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长出很长的距离，利用中间目标将它们的导航简化为较短的、可控制的部分。这些中间靶点之一是神经系统的腹中线。穿过中线的轴突生长锥体改变了它们对分泌的中线指导线索的反应：它们被缝隙排斥，同时失去了对最初引导它们到中线的吸引剂netrin-1的反应性。这些相辅相成的变化使曾经具有吸引力的环境看起来令人厌恶，从而有助于将增长锥体从中线驱逐出去。初步研究表明，这两种变化是相互关联的，因此缝隙受体Robo1的激活可以通过直接结合Netrin-1的胞内结构域和Netrin受体DCC的结构域来抑制Netrin-1的吸引作用，但不能抑制Netrin-1的生长刺激作用。有趣的是，最近的遗传证据支持Robo1在中线指导中的关键作用，并暗示Robo1介导的排斥力对于从中线驱逐生长锥是必不可少的。斯坦博士假设，缝隙受体Robo1参与沉默Netrin介导的吸引，以及从脊椎动物中线驱逐生长锥，Robo1细胞质区域按模块组织，不同的模块对于发出依赖于Robo1的沉默和排斥信号是必不可少的。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。斯坦实验室将首先阐明Robo1用来发出斥力信号的受体机制，并将使用生化和功能分析进一步研究已知和最近发现的与Robo1细胞内结构域相关的伙伴对发出Robo1斥力信号的功能贡献。这项研究的结果将使我们深入了解缝隙受体Robo1连接哪些细胞内信号通路来发出排斥信号，并将成为理解沉默的分子基础的一个切入点。这项工作将有助于巩固对轴突引导受体(如Robo1)耦合到信号排斥的细胞内信号通路的一般理解，并可能阐明这些信息是如何转化为中线定向引导的。斯坦正在她的实验室里培训本科生、研究生、博士后研究员和研究助理，包括代表不足的少数族裔群体的成员。该项目将支持对学生进行生物化学、细胞和发育神经生物学领域的培训，为他们从事高级科学职业做准备。斯坦博士还将把教育与她的研究项目结合起来，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大学范围内讨论现代发育生物学突破性技术的专题研讨会编写最新教材，并发起一个提高大脑意识的社区推广计划。最后，关于管理神经系统连接的分子和细胞机制的基础知识为开发未来普遍有益于社会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.