Researches

We produced monoclonal antibody lot1 that recognizes a specific subset of early-generated neurons designated as lot cells. The lot cells mark the future site of the LOT in the telencephalon before the first mitral cell axons grow out of the olfactory bulb. Mitral cell axons follow the pathway that lot cells lay out in the telencephalon and maintain a close contact with lot cells until the LOT is formed. When lot cells are pharmacologically ablated in organotypic culture, mitral cell axons stall as if they loose the pathway. These results suggest that lot cells serve as "guidepost" for mitral cell axons. Although lot cells are the first example of the guidepost scaffold stretching the entire tract region, we think a similar mechanism would work in other neuronal projections.

(left) Whole-mount immunostaining of the mouse telencephalon at embryonic day 12 (E12) with monoclonal antibody lot1. The lot cells that this antibody recognizes align in the future pathway of mitral cell axons. (right) Lot cells guide mitral cell axons into the LOT pathway.

To clarify molecular mechanisms of the mitral cell guidance, we have been searching for molecules that regulate growth of mitral cell axons. We screened membrane proteins that promote the axonal growth (publication 2) and examined effects of known guiding molecules on mitral cell axons (publication 6). A monoclonal antibody that inhibits growth of mitral cell axons was isolated, and investigation of the antigen that this antibody recognizes is now in progress. We are also trying to isolate molecules that lot cells specifically express, because these cells express essential molecules for the guidance of mitral cell axons.

Why do lot cells align in the future site of the LOT? The question led to this project. We investigated mechanisms of where lot cells develop and how they get localized in the LOT position under various cultures. The telencephalon is divided into two compartments, the neocortex and ganglionic eminence. Lot cells differentiated from all regions of the neocortex but not ganglionic eminence. Cell tracing analyses demonstrated that lot cells generated from the neocortex subsequently followed a tangential migration stream ventrally toward the future site of the LOT. Although some neurons migrate for a long distance during the development, this tangential ventral migration of neurons had not been described. Thus, these results reveal a new type of neuronal migration in the telencephalon and introduce an unexpected dramatic feature of the earliest regionalization in the telencephalon.

(left) Neuronal migration in the E10 mouse telencephalon. Fluorescent dye was injected into the neocortex (asterisk), and the cells labeled with the dye were traced under whole embryo culture. Most of the dye-labeled cells migrate ventrally and align in the future LOT position, drawing an inverted ÒTÓ shape. These cells eventually differentiate into lot cells. (right) Lot cells differentiate from all regions of the neocortex and migrate tangentially and ventrally to the LOT position.

Collateral branching of axons is a poorly characterized process, although the process is prevailing in developing vertebrate neural system. Mitral cell axons give off collateral branches from the LOT and innervate the target olfactory cortex. To investigate the mechanism of collateral branching, a series of heterochronic and heterotopic co-cultures of olfactory bulbs with various olfactory cortical strips were conducted. These experiments indicated that development of collateral branches is triggered by environmental cues but not by intrinsic mechanisms in mitral cells. The collateral-inducing cues are apparently different from the cues directing outgrowth of primary mitral cell axons.

(left) Collateral projection of mitral cell axons in the E17 mouse telencephalon. A number of collateral branches sprout from the LOT and invade the olfactory cortex. (right) Fluorescent dye-labeled mitral cell axons and their collateral branches.

(1); N. Sugisaki, T. Hirata, I. Naruse, A. Kawakami, T. Kitsukawa and H. Fujisawa (1996): Positional cues that are strictly localized in the telencephalon induce preferential growth of mitral cell axons. J. Neurobiol. 29, 127-137.

(2); T. Hirata, and H. Fujisawa (1997): Cortex-specific distribution of membrane-bound factors that promote neurite outgrowth of mitral cells in culture. J. Neurobiol. 32, 415-425.

(3); Y. Sato, T. Hirata, M. Ogawa and H. Fujisawa (1998): Requirement for early-generated neurons recognized by monoclonal antibody lot1 in the formation of lateral olfactory tract. J. Neurosci. 18, 7800-7810.

(4); T. Hirata and H. Fujisawa (1999): Environmental control of collateral branching and target invasion of mitral cell axons during development. J. Neurobiol. 38, 93-104.

(5); N. Tomioka, N. Osumi, Y. Sato, H. Fujisawa and T. Hirata (2000): Neocortical origin and tangential migration of guidepost neurons in the lateral olfactory tract. J. Neurosci. 20, 5802-5812.

(6); T. Hirata, H. Fujisawa, J. Y. Wu and Y. Rao (2001): Short-range guidance of olfactory bulb axons is independent of repulsive factor slit. J. Neurosci. 21, 2373-2379.

(7); T. Hirata, T. Nomura, Y. Takagi, Y. Sato, N. Tomioka, H. Fujisawa and N. Osumi (2002): Mosaic development of the olfactory cortex with Pax6-dependent and -independent components. Brain Res. Dev. Brain Res. 136, 17-26.

(8); H. Tozaki, T. Kawasaki, Y Takagi and T. Hirata (2002): Expression of Nogo protein by growing axons in the developing nervous system. Brain Res. Mol. Brain Res. 15, 111-119.

(9); H. Yamatani, Y. Sato, H. Fujisawa and T. Hirata (2004): Chronotopic organization of olfactory bulb axons in the lateral olfactory tract. J. Comp. Neurol. 475, 247-260.

(10); H. Tozaki, S. Tanaka and T. Hirata (2004): Theoretical consideration of olfactory axon projection with an activity-dependent neural network model. Mol. Cell. Neurosci. 26, 503-517.

(11); T. Kawasaki, Y. Takagi, H. Yamatani and T. Hirata (2004) Systematic screening and identification of antigens recognized by monoclonal antibodies raised against the developing lateral olfactory tract. J. Neurobiol. (Articles online in advance of print)