Mutant Ahi1 Affects Retinal Axon Projection In Zebrafish Via Toxic Gain Of Function

AHI1 is an evolutionary conserved gene that is widely expressed in a n?mber of animals from Xenopus to h?mans.The h?man AHI1 protein contained three domains: N-terminal coiled-coil domain,middle WD40 repeat domain,and C-terminal SH3 domain.The WD40 repeat domain and SH3 domain are conserved in Ahi1 in different species.Mutations in the N-terminal region of h?man AHI1 were found to affect early development and cause Joubert Syndrome.However,it remains unknown whether AHI1 mutations affect early brain development via loss of normal function or gain of toxic function.Compared to the mouse Ahi1,the zebrafish Ahi1 protein is closer to the h?man AHI1 in its structure.Thus,we used zebrafish as a model to explore Ahi1 function in early brain development.Through in-situ hybridization,we found that ahi1 is expressed at early developmental stages in zebrafish embryos with abundant expression in embryonic brain and is gradually decreased during development process.We injected morpholino into one-cell stage embryos to knock down ahi1 expression and detected a truncated ahi1 mRNA that has deleted two exons.The 4 dpf injected embryos exhibited abnormal optic nerve projection phenotypes including abnormal middle crossing and elongation.In addition,ocular tissues area was also reduced when compared to wild type embryos.Using p53 or control morpholino,we found that the above phenotypes are specific to ahi1 morpholino and were not due to morpholino toxicity or injection injure.We examined the 4 dpf ahi1 null zebrafish embryos but did not find any abnormal optic nerve projection phenotypes or abnormal eye structures.We then designed two gRNAs to target the zebrafish ahi1 via CRISPR/Cas9 and generated mutant zebrafish expressing truncated ahi1.The mutant zebrafish displayed abnormal optical axon projection similar to that caused by ahi1 morpholino injection.Thus,using both morpholino and CRISPR/Cas9,we found the truncated Ahi1,which does not contain the intact WD domain,could affect optical axon projection in zebrafish via gain-of-function toxicity.