Mutational Analysis Of Dishevelled Genes In Zebrafish Reveals Distinct Functions In Embryonic Patterning And Gastrulation Cell Movements

Wnt signaling pathway consists of canonical Wnt signaling pathway and noncanonicalWnt/planar cell polarity(PCP)signaling pathway.Canonical Wnt signaling pathway stabilizes P-catenin,the subsequent accumulation and nuclear localization of β-catenin activates target genes expression,accomplishing cell fate determination.Noncanonical Wntsignaling pathway triggers the rearrangement of cell skeleton,regulating the cell movement.Wnt signaling pathway plays critical roles in dorsoventral fate specification and anteroposterior patterning,as well as in morphogenetic cell movements of vertebrate embryonic development.Categoried by the time and source,conanical Wnt signaling pathway in zebrafish can be devided into maternal Wnt signaling pathway and zygotic Wnt signaling pathway.Maternal"Wnt signaling pathway is activated by maternally supplied factors at blastula stage,which determines the dorso-anterior cell fate.Zygotic Wnt signaling pathwayis activated by zygotically supplied factors at gastrula stage,which determines the ventro-posterior cell fate.PCP signaling pathway regulates the cell polarity,defect of which causes convergence extension(CE)movement and craniofacial abnormalities.Scaffold protein Dishevelled,or Dvl,mediates the activation of Wnt/β-catenin and Wnt/planar cell polarity pathways.There are 5 highly conserved Dv1 proteins in zebrafish,but the implication of each Dvlin keyearly developmental processes remains poorly understood.No investigation suggests how the 5 Dvl proteins and their maternal and zygotic dosage participate in the canonical and noncanonical Wnt signaling pathway activation.In this study,we used genome-editing approach to generate different combinations of maternal and zygotic dvlmutants inzebrafish,and examined their functions during early development.Maternal transcripts fordvl2and dvl3aare most abundantly expressed,whereas the transcriptlevels of other dvlgenes are negligible,suggesting dvl2 and dvl3a may play a major role in zebrafih early embryo development.Among 5 maternal-zygotic(MZ)dvl homologue mutants,only MZdvl2 displays obvious convergence extension movement defect and craniofacial defect.MZdvl3a mutant displays slight convergence extension movement defect.MZdvlla,MZdvl1b and MZdvl3b develop roughly normal.These observations suggest that dvl2 plays predominant role cmparing with its paralogues in zebrafish early embryo development.We then further reduced Dvl dosage by double mutating.Zygotic dvl2and dvl3adouble mutants display mild axisextension defect with correct anteroposterior patterning.Zygotic dvl2 and maternal-zygotic dvl3a displays severe CE defect and posterior developmental abnormality.By construction of mosiac fish,we obtained maternal and zygotic dvl2and dvl3adouble mutants,albeit the lethality of zygotic mutants.Maternal and zygoticdouble mutants exhibit most strongly impaired convergence and extension movements,severe trunk and posterior deficiencies,and frequent occurrence of cyclopia and craniofacial defects.Phenotypic and molecular analyses show that early dorsal fate specification is not affected in maternal and zygotic dvl2and dvl3adouble mutants,suggestingthat the two proteins may be dispensable for the activation of maternal Wnt/β-catenin signaling.Our results suggest that maternal and zygotic Dv12 and Dv13 a products are required for the activationof zygotic Wnt/β-catenin signaling and Wnt/planar cell polarity pathway in a dosage-dependent manner,while Dv12 plays a predominant role.This work providesinsight into the mechanisms of Dvl-mediated Wntsignaling pathways during early vertebratedevelopment and a more comprehensive understanding of the essence of dorsal determinants.