The effects of morpholino-based knockdown of LNX1 (ligand of numb protein X1) on zebrafish vascular development
2015
Hochschulschrift
Zugriff:
104
Because establishment of blood vessels in vertebrates is highly conserved in evolution, and extremely important in embryogenesis, zebrafish have become a widely accepted model that has been used to identify many genes involved in vascular development. Using zebrafish, we found that transcription factors islet2 and coup-TF1b act via the Notch signaling pathway to control vein and intersegmental vessel (ISV) growth. We further investigated the downstream targets of islet2 and coup-TF1b by microarray, and identified ligand of numb-protein X 1 (lnx1) as a potential downstream target that may mediate vasculogenesis. Amino acid sequence alignment and phylogenetic analysis revealed that lnx1 is highly conserved in vertebrates. To study the effects of lnx1 on blood vessel formation, we first showed that lnx1 mRNA is expressed in developing vessels. Morpholino knockdown of lnx1 led to vascular growth defects wherein a mesh-like pattern was unable to form in the tail vein plexus, suggesting a necessity for lnx1 in promoting ISV and caudal vein plexus (CVP) growth. We further demonstrated that the defects in vessel development were associated with edema and impaired circulation. To address whether cell death contributed to the inhibition of ISV and CVP growth, we performed acridine orange stain (AO stain) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. The data showed no increase in endothelial cell apoptosis after morpholino injection, suggesting that cell death is not the cause of the observed vascular phenotype. Moreover, we examined the expression of vascular markers (flt4, flk, ephrin B2, mrc1, stabilin) and found the expression was decreased, demonstrating the effect of lnx1 morpholino on blood vessels. Finally, we examined the interaction between various signaling pathways and lnx1. We found inactivation of VEGF and Notch signals reduced the expression of lnx1, and knockdown of lnx1 caused reductions in vegfaa and ERK expression. Overall, we conclude that the loss of lnx1 impairs vascular development, and that this effect is mediated by VEGF-ERK/Notch signaling in zebrafish.
Titel: |
The effects of morpholino-based knockdown of LNX1 (ligand of numb protein X1) on zebrafish vascular development
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Autor/in / Beteiligte Person: | Huang, Mei-feng ; 黃美鳳 |
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Veröffentlichung: | 2015 |
Medientyp: | Hochschulschrift |
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