Subcortically and callosally projecting neurons are distinct neuronal pools in the motor cortex of the reeler mouse.

Subcortically projecting neurons and callosally projecting ones are distinct neuronal pools in the cerebral cortex of the rodents. However, cortical efferent neurons are known to project multiple targets transiently by plural collateral axons. These plural axons are eliminated during prenatal and postnatal development. In the cerebral cortex of the Reelin-deficient mouse, reeler, which is caused by mutation of the reelin gene, cortical efferent neurons are ectopically distributed. However, it is still unknown whether cortical efferent neurons in the reeler mouse lose surplus collateral axons or maintain them during developmental periods. If surplus collaterals of malpositioned cortical neurons are not eliminated, neurons projecting subcortically may project their axons to the contralateral hemisphere. To test this plausible hypothesis, we made double injections of two fluorescent dyes, Fast Blue and Diamidino yellow dihydrochloride into two of three regions, i.e., upper cervical cord, ventral lateral thalamic nucleus, and contralateral motor cortex of the normal and reeler mice, to label corticospinal, corticothalamic and callosal commissure neurons in the motor cortex, retrogradely. No double labeled neurons were identified in the motor cortex of the normal and reeler mice, although the distribution patterns of these cortical efferent neurons were completely different between normal and reeler mice. These findings strongly suggest that collateral elimination of cortical efferent neurons during developing periods are not affected in this mutant mouse.

Rac1 in cortical projection neurons is selectively required for midline crossing of commissural axonal formation.

Rac1 is a member of Rho family GTPases and regulates multiple cellular functions through actin cytoskeleton reorganization. During cerebral corticogenesis, Rac1 has been assumed to be involved in neuronal migration, neurite formation, polarization and axonal guidance. Here we show the specific role of Rac1, regulating midline crossing of commissural axons during cortical development by using cortex-restricted Rac1-knockout mice. In the knockout mice, Rac1 was eliminated from the beginning of corticogenesis exclusively in the dorsal telencephalon where progenitors of cortical projection neurons are located. Cortical lamination was distorted only mildly in the knockout mice, being preserved with six layers of neurons. However, cortex-restricted Rac1 deletion exhibited striking agenesis of commissural axons including the corpus callosum and anterior commissure without affecting other corticofugal axons including corticospinal and corticothalamic projections. Of note, the commissural axons of the knockout mice were potent in extending their process, but failed to cross the midline. Therefore, these findings indicate that Rac1 specifically controls the midline crossing of the commissural fibers, but not axonal formation of corticospinal or corticothalamic fibers during cortical development.

Unusual patch-matrix organization in the retrosplenial cortex of the reeler mouse and Shaking rat Kawasaki.

The rat granular retrosplenial cortex (GRS) is a simplified cortex, with distinct stratification and, in the uppermost layers, distinct modularity. Thalamic and cortical inputs are segregated by layers and in layer 1 colocalize, respectively, with apical dendritic bundles originating from neurons in layers 2 or 5. To further investigate this organization, we turned to reelin-deficient reeler mouse and Shaking rat Kawasaki. We found that the disrupted lamination, evident in Nissl stains in these rodents, is in fact a patch-matrix mosaic of segregated afferents and dendrites. Patches consist of thalamocortical connections, visualized by vesicular glutamate transporter 2 (VGluT2) or AChE. The surrounding matrix consists of corticocortical terminations, visualized by VGluT1 or zinc. Dendrites concentrate in the matrix or patches, depending on whether they are OCAM positive (matrix) or negative (patches). In wild-type rodents and, presumably, mutants, OCAM(+) structures originate from layer 5 neurons. By double labeling for dendrites (filled by Lucifer yellow in fixed slice) and OCAM immunofluorescence, we ascertained 2 populations in reeler: dendritic branches either preferred (putative layer 5 neurons) or avoided (putative supragranular neurons) the OCAM(+) matrix. We conclude that input-target relationships are largely preserved in the mutant GRS and that dendrite-dendrite interactions involving OCAM influence the formation of the mosaic configuration.

Expression of zebrafish ROR alpha gene in cerebellar-like structures.

Mouse genetic studies have identified several genes involved in cerebellar development. The mouse mutants staggerer and lurcher are functionally deficient for the retinoid-related orphan receptor alpha (ROR alpha) and glutamate receptor delta2 (Grid2) genes, respectively, and they show similar functional and developmental abnormalities in the cerebellum. Here, we report the cloning and expression pattern of zebrafish ROR alpha orthologues rora1 and rora2, and compare their expression pattern with that of grid2. Expression of rora1 and rora2 is initiated at late gastrula and pharyngula stages, respectively. Both rora1 and rora2 are spatially expressed in the retina and tectum. Expression of rora2 was further observed in the cerebellum, as reported for mammalian ROR alpha. In the adult brain, rora2 and grid2 are coexpressed in brain regions, designated as cerebellar-like structures. These observations suggest an evolutionarily conserved function of ROR alpha orthologues in the vertebrate brain.

Ectopic corticospinal tract and corticothalamic tract neurons in the cerebral cortex of yotari and reeler mice.

Reeler and yotari mice, which are mutant for Reelin or Dab1, respectively, show disorders of cerebral cortical lamination. We injected horseradish peroxidase (HRP) into the upper lumbar enlargement to label corticospinal tract (CST) neurons and wheat germ agglutinin-conjugated HRP (WGA-HRP) into the ventral lateral nucleus of the thalamus to label corticothalamic tract (CTT) neurons in both 19-day-old yotari and reeler mice with the aim of discovering whether or not they show differences in the distribution pattern of layer V or layer VI neurons. Similar injections of tracers were made in normal controls. HRP-labeled CST neurons, which were exclusively distributed in layer V of the normal cortex, were radially scattered in the cortex of both mutants, but those in reeler were more deeply distributed than in yotari. WGA-labeled CTT neurons, which were mainly located in layer VI in the normal cortex, were superficially distributed just beneath the pia mater in both reeler and yotari cortex. The present quantitative study shows that the distribution pattern of layer V neurons, but not layer VI neurons, differs between reeler and yotari mice, suggesting that the Reelin and Dab1 proteins may play different roles in the migration and cell positioning of layer V neurons.

Neuronal lineage-specific induction of phospholipase Cepsilon expression in the developing mouse brain.

Phospholipase C is a key enzyme of intracellular signal transduction in the central nervous system. We and others recently discovered a novel class of phospholipase C, phospholipase Cepsilon, which is regulated by Ras and Rap small GTPases. As a first step toward analysis of its function, we have examined the spatial and temporal expression patterns of phospholipase Cepsilon during mouse development by in situ hybridization and immunohistochemistry. Around embryonic day 10.5, abundant expression of phospholipase Cepsilon is observed specifically in the outermost layer of the neural tube. On embryonic day 12 and later, it is observed mainly in the marginal zone of developing brain and spinal cord as well as in other regions undergoing neuronal differentiation, such as the retina and olfactory epithelium. The phospholipase Cepsilon-expressing cells almost invariably express microtubule-associated protein 2, but hardly express nestin or glial fibrillary acidic protein, indicating that the expression of phospholipase Cepsilon is induced specifically in cells committed to the neuronal lineage. The expression of phospholipase Cepsilon persists in the terminally differentiated neurons and exhibits no regional specificity. Further, an in vitro culture system of neuroepithelial stem cells is employed to show that abundant expression of phospholipase Cepsilon occurs in parallel with the loss of nestin expression as well as with the induction of microtubule-associated protein 2 expression and neuronal morphology. Also, glial fibrillary acidic protein-positive glial lineage cells do not exhibit the high phospholipase Cepsilon expression. These results suggest that the induction of phospholipase Cepsilon expression may be a specific event associated with the commitment of the neural precursor cells to the neuronal lineage.