| The normal extension and targeting of neuronal axons and dendrites,as well as the mutual recognition and interaction between neurons,are crucial for the assembly of neural circuits.This complex process requires the involvement of different cell surface recognition molecules.In Drosophila melanogaster,the Down syndrome cell adhesion molecule 1（Dscam1）serves as a cell surface molecule extensively involved in regulating the normal development of various types of neurons.One classical model proposes that each neuron expresses dozens of Dscam1 protein isoforms,with isoforms sharing the same extracellular domains engaging in homophilic interactions,thereby transmitting repulsive signals that enable neuronal arborization to undergo self-avoidance.In this self-avoidance model,the number of Dscam1 isoforms is important,while the specific characteristics of a particular Dscam1 isoform are not.This classic self-avoidance model reasonably explains self-avoidance/non-self coexistence patterns of dendritic arborization neurons and the separation of mushroom body sister axons.However,the extent to which the classical self-avoidance model can explain the role of Dscam1 isoform diversity in neuronal development is unclear.Since the Dscam1 diversity is due to the mutually exclusive alternative splicing of exon clusters 4,6,and 9,which encode half of the immunoglobulin2（Ig2）,half of the Ig3,and the entire Ig7 domain,respectively,this study focuses on manipulating the exon 9 splicing patterns to investigate the crucial role of Dscam1 isoforms.This study constructed a series of cis-allelic mutants by CRISPR/Cas9-mediated deletion of the conserved sequence of Dscam1 intron 8.These mutants were named Dscam1Δ9u D1-Δ9u D5 based on the different deletion sequences.Molecular identification of Dscam1Δ9u D1-Δ9u D5 was conducted by RT-PCR,Western blot and RNA-seq,and the results showed that Dscam1Δ9u D1-Δ9u D5 mutants exhibited altered composition of Dscam1 Ig7 variants but unchanged protein expression levels and isoform diversity.This characteristic makes them suitable for studying the importance of Dscam1 isoform composition in Drosophila growth and neural development.Firstly,phenotypic analysis showed that altering the composition of Dscam1 Ig7 variants affected the egg-laid and survival rate of Drosophila,and also slightly affected the average lifespan of Dscam1Δ9u D2-Δ9u D5 mutants,but had little effect on the locomotor ability.These results suggest that the composition of Dscam1 Ig7 variants plays a role in the growth and development of Drosophila.Secondly,phenotypic analysis of dendritic morphology in dendritic arborization neurons revealed that altering the composition of Dscam1 Ig7 variants does not affect the self-avoidance of self-dendrites or the coexistence of Class I and Class III dendritic arborization neurons,supporting previous models that Dscam1 isoform diversity mediates self-avoidance/non-self coexistence patterns in dendritic arborization neurons.Interestingly,further analysis showed that altering the composition of Dscam1 Ig7 variants led to excessive branching and growth of dendrites in Class I dendritic arborization neurons.This result reveals a distinctive function of Dscam1 isoforms in the development of dendritic arborization neurons is different from the classical self-avoidance mechanism.Importantly,phenotype analysis of the mushroom body revealed that changing the composition of Dscam1 Ig7 variants resulted in up to 60%defects in Dscam1Δ9u D4 and Dscam1Δ9u D5 mutants.Further analysis using the MARCM technique to label single mushroom body neurons showed that axons of individual mutant neurons primarily exhibited single branch or growth defects.These findings do not support the previous model that diversity of Dscam1isoforms mediates axonal growth in the mushroom body.Similarly,comparative analysis of mechanosensory neurons indicated that altering the composition of the Dscam1 Ig7 variants resulted in mild defects in the extension and branching of mechanosensory neuron axons.Finally,this study analyzed the effects of reducing the expression level of Dscam1 on the phenotypes resulting from altered Dscam1 Ig7 variants composition.The results showed that when Dscam1 expression level was halved,the axonal development defects in the mushroom body and mechanosensory neurons were exacerbated,but dendritic branching and growth defects in dendritic arborization neurons were partially reversed.These results further demonstrate that the mechanisms by which Dscam1 isoforms regulate axonal and dendritic growth and branching in neurons are significantly different.In summary,this study generated a series of Dscam1 mutants with altered isoform compositions by deleting non-coding cis-elements,enabling functional investigation of Dscam1isoform diversity.Through analysis of three different neuronal phenotypes,this study found that changes in Dscam1 Ig7 variants caused distinct and significant phenotypic defects:up to 60%defects in mushroom body,significant overgrowth and branching of dendritic arborization neurons but normal self/non-self discrimination,and slight growth and branching defects in mechanosensory neurons.These results revealed the widespread nonclassical functions of Dscam1 diversity in neuronal morphogenesis,challenged the classical self-avoidance model of Dscam1 in mushroom body development,and expanded our understandings of the diverse functions of Dscam1 diversity in the nervous system. |
