Expression And Function Of Pax2 And Its Upstream LncRNA-CT025909.4 During Zebrafish Embryonic Development

The Pax is highly evolutionally conserved gene family which can be found in organisms ranging from drosophila to vertebrates.As one key member of this family,Pax2 is an important molecular marker of the mid-hindbrain boundary and participates in the development of the central nervous system,but its regulatory mechanism is not fully understood.Interestingly,we found a long non-coding RNA�lncRNA CT025909.4,which is in the upstream of Pax2 by analyzing the zebrafish genome database.The present study aimed to explore the expression and possible functions of Pax2 and lncRNACT025909.4 in the embryonic development of zebrafish.Firstly,we analyzed bioinformatics and confirmed the existence of lncRNA CT025909.4 by RT-PCR combined with sequencing.The result of q PCR and in situ hybridization determined its specific expression pattern.Subsequently,antisense morpholinos(MOs)were designed to block the splicing of lncRNA CT025909.4.After injection of MO-lncRNA CT to the one-cell zygotes,the effect of embryonic development and the expression of related genes were determined including midbrain-hindbrain boundary organizers,pax gene family and neuro-development genes.Pax2 mutant alleles were generated by CRISPR/Cas9-mediated genome editing to further investigate the expression and function of Pax2.The main experimental results obtained are as follows:1.According to the ensembl online database,we found that lncRNA CT025909.4 was located in the upstream of Pax2 gene on the chromosome 13 of zebrafish,with a length of 1 283 bp and 3 exons,belonging to intergene lncRNA(lincRNA).LncRNA CT025909.4 was amplified by RT-PCR and the sequencing result was blast with zebrafish genome database,which was perfectly matched to zebrafish chromosome 13.2.The result of q PCR showed that Pax2 and lncRNA CT025909.4 expressed in different stages of zebrafish embryonic development,including 2,6,10,12,24,48,72,and 96 hours post fertilization(hpf).Meanwhile,the result of in situ hybridization showed that Pax2 was specifically expressed in the eyes,ear vesicles,mid-hindbrain boundary,spinal cord,and pronephric duct while lncRNA CT025909.4 showed general expression pattern,but mainly expressed in the fish head.3.Pax2 and lncRNA CT025909.4 were expressed in different adult fish tissues,and both of them had the highest expression level in the fish brain.4.After knockdown of lncRNA CT025909.4(CT-MO)by morpholino,q PCR and in situ hybridization results both confirmed that the expression level of lncRNA CT025909.4 was decreased.The statistics of embryo mortality and malformation rate showed that the mortality and malformation rate of embryos in the CT-MO group were higher than those in the control-MO group(Con-MO),and much higher than those from the wild type embryos.5.After the injection of lncRNA CT-MO,embryos generated abnormal phenotypes such as developmental delay,heart swelling,and spinal curvature.At 72 h after injection,the heart rate of larva in the CT-MO group was significantly lower than that in the untreated group.6.After knockdown of lncRNA CT025909.4 expression in embryos,expressions of the Pax family(Pax2,Pax5,Pax8,Pax3 and Pax6),mid-hindbrain organizer(Otx2,Gbx2,Engrailed,and Fgf8)and neurodevelopment-related genes(neurod4,Ephrin B3,Kreisler,and Erb B4)were determined by q PCR,and the result showed that the expressions of Pax5,Gbx2,Engrailed,and Fgf8 decreased;Pax2,neurod4,Ephrin B3,Kreisler,and Erb B4 were significantly down-regulated compared with the control injection group.The expression levels of Pax8,Pax3,and Pax6 did not change between the CT-MO group and the Con-MO group.7.After lncRNA CT025909.4 knockdown,expressions of the nearby genes were detected and the result showed that the level of cdhr1 a,chst3a,and Iit2 was extremely down-regulated,but the expression of hiflan and cued2 was up-regulated.8.After lncRNA CT025909.4 knockdown,the expressions of cardiac development marker genes were determined by q PCR,and the result showed that the expressions of NKX2.5,NKX2.7 and vmhc were markedly significantly down-regulated,but Bmp4 and myl7 remarkably increased.The expression of gata4 increased,but no significant difference was observed when compared to the control-MO group.9.Using CRISPR/Cas9 system to knock out Pax2 gene,genomic DNA was extracted at 24 hpf and PCR sequencing results showed that there were evenly distributed nested peaks after the g RNA sequence.The result of in situ hybridization showed that the expression of Pax2 in the optic vesicles was reduced or absent from the injected embryos.Moreover,the expression of the mid-hindbrain boundary was also reduced.The larva showed defects in ear,such as smaller otic capsule and otoliths,the two otoliths disappeared,only with one or no separation.The results of this study showed that Pax2 was mainly expressed in eyes,ears,mid-hindbrain boundary,and spinal cord during the embryonic development of zebrafish.LncRNA CT025909.4presented a generic-expression pattern,but mainly expressed in the fish brain.After lncRNA CT025909.4knockdown,the expressions of mid-hindbrain organizer,neurodevelopment-related genes and neighboring genes were affected to a certain extent.This result indicates that lncRNA-CT may play an important role in the development of the fish embryonic nervous system by regulating related genes.However,the mechanism of the regulation and whether it interacts with Pax2 needs further investigation.The result of this study further confirmed the irreplaceable role of Pax2 in the zebrafish embryonic development.The work provides an experimental basis for further exploring the role and its regulatory mechanism of lncRNA CT025909.4 in zebrafish embryonic development.The present study is important for revealing the mechanism of zebrafish early embryonic development and it also enriches the research content of fish genomics and developmental biology.