CAREER: Molecular Analysis of Midline Crossing in Vertebrates

在发育中的神经系统中，轴突在复杂的地形上生长很长的距离，利用中间目标将它们的导航简化为短的、可控的片段。其中一个中间目标是神经系统的腹中线。穿过中线的轴突生长锥改变了它们对分泌的中线引导信号的反应性：它们被Slit排斥，同时失去了对引诱剂netrin-1的反应性，而正是netrin-1最初引导它们到达中线。这些相互加强的变化使曾经有吸引力的环境变得令人反感，从而有助于将生长锥从中线排出。最初的研究表明，这两种变化是连锁的，因此，通过将Robo1的胞内结构域直接结合到netrin受体DCC的结构域，激活Slit受体Robo1可以抑制netrin-1的吸引作用，但不能抑制其刺激生长的作用。有趣的是，最近的遗传证据支持Robo1在中线引导中的关键作用，并表明Robo1介导的排斥对于将生长锥从中线排出至关重要。Stein博士假设，Slit受体Robo1参与了网络介导的吸引力沉默，以及从脊椎动物中线排出生长锥，Robo1细胞质结构域以模块的形式组织，不同的模块对于Robo1依赖性沉默和排斥的信号传导至关重要。斯坦博士进一步提出，沉默和排斥是通过不同但重叠的途径发出信号的。斯坦计划使用生化和功能方法来了解Robo1是如何发出排斥信号的。Stein实验室将首先阐明Robo1用于排斥力信号的受体机制，并将进一步研究已知的和新的，最近发现的与Robo1细胞内结构域相关的伙伴在脊椎动物中对Robo1排斥力信号的功能贡献，使用生化和功能分析。这项研究的结果将深入了解狭缝受体Robo1结合的细胞内信号通路，并将成为了解沉默分子基础的切入点。这项工作将有助于巩固对轴突引导受体（如Robo1）与信号排斥相结合的细胞内信号传导途径的一般理解，并可能阐明这些信息是如何转化为中线的定向引导的。斯坦正在她的实验室培训本科生、研究生、博士后研究员和研究助理，其中包括未被充分代表的少数群体的成员。该项目将支持学生在生物化学、细胞和发育神经生物学领域的培训，为他们从事高级科学事业做好准备。斯坦博士还将通过为神经细胞生物学和发育神经生物学的高级课程开发最新的教学材料、为本科生举办的研究研讨会和全校范围的专题研讨会，讨论现代发育生物学的突破性技术，以及发起一个关于大脑意识的社区外展计划，将教育与她的研究项目结合起来。最后，关于控制神经系统连接的分子和细胞机制的基本知识为开发未来对社会普遍有益的神经生物学应用提供了坚实的基础。

In the developing nervous system, axons grow long distances over complex terrains, making use of intermediate targets to simplify their navigation into short, controllable segments. One of these intermediate targets is the ventral midline of the nervous system. Axonal growth cones that cross the midline change their responsiveness to secreted midline guidance cues: They become repelled by Slit and simultaneously lose responsiveness to the attractant netrin-1, which initially guided them to the midline. These mutually reinforcing changes then help to expel growth cones from the midline by making a once-attractive environment appear repulsive. Initial studies revealed that these two changes are interlocked, and thus activation of the Slit receptor Robo1 can silence the attractive effect of netrin-1, but not its growth-stimulatory effect, through direct binding of the intracellular domain of Robo1 to that of the netrin receptor DCC. Interestingly, recent genetic evidence supports a crucial role for Robo1 in midline guidance and implies that Robo1-mediated repulsion is essential for expelling growth cones from the midline. Dr. Stein hypothesizes that the Slit receptor Robo1 participates in silencing netrin-mediated attraction, as well as in expelling growth cones from the vertebrate midline, that the Robo1 cytoplasmic domain is organized in modules and that distinct modules are essential for signaling Robo1-dependent silencing and repulsion. Dr. Stein further proposes that silencing and repulsion are signaled through distinct, but overlapping, pathways.Dr. Stein plans to use biochemical and functional approaches to learn how Robo1 signals repulsion. The Stein laboratory will first elucidate the receptor mechanism that Robo1 utilizes to signal repulsion and will further investigate the functional contribution of known and novel, recently identified partners associating with the intracellular domain of Robo1 to signaling Robo1 repulsion in vertebrates, using biochemical and functional assays. The results of this study will give insights into which intracellular signaling pathways the Slit receptor Robo1 couples to signal repulsion and will be an entry point to understand the molecular basis of silencing. The work will help to consolidate the general understanding of the intracellular signaling pathways to which axonal guidance receptors, such as Robo1, couple to signal repulsion and may elucidate how this information is translated into directed guidance at the midline.Dr. Stein is training undergraduates, graduate students, postdoctoral fellows and research assistant in her laboratory, including members of underrepresented minority groups. The project will support training of students in areas of biochemistry, cellular and developmental neurobiology, preparing them for advanced scientific careers. Dr. Stein will also integrate education with her research program by developing up-to-date instructional material for an advanced course in neuronal cell biology and developmental neurobiology, a research seminar for undergraduates and a University-wide special-topic seminar discussing breakthrough technologies in modern developmental biology, as well as initiating a community outreach program for brain awareness. Finally, fundamental knowledge about the molecular and cellular mechanisms governing nervous-system wiring provides a firm foundation for developing future neurobiological applications of general benefit to society.