Rules Of Zebrafish RNase1and5in Early Embryonic Development

Zebrafish (Danio rerio) RNase A superfamily was first put forward by Elio Pizzo et al. in2006, members of which are identified and characterized as orthologous genes of human angiogenin (hANG) and named as zebrafish RNase-1/2/3/4/5(ZF-RNase-1/2/3/4/5) respectively. Although their protein structures and in vitro functions are remarkably similar to hANG, in vivo functional characterization of them is still lacking. Here, we reveal not only temporal and spatial pattern of two members---ZF-RNase-1and ZF-RNase-5in early embryonic development, but also in vivo functions of both.Initially, we cloned expressed sequences tags (ESTs) of ZF-RNases from AB wild type embryos. With the help of semi-quantitative reverse transcription and PCR (semi-quantitative RT-PCR), we found that only ZF-RNase-1/3/5were expressed during early embryonic development. Then, we performed real-time PCR (qRT-PCR) to build detailed temporal expression pattern of these3genes from0hours post fertilization (hpf) to72hpf. Results showed that ZF-RNase-1was high-expressed at24hpf specifically, while expression of ZF-RNase-3/5was gradually up-regulated during this period, suggesting they may take part in early embryonic development. Due to distinct differences in both protein structures and in vitro functions between ZF-RNase-3and hANG in previous reports, we focused on ZF-RNase-1and ZF-RNase-5in our following research. In order to build the spatial expression pattern of ZF-RNase-1/5, we performed whole-mount in situ hybridisation (WISH). Results showed that both ZF-RNase-1and ZF-RNase-5mRNA were abundant in primary head and ventral trunk where the two major blood vessels-dorsal aorta (DA) and posterior cardinal vein (PCV) located, suggesting they may participate in development of vascular system. In addition, ZF-RNase-5mRNA was enriched around yolk sac as well. Furthermore, by means of rapid-amplification of cDNA ends (RACE), we first sequenced full-length cDNA of both.To investigate functions, specific morpholino phosphorodiamidate antisense oligonucleotides (MOs) were designed and injected into zygote at single-cell stage to block their translation respectively. Interestingly, we found distinct and obvious defects in both morphants, suggesting their roles in different stages and diverse tissues in early embryonic development.Firstly, we revealed ZF-RNase-1promotes angiogenesis in embryonic development. With the help of Tg(flil:EGFP)y1, a strain of transgenic fish in which enhanced green fluorescent protein (EGFP) is driven by the promoter of fli, one of vascular endothelial cell markers, significant intersegmental vessels (ISVs) defects were observed in ZF-RNase-1morphants at36hpf, which could be partially rescued by co-injected ZF-RNase-1mRNA transcribed in vitro. Specifically speaking, ISVs failed to extend upward to the top of dorsum, let alone interconnected with each other to form the dorsal longitudinal anastomotic vessels (DLAVs) in ZF-RNase-1morphants. Meanwhile, ectopic ISV formed in ZF-RNase-1over-expressed embryos, branching just over the horizontal septum (HS) rather than at the top of dorsum, though DLAVs formed in the correct location. In our further study, when we knocked down or over-expressed ZF-RNase-1in embryos, corresponding changes of expression level of kinase insert domain receptor like (kdrl), the specific receptor of pro-angiogenic vascular endothelial growth factor (VEGF) signals on membrane of vascular endothelial cells were detected. Nevertheless, that of dll4, ligands of anti-angiogenic Notch signals, was of no change under the same condition. Thus, we demonstrated ZF-RNase-1regulates growth of ISV through VEGF signaling pathway in early embryonic development.Secondly, we found ZF-RNase-5not only promotes angiogenesis, but also be a new yolk extension regulator in embryonic development. The suppressed sprouting of ISVs could be partially rescued by ZF-RNase-5mRNA transcribed in vitro in ZF-RNase-5morphants. It is known that yolk extension (YE), a cylindrical mass extended posteriorly from the spherical yolk ball, forms through processes of tabulation and elongation from14-somite stage at16hpf hpf to22-somite stage at24hpf, providing trunk with nutrients in early embryonic development. Here, severe yolk extension defects as well as especially lower hatching rate and survival rate during2-5days post fertilization (dpf) were observed in ZF-RNase-5morphants, which were partially rescued by ZF-RNase-5mRNA transcribed in vitro, suggesting its importance and necessity for early embryonic development. In our further study, we performed TUNEL assay to detect specific increased DNA damage in yolk syncytial layer (YSL) which plays crucial roles in not only formation and maintenance of yolk extension but also nutrient transport and metabolism between trunk and yolk sac. In addition, the protein level of p53, a well-known apoptotic protein, was significantly up-regulated in ZF-RNase-5morphants. To confirm above-mentioned yolk extension defects above-mentioned were related to activated p53pathway, we rescued the defects in two ways:knocking down the expression of p53with its specific Morpholino and utilizing tp53M214K transgenic fish line with a mutant amino acid in DNA-binding domain of p53. Therefore, we concluded that ZF-RNase-5regulates formation and maintenance of yolk extension through a p53-dependent DNA damage response pathway, as well as sprouting of ISVs in early embryonic development.In conclusion, ZF-RNase-1and ZF-RNase-5, two members of zebrafish RNase A superfamily were expressed significantly in early embryonic development, even if ZF-RNase-1was up-regulated at24hpf specifically. Moreover, both of their mRNA were abundant in primary head and ventral trunk; ZF-RNase-5mRNA was enriched around the yolk sac too. Then, we revealed that ZF-RNase-1promotes growth of ISVs through VEGF signaling pathway, as well as ZF-RNase-5regulates formation and maintenance of zebrafish yolk extension through a p53-dependent DNA damage response pathway in vivo in early embryonic development. Interestingly, according to current reports, hANG is high-expressed in early human placental development, suggesting its roles in placental vasculogenesis and organogenesis. Therefore, our results imply potential functions of hANG in vivo-promoting angiogenesis and maintain normal shape or functions of placenta.