Mechanisms of Sema-1a dependent midline crossing in the Drosophila CNS

果蝇 CNS 中 Sema-1a 依赖性中线穿越的机制

• 批准号: 8652151
• 负责人: Melissa Courtney Hernandez
• 金额: $ 4.22万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-03-01 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8652151
• 关键词: Address; Axon; Binding; Biological Assay; Cells; Cues; Cytoplasmic Tail; DCC gene; Data; Defect; Development; Drosophila genus; Elements; Embryo; Excision; Exhibits; Failure; Genes; Genetic; Genetic Screening; Human; Injury; Left; Ligands; Mediating; Modeling; Motor; Movement; Movement Disorders; Mutation; Nerve Regeneration; Nervous System Physiology; Neurons; Oncogenes; Pathway interactions; Process; Role; Semaphorins; Side; Signal Transduction; System; Testing; Transgenic Organisms; axon guidance; in vivo; insight; mutant; nervous system development; novel; plexin; public health relevance; receptor; research study; screening

DESCRIPTION (provided by applicant): For the majority of developing neurons an essential step in proper guidance involves crossing the midline, and failure to do so often results in an inability to coordinate movement. This is further evidenced by the association between mirror movement disorders and mutations in the deleted in colorectal cancer (DCC) gene, which is a highly conserved guidance receptor that mediates midline crossing. However, DCC mutants (or frazzled in Drosophila) still exhibit significant midline crossing, implicating additional pro-crossing mechanisms. To identify these alternative pathways and their potential contribution to motor coordination, a genetic screen was performed and the transmembrane Sema, sema-1a, was identified as a novel pro-crossing gene. This proposal aims to determine the mechanism of sema-1a in midline crossing and the potential contribution of its canonical receptor, plexin A. Semas traditionally signal repulsion as ligands for Plexin receptors, however, transmembrane Semas can also act as receptors themselves and signal in reverse. Preliminary data indicates that Sema-1a requires its cytoplasmic domain to promote midline crossing, suggesting it may function through reverse signaling. Aim 1 will determine if Sema-1a promotes midline crossing through forward or reverse signaling. Sema-1a reverse signaling would imply a cell autonomous requirement in commissural neurons, which can be tested in defined subsets through in vivo rescue assays. Additionally, the elements within the cytoplasmic region that confer midline crossing activity will be identified. Downstream effector molecules, which bind Sema-1a's cytoplasmic region, will also be examined for defects in midline crossing. Plexin A (PlexA) is the canonical binding partner of Sema-1a repulsive signaling, however, Sema- 1a can also function in a Plexin-independent manner. Mutations in plexA significantly enhance the crossing defects in the sensitized screening background suggesting that PlexA also promotes midline crossing. It is unclear whether PlexA functions as the binding partner for Sema-1a in this process. Aim 2 will determine if PlexA acts as the binding partner for Sema-1a mediated midline crossing. It will be determined if Sema-1a mediated midline crossing is PlexA dependent. Furthermore, the signaling mechanism can be determined by defining which neurons require PlexA for midline crossing. As both Sema-1a and PlexA traditionally function through repulsive signaling, this mechanism will be tested with in vivo rescue assays. Together with the structural requirements identified in Aim1, these experiments will establish a mechanism for Sema-1a mediated midline crossing.

描述（由申请人提供）：对于大多数发育中的神经元来说，正确引导的一个重要步骤是跨越中线，如果不能做到这一点，通常会导致无法协调运动。这进一步证明了镜像运动障碍与结肠直肠癌（DCC）基因缺失突变之间的关联，DCC基因是一种高度保守的介导中线穿越的指导受体。然而，DCC突变体（或果蝇中的frazzled）仍然表现出显着的中线交叉，涉及额外的亲交叉机制。为了确定这些替代途径及其对运动协调的潜在贡献，进行了遗传筛选，并将跨膜Sema，sema-1a，鉴定为新的亲交叉基因。本研究旨在探讨sema-1a在中线穿越中的作用机制及其经典受体plexin A的作用。 Sema传统上作为丛状蛋白受体的配体发出排斥信号，然而，跨膜Sema本身也可以作为受体并反向发出信号。初步数据表明，Sema-1a需要其胞质结构域来促进中线交叉，这表明它可能通过反向信号传导发挥作用。目的1将确定Sema-1a是否通过正向或反向信号传导促进中线穿越。Sema-1a反向信号传导将意味着连合神经元中的细胞自主要求，这可以通过体内救援测定在定义的子集中进行测试。此外，将鉴定胞质区域内赋予中线穿越活性的元件。下游效应分子，结合Sema-1a的细胞质区域，也将检查中线交叉的缺陷。 丛蛋白A（PlexA）是 Sema-1a排斥性信号传导的典型结合配偶体，然而，Sema- 1a也可以以丛状蛋白非依赖性方式起作用。plexA中的突变显著增强了敏化筛选背景中的交叉缺陷，表明PlexA也促进中线交叉。目前尚不清楚PlexA是否在此过程中作为Sema-1a的结合伴侣发挥作用。目的2将确定PlexA是否作为Sema-1a介导的中线穿越的结合伴侣。将确定Sema-1a介导的中线穿越是否为丛A依赖性。此外，可以通过定义哪些神经元需要PlexA进行中线穿越来确定信号传导机制。由于Sema-1a和PlexA两者传统上通过排斥信号传导起作用，因此将用体内拯救测定来测试该机制。结合Aim 1中确定的结构要求，这些实验将建立Sema-1a介导的中线穿越机制。