SEC24B Function Of Genes In Zebrafish Embryonic Development And Correlation Analysis And Human Neural Tube Defects

Neural tube defects(NTDs) present a series of severe birth defects caused by failure of neural tube closure during embryogenesis at any level of the rostrocaudal axis. Both environmental and genetic factors contribute to the development of NTD. It has been confirmed that Vangl2gene of the planar cell polarity (PCP) signaling pathway is closely related to this disease. Evidences in mice model suggested that Sec24b, a core member of endoplasmic reticulum (ER)-to-Golgi transport vesicle(COPII), can bind to Vangl2and mediate the transports from the ER to the Golgi. Recent studies revealed that Sec24b mutation caused NTD through the transport failure of Vangl2. In the previous study, we found four disease-specific SEC24B missense mutations (F227S, F682L R1248Q, A1251G) in human NTD patients with case-control method. But the function alteration is unclear. So a platform to validate the function of SEC24B missense mutations is needed, and zebrafish become a candidate.In the present study, SEC24B and Vangl2gene were investigated, the details and results are as following:1. Bioinformatics analysis of Sec24b genes:Download the gene sequences of Sec24family of human and zerberfish from the NCBI database, analyzed with different bioinformatics soft wares. The results showed that zebrafish Sec24b gene shared77%identity with human.2. Cloning the Sec24b gene of zebrafish and analyzing the functions:(1)Total RNA was extracted from zebrafish and, Sec24b gene was cloned by RT-PCR and verified by DNA sequencing;(2)Using RNA Whole-Mount in situ hybridization and frozen section to profile the spatiotemporal expression of Sec24b during development;(3) Suppressing the expression of Sec24b:To block the translation of Sec24b mRNA using antisense oligonucleotide labeled with MorpholinoMO) and,observe the phenotype variation;(4)Over-expression experiment:injected zeberfish zygotes with SEC24B-W、SEC24B-Y613and pCMV6plasmids, observed the phenotype variation caused by wide tpye and mutant SEC24B.(5)Function rescue experiment in zerberfish:co-injected zeberfish zygotes with human-SEC24B-GFP, SEC24B-Y613, Sec24b-EGFP plasmids and Sec24b-MO, respectively, observed the embryonic phenotypes and performed statistic analysis.Results:①Zebrafish Sec24b gene was successfully cloned,the length is495bp as verified by DNA sequencing, It can be used as the template to synthesize mRNA probe;②Whole embryo in situ hybridization revealed that Sec24b is expressed maternally and zygotically, with an expression peak in the neurula stage. After one day of development, Sec24b expression is detected in brain and anterius segmentum of body, which suggested the expression is specific in some regions. After fertilization36hpf, stained signal was only viewed at the head region and optic vesicles;③Knockdown of the gene with antisense MO caused a well described CE defect depicted by marked shortening of the anterior-posterior axis, smaller head and some severity of the extension defects;④Over-expression of Sec24B brought an abnormal convergence. Indicating that convergence was a precise dosage control process, inadequate or excessive Sec24B level can cause extended function.⑤In addition, the result of rescued experiment in zerbrafish showed that human SEC24B proteins can rescue the deformity caused by Sec24b knockout. These results validated zebrafish as a good platform for the further research of human SEC24B gene mutation and functions.3. Functional analysis of human SEC24B missense mutations in NTD.Zebrafish system and cultured cell system were used to detect the expression level of SEC24B, the protein interaction with VANGL2and sub-cellular localization. The details are as following:①Sixexpression plasmids with myc-tag containing wild type and mutantSec24b(pCMV6-SEC24B-F227S-myc, pCMV6-SEC24B-F682L-myc, pCMV-SEC24B-R1248Q-myc, pCMV6-SEC24B-A1251G-myc)were constructed.They wereadopted to transfect HEK293T cells, then the expression of different SEC24B proteins were detected by western blot,②The physical interaction with VANGL2were confirmed by Co-IP;③Endogenous Sec24b gene was silenced by using siRNA, exogenous Sec24b and mutants were expressed by wild type and mutant Sec24b plasmids transfection. Cellular location of VANGL2were visualized by fluorescent co-localization.④Co-injected1-2cell zebrafish embryos with different concentration of wild type and mutant plasmid and Sec24b-MO respectively, statisised and annalysed abnormal phenotypes.Results:.(1)The results demonstrated that:the SEC24B variants will influence its stability and its physical interaction with VANGL2; The expression level of P227S and F682Lwere lower than WT, Co-IP assay also indicated that mutants F227S and F682L alter their interactions with VANGL2, their interactions disappeared or lowered.(2)Subcellular localization showed that mutants Phe227Ser and R1248Q would dramatically affect SEC24B function by the sorting and the delivering of VANGL2out of the ER, VANGL2accumulated in ER. A1251G, VANGL2proteins were partially trapped in the ER,F682L was uniformly distributed in cytoplasm, neither trapped.(3) Rescue assays demonstrated that four variants cann’t rescue abnormality caused by Sec24b-MO obviously, but F682L and R1248Q could recese partially.In conclusion, loss of function of Sec24b gene could effcet VANGL2protein transport from the ER to the Golgi, caused NTD indirectly. Functional mutations in SEC24B might contribute to the etiology of a subset of human NTD. Human SEC24B gene could partially recsue the function of Sec24b loss in zerberfish; F227S mutation displayed loss-of-function effects completely. So zerberfish could work as a well-suited platform for furtherinvestigation on the relationhip between human NTD and function and mutation of core genes in of PCP.