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.
The most fascinating and challenging question in neurobiology is to understand how our nervous system forms and function, in particular how the billions of neurons that encompass our functional mature circuit find their appropriate partners to establish functional connection. In the developing nervous system, neuronal extensions, axons, grow long distances over complex terrains, making use of intermediate targets to simplify their navigation into short, controllable segments. The research of Dr. Stein focuses on one of these intermediate targets, the ventral midline of the nervous system. Here, axonal growth cones that cross the midline change their responsiveness to secreted midline guidance molecules: They become repelled by the protein Slit and simultaneously lose responsiveness to the attractant netrin-1, which initially guided them to this intermediate target. These mutually reinforcing changes than 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. However, previous studies suggested that may other guidance receptors and cues are involved in avigating commissural axons towards and across the midline. Research supported by this application revealed not only the identity of this additional receptor, which encodes the protein Down Syndrome Cell Adhesion Molecule (DSCAM), but also revealed that DSCAM is a netrin receptor that is essential for the turning of commissural axons at the vertebrate midline, their first intermediate target. In addition, the lab has indentified intracellular targets that selectively associate with DSCAM to propagate axonal growth responses. Taken together, studies under this application have further contributed to the better understanding how axonal growth cones find their targets. Dr. Stein has engaged ten undergraduate students in neurobiology research, including members from underrepresented minority groups. In addition, two graduate students were actively involved in the research described above. Under this project students were trained in areas of biochemistry, cellular and developmental neurobiology, that prepared them for advanced scientific careers. Dr. Stein has also integrate education with this research program by launching an advanced course in neuronal cell biology and developmental neurobiology, a research seminar for undergraduates and University wide special topic seminar discussing breakthrough technologies in modern developmental biology.
