Function Of Amphioxus Pax1/9 Gene In Pharynx Development And Characteristric Of Amphioxus Hsp70 Promoter And Application In Vivo

In vertebrates,Pax1 and Pax9 genes play important roles in regulating pharyngeal development as transcription factors interacting with Eya,Six1,Tbx1 composing a complicated regulating network in pharyngeal endoderm.Amphioxus Pax1/9 gene has strong expression signals in pharyngeal endoderm from neurula stage to larva stage.However,how the gene participates in the development of pharynx apparatus still remains to be revealed.In this study,we knocked out amphioxus Pax1/9 gene using TALEN method.After detecting the mutation type and genotype of F0 and Fi,we obtained Pax1/9 homozygous mutant embryos.Observation under optical microscope and paraffin section showed that the first gill slit had smaller cleft along with the second and third gill slit completely disappeared in Pax1/9-/-mutants.Furthermore,Paxl/9-/-mutants were died in 13 days after fertilization because they were unable fed all the time.Given these results,we concluded that Pax1/9 gene were essential for amphioxus normally developing.In Paxl/9 mutants,WISH indicated that the expression signals of Eya and Six1/2 reduced in the first gill slit primordium and almost disappeared in the second primordium.The expression of Tbx1/10 were disappeared in the second and third pharyngeal arch.These results suggested that Pax1/9 gene played a key role in patterning first gill slit and generating the posterior gill slits by regulating Eya,Six1/2 and Tbx1/10 expression.In Paxl/9-/-mutants,the expression of Nkx2.1 was declined and the endostyle marked by Nkx2.1 was abnormal.This showed that Pax1/9 gene might regulate the development of amphioxus endostyle.In order to study whether Pax1/9 gene is upstream of Eya,Six1/2 and Tbx1/10,we injected Pax1/9 mRNA,Six1/2 mRNA,Eya mRNA and Tbx1/10 mRNA respectively into Pax1/9 F2 embryos.At 3-gill slits larva stage,we found that the experimental group injected Pax1/9 mRNA or Six1/2mRNA was a certain proportion deformity,in which the larva had one mouth on both sides of the body and the cleft of first gill slit was restored,but the posterior gill slits were still disappeared.Groups injected with Eya mRNA or Six1/2 mRNA did not display any rescued phenotype.WISH further clarified the results of phenotypic observation.The possible reasons may be mechanism patterning the first gill slit is different from posterior gill slits and overexpression of Paxl/9 gene may activate other signal related with amphioxus left-right asymmetry.Amphioxus is a favorable material for investigating the evolution from invertebrate to vertebrate and development,but relevant instruments for amphioxus are required in experimental researches.Hsp70 promoter could activate boost expression of the downstream gene after heat shock.This thermal-inducible system has been widely used in many model animals,but not in amphioxus.In this study,we first set different heat shock conditions and heat shocked at five stages:4-cell stage,128-cell stage,late blastula,middle gastrula and 3-somite stage.Then we determined preliminary heat shock condition on different developmental stages,according to the statistics of embryonic deformity rate after heat shocked.RT-qPCR analysis showed that endogenous Hsp70 gene was rising rapidly in one hour after heat shocked at late blastula and posterior stages,and then down to normal level after 5 hours.However,Hsp70 gene was almost unchanged in embryos heat shocked at 4-cell stage.When we heat shocked embryos at 4-cell stage,expression of endogenous Hsp70 gene was unchanged.To investigate the possible reasons,we increased the heat shock temperature of 4-cell stage from 31 ? to 33 ?,and kept heating time for 30 min.We also heat shocked embryos at late blastula stage under 33 ? for 30 min.RT-qPCR showed that Hsp70 gene was intensely expressed within one hour after heat shocked at late blastula stage,however,Hsp70 gene was still not largely induced in embryos heat shocked at 4-cell stage by heat shock.Therefore,embryos heat shocked at 4-cell stage could not induce stress response.Finally,we heat-shocked the embyos at six developmental stages before late blastula stage(included late blastula stage)under 33 ? for 30 min.RT-qPCR showed that,embryos at 256-cell stage began to response to thermal stimuli.WISH and cross section analysis showed that Hsp70 gene expressed in all layers,in which the epidermal ectoderm had stronger expressional signals.In order to analysis the activity of exogenous Hsp70 promoter in vivo,we injected recombined plasmids Hsp70::mCherry and Hsp70::LacZ respectively into amphioxus unfertilized eggs,and then heat-shocked the embryos at late blastula,middle gastrula and 3-somites stages.We found that there were strong red fluorescence signals and LacZ gene expressing signals within two hours after heat shocked.These results indicated that exogenous Hsp70 promoter had a high activity in amphioxus embryos.We also injected recombined plasmids Hsp70::Vgl and Hsp70::Cer respectively into amphioxus unfertilized eggs and then heat-shocked the embryos at late blastula,middle gastrula and 3-somites stages.WISH analysis showed that the expression of Vg1 gene was efficiently activated after heat shocked.Observation on 3-gill-slit larvae revealed that ectopic Vg1 expression led to two-left-side phenotype or left-right reverse phenotype,but ectopic Cer expression caused two-right-side phenotype.Given these results,we concluded that Cer and Vg1 might play reverse role in amphioxus asymmetry mechanism.In summary,we analyzed the characteristric of amphioxus endogenous Hsp70 gene and established an efficient thermo-inducible system,which was successfully applied into studying amphioxus gene function.