gata2a

The zebrafish is amenable to a variety of genetic approaches. However, lack of conditional deletion alleles limits stage- or cell-specific gene knockout. Here, we applied an existing protocol to establish a floxed allele for gata2a but failed to do so due to off-target integration and incomplete knockin. To address these problems, we applied simultaneous co-targeting with Cas12a to insert loxP sites in cis, together with transgenic counterscreening and comprehensive molecular analysis, to identify off-target insertions and confirm targeted knockins...

Inherited bone marrow failure associated with heterozygous mutations in GATA2 predisposes toward hematological malignancies, but the mechanisms remain poorly understood. Here, we investigate the mechanistic basis of marrow failure in a zebrafish model of GATA2 deficiency. Single-cell transcriptomics and chromatin accessibility assays reveal that loss of gata2a leads to skewing toward the erythroid lineage at the expense of myeloid cells, associated with loss of cebpa expression and decreased PU.1 and CEBPA transcription factor accessibility in hematopoietic stem and progenitor cells (HSPCs)...

The normal development of lens fiber cells plays a critical role in lens morphogenesis and maintaining transparency. Factors involved in the development of lens fiber cells are largely unknown in vertebrates. In this study, we reported that GATA2 is essential for lens morphogenesis in Nile tilapia ( Oreochromis niloticus ). In this study, Gata2a was detected in the primary and secondary lens fiber cells, with the highest expression in primary fiber cells. gata2a homozygous mutants of tilapia were obtained using CRISPR/Cas9...

BACKGROUND: In the zebrafish midbrain, GABAergic neurons develop from precursors located in the nucleus of the medial longitudinal fasciculus (nMLF). However, the precise mechanisms that underline generation of the nMLF GABAergic neuron are poorly understood. RESULTS: GABAergic neurons in the nMLF co-express transcription factors tal2, gata2a, gata3, and nkx1.2lb. The Nodal-related gene and shh signaling are required for differentiation of nMLF GABAergic neuron precursors...

The maintenance and proliferation of hematopoietic stem cells (HSCs) are tightly regulated by their niches in the bone marrow. The analysis of niche cells or stromal cell lines that can support HSCs has facilitated the finding of novel supporting factors for HSCs. Despite large efforts in the murine bone marrow; however, HSC expansion is still difficult ex vivo, highlighting the need for new approaches to elucidate the molecular elements that regulate HSCs. The zebrafish provides a unique model to study hematopoietic niches as HSCs are maintained in the kidney, allowing for a parallel view of hematopoietic niches over evolution...

Epigenetic regulation is integral in orchestrating the spatiotemporal regulation of gene expression which underlies tissue development. The emergence of new tools to assess genome-wide epigenetic modifications has enabled significant advances in the field of vascular biology in zebrafish. Zebrafish represents a powerful model to investigate the activity of cis -regulatory elements in vivo by combining technologies such as ATAC-seq, ChIP-seq and CUT&Tag with the generation of transgenic lines and live imaging to validate the activity of these regulatory elements...

Zebrafish lateral line system which is derived from neurogenic placodes has become a popular model for developmental biology since its formation involves cell migration, pattern formation, organogenesis, and hair cell regeneration. Transgenic lines play a crucial role in lateral line system study. Here, we identified an enhancer trap transgenic zebrafish line Et(gata2a:EGFP)189b (ET189b for short), which expressed enhanced green fluorescent protein (EGFP) in the pituitary, otic, and lateral line placodes and their derivatives...

Germline loss or mutation of one copy of the transcription factor GATA2 in humans leads to a range of clinical phenotypes affecting hematopoietic, lymphatic and vascular systems. GATA2 heterozygous mice show only a limited repertoire of the features observed in humans. Zebrafish have two copies of the Gata2 gene as a result of an additional round of ancestral whole genome duplication. These genes, Gata2a and Gata2b, show distinct but overlapping expression patterns, and between them, highlight a significantly broader range of the phenotypes observed in GATA2 deficient syndromes, than each one alone...

RUNX1 is essential for the generation of hematopoietic stem cells (HSCs). Runx1-null mouse embryos lack definitive hematopoiesis and die in mid-gestation. However, although zebrafish embryos with a runx1 W84X mutation have defects in early definitive hematopoiesis, some runx1W84X/W84X embryos can develop to fertile adults with blood cells of multilineages, raising the possibility that HSCs can emerge without RUNX1. Here, using 3 new zebrafish runx1-/- lines, we uncovered the compensatory mechanism for runx1-independent hematopoiesis...

The differentiation of hematopoietic stem cells (HSCs) is tightly controlled to ensure a proper balance between myeloid and lymphoid cell output. GATA2 is a pivotal hematopoietic transcription factor required for generation and maintenance of HSCs. GATA2 is expressed throughout development, but because of early embryonic lethality in mice, its role during adult hematopoiesis is incompletely understood. Zebrafish contains 2 orthologs of GATA2: Gata2a and Gata2b, which are expressed in different cell types. We show that the mammalian functions of GATA2 are split between these orthologs...

Gata2 is a key transcription factor required to generate Haematopoietic Stem and Progenitor Cells (HSPCs) from haemogenic endothelium (HE); misexpression of Gata2 leads to haematopoietic disorders. Here we deleted a conserved enhancer (i4 enhancer) driving pan-endothelial expression of the zebrafish gata2a and showed that Gata2a is required for HE programming by regulating expression of runx1 and of the second Gata2 orthologue, gata2b. By 5 days, homozygous gata2aΔi4/Δi4 larvae showed normal numbers of HSPCs, a recovery mediated by Notch signalling driving gata2b and runx1 expression in HE...

Hematopoietic stem cells (HSCs) maintain the entire blood system throughout life and are utilized in therapeutic approaches for blood diseases. Prospective isolation of highly purified HSCs is crucial to understand the molecular mechanisms underlying regulation of HSCs. The zebrafish is an elegant genetic model for the study of hematopoiesis due to its many unique advantages. It has not yet been possible, however, to purify HSCs in adult zebrafish due to a lack of specific HSC markers. Here we show the enrichment of zebrafish HSCs by a combination of two HSC-related transgenes, gata2a:GFP and runx1:mCherry...

Specification of neurons in the spinal cord relies on extrinsic and intrinsic signals, which in turn are interpreted by expression of transcription factors. V2 interneurons develop from the ventral aspects of the spinal cord. We report here a novel neuronal V2 subtype, named V2s, in zebrafish embryos. Formation of these neurons depends on the transcription factors sox1a and sox1b. They develop from common gata2a - and gata3 -dependent precursors co-expressing markers of V2b and V2s interneurons. Chemical blockage of Notch signalling causes a decrease in V2s and an increase in V2b cells...

Tetracycline hydrochloride (TH), indomethacin (IM), and bezafibrate (BF) belong to the three different important classes of pharmaceuticals, which are well known for their toxicity and environmental concerns. However, studies are still elusive to highlight the mechanistic toxicity of these pharmaceuticals and rank them using both, the toxicity prediction and confirmation approaches. Therefore, we employed the next generation toxicity testing in 21st century (TOX21) tools and estimated the in vitro/vivo toxic endpoints of mentioned pharmaceuticals, and then confirmed them using in vitro/vivo assays...

Vertebrate locomotor circuitry contains distinct classes of ventral spinal cord neurons which each have particular functional properties. While we know some of the genes expressed by each of these cell types, we do not yet know how several of these neurons are specified. Here, we investigate the functions of Tal1, Gata2a, and Gata3 transcription factors in the development of two of these populations of neurons with important roles in locomotor circuitry: V2b neurons and cerebrospinal fluid-contacting Kolmer-Agduhr (KA) neurons (also called CSF-cNs)...

Background. Following the two rounds of whole-genome duplication that occurred during deuterostome evolution, a third genome duplication occurred in the lineage of teleost fish and is considered to be responsible for much of the biological diversification within the lineage. GATA2, a member of GATA family of transcription factors, is an important regulator of gene expression in hematopoietic cell in mammals, yet the role of this gene or its putative paralogs in ray-finned fishes remains relatively unknown. Methods...

The adult blood system is established by hematopoietic stem cells (HSCs), which arise during development from an endothelial-to-hematopoietic transition of cells comprising the floor of the dorsal aorta. Expression of aortic runx1 has served as an early marker of HSC commitment in the zebrafish embryo, but recent studies have suggested that HSC specification begins during the convergence of posterior lateral plate mesoderm (PLM), well before aorta formation and runx1 transcription. Further understanding of the earliest stages of HSC specification necessitates an earlier marker of hemogenic endothelium...

Congenital diseases caused by abnormal development of the cranial neural crest usually present craniofacial malformations and heart defects while the precise mechanism is not fully understood. Here, we show that the zebrafish eif3ba mutant caused by pseudo-typed retrovirus insertion exhibited a similar phenotype due to the hypogenesis of cranial neural crest cells (NCCs). The derivatives of cranial NCCs, including the NCC-derived cell population of pharyngeal arches, craniofacial cartilage, pigment cells and the myocardium derived from cardiac NCCs, were affected in this mutant...

Genomic deletions induced by imprecise excision of transposons have been used to disrupt gene functions in Drosophila. To determine the excision properties of Tol2, a popular transposon in zebrafish, we took advantage of two transgenic zebrafish lines Et(gata2a:EGFP)pku684 and Et(gata2a:EGFP)pku760, and mobilized the transposon by injecting transposase mRNA into homozygous transgenic embryos. Footprint analysis showed that the Tol2 transposons were excised in either a precise or an imprecise manner. Furthermore, we identified 1093-bp and 1253-bp genomic deletions in Et(gata2a:EGFP)pku684 founder embryos flanking the 5' end of the original Tol2 insertion site, and a 1340-bp deletion in the Et(gata2a:EGFP)pku760 founder embryos flanking the 3' end of the insertion site...

The pituitary is a complex gland comprising different cell types each secreting specific hormones. The extensive network of signaling molecules and transcription factors required for determination and terminal differentiation of specific cell types is still not fully understood. The SRY-like HMG-box (SOX) transcription factor Sox4 plays important roles in many developmental processes and has two homologs in zebrafish, Sox4a and Sox4b. We show that the sox4b gene is expressed in the pituitary anlagen starting at 24 h after fertilization (hpf) and later in the entire head region including the pituitary...