Project description:GATA2 is a pivotal hematopoietic transcription factor required for generation and maintenance of hematopoietic stem cells (HSCs). Due to early embryonic lethality of Gata2 deficiency in mice, its role during adult hematopoiesis is incompletely understood. In zebrafish, mammalian functions of Gata2 are split between two orthologues: Gata2a and Gata2b. Previous studies have shown that Gata2b is prominently expressed in hematopoietic stem and progenitor cells (HSPCs), whereas Gata2a is mainly expressed in the vasculature. We found that Gata2b deficient zebrafish have a reduction in embryonic definitive HSPC numbers and have impaired myeloid lineage differentiation, but are viable. This allowed us to study the role of Gata2b in adult hematopoiesis. To assess the impact of Gata2b deficiency on the transcriptional profile of HSPCs and differentiated cells, we sorted the entire progenitor and HSPC population including the lymphoid population from kidney marrow (KM) of WT and germline Gata2b deficient zebrafish based on scatter profiles and processed for single-cell RNA sequencing. To enrich the scarce HSC population, we used pooled KM from two WT and Gata2b deficient Tg(CD41:GFP) zebrafish per sample and included all CD41:GFPlow expressing cells present in the kidney marrow pool as these cells were shown to contain transplantable HSCs.
Project description:Single cell transcriptome analyses of whole kidney marrow cells isolated from wild type and rad21 heterozygous mutant adult zebrafish.
Project description:The transcription factor GATA2 plays a major role in the generation and maintenance of the hematopoietic system. In humans, heterozygous germline mutations in GATA2 often lead to a loss of function of one allele, causing GATA2 haploinsufficiency. In mice, Gata2 has an essential regulatory function in hematopoietic stem cell (HSC) generation and maintenance. However, whereas Gata2-null mice are lethal at embryonic day (E) 10.53, Gata2 heterozygous (Gata2+/-) mice survive to adulthood with normal blood values. However, mouse models thus emerged as a useful source to identify the function of GATA2 in HSC generation and fitness, they leave the mechanisms causing the different aspects of GATA2 deficiency syndrome largely undiscovered. Zebrafish have the advantage of having two GATA2 orthologues; Gata2a and Gata2b. Gata2a is expressed predominantly in the vasculature and is required for programming of the hemogenic endothelium. Gata2b is expressed in hematopoietic stem/progenitor cells (HSPCs) and homozygous deletion (gata2b-/-) redirects HSPCs differentiation bias, thus mimicking one of the GATA2 haploinsufficiency phenotypes found in patients. But patients carry heterozygous rather than homozygous GATA2 mutations, we specifically focused on how heterozygous Gata2b mutations could be mechanistically linked to erythro-myelodysplasia, a major clinical hallmark of GATA2 patients. To investigate the mechanisms of heterozygous GATA2 mutation caused GATA2 deficiency syndrome, we created a heterozygous gata2b mutation zebrafish model and sorted the entire progenitor and HSPC population including the lymphoid population from kidney marrow (KM) of WT and mutated zebrafish based on the scatter profile of flow cytometry for single-cell RNA (scRNA) sequencing.
Project description:Kit ligand (Kitlg) is a pleiotropic cytokine with a prominent role in vertebrate erythropoiesis. Due to whole genome duplication, zebrafish possess two copies of Kit ligand (Kitlga and Kitlgb). We generated a recombinant version of Kitlga, tested its biological activity in culture of zebrafish kidney marrow cells and performed RNA-seq analysis. Using this assay, we demonstrate that Kitlga cooperates with other factors to promote erythroid cell expansion ex vivo.
Project description:hematopoiesis and myelopoiesis was tightly controled by microRNAs. In the zebrafish adult kidney, specific sets of genes were dysregulated in myelomonocytes or whole kidney marrow after deletion of miR-142-3p. microarrays were used to clarified the miR-142-3p regulatory network in myelopoiesis in miR-142-3p knockout zebrafish kidney and we identified distinct classes of up-regulated genes in zebrafish myelopoiesis or hematopoiesis after deletion of miR-142-3p. The myelomonocytes and whole kidney marrow (without erythrocytes) were sorted from wild-type or miR-142-3p double knockout zebrafish kidney at 60 dpf (four zebrafish kidneys and two independent repeats for each sample). Total RNA was extracted and hybridization on Affymetrix microarrays.
Project description:hematopoiesis and myelopoiesis was tightly controled by microRNAs. In the zebrafish adult kidney, specific sets of genes were dysregulated in myelomonocytes or whole kidney marrow after deletion of miR-142-3p. microarrays were used to clarified the miR-142-3p regulatory network in myelopoiesis in miR-142-3p knockout zebrafish kidney and we identified distinct classes of up-regulated genes in zebrafish myelopoiesis or hematopoiesis after deletion of miR-142-3p.