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Crossing the midline • Lect 11 Many species have neurons that project in two directions: 1. Those that cross the midline (contralateral) 2. Those that don’t (ipsilateral) Netrin is important for commissure development Netrin RNA expression Netrin mutant Mitchell et al. 1996 Neuron 17:203-215 1 Answers to all 3 questions provided by considering Slit / Robo Drosophila embryo CNS 1. Why do commissural axons cross the midline but not other axons? 2. If the midline is attractive to commissural axons, why do they leave the midline? 3. Why do commissural axons make longitudinal projections, rather than turning back into the midline? Slit mut commisures longitudinal Analysis of Robo 1. Robo RNA expression is uniform across all neurons 2. Robo protein is not uniform Robo mut Too many axons enter the midline & stay there axons collapse into the midline • Longitudinal growth cones have Robo, but growth cones in the commissures do not • Suggests that maybe Robo is dynamically regulated in commissural growth cones During commissure formation there is little Robo in the commissures, but lots in the longitudinals Kidd et al., 1998 Cell 92:205-215 2 • What keeps Robo levels low in commissural axons, allowing them to cross the midline? Comm is expressed in neurons that cross the midline, but not in other neurons Red = neural marker Green = Comm RNA • Identification of commissureless (comm) – A Drosophila mutant lacking commissures WT Comm mutant Comm protein in WT Tear et al. 1996 Neuron 16:501-514 Yellow indicates comm expressed in those neurons Keleman et al. 2002, Cell 110:415-427 Model for Comm regulation of Robo To keep Robo low, Comm diverts Robo delivery from the growth cone into endosomes – Secretion of Slit is by midline glial cells After growth cones cross the midline, Comm function /expression is reduced, so Robo delivery to the growth cone increases Robo mutant missing midline glia Dickson & Gilestro, Ann. Rev. Cell Biol 2006, 22:651-75 3 Short Summary • What controls crossing the midline? – Ipsilateral axons are always repelled from midline by slit / robo – Contralateral axons are initially insensitive to Slit / Robo (controlled by Comm) and are attracted to midline by Netrin – After crossing, Robo presence on contralateral axons increases and axons are prevented from re-crossing Open Questions: 1. Do (and how) contralateral axons lose their attaction to the midline after crossing? 2. How do contralateral axons acquire sensitivity to Slit after crossing? (not likely a Comm regulated event) What about vertebrates? • General principles seem similar – Differential sensitivity to repellents • But, there doesn’t seem to be a vertebrate equivalent of Comm! WT Robo3 mutant • Vert have 3 Robo orthologs Sabatier et al. Cell 2004, 117:157-169 4 5