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1.
Int J Mol Sci ; 20(21)2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31671683

RESUMO

The exponential increase of patients with diabetes mellitus urges for novel therapeutic strategies to reduce the socioeconomic burden of this disease. The loss or dysfunction of insulin-producing ß-cells, in patients with type 1 and type 2 diabetes respectively, put these cells at the center of the disease initiation and progression. Therefore, major efforts have been taken to restore the ß-cell mass by cell-replacement or regeneration approaches. Implementing novel therapies requires deciphering the developmental mechanisms that generate ß-cells and determine the acquisition of their physiological phenotype. In this review, we summarize the current understanding of the mechanisms that coordinate the postnatal maturation of ß-cells and define their functional identity. Furthermore, we discuss different routes by which ß-cells lose their features and functionality in type 1 and 2 diabetic conditions. We then focus on potential mechanisms to restore the functionality of those ß-cell populations that have lost their functional phenotype. Finally, we discuss the recent progress and remaining challenges facing the generation of functional mature ß-cells from stem cells for cell-replacement therapy for diabetes treatment.

2.
Stem Cell Res ; 39: 101531, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31419739

RESUMO

Induced pluripotent stem cells (iPSCs) can be used to generate different somatic cell types in vitro, including insulin-producing pancreatic ß-cells. Here, we have generated iPSCs from a healthy male individual using an episomal reprogramming method. The resulting iPSCs are integration-free, have a normal karyotype and are pluripotent in vitro and in vivo. Furthermore, we show that this iPSC line can be differentiated into pancreatic lineage cells. Taken together, this iPSC line will be useful to test differentiation protocols towards ß-cell as well as other cell types and will also serve as a control for drug development and disease modelling studies.

3.
Curr Opin Cell Biol ; 61: 48-55, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31377680

RESUMO

The pancreas is derived from the foregut endoderm during embryonic development. After gastrulation and endoderm germ layer formation complex morphogenetic events coupled with cell differentiation programs pattern the gut tube and induce pancreas organogenesis. This results in formation of exocrine, ductal and hormone-producing endocrine cells. Among these, endocrine cells are responsible for blood glucose homeostasis and their malfunction leads to diabetes mellitus, which cannot be stopped or reversed by the current standard treatments. Thus, intense efforts to regenerate or replace the lost or dysfunctional insulin-producing ß-cells are on the way. This depends on identifying the factors that coordinate pancreas organogenesis. Here, we highlight the contribution of canonical and non-canonical Wnt signaling branches in orchestrating endoderm formation, pancreatic morphogenesis as well as endocrine cell formation and function.

4.
Sci Rep ; 9(1): 11953, 2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31420575

RESUMO

The endocardium is the endothelial component of the vertebrate heart and plays a key role in heart development. Where, when, and how the endocardium segregates during embryogenesis have remained largely unknown, however. We now show that Nkx2-5+ cardiac progenitor cells (CPCs) that express the Sry-type HMG box gene Sox17 from embryonic day (E) 7.5 to E8.5 specifically differentiate into the endocardium in mouse embryos. Although Sox17 is not essential or sufficient for endocardium fate, it can bias the fate of CPCs toward the endocardium. On the other hand, Sox17 expression in the endocardium is required for heart development. Deletion of Sox17 specifically in the mesoderm markedly impaired endocardium development with regard to cell proliferation and behavior. The proliferation of cardiomyocytes, ventricular trabeculation, and myocardium thickening were also impaired in a non-cell-autonomous manner in the Sox17 mutant, likely as a consequence of down-regulation of NOTCH signaling. An unknown signal, regulated by Sox17 and required for nurturing of the myocardium, is responsible for the reduction in NOTCH-related genes in the mutant embryos. Our results thus provide insight into differentiation of the endocardium and its role in heart development.

5.
Nucleic Acids Res ; 47(17): 9069-9086, 2019 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-31350899

RESUMO

Pioneer transcription factors (PTF) can recognize their binding sites on nucleosomal DNA and trigger chromatin opening for recruitment of other non-pioneer transcription factors. However, critical properties of PTFs are still poorly understood, such as how these transcription factors selectively recognize cell type-specific binding sites and under which conditions they can initiate chromatin remodelling. Here we show that early endoderm binding sites of the paradigm PTF Foxa2 are epigenetically primed by low levels of active chromatin modifications in embryonic stem cells (ESC). Priming of these binding sites is supported by preferential recruitment of Foxa2 to endoderm binding sites compared to lineage-inappropriate binding sites, when ectopically expressed in ESCs. We further show that binding of Foxa2 is required for chromatin opening during endoderm differentiation. However, increased chromatin accessibility was only detected on binding sites which are synergistically bound with other endoderm transcription factors. Thus, our data suggest that binding site selection of PTFs is directed by the chromatin environment and that chromatin opening requires collaboration of PTFs with additional transcription factors.

6.
Development ; 146(12)2019 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-31160421

RESUMO

Deciphering mechanisms of endocrine cell induction, specification and lineage allocation in vivo will provide valuable insights into how the islets of Langerhans are generated. Currently, it is ill defined how endocrine progenitors segregate into different endocrine subtypes during development. Here, we generated a novel neurogenin 3 (Ngn3)-Venus fusion (NVF) reporter mouse line, that closely mirrors the transient endogenous Ngn3 protein expression. To define an in vivo roadmap of endocrinogenesis, we performed single cell RNA sequencing of 36,351 pancreatic epithelial and NVF+ cells during secondary transition. This allowed Ngn3 low endocrine progenitors, Ngn3 high endocrine precursors, Fev+ endocrine lineage and hormone+ endocrine subtypes to be distinguished and time-resolved, and molecular programs during the step-wise lineage restriction steps to be delineated. Strikingly, we identified 58 novel signature genes that show the same transient expression dynamics as Ngn3 in the 7260 profiled Ngn3-expressing cells. The differential expression of these genes in endocrine precursors associated with their cell-fate allocation towards distinct endocrine cell types. Thus, the generation of an accurately regulated NVF reporter allowed us to temporally resolve endocrine lineage development to provide a fine-grained single cell molecular profile of endocrinogenesis in vivo.

7.
Development ; 146(12)2019 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-31249007

RESUMO

Single cell genomics has become a popular approach to uncover the cellular heterogeneity of progenitor and terminally differentiated cell types with great precision. This approach can also delineate lineage hierarchies and identify molecular programmes of cell-fate acquisition and segregation. Nowadays, tens of thousands of cells are routinely sequenced in single cell-based methods and even more are expected to be analysed in the future. However, interpretation of the resulting data is challenging and requires computational models at multiple levels of abstraction. In contrast to other applications of single cell sequencing, where clustering approaches dominate, developmental systems are generally modelled using continuous structures, trajectories and trees. These trajectory models carry the promise of elucidating mechanisms of development, disease and stimulation response at very high molecular resolution. However, their reliable analysis and biological interpretation requires an understanding of their underlying assumptions and limitations. Here, we review the basic concepts of such computational approaches and discuss the characteristics of developmental processes that can be learnt from trajectory models.

8.
Mol Cell ; 74(5): 951-965.e13, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31047794

RESUMO

RNA-binding proteins (RBPs) and long non-coding RNAs (lncRNAs) are key regulators of gene expression, but their joint functions in coordinating cell fate decisions are poorly understood. Here we show that the expression and activity of the RBP TDP-43 and the long isoform of the lncRNA Neat1, the scaffold of the nuclear compartment "paraspeckles," are reciprocal in pluripotent and differentiated cells because of their cross-regulation. In pluripotent cells, TDP-43 represses the formation of paraspeckles by enhancing the polyadenylated short isoform of Neat1. TDP-43 also promotes pluripotency by regulating alternative polyadenylation of transcripts encoding pluripotency factors, including Sox2, which partially protects its 3' UTR from miR-21-mediated degradation. Conversely, paraspeckles sequester TDP-43 and other RBPs from mRNAs and promote exit from pluripotency and embryonic patterning in the mouse. We demonstrate that cross-regulation between TDP-43 and Neat1 is essential for their efficient regulation of a broad network of genes and, therefore, of pluripotency and differentiation.


Assuntos
Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias Murinas/metabolismo , RNA Longo não Codificante/genética , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , MicroRNAs/genética , Células-Tronco Pluripotentes/metabolismo , Poliadenilação/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
9.
Nature ; 569(7756): 342-343, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31076731
10.
Nat Biotechnol ; 37(4): 461-468, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30936567

RESUMO

Recent single-cell RNA-sequencing studies have suggested that cells follow continuous transcriptomic trajectories in an asynchronous fashion during development. However, observations of cell flux along trajectories are confounded with population size effects in snapshot experiments and are therefore hard to interpret. In particular, changes in proliferation and death rates can be mistaken for cell flux. Here we present pseudodynamics, a mathematical framework that reconciles population dynamics with the concepts underlying developmental trajectories inferred from time-series single-cell data. Pseudodynamics models population distribution shifts across trajectories to quantify selection pressure, population expansion, and developmental potentials. Applying this model to time-resolved single-cell RNA-sequencing of T-cell and pancreatic beta cell maturation, we characterize proliferation and apoptosis rates and identify key developmental checkpoints, data inaccessible to existing approaches.


Assuntos
Diferenciação Celular/genética , Análise de Sequência de RNA/estatística & dados numéricos , Análise de Célula Única/estatística & dados numéricos , Animais , Apoptose/genética , Biotecnologia , Proliferação de Células/genética , Feminino , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Funções Verossimilhança , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Fatores de Tempo
11.
Dev Cell ; 49(1): 1-3, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30965032

RESUMO

How organ morphogenesis specifies cell fate and whether organ progenitors are predetermined or specified via niche signals are critical developmental biology questions. In this issue of Developmental Cell, Nyeng et al. (2019) modulate cell-cell adhesion in the pancreas and provide evidence that progenitors are plastic and instructed by niche signals.


Assuntos
Pâncreas , Cateninas , Diferenciação Celular , Movimento Celular , Morfogênese
12.
Mol Metab ; 24: 80-97, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30930126

RESUMO

OBJECTIVE: Hundreds of missense mutations in the coding region of PDX1 exist; however, if these mutations predispose to diabetes mellitus is unknown. METHODS: In this study, we screened a large cohort of subjects with increased risk for diabetes and identified two subjects with impaired glucose tolerance carrying common, heterozygous, missense mutations in the PDX1 coding region leading to single amino acid exchanges (P33T, C18R) in its transactivation domain. We generated iPSCs from patients with heterozygous PDX1P33T/+, PDX1C18R/+ mutations and engineered isogenic cell lines carrying homozygous PDX1P33T/P33T, PDX1C18R/C18R mutations and a heterozygous PDX1 loss-of-function mutation (PDX1+/-). RESULTS: Using an in vitro ß-cell differentiation protocol, we demonstrated that both, heterozygous PDX1P33T/+, PDX1C18R/+ and homozygous PDX1P33T/P33T, PDX1C18R/C18R mutations impair ß-cell differentiation and function. Furthermore, PDX1+/- and PDX1P33T/P33T mutations reduced differentiation efficiency of pancreatic progenitors (PPs), due to downregulation of PDX1-bound genes, including transcription factors MNX1 and PDX1 as well as insulin resistance gene CES1. Additionally, both PDX1P33T/+ and PDX1P33T/P33T mutations in PPs reduced the expression of PDX1-bound genes including the long-noncoding RNA, MEG3 and the imprinted gene NNAT, both involved in insulin synthesis and secretion. CONCLUSIONS: Our results reveal mechanistic details of how common coding mutations in PDX1 impair human pancreatic endocrine lineage formation and ß-cell function and contribute to the predisposition for diabetes.

13.
SLAS Discov ; 24(3): 398-413, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30616481

RESUMO

Compound screening in biological assays and subsequent optimization of hits is indispensable for the development of new molecular research tools and drug candidates. To facilitate such discoveries, the European Research Infrastructure EU-OPENSCREEN was founded recently with the support of its member countries and the European Commission. Its distributed character harnesses complementary knowledge, expertise, and instrumentation in the discipline of chemical biology from 20 European partners, and its open working model ensures that academia and industry can readily access EU-OPENSCREEN's compound collection, equipment, and generated data. To demonstrate the power of this collaborative approach, this perspective article highlights recent projects from EU-OPENSCREEN partner institutions. These studies yielded (1) 2-aminoquinazolin-4(3 H)-ones as potential lead structures for new antimalarial drugs, (2) a novel lipodepsipeptide specifically inducing apoptosis in cells deficient for the pVHL tumor suppressor, (3) small-molecule-based ROCK inhibitors that induce definitive endoderm formation and can potentially be used for regenerative medicine, (4) potential pharmacological chaperones for inborn errors of metabolism and a familiar form of acute myeloid leukemia (AML), and (5) novel tankyrase inhibitors that entered a lead-to-candidate program. Collectively, these findings highlight the benefits of small-molecule screening, the plethora of assay designs, and the close connection between screening and medicinal chemistry within EU-OPENSCREEN.

14.
Nat Rev Endocrinol ; 2018 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-30504925

RESUMO

Diabetes mellitus is a multifactorial disease affecting increasing numbers of patients worldwide. Progression to insulin-dependent diabetes mellitus is characterized by the loss or dysfunction of pancreatic ß-cells, but the pathomechanisms underlying ß-cell failure in type 1 diabetes mellitus and type 2 diabetes mellitus are still poorly defined. Regeneration of ß-cell mass from residual islet cells or replacement by ß-like cells derived from stem cells holds great promise to stop or reverse disease progression. However, the development of new treatment options is hampered by our limited understanding of human pancreas organogenesis due to the restricted access to primary tissues. Therefore, the challenge is to translate results obtained from preclinical model systems to humans, which requires comparative modelling of ß-cell biology in health and disease. Here, we discuss diverse modelling systems across different species that provide spatial and temporal resolution of cellular and molecular mechanisms to understand the evolutionary conserved genotype-phenotype relationship and translate them to humans. In addition, we summarize the latest knowledge on organoids, stem cell differentiation platforms, primary micro-islets and pseudo-islets, bioengineering and microfluidic systems for studying human pancreas development and homeostasis ex vivo. These new modelling systems and platforms have opened novel avenues for exploring the developmental trajectory, physiology, biology and pathology of the human pancreas.

15.
Dev Biol ; 2018 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-30472119

RESUMO

The enteric nervous system is thought to originate solely from the neural crest. Transgenic lineage tracing revealed a novel population of clonal pancreatic duodenal homeobox-1 (Pdx1)-Cre lineage progenitor cells in the tunica muscularis of the gut that produced pancreatic descendants as well as neurons upon differentiation in vitro. Additionally, an in vivo subpopulation of endoderm lineage enteric neurons, but not glial cells, was seen especially in the proximal gut. Analysis of early transgenic embryos revealed Pdx1-Cre progeny (as well as Sox-17-Cre and Foxa2-Cre progeny) migrating from the developing pancreas and duodenum at E11.5 and contributing to the enteric nervous system. These results show that the mammalian enteric nervous system arises from both the neural crest and the endoderm. Moreover, in adult mice there are separate Wnt1-Cre neural crest stem cells and Pdx1-Cre pancreatic progenitors within the muscle layer of the gut.

16.
Development ; 145(17)2018 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-30093553

RESUMO

Mutations in Hes1, a target gene of the Notch signalling pathway, lead to ectopic pancreas by a poorly described mechanism. Here, we use genetic inactivation of Hes1 combined with lineage tracing and live imaging to reveal an endodermal requirement for Hes1, and show that ectopic pancreas tissue is derived from the dorsal pancreas primordium. RNA-seq analysis of sorted E10.5 Hes1+/+ and Hes1-/- Pdx1-GFP+ cells suggested that upregulation of endocrine lineage genes in Hes1-/- embryos was the major defect and, accordingly, early pancreas morphogenesis was normalized, and the ectopic pancreas phenotype suppressed, in Hes1-/-Neurog3-/- embryos. In Mib1 mutants, we found a near total depletion of dorsal progenitors, which was replaced by an anterior Gcg+ extension. Together, our results demonstrate that aberrant morphogenesis is the cause of ectopic pancreas and that a part of the endocrine differentiation program is mechanistically involved in the dysgenesis. Our results suggest that the ratio of endocrine lineage to progenitor cells is important for morphogenesis and that a strong endocrinogenic phenotype without complete progenitor depletion, as seen in Hes1 mutants, provokes an extreme dysgenesis that causes ectopic pancreas.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Coristoma/genética , Morfogênese/genética , Proteínas do Tecido Nervoso/genética , Pâncreas/anormalidades , Pâncreas/embriologia , Fatores de Transcrição HES-1/genética , Animais , Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ubiquitina-Proteína Ligases/genética
17.
Metabolites ; 8(3)2018 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-30134533

RESUMO

Night shift work can have a serious impact on health. Here, we assess whether and how night shift work influences the metabolite profiles, specifically with respect to different chronotype classes. We have recruited 100 women including 68 nurses working both, day shift and night shifts for up to 5 consecutive days and collected 3640 spontaneous urine samples. About 424 waking-up urine samples were measured using a targeted metabolomics approach. To account for urine dilution, we applied three methods to normalize the metabolite values: creatinine-, osmolality- and regression-based normalization. Based on linear mixed effect models, we found 31 metabolites significantly (false discovery rate <0.05) affected in nurses working in night shifts. One metabolite, acylcarnitine C10:2, was consistently identified with all three normalization methods. We further observed 11 and 4 metabolites significantly associated with night shift in early and late chronotype classes, respectively. Increased levels of medium- and long chain acylcarnitines indicate a strong impairment of the fatty acid oxidation. Our results show that night shift work influences acylcarnitines and BCAAs, particularly in nurses in the early chronotype class. Women with intermediate and late chronotypes appear to be less affected by night shift work.

18.
Mol Metab ; 9: 57-68, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29396371

RESUMO

OBJECTIVE: Homozygous loss-of-function mutations in the gene coding for the homeobox transcription factor (TF) PDX1 leads to pancreatic agenesis, whereas heterozygous mutations can cause Maturity-Onset Diabetes of the Young 4 (MODY4). Although the function of Pdx1 is well studied in pre-clinical models during insulin-producing ß-cell development and homeostasis, it remains elusive how this TF controls human pancreas development by regulating a downstream transcriptional program. Also, comparative studies of PDX1 binding patterns in pancreatic progenitors and adult ß-cells have not been conducted so far. Furthermore, many studies reported the association between single nucleotide polymorphisms (SNPs) and T2DM, and it has been shown that islet enhancers are enriched in T2DM-associated SNPs. Whether regions, harboring T2DM-associated SNPs are PDX1 bound and active at the pancreatic progenitor stage has not been reported so far. METHODS: In this study, we have generated a novel induced pluripotent stem cell (iPSC) line that efficiently differentiates into human pancreatic progenitors (PPs). Furthermore, PDX1 and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify PDX1 transcriptional targets and active enhancer and promoter regions. To address potential differences in the function of PDX1 during development and adulthood, we compared PDX1 binding profiles from PPs and adult islets. Moreover, combining ChIP-seq and GWAS meta-analysis data we identified T2DM-associated SNPs in PDX1 binding sites and active chromatin regions. RESULTS: ChIP-seq for PDX1 revealed a total of 8088 PDX1-bound regions that map to 5664 genes in iPSC-derived PPs. The PDX1 target regions include important pancreatic TFs, such as PDX1 itself, RFX6, HNF1B, and MEIS1, which were activated during the differentiation process as revealed by the active chromatin mark H3K27ac and mRNA expression profiling, suggesting that auto-regulatory feedback regulation maintains PDX1 expression and initiates a pancreatic TF program. Remarkably, we identified several PDX1 target genes that have not been reported in the literature in human so far, including RFX3, required for ciliogenesis and endocrine differentiation in mouse, and the ligand of the Notch receptor DLL1, which is important for endocrine induction and tip-trunk patterning. The comparison of PDX1 profiles from PPs and adult human islets identified sets of stage-specific target genes, associated with early pancreas development and adult ß-cell function, respectively. Furthermore, we found an enrichment of T2DM-associated SNPs in active chromatin regions from iPSC-derived PPs. Two of these SNPs fall into PDX1 occupied sites that are located in the intronic regions of TCF7L2 and HNF1B. Both of these genes are key transcriptional regulators of endocrine induction and mutations in cis-regulatory regions predispose to diabetes. CONCLUSIONS: Our data provide stage-specific target genes of PDX1 during in vitro differentiation of stem cells into pancreatic progenitors that could be useful to identify pathways and molecular targets that predispose for diabetes. In addition, we show that T2DM-associated SNPs are enriched in active chromatin regions at the pancreatic progenitor stage, suggesting that the susceptibility to T2DM might originate from imperfect execution of a ß-cell developmental program.

19.
Dev Biol ; 435(2): 130-137, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29397878

RESUMO

Failure of neural tube closure leads to neural tube defects (NTDs), common congenital abnormalities in humans. Among the genes whose loss of function causes NTDs in mice, Grainyhead-like3 (Grhl3) is essential for spinal neural tube closure, with null mutants exhibiting fully penetrant spina bifida. During spinal neurulation Grhl3 is initially expressed in the surface (non-neural) ectoderm, subsequently in the neuroepithelial component of the neural folds and at the node-streak border, and finally in the hindgut endoderm. Here, we show that endoderm-specific knockout of Grhl3 causes late-arising spinal NTDs, preceded by increased ventral curvature of the caudal region which was shown previously to suppress closure of the spinal neural folds. This finding supports the hypothesis that diminished Grhl3 expression in the hindgut is the cause of spinal NTDs in the curly tail, carrying a hypomorphic Grhl3 allele. Complete loss of Grhl3 function produces a more severe phenotype in which closure fails earlier in neurulation, before the stage of onset of expression in the hindgut of wild-type embryos. This implicates additional tissues and NTD mechanisms in Grhl3 null embryos. Conditional knockout of Grhl3 in the neural plate and node-streak border has minimal effect on closure, suggesting that abnormal function of surface ectoderm, where Grhl3 transcripts are first detected, is primarily responsible for early failure of spinal neurulation in Grhl3 null embryos.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Defeitos do Tubo Neural/genética , Tubo Neural/fisiologia , Neurulação/genética , Fatores de Transcrição/fisiologia , Animais , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Camadas Germinativas/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Placa Neural/metabolismo , Defeitos do Tubo Neural/embriologia , Defeitos do Tubo Neural/patologia , Especificidade de Órgãos , RNA Mensageiro/biossíntese , Disrafismo Espinal/embriologia , Disrafismo Espinal/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética
20.
Exp Cell Res ; 365(1): 145-155, 2018 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-29458175

RESUMO

The aorta-gonad-mesonephros region, from which definitive hematopoiesis first arises in midgestation mouse embryos, has intra-aortic hematopoietic clusters (IAHCs) containing hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). We previously reported expression of the transcription factor Sox17 in IAHCs, and overexpression of Sox17 in CD45lowc-KIThigh cells comprising IAHCs maintains the formation of cell clusters and their multipotency in vitro over multiple passages. Here, we demonstrate the importance of NOTCH1 in IAHC formation and maintenance of the HSC/HPC phenotype. We further show that Notch1 expression is positively regulated by SOX17 via direct binding to its gene promoter. SOX17 and NOTCH1 were both found to be expressed in vivo in cells of IAHCs by whole mount immunostaining. We found that cells transduced with the active form of NOTCH1 or its downstream target, Hes1, maintained their multipotent colony-forming capacity in semisolid medium. Moreover, cells stimulated by NOTCH1 ligand, Jagged1, or Delta-like protein 1, had the capacity to form multilineage colonies. Conversely, knockdown of Notch1 and Hes1 led to a reduction of their multipotent colony-forming capacity. These results suggest that the Sox17-Notch1-Hes1 pathway is critical for maintaining the undifferentiated state of IAHCs.

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