Functional Analysis Of Lurapl In The Morphogenetic Movements Of Early Zebrafish Embryos

Lurapl(leucine repeat adaptor protein 1),also known as Lrap35a,is an adaptor protein with two leucine-rich repeats at its N-terminal region and a PDZ-binding motif at the extreme C-terminus.In cultured cells,it has been shown that Lurapl regulates actomyosin retrograde flow and cell migration by forming a tripartite complex with myotonic dystrophy kinase-related Rac/Cdc42-binding kinase(MRCK),and the unconventional MYO18A through the leucine-rich repeats and the PDZ-binding motif,respectively.Although this protein is highly conserved among vertebrate species,its implication in regulating cell movements during early development has never been reported.Here,we generated a null mutation of lurapl in zebrafish using TALENs technique.We found that the heterozygous mutant embryos develop normally,and the zygotic homozygous mutant embryos exhibit weak morphogenesis defects,but is viable and fertile.The MZlurapl embryos,obtained by intercrossing homozygous mutant fish,however,displayed obvious epiboly delay and convergence and extension(C&E)defects.lurapl mRNA overexpression can specifically rescue these morphogenetic defects of MZlurapl mutant embryos,suggesting that maternal lurapl should participate epiboly and C&E movements before and during gastrula stage.The driving force of epiboly in zebrafish embryos is originated from two sources:one is the pulling force from the cortical F-actin of the vegetal yolk cell,and the other is from the radial intercalation of deep cells in the blastodisc.In MZlurapl embryos,the shape of marginal EVL cells tends to be less elongated in the animal-vegetal direction,and significantly reduced E-YSNs were pulled out vegetally from the YSL.This is partially due to the disorganized assembly of the cortical F-actin of the vegetal yolk cell in MZlurapl gastrula.This defective organization of cortical F-actin may result in the decrease of tension on the EVL leading edge and the abnormal E-YSN behaviour.The reason why epiboly delay can only be partially interpreted by the F-actin assembly defect is that this defect is highly penetrated in embryos from one spawn or between multiple batches,while all these embryos displayed similar extent of epiboly defect.This leads us to investigate the cell behavior in the blastodisc.Loss of function of Lurapl also leads to the aberrant radial intercalation behaviour of deep cells.Further analysis revealed that the MZlurapl cells can sort from the wild-type,which is due to the weakened cohesion between mutant cells.In conclusion,these results indicate that the Lurap1 regulates epiboly movement by promoting cell cohesion as well as the assembly and organization of cortical F-actin in the vegetal yolk cell.Gastrulation is a fundamental morphogenetic event that requires polarised cell behaviours for coordinated asymmetric cell movements.Wnt/PCP signalling plays a critical role in this process,Dishevelled is an important conserved scaffold protein that relays Wnt/PCP signals from membrane receptors to the modulation of cytoskeleton organisation.However,it remains unclear how its activity is regulated for the activation of downstream effectors.Here we report that Lurapl is a Dishevelled-interacting protein that regulates Wnt/PCP signalling in convergence and extension movements during vertebrate gastrulation.Its loss-of-function leads to enhanced Dishevelled membrane localisation and increased JNK activity.In addition,Lurapl functionally interacts with Dishevelled and JNK respectively to regulate convergence and extension movements.These findings demonstrate that Lurap1 functions to regulate cellular polarisation and motile behaviours during gastrulation movements.