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1.
PLoS Biol ; 19(9): e3001394, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34550965

RESUMO

The ZEB2 transcription factor has been demonstrated to play important roles in hematopoiesis and leukemic transformation. ZEB1 is a close family member of ZEB2 but has remained more enigmatic concerning its roles in hematopoiesis. Here, we show using conditional loss-of-function approaches and bone marrow (BM) reconstitution experiments that ZEB1 plays a cell-autonomous role in hematopoietic lineage differentiation, particularly as a positive regulator of monocyte development in addition to its previously reported important role in T-cell differentiation. Analysis of existing single-cell (sc) RNA sequencing (RNA-seq) data of early hematopoiesis has revealed distinctive expression differences between Zeb1 and Zeb2 in hematopoietic stem and progenitor cell (HSPC) differentiation, with Zeb2 being more highly and broadly expressed than Zeb1 except at a key transition point (short-term HSC [ST-HSC]➔MPP1), whereby Zeb1 appears to be the dominantly expressed family member. Inducible genetic inactivation of both Zeb1 and Zeb2 using a tamoxifen-inducible Cre-mediated approach leads to acute BM failure at this transition point with increased long-term and short-term hematopoietic stem cell numbers and an accompanying decrease in all hematopoietic lineage differentiation. Bioinformatics analysis of RNA-seq data has revealed that ZEB2 acts predominantly as a transcriptional repressor involved in restraining mature hematopoietic lineage gene expression programs from being expressed too early in HSPCs. ZEB1 appears to fine-tune this repressive role during hematopoiesis to ensure hematopoietic lineage fidelity. Analysis of Rosa26 locus-based transgenic models has revealed that Zeb1 as well as Zeb2 cDNA-based overexpression within the hematopoietic system can drive extramedullary hematopoiesis/splenomegaly and enhance monocyte development. Finally, inactivation of Zeb2 alone or Zeb1/2 together was found to enhance survival in secondary MLL-AF9 acute myeloid leukemia (AML) models attesting to the oncogenic role of ZEB1/2 in AML.

2.
Genes (Basel) ; 12(7)2021 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-34356053

RESUMO

After its publication in 1999 as a DNA-binding and SMAD-binding transcription factor (TF) that co-determines cell fate in amphibian embryos, ZEB2 was from 2003 studied by embryologists mainly by documenting the consequences of conditional, cell-type specific Zeb2 knockout (cKO) in mice. In between, it was further identified as causal gene causing Mowat-Wilson Syndrome (MOWS) and novel regulator of epithelial-mesenchymal transition (EMT). ZEB2's functions and action mechanisms in mouse embryos were first addressed in its main sites of expression, with focus on those that helped to explain neurodevelopmental and neural crest defects seen in MOWS patients. By doing so, ZEB2 was identified in the forebrain as the first TF that determined timing of neuro-/gliogenesis, and thereby also the extent of different layers of the cortex, in a cell non-autonomous fashion, i.e., by its cell-intrinsic control within neurons of neuron-to-progenitor paracrine signaling. Transcriptomics-based phenotyping of Zeb2 mutant mouse cells have identified large sets of intact-ZEB2 dependent genes, and the cKO approaches also moved to post-natal brain development and diverse other systems in adult mice, including hematopoiesis and various cell types of the immune system. These new studies start to highlight the important adult roles of ZEB2 in cell-cell communication, including after challenge, e.g., in the infarcted heart and fibrotic liver. Such studies may further evolve towards those documenting the roles of ZEB2 in cell-based repair of injured tissue and organs, downstream of actions of diverse growth factors, which recapitulate developmental signaling principles in the injured sites. Evident questions are about ZEB2's direct target genes, its various partners, and ZEB2 as a candidate modifier gene, e.g., in other (neuro)developmental disorders, but also the accurate transcriptional and epigenetic regulation of its mRNA expression sites and levels. Other questions start to address ZEB2's function as a niche-controlling regulatory TF of also other cell types, in part by its modulation of growth factor responses (e.g., TGFß/BMP, Wnt, Notch). Furthermore, growing numbers of mapped missense as well as protein non-coding mutations in MOWS patients are becoming available and inspire the design of new animal model and pluripotent stem cell-based systems. This review attempts to summarize in detail, albeit without discussing ZEB2's role in cancer, hematopoiesis, and its emerging roles in the immune system, how intense ZEB2 research has arrived at this exciting intersection.

3.
Methods Mol Biol ; 2351: 165-179, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382189

RESUMO

Targeted chromatin capture (T2C) is a 3C-based method and is used to study the 3D chromatin organization, interactomes and structural changes associated with gene regulation, progression through the cell cycle, and cell survival and development. Low input targeted chromatin capture (low-T2C) is an optimized version of the T2C protocol for low numbers of cells. Here, we describe the protocol for low-T2C, including all experimental steps and bioinformatics tools in detail.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/genética , Biologia Computacional/métodos , Cromatina/química , Cromatina/metabolismo , Mapeamento Cromossômico , Regulação da Expressão Gênica , Biblioteca Gênica , Genômica/métodos , Reprodutibilidade dos Testes
4.
Cardiovasc Res ; 2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33909875

RESUMO

AIMS: Hepatic capillaries are lined with specialised liver sinusoidal endothelial cells (LSECs) which support macromolecule passage to hepatocytes and prevent fibrosis by keeping hepatic stellate cells (HSCs) quiescent. LSEC specialisation is co-determined by transcription factors. The Zinc-Finger E-Box-binding Homeobox (Zeb)2 transcription factor is enriched in LSECs. Here, we aimed to elucidate the endothelium-specific role of Zeb2 during maintenance of the liver and in liver fibrosis. METHODS AND RESULTS: To study the role of Zeb2 in liver endothelium we generated EC-specific Zeb2 knock-out (ECKO) mice. Sequencing of liver EC RNA revealed that deficiency of Zeb2 results in prominent expression changes in angiogenesis-related genes. Accordingly, the vascular area was expanded and the presence of pillars inside ECKO liver vessels indicated that this was likely due to increased intussusceptive angiogenesis. LSEC marker expression was not profoundly affected and fenestrations were preserved upon Zeb2 deficiency. However, an increase in continuous EC markers suggested that Zeb2-deficient LSECs are more prone to dedifferentiation, a process called 'capillarisation'. Changes in the endothelial expression of ligands that may be involved in HSC quiescence together with significant changes in the expression profile of HSCs showed that Zeb2 regulates LSEC-HSC communication and HSC activation. Accordingly, upon exposure to the hepatotoxin carbon tetrachloride (CCl4), livers of ECKO mice showed increased capillarisation, HSC activation and fibrosis compared to livers from wild-type littermates. The vascular maintenance and anti-fibrotic role of endothelial Zeb2 was confirmed in mice with EC-specific overexpression of Zeb2, as the latter resulted in reduced vascularity and attenuated CCl4-induced liver fibrosis. CONCLUSION: Endothelial Zeb2 preserves liver angioarchitecture and protects against liver fibrosis. Zeb2 and Zeb2-dependent genes in liver ECs may be exploited to design novel therapeutic strategies to attenuate hepatic fibrosis.

5.
Nat Commun ; 12(1): 84, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33398012

RESUMO

The disruption in blood supply due to myocardial infarction is a critical determinant for infarct size and subsequent deterioration in function. The identification of factors that enhance cardiac repair by the restoration of the vascular network is, therefore, of great significance. Here, we show that the transcription factor Zinc finger E-box-binding homeobox 2 (ZEB2) is increased in stressed cardiomyocytes and induces a cardioprotective cross-talk between cardiomyocytes and endothelial cells to enhance angiogenesis after ischemia. Single-cell sequencing indicates ZEB2 to be enriched in injured cardiomyocytes. Cardiomyocyte-specific deletion of ZEB2 results in impaired cardiac contractility and infarct healing post-myocardial infarction (post-MI), while cardiomyocyte-specific ZEB2 overexpression improves cardiomyocyte survival and cardiac function. We identified Thymosin ß4 (TMSB4) and Prothymosin α (PTMA) as main paracrine factors released from cardiomyocytes to stimulate angiogenesis by enhancing endothelial cell migration, and whose regulation is validated in our in vivo models. Therapeutic delivery of ZEB2 to cardiomyocytes in the infarcted heart induces the expression of TMSB4 and PTMA, which enhances angiogenesis and prevents cardiac dysfunction. These findings reveal ZEB2 as a beneficial factor during ischemic injury, which may hold promise for the identification of new therapies.


Assuntos
Isquemia/patologia , Miócitos Cardíacos/metabolismo , Neovascularização Fisiológica , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Animais , Movimento Celular/genética , Proliferação de Células/genética , Dependovirus/metabolismo , Regulação da Expressão Gênica , Humanos , Isquemia/genética , Camundongos Knockout , Modelos Biológicos , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Miócitos Cardíacos/patologia , Neovascularização Fisiológica/genética , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Timosina/análogos & derivados , Timosina/genética , Timosina/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética
6.
Sci Immunol ; 6(55)2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514640

RESUMO

Group 2 innate lymphoid cells (ILC2s) orchestrate protective type 2 immunity and have been implicated in various immune disorders. In the mouse, circulatory inflammatory ILC2s (iILC2s) were identified as a major source of type 2 cytokines. The human equivalent of the iILC2 subset remains unknown. Here, we identify a human inflammatory ILC2 population that resides in inflamed mucosal tissue and is specifically marked by surface CD45RO expression. CD45RO+ ILC2s are derived from resting CD45RA+ ILC2s upon activation by epithelial alarmins such as IL-33 and TSLP, which is tightly linked to STAT5 activation and up-regulation of the IRF4/BATF transcription factors. Transcriptome analysis reveals marked similarities between human CD45RO+ ILC2s and mouse iILC2s. Frequencies of CD45RO+ inflammatory ILC2 are increased in inflamed mucosal tissue and in the circulation of patients with chronic rhinosinusitis or asthma, correlating with disease severity and resistance to corticosteroid therapy. CD45RA-to-CD45RO ILC2 conversion is suppressed by corticosteroids via induction of differentiation toward an immunomodulatory ILC2 phenotype characterized by low type 2 cytokine and high amphiregulin expression. Once converted, however, CD45RO+ ILC2s are resistant to corticosteroids, which is associated with metabolic reprogramming resulting in the activation of detoxification pathways. Our combined data identify CD45RO+ inflammatory ILC2s as a human analog of mouse iILC2s linked to severe type 2 inflammatory disease and therapy resistance.

7.
Dev Biol ; 468(1-2): 80-92, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32950463

RESUMO

The interplay between signaling molecules and transcription factors during retinal development is key to controlling the correct number of retinal cell types. Zeb2 (Sip1) is a zinc-finger multidomain transcription factor that plays multiple roles in central and peripheral nervous system development. Haploinsufficiency of ZEB2 causes Mowat-Wilson syndrome, a congenital disease characterized by intellectual disability, epilepsy and Hirschsprung disease. In the developing retina, Zeb2 is required for generation of horizontal cells and the correct number of interneurons; however, its potential function in controlling gliogenic versus neurogenic decisions remains unresolved. Here we present cellular and molecular evidence of the inhibition of Müller glia cell fate by Zeb2 in late stages of retinogenesis. Unbiased transcriptomic profiling of control and Zeb2-deficient early-postnatal retina revealed that Zeb2 functions in inhibiting Id1/2/4 and Hes1 gene expression. These neural progenitor factors normally inhibit neural differentiation and promote Müller glia cell fate. Chromatin immunoprecipitation (ChIP) supported direct regulation of Id1 by Zeb2 in the postnatal retina. Reporter assays and ChIP analyses in differentiating neural progenitors provided further evidence that Zeb2 inhibits Id1 through inhibition of Smad-mediated activation of Id1 transcription. Together, the results suggest that Zeb2 promotes the timely differentiation of retinal interneurons at least in part by repressing BMP-Smad/Notch target genes that inhibit neurogenesis. These findings show that Zeb2 integrates extrinsic cues to regulate the balance between neuronal and glial cell types in the developing murine retina.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Células Ependimogliais/metabolismo , Interneurônios/metabolismo , Retina/embriologia , Transdução de Sinais , Proteínas Smad/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/genética , Camundongos , Camundongos Transgênicos , Proteínas Smad/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética
8.
Sci Immunol ; 5(50)2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826341

RESUMO

Inflammatory bowel disease (IBD) encompasses a spectrum of gastrointestinal disorders driven by dysregulated immune responses against gut microbiota. We integrated single-cell RNA and antigen receptor sequencing to elucidate key components, cellular states, and clonal relationships of the peripheral and gastrointestinal mucosal immune systems in health and ulcerative colitis (UC). UC was associated with an increase in IgG1+ plasma cells in colonic tissue, increased colonic regulatory T cells characterized by elevated expression of the transcription factor ZEB2, and an enrichment of a γδ T cell subset in the peripheral blood. Moreover, we observed heterogeneity in CD8+ tissue-resident memory T (TRM) cells in colonic tissue, with four transcriptionally distinct states of differentiation observed across health and disease. In the setting of UC, there was a marked shift of clonally related CD8+ TRM cells toward an inflammatory state, mediated, in part, by increased expression of the T-box transcription factor Eomesodermin. Together, these results provide a detailed atlas of transcriptional changes occurring in adaptive immune cells in the context of UC and suggest a role for CD8+ TRM cells in IBD.

9.
Hum Mol Genet ; 29(15): 2535-2550, 2020 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-32628253

RESUMO

The transcription factor zinc finger E-box binding protein 2 (ZEB2) controls embryonic and adult cell fate decisions and cellular maturation in many stem/progenitor cell types. Defects in these processes in specific cell types underlie several aspects of Mowat-Wilson syndrome (MOWS), which is caused by ZEB2 haplo-insufficiency. Human ZEB2, like mouse Zeb2, is located on chromosome 2 downstream of a ±3.5 Mb-long gene-desert, lacking any protein-coding gene. Using temporal targeted chromatin capture (T2C), we show major chromatin structural changes based on mapping in-cis proximities between the ZEB2 promoter and this gene desert during neural differentiation of human-induced pluripotent stem cells, including at early neuroprogenitor cell (NPC)/rosette state, where ZEB2 mRNA levels increase significantly. Combining T2C with histone-3 acetylation mapping, we identified three novel candidate enhancers about 500 kb upstream of the ZEB2 transcription start site. Functional luciferase-based assays in heterologous cells and NPCs reveal co-operation between these three enhancers. This study is the first to document in-cis Regulatory Elements located in ZEB2's gene desert. The results further show the usability of T2C for future studies of ZEB2 REs in differentiation and maturation of multiple cell types and the molecular characterization of newly identified MOWS patients that lack mutations in ZEB2 protein-coding exons.

10.
Cancer Res ; 80(14): 2983-2995, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32503808

RESUMO

Epithelial-to-mesenchymal transition (EMT)-inducing transcription factors (TF) are well known for their ability to induce mesenchymal states associated with increased migratory and invasive properties. Unexpectedly, nuclear expression of the EMT-TF ZEB2 in human primary melanoma has been shown to correlate with reduced invasion. We report here that ZEB2 is required for outgrowth for primary melanomas and metastases at secondary sites. Ablation of Zeb2 hampered outgrowth of primary melanomas in vivo, whereas ectopic expression enhanced proliferation and growth at both primary and secondary sites. Gain of Zeb2 expression in pulmonary-residing melanoma cells promoted the development of macroscopic lesions. In vivo fate mapping made clear that melanoma cells undergo a conversion in state where ZEB2 expression is replaced by ZEB1 expression associated with gain of an invasive phenotype. These findings suggest that reversible switching of the ZEB2/ZEB1 ratio enhances melanoma metastatic dissemination. SIGNIFICANCE: ZEB2 function exerts opposing behaviors in melanoma by promoting proliferation and expansion and conversely inhibiting invasiveness, which could be of future clinical relevance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/14/2983/F1.large.jpg.


Assuntos
Proliferação de Células , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/secundário , Melanoma/patologia , Fatores de Transcrição/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Animais , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Melanoma/genética , Melanoma/metabolismo , Camundongos , Invasividade Neoplásica , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética
11.
Development ; 147(10)2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32253238

RESUMO

The transcription factor Zeb2 controls fate specification and subsequent differentiation and maturation of multiple cell types in various embryonic tissues. It binds many protein partners, including activated Smad proteins and the NuRD co-repressor complex. How Zeb2 subdomains support cell differentiation in various contexts has remained elusive. Here, we studied the role of Zeb2 and its domains in neurogenesis and neural differentiation in the young postnatal ventricular-subventricular zone (V-SVZ), in which neural stem cells generate olfactory bulb-destined interneurons. Conditional Zeb2 knockouts and separate acute loss- and gain-of-function approaches indicated that Zeb2 is essential for controlling apoptosis and neuronal differentiation of V-SVZ progenitors before and after birth, and we identified Sox6 as a potential downstream target gene of Zeb2. Zeb2 genetic inactivation impaired the differentiation potential of the V-SVZ niche in a cell-autonomous fashion. We also provide evidence that its normal function in the V-SVZ also involves non-autonomous mechanisms. Additionally, we demonstrate distinct roles for Zeb2 protein-binding domains, suggesting that Zeb2 partners co-determine neuronal output from the mouse V-SVZ in both quantitative and qualitative ways in early postnatal life.


Assuntos
Ventrículos Laterais/embriologia , Ventrículos Laterais/crescimento & desenvolvimento , Neurogênese/genética , Bulbo Olfatório/embriologia , Bulbo Olfatório/crescimento & desenvolvimento , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Animais , Apoptose/genética , Movimento Celular/genética , Proliferação de Células/genética , Técnicas de Inativação de Genes , Interneurônios/metabolismo , Ventrículos Laterais/metabolismo , Camundongos , Camundongos Knockout , Células-Tronco Neurais/metabolismo , Bulbo Olfatório/metabolismo , Fatores de Transcrição SOXD/metabolismo , Transdução de Sinais/imunologia , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética
12.
Int J Mol Sci ; 21(7)2020 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-32260521

RESUMO

Skeletal muscle differentiation is triggered by a unique family of myogenic basic helix-loop-helix transcription factors, including MyoD, MRF-4, Myf-5, and Myogenin. These transcription factors bind promoters and distant regulatory regions, including E-box elements, of genes whose expression is restricted to muscle cells. Other E-box binding zinc finger proteins target the same DNA response elements, however, their function in muscle development and regeneration is still unknown. Here, we show that the transcription factor zinc finger E-box-binding homeobox 2 (Zeb2, Sip-1, Zfhx1b) is present in skeletal muscle tissues. We investigate the role of Zeb2 in skeletal muscle differentiation using genetic tools and transgenic mouse embryonic stem cells, together with single-cell RNA-sequencing and in vivo muscle engraftment capability. We show that Zeb2 over-expression has a positive impact on skeletal muscle differentiation in pluripotent stem cells and adult myogenic progenitors. We therefore propose that Zeb2 is a novel myogenic regulator and a possible target for improving skeletal muscle regeneration. The non-neural roles of Zeb2 are poorly understood.


Assuntos
Diferenciação Celular , Desenvolvimento Muscular , Células-Tronco Pluripotentes/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Animais , Linhagem Celular , Masculino , Camundongos , Camundongos Nus , Músculo Esquelético/citologia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Células-Tronco Pluripotentes/citologia , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética
13.
J Innate Immun ; 12(1): 47-62, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30726833

RESUMO

Recent studies using animal models have generated profound insight into the functions of various subsets of innate lymphoid cells (ILCs). The group 2 ILC subset (ILC2) has been implicated in tissue homeostasis, defense responses against parasites, tissue repair, and immunopathology associated with type-2 immunity. In addition, progress has also been made in translating these findings from animal studies into a context of human immunity. Importantly, recent observations strongly support a role for ILC2s in several diseases of the human respiratory system. However, many aspects of human ILC2 biology are still unclear, including how these cells develop and which signals control their activity. As a result, the exact role played by ILCs in human health and disease remains poorly understood. Here, we summarize our current understanding of human ILC2 biology and focus on their potential involvement in various human respiratory disorders.


Assuntos
Pneumopatias/imunologia , Linfócitos/imunologia , Células Th2/imunologia , Animais , Diferenciação Celular , Plasticidade Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Imunidade Inata
14.
Brief Funct Genomics ; 19(2): 71-82, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-31819944

RESUMO

Chromosomes have a complex three-dimensional (3D) architecture comprising A/B compartments, topologically associating domains and promoter-enhancer interactions. At all these levels, the 3D genome has functional consequences for gene transcription and therefore for cellular identity. The development and activation of lymphocytes involves strict control of gene expression by transcription factors (TFs) operating in a three-dimensionally organized chromatin landscape. As lymphocytes are indispensable for tissue homeostasis and pathogen defense, and aberrant lymphocyte activity is involved in a wide range of human morbidities, acquiring an in-depth understanding of the molecular mechanisms that control lymphocyte identity is highly relevant. Here we review current knowledge of the interplay between 3D genome organization and transcriptional control during B and T lymphocyte development and antigen-dependent activation, placing special emphasis on the role of TFs.

15.
Stem Cells ; 38(2): 202-217, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31675135

RESUMO

Cooperative actions of extrinsic signals and cell-intrinsic transcription factors alter gene regulatory networks enabling cells to respond appropriately to environmental cues. Signaling by transforming growth factor type ß (TGFß) family ligands (eg, bone morphogenetic proteins [BMPs] and Activin/Nodal) exerts cell-type specific and context-dependent transcriptional changes, thereby steering cellular transitions throughout embryogenesis. Little is known about coordinated regulation and transcriptional interplay of the TGFß system. To understand intrafamily transcriptional regulation as part of this system's actions during development, we selected 95 of its components and investigated their mRNA-expression dynamics, gene-gene interactions, and single-cell expression heterogeneity in mouse embryonic stem cells transiting to neural progenitors. Interrogation at 24 hour intervals identified four types of temporal gene transcription profiles that capture all stages, that is, pluripotency, epiblast formation, and neural commitment. Then, between each stage we performed esiRNA-based perturbation of each individual component and documented the effect on steady-state mRNA levels of the remaining 94 components. This exposed an intricate system of multilevel regulation whereby the majority of gene-gene interactions display a marked cell-stage specific behavior. Furthermore, single-cell RNA-profiling at individual stages demonstrated the presence of detailed co-expression modules and subpopulations showing stable co-expression modules such as that of the core pluripotency genes at all stages. Our combinatorial experimental approach demonstrates how intrinsically complex transcriptional regulation within a given pathway is during cell fate/state transitions.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Células-Tronco Embrionárias/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Diferenciação Celular , Humanos
16.
Cells ; 8(8)2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31357500

RESUMO

The exposure of mouse embryos in utero and primary cortical neurons to ionizing radiation results in the P53-dependent activation of a subset of genes that is highly induced during brain development and neuronal maturation, a feature that these genes reportedly share with circular RNAs (circRNAs). Interestingly, some of these genes are predicted to express circular transcripts. In this study, we validated the abundance of the circular transcript variants of four P53 target genes (Pvt1, Ano3, Sec14l5, and Rnf169). These circular variants were overall more stable than their linear counterparts. They were furthermore highly enriched in the brain and their transcript levels continuously increase during subsequent developmental stages (from embryonic day 12 until adulthood), while no further increase could be observed for linear mRNAs beyond post-natal day 30. Finally, whereas radiation-induced expression of P53 target mRNAs peaks early after exposure, several of the circRNAs showed prolonged induction in irradiated embryonic mouse brain, primary mouse cortical neurons, and mouse blood. Together, our results indicate that the circRNAs from these P53 target genes are induced in response to radiation and they corroborate the findings that circRNAs may represent biomarkers of brain age. We also propose that they may be superior to mRNA as long-term biomarkers for radiation exposure.


Assuntos
Encéfalo/metabolismo , Encéfalo/efeitos da radiação , Neurônios/metabolismo , Neurônios/efeitos da radiação , RNA Circular , Radiação Ionizante , Processamento Alternativo , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Masculino , Neurônios/citologia , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
18.
Development ; 145(13)2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29884675

RESUMO

Upon gastrulation, the mammalian conceptus transforms rapidly from a simple bilayer into a multilayered embryo enveloped by its extra-embryonic membranes. Impaired development of the amnion, the innermost membrane, causes major malformations. To clarify the origin of the mouse amnion, we used single-cell labelling and clonal analysis. We identified four clone types with distinct clonal growth patterns in amniotic ectoderm. Two main types have progenitors in extreme proximal-anterior epiblast. Early descendants initiate and expand amniotic ectoderm posteriorly, while descendants of cells remaining anteriorly later expand amniotic ectoderm from its anterior side. Amniogenesis is abnormal in embryos deficient in the bone morphogenetic protein (BMP) signalling effector SMAD5, with delayed closure of the proamniotic canal, and aberrant amnion and folding morphogenesis. Transcriptomics of individual Smad5 mutant amnions isolated before visible malformations and tetraploid chimera analysis revealed two amnion defect sets. We attribute them to impairment of progenitors of the two main cell populations in amniotic ectoderm and to compromised cuboidal-to-squamous transition of anterior amniotic ectoderm. In both cases, SMAD5 is crucial for expanding amniotic ectoderm rapidly into a stretchable squamous sheet to accommodate exocoelom expansion, axial growth and folding morphogenesis.


Assuntos
Âmnio/embriologia , Ectoderma/embriologia , Morfogênese/fisiologia , Transdução de Sinais/fisiologia , Proteína Smad5/metabolismo , Células-Tronco/metabolismo , Âmnio/citologia , Animais , Ectoderma/citologia , Camundongos , Proteína Smad5/genética , Células-Tronco/citologia
19.
Nat Commun ; 8(1): 1249, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-29093487

RESUMO

Muscular dystrophies (MDs) are often characterized by impairment of both skeletal and cardiac muscle. Regenerative strategies for both compartments therefore constitute a therapeutic avenue. Mesodermal iPSC-derived progenitors (MiPs) can regenerate both striated muscle types simultaneously in mice. Importantly, MiP myogenic propensity is influenced by somatic lineage retention. However, it is still unknown whether human MiPs have in vivo potential. Furthermore, methods to enhance the intrinsic myogenic properties of MiPs are likely needed, given the scope and need to correct large amounts of muscle in the MDs. Here, we document that human MiPs can successfully engraft into the skeletal muscle and hearts of dystrophic mice. Utilizing non-invasive live imaging and selectively induced apoptosis, we report evidence of striated muscle regeneration in vivo in mice by human MiPs. Finally, combining RNA-seq and miRNA-seq data, we define miRNA cocktails that promote the myogenic potential of human MiPs.


Assuntos
Coração/crescimento & desenvolvimento , Células-Tronco Pluripotentes Induzidas/citologia , Mesoderma/citologia , MicroRNAs/genética , Desenvolvimento Muscular/genética , Músculo Esquelético/crescimento & desenvolvimento , Distrofia Muscular Animal/patologia , Miocárdio/citologia , Animais , Diferenciação Celular , Ecocardiografia , Coração/diagnóstico por imagem , Humanos , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/diagnóstico por imagem , Músculo Esquelético/patologia , Distrofia Muscular Animal/diagnóstico por imagem , Miocárdio/patologia , Regeneração
20.
Sci Rep ; 7(1): 8568, 2017 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-28819210

RESUMO

Neural connectivity requires neuronal differentiation, axon growth, and precise target innervation. Midbrain dopaminergic neurons project via the nigrostriatal pathway to the striatum to regulate voluntary movement. While the specification and differentiation of these neurons have been extensively studied, the molecular mechanisms that regulate midbrain dopaminergic axon growth and target innervation are less clear. Here we show that the transcription factor Zeb2 cell-autonomously represses Smad signalling to limit midbrain dopaminergic axon growth and target innervation. Zeb2 levels are downregulated in the embryonic rodent midbrain during the period of dopaminergic axon growth, when BMP pathway components are upregulated. Experimental knockdown of Zeb2 leads to an increase in BMP-Smad-dependent axon growth. Consequently there is dopaminergic hyperinnervation of the striatum, without an increase in the numbers of midbrain dopaminergic neurons, in conditional Zeb2 (Nestin-Cre based) knockout mice. Therefore, these findings reveal a new mechanism for the regulation of midbrain dopaminergic axon growth during central nervous system development.


Assuntos
Axônios/metabolismo , Neurônios Dopaminérgicos/metabolismo , Mesencéfalo/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Animais , Linhagem Celular Tumoral , Corpo Estriado/citologia , Corpo Estriado/metabolismo , Feminino , Humanos , Mesencéfalo/citologia , Camundongos Knockout , Camundongos Transgênicos , Nestina/genética , Nestina/metabolismo , Interferência de RNA , Ratos Sprague-Dawley , Substância Negra/citologia , Substância Negra/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética
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