Functional Studies On The Diversity Of Mutually Exclusive Alternative Splicing Variants Of Drosophila Melanogaster Dscam1

The Drosophila melanogaster Down syndrome cell adhesion molecule 1（Dscam1）gene contains 12 variable exons 4,48 variable exons 6,and 33 variable exons 9,which can generate 19,008 isoforms with homophilic binding specificity through mutually exclusive alternative splicing.Each neuron randomly expresses dozens of unique combinations of Dscam1 protein isoforms,which in turn can precisely distinguish between self and non-self during neural assembly.Current studies on Dscam1ectodomain diversity in the nervous system have focused on variable exon 4 and 9clusters,but the effect of variable exon 6 diversity on neural development in Drosophila is unclear.Meanwhile,it has been shown that thousands of Dscam1 diversities are sufficient for recognition between self and non-self,but whether there are redundancies in tens of thousands of Dscam1 diversities and whether there are differences among these Dscam1 isoforms are yet to be elucidated.To address the above scientific questions,a more systematic study on the diversity and specificity of exon 6 was carried out in this project.The results are as follows.1.This study constructs six deletion mutants with 27,18,8,6,3,and 2 exon 6variants respectively encoding 10,692,7,128,3,168,2,376,1,188,792 Dscam1isoforms(Dscam^Δ6.y-6.y’)and 10 mutants with single exon 6 variant encoding 396Dscam1 isoforms(Dscam^single6.y)using CRISPR/Cas9 technology.RT-PCR results indicated that the Dscam1 transcript levels of these mutants were similar to that of the wild-type.2.Phenotypic analysis for all mutants showed that as Dscam1 diversity decreased,the degree of defects in embryo hatching,locomotion,reproduction,longevity,dendritic coexistence,and mushroom bodies began to increase.Reproduction and longevity were more sensitive to the effects of reduced Dscam1 diversity,suggesting that they may need more diversity.The results suggest that high Dscam1 diversity has an important role in Drosophila growth and neurodevelopment.3.Pair-wise comparisons of phenotypes revealed Dscam^single6.y showed differences in embryonic hatchability,locomotion,reproduction,dendritic coexistence,and mushroom body phenotypes.In particular,as for mushroom body phenotypes,Dscam^single6.y mutants exhibited not only significantly different defect ratios but also different compositions of defect types.These results suggest a functional difference between Dscam1 isoforms.4.In this study,two unreported defect phenotypes were found in Dscam^single6.ymushroom bodies:shortened lobes and two-lobe thinning,and mushroom body single neurons exhibited single-branch and growth defects.These defects could not be explained by Dscam1-mediated self-avoidance mechanisms.The results of this study suggest that self-avoidance alone cannot explain the function of Dscam1 protein in mushroom body development.5.This study found that survival,locomotion,dendritic coexistence abnormalities,and mushroom body defects were significantly improved in Dscam^Single6.y/Dscam^nullcompared to Dscam^Single6.y.The results of this study suggest that reducing the expression level of Dscam1 ameliorates the defects caused by reduced diversity.In conclusion,the results of this paper demonstrate the important role of Dscam1diversity and specificity in Drosophila development,reveal the vital function of Dscam1-mediated non-repulsive signaling in mushroom body development,elucidate the functional link between Dscam1 diversity and expression levels,and provide new ideas and new insights for studying Dscam1.