RESUMO
Genetic drivers of cancer can be dysregulated through epigenetic modifications of DNA. Although the critical role of DNA 5-methylcytosine (5mC) in the regulation of transcription is recognized, the functions of other non-canonical DNA modifications remain obscure. Here, we report the identification of novel N6-methyladenine (N6-mA) DNA modifications in human tissues and implicate this epigenetic mark in human disease, specifically the highly malignant brain cancer glioblastoma. Glioblastoma markedly upregulated N6-mA levels, which co-localized with heterochromatic histone modifications, predominantly H3K9me3. N6-mA levels were dynamically regulated by the DNA demethylase ALKBH1, depletion of which led to transcriptional silencing of oncogenic pathways through decreasing chromatin accessibility. Targeting the N6-mA regulator ALKBH1 in patient-derived human glioblastoma models inhibited tumor cell proliferation and extended the survival of tumor-bearing mice, supporting this novel DNA modification as a potential therapeutic target for glioblastoma. Collectively, our results uncover a novel epigenetic node in cancer through the DNA modification N6-mA.
Assuntos
Adenina/análogos & derivados , Neoplasias Encefálicas/patologia , Metilação de DNA , Glioblastoma/patologia , Adenina/análise , Adenina/química , Adulto , Idoso , Homólogo AlkB 1 da Histona H2a Dioxigenase/antagonistas & inibidores , Homólogo AlkB 1 da Histona H2a Dioxigenase/genética , Homólogo AlkB 1 da Histona H2a Dioxigenase/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidade , Hipóxia Celular , Criança , Epigenômica , Feminino , Glioblastoma/metabolismo , Glioblastoma/mortalidade , Heterocromatina/metabolismo , Histonas/metabolismo , Humanos , Estimativa de Kaplan-Meier , Masculino , Camundongos , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Compensatory proliferation triggered by hepatocyte loss is required for liver regeneration and maintenance but also promotes development of hepatocellular carcinoma (HCC). Despite extensive investigation, the cells responsible for hepatocyte restoration or HCC development remain poorly characterized. We used genetic lineage tracing to identify cells responsible for hepatocyte replenishment following chronic liver injury and queried their roles in three distinct HCC models. We found that a pre-existing population of periportal hepatocytes, located in the portal triads of healthy livers and expressing low amounts of Sox9 and other bile-duct-enriched genes, undergo extensive proliferation and replenish liver mass after chronic hepatocyte-depleting injuries. Despite their high regenerative potential, these so-called hybrid hepatocytes do not give rise to HCC in chronically injured livers and thus represent a unique way to restore tissue function and avoid tumorigenesis. This specialized set of pre-existing differentiated cells may be highly suitable for cell-based therapy of chronic hepatocyte-depleting disorders.
Assuntos
Hepatócitos/transplante , Fígado/citologia , Fígado/fisiologia , Animais , Ductos Biliares/citologia , Proliferação de Células , Transplante de Células/métodos , Hepatócitos/classificação , Hepatócitos/citologia , Fígado/lesões , Neoplasias Hepáticas , Camundongos , Regeneração , Fatores de Transcrição SOX9/genética , TranscriptomaRESUMO
Genetic risk variants that have been identified in genome-wide association studies of complex diseases are primarily non-coding1. Translating these risk variants into mechanistic insights requires detailed maps of gene regulation in disease-relevant cell types2. Here we combined two approaches: a genome-wide association study of type 1 diabetes (T1D) using 520,580 samples, and the identification of candidate cis-regulatory elements (cCREs) in pancreas and peripheral blood mononuclear cells using single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) of 131,554 nuclei. Risk variants for T1D were enriched in cCREs that were active in T cells and other cell types, including acinar and ductal cells of the exocrine pancreas. Risk variants at multiple T1D signals overlapped with exocrine-specific cCREs that were linked to genes with exocrine-specific expression. At the CFTR locus, the T1D risk variant rs7795896 mapped to a ductal-specific cCRE that regulated CFTR; the risk allele reduced transcription factor binding, enhancer activity and CFTR expression in ductal cells. These findings support a role for the exocrine pancreas in the pathogenesis of T1D and highlight the power of large-scale genome-wide association studies and single-cell epigenomics for understanding the cellular origins of complex disease.
Assuntos
Diabetes Mellitus Tipo 1/genética , Epigenômica , Predisposição Genética para Doença , Análise de Célula Única , Cromatina/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Feminino , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Imunidade/genética , Masculino , Ductos Pancreáticos/metabolismo , Ductos Pancreáticos/patologiaRESUMO
Many sequence variants have been linked to complex human traits and diseases1, but deciphering their biological functions remains challenging, as most of them reside in noncoding DNA. Here we have systematically assessed the binding of 270 human transcription factors to 95,886 noncoding variants in the human genome using an ultra-high-throughput multiplex protein-DNA binding assay, termed single-nucleotide polymorphism evaluation by systematic evolution of ligands by exponential enrichment (SNP-SELEX). The resulting 828 million measurements of transcription factor-DNA interactions enable estimation of the relative affinity of these transcription factors to each variant in vitro and evaluation of the current methods to predict the effects of noncoding variants on transcription factor binding. We show that the position weight matrices of most transcription factors lack sufficient predictive power, whereas the support vector machine combined with the gapped k-mer representation show much improved performance, when assessed on results from independent SNP-SELEX experiments involving a new set of 61,020 sequence variants. We report highly predictive models for 94 human transcription factors and demonstrate their utility in genome-wide association studies and understanding of the molecular pathways involved in diverse human traits and diseases.
Assuntos
Polimorfismo de Nucleotídeo Único/genética , Técnica de Seleção de Aptâmeros , Máquina de Vetores de Suporte , Fatores de Transcrição/metabolismo , Sítios de Ligação/genética , Doença/genética , Genoma Humano/genética , Humanos , Ligantes , Ligação ProteicaRESUMO
The pancreas is an essential organ for proper nutrient metabolism and has both endocrine and exocrine function. In the past two decades, knowledge of how the pancreas develops during embryogenesis has significantly increased, largely from developmental studies in model organisms. Specifically, the molecular basis of pancreatic lineage decisions and cell differentiation is well studied. Still not well understood are the mechanisms governing three-dimensional morphogenesis of the organ. Strategies to derive transplantable ß-cells in vitro for diabetes treatment have benefited from the accumulated knowledge of pancreas development. In this review, we provide an overview of the current understanding of pancreatic lineage determination and organogenesis, and we examine future implications of these findings for treatment of diabetes mellitus through cell replacement.
Assuntos
Organogênese , Pâncreas/citologia , Pâncreas/embriologia , Animais , Diferenciação Celular , Humanos , Pâncreas/metabolismo , Transdução de SinaisRESUMO
A comprehensive understanding of mechanisms that underlie the development and function of human cells requires human cell models. For the pancreatic lineage, protocols have been developed to differentiate human pluripotent stem cells (hPSCs) into pancreatic endocrine and exocrine cells through intermediates resembling in vivo development. In recent years, this differentiation system has been employed to decipher mechanisms of pancreatic development, congenital defects of the pancreas, as well as genetic forms of diabetes and exocrine diseases. In this review, we summarize recent insights gained from studies of pancreatic hPSC models. We discuss how genome-scale analyses of the differentiation system have helped elucidate roles of chromatin state, transcription factors, and noncoding RNAs in pancreatic development and how the analysis of cells with disease-relevant mutations has provided insight into the molecular underpinnings of genetically determined diseases of the pancreas.
Assuntos
Modelos Biológicos , Pâncreas/citologia , Pâncreas/crescimento & desenvolvimento , Células-Tronco Pluripotentes/citologia , Diferenciação Celular , Estudo de Associação Genômica Ampla , Humanos , Pâncreas/patologia , Pancreatopatias/genética , Pancreatopatias/fisiopatologia , Células-Tronco Pluripotentes/fisiologiaRESUMO
It is now recognized that cell identity is more fluid, and tissues more plastic, than previously thought. The plasticity of cells is relevant to diverse fields, most notably developmental and stem cell biology, regenerative medicine, and cancer biology. To date, a comprehensive and uniform nomenclature to define distinct cell states and their injury-induced interconversions has been elusive. The first Keystone Symposium devoted exclusively to cellular plasticity in regeneration and tumorigenesis was held on January 2019 in Keystone, Colorado, and featured a workshop on terminology in the cell plasticity field. Definitions for terms such as plasticity, de- and transdifferentiation, reversion, and paligenosis were discussed. Here, we summarize the content and tenor of the symposium and nomenclature-focused workshop with regard to terms in the field. We outline the challenges with current definitions and recommend best practices and approaches to developing an accurate and acceptable nomenclature in the future.
Assuntos
Plasticidade Celular , Terminologia como Assunto , Animais , Carcinogênese , Congressos como Assunto , Humanos , Plásticos , Medicina RegenerativaRESUMO
Pancreatic intraepithelial neoplasia is a pre-malignant lesion that can progress to pancreatic ductal adenocarcinoma, a highly lethal malignancy marked by its late stage at clinical presentation and profound drug resistance. The genomic alterations that commonly occur in pancreatic cancer include activation of KRAS2 and inactivation of p53 and SMAD4 (refs 2-4). So far, however, it has been challenging to target these pathways therapeutically; thus the search for other key mediators of pancreatic cancer growth remains an important endeavour. Here we show that the stem cell determinant Musashi (Msi) is a critical element of pancreatic cancer progression both in genetic models and in patient-derived xenografts. Specifically, we developed Msi reporter mice that allowed image-based tracking of stem cell signals within cancers, revealing that Msi expression rises as pancreatic intraepithelial neoplasia progresses to adenocarcinoma, and that Msi-expressing cells are key drivers of pancreatic cancer: they preferentially harbour the capacity to propagate adenocarcinoma, are enriched in circulating tumour cells, and are markedly drug resistant. This population could be effectively targeted by deletion of either Msi1 or Msi2, which led to a striking defect in the progression of pancreatic intraepithelial neoplasia to adenocarcinoma and an improvement in overall survival. Msi inhibition also blocked the growth of primary patient-derived tumours, suggesting that this signal is required for human disease. To define the translational potential of this work we developed antisense oligonucleotides against Msi; these showed reliable tumour penetration, uptake and target inhibition, and effectively blocked pancreatic cancer growth. Collectively, these studies highlight Msi reporters as a unique tool to identify therapy resistance, and define Msi signalling as a central regulator of pancreatic cancer.
Assuntos
Carcinoma Ductal Pancreático/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Imagem Molecular , Proteínas do Tecido Nervoso/genética , Neoplasias Pancreáticas/tratamento farmacológico , Proteínas de Ligação a RNA/genética , Animais , Carcinoma in Situ/genética , Carcinoma in Situ/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Deleção de Genes , Genes Reporter/genética , Humanos , Masculino , Camundongos , Modelos Genéticos , Células Neoplásicas Circulantes/metabolismo , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/metabolismo , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacocinética , Oligonucleotídeos Antissenso/uso terapêutico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Taxa de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Co-localized combinations of histone modifications ("chromatin states") have been shown to correlate with promoter and enhancer activity. Changes in chromatin states over multiple time points ("chromatin state trajectories") have previously been analyzed at promoter and enhancers separately. With the advent of time series Hi-C data it is now possible to connect promoters and enhancers and to analyze chromatin state trajectories at promoter-enhancer pairs. RESULTS: We present TimelessFlex, a framework for investigating chromatin state trajectories at promoters and enhancers and at promoter-enhancer pairs based on Hi-C information. TimelessFlex extends our previous approach Timeless, a Bayesian network for clustering multiple histone modification data sets at promoter and enhancer feature regions. We utilize time series ATAC-seq data measuring open chromatin to define promoters and enhancer candidates. We developed an expectation-maximization algorithm to assign promoters and enhancers to each other based on Hi-C interactions and jointly cluster their feature regions into paired chromatin state trajectories. We find jointly clustered promoter-enhancer pairs showing the same activation patterns on both sides but with a stronger trend at the enhancer side. While the promoter side remains accessible across the time series, the enhancer side becomes dynamically more open towards the gene activation time point. Promoter cluster patterns show strong correlations with gene expression signals, whereas Hi-C signals get only slightly stronger towards activation. The code of the framework is available at https://github.com/henriettemiko/TimelessFlex . CONCLUSIONS: TimelessFlex clusters time series histone modifications at promoter-enhancer pairs based on Hi-C and it can identify distinct chromatin states at promoter and enhancer feature regions and their changes over time.
Assuntos
Cromatina , Elementos Facilitadores Genéticos , Teorema de Bayes , Cromatina/genética , Cromossomos , Regiões Promotoras GenéticasRESUMO
OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumour thought to arise from ductal cells via pancreatic intraepithelial neoplasia (PanIN) precursor lesions. Modelling of different genetic events in mice suggests both ductal and acinar cells can give rise to PDAC. However, the impact of cellular context alone on tumour development and phenotype is unknown. DESIGN: We examined the contribution of cellular origin to PDAC development by inducing PDAC-associated mutations, KrasG12D expression and Trp53 loss, specifically in ductal cells (Sox9CreER;KrasLSL-G12D;Trp53flox/flox ('Duct:KPcKO ')) or acinar cells (Ptf1aCreER;KrasLSL-G12D;Trp53flox/flox ('Acinar:KPcKO ')) in mice. We then performed a thorough analysis of the resulting histopathological changes. RESULTS: Both mouse models developed PDAC, but Duct:KPcKO mice developed PDAC earlier than Acinar:KPcKO mice. Tumour development was more rapid and associated with high-grade murine PanIN (mPanIN) lesions in Duct:KPcKO mice. In contrast, Acinar:KPcKO mice exhibited widespread metaplasia and low-grade as well as high-grade mPanINs with delayed progression to PDAC. Acinar-cell-derived tumours also had a higher prevalence of mucinous glandular features reminiscent of early mPanIN lesions. CONCLUSION: These findings indicate that ductal cells are primed to form carcinoma in situ that become invasive PDAC in the presence of oncogenic Kras and Trp53 deletion, while acinar cells with the same mutations appear to require a prolonged period of transition or reprogramming to initiate PDAC. Our findings illustrate that PDAC can develop in multiple ways and the cellular context in which mutations are acquired has significant impact on precursor lesion initiation, disease progression and tumour phenotype.
Assuntos
Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/patologia , Células Acinares/metabolismo , Células Acinares/patologia , Animais , Carcinoma in Situ/genética , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Gradação de Tumores , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fenótipo , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fatores de Tempo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND & AIMS: Intraductal papillary mucinous neoplasias (IPMNs) are precancerous cystic lesions that can develop into pancreatic ductal adenocarcinomas (PDACs). These large macroscopic lesions are frequently detected during medical imaging, but it is unclear how they form or progress to PDAC. We aimed to identify cells that form IPMNs and mutations that promote IPMN development and progression. METHODS: We generated mice with disruption of Pten specifically in ductal cells (Sox9CreERT2;Ptenflox/flox;R26RYFP or PtenΔDuct/ΔDuct mice) and used PtenΔDuct/+ and Pten+/+ mice as controls. We also generated KrasG12D;PtenΔDuct/ΔDuct and KrasG12D;PtenΔDuct/+ mice. Pancreata were collected when mice were 28 weeks to 14.5 months old and analyzed by histology, immunohistochemistry, and electron microscopy. We performed multiplexed droplet digital polymerase chain reaction to detect spontaneous Kras mutations in PtenΔDuct/ΔDuct mice and study the effects of Ras pathway activation on initiation and progression of IPMNs. We obtained 2 pancreatic sections from a patient with an invasive pancreatobiliary IPMN and analyzed the regions with and without the invasive IPMN (control tissue) by immunohistochemistry. RESULTS: Mice with ductal cell-specific disruption of Pten but not control mice developed sporadic, macroscopic, intraductal papillary lesions with histologic and molecular features of human IPMNs. PtenΔDuct/ΔDuct mice developed IPMNs of several subtypes. In PtenΔDuct/ΔDuct mice, 31.5% of IPMNs became invasive; invasion was associated with spontaneous mutations in Kras. KrasG12D;PtenΔDuct/ΔDuct mice all developed invasive IPMNs within 1 month. In KrasG12D;PtenΔDuct/+ mice, 70% developed IPMN, predominately of the pancreatobiliary subtype, and 63.3% developed PDAC. In all models, IPMNs and PDAC expressed the duct-specific lineage tracing marker yellow fluorescent protein. In immunohistochemical analyses, we found that the invasive human pancreatobiliary IPMN tissue had lower levels of PTEN and increased levels of phosphorylated (activated) ERK compared with healthy pancreatic tissue. CONCLUSIONS: In analyses of mice with ductal cell-specific disruption of Pten, with or without activated Kras, we found evidence for a ductal cell origin of IPMNs. We also showed that PTEN loss and activated Kras have synergistic effects in promoting development of IPMN and progression to PDAC.
Assuntos
Carcinoma Ductal Pancreático/enzimologia , Transformação Celular Neoplásica/metabolismo , Neoplasias Císticas, Mucinosas e Serosas/enzimologia , PTEN Fosfo-Hidrolase/deficiência , Ductos Pancreáticos/enzimologia , Neoplasias Pancreáticas/enzimologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem da Célula , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Progressão da Doença , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Invasividade Neoplásica , Neoplasias Císticas, Mucinosas e Serosas/genética , Neoplasias Císticas, Mucinosas e Serosas/patologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fenótipo , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais , Fatores de TempoRESUMO
The molecular identification of adult hepatic stem/progenitor cells has been hampered by the lack of truly specific markers. To isolate putative adult liver progenitor cells, we used cell surface-marking antibodies, including MIC1-1C3, to isolate subpopulations of liver cells from normal adult mice or those undergoing an oval cell response and tested their capacity to form bilineage colonies in vitro. Robust clonogenic activity was found to be restricted to a subset of biliary duct cells antigenically defined as CD45(-)/CD11b(-)/CD31(-)/MIC1-1C3(+)/CD133(+)/CD26(-), at a frequency of one of 34 or one of 25 in normal or oval cell injury livers, respectively. Gene expression analyses revealed that Sox9 was expressed exclusively in this subpopulation of normal liver cells and was highly enriched relative to other cell fractions in injured livers. In vivo lineage tracing using Sox9creER(T2)-R26R(YFP) mice revealed that the cells that proliferate during progenitor-driven liver regeneration are progeny of Sox9-expressing precursors. A comprehensive array-based comparison of gene expression in progenitor-enriched and progenitor-depleted cells from both normal and DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine or diethyl1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate)-treated livers revealed new potential regulators of liver progenitors.
Assuntos
Separação Celular/métodos , Fígado/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Clonais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Hepatócitos/citologia , Hepatócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco/metabolismoRESUMO
BACKGROUND: The plasticity of pancreatic acinar cells to undergo acinar to ductal metaplasia (ADM) has been demonstrated to contribute to the regeneration of the pancreas in response to injury. Sox9 is critical for ductal cell fate and important in the formation of ADM, most likely in concert with a complex hierarchy of, as yet, not fully elucidated transcription factors. RESULTS: By using a mouse model of acute pancreatitis and three dimensional organoid culture of primary pancreatic ductal cells, we herein characterize the Ets-transcription factor Etv5 as a pivotal regulator of ductal cell identity and ADM that acts upstream of Sox9 and is essential for Sox9 expression in ADM. Loss of Etv5 is associated with increased severity of acute pancreatitis and impaired ADM formation leading to delayed tissue regeneration and recovery in response to injury. CONCLUSIONS: Our data provide new insights in the regulation of ADM with implications in our understanding of pancreatic homeostasis, pancreatitis and epithelial plasticity. Developmental Dynamics 247:854-866, 2018. © 2018 Wiley Periodicals, Inc.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Ductos Pancreáticos/metabolismo , Ductos Pancreáticos/fisiologia , Pancreatite/metabolismo , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição/metabolismo , Células Acinares/citologia , Células Acinares/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Proteínas de Ligação a DNA/genética , Camundongos , Camundongos Knockout , Pâncreas/embriologia , Pâncreas/metabolismo , Pancreatite/genética , Fatores de Transcrição SOX9/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fatores de Transcrição/genéticaRESUMO
Heterozygous mutations in the human HNF1B gene are associated with maturity-onset diabetes of the young type 5 (MODY5) and pancreas hypoplasia. In mouse, Hnf1b heterozygous mutants do not exhibit any phenotype, whereas the homozygous deletion in the entire epiblast leads to pancreas agenesis associated with abnormal gut regionalization. Here, we examine the specific role of Hnf1b during pancreas development, using constitutive and inducible conditional inactivation approaches at key developmental stages. Hnf1b early deletion leads to a reduced pool of pancreatic multipotent progenitor cells (MPCs) due to decreased proliferation and increased apoptosis. Lack of Hnf1b either during the first or the secondary transitions is associated with cystic ducts. Ductal cells exhibit aberrant polarity and decreased expression of several cystic disease genes, some of which we identified as novel Hnf1b targets. Notably, we show that Glis3, a transcription factor involved in duct morphogenesis and endocrine cell development, is downstream Hnf1b. In addition, a loss and abnormal differentiation of acinar cells are observed. Strikingly, inactivation of Hnf1b at different time points results in the absence of Ngn3(+) endocrine precursors throughout embryogenesis. We further show that Hnf1b occupies novel Ngn3 putative regulatory sequences in vivo. Thus, Hnf1b plays a crucial role in the regulatory networks that control pancreatic MPC expansion, acinar cell identity, duct morphogenesis and generation of endocrine precursors. Our results uncover an unappreciated requirement of Hnf1b in endocrine cell specification and suggest a mechanistic explanation of diabetes onset in individuals with MODY5.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fator 1-beta Nuclear de Hepatócito/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Pâncreas/citologia , Pâncreas/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/fisiologia , Imunoprecipitação da Cromatina , Ducto Cístico/citologia , Ducto Cístico/metabolismo , Proteínas de Ligação a DNA , Feminino , Fator 1-beta Nuclear de Hepatócito/genética , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Proteínas do Tecido Nervoso/genética , Gravidez , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
V1 interneurons are inhibitory neurons that play an essential role in vertebrate locomotion. The molecular mechanisms underlying their genesis remain, however, largely undefined. Here, we show that the transcription factor Prdm12 is selectively expressed in p1 progenitors of the hindbrain and spinal cord in the frog embryo, and that a similar restricted expression profile is observed in the nerve cord of other vertebrates as well as of the cephalochordate amphioxus. Using frog, chick and mice, we analyzed the regulation of Prdm12 and found that its expression in the caudal neural tube is dependent on retinoic acid and Pax6, and that it is restricted to p1 progenitors, due to the repressive action of Dbx1 and Nkx6-1/2 expressed in the adjacent p0 and p2 domains. Functional studies in the frog, including genome-wide identification of its targets by RNA-seq and ChIP-Seq, reveal that vertebrate Prdm12 proteins act as a general determinant of V1 cell fate, at least in part, by directly repressing Dbx1 and Nkx6 genes. This probably occurs by recruiting the methyltransferase G9a, an activity that is not displayed by the amphioxus Prdm12 protein. Together, these findings indicate that Prdm12 promotes V1 interneurons through cross-repressive interactions with Dbx1 and Nkx6 genes, and suggest that this function might have only been acquired after the split of the vertebrate and cephalochordate lineages.
Assuntos
Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Morfogênese/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Células de Renshaw/fisiologia , Xenopus/embriologia , Animais , Sequência de Bases , Embrião de Galinha , Imunoprecipitação da Cromatina , Biologia Computacional , Primers do DNA/genética , DNA Complementar/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Rombencéfalo/metabolismo , Análise de Sequência de RNA , Especificidade da Espécie , Medula Espinal/metabolismoRESUMO
The study of hematopoietic colony-forming units using semisolid culture media has greatly advanced the knowledge of hematopoiesis. Here we report that similar methods can be used to study pancreatic colony-forming units. We have developed two pancreatic colony assays that enable quantitative and functional analyses of progenitor-like cells isolated from dissociated adult (2-4 mo old) murine pancreas. We find that a methylcellulose-based semisolid medium containing Matrigel allows growth of duct-like "Ring/Dense" colonies from a rare (â¼1%) population of total pancreatic single cells. With the addition of roof plate-specific spondin 1, a wingless-int agonist, Ring/Dense colony-forming cells can be expanded more than 100,000-fold when serially dissociated and replated in the presence of Matrigel. When cells grown in Matrigel are then transferred to a Matrigel-free semisolid medium with a unique laminin-based hydrogel, some cells grow and differentiate into another type of colony, which we name "Endocrine/Acinar." These Endocrine/Acinar colonies are comprised mostly of endocrine- and acinar-like cells, as ascertained by RNA expression analysis, immunohistochemistry, and electron microscopy. Most Endocrine/Acinar colonies contain beta-like cells that secrete insulin/C-peptide in response to D-glucose and theophylline. These results demonstrate robust self-renewal and differentiation of adult Ring/Dense colony-forming units in vitro and suggest an approach to producing beta-like cells for cell replacement of type 1 diabetes. The methods described, which include microfluidic expression analysis of single cells and colonies, should also advance study of pancreas development and pancreatic progenitor cells.
Assuntos
Ensaio de Unidades Formadoras de Colônias/métodos , Pâncreas/citologia , Células Acinares/citologia , Células Acinares/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Colágeno , Meios de Cultura , Combinação de Medicamentos , Hidrogéis , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Laminina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Proteínas do Tecido Nervoso/metabolismo , Pâncreas/crescimento & desenvolvimento , Pâncreas/metabolismo , Proteoglicanas , Via de Sinalização WntRESUMO
Lung branching morphogenesis is a highly orchestrated process that gives rise to the complex network of gas-exchanging units in the adult lung. Intricate regulation of signaling pathways, transcription factors, and epithelial-mesenchymal cross-talk are critical to ensuring branching morphogenesis occurs properly. Here, we describe a role for the transcription factor Sox9 during lung branching morphogenesis. Sox9 is expressed at the distal tips of the branching epithelium in a highly dynamic manner as branching occurs and is down-regulated starting at embryonic day 16.5, concurrent with the onset of terminal differentiation of type 1 and type 2 alveolar cells. Using epithelial-specific genetic loss- and gain-of-function approaches, our results demonstrate that Sox9 controls multiple aspects of lung branching. Fine regulation of Sox9 levels is required to balance proliferation and differentiation of epithelial tip progenitor cells, and loss of Sox9 leads to direct and indirect cellular defects including extracellular matrix defects, cytoskeletal disorganization, and aberrant epithelial movement. Our evidence shows that unlike other endoderm-derived epithelial tissues, such as the intestine, Wnt/ß-catenin signaling does not regulate Sox9 expression in the lung. We conclude that Sox9 collectively promotes proper branching morphogenesis by controlling the balance between proliferation and differentiation and regulating the extracellular matrix.
Assuntos
Matriz Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Pulmão/embriologia , Organogênese/fisiologia , Mucosa Respiratória/metabolismo , Fatores de Transcrição SOX9/metabolismo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células , Imunoprecipitação da Cromatina , Doxiciclina/farmacologia , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento/genética , Imuno-Histoquímica , Hibridização In Situ , Pulmão/citologia , Camundongos , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase em Tempo Real , Mucosa Respiratória/fisiologia , Tamoxifeno/farmacologiaRESUMO
All pancreatic endocrine cell types arise from a common endocrine precursor cell population, yet the molecular mechanisms that establish and maintain the unique gene expression programs of each endocrine cell lineage have remained largely elusive. Such knowledge would improve our ability to correctly program or reprogram cells to adopt specific endocrine fates. Here, we show that the transcription factor Nkx6.1 is both necessary and sufficient to specify insulin-producing beta cells. Heritable expression of Nkx6.1 in endocrine precursors of mice is sufficient to respecify non-beta endocrine precursors towards the beta cell lineage, while endocrine precursor- or beta cell-specific inactivation of Nkx6.1 converts beta cells to alternative endocrine lineages. Remaining insulin(+) cells in conditional Nkx6.1 mutants fail to express the beta cell transcription factors Pdx1 and MafA and ectopically express genes found in non-beta endocrine cells. By showing that Nkx6.1 binds to and represses the alpha cell determinant Arx, we identify Arx as a direct target of Nkx6.1. Moreover, we demonstrate that Nkx6.1 and the Arx activator Isl1 regulate Arx transcription antagonistically, thus establishing competition between Isl1 and Nkx6.1 as a critical mechanism for determining alpha versus beta cell identity. Our findings establish Nkx6.1 as a beta cell programming factor and demonstrate that repression of alternative lineage programs is a fundamental principle by which beta cells are specified and maintained. Given the lack of Nkx6.1 expression and aberrant activation of non-beta endocrine hormones in human embryonic stem cell (hESC)-derived insulin(+) cells, our study has significant implications for developing cell replacement therapies.
Assuntos
Células Endócrinas , Proteínas de Homeodomínio , Células Secretoras de Insulina , Insulina , Animais , Diferenciação Celular/genética , Linhagem da Célula , Terapia Baseada em Transplante de Células e Tecidos , Células Endócrinas/citologia , Células Endócrinas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Insulina/genética , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Fatores de Transcrição Maf Maior/genética , Fatores de Transcrição Maf Maior/metabolismo , Camundongos , Pâncreas/citologia , Células-Tronco , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In the pancreas, Notch signaling is thought to prevent cell differentiation, thereby maintaining progenitors in an undifferentiated state. Here, we show that Notch renders progenitors competent to differentiate into ductal and endocrine cells by inducing activators of cell differentiation. Notch signaling promotes the expression of Sox9, which cell-autonomously activates the pro-endocrine gene Ngn3. However, at high Notch activity endocrine differentiation is blocked, as Notch also induces expression of the Ngn3 repressor Hes1. At the transition from high to intermediate Notch activity, only Sox9, but not Hes1, is maintained, thus de-repressing Ngn3 and initiating endocrine differentiation. In the absence of Sox9 activity, endocrine and ductal cells fail to differentiate, resulting in polycystic ducts devoid of primary cilia. Although Sox9 is required for Ngn3 induction, endocrine differentiation necessitates subsequent Sox9 downregulation and evasion from Notch activity via cell-autonomous repression of Sox9 by Ngn3. If high Notch levels are maintained, endocrine progenitors retain Sox9 and undergo ductal fate conversion. Taken together, our findings establish a novel role for Notch in initiating both ductal and endocrine development and reveal that Notch does not function in an on-off mode, but that a gradient of Notch activity produces distinct cellular states during pancreas development.
Assuntos
Pâncreas/citologia , Pâncreas/metabolismo , Receptores Notch/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Citometria de Fluxo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores Notch/genética , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição HES-1RESUMO
All mature pancreatic cell types arise from organ-specific multipotent progenitor cells. Although previous studies have identified cell-intrinsic and -extrinsic cues for progenitor cell expansion, it is unclear how these cues are integrated within the niche of the developing organ. Here, we present genetic evidence in mice that the transcription factor Sox9 forms the centerpiece of a gene regulatory network that is crucial for proper organ growth and maintenance of organ identity. We show that pancreatic progenitor-specific ablation of Sox9 during early pancreas development causes pancreas-to-liver cell fate conversion. Sox9 deficiency results in cell-autonomous loss of the fibroblast growth factor receptor (Fgfr) 2b, which is required for transducing mesenchymal Fgf10 signals. Likewise, Fgf10 is required to maintain expression of Sox9 and Fgfr2 in epithelial progenitors, showing that Sox9, Fgfr2 and Fgf10 form a feed-forward expression loop in the early pancreatic organ niche. Mirroring Sox9 deficiency, perturbation of Fgfr signaling in pancreatic explants or genetic inactivation of Fgf10 also result in hepatic cell fate conversion. Combined with previous findings that Fgfr2b or Fgf10 are necessary for pancreatic progenitor cell proliferation, our results demonstrate that organ fate commitment and progenitor cell expansion are coordinately controlled by the activity of a Sox9/Fgf10/Fgfr2b feed-forward loop in the pancreatic niche. This self-promoting Sox9/Fgf10/Fgfr2b loop may regulate cell identity and organ size in a broad spectrum of developmental and regenerative contexts.