Functional analysis of DSCAM isoforms during development of neuronal wiring in Drosophila

2005 Fiscal Year Final Research Report Summary

• Project/Area Number: 16570061
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Animal physiology/Animal behavior
• Research Institution: Shinshu University
• Principal Investigator: SAKAGUCHI Masahiko Shinshu University, Faculty of Education, Associate Professor, 教育学部, 助教授 (30221998)
• Project Period (FY): 2004 – 2005
• Keywords: Drosophila / DSCAM / splicing / genetics / Down-syndrome / cell adhesion molecule / mutant / neural wiring

Research Abstract

Down syndrome cell adhesion molecule (DSCAM) is an immunoglobulin superfamily protein required for the formation of neuronal connections in Drosophila. The DSCAM consists of 24 exons. Mutually exclusive alternative splicing occurs for exon 4,6,9, and 17.1 of 12 exon 4 alternatives, 1 of 48 exon 6 alternatives, 1 of 33 exon 9 alternatives, and 1 of 2 exon 17 alternatives are retained in each mRNA. Thus, alternative splicing of DSCAM generates an enormous molecular diversity with maximally 38,016 different receptors in Drosophila. Whether this large diversity is required in vivo for the neuronal wiring specificity is currently unknown. Previous experiments have shown that single DSCAM isoforms could rescue defects in DSCAM null mushroom body neurons during neuronal differentiation or axon bifurcation. Different isoforms rescued the phenotypic defects equally well, questioning the need for the molecular diversity of DSCAM in this system. Genetic analysis of DSCAM has been done in DSCAM he terozygous null mutant flies with MARCM method, or in fly strains which selectively lack some alternative exons due to imprecise P-element excision of the P-element inserted DSCAM allele. I planed to get homozygous lethal mutant flies specific for single alternative exon by EMS mutagenesis. First, DSCAM[KG00799a] DSCAM[KG00799b] fly (BL-12956 derived) was crossed to delta2-3 fly to excise the small genome between two P-elements. Because the start Met codon region is between the two P-elements, rigid DSCAM null (DSCAM-Df)/CyO was expected among white eye flies. EMS-treated lines which were homozygous lethal and failed to complement with DSCAM-Df were collected as DSCAM mutant allele/CyO. Mutant flies specific for different alternative exon should be able to complement with each other. For example, 6.1 mutant/CyO and 6.4 mutant/CyO can generate the next generation with 6.1 mutant/6.4 mutant(Cy+). A total of 2000 independent lines were analysed. Four lines were established. Unfortunately, these lines did not show any complementation each other. The phenotypes of MARCM clones were almost the same.