Cellular and molecular analyses of vascular tube and lumen formation in zebrafish

Abstract

Tube and lumen formation are essential steps in forming a functional vasculature. Despite their significance, our understanding of these processes remains limited, especially at the cellular and molecular levels. In this study, we analyze mechanisms of angioblast coalescence in the zebrafish embryonic midline and subsequent vascular tube formation. To facilitate these studies, we generated a transgenic line where EGFP expression is controlled by the zebrafish flk1 promoter. We find that angioblasts migrate as individual cells to form a vascular cord at the midline. This transient structure is stabilized by endothelial cell-cell junctions, and subsequently undergoes lumen formation to form a fully patent vessel. Downregulating the VEGF signaling pathway, while affecting the number of angioblasts, does not appear to affect their migratory behavior. Our studies also indicate that the endoderm, a tissue previously implicated in vascular development, provides a substratum for endothelial cell migration and is involved in regulating the timing of this process, but that it is not essential for the direction of migration. In addition, the endothelial cells in endodermless embryos form properly lumenized vessels, contrary to what has been previously reported in Xenopus and avian embryos. These studies provide the tools and a cellular framework for the investigation of mutations affecting vasculogenesis in zebrafish.

LSD1/KDM1A promotes hematopoietic commitment of hemangioblasts through downregulation of Etv2

“…AB and Tupfel long fin (TL) strains were used as WT zebrafish. The Tg(kdrl:EGFP) s843 transgenic line was described previously (38). For genotyping lsd1 it627 , genomic DNA at the mutation sites was amplified by PCR using the primers 5′-ctggcagaggagggacaaactgcacggccggcggcagct and 5′-gcagtcatctgactgtgcg, and was digested with PvuII.…”

“…To answer the question of whether excess endothelialization occurs in LSD1 mutants, we crossed the lsd1 it627 mutant with the Tg(kdrl:EGFP) s843 line, which can visualize vasculogenesis (38), and analyzed the GFP expression in progeny lines. No obvious difference in GFP expression was observed between lsd1 it627 mutants and WT embryos (Fig.…”

Zebrafish scube1 (Signal Peptide-CUB (Complement Protein C1r/C1s, Uegf, and Bmp1)-EGF (Epidermal Growth Factor) Domain-containing Protein 1) Is Involved in Primitive Hematopoiesis

“…Zebrafish Maintenance-Wild-type AB strain, Tg(kdr:EGFP) (22), and Tg(gata1:dsRed) (23) transgenic zebrafish were maintained in a 14-h light/10-h dark cycle at 28.5°C. Zebrafish embryos were collected by natural spawning and incubated in 0.3ϫ Danieau's buffer (1ϫ Danieau's buffer: 58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO 4 , 0.6 mM Ca(NO 3 ) 2 , and 5 mM HEPES (pH 7.6) with double-distilled water) until observation or fixation.…”

pak2a mutations cause cerebral hemorrhage in redhead zebrafish

“…Michigan pet store fish were used for the mutagenesis and crosses were performed with TL for meiotic mapping. A doubly homozygous Tg(flk1:EGFP);Tg(gata1:dsred) line was used for simultaneous imaging of vascular pattern and vascular integrity (31,32).…”

“…7). We next used a double transgenic Tg(flk1:EGFP);Tg(gata1:dsred) line to simultaneously assess vascular pattern (green fluorescent vessels) and vascular integrity (red fluorescent erythrocytes) (31,32). Massive cranial hemorrhage was readily apparent in confocal images of pak2a MO-injected animals compared with control MO-injected animals ( Fig.…”