, yet unknown, PKCι function that cannot be compensated by the overexpression of PKCζ at E9.5.