Takahiko Kawasaki

The Temporal Sequence of the Mammalian Neocortical Neurogenetic Program Drives Mediolateral Pattern in the Chick Pallium

The six-layered neocortex permits complex information processing in all mammalian species. Because its homologous region (the pallium) in nonmammalian amniotes has a different architecture, the ability of neocortical progenitors to generate an orderly sequence of distinct cell types was thought to have arisen in the mammalian lineage. This study, however, shows that layer-specific neuron subtypes do exist in the chick pallium. Deep- and upper-layer neurons are not layered but are segregated in distinct mediolateral domains in vivo. Surprisingly, cultured chick neural progenitors produce multiple layer-specific neuronal subtypes in the same chronological sequence as seen in mammals. These results suggest that the temporal sequence of the neocortical neurogenetic program was already inherent in the last common ancestor of mammals and birds and that mammals use this conserved program to generate a uniformly layered neocortex, whereas birds impose spatial constraints on the sequence to pattern the pallium.

Expression of Nogo protein by growing axons in the developing nervous system

We produced monoclonal antibody NG1 that strongly binds growing axons in the developing nervous system of mice. This antibody intensely labeled the growth cone of cultured neurons. Although these immunostaining patterns suggested the association of growing axons with the antigen recognized by this antibody, the antigen was identified as Nogo protein, an axonal repulsive factor isolated from the myelin. On the basis of this unexpected finding, we discuss the possible functions of Nogo in the developing nervous system.

Cartilage Acidic Protein–1B (LOTUS), an Endogenous Nogo Receptor Antagonist for Axon Tract Formation

A molecule that functions in normal olfactory tract development could provide clues to failed neuronal regeneration in adults. Neural circuitry formation depends on the molecular control of axonal projection during development. By screening with fluorophore-assisted light inactivation in the developing mouse brain, we identified cartilage acidic protein–1B as a key molecule for lateral olfactory tract (LOT) formation and named it LOT usher substance (LOTUS). We further identified Nogo receptor–1 (NgR1) as a LOTUS-binding protein. NgR1 is a receptor of myelin-derived axon growth inhibitors, such as Nogo, which prevent neural regeneration in the adult. LOTUS suppressed Nogo-NgR1 binding and Nogo-induced growth cone collapse. A defasciculated LOT was present in lotus-deficient mice but not in mice lacking both lotus- and ngr1. These findings suggest that endogenous antagonism of NgR1 by LOTUS is crucial for normal LOT formation.

Netrin 1 regulates ventral tangential migration of guidepost neurons in the lateral olfactory tract

In the developing nervous system, functional neural networks are constructed with intricate coordination of neuronal migrations and axonal projections. We have previously reported a ventral tangential migration of a special type of cortical neurons, lot cells, in the mouse embryo. These neurons originate from the ventricular zone of the entire neocortex, tangentially migrate in the surface layer of the neocortex into the ventral direction, align in the future pathway of the lateral olfactory tract (LOT) and eventually guide the projection of LOT axons. In this study, we developed an organotypic culture system to investigate the regulation of this cell migration in the developing telencephalon. Our data show that the neocortex contains the signals that direct lot cells ventrally, that the ganglionic eminence excludes lot cells by repelling the migration and that lot cells are attracted to netrin 1, an axon guidance factor. Furthermore, we demonstrate that mutations in the genes encoding netrin 1 and its functional receptor Dcc lead to inappropriate distribution of lot cells and subsequent partial disruption of LOT projection. These results suggest that netrin 1 regulates the migration of lot cells and LOT projections, possibly by ensuring the correct distribution of these guidepost neurons.

Robo1 and Robo2 Control the Development of the Lateral Olfactory Tract

The development of olfactory bulb projections that form the lateral olfactory tract (LOT) is still poorly understood. It is known that the septum secretes Slit1 and Slit2 which repel olfactory axons in vitro and that in Slit1−/−;Slit2−/− mutant mice, the LOT is profoundly disrupted. However, the involvement of Slit receptors, the roundabout (Robo) proteins, in guiding LOT axons has not been demonstrated. We show here that both Robo1 and Robo2 receptors are expressed on early developing LOT axons, but that only Robo2 is present at later developmental stages. Olfactory bulb axons from Robo1−/−;Robo2−/− double-mutant mice are not repelled by Slit in vitro. The LOT develops normally in Robo1−/− mice, but is completely disorganized in Robo2−/− and Robo1−/−;Robo2−/− double-mutant embryos, with many LOT axons spreading along the ventral surface of the telencephalon. Finally, the position of lot1-expressing cells, which have been proposed to be the LOT guidepost cells, appears unaffected in Slit1−/−;Slit2−/− mice and in Robo1−/−;Robo2−/− mice. Together, our results indicate that Robo1 and Robo2 directly mediate the repulsive activity of Slit receptors on LOT axons, and are required for normal guidance of these axons in vivo.

Semaphorin 3F Confines Ventral Tangential Migration of Lateral Olfactory Tract Neurons onto the Telencephalon Surface

Ventral tangential migration of neurons is the most prominent mode of neuronal translocation during earliest neurogenesis in the mouse telencephalon. A typical example of the neurons that adopt this migration mode is guidepost neurons in the lateral olfactory tract designated as lot cells. These neurons are generated from the neocortical neuroepithelium and migrate tangentially down to the ventral edge of the neocortex abutting the ganglionic eminence, on which the future lateral olfactory tract develops. We show here that this migration stream is repelled by a secreted axon guidance molecule, semaphorin 3F through interaction with its specific receptor, neuropilin-2. Accordingly, in mutant mice for semaphorin 3F or neuropilin-2, lot cells ectopically penetrated into the deep brain domain, which normally expresses semaphorin 3F. These results reveal that semaphorin 3F is an important regulator of the ventral tangential migration stream, confining the migrating neurons on the telencephalon surface by repelling from the deeper domain.

Proteomics analysis of the temporal changes in axonal proteins during maturation

After the initial primary projection, axons undergo various structural and functional changes to establish mature neural circuits. The changes in protein expression associated with this maturation were investigated in lateral olfactory tract axons using two‐dimensional gel electrophoresis. The most prominent group upregulated during the period consisted of calcium‐dependent membrane‐binding proteins including VILIP1, neurocalcin δ, copine 6, and annexin A6 from three structurally different families. During maturation of primary cultured neurons, annexin A6 gradually became concentrated on the axon initial segment, and its overexpression significantly enhanced axon branching. On the other hand, overexpression of VILIP1 and neurocalcin δ reduced axon outgrowth and branching. The second group upregulated during axon maturation comprised tubulin‐ and microtubule‐binding proteins including CRMP2, guanine deaminase, MAP1B, and fibronectin type3 SPRY domain‐containing protein. Because the maturation of lateral olfactory axons involves massive extension of secondary collateral branches, the augmentation of these proteins during these stages may underlie the drastic restructuring of the axon cytoskeleton.

Transcallosal Projections Require Glycoprotein M6-Dependent Neurite Growth and Guidance

The function of mature neurons critically relies on the developmental outgrowth and projection of their cellular processes. It has long been postulated that the neuronal glycoproteins M6a and M6b are involved in axon growth because these four-transmembrane domain-proteins of the proteolipid protein family are highly enriched on growth cones, but in vivo evidence has been lacking. Here, we report that the function of M6 proteins is required for normal axonal extension and guidance in vivo. In mice lacking both M6a and M6b, a severe hypoplasia of axon tracts was manifested. Most strikingly, the corpus callosum was reduced in thickness despite normal densities of cortical projection neurons. In single neuron tracing, many axons appeared shorter and disorganized in the double-mutant cortex, and some of them were even misdirected laterally toward the subcortex. Probst bundles were not observed. Upon culturing, double-mutant cortical and cerebellar neurons displayed impaired neurite outgrowth, indicating a cell-intrinsic function of M6 proteins. A rescue experiment showed that the intracellular loop of M6a is essential for the support of neurite extension. We propose that M6 proteins are required for proper extension and guidance of callosal axons that follow one of the most complex trajectories in the mammalian nervous system.

The COUP-TFII/Neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain

Significance Recently the preoptic area (POa) has been shown to be a source of GABAergic neurons in the medial amygdala and cerebral cortex, where they are thought to play a pivotal role in emotions and intelligence, respectively. However, it is unknown how the POa-derived neurons migrate and selectively segregate into either the amygdala or cortex. By using focal in utero labeling of the POa, we show that switching on/off the transcription factor COUP-TFII (Chicken ovalbumin upstream promoter transcription factor II) and the receptor Neuropilin-2 (Nrp2) directs the POa-derived neurons toward either the amygdala or cortex. Our study revealed an essential role of COUP-TFII/Nrp2 expression dynamics in the development of the amygdala and cortex. The preoptic area (POa) of the rostral diencephalon supplies the neocortex and the amygdala with GABAergic neurons in the developing mouse brain. However, the molecular mechanisms that determine the pathway and destinations of POa-derived neurons have not yet been identified. Here we show that Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII)–induced expression of Neuropilin-2 (Nrp2) and its down-regulation control the destination of POa-derived GABAergic neurons. Initially, a majority of the POa-derived migrating neurons express COUP-TFII and form a caudal migratory stream toward the caudal subpallium. When a subpopulation of cells steers toward the neocortex, they exhibit decreased expression of COUP-TFII and Nrp2. The present findings show that suppression of COUP-TFII/Nrp2 changed the destination of the cells into the neocortex, whereas overexpression of COUP-TFII/Nrp2 caused cells to end up in the medial part of the amygdala. Taken together, these results reveal that COUP-TFII/Nrp2 is a molecular switch determining the pathway and destination of migrating GABAergic neurons born in the POa.

Guidepost Neurons for the Lateral Olfactory Tract: Expression of Metabotropic Glutamate Receptor 1 and Innervation by Glutamatergic Olfactory Bulb Axons

The guidepost neurons for the lateral olfactory tract, which are called lot cells, are the earliest‐generated neurons in the neocortex. They migrate tangentially and ventrally further down this tract, and provide scaffolding for the olfactory bulb axons projecting into this pathway. The molecular profiles of the lot cells are largely uncharacterized. We found that lot cells specifically express metabotropic glutamate receptor subtype‐1 at a very early stage of development. This receptor is functionally competent and responds to a metabotropic glutamate receptor agonist with a transient increase in the intracellular calcium ion concentration. When the glutamatergic olfactory bulb axons were electrically stimulated, lot cells responded to the stimulation with a calcium increase mainly via ionotropic glutamate receptors, suggesting potential neurotransmission between the axons and lot cells during early development. Together with the finding that lot cells themselves are glutamatergic excitatory neurons, our results provide another notable example of precocious interactions between the projecting axons and their intermediate targets.