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1.
Hepatology ; 72(5): 1786-1799, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32060934

RESUMO

BACKGROUND AND AIMS: During liver development, bipotent progenitor cells differentiate into hepatocytes and biliary epithelial cells to ensure a functional liver required to maintain organismal homeostasis. The developmental cues controlling the differentiation of committed progenitors into these cell types, however, are incompletely understood. Here, we discover an essential role for estrogenic regulation in vertebrate liver development to affect hepatobiliary fate decisions. APPROACH AND RESULTS: Exposure of zebrafish embryos to 17ß-estradiol (E2) during liver development significantly decreased hepatocyte-specific gene expression, liver size, and hepatocyte number. In contrast, pharmacological blockade of estrogen synthesis or nuclear estrogen receptor (ESR) signaling enhanced liver size and hepatocyte marker expression. Transgenic reporter fish demonstrated nuclear ESR activity in the developing liver. Chemical inhibition and morpholino knockdown of nuclear estrogen receptor 2b (esr2b) increased hepatocyte gene expression and blocked the effects of E2 exposure. esr2b-/- mutant zebrafish exhibited significantly increased expression of hepatocyte markers with no impact on liver progenitors, other endodermal lineages, or vasculature. Significantly, E2-stimulated Esr2b activity promoted biliary epithelial differentiation at the expense of hepatocyte fate, whereas loss of esr2b impaired biliary lineage commitment. Chemical and genetic epistasis studies identified bone morphogenetic protein (BMP) signaling as a mediator of the estrogen effects. The divergent impact of estrogen on hepatobiliary fate was confirmed in a human hepatoblast cell line, indicating the relevance of this pathway for human liver development. CONCLUSIONS: Our studies identify E2, esr2b, and downstream BMP activity as important regulators of hepatobiliary fate decisions during vertebrate liver development. These results have significant clinical implications for liver development in infants exposed to abnormal estrogen levels or estrogenic compounds during pregnancy.


Assuntos
Sistema Biliar/embriologia , Estradiol/metabolismo , Receptor beta de Estrogênio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fígado/embriologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Sistema Biliar/citologia , Sistema Biliar/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Embrião não Mamífero , Estradiol/administração & dosagem , Receptor beta de Estrogênio/genética , Feminino , Técnicas de Silenciamento de Genes , Hepatócitos/fisiologia , Fígado/citologia , Fígado/metabolismo , Masculino , Modelos Animais , Morfolinos/administração & dosagem , Morfolinos/genética , Transdução de Sinais/genética , Células-Tronco/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
2.
Genome Res ; 22(8): 1541-8, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22555591

RESUMO

Genetic mapping of mutations in model systems has facilitated the identification of genes contributing to fundamental biological processes including human diseases. However, this approach has historically required the prior characterization of informative markers. Here we report a fast and cost-effective method for genetic mapping using next-generation sequencing that combines single nucleotide polymorphism discovery, mutation localization, and potential identification of causal sequence variants. In contrast to prior approaches, we have developed a hidden Markov model to narrowly define the mutation area by inferring recombination breakpoints of chromosomes in the mutant pool. In addition, we created an interactive online software resource to facilitate automated analysis of sequencing data and demonstrate its utility in the zebrafish and mouse models. Our novel methodology and online tools will make next-generation sequencing an easily applicable resource for mutation mapping in all model systems.


Assuntos
Análise Mutacional de DNA/métodos , Software , Peixe-Zebra/genética , Alelos , Animais , Mapeamento Cromossômico/métodos , Cromossomos/genética , Cruzamentos Genéticos , Feminino , Frequência do Gene , Genômica/métodos , Homozigoto , Masculino , Cadeias de Markov , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Polimorfismo de Nucleotídeo Único , Recombinação Genética , Fatores de Tempo
3.
Blood ; 121(13): 2483-93, 2013 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-23341543

RESUMO

Many pathways regulating blood formation have been elucidated, yet how each coordinates with embryonic biophysiology to modulate the spatiotemporal production of hematopoietic stem cells (HSCs) is currently unresolved. Here, we report that glucose metabolism impacts the onset and magnitude of HSC induction in vivo. In zebrafish, transient elevations in physiological glucose levels elicited dose-dependent effects on HSC development, including enhanced runx1 expression and hematopoietic cluster formation in the aorta-gonad-mesonephros region; embryonic-to-adult transplantation studies confirmed glucose increased functional HSCs. Glucose uptake was required to mediate the enhancement in HSC development; likewise, metabolic inhibitors diminished nascent HSC production and reversed glucose-mediated effects on HSCs. Increased glucose metabolism preferentially impacted hematopoietic and vascular targets, as determined by gene expression analysis, through mitochondrial-derived reactive oxygen species (ROS)-mediated stimulation of hypoxia-inducible factor 1α (hif1α). Epistasis assays demonstrated that hif1α regulates HSC formation in vivo and mediates the dose-dependent effects of glucose metabolism on the timing and magnitude of HSC production. We propose that this fundamental metabolic-sensing mechanism enables the embryo to respond to changes in environmental energy input and adjust hematopoietic output to maintain embryonic growth and ensure viability.


Assuntos
Metabolismo dos Carboidratos/fisiologia , Indução Embrionária , Glucose/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Animais , Animais Geneticamente Modificados , Metabolismo dos Carboidratos/genética , Proliferação de Células/efeitos dos fármacos , Embrião não Mamífero , Indução Embrionária/efeitos dos fármacos , Indução Embrionária/genética , Regulação da Expressão Gênica no Desenvolvimento , Glucose/farmacologia , Glicólise/efeitos dos fármacos , Glicólise/genética , Glicólise/fisiologia , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Fosforilação Oxidativa , Fatores de Tempo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
4.
PLoS Genet ; 7(9): e1002264, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21931561

RESUMO

Chronic kidney disease (CKD) is an increasing global public health concern, particularly among populations of African ancestry. We performed an interrogation of known renal loci, genome-wide association (GWA), and IBC candidate-gene SNP association analyses in African Americans from the CARe Renal Consortium. In up to 8,110 participants, we performed meta-analyses of GWA and IBC array data for estimated glomerular filtration rate (eGFR), CKD (eGFR <60 mL/min/1.73 m(2)), urinary albumin-to-creatinine ratio (UACR), and microalbuminuria (UACR >30 mg/g) and interrogated the 250 kb flanking region around 24 SNPs previously identified in European Ancestry renal GWAS analyses. Findings were replicated in up to 4,358 African Americans. To assess function, individually identified genes were knocked down in zebrafish embryos by morpholino antisense oligonucleotides. Expression of kidney-specific genes was assessed by in situ hybridization, and glomerular filtration was evaluated by dextran clearance. Overall, 23 of 24 previously identified SNPs had direction-consistent associations with eGFR in African Americans, 2 of which achieved nominal significance (UMOD, PIP5K1B). Interrogation of the flanking regions uncovered 24 new index SNPs in African Americans, 12 of which were replicated (UMOD, ANXA9, GCKR, TFDP2, DAB2, VEGFA, ATXN2, GATM, SLC22A2, TMEM60, SLC6A13, and BCAS3). In addition, we identified 3 suggestive loci at DOK6 (p-value = 5.3×10(-7)) and FNDC1 (p-value = 3.0×10(-7)) for UACR, and KCNQ1 with eGFR (p = 3.6×10(-6)). Morpholino knockdown of kcnq1 in the zebrafish resulted in abnormal kidney development and filtration capacity. We identified several SNPs in association with eGFR in African Ancestry individuals, as well as 3 suggestive loci for UACR and eGFR. Functional genetic studies support a role for kcnq1 in glomerular development in zebrafish.


Assuntos
População Negra/genética , Loci Gênicos , Taxa de Filtração Glomerular/genética , Canal de Potássio KCNQ1/genética , Falência Renal Crônica/genética , Rim/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular/genética , Adulto , Idoso , Animais , Feminino , Técnicas de Silenciamento de Genes , Estudos de Associação Genética , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
5.
Dev Biol ; 372(2): 178-89, 2012 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-22982668

RESUMO

Developmental signals determine organ morphology and position during embryogenesis. To discover novel modifiers of liver development, we performed a chemical genetic screen in zebrafish and identified retinoic acid as a positive regulator of hepatogenesis. Knockdown of the four RA receptors revealed that all receptors affect liver formation, however specific receptors exert differential effects. Rargb knockdown results in bilateral livers but does not impact organ size, revealing a unique role for Rargb in conferring left-right positional information. Bilateral populations of hepatoblasts are detectable in rargb morphants, indicating Rargb acts during hepatic specification to position the liver, and primitive endoderm is competent to form liver on both sides. Hearts remain at the midline and gut looping is perturbed in rargb morphants, suggesting Rargb affects lateral plate mesoderm migration. Overexpression of Bmp during somitogenesis similarly results in bilateral livers and midline hearts, and inhibition of Bmp signaling rescues the rargb morphant phenotype, indicating Rargb functions upstream of Bmp to regulate organ sidedness. Loss of rargb causes biliary and organ laterality defects as well as asplenia, paralleling symptoms of the human condition right atrial isomerism. Our findings uncover a novel role for RA in regulating organ laterality and provide an animal model of one form of human heterotaxia.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Receptores do Ácido Retinoico/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Padronização Corporal , Embrião não Mamífero/metabolismo , Fígado/embriologia , Fígado/metabolismo , Modelos Animais , Proteína Nodal/metabolismo , Fenótipo , Receptores do Ácido Retinoico/genética , Transdução de Sinais , Tretinoína/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Receptor gama de Ácido Retinoico
6.
Blood ; 115(1): 133-9, 2010 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-19880500

RESUMO

Recently, messenger RNAs in eukaryotes have shown to associate with antisense (AS) transcript partners that are often referred to as long noncoding RNAs (lncRNAs) whose function is largely unknown. Here, we have identified a natural AS transcript for tyrosine kinase containing immunoglobulin and epidermal growth factor homology domain-1 (tie-1), tie-1AS lncRNA in zebrafish, mouse, and humans. In embryonic zebrafish, tie-1AS lncRNA transcript is expressed temporally and spatially in vivo with its native target, the tie-1 coding transcript and in additional locations (ear and brain). The tie-1AS lncRNA selectively binds tie-1 mRNA in vivo and regulates tie-1 transcript levels, resulting in specific defects in endothelial cell contact junctions in vivo and in vitro. The ratio of tie-1 versus tie-1AS lncRNA is altered in human vascular anomaly samples. These results directly implicate noncoding RNA-mediated transcriptional regulation of gene expression as a fundamental control mechanism for physiologic processes, such as vascular development.


Assuntos
Loci Gênicos/genética , RNA Antissenso/genética , RNA não Traduzido/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Animais , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Endotélio/efeitos dos fármacos , Endotélio/metabolismo , Endotélio/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Junções Intercelulares/efeitos dos fármacos , Junções Intercelulares/metabolismo , Junções Intercelulares/patologia , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos , Fenótipo , RNA Antissenso/metabolismo , RNA não Traduzido/metabolismo , Receptor de TIE-1/genética , Receptor de TIE-1/metabolismo , Especificidade da Espécie , Doenças Vasculares/genética , Doenças Vasculares/patologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Proteínas de Peixe-Zebra/genética
7.
Blood ; 113(5): 1184-91, 2009 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-18927432

RESUMO

Mitogen-activated protein kinases play an integral role in several cellular processes. To regulate mitogen-activated protein kinases, cells express members of a counteracting group of proteins called phosphatases. In this study, we have identified a specific role that one member of this family of phosphatases, dual-specific phosphatase-5 (Dusp-5) plays in vascular development in vivo. We have determined that dusp-5 is expressed in angioblasts and in established vasculature and that it counteracts the function of a serine threonine kinase, Snrk-1, which also plays a functional role in angioblast development. Together, Dusp-5 and Snrk-1 control angioblast populations in the lateral plate mesoderm with Dusp-5 functioning downstream of Snrk-1. Importantly, mutations in dusp-5 and snrk-1 have been identified in affected tissues of patients with vascular anomalies, implicating the Snrk-1-Dusp-5 signaling pathway in human disease.


Assuntos
Vasos Sanguíneos/embriologia , Fosfatases de Especificidade Dupla/biossíntese , Regulação da Expressão Gênica , Hemangioma/enzimologia , Mesoderma/irrigação sanguínea , Proteínas de Neoplasias/biossíntese , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas de Peixe-Zebra/biossíntese , Peixe-Zebra/embriologia , Animais , Vasos Sanguíneos/patologia , Fosfatases de Especificidade Dupla/genética , Regulação da Expressão Gênica/genética , Hemangioma/genética , Hemangioma/patologia , Humanos , Mesoderma/embriologia , Mesoderma/patologia , Mutação , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Proteínas de Peixe-Zebra/genética
8.
Blood ; 113(5): 1192-9, 2009 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-18723694

RESUMO

In vertebrates, molecular mechanisms dictate angioblasts' migration and subsequent differentiation into arteries and veins. In this study, we used a microarray screen to identify a novel member of the sucrose nonfermenting related kinase (snrk-1) family of serine/threonine kinases expressed specifically in the embryonic zebrafish vasculature and investigated its function in vivo. Using gain- and loss-of-function studies in vivo, we show that Snrk-1 plays an essential role in the migration, maintenance, and differentiation of angioblasts. The kinase function of Snrk-1 is critical for migration and maintenance, but not for the differentiation of angioblasts. In vitro, snrk-1 knockdown endothelial cells show only defects in migration. The snrk-1 gene acts downstream or parallel to notch and upstream of gridlock during artery-vein specification, and the human gene compensates for zebrafish snrk-1 knockdown, suggesting evolutionary conservation of function.


Assuntos
Artérias/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas Serina-Treonina Quinases/biossíntese , Veias/embriologia , Peixe-Zebra/embriologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Movimento Celular/fisiologia , Células Endoteliais/enzimologia , Técnicas de Silenciamento de Genes , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Serina-Treonina Quinases/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
9.
Circ Res ; 103(7): 710-6, 2008 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-18757825

RESUMO

Rho GTPases play an important and versatile role in several biological processes. In this study, we identified the zebrafish ortholog of the mammalian Rho A guanine exchange factor, synectin-binding guanine exchange factor (Syx), and determined its in vivo function in the zebrafish and the mouse. We found that Syx is expressed specifically in the vasculature of these organisms. Loss-of-function studies in the zebrafish and mouse point to a specific role for Syx in angiogenic sprouting in the developing vascular bed. Importantly, vasculogenesis and angioblast differentiation steps were unaffected in syx knockdown zebrafish embryos, and the vascular sprouting defects were partially rescued by the mouse ortholog. Syx knockdown in vitro impairs vascular endothelial growth factor-A-induced endothelial cell migration and angiogenesis. We have also uncovered a potential mechanism of endothelial sprout guidance in which angiomotin, a component of endothelial cell junctions, plays an additive role with Syx in directing endothelial sprouts. These results identify Syx as an essential contributor to angiogenesis in vivo.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neovascularização Fisiológica , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Proteína rhoA de Ligação ao GTP/metabolismo , Angiomotinas , Animais , Movimento Celular/fisiologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Junções Intercelulares/genética , Junções Intercelulares/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteína rhoA de Ligação ao GTP/genética
10.
Nat Commun ; 6: 6023, 2015 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-25599650

RESUMO

The ciliary kinase NEK8 plays a critical role in situs determination and cystic kidney disease, yet its exact function remains unknown. In this study, we identify ANKS6 as a target and activator of NEK8. ANKS6 requires NEK8 for localizing to the ciliary inversin compartment (IC) and activates NEK8 by binding to its kinase domain. Here we demonstrate the functional importance of this interaction through the analysis of two novel mouse mutations, Anks6(Streaker) and Nek8(Roc). Both display heterotaxy, cardiopulmonary malformations and cystic kidneys, a syndrome also characteristic of mutations in Invs and Nphp3, the other known components of the IC. The Anks6(Strkr) mutation decreases ANKS6 interaction with NEK8, precluding NEK8 activation. The Nek8(Roc) mutation inactivates NEK8 kinase function while preserving ANKS6 localization to the IC. Together, these data reveal the crucial role of NEK8 kinase activation within the IC, promoting proper left-right patterning, cardiopulmonary development and renal morphogenesis.


Assuntos
Padronização Corporal/fisiologia , Proteínas de Transporte/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Western Blotting , Padronização Corporal/genética , Proteínas de Transporte/genética , Linhagem Celular , Feminino , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Masculino , Camundongos , Morfogênese/genética , Morfogênese/fisiologia , Mutação , Quinases Relacionadas a NIMA , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Ratos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
11.
Dev Cell ; 29(4): 437-53, 2014 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-24871948

RESUMO

Genetic control of hematopoietic stem and progenitor cell (HSPC) function is increasingly understood; however, less is known about the interactions specifying the embryonic hematopoietic niche. Here, we report that 17ß-estradiol (E2) influences production of runx1+ HSPCs in the AGM region by antagonizing VEGF signaling and subsequent assignment of hemogenic endothelial (HE) identity. Exposure to exogenous E2 during vascular niche development significantly disrupted flk1+ vessel maturation, ephrinB2+ arterial identity, and specification of scl+ HE by decreasing expression of VEGFAa and downstream arterial Notch-pathway components; heat shock induction of VEGFAa/Notch rescued E2-mediated hematovascular defects. Conversely, repression of endogenous E2 activity increased somitic VEGF expression and vascular target regulation, shifting assignment of arterial/venous fate and HE localization; blocking E2 signaling allowed venous production of scl+/runx1+ cells, independent of arterial identity acquisition. Together, these data suggest that yolk-derived E2 sets the ventral boundary of hemogenic vascular niche specification by antagonizing the dorsal-ventral regulatory limits of VEGF.


Assuntos
Antagonistas de Estrogênios/farmacologia , Hemangioblastos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Proteínas de Peixe-Zebra/biossíntese , Peixe-Zebra/embriologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Compostos Benzidrílicos/farmacologia , Subunidade alfa 2 de Fator de Ligação ao Core/biossíntese , Efrina-B2/antagonistas & inibidores , Estradiol/análogos & derivados , Estradiol/farmacologia , Estrogênios/farmacologia , Etinilestradiol/farmacologia , Fulvestranto , Genisteína/farmacologia , Resposta ao Choque Térmico , Morfolinos/genética , Fenóis/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/biossíntese , Receptores de Estradiol/genética , Receptores Notch/biossíntese , Transdução de Sinais , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/antagonistas & inibidores
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