Regulatory Effects Of Retinoic Acid On The Early Neurodevelopment Of Zebrafish Embryos

Objective:Retinoic acid(RA)is an active derivative of vitamin A.During the development of vertebrate nervous system,RA signaling pathway is indispensable.The aim of this study is to explore the regulatory effects of RA signaling on embryonic morphogenesis,neural differentiation and neurodevelopment at the early developmental stages of zebrafish embryos,and to reveal the mechanisms of its actions.Methods:(1)From 0 hpf to 72 hpf,the zebra fish embryos were exposed to the 0.1?m RA or 2 ?m BMS493,a RA receptor antagonist,respectively,The morphological changes of embryos were observed under the stereomicroscope,and the embryonic mortality rate,deformity rate and hatching rate were recorded.(2)The embryos were collected from 2 hpf to72 hpf for the extraction of total RNA.qPCR method was used to quantitatively analyze the expression of stem cell(SC)and neural marker genes(Sox2,Shha,Nestin,Elav13,Eno2,GFAP and O1ig2),apoptosis-related genes(Bax and BCL2)and transcription coactivator RBM14.Whole mount in situ hybridization experiments were used to localize the expression of some of the above-mentioned genes(Sox2,Shha,GFAP,RBM14 and Eno2)in 12 hpf-48 hpf embryos,and the expression of Elavl3,activated Caspase 3 and PH3 was localized by whole mount fluorescent immunostaining.(3)Morpholino(MO)against RBM14 was microinjected into one-cell stage embryos to knockdown the expression of RBM14.Three hours later,the injected embryos were exposed to BMS493,and then the effects of two treatments on the embryonic development was observed.Results:(1)The up-regulation and down-regulation of RA signal respectively induced by exogenous RA and BMS493 both significantly inhibited embryo hatching rate and increased the embryo malformation rate.(2)Our qPCR data showed that exogenous RA inhibited the expression of the SC marker Sox2 and the morphogenetic gene Shha,whereas induced expression of genes involved in neural differentiation.(neural SC marker Nestin,early neuronal marker Elavl3,neuronal marker Eno2,astrocyte marker GFAP,oligodendrocyte marker Olig2).The results of in situ hybridization and fluorescent immunostaing experiments suggested that RA induced abnormal differentiation of SCs to neurons and glial cells in the developing central nervous system(Sox2,Elavl3 and GFAP).(3)The quantitive analysis of gene expression showed that RA receptor antagonist BMS493 had opposite effects on most of the SC and neural marker genes we examined to that of exogenous RA:BMS493 promoted Sox2 expression,while inhibited the expression of Nestin,Elavl3,Eno2,GFAP and Olig2.The expression of Shha was not obviously changed upon BMS493 exposure.The in situ hybridization and immunostaing data suggested that the embryonic spinal cord was more sensitive to the loss of RA signal than the central nervous system(Elav13 and GFAP).(4)Both RA and BMS493 induced apoptosis in zebrafish embryos.The level of active Caspase 3 and the expression ratio of Bax to Bc12 were upregulated by both molecules.However,the effects of RA was stronger than that of BMS493 and its effects also took place earlier.RAand BMS493 showed no obvious interference on cell proliferation in zebrafish embryos.(5)RA signal inhibited the expression of RBM14,meanwhile RBM14 negatively regulated the expression of RARs,vital molecules in RA signaling pathway.The down-regulation of RA signal induced by BMS493 can antagonize the effects of RBM14 MO,which was designed to specifically knock down the expression of RBM14,on embryonic malformation and cell apoptosis.Conclusion:(1)The appropriate level of RA signal is critical to stem cell differentiation and embryonic development.(2)During the early development of zebrafish embryos,RA signal is capable to induce SCs to differentiate to neurons and glial cells;(3)Abnormal RA signal level is responsible to the upregulation of apoptosis in zebrafish embryos.The effects of RA signal on cell apoptosis in embryonic nervous system might contribute to its function in the regulation of neurodevelopment.(4)The negative interaction between RA signal and transcription coactivator RBM14 is involved in regulation of embryonic development as a crucial regulatory element.