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1.
Hepatology ; 72(5): 1786-1799, 2020 11.
Article in English | MEDLINE | ID: mdl-32060934

ABSTRACT

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.


Subject(s)
Biliary Tract/embryology , Estradiol/metabolism , Estrogen Receptor beta/metabolism , Gene Expression Regulation, Developmental , Liver/embryology , Zebrafish Proteins/metabolism , Animals , Animals, Genetically Modified , Biliary Tract/cytology , Biliary Tract/metabolism , Cell Differentiation/genetics , Cell Line , Embryo, Nonmammalian , Estradiol/administration & dosage , Estrogen Receptor beta/genetics , Female , Gene Knockdown Techniques , Hepatocytes/physiology , Liver/cytology , Liver/metabolism , Male , Models, Animal , Morpholinos/administration & dosage , Morpholinos/genetics , Signal Transduction/genetics , Stem Cells/physiology , Zebrafish , Zebrafish Proteins/genetics
2.
Circ Res ; 126(1): 6-24, 2020 01 03.
Article in English | MEDLINE | ID: mdl-31730408

ABSTRACT

RATIONALE: Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is evolving rapidly. Recently, second-generation CRISPR/Cas9 activation systems based on nuclease inactive dead (d)Cas9 fused to transcriptional transactivation domains were developed for directing specific guide (g)RNAs to regulatory regions of any gene of interest, to enhance transcription. The application of dCas9 to activate cardiomyocyte transcription in targeted genomic loci in vivo has not been demonstrated so far. OBJECTIVE: We aimed to develop a mouse model for cardiomyocyte-specific, CRISPR-mediated transcriptional modulation, and to demonstrate its versatility by targeting Mef2d and Klf15 loci (2 well-characterized genes implicated in cardiac hypertrophy and homeostasis) for enhanced transcription. METHODS AND RESULTS: A mouse model expressing dCas9 with the VPR transcriptional transactivation domains under the control of the Myh (myosin heavy chain) 6 promoter was generated. These mice innocuously expressed dCas9 exclusively in cardiomyocytes. For initial proof-of-concept, we selected Mef2d, which when overexpressed, led to hypertrophy and heart failure, and Klf15, which is lowly expressed in the neonatal heart. The most effective gRNAs were first identified in fibroblast (C3H/10T1/2) and myoblast (C2C12) cell lines. Using an improved triple gRNA expression system (TRISPR [triple gRNA expression construct]), up to 3 different gRNAs were transduced simultaneously to identify optimal conditions for transcriptional activation. For in vivo delivery of the validated gRNA combinations, we employed systemic administration via adeno-associated virus serotype 9. On gRNA delivery targeting Mef2d expression, we recapitulated the anticipated cardiac hypertrophy phenotype. Using gRNA targeting Klf15, we could enhance its transcription significantly, although Klf15 is physiologically silenced at that time point. We further confirmed specific and robust dCas9VPR on-target effects. CONCLUSIONS: The developed mouse model permits enhancement of gene expression by using endogenous regulatory genomic elements. Proof-of-concept in 2 independent genomic loci suggests versatile applications in controlling transcription in cardiomyocytes of the postnatal heart.


Subject(s)
CRISPR-Cas Systems , Gene Expression Regulation , Myocardium/metabolism , Transcriptional Activation , Animals , Cell Line , Dependovirus/genetics , Fibroblasts/metabolism , Gene Expression Regulation/genetics , Genes, Synthetic , Genetic Vectors/genetics , Heart/growth & development , Kruppel-Like Transcription Factors/biosynthesis , Kruppel-Like Transcription Factors/genetics , MEF2 Transcription Factors/biosynthesis , MEF2 Transcription Factors/genetics , Mice , Mice, Transgenic , Myocytes, Cardiac/metabolism , Myosin Heavy Chains/genetics , Promoter Regions, Genetic , Protein Domains , RNA Polymerase III/genetics , RNA, Guide, Kinetoplastida/genetics
4.
EMBO J ; 35(21): 2315-2331, 2016 11 02.
Article in English | MEDLINE | ID: mdl-27638855

ABSTRACT

During development, hematopoietic stem cells (HSCs) emerge from aortic endothelial cells (ECs) through an intermediate stage called hemogenic endothelium by a process known as endothelial-to-hematopoietic transition (EHT). While Notch signaling, including its upstream regulator Vegf, is known to regulate this process, the precise molecular control and temporal specificity of Notch activity remain unclear. Here, we identify the zebrafish transcriptional regulator evi1 as critically required for Notch-mediated EHT In vivo live imaging studies indicate that evi1 suppression impairs EC progression to hematopoietic fate and therefore HSC emergence. evi1 is expressed in ECs and induces these effects cell autonomously by activating Notch via pAKT Global or endothelial-specific induction of notch, vegf, or pAKT can restore endothelial Notch and HSC formations in evi1 morphants. Significantly, evi1 overexpression induces Notch independently of Vegf and rescues HSC numbers in embryos treated with a Vegf inhibitor. In sum, our results unravel evi1-pAKT as a novel molecular pathway that, in conjunction with the shh-vegf axis, is essential for activation of Notch signaling in VDA endothelial cells and their subsequent conversion to HSCs.


Subject(s)
DNA-Binding Proteins/metabolism , Hematopoietic Stem Cells/metabolism , Proto-Oncogenes/physiology , Transcription Factors/metabolism , Vascular Endothelial Growth Factor A/metabolism , Zebrafish Proteins/metabolism , Animals , Animals, Genetically Modified , Aorta/metabolism , DNA-Binding Proteins/genetics , Diamines/pharmacology , Embryo, Nonmammalian , Endothelial Cells/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogenes/genetics , Receptors, Notch/metabolism , Thiazoles/pharmacology , Transcription Factors/genetics , Zebrafish , Zebrafish Proteins/genetics
5.
Proc Natl Acad Sci U S A ; 113(2): 338-43, 2016 Jan 12.
Article in English | MEDLINE | ID: mdl-26719419

ABSTRACT

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)9 genomic editing has revolutionized the generation of mutant animals by simplifying the creation of null alleles in virtually any organism. However, most current approaches with this method require zygote injection, making it difficult to assess the adult, tissue-specific functions of genes that are widely expressed or which cause embryonic lethality when mutated. Here, we describe the generation of cardiac-specific Cas9 transgenic mice, which express high levels of Cas9 in the heart, but display no overt defects. In proof-of-concept experiments, we used Adeno-Associated Virus 9 (AAV9) to deliver single-guide RNA (sgRNA) that targets the Myh6 locus exclusively in cardiomyocytes. Intraperitoneal injection of postnatal cardiac-Cas9 transgenic mice with AAV9 encoding sgRNA against Myh6 resulted in robust editing of the Myh6 locus. These mice displayed severe cardiomyopathy and loss of cardiac function, with elevation of several markers of heart failure, confirming the effectiveness of this method of adult cardiac gene deletion. Mice with cardiac-specific expression of Cas9 provide a tool that will allow rapid and accurate deletion of genes following a single injection of AAV9-sgRNAs, thereby circumventing embryonic lethality. This method will be useful for disease modeling and provides a means of rapidly editing genes of interest in the heart.


Subject(s)
Aging/genetics , CRISPR-Cas Systems/genetics , Gene Deletion , Myocardium/metabolism , Animals , Cardiomegaly/complications , Cardiomegaly/pathology , Cell Separation , Dependovirus/metabolism , Gene Knockdown Techniques , Heart Failure/complications , Heart Failure/pathology , Mice, Transgenic , Models, Animal , Myocytes, Cardiac/metabolism , Myosin Heavy Chains/genetics , Organ Specificity/genetics , RNA, Guide, Kinetoplastida/metabolism
6.
Dev Biol ; 406(2): 109-16, 2015 Oct 15.
Article in English | MEDLINE | ID: mdl-26386146

ABSTRACT

Myocardin-Related Transcription Factors A and B (MRTF-A and MRTF-B) are highly homologous proteins that function as powerful coactivators of serum response factor (SRF), a ubiquitously expressed transcription factor essential for cardiac development. The SRF/MRTF complex binds to CArG boxes found in the control regions of genes that regulate cytoskeletal dynamics and muscle contraction, among other processes. While SRF is required for heart development and function, the role of MRTFs in the developing or adult heart has not been explored. Through cardiac-specific deletion of MRTF alleles in mice, we show that either MRTF-A or MRTF-B is dispensable for cardiac development and function, whereas deletion of both MRTF-A and MRTF-B causes a spectrum of structural and functional cardiac abnormalities. Defects observed in MRTF-A/B null mice ranged from reduced cardiac contractility and adult onset heart failure to neonatal lethality accompanied by sarcomere disarray. RNA-seq analysis on neonatal hearts identified the most altered pathways in MRTF double knockout hearts as being involved in cytoskeletal organization. Together, these findings demonstrate redundant but essential roles of the MRTFs in maintenance of cardiac structure and function and as indispensible links in cardiac cytoskeletal gene regulatory networks.


Subject(s)
Gene Regulatory Networks/physiology , Heart/embryology , Morphogenesis/physiology , Sarcomeres/physiology , Serum Response Factor/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Animals , Base Sequence , Cytoskeleton/physiology , Echocardiography , Heart/physiology , Histological Techniques , Mice , Mice, Knockout , Microscopy, Electron, Transmission , Molecular Sequence Data , Real-Time Polymerase Chain Reaction , Sarcomeres/metabolism , Sequence Analysis, RNA , Trans-Activators/deficiency , Transcription Factors/deficiency
7.
Stem Cells ; 33(8): 2596-612, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25931248

ABSTRACT

Cannabinoids (CB) modulate adult hematopoietic stem and progenitor cell (HSPCs) function, however, impact on the production, expansion, or migration of embryonic HSCs is currently uncharacterized. Here, using chemical and genetic approaches targeting CB-signaling in zebrafish, we show that CB receptor (CNR) 2, but not CNR1, regulates embryonic HSC development. During HSC specification in the aorta-gonad-mesonephros (AGM) region, CNR2 stimulation by AM1241 increased runx1;cmyb(+) HSPCs, through heightened proliferation, whereas CNR2 antagonism decreased HSPC number; FACS analysis and absolute HSC counts confirmed and quantified these effects. Epistatic investigations showed AM1241 significantly upregulated PGE2 synthesis in a Ptgs2-dependent manner to increase AGM HSCs. During the phases of HSC production and colonization of secondary niches, AM1241 accelerated migration to the caudal hematopoietic tissue (CHT), the site of embryonic HSC expansion, and the thymus; however these effects occurred independently of PGE2. Using a candidate approach for HSC migration and retention factors, P-selectin was identified as the functional target of CNR2 regulation. Epistatic analyses confirmed migration of HSCs into the CHT and thymus was dependent on CNR2-regulated P-selectin activity. Together, these data suggest CNR2-signaling optimizes the production, expansion, and migration of embryonic HSCs by modulating multiple downstream signaling pathways.


Subject(s)
Dinoprostone/metabolism , Hematopoietic Stem Cells/metabolism , P-Selectin/metabolism , Receptor, Cannabinoid, CB1/metabolism , Receptor, Cannabinoid, CB2/metabolism , Zebrafish Proteins/metabolism , Zebrafish/embryology , Animals , Hematopoietic Stem Cells/cytology , Signal Transduction/physiology
8.
Dev Cell ; 29(4): 437-53, 2014 May 27.
Article in English | MEDLINE | ID: mdl-24871948

ABSTRACT

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.


Subject(s)
Estrogen Antagonists/pharmacology , Hemangioblasts/metabolism , Hematopoietic Stem Cells/metabolism , Vascular Endothelial Growth Factor A/biosynthesis , Zebrafish Proteins/biosynthesis , Zebrafish/embryology , Animals , Basic Helix-Loop-Helix Transcription Factors/antagonists & inhibitors , Basic Helix-Loop-Helix Transcription Factors/biosynthesis , Benzhydryl Compounds/pharmacology , Core Binding Factor Alpha 2 Subunit/biosynthesis , Ephrin-B2/antagonists & inhibitors , Estradiol/analogs & derivatives , Estradiol/pharmacology , Estrogens/pharmacology , Ethinyl Estradiol/pharmacology , Fulvestrant , Genistein/pharmacology , Heat-Shock Response , Morpholinos/genetics , Phenols/pharmacology , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/biosynthesis , Receptors, Estradiol/genetics , Receptors, Notch/biosynthesis , Signal Transduction , T-Cell Acute Lymphocytic Leukemia Protein 1 , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Zebrafish/genetics , Zebrafish Proteins/antagonists & inhibitors
9.
Exp Hematol ; 42(8): 684-96, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24816275

ABSTRACT

Exploitation of the zebrafish model in hematology research has surged in recent years, becoming one of the most useful and tractable systems for understanding regulation of hematopoietic development, homeostasis, and malignancy. Despite the evolutionary distance between zebrafish and humans, remarkable genetic and phenotypic conservation in the hematopoietic system has enabled significant advancements in our understanding of blood stem and progenitor cell biology. The strengths of zebrafish in hematology research lie in the ability to perform real-time in vivo observations of hematopoietic stem, progenitor, and effector cell emergence, expansion, and function, as well as the ease with which novel genetic and chemical modifiers of specific hematopoietic processes or cell types can be identified and characterized. Further, myriad transgenic lines have been developed including fluorescent reporter systems to aid in the visualization and quantification of specified cell types of interest and cell-lineage relationships, as well as effector lines that can be used to implement a wide range of experimental manipulations. As our understanding of the complex nature of blood stem and progenitor cell biology during development, in response to infection or injury, or in the setting of hematologic malignancy continues to deepen, zebrafish will remain essential for exploring the spatiotemporal organization and integration of these fundamental processes, as well as the identification of efficacious small molecule modifiers of hematopoietic activity. In this review, we discuss the biology of the zebrafish hematopoietic system, including similarities and differences from mammals, and highlight important tools currently utilized in zebrafish embryos and adults to enhance our understanding of vertebrate hematology, with emphasis on findings that have impacted our understanding of the onset or treatment of human hematologic disorders and disease.


Subject(s)
Hematopoiesis , Zebrafish/physiology , Animals , Cell Lineage , Hematopoietic Stem Cells/physiology , Models, Animal , Zebrafish/embryology , Zebrafish/genetics
10.
Dev Cell ; 28(4): 423-37, 2014 Feb 24.
Article in English | MEDLINE | ID: mdl-24530296

ABSTRACT

The liver and pancreas arise from common endodermal progenitors. How these distinct cell fates are specified is poorly understood. Here we describe prostaglandin E2 (PGE2) as a regulator of endodermal fate specification during development. Modulating PGE2 activity has opposing effects on liver versus pancreas specification in zebrafish embryos as well as mouse endodermal progenitors. The PGE2 synthetic enzyme cox2a and receptor ep2a are patterned such that cells closest to PGE2 synthesis acquire a liver fate, whereas more distant cells acquire a pancreas fate. PGE2 interacts with the bmp2b pathway to regulate fate specification. At later stages of development, PGE2 acting via the ep4a receptor promotes outgrowth of both the liver and pancreas. PGE2 remains important for adult organ growth, as it modulates liver regeneration. This work provides in vivo evidence that PGE2 may act as a morphogen to regulate cell-fate decisions and outgrowth of the embryonic endodermal anlagen.


Subject(s)
Cell Lineage , Dinoprostone/metabolism , Endoderm/metabolism , Liver/metabolism , Pancreas/metabolism , Animals , Bone Morphogenetic Protein 2/metabolism , Cell Differentiation/physiology , Endoderm/cytology , Liver/cytology , Liver/embryology , Mice , Organogenesis , Pancreas/cytology , Pancreas/embryology , Signal Transduction/physiology , Zebrafish , Zebrafish Proteins/metabolism
11.
Blood ; 121(13): 2483-93, 2013 Mar 28.
Article in English | MEDLINE | ID: mdl-23341543

ABSTRACT

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.


Subject(s)
Carbohydrate Metabolism/physiology , Embryonic Induction , Glucose/metabolism , Hematopoietic Stem Cells/physiology , Animals , Animals, Genetically Modified , Carbohydrate Metabolism/genetics , Cell Proliferation/drug effects , Embryo, Nonmammalian , Embryonic Induction/drug effects , Embryonic Induction/genetics , Gene Expression Regulation, Developmental , Glucose/pharmacology , Glycolysis/drug effects , Glycolysis/genetics , Glycolysis/physiology , Hematopoiesis/drug effects , Hematopoiesis/genetics , Hematopoiesis/physiology , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/physiology , Oxidative Phosphorylation , Time Factors , Zebrafish/embryology , Zebrafish/genetics , Zebrafish/metabolism
12.
Dev Biol ; 373(2): 431-41, 2013 Jan 15.
Article in English | MEDLINE | ID: mdl-22960038

ABSTRACT

Growth Factor Independence (Gfi) transcription factors play essential roles in hematopoiesis, differentially activating and repressing transcriptional programs required for hematopoietic stem/progenitor cell (HSPC) development and lineage specification. In mammals, Gfi1a regulates hematopoietic stem cells (HSC), myeloid and lymphoid populations, while its paralog, Gfi1b, regulates HSC, megakaryocyte and erythroid development. In zebrafish, gfi1aa is essential for primitive hematopoiesis; however, little is known about the role of gfi1aa in definitive hematopoiesis or about additional gfi factors in zebrafish. Here, we report the isolation and characterization of an additional hematopoietic gfi factor, gfi1b. We show that gfi1aa and gfi1b are expressed in the primitive and definitive sites of hematopoiesis in zebrafish. Our functional analyses demonstrate that gfi1aa and gfi1b have distinct roles in regulating primitive and definitive hematopoietic progenitors, respectively. Loss of gfi1aa silences markers of early primitive progenitors, scl and gata1. Conversely, loss of gfi1b silences runx-1, c-myb, ikaros and cd41, indicating that gfi1b is required for definitive hematopoiesis. We determine the epistatic relationships between the gfi factors and key hematopoietic transcription factors, demonstrating that gfi1aa and gfi1b join lmo2, scl, runx-1 and c-myb as critical regulators of teleost HSPC. Our studies establish a comparative paradigm for the regulation of hematopoietic lineages by gfi transcription factors.


Subject(s)
DNA-Binding Proteins/genetics , Gene Expression Regulation, Developmental , Hematopoiesis/genetics , Zebrafish Proteins/genetics , Zebrafish/genetics , Amino Acid Sequence , Animals , Cloning, Molecular , Conserved Sequence/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Embryo, Nonmammalian/metabolism , Epistasis, Genetic , Erythropoiesis/genetics , Evolution, Molecular , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Hematopoietic System/embryology , Hematopoietic System/metabolism , Models, Biological , Molecular Sequence Data , Zebrafish/embryology , Zebrafish Proteins/chemistry , Zebrafish Proteins/metabolism
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