The Role Of Sox2 In Zebrafish Development

Sox2,belonging to the HMG box family,is one of pluripotent factors that play essential roles in vertebrate development.Sox2-null mouse embryos die before implantation and the mutation in SOX2 is associated with human microphthalmia.Drosophila Sox protein Sox70D/fish-hook/Dichaete is closely related to the vertebrate Sox2 with a 42%overall amino acid identity.Loss function of Sox70D leads to segmentation defects including loss or fusion of abdominal denticle belts and to a defective organization of head structures.Over-expression of dominant-negative Sox2 in Xenopus embryos inhibits neural differentiation.Knocking down sox2 in zebrafish causes mild up-turned tail phenotype at 32 hpf.Although the role of sox2 have been studied in many animals,the function and molecular mechanism underlying vertebrate early development remain largely unknown.To reveal the function and mechanism of Sox2 in vertebrate development,we use zebrafish as a model organism.Using TALEN technology,we created sox2 knockout zebrafish.Analyzing the phenotype of sox2 null embryos,we found that the homozygous mutants exhibited up-turned tail defect from the later period of pharyngula(32hpf)and shorter body length from 48hpf.Results form further study revealed that the decreased body length results from shortened truck that occurs at segmentation stage.The swimbladder is a unique organ in fish and it functions to maintain the fish balance at a certain position under water.Observing the morphogenesis of swimbladder in the sox2 null embryos,we found that the posterior chamber of the swimbladder was uninflated between 4dpf to 5dpf.Performing video-recording experiment,we found that the tail flick behavior is destroyed in the mutant zebrafish embryos and then the swim up behavior is unable to accomplish.The mutant larve therefore cannot reach the water surface to gulp air and ultimately leads to the uninflation of the swimbladder.To understand the mechanism underlying the swim-up defects,we cross the sox2 null allele in the background of Tg(huc:eGFP)and Tg(hb9:GFP).Observing the mutant embryos under the confocal microscope,we found the mutant embryos lost the neuron filipodia in the tail region and had growth defects of motor neuron axons in the regions of truck,tail and swimbladder.In addition,abnormal expressions of the skeletal muscle markers were found in the mutant embryos,suggesting the developmental defects of the muscle in the mutant embryos.To identify the downstream target gene of sox2 to control the motor neuron development,we performed deep sequencing on the transcriber of mutant embryos vs wild type embryos at 72hpf.Results from bioinformatic analyses on the transciptome data revealed that the guidance pathway was abnormal in the mutant embryos.Performing real-time PCR,we found the expression of both sema3bl,encoding a member belonging to the the Semaphorins family of the axon guidance repulsion factors,and ntnlb,encoding one of the axon guidance attraction factors,were decreased in the knockout embryos.Results from whole mount in situ hybridization revealed that sema3bl is down-regulated in the pectoral fin of the mutant embryos.Consistent with this,the axon branch and length projecting to the region of pectoral fin is much more and much longer in mutant embryos than wild-type embryos.Also,ntn1b is obviously down-regulated its expression in the midbrain,hindbrain and spinal cord of the mutant embryos.To verify whether sema3bl and ntn1b are direct targets of sox2,we analyzed the bioinformatic features of the promoter of sema3bl and ntn1b.It turns out that both of the regulatory sequence in the 5’flanking region of the two genes have more than three Sox2 binding sites.Employing dual luciferase assay,we demonstrated that overexpression of zebrafish Sox2 can activate the activity of the sema3bl promoter.Besides,we found that the promoter of the sema3bl has two Sox2 binding sites through ChIP analysis.These data suggest that sox2 controls the motor neuron development and finally affects the swim up behavior by directly regulating the expression of sema3bl and ntn1bIn summary,our study demonstrated for the first time that sox2 controls axon guidance of the motor neuron and thereby controls the swim up behavior by directly regulating the expression of sema3bl and ntn1b.The results lay the foundation for investigating the roles of sox2 in vertebrate neurogenesis.