Mechanistic Analysis Of Dishevelled-binding Protein Lurapl In The Regulation Of Cell Polarity And Wnt Receptor Frizzled8a In The Modulation Of Retinal Cell Differentiation

The canonical Wnt/?-catenin and the non-canonical Wnt/PCP signaling pathways regulate a myriad of biological processes throughout the early embryonic development and adult life of all animals.They also play key roles in various human diseases.In these pathways,Dishevelled interacts with Frizzled receptors to relay signals that control many cellular events including cell proliferation,cell differentiation,cell polarity,and stem cell renewal.During early development,both Frizzled and Dishevelled function in Wnt/?-catenin and Wnt/PCP pathways to orchestrate cell differentiation and asymmetric cell movements.However,the mechanism by which that coordinates these distinct and related developmental process remains elusive.Here,we use zebrafish as a model to study the regulation of Dishevelled activity in cell polarity during convergence and extension,which is dependent on Wnt/PCP signaling,and the implication of Frizzled8a in retina differentiation that requires "Wnt/?-catenin signaling.The Wnt/PCP signaling pathway plays a key role in regulating convergence and extension movements during gastrulation.This pathway contains several core proteins including Frizzled receptor and Dishevelled.Dishevelled functions as a scaffold protein that transduces Wnt/PCP signal from membrane receptors to downstream effector molecules,regulating cellular polarisation.It posseses three well conserved domains:DIX,PDZ and DEP.DIX domain regulates the polymerization of Dishevelled,while PDZ domain directly interacts with Frizzled receptors as well as other cytoplamic proteins.The DEP at the C-terminus also contains interaction sites with other proteins.Functional studies have shown that the PDZ and DEP domains are required for activation of the Wnt/PCP signaling pathway to establish and maintain cell polarity during gastrulation.However,the mechanism regulating Dishevelled activity in Wnt/PCP signaling still needs further detailed investigation.We have identified a novel protein,Lurapl(Leucine-repeat adaptor protein 1),which interacts with the PDZ domain of Dishevelled through its C-termnus PDZ-binding motif.By in vivo time-lapse imaging,we found that,in maternal-zygotic lurapl mutant(MZlurap1),the movement of lateral cells toward dorsal midline and the elongation of dorsal midline cells along anteroposterior axis were defective,thus resulting in impaired convergence and extension movements.Further analyses indicated that Lurapl regulates the formation of oriented lamellipodia and filopodia in movement cells.Moreover,Lurapl was required for the correct positioning of microtubule organisation center/centriole,which is a read-out of Wnt/PCP signaling.Thus,Lurapl is likely involved in cytoskeletal rearragement required for cellular protrusive activity and directional movement.Epistatic study showed that Lurapl regulates the activity of Dishevelled and acts upstream of JNK in Wnt/PCP signaling.Together,our results demonstrate that Lurapl,likely by modulating Dishevelled membrane recruitement,restricts the local activation of Wnt/PCP signaling and prevents the random formation of cellular protrusion.This work thus identified a novel component of Wnt/PCP pathway,implicated in the regulation of cell polarity and movement behaviors during gastrulation.During vertebrate development,eye formation involves highly coordinated processes.The early eye field specification and development of various eye tissues are regulated by several transcription factors and signaling pathways.Wnt/p-catenin signaling plays a key role in different aspects of eye development.It is activated in retinal mitotic cells,and regulates the proliferation of retinal precursor cells.Although several Wnt receptors of the Frizzled family were shown to be involved in eye development,their precise function during eye differentiation remains elusive.The expression of zebrafish frizzled8a gene was found first in the eye field,and then in the retina.We used the TALENs gene-editing technology to generate a targeted mutation in frizzled8a gene,and found that loss-of-function of this gene leads to microphthalmia.Histological,cellular and molecular analyses showed that retinal progenitor cells in microphthalmic embryos failed to exit normally from the cell cycle and to enter into the post-mitotic state.They subsequently exhibited delayed differentiation associated with enhanced apoptosis,which results in an abnormal lamination of retinal layers,progressively decreased retinal cell number,and small eye phenotype.These findings suggest that Frizzled8a may play a specific role in regulating cell cycle progression during the differentiation process of retinal progenitor cells.The Wnt signaling pathway plays critical roles in the whole life of all animals,and its dysfunction leads to various human diseases.Dishevelled functions in the crossroad of different Wnt pathways,it receives extracellular Wnt inputs and transduces distinct signals.Further identification of novel Dishevelled-interacting partners should provide insight into the mechanism regulating the activation of different Wnt pathways.In addition,different Frizzled receptors display both signaling and tissue expression specificities.Analysis of the tissue-specific function of Frizzled receptors should help to understand the implication of Wnt signaling in key developmental processes.The abnormal differentiation of retinal progenitor cells followngfrizzled8a loss-of-function has the potential to provide insight into the molecular pathways involved in human ocular pathologies.