Regulation of smad gene by Notch signal pathway ligand delta-like 4 in zebrafish (Danio rerio)
CHENG Yao, WANG Zirui, ZHOU Zebin, ZHANG Peng, QIU Junqiang, LI Weiming, ZHANG Qinghua*
1. International Research Center for Marine Biosciences, Ministry of Science and Technology, Shanghai Ocean University, Shanghai 201306, China; 2.Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai 201306, China; 3.Department of Fisheries and Wildlife, Michigan State University, East Lansing 48824, USA
Abstract: To investigate the regulation of Notch signal pathway ligand delta-like 4 (dll4) on smad genes (smad1 and smad7) in zebrafish (Daniorerio), the expression of smad1 and smad7 genes of Smad family was conducted in 2 dpf(day post fertilization) zebrafish dll4 homozygous mutants using qRT-PCR method. The transcriptional binding sites and CpG islands of smad1 and smad7 promoter sequences were predicted by bioinformatics software. The regulation of dll4 on smad1 and smad7 was verified in HEK293T cells by double luciferase assay. The results showed that the expression levels of smad1 and smad7 in dll4 homozygous mutant zebrafish were significantly down-regulated (P<0.000 1), only smad7 gene promoter had CpG island, and both gene promoters contained transcription factor binding sites such as HNF-3, GATA-1 and Oct-1. The activity of pGL3-smad1-Luc and pGL3-smad7-Luc reporter gene vectors in HEK293T cells was about 7.3 times and 142.7 times higher than that in the control group, respectively. Furthermore, p3×Flag-CMV-dll4 was co-transfected with pGL3-smad1-Luc and pGL3-smad7-Luc into HEK293T cells, and the luciferase activity of p3×Flag-CMV-dll4 was 2.8 and 2.2 times comparing with the control group, respectively. These findings indicate that the ligand dll4 of Notch signaling pathway in zebrafish can play a positive regulatory role in angiogenesis through smad gene family, and provide a specific biological target for the repair of damaged blood vessels and tumor angiogenesis.
2023, Vol. 38 
