Zebrafish runx1 promoter-EGFP transgenics mark discrete sites of definitive blood progenitors

Blood ◽  
2009 ◽  
Vol 113 (6) ◽  
pp. 1241-1249 ◽  
Author(s):  
Enid Yi Ni Lam ◽  
Jackie Y. M. Chau ◽  
Maggie L. Kalev-Zylinska ◽  
Timothy M. Fountaine ◽  
R. Scott Mead ◽  
...  
Keyword(s):  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Hematopoietic Stem ◽  
Blood Island ◽  
Transgenic Lines ◽  
Complex Regulation ◽  
Green Fluorescent ◽  
And Function ◽  
Definitive Hematopoiesis ◽  
Insight Into

Abstract The transcription factor Runx1 is essential for the development of definitive hematopoietic stem cells (HSCs) during vertebrate embryogenesis and is transcribed from 2 promoters, P1 and P2, generating 2 major Runx1 isoforms. We have created 2 stable runx1 promoter zebrafish-transgenic lines that provide insight into the roles of the P1 and P2 isoforms during the establishment of definitive hematopoiesis. The Tg(runx1P1:EGFP) line displays fluorescence in the posterior blood island, where definitive erythromyeloid progenitors develop. The Tg(runx1P2:EGFP) line marks definitive HSCs in the aorta-gonad-mesonephros, with enhanced green fluorescent protein–labeled cells later populating the pronephros and thymus. This suggests that a function of runx1 promoter switching is associated with the establishment of discrete definitive blood progenitor compartments. These runx1 promoter–transgenic lines are novel tools for the study of Runx1 regulation and function in normal and malignant hematopoiesis. The ability to visualize and isolate fluorescently labeled HSCs should contribute to further elucidating the complex regulation of HSC development.

Characterization of the mononuclear phagocyte system in zebrafish

Blood ◽  
2011 ◽  
Vol 117 (26) ◽  
pp. 7126-7135 ◽  
Author(s):  
Valerie Wittamer ◽  
Julien Y. Bertrand ◽  
Patrick W. Gutschow ◽  
David Traver
Keyword(s):  
Fluorescent Protein ◽  
Transgenic Animals ◽  
Regulatory Elements ◽  
Mononuclear Phagocyte ◽  
Mononuclear Phagocytes ◽  
Reporter Line ◽  
Transgenic Lines ◽  
Green Fluorescent ◽  
And Function ◽  
Antigen Presenting

Abstract The evolutionarily conserved immune system of the zebrafish (Danio rerio), in combination with its genetic tractability, position it as an excellent model system in which to elucidate the origin and function of vertebrate immune cells. We recently reported the existence of antigen-presenting mononuclear phagocytes in zebrafish, namely macrophages and dendritic cells (DCs), but have been impaired in further characterizing the biology of these cells by the lack of a specific transgenic reporter line. Using regulatory elements of a class II major histocompatibility gene, we generated a zebrafish reporter line expressing green fluorescent protein (GFP) in all APCs, macrophages, DCs, and B lymphocytes. Examination of mhc2dab:GFP; cd45:DsRed double-transgenic animals demonstrated that kidney mhc2dab:GFPhi; cd45:DsRedhi cells were exclusively mature monocytes/macrophages and DCs, as revealed by morphologic and molecular analyses. Mononuclear phagocytes were found in all hematolymphoid organs, but were most abundant in the intestine and spleen, where they up-regulate the expression of inflammatory cytokines upon bacterial challenge. Finally, mhc2dab:GFP and cd45:DsRed transgenes mark mutually exclusive cell subsets in the lymphoid fraction, enabling the delineation of the major hematopoietic lineages in the adult zebrafish. These findings suggest that mhc2dab:GFP and cd45:DsRed transgenic lines will be instrumental in elucidating the immune response in the zebrafish.

scholarly journals Development of multilineage adult hematopoiesis in the zebrafish with a runx1 truncation mutation

Blood ◽  
2010 ◽  
Vol 115 (14) ◽  
pp. 2806-2809 ◽  
Author(s):  
Raman Sood ◽  
Milton A. English ◽  
Christiane L. Belele ◽  
Hao Jin ◽  
Kevin Bishop ◽  
...  
Keyword(s):  
Stem Cells ◽  
Green Fluorescent Protein ◽  
Hematopoietic Stem Cells ◽  
Fluorescent Protein ◽  
Lineage Tracing ◽  
Transgenic Zebrafish ◽  
Hematopoietic Stem ◽  
Truncation Mutation ◽  
Green Fluorescent ◽  
Definitive Hematopoiesis

Abstract Runx1 is required for the emergence of hematopoietic stem cells (HSCs) from hemogenic endothelium during embryogenesis. However, its role in the generation and maintenance of HSCs during adult hematopoiesis remains uncertain. Here, we present analysis of a zebrafish mutant line carrying a truncation mutation, W84X, in runx1. The runx1W84X/W84X embryos showed blockage in the initiation of definitive hematopoiesis, but some embryos were able to recover from a larval “bloodless” phase and develop to fertile adults with multilineage hematopoiesis. Using cd41–green fluorescent protein transgenic zebrafish and lineage tracing, we demonstrated that the runx1W84X/W84X embryos developed cd41+ HSCs in the aorta-gonad-mesonephros region, which later migrated to the kidney, the site of adult hematopoiesis. Overall, our data suggest that in zebrafish adult HSCs can be formed without an intact runx1.

scholarly journals Localization and Function of Early Cell Division Proteins in Filamentous Escherichia coli Cells Lacking Phosphatidylethanolamine

1998 ◽  
Vol 180 (16) ◽  
pp. 4252-4257 ◽  
Author(s):  
Eugenia Mileykovskaya ◽  
Qin Sun ◽  
William Margolin ◽  
William Dowhan
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Ring Structure ◽  
Whole Cells ◽  
Nucleation Sites ◽  
Spiral Structures ◽  
Green Fluorescent ◽  
And Function ◽  
Insight Into

ABSTRACT Escherichia coli cells that contain thepss-93 null mutation are completely deficient in the major membrane phospholipid phosphatidylethanolamine (PE). Such cells are defective in cell division. To gain insight into how a phospholipid defect could block cytokinesis, we used fluorescence techniques on whole cells to investigate which step of the cell division cycle was affected. Several proteins essential for early steps in cytokinesis, such as FtsZ, ZipA, and FtsA, were able to localize as bands to potential division sites in pss-93 filaments, indicating that the generation and localization of potential division sites was not grossly affected by the absence of PE. However, there was no evidence of constriction at most of these potential division sites. FtsZ and green fluorescent protein (GFP) fusions to FtsZ and ZipA often formed spiral structures in these mutant filaments. This is the first report of spirals formed by wild-type FtsZ expressed at normal levels and by ZipA-GFP. The results suggest that the lack of PE may affect the correct interaction of FtsZ with membrane nucleation sites and alter FtsZ ring structure so as to prevent or delay its constriction.

scholarly journals The Making of Transgenic Drosophila guttifera

2020 ◽  
Author(s):  
Mujeeb Shittu ◽  
Tessa Steenwinkel ◽  
Shigeyuki Koshikawa ◽  
Thomas Werner
Keyword(s):  
Egg Production ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Model Systems ◽  
Comprehensive Method ◽  
Transgenic Lines ◽  
Egg Production Rate ◽  
Green Fluorescent ◽  
Transgenic Drosophila ◽  
Entire Procedure

The complex color patterns on the wings and body of Drosophila guttifera (D. guttifera) are emerging as model systems for studying evolutionary and developmental processes. Studies regarding these processes depend on overexpression and down-regulation of developmental genes, which ultimately rely upon an effective transgenic system. Methods describing transgenesis in Drosophila melanogaster (D. melanogaster) have been extensively reported in several studies. However, these methods cannot be directly applied to D. guttifera due to the low egg production rate and very delicate embryos’ pre-injection treatment requirements. In this protocol, we describe extensively a comprehensive method used for generating transgenic D. guttifera. Using the protocol described here, we are able to establish transgenic lines, identifiable by the expression of Enhanced Green Fluorescent Protein (EGFP) in the eye disks of D. guttifera larvae. The entire procedure, from injection to screening for transgenic larvae, can be completed in approximately 30 days and should be relatively easy to adapt to other non-model Drosophila species, for which no white-eyed mutants exist.

scholarly journals The Making of Transgenic Drosophila guttifera

2020 ◽  
Vol 3 (2) ◽  
pp. 31 ◽  
Author(s):  
Mujeeb Shittu ◽  
Tessa Steenwinkel ◽  
Shigeyuki Koshikawa ◽  
Thomas Werner
Keyword(s):  
Egg Production ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Model Systems ◽  
Comprehensive Method ◽  
Transgenic Lines ◽  
Egg Production Rate ◽  
Green Fluorescent ◽  
Transgenic Drosophila ◽  
Entire Procedure

The complex color patterns on the wings and body of Drosophila guttifera (D. guttifera) are emerging as model systems for studying evolutionary and developmental processes. Studies regarding these processes depend on overexpression and downregulation of developmental genes, which ultimately rely upon an effective transgenic system. Methods describing transgenesis in Drosophila melanogaster (D. melanogaster) have been reported in several studies, but they cannot be applied to D. guttifera due to the low egg production rate and the delicacy of the eggs. In this protocol, we describe extensively a comprehensive method used for generating transgenic D. guttifera. Using the protocol described here, we are able to establish transgenic lines, identifiable by the expression of enhanced green fluorescent protein (EGFP) in the eye disks of D. guttifera larvae. The entire procedure, from injection to screening for transgenic larvae, can be completed in approximately 30 days and should be relatively easy to adapt to other non-model Drosophila species, for which no white-eyed mutants exist.

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