Regulatory Effects Of Wnt/?-catenin Signaling Pathway On The Early Neurodevelopment Of Zebrafish Embryos

Objectives: Wnt signaling is an important signaling pathway,which plays a vital role in the organogenesis and embryonic development,and is also closely related to the self-renewal and differentiation of tissue stem cells(SCs).In this study,we used zebrafish as a model to study the effects of Wnt signaling on embryonic morphology,the functions of neural SCs and the expression of neural transcription factors.We also intended to explore the mechanism of the actions of Wnt signaling on neural differentiation and neurodevelopment during the early embryonic development.Methods:(1)At 3-72 hpf,zebrafish embryos were exposed to 3 ?M BIO,a Wnt activator or 10 ?M IWR,a Wnt inhibitor,respectively to up-regulate or down-regulate Wnt signaling,and then the mortality rate,malformation rate and hatchability rate of the embryos were recorded.(2)After the exposure to BIO or IWR,the embryos were collected at 2,12,24,48 and 72 hpf for the extraction of total RNA,which was used for the quantitative PCR analysis of the expression of Wnt target genes(Axin2 and Lgr6),embryonic SC and neural differentiation marker genes(Sox2,Shha,Nestin,Eno2 and GFAP)and pro-and anti-apoptotic genes(Baxa and Bcl2).12-48 hpf embryos were used for the whole-embryo in situ hybridization to measure the spatial expression of Axin2,Sox2,Shha,Eno2 and GFAP.Whole-embryo immunofluorescent staining was applied to detect activated Caspase 3 and Phospho-Histone H3(PH3)to observe the changes in apoptosis and proliferation in 12-48 hpf embryos.(3)Upon up-and down-regulation of Wnt signaling caused by BIO and IWR respectively,the expression of radial glial progenitor marker,Pax6,intermediate progenitor marker,Tbr2,and transcription coactivator,RBM14,was measured by qPCR(12-72 hpf)and wholeembryo in situ hybridization(12-48 hpf)in the central nervous system of zebrafish embryos.To knock-down the expression RBM14,a morpholino(MO)oligonucleotide,specifically blocking the translation of RBM14,was delivered into the yolk of embryos at one cell stage by microinjection,and then whole-embryo in situ hybridization was applied to measure the expression of Pax6 and Tbr2 in the zebrafish embryos.Result:(1)Both BIO and IWR significantly increased embryonic mortality,induced embryonic malformation and inhibited embryo hatching.(2)Activation of Wnt signaling caused by BIO significantly induced the expression of Aixn2,a Wnt target gene,whereas inhibited the expression of another Wnt target gene,Lgr6.This opposite change in the expression of these two genes might be due to the different roles they play in Wnt pathway.Up-regulation of Wnt signaling inhibited the expression of SC marker,Sox2,neural SC marker,Nestin,morphogenetic gene,Shha,and mature neuron marker,Eno2,while astrocyte marker,GFAP,showed early down-regulation and late up-regulation upon BIO treatment.(3)Among all the genes tested,IWR caused the opposite changes in the expression of most genes from that induced by BIO,such as Axin2,Lgr6,Sox2,Shha,Nestin and Eno2,nevertheless the expressional changes of GFAP caused by IWR appears to be consistent with the BIO-induced.The possible reason for the similar responses of GFAP to both BIO and IWR might be related to the higher susceptibility of astrocytes to apoptosis than that of other cell lineages.(4)Both BIO and IWR significantly induced the increasing of the level of activated caspase 3 and the expression ratio of Bax to Bcl2,and caused the inhibition of cell proliferation marker PH3.However,the effects of BIO were stronger than that of IWR.These results suggest that abnormal Wnt signaling promotes apoptosis and inhibition of cell proliferation in zebrafish embryos.(5)The excess Wnt signaling remarkably inhibited the expression of Pax6 a,Tbr2 and RBM14,while the inhibition of Wnt caused the opposite changes in the expression of these genes.Knocking down of RBM14 expression caused by of the microinjection of RBM14 MO induced the downregulation of the expression of Pax6 a in the telencephalon,forebrain and eyes,and caused the inhibition of the expression of Tbr2 in the dorsal telencephalon and dorsal diencephalon(olfactory bulb).Therefore,down-regulation of RBM14 showed similar,but weaker,inhibitory effects on Pax6 and Tbr2 as that induced by excess Wnt signaling induced by BIO.Conclusion:(1)At the early developmental stages of zebrafish embryo,abnormal up-and down-regulation of Wnt signaling induced massive death and severe deformities of survival embryos;(2)Abnormal Wnt signaling severely disturbed the process of SC differentiation,and caused the aberrant expression patterns of SC markers and neural differentiation markers;(3)Anomalous changes in Wnt signaling pathway significantly induced apoptosis and inhibited cell proliferation in zebrafish embryos;(4)Down-regulation of RBM14 might be involved in the negative regulatory effects of Wnt on the expression of radial glial progenitor marker,Pax6 a,and intermediate progenitor marker,Tbr2;(5)The physiological level of Wnt signaling is essential for early embryonic development.If the level of Wnt signaling is beyond the physiological range,the embryonic development would be seriously disrupted that could result in developmental disorders of multiple organs including the nervous system.