SCL-GFP transgenic zebrafish: In vivo imaging of blood and endothelial development and identification of the initial site of definitive hematopoiesis

Abstract

The bHLH transcription factor SCL plays a central role in the generation of hematopoietic cells in vertebrates. We modified a PAC containing the whole zebrafish scl locus, inserting GFP into the first coding exon of scl. In germline-transgenic zebrafish generated using this construct, GFP expression completely recapitulates the endogenous expression of scl in blood, endothelium and CNS. We performed in vivo timelapse imaging of blood and endothelial precursor migration at the single-cell level and show that these cells migrate from the posterior lateral plate mesoderm to their site of differentiation in the intermediate cell mass. The anterior lateral plate domain of GFP expression gives rise to primitive macrophages and the blood vessels of the head. In later embryos, GFP expression identifies clusters of hematopoietic cells that develop between the dorsal aorta and posterior cardinal veins after primitive erythrocytes have entered circulation. Two treatments that block definitive hematopoiesis (treatment with dioxin (TCDD), and injection of an antisense morpholino oligonucleotide targeted to runx1) ablate these hematopoietic clusters. This indicates that these clusters represent the first site of definitive hematopoiesis in zebrafish. This site is anatomically homologous to the proposed source of hematopoietic stem cells in amniotes, the aorta-gonad-mesonephros (AGM) region. A second transgenic line, containing the promoter of scl driving GFP, lacks expression in the definitive clusters.

Genetic control of hematopoietic development in Xenopus and zebrafish

“…5G). Thus, clearly the PLM contains precursors of both blood and endothelium at early somitogenesis stages (as already shown by Vogeli et al, 2006;Zhang and Rodaway, 2007). However, the existence of 'hemangioblasts' in the PLM awaits the labeling of single cells within this population.…”

Ddx46 Is Required for Multi-Lineage Differentiation of Hematopoietic Stem Cells in Zebrafish

“…Approximately 8 kb of the 5¢ upstream sequence of tal1 [24,25] was polymerase chain reaction (PCR)-amplified from zebrafish genomic DNA. The amplified tal1 promoter, EGFP, and SV40 poly(A) were placed in the pT2KXIGDin vector that has Tol2 transposable elements [26].…”

Regulation of Stem Cells in the Zebra Fish Hematopoietic System

“…The CHT is similar to mouse placenta or fetal liver, and developing lymphoid and myeloid cells in this region can be identified by markers such as c-myb, scl, runx1, and ikaros (Murayama et al 2006;Zhang and Rodaway 2007). Thymic immigrants differentiate into rag1 + lymphoid cells (Murayama et al 2006;Jin et al 2007;Kissa et al 2008).…”

“…Live imaging of cells labeled with green fluorescent protein (GFP) driven by HSC-specific promoters, such as CD41 and c-myb, and caged fluorescein-dextran cell-tracing experiments have shown that HSCs from the AGM region migrate to colonize the CHT, thymus, and pronephros (Murayama et al 2006;Jin et al 2007;Zhang and Rodaway 2007;Bertrand et al 2008;Kissa et al 2008). CD41 marks HSCs in the mouse (Mitjavila-Garcia et al 2002;Ferkowicz et al 2003;Mikkola et al 2003).…”