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1.
Development ; 151(3)2024 Feb 01.
Article in English | MEDLINE | ID: mdl-37982461

ABSTRACT

Early organogenesis represents a key step in animal development, during which pluripotent cells diversify to initiate organ formation. Here, we sampled 300,000 single-cell transcriptomes from mouse embryos between E8.5 and E9.5 in 6-h intervals and combined this new dataset with our previous atlas (E6.5-E8.5) to produce a densely sampled timecourse of >400,000 cells from early gastrulation to organogenesis. Computational lineage reconstruction identified complex waves of blood and endothelial development, including a new programme for somite-derived endothelium. We also dissected the E7.5 primitive streak into four adjacent regions, performed scRNA-seq and predicted cell fates computationally. Finally, we defined developmental state/fate relationships by combining orthotopic grafting, microscopic analysis and scRNA-seq to transcriptionally determine cell fates of grafted primitive streak regions after 24 h of in vitro embryo culture. Experimentally determined fate outcomes were in good agreement with computationally predicted fates, demonstrating how classical grafting experiments can be revisited to establish high-resolution cell state/fate relationships. Such interdisciplinary approaches will benefit future studies in developmental biology and guide the in vitro production of cells for organ regeneration and repair.


Subject(s)
Gastrulation , Organogenesis , Mice , Animals , Cell Differentiation , Organogenesis/genetics , Primitive Streak , Endothelium , Embryo, Mammalian , Mammals
2.
bioRxiv ; 2023 Aug 25.
Article in English | MEDLINE | ID: mdl-37662317

ABSTRACT

During embryogenesis, yolk-sac and intra-embryonic-derived hematopoietic progenitors, comprising the precursors of adult hematopoietic stem cells, converge into the fetal liver. With a new staining strategy, we defined all non-hematopoietic components of the fetal liver and found that hepatoblasts are the major producers of hematopoietic growth factors. We identified mesothelial cells, a novel component of the stromal compartment, producing Kit ligand, a major hematopoietic cytokine. A high-definition imaging dataset analyzed using a deep-learning based pipeline allowed the unambiguous identification of hematopoietic and stromal populations, and enabled determining a neighboring network composition, at the single cell resolution. Throughout active hematopoiesis, progenitors preferentially associate with hepatoblasts, but not with stellate or endothelial cells. We found that, unlike yolk sac-derived progenitors, intra-embryonic progenitors respond to a chemokine gradient created by CXCL12-producing stellate cells. These results revealed that FL hematopoiesis is a spatiotemporal dynamic process, defined by an environment characterized by low cytokine concentrations.

3.
Nat Commun ; 14(1): 4645, 2023 08 14.
Article in English | MEDLINE | ID: mdl-37580379

ABSTRACT

In mitosis, most transcription factors detach from chromatin, but some are retained and bookmark genomic sites. Mitotic bookmarking has been implicated in lineage inheritance, pluripotency and reprogramming. However, the biological significance of this mechanism in vivo remains unclear. Here, we address mitotic retention of the hemogenic factors GATA2, GFI1B and FOS during haematopoietic specification. We show that GATA2 remains bound to chromatin throughout mitosis, in contrast to GFI1B and FOS, via C-terminal zinc finger-mediated DNA binding. GATA2 bookmarks a subset of its interphase targets that are co-enriched for RUNX1 and other regulators of definitive haematopoiesis. Remarkably, homozygous mice harbouring the cyclin B1 mitosis degradation domain upstream Gata2 partially phenocopy knockout mice. Degradation of GATA2 at mitotic exit abolishes definitive haematopoiesis at aorta-gonad-mesonephros, placenta and foetal liver, but does not impair yolk sac haematopoiesis. Our findings implicate GATA2-mediated mitotic bookmarking as critical for definitive haematopoiesis and highlight a dependency on bookmarkers for lineage commitment.


Subject(s)
Chromatin , GATA2 Transcription Factor , Mitosis , Animals , Mice , Chromosomes/metabolism , DNA , Hematopoiesis/genetics , GATA2 Transcription Factor/genetics
4.
Science ; 381(6659): eadd7564, 2023 08 18.
Article in English | MEDLINE | ID: mdl-37590359

ABSTRACT

The extraembryonic yolk sac (YS) ensures delivery of nutritional support and oxygen to the developing embryo but remains ill-defined in humans. We therefore assembled a comprehensive multiomic reference of the human YS from 3 to 8 postconception weeks by integrating single-cell protein and gene expression data. Beyond its recognized role as a site of hematopoiesis, we highlight roles in metabolism, coagulation, vascular development, and hematopoietic regulation. We reconstructed the emergence and decline of YS hematopoietic stem and progenitor cells from hemogenic endothelium and revealed a YS-specific accelerated route to macrophage production that seeds developing organs. The multiorgan functions of the YS are superseded as intraembryonic organs develop, effecting a multifaceted relay of vital functions as pregnancy proceeds.


Subject(s)
Embryonic Development , Yolk Sac , Female , Humans , Pregnancy , Blood Coagulation/genetics , Macrophages , Yolk Sac/cytology , Yolk Sac/metabolism , Embryonic Development/genetics , Atlases as Topic , Gene Expression , Gene Expression Profiling , Hematopoiesis/genetics , Liver/embryology
5.
Nat Commun ; 13(1): 773, 2022 02 09.
Article in English | MEDLINE | ID: mdl-35140205

ABSTRACT

The transcription factor RUNX1 is a critical regulator of developmental hematopoiesis and is frequently disrupted in leukemia. Runx1 is a large, complex gene that is expressed from two alternative promoters under the spatiotemporal control of multiple hematopoietic enhancers. To dissect the dynamic regulation of Runx1 in hematopoietic development, we analyzed its three-dimensional chromatin conformation in mouse embryonic stem cell (ESC) differentiation cultures. Runx1 resides in a 1.1 Mb topologically associating domain (TAD) demarcated by convergent CTCF motifs. As ESCs differentiate to mesoderm, chromatin accessibility, Runx1 enhancer-promoter (E-P) interactions, and CTCF-CTCF interactions increase in the TAD, along with initiation of Runx1 expression from the P2 promoter. Differentiation to hematopoietic progenitor cells is associated with the formation of tissue-specific sub-TADs over Runx1, a shift in E-P interactions, P1 promoter demethylation, and robust expression from both Runx1 promoters. Deletion of promoter-proximal CTCF sites at the sub-TAD boundaries has no obvious effects on E-P interactions but leads to partial loss of domain structure, mildly affects gene expression, and delays hematopoietic development. Together, our analysis of gene regulation at a large multi-promoter developmental gene reveals that dynamic sub-TAD chromatin boundaries play a role in establishing TAD structure and coordinated gene expression.


Subject(s)
Chromatin/metabolism , Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor Alpha 2 Subunit/metabolism , Gene Expression , Animals , Cell Cycle Proteins/metabolism , Cell Differentiation , DNA/chemistry , Gene Expression Regulation, Developmental , Hematopoietic Stem Cells/metabolism , Mesoderm/metabolism , Mice , Nucleic Acid Conformation , Promoter Regions, Genetic
6.
Nat Commun ; 12(1): 7019, 2021 12 02.
Article in English | MEDLINE | ID: mdl-34857757

ABSTRACT

Yolk sac (YS) hematopoiesis is critical for the survival of the embryo and a major source of tissue-resident macrophages that persist into adulthood. Yet, the transcriptional and epigenetic regulation of YS hematopoiesis remains poorly characterized. Here we report that the epigenetic regulator Ezh2 is essential for YS hematopoiesis but dispensable for subsequent aorta-gonad-mesonephros (AGM) blood development. Loss of EZH2 activity in hemogenic endothelium (HE) leads to the generation of phenotypically intact but functionally deficient erythro-myeloid progenitors (EMPs), while the generation of primitive erythroid cells is not affected. EZH2 activity is critical for the generation of functional EMPs at the onset of the endothelial-to-hematopoietic transition but subsequently dispensable. We identify a lack of Wnt signaling downregulation as the primary reason for the production of non-functional EMPs. Together, our findings demonstrate a critical and stage-specific role of Ezh2 in modulating Wnt signaling during the generation of EMPs from YS HE.


Subject(s)
Enhancer of Zeste Homolog 2 Protein/genetics , Erythroid Cells/metabolism , Gene Expression Regulation, Developmental , Mouse Embryonic Stem Cells/metabolism , Myeloid Progenitor Cells/metabolism , Vesicular Transport Proteins/genetics , Yolk Sac/metabolism , Animals , Cell Differentiation , Embryo, Mammalian , Enhancer of Zeste Homolog 2 Protein/deficiency , Epigenesis, Genetic , Erythroid Cells/cytology , Female , Fetus , Genes, Reporter , Hematopoiesis/genetics , Liver/cytology , Liver/growth & development , Liver/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mouse Embryonic Stem Cells/cytology , Myeloid Progenitor Cells/pathology , Primary Cell Culture , Vesicular Transport Proteins/metabolism , Wnt Signaling Pathway , Yolk Sac/cytology , Yolk Sac/growth & development , Red Fluorescent Protein
7.
Cell Rep ; 37(11): 110103, 2021 12 14.
Article in English | MEDLINE | ID: mdl-34910918

ABSTRACT

Hematopoietic stem cells (HSCs) emerge during development from the vascular wall of the main embryonic arteries. The onset of circulation triggers several processes that provide critical external factors for HSC generation. Nevertheless, it is not fully understood how and when the onset of circulation affects HSC emergence. Here we show that in Ncx1-/- mouse embryos devoid of circulation the HSC lineage develops until the phenotypic pro-HSC stage. However, these cells reside in an abnormal microenvironment, fail to activate the hematopoietic program downstream of Runx1, and are functionally impaired. Single-cell transcriptomics shows that during the endothelial-to-hematopoietic transition, Ncx1-/- cells fail to undergo a glycolysis to oxidative phosphorylation metabolic switch present in wild-type cells. Interestingly, experimental activation of glycolysis results in decreased intraembryonic hematopoiesis. Our results suggest that the onset of circulation triggers metabolic changes that allow HSC generation to proceed.


Subject(s)
Cell Differentiation , Cell Lineage , Endothelium, Vascular/pathology , Glycolysis , Hematopoiesis , Hematopoietic Stem Cells/pathology , Sodium-Calcium Exchanger/physiology , Animals , Endothelium, Vascular/metabolism , Female , Hematopoietic Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Oxidative Phosphorylation , Single-Cell Analysis , Transcriptome
8.
Genome Res ; 31(7): 1159-1173, 2021 Jul.
Article in English | MEDLINE | ID: mdl-34088716

ABSTRACT

Regulatory interactions mediated by transcription factors (TFs) make up complex networks that control cellular behavior. Fully understanding these gene regulatory networks (GRNs) offers greater insight into the consequences of disease-causing perturbations than can be achieved by studying single TF binding events in isolation. Chromosomal translocations of the lysine methyltransferase 2A (KMT2A) gene produce KMT2A fusion proteins such as KMT2A-AFF1 (previously MLL-AF4), causing poor prognosis acute lymphoblastic leukemias (ALLs) that sometimes relapse as acute myeloid leukemias (AMLs). KMT2A-AFF1 drives leukemogenesis through direct binding and inducing the aberrant overexpression of key genes, such as the anti-apoptotic factor BCL2 and the proto-oncogene MYC However, studying direct binding alone does not incorporate possible network-generated regulatory outputs, including the indirect induction of gene repression. To better understand the KMT2A-AFF1-driven regulatory landscape, we integrated ChIP-seq, patient RNA-seq, and CRISPR essentiality screens to generate a model GRN. This GRN identified several key transcription factors such as RUNX1 that regulate target genes downstream of KMT2A-AFF1 using feed-forward loop (FFL) and cascade motifs. A core set of nodes are present in both ALL and AML, and CRISPR screening revealed several factors that help mediate response to the drug venetoclax. Using our GRN, we then identified a KMT2A-AFF1:RUNX1 cascade that represses CASP9, as well as KMT2A-AFF1-driven FFLs that regulate BCL2 and MYC through combinatorial TF activity. This illustrates how our GRN can be used to better connect KMT2A-AFF1 behavior to downstream pathways that contribute to leukemogenesis, and potentially predict shifts in gene expression that mediate drug response.

9.
Nat Commun ; 12(1): 821, 2021 02 05.
Article in English | MEDLINE | ID: mdl-33547282

ABSTRACT

Down syndrome is associated with genome-wide perturbation of gene expression, which may be mediated by epigenetic changes. We perform an epigenome-wide association study on neonatal bloodspots comparing 196 newborns with Down syndrome and 439 newborns without Down syndrome, adjusting for cell-type heterogeneity, which identifies 652 epigenome-wide significant CpGs (P < 7.67 × 10-8) and 1,052 differentially methylated regions. Differential methylation at promoter/enhancer regions correlates with gene expression changes in Down syndrome versus non-Down syndrome fetal liver hematopoietic stem/progenitor cells (P < 0.0001). The top two differentially methylated regions overlap RUNX1 and FLI1, both important regulators of megakaryopoiesis and hematopoietic development, with significant hypermethylation at promoter regions of these two genes. Excluding Down syndrome newborns harboring preleukemic GATA1 mutations (N = 30), identified by targeted sequencing, has minimal impact on the epigenome-wide association study results. Down syndrome has profound, genome-wide effects on DNA methylation in hematopoietic cells in early life, which may contribute to the high frequency of hematological problems, including leukemia, in children with Down syndrome.


Subject(s)
Core Binding Factor Alpha 2 Subunit/genetics , Down Syndrome/genetics , Epigenesis, Genetic , Hematopoiesis/genetics , Hematopoietic Stem Cells/metabolism , Proto-Oncogene Protein c-fli-1/genetics , Case-Control Studies , Core Binding Factor Alpha 2 Subunit/metabolism , CpG Islands , DNA Methylation , Down Syndrome/metabolism , Down Syndrome/pathology , Female , Fetus , GATA1 Transcription Factor/genetics , GATA1 Transcription Factor/metabolism , Genome, Human , Genome-Wide Association Study , Hematopoietic Stem Cells/pathology , Humans , Infant, Newborn , Liver/metabolism , Liver/pathology , Male , Promoter Regions, Genetic , Proto-Oncogene Protein c-fli-1/metabolism
11.
Nat Cell Biol ; 23(1): 61-74, 2021 01.
Article in English | MEDLINE | ID: mdl-33420489

ABSTRACT

Extra-embryonic mesoderm (ExM)-composed of the earliest cells that traverse the primitive streak-gives rise to the endothelium as well as haematopoietic progenitors in the developing yolk sac. How a specific subset of ExM becomes committed to a haematopoietic fate remains unclear. Here we demonstrate using an embryonic stem cell model that transient expression of the T-box transcription factor Eomesodermin (Eomes) governs haemogenic competency of ExM. Eomes regulates the accessibility of enhancers that the transcription factor stem cell leukaemia (SCL) normally utilizes to specify primitive erythrocytes and is essential for the normal development of Runx1+ haemogenic endothelium. Single-cell RNA sequencing suggests that Eomes loss of function profoundly blocks the formation of blood progenitors but not specification of Flk-1+ haematoendothelial progenitors. Our findings place Eomes at the top of the transcriptional hierarchy regulating early blood formation and suggest that haemogenic competence is endowed earlier during embryonic development than was previously appreciated.


Subject(s)
Embryonic Stem Cells/cytology , Hemangioblasts/cytology , Mesoderm/cytology , T-Box Domain Proteins/physiology , Yolk Sac/cytology , Animals , Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor Alpha 2 Subunit/metabolism , Embryonic Stem Cells/metabolism , Female , Hemangioblasts/metabolism , Male , Mesoderm/metabolism , Mice, Knockout , Pregnancy , RNA-Seq , Single-Cell Analysis , T-Cell Acute Lymphocytic Leukemia Protein 1/genetics , T-Cell Acute Lymphocytic Leukemia Protein 1/metabolism , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolism , Yolk Sac/metabolism
12.
IUBMB Life ; 72(1): 45-52, 2020 01.
Article in English | MEDLINE | ID: mdl-31634421

ABSTRACT

Runx1 is an important haematopoietic transcription factor as stressed by its involvement in a number of haematological malignancies. Furthermore, it is a key regulator of the emergence of the first haematopoietic stem cells (HSCs) during development. The transcription factor Gata3 has also been linked to haematological disease and was shown to promote HSC production in the embryo by inducing the secretion of important niche factors. Both proteins are expressed in several different cell types within the aorta-gonads-mesonephros (AGM) region, in which the first HSCs are generated; however, a direct interaction between these two key transcription factors in the context of embryonic HSC production has not formally been demonstrated. In this current study, we have detected co-localisation of Runx1 and Gata3 in rare sub-aortic mesenchymal cells in the AGM. Furthermore, the expression of Runx1 is reduced in Gata3 -/- embryos, which also display a shift in HSC emergence. Using an AGM-derived cell line as a model for the stromal microenvironment in the AGM and performing ChIP-Seq and ChIP-on-chip experiments, we demonstrate that Runx1, together with other key niche factors, is a direct target gene of Gata3. In addition, we can pinpoint Gata3 binding to the Runx1 locus at specific enhancer elements which are active in the microenvironment. These results reveal a direct interaction between Gata3 and Runx1 in the niche that supports embryonic HSCs and highlight a dual role for Runx1 in driving the transdifferentiation of haemogenic endothelial cells into HSCs as well as in the stromal cells that support this process.


Subject(s)
Core Binding Factor Alpha 2 Subunit/metabolism , Embryo, Mammalian/cytology , Embryonic Development , Endothelium, Vascular/cytology , GATA3 Transcription Factor/metabolism , Hematopoietic Stem Cells/cytology , Animals , Aorta/cytology , Aorta/metabolism , Core Binding Factor Alpha 2 Subunit/genetics , Embryo, Mammalian/metabolism , Endothelium, Vascular/metabolism , Female , GATA3 Transcription Factor/genetics , Gonads/cytology , Gonads/metabolism , Hematopoiesis , Hematopoietic Stem Cells/metabolism , Mesonephros/cytology , Mesonephros/metabolism , Mice , Mice, Inbred C57BL
13.
Nat Commun ; 10(1): 3577, 2019 08 08.
Article in English | MEDLINE | ID: mdl-31395869

ABSTRACT

Haematopoietic stem cells are generated from the haemogenic endothelium (HE) located in the floor of the dorsal aorta (DA). Despite being integral to arteries, it is controversial whether HE and arterial endothelium share a common lineage. Here, we present a transgenic zebrafish runx1 reporter line to isolate HE and aortic roof endothelium (ARE)s, excluding non-aortic endothelium. Transcriptomic analysis of these populations identifies Runx1-regulated genes and shows that HE initially expresses arterial markers at similar levels to ARE. Furthermore, runx1 expression depends on prior arterial programming by the Notch ligand dll4. Runx1-/- mutants fail to downregulate arterial genes in the HE, which remains integrated within the DA, suggesting that Runx1 represses the pre-existing arterial programme in HE to allow progression towards the haematopoietic fate. These findings strongly suggest that, in zebrafish, aortic endothelium is a precursor to HE, with potential implications for pluripotent stem cell differentiation protocols for the generation of transplantable HSCs.


Subject(s)
Arteries/embryology , Endothelium, Vascular/embryology , Hemangioblasts/physiology , Zebrafish/embryology , Animals , Animals, Genetically Modified , Arteries/cytology , Arteries/metabolism , Cell Lineage , Core Binding Factor Alpha 2 Subunit/genetics , Embryo, Nonmammalian , Embryonic Development , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Gene Knockout Techniques , Intracellular Signaling Peptides and Proteins/metabolism , Zebrafish/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism
14.
Nucleic Acids Res ; 47(14): 7402-7417, 2019 08 22.
Article in English | MEDLINE | ID: mdl-31127293

ABSTRACT

The CRISPR system is widely used in genome editing for biomedical research. Here, using either dual paired Cas9D10A nickases or paired Cas9 nuclease we characterize unintended larger deletions at on-target sites that frequently evade common genotyping practices. We found that unintended larger deletions are prevalent at multiple distinct loci on different chromosomes, in cultured cells and mouse embryos alike. We observed a high frequency of microhomologies at larger deletion breakpoint junctions, suggesting the involvement of microhomology-mediated end joining in their generation. In populations of edited cells, the distribution of larger deletion sizes is dependent on proximity to sgRNAs and cannot be predicted by microhomology sequences alone.


Subject(s)
CRISPR-Cas Systems , Chromosome Deletion , Chromosomes, Mammalian/genetics , Gene Editing/methods , Sequence Deletion , Animals , Cell Line , Chromosome Breakpoints , Chromosomes, Mammalian/metabolism , DNA End-Joining Repair , Deoxyribonuclease I/genetics , Deoxyribonuclease I/metabolism , Endonucleases/genetics , Endonucleases/metabolism , Mice , Models, Genetic , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolism
15.
EMBO Rep ; 19(10)2018 10.
Article in English | MEDLINE | ID: mdl-30166337

ABSTRACT

Few studies report on the in vivo requirement for hematopoietic niche factors in the mammalian embryo. Here, we comprehensively analyze the requirement for Kit ligand (Kitl) in the yolk sac and aorta-gonad-mesonephros (AGM) niche. In-depth analysis of loss-of-function and transgenic reporter mouse models show that Kitl-deficient embryos harbor decreased numbers of yolk sac erythro-myeloid progenitor (EMP) cells, resulting from a proliferation defect following their initial emergence. This EMP defect causes a dramatic decrease in fetal liver erythroid cells prior to the onset of hematopoietic stem cell (HSC)-derived erythropoiesis, and a reduction in tissue-resident macrophages. Pre-HSCs in the AGM require Kitl for survival and maturation, but not proliferation. Although Kitl is expressed widely in all embryonic hematopoietic niches, conditional deletion in endothelial cells recapitulates germline loss-of-function phenotypes in AGM and yolk sac, with phenotypic HSCs but not EMPs remaining dependent on endothelial Kitl upon migration to the fetal liver. In conclusion, our data establish Kitl as a critical regulator in the in vivoAGM and yolk sac endothelial niche.


Subject(s)
Embryonic Development/genetics , Hematopoiesis/genetics , Hematopoietic Stem Cells/cytology , Stem Cell Factor/genetics , Animals , Aorta/growth & development , Cell Lineage/genetics , Cell Proliferation/genetics , Endothelial Cells/cytology , Endothelial Cells/metabolism , Erythropoiesis/genetics , Gene Expression Regulation, Developmental/genetics , Gonads/growth & development , Mesonephros/growth & development , Mice , Mice, Transgenic , Stem Cell Niche/genetics , Yolk Sac/growth & development
16.
Blood ; 131(20): 2223-2234, 2018 05 17.
Article in English | MEDLINE | ID: mdl-29555646

ABSTRACT

Despite the well-established cell-intrinsic role of epigenetic factors in normal and malignant hematopoiesis, their cell-extrinsic role remains largely unexplored. Herein we investigated the hematopoietic impact of inactivating Ezh2, a key component of polycomb repressive complex 2 (PRC2), in the fetal liver (FL) vascular niche. Hematopoietic specific (Vav-iCre) Ezh2 inactivation enhanced FL hematopoietic stem cell (HSC) expansion with normal FL erythropoiesis. In contrast, endothelium (Tie2-Cre) targeted Ezh2 inactivation resulted in embryonic lethality with severe anemia at embryonic day 13.5 despite normal emergence of functional HSCs. Ezh2-deficient FL endothelium overexpressed Mmp9, which cell-extrinsically depleted the membrane-bound form of Kit ligand (mKitL), an essential hematopoietic cytokine, in FL. Furthermore, Mmp9 inhibition in vitro restored mKitL expression along with the erythropoiesis supporting capacity of FL endothelial cells. These data establish that Ezh2 is intrinsically dispensable for FL HSCs and provides proof of principle that modulation of epigenetic regulators in niche components can exert a marked cell-extrinsic impact.


Subject(s)
Endothelial Cells/cytology , Endothelial Cells/metabolism , Enhancer of Zeste Homolog 2 Protein/genetics , Fetus , Hematopoiesis, Extramedullary , Liver/physiology , Anemia/genetics , Anemia/metabolism , Animals , Biomarkers , Cells, Cultured , Enhancer of Zeste Homolog 2 Protein/metabolism , Fluorescent Antibody Technique , Gene Expression , Gene Silencing , Hematopoiesis, Extramedullary/genetics , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Immunohistochemistry , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Mice , Phenotype , Receptor, TIE-2/genetics , Receptor, TIE-2/metabolism , Stem Cell Factor/metabolism
17.
Int J Dev Biol ; 61(3-4-5): 329-335, 2017.
Article in English | MEDLINE | ID: mdl-28621430

ABSTRACT

In vertebrates, definitive hematopoietic stem cells (HSCs) first emerge in the ventral wall of the aorta in the Aorta-Gonad-Mesonephros (AGM) region of the embryo, where they differentiate from a specialized type of endothelium termed Hemogenic Endothelium (HE). While the transition from HE to hematopoietic tissue has received much experimental attention, much less is known regarding generation of HE itself. The current study investigates the emergence of the HE in the chick embryo aorta. Using the HE marker Runx1 as well as a new chicken-reactive antibody to the endothelial marker VE-Cadherin, we document the relationship between the emerging HE and surrounding tissues, particularly the coelomic epithelium (CE) and CE-derived sub-aortic mesenchyme. In addition, the fate of the CE cells was traced by electroporation of a GFP-expressing plasmid into the CE, followed by analysis using immunofluorescence and in situ hybridization. We make the novel observation that CE-derived mesenchyme transiently invades through the ventral wall of the aorta during the period of establishment of HE and just prior to the emergence of hematopoietic cell clusters in the ventral aortic wall. These observations emphasize a hitherto unappreciated dynamism in the aortic wall during the period of HE generation, and open the door to future studies regarding the role of invasive CE-derived cells during aortic hematopoiesis.


Subject(s)
Aorta/embryology , Aorta/physiology , Hemangioblasts/physiology , Hematopoiesis , Mesenchymal Stem Cells/cytology , Animals , Antigens, CD/metabolism , Cadherins/metabolism , Cell Lineage , Chick Embryo , Core Binding Factor Alpha 2 Subunit/metabolism , Endothelium, Vascular/metabolism , Green Fluorescent Proteins/metabolism , Hematopoietic Stem Cells/cytology , Mesoderm/metabolism , Mesonephros , Microscopy, Fluorescence
18.
Adv Exp Med Biol ; 962: 47-64, 2017.
Article in English | MEDLINE | ID: mdl-28299650

ABSTRACT

The de novo generation of hematopoietic stem and progenitor cells (HSPC) occurs solely during embryogenesis from a population of epithelial cells called hemogenic endothelium (HE). During midgestation HE cells in multiple intra- and extraembryonic vascular beds leave the vessel wall as they transition into HSPCs in a process termed the endothelial to hematopoietic transition (EHT). Runx1 expression in HE cells orchestrates the transcriptional switch necessary for the transdifferentiation of endothelial cells into functional HSPCs. Runx1 is widely considered the master regulator of developmental hematopoiesis because it plays an essential function during specification of the hematopoietic lineage during embryogenesis. Here we review the role of Runx1 in embryonic HSPC formation, with a particular focus on its role in hemogenic endothelium.


Subject(s)
Blood Cells/metabolism , Core Binding Factor Alpha 2 Subunit/metabolism , Embryonic Development/physiology , Hemangioblasts/metabolism , Animals , Cell Transdifferentiation/physiology , Embryonic Stem Cells/metabolism , Embryonic Stem Cells/physiology , Endothelium, Vascular/metabolism , Hematopoiesis/physiology , Hematopoietic Stem Cells/metabolism , Stem Cells/metabolism
19.
Blood ; 129(15): 2061-2069, 2017 04 13.
Article in English | MEDLINE | ID: mdl-28179276

ABSTRACT

The Runx family of transcription factors (Runx1, Runx2, and Runx3) are highly conserved and encode proteins involved in a variety of cell lineages, including blood and blood-related cell lineages, during developmental and adult stages of life. They perform activation and repressive functions in the regulation of gene expression. The requirement for Runx1 in the normal hematopoietic development and its dysregulation through chromosomal translocations and loss-of-function mutations as found in acute myeloid leukemias highlight the importance of this transcription factor in the healthy blood system. Whereas another review will focus on the role of Runx factors in leukemias, this review will provide an overview of the normal regulation and function of Runx factors in hematopoiesis and focus particularly on the biological effects of Runx1 in the generation of hematopoietic stem cells. We will present the current knowledge of the structure and regulatory features directing lineage-specific expression of Runx genes, the models of embryonic and adult hematopoietic development that provide information on their function, and some of the mechanisms by which they affect hematopoietic function.


Subject(s)
Core Binding Factor Alpha 2 Subunit , Gene Expression Regulation, Leukemic , Hematopoiesis , Hematopoietic Stem Cells , Leukemia , Mutation , Neoplasm Proteins , Acute Disease , Animals , Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor Alpha 2 Subunit/metabolism , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/pathology , Humans , Leukemia/genetics , Leukemia/metabolism , Leukemia/pathology , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism
20.
Dev Biol ; 424(2): 236-245, 2017 04 15.
Article in English | MEDLINE | ID: mdl-28189604

ABSTRACT

Hematopoietic stem cells (HSCs) emerge during development via an endothelial-to-hematopoietic transition from hemogenic endothelium of the dorsal aorta (DA). Using in situ hybridization and analysis of a knock-in RedStar reporter, we show that the transcriptional regulator Hhex is expressed in endothelium of the dorsal aorta (DA) and in clusters of putative HSCs as they are specified during murine development. We exploited this observation, using the Hhex locus to define cis regulatory elements, enhancers and interacting transcription factors that are both necessary and sufficient to support gene expression in the emerging HSC. We identify an evolutionarily conserved non-coding region (ECR) in the Hhex locus with the capacity to bind the hematopoietic-affiliated transcriptional regulators Gata2, SCL, Fli1, Pu.1 and Ets1/2. This region is sufficient to drive the expression of a transgenic GFP reporter in the DA endothelium and intra-aortic hematopoietic clusters. GFP-positive AGM cells co-expressed HSC-associated markers c-Kit, CD34, VE-Cadherin, and CD45, and were capable of multipotential differentiation and long term engraftment when transplanted into myelo-ablated recipients. The Hhex ECR was also sufficient to drive expression at additional blood sites including the yolk sac blood islands, fetal liver, vitelline and umbilical arteries and the adult bone marrow, suggesting a common mechanism for Hhex regulation throughout ontogenesis of the blood system. To explore the physiological requirement for the Hhex ECR region during hematoendothelial development, we deleted the ECR element from the endogenous locus in the context of a targeted Hhex-RedStar reporter allele. Results indicate a specific requirement for the ECR in blood-associated Hhex expression during development and further demonstrate a requirement for this region in the adult HSC compartment. Taken together, our results identified the ECR region as an enhancer both necessary and sufficient for gene expression in HSC development and homeostasis. The Hhex ECR thus appears to be a core node for the convergence of the transcription factor network that governs the emergence of HSCs.


Subject(s)
Gene Expression Regulation , Hematopoiesis/genetics , Hematopoietic Stem Cells/metabolism , Homeodomain Proteins/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Animals , Cell Compartmentation , Cell Lineage/genetics , Colony-Forming Units Assay , Conserved Sequence/genetics , Embryo, Mammalian/metabolism , Genetic Loci , Green Fluorescent Proteins/metabolism , Hematopoietic Stem Cells/cytology , Homeodomain Proteins/genetics , Mice, Inbred C57BL , Mice, Transgenic , Regulatory Sequences, Nucleic Acid/genetics , Transcription Factors/genetics
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