Unraveling cellular and molecular mechanisms that coordinate pre- and postsynaptic maturation by intravital imaging in the Drosophila central nervous system

我们的目标是了解中枢神经系统(CNS)神经网络发展的细胞和分子机制。瞬时的细胞间相互作用和突触蛋白支架的动态组装是网络形成的标志。对这些相互作用过程的适当调控是胚胎发生过程中网络功能出现并在胚胎后生长和成熟过程中保持的先决条件。我们在基因筛查中发现了受体酪氨酸激酶间变性淋巴瘤激酶(ALK)，以调节果蝇运动系统中突触后生长和突触的形成。ALK在发育中的肠道和神经系统中表达；有趣的是，这种表达模式在脊椎动物(老鼠)和苍蝇之间是保守的。ALK信号在调节神经元生长和突触发生中的作用尚不完全清楚，特别是它如何影响突触后细胞的发育和它们的连接性。在这个建议中，我们具体讨论了i)突触前和突触后终末之间的细胞相互作用如何导致神经元分支的稳定和中枢神经系统突触的发展；ii)在这些分支上形成的新生突触的分子成熟；以及iii)ALK信号在调节这些相互作用中的作用。为了以最小的干扰研究网络形成的动力学机制，我们开发了两种新的技术：i)果蝇胚胎和幼虫CNS中伙伴神经元(运动神经元和间神经元)之间的细胞和分子相互作用的活体成像；ii)单个神经元内源性表达蛋白的荧光标记的靶向激活。这项工作计划将极大地促进我们对发育中的中央网络中伙伴神经元之间的细胞相互作用、突触前和突触后支架形成的分子动力学以及这些分子是如何调控的理解。此外，它将产生一个丰富的遗传工具包，很容易应用于研究神经系统其他部分的突触生物学，例如研究记忆形成或神经退行性疾病。

We aim to understand the cellular and molecular mechanisms that underlie the development of neural networks in the central nervous system (CNS). Transient cell-cell interactions and the dynamic assembly of synaptic protein scaffolds are hallmarks of network formation. Appropriate regulation of these interactive processes is a prerequisite for network function to emerge during embryogenesis and to be maintained during postembryonic growth and maturation. We identified the receptor tyrosine kinase Anaplastic lymphoma kinase (Alk) in a genetic screen to regulate postsynaptic growth and synapse formation in the motor system of the fruit fly Drosophila melanogaster. Alk is expressed in the developing gut and nervous system; intriguingly, this expression pattern is conserved between vertebrates (mouse) and the fly. The role of Alk signalling in regulating neuronal growth and synaptogenesis is incompletely understood, particularly how it affects the development of postsynaptic cells and their connectivity. In this proposal we specifically address i) how the cellular interactions between pre- and postsynaptic terminals lead to the stabilisation of neuronal branches and the development of synapses in the CNS; ii) the molecular maturation of nascent synapses that form on these branches; and iii) the role of Alk signalling in regulating these interactions. To investigate the dynamic mechanisms of network formation with minimal disturbance, we developed 2 novel techniques: i) intravital imaging of cellular and molecular interactions between partner neurons (inter- and motorneurons) in the embryonic and larval CNS of fruit flies and ii) targeted activation of fluorescent tagging of endogenously expressed proteins in individual neurons. The programme of work will significantly advance our understanding of the cellular interactions between partner neurons in a developing central network, the molecular dynamics of pre- and postsynaptic scaffold formation and how these are regulated. Further, it will generate a rich genetic tool kit that is readily applicable to study synapse biology in other parts of the nervous system, to investigate for example memory formation or neurodegenerative diseases.