Targeting the hemangioblast with a novel cell type-specific enhancer
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ABSTRACT:
ORGANISM(S): Gallus gallus
PROVIDER: GSE32494 | GEO | 2011/09/30
SECONDARY ACCESSION(S): PRJNA147765
REPOSITORIES: GEO
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Project description:Hemangioblasts are known as the common precursors for primitive hematopoietic and endothelial lineages. Their existence has been supported mainly by the observation that both cell types develop in close proximity and by in vitro differentiation and genetic studies. However, more compelling evidence will arise from tracking their cell fates using a lineage-specific marker. We report the identification of a hemangioblast-specific enhancer (Hb) located in the cis-regulatory region of chick Cerberus gene (cCer) that is able to direct the expression of enhanced green fluorescent protein (eGFP) to the precursors of yolk sac blood and endothelial cells in electroporated chick embryos. Moreover, we present the Hb-eGFP reporter as a powerful live imaging tool for visualizing hemangioblast cell fate and blood island morphogenesis. We hereby introduce the Hb enhancer as a valuable resource for genetically targeting the hemangioblast population as well as for studying the dynamics of vascular and blood cell development. We used microarray analysis of Hb-eGFP expressing cells to verify the expression of hemangioblast-specific genes in this cell population. In order to characterize the gene expression profile of the Hb-eGFP-positive population, we have isolated Hb-eGFP-positive cells and compared their expression profile with the control RFP-positive population. In brief, chicken embryos were electroporated at HH3 with Hb-eGFP and pCAGGS-RFP reporter constructs, observed under a fluorescence stereoscope and harvested at stage HH5-6 into three groups of four embryos each. Embryos were dissociated into single cell suspensions and the eGFP-positive and eGFP-negative/RFP-positive cell populations were sorted by Fluorescence Activated Cell Sorting (FACS). Cell populations were collected simultaneously into two different tubes, containing RNAlater (Ambion) and subsequently used for RNA extraction. Total RNA was isolated from triplicates of each population, evaluated for RNA integrity, reverse transcribed, amplified and hybridized against six Affymetrix Chicken Genome microarrays.
2011-09-29 | E-GEOD-32494 | biostudies-arrayexpress
Project description:Successful derivation of a specific cell lineage from pluripotent stem cells will tremendously facilitate the clinical usage of pluripotent stem derived somatic cells. Herein, we demonstrate that ER71/Etv2, GATA2 and Scl form a core network in hemangioblast development and that transient co-expression of these three factors robustly induced hemangioblasts from ES cells. Such induced hemangioblasts potently generated hematopoietic and endothelial cells in culture as well as in vivo, warranting the evaluation of these cells in the future for repairing and/or regenerating hematopoietic and/or angiogenic defects.
2014-02-03 | GSE45147 | GEO
Project description:Genetic evidence has implicated several genes as being critical for heart development. However, the inducers of these genes as well as their other targets and the pathways they constitute, remain largely unknown. In the avian embryo, Cerberus (cCer) transcripts are detected in the anterior mesendoderm including the heart precursor cells and in the left lateral plate mesoderm. We have identified a promoter element of chick cCer able to drive EGFP expression into the heart and the hemangioblast precursor cells, allowing us to identify a population of cells that consistently exit from the anterior primitive streak region from as early as stage HH3+ and that later will populate the heart. In order to identify and study novel genes expressed and involved in the correct development and differentiation of the vertebrate H/HPC (Heart/ Hemangioblast Precursor Cells) lineages, a differential screening using Affymetrix GeneChip system technologies was performed. Remarkably, this screening led to the identification of more than 800 transcripts potentially expressed in these precursor lineages. We have identified unknown genes that are differentially expressed in the H/HPC precursor lineages. By developing a procedure to isolate the heart precursor cells using the cer2.5-EGFP construct, we were able to specifically isolate a population of H/HPC expressing already known cardiac markers, and a long list of still uncharacterized genes. From those, we defined by WISH that at least some of these in silico identified transcripts are in fact expressed in these cell populations and more importantly, functionally required for heart formation.More importantly, our study unveiled several uncharacterized genes that can now be used for further studies.
2010-08-04 | GSE23389 | GEO
Project description:Genetic evidence has implicated several genes as being critical for heart development. However, the inducers of these genes as well as their other targets and the pathways they constitute, remain largely unknown. In the avian embryo, Cerberus (cCer) transcripts are detected in the anterior mesendoderm including the heart precursor cells and in the left lateral plate mesoderm. We have identified a promoter element of chick cCer able to drive EGFP expression into the heart and the hemangioblast precursor cells, allowing us to identify a population of cells that consistently exit from the anterior primitive streak region from as early as stage HH3+ and that later will populate the heart. In order to identify and study novel genes expressed and involved in the correct development and differentiation of the vertebrate H/HPC (Heart/ Hemangioblast Precursor Cells) lineages, a differential screening using Affymetrix GeneChip system technologies was performed. Remarkably, this screening led to the identification of more than 800 transcripts potentially expressed in these precursor lineages. We have identified unknown genes that are differentially expressed in the H/HPC precursor lineages. By developing a procedure to isolate the heart precursor cells using the cer2.5-EGFP construct, we were able to specifically isolate a population of H/HPC expressing already known cardiac markers, and a long list of still uncharacterized genes. From those, we defined by WISH that at least some of these in silico identified transcripts are in fact expressed in these cell populations and more importantly, functionally required for heart formation.More importantly, our study unveiled several uncharacterized genes that can now be used for further studies. Portions of chick embryos were selected and excised at early stages of development for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain homogeneous populations of embryos at early gastrula stage in order to increase the temporal resolution of expression profiles. To that end, we hand-selected and excised portions of the embryos according to morphological criteria and EGFP expression. Four GeneChip chicken arrays used for another study were added to the two arrays in this study to ensure a robust model for expression value computation (Note: The 4 samples chick_T2-1, chick_T2-2, chick_T2-3, chick_T2-4, were performed by another group and for a different array, and nothing has to do with our data. They were only run at the same time and used for normalization. Raw data unavailable.). The arrays were normalized to a baseline array with median CEL intensity by applying an Invariant Set Normalization Method. Normalized CEL intensities of the six arrays were used to obtain model-based gene expression indices (MBEI) based on a PM (Perfect Match)-only model. Processed data for all 6 samples can be found in supplementary file linked below.
2010-08-04 | E-GEOD-23389 | biostudies-arrayexpress
Project description:Successful derivation of a specific cell lineage from pluripotent stem cells will tremendously facilitate the clinical usage of pluripotent stem derived somatic cells. Herein, we demonstrate that ER71/Etv2, GATA2 and Scl form a core network in hemangioblast development and that transient co-expression of these three factors robustly induced hemangioblasts from ES cells. Such induced hemangioblasts potently generated hematopoietic and endothelial cells in culture as well as in vivo, warranting the evaluation of these cells in the future for repairing and/or regenerating hematopoietic and/or angiogenic defects. We have established a doxycycline inducible ES cell, iEGS, in which ER71/Etv2, GATA2 three transcription factors can be transiently co-expressed by doxycycline induction. We further analyzed the downstream target genes and signaling pathways at 6, 12 and 24hrs after ER71/Etv2, GATA2 induction. These data were obtained from three independent experiments.
2014-02-03 | E-GEOD-45147 | biostudies-arrayexpress
Project description:The first wave of hematopoiesis is the primitive hematopoiesis, which produces embryonic erythroid and myeloid cells. Primitive erythrocytes are thought to be generated from bipotent hemangioblasts, but the molecular basis remains unclear. Transcriptional repressors Gfi1aa and Gfi1b have been shown to cooperatively promote primitive erythrocytes differentiation from hemangioblasts in zebrafish. However, the mechanism of these repressors during the primitive wave is largely unknown. Herein, by functional analysis of zebrafish gfi1aasmu10, gfi1bsmu11, gfi1absmu12 single, double, and triple mutants, we found that Gfi1aa not only plays a predominant role in primitive erythropoiesis but also synergizes with Gfi1ab. To screen Gfi1aa downstream targets, we performed RNA-seq and ChIP-seq analysis and found two endothelial transcription factors, etv2 and sox7, to be repressed by Gfi1aa. Genetic analysis demonstrated Gfi1aa to promote hemangioblast differentiation into primitive erythrocytes by inhibiting both etv2 and sox7 in a Lsd1-dependent manner. Moreover, the H3K4me1 level of etv2 and sox7 were increased in gfi1aa mutant. Taken together, these results suggest that Gfi1aa/Lsd1-dependent etv2/sox7 downregulation is critical for hemangioblast differentiation during primitive hematopoiesis by inhibition of endothelialization. The different and redundant roles for Gfi1(s), as well as their genetic and epigenetic regulation during primitive hematopoiesis, help us to better know the molecular basis of the primitive hematopoiesis and sheds light on the understanding the Gfi1(s) related pathogenesis.
2021-12-17 | GSE181395 | GEO
Project description:The first site exhibiting hematopoietic activity in mammalian development is the yolk sac blood island, which originates from the hemangioblast. Here we performed differentiation assays, as well as genome-wide molecular and functional studies in BL-CFCs to gain insight into the function of the essential Ldb1 factor in early primitive hematopoietic development. We show that the previously reported lack of yolk sac hematopoiesis and vascular development in Ldb1-/- mouse result from a decreased number of hemangioblasts and a block in their ability to differentiate into erythroid and endothelial progenitor cells. Transcriptome analysis and correlation with the genome wide binding pattern of Ldb1 in hemangioblasts revealed a number of direct target genes and pathways misregulated in the absence of Ldb1. The regulation of essential developmental factors by Ldb1 defines it as an upstream transcriptional regulator of hematopoietic/endothelial development. We show the complex interplay that exists between transcription factors and signaling pathways during the very early stages of hematopoietic/endothelial development and the specific signalling occurring in hemangioblasts in contrast to more advanced hematopoietic developmental stages. Finally, by revealing novel genes and pathways, not previously associated with early development, our study provides novel candidate targets to manipulate the differentiation of hematopoietic and/or endothelial cells.
2013-02-09 | GSE43041 | GEO
Project description:The first site exhibiting hematopoietic activity in mammalian development is the yolk sac blood island, which originates from the hemangioblast. Here we performed differentiation assays, as well as genome-wide molecular and functional studies in BL-CFCs to gain insight into the function of the essential Ldb1 factor in early primitive hematopoietic development. We show that the previously reported lack of yolk sac hematopoiesis and vascular development in Ldb1-/- mouse result from a decreased number of hemangioblasts and a block in their ability to differentiate into erythroid and endothelial progenitor cells. Transcriptome analysis and correlation with the genome wide binding pattern of Ldb1 in hemangioblasts revealed a number of direct target genes and pathways misregulated in the absence of Ldb1. The regulation of essential developmental factors by Ldb1 defines it as an upstream transcriptional regulator of hematopoietic/endothelial development. We show the complex interplay that exists between transcription factors and signaling pathways during the very early stages of hematopoietic/endothelial development and the specific signalling occurring in hemangioblasts in contrast to more advanced hematopoietic developmental stages. Finally, by revealing novel genes and pathways, not previously associated with early development, our study provides novel candidate targets to manipulate the differentiation of hematopoietic and/or endothelial cells. We used microarrays to detail the global programme of gene expression underlying the Ldb1+/+ and Ldb1-/- in Flk1+ cells.
2013-02-09 | GSE43042 | GEO
Project description:The first site exhibiting hematopoietic activity in mammalian development is the yolk sac blood island, which originates from the hemangioblast. Here we performed differentiation assays, as well as genome-wide molecular and functional studies in BL-CFCs to gain insight into the function of the essential Ldb1 factor in early primitive hematopoietic development. We show that the previously reported lack of yolk sac hematopoiesis and vascular development in Ldb1-/- mouse result from a decreased number of hemangioblasts and a block in their ability to differentiate into erythroid and endothelial progenitor cells. Transcriptome analysis and correlation with the genome wide binding pattern of Ldb1 in hemangioblasts revealed a number of direct target genes and pathways misregulated in the absence of Ldb1. The regulation of essential developmental factors by Ldb1 defines it as an upstream transcriptional regulator of hematopoietic/endothelial development. We show the complex interplay that exists between transcription factors and signaling pathways during the very early stages of hematopoietic/endothelial development and the specific signalling occurring in hemangioblasts in contrast to more advanced hematopoietic developmental stages. Finally, by revealing novel genes and pathways, not previously associated with early development, our study provides novel candidate targets to manipulate the differentiation of hematopoietic and/or endothelial cells. Examination of endogenous Ldb1 genome-wide binding sites comparsion between ChIP and Control on Flk1+ BL-CFCs RNA was isolated from Ldb1+/+ and Ldb1-/- Flk1+ cells with the QIAGEN RNeasy Mini Kit and integrity was checked on the Agilent 2100 Bioanalyzer. RNA sequencing was performed on Illumina HiSeq 2000 platform according to the manufacturer instructions.
2013-02-09 | E-GEOD-43041 | biostudies-arrayexpress
Project description:The first site exhibiting hematopoietic activity in mammalian development is the yolk sac blood island, which originates from the hemangioblast. Here we performed differentiation assays, as well as genome-wide molecular and functional studies in BL-CFCs to gain insight into the function of the essential Ldb1 factor in early primitive hematopoietic development. We show that the previously reported lack of yolk sac hematopoiesis and vascular development in Ldb1-/- mouse result from a decreased number of hemangioblasts and a block in their ability to differentiate into erythroid and endothelial progenitor cells. Transcriptome analysis and correlation with the genome wide binding pattern of Ldb1 in hemangioblasts revealed a number of direct target genes and pathways misregulated in the absence of Ldb1. The regulation of essential developmental factors by Ldb1 defines it as an upstream transcriptional regulator of hematopoietic/endothelial development. We show the complex interplay that exists between transcription factors and signaling pathways during the very early stages of hematopoietic/endothelial development and the specific signalling occurring in hemangioblasts in contrast to more advanced hematopoietic developmental stages. Finally, by revealing novel genes and pathways, not previously associated with early development, our study provides novel candidate targets to manipulate the differentiation of hematopoietic and/or endothelial cells. We used microarrays to detail the global programme of gene expression underlying the Ldb1+/+ and Ldb1-/- in Flk1+ cells. RNA was isolated from Ldb1+/+ and Ldb1-/- Flk1+ cells with the QIAGEN RNeasy Mini Kit and integrity was checked on the Agilent 2100 Bioanalyzer. RNA was converted to biotin-labelled cRNA, hybridised on the Mouse Genome 430 2.0 Array and analyzed with the Affymetrix GeneChip® Scanner 3000 according to the manufacturer protocol.
2013-02-09 | E-GEOD-43042 | biostudies-arrayexpress