Transcriptional Regulation Of Cerberus Gene During Amphioxus Embryonic Development

Left-right(L-R)asymmetry,established during early embryogenesis,is an essential feature of bilaterians.In vertebrate embryos,L-R asymmetry is regulated by genes of asymmetric expression patterns including Cerberus(Cer),Nodal and Lefty.Among these genes,Cer is the first one showing asymmetric expression pattern which occurs at the early neurula stage and manifests by a higher level of expression in the right side of the Left-Right Organizer(LRO)than in the left side.Since Cer is an inhibitor of Nodal protein,its asymmetric expression could then lead to an L>R asymmetric Nodal signaling activity,through which and its downstream effectors a L-R asymmetric morphology is eventually established.In most vertebrates,asymmetric expression of Cer is established by a cilia-driven leftwards flow within the LRO,but the precise mechanism underlying this remains unclear.A study in mice indicated that the asymmetric Cer expression is mainly determined post-transcriptionally by decay of Cer mRNA in a manner dependent on its 3’-UTR region,although this has not been proved in other vertebrates.Besides,studies in vertebrate embryos also indicated that transcription of Cer gene is regulated by Notch signaling and Wnt-beta-catenin-Foxj1 cascade.Amphioxus belongs to Cephalochordates and occupies a key phylogenetic position in the evolutionary path that leads to the transition from invertebrates to vertebrates.Our recent studies demonstrated that Cer shows a similar expression pattern in amphioxus embryos like its cognates in vertebrates,and acts upstream of Nodal signaling pathway during amphioxus L-R development.This indicates that asymmetric Cer expression is probably regulated by a conserved mechanism among chordates.To dissect how asymmetric expression of Cer gene is established,we made 12 transgenic mCherry reporter constructs containing series deletions of the sequence upstream of amphioxus Cer start codon and its 3’-UTR,and generated amphioxus transgenic lines carrying 11 of these constructs.In situ analysis of mCherry expression on these transgenic embryos showed that 1)the 0.15kb and 0.6kb sequences around the transcription start site(TSS)of Cer gene is unable to drive mCherry expression in any cells of amphioxus embryos;2)the 0.9kb sequence around the TSS and the 0.8kb and 1.1kb sequences upstream of the start codon have the ability to drive mCherry expression,but in a different pattern(more anterior and in scattered cells)from endogenous Cer gene;3)the 3.3kb and 5.3kb regions upstream of the start codon could recover endogenous Cer expression to some extent,with the 3.3kb driving mCherry expression in fewer cells and more anterior region and the 5.3kb driving mCherry expression in more cells and both of them driving asymmetric mCherry expression in some embryos,compared to endogenous Cer expression;4)the 3.3kb+3’-UTR and 5.3kb+3’-UTR show similar transcription activities as the 3.3kb and 5.3kb regions,but drive mCherry expression more frequently only in the right side of the embryo;5)the combination of three ATAC-seq peaks within the 5.3kb and the 3’-UTR is able to completely recover mCherry expression as endogenous Cer gene.Together,these results indicate that the core regulatory sequence controlling asymmetric Cer expression is located with the three ATAC-seq peaks and the 3’-UTR.To clarify if asymmetric Cer expression is regulated post-transcriptionally in amphioxus,we analyzed Cer pre-mRNA expression pattern using 3 antisence RNA probes from Cer intron region.We detected no signal,and speculate that this might be caused by an extremely short half-life of the pre-mRNA.To overcome this problem,we synthesized a morpholino to block Cer pre-mRNA splicing and injected it into amphioxus embryos.The injection caused all embryos to develop a two-left phenotype like Cer mutation.In the injected embryos,both Cer mRNA and its pre-mRNA are asymmetricall expressed like Cer mRNA in the unjected embryos.This result suggested that the asymmetric Cer expression in amphioxus is mainly determined at transcriptional level,but not post-transcriptionally like Cer gene in mice.