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

描述（由申请人提供）：对于大多数发育中的神经元来说，正确引导的重要步骤包括越过中线，如果不能这样做，通常会导致无法协调运动。镜像运动障碍与结肠直肠癌（DCC）基因缺失突变之间的关联进一步证明了这一点，DCC基因是一种高度保守的引导受体，介导中线交叉。然而，DCC突变体（或果蝇中的疲劳突变体）仍然表现出显著的中线杂交，这暗示了额外的亲杂交机制。为了确定这些替代途径及其对运动协调的潜在贡献，进行了基因筛选，并将跨膜Sema， Sema -1a，鉴定为一种新的前交叉基因。本研究旨在确定sema-1a在中线交叉中的机制及其典型受体丛蛋白a的潜在作用。Semas传统上作为丛蛋白受体的配体发出排斥信号，然而，跨膜Semas本身也可以作为受体并反向发出信号。初步数据表明，Sema-1a需要其细胞质结构域促进中线交叉，提示其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促进中线交叉。Sema-1a反向信号可能意味着在交联神经元中存在细胞自主需求，这可以通过体内救援试验在定义的亚群中进行测试。此外，细胞质区域内赋予中线交叉活性的元素将被确定。下游效应分子，结合Sema-1a的细胞质区域，也将检查中线交叉缺陷。Plexin A （PlexA）是

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.