Functional Study Of Vegetally Localized Maternal Factor Vrtn During Dorsoventral Patterning In Zebrafish

The formation of body plan is crucial for individual development.In particular,the establishment of dorsoventral(DV)axis is one of the earliest developmental processes that breaks radial symmetry of the fertilized egg,determines the order of organ anlages along the dorsoventral direction and is crucial for the formation of basic bilateral vertebrate body plan.The vegetal pole cytoplasm is a critical source of maternal signals implicated in the patterning of the primary dorsoventral axis both in fish and amphibians.It is essential for the formation of DV axis.Triggered by fertilization and controlled by microtubules,the transportation of dorsal determinants(DDs)is asymmetrical,from vegetal pole to the perspective dorsal side in fish and in amphibians.Then,DDs activate Wnt/?-catenin signalling in the dorsal region of the blastula,causing the stabilization and nuclear localization of ?-catenin 2,which is essential for zygotic expression of Spemann organizer genes.Several candidate DDs including Dishevelled,G13P,Wntl I in Xenopus,and Wnt8a in zebrafish,have been suggested to be required for dorsal axis formation.The dorsal organizer is thought to play a central role in the formation of the dorsal ventral axis.It is well established that the Spemann organizer expresses both BMP signalling antagonists and BMP ligands,which is required for establishing a correct BMP Oradient and represents a mechanism of self-regulation of the morphogenetic field.The role and regulation of F3MP signalling in the dorsal region have been well characterized,however,how this pathway is regulated in other regions of the early embryo remains largely unknown.Several maternal factors,including Gdf6a and Pou5f3.have been shown to play a critical role for zygotic transcription of bmp genes,but no repressive transcription factors targeting bmp genes have been found in the lateral and ventral regions.Previous work of our lab has suggested the existence of vegetally localized bmp inhibitory factor functioning independent of the Wnt/?-catenin signaling.In this study,we identified a novel vegetally localized factor,Vrtn,through high throughput RNA-seq of animal and vegetal explants of zebrafish fertilized eggs.The transcripts of vrtn migrate rapidly from vegetal pole to the margin of the blastodisc.Overexpression of vrtn by injecting the synthetic mRNA(100 pg)resulted in varied degrees of dorsalization.This dorsalization is specifically caused by BMP inhibition,and irrelevant with the activation of Wnt/?-catenin.We next successfully knocked out vrtn to further examine its endogenous function by CRISPR/Cas9 mediated genome editing.Maternal and zygotic mutant embryos(MZ-vrtn)displayed a mildly ventralized phenotype.When?-catenin 2 was knocked down in MZ-vrtn embryos,severely ventralized embryos was synergestically increased.We then aligned vrtn amino acid sequences from different species and identified five conserved domains(CDs).The subcellular localization of Vrtn and its truncated mutants were first analysed and the results showed that CD1 and CD3 contain motifs critical for the nuclear localization of this protein.We then carried out one-hybrid analyses using the UAS-Gal4 system to investigate how Vrtn functions as a transcriptional regulator,and found that Vrtn acts as a potent transcriptional repressor.Moreover,an N-terminal domain was identified to be important for its repressor activity.Further analysis by CHIP-qPCR confirmed that Vrtn can bind to the upstream sequence of bmp2b,and thus can repress the transcription of bmp2b gene.In summary,we have identified and characterized a novel vegetally localized transcriptional repressor,Vrtn,which binds bmp2h upstream sequence and inhibits its transcription independently of maternal Wnt/?-catenin signaling.Our work thus unveils a novel maternal mechanism regulating BMP gradient for the formation of dorsoventral axis.