Development of the mouse amygdala as revealed by enhanced green fluorescent protein gene transfer by means of in utero electroporation.

The amygdala is located in the caudal part of the ventral telencephalon. It is composed of many subdivisions and is involved in the control of emotion. It is important to know the mechanisms of amygdalar development in order to analyze the pathogenesis of emotional disorders, but they are still not adequately understood. In the present study the migration, differentiation, and distribution of amygdalar neurons in the mouse embryo were investigated by means of in utero electroporation. Ventricular zone cells in restricted regions, that is, the caudal ganglionic eminence (CGE), the ventral pallium, the lateral pallium, and the diencephalon, were labeled with an expression vector of the enhanced green fluorescent protein (EGFP) gene. Labeling at embryonic day (E)10 revealed that the central nucleus originates from the neuroepithelium in the ganglionic eminence and the labeling at E11 and E12 revealed that the basolateral complex originates from the neuroepithelium of the ventral and lateral pallia. The introduction of the EGFP gene into the neuroepithelium of the third ventricle at E11 showed that the medial nucleus originates, at least in part, from the neuroepithelium of the diencephalon and migrates over the diencephalo-telencephalic boundary. The radial glial arrangement corresponded well with the initial migration of amygdalar neurons, and the radial processes later formed the boundary demarcating the basolateral complex. These findings indicate that the neurons originating from the temporally and spatially restricted neuroepithelium in both the telencephalon and diencephalon migrate and differentiate to form the mosaic of amygdalar subdivisions.

Induction of oligodendrocyte progenitors in dorsal forebrain by intraventricular microinjection of FGF-2.

During embryonic development, oligodendrocyte progenitors (OLPs) originate from the ventral forebrain under the regulation of Sonic hedgehog (Shh). Shh controls the expression of transcription factor Olig2, which is strongly implicated in OLP generation. Studies of mice deficient in Shh expression suggest, however, that an alternative pathway for OLP generation may exist. The generation of OLPs in dorsal forebrain has been suggested since treatment of dorsal-neural progenitor cells in culture with fibroblast growth factor (FGF-2) results in OLP induction. To ask if dorsal induction of OLPs in embryonic forebrain can occur in vivo and if FGF-2 could initiate an alternative pathway of regulation, we used in utero microinjection of FGF-2 into the lateral ventricles of mouse fetal forebrain. A single injection of FGF-2 at E13.5 resulted in the expression of the OLP markers Olig2 and PDGFRalpha mRNA in dorsal forebrain ventricular and intermediate zones. However, FGF-2 did not induce dorsal expression of Shh, Patched1 or Nkx2.1, and co-injection of FGF-2 and a Shh inhibitor did not attenuate the induction of Olig2 and PDGFRalpha, suggesting that Shh signaling was not involved in this FGF-2-mediated dorsal induction. These results demonstrate that the dorsal embryonic forebrain in vivo has the potential to generate OLPs in the presence of normal positional cues and that this can be driven by FGF-2 independent of Shh signaling.

Altered gene expression in the subdivisions of the amygdala of Fyn-deficient mice as revealed by laser capture microdissection and mKIAA cDNA array analysis.

Fyn-tyrosine-kinase-deficient mice exhibit increased fearfulness and display enhanced excitability in the amygdala. To gain insight into the molecular changes associated with the increased excitability of the amygdala, we used a newly developed cDNA array system comprising mouse KIAA cDNA clones to identify novel genes differentially expressed in the amygdala of fyn(-/-) and fyn(+/-) mice following administration of N-methyl-D-aspartate (NMDA). Laser capture microdissection in combination with PCR-based cDNA amplification allowed us to analyze gene expression in each amygdalar subdivision. The statistical significance of the differential expressions was tested by one-way analysis of variance (ANOVA) by the false discovery rate controlling approach. Among the 805 mKIAA cDNA clones tested, only the expression level of mKIAA1577 (Zinc finger SWIM domain containing protein 6; gene name, Zswim6) showed statistically significant change in regard to the genotype and amygdalar subdivision. Namely, only the lowered expression of mKIAA1577 in the central nucleus of fyn(-/-) mice 1 h after NMDA administration (2.1-fold lower relative to fyn(+/-) mice) was statistically significant. In situ hybridization analysis confirmed the downregulation of the mRNA in the central nucleus of the fyn(-/-) mice 1 h after NMDA administration (3.2-fold lower relative to fyn(+/-) mice). The NMDA-induced change in gene expression was partially blocked by the NMDA antagonist D-AP-5. These results suggest that Fyn deficiency was responsible for the NMDA-induced downregulation of a specific gene in the amygdalar central nucleus.

Direct evidence that ventral forebrain cells migrate to the cortex and contribute to the generation of cortical myelinating oligodendrocytes.

Cortical neuroepithelial cells generate neurons, astrocytes, and oligodendrocytes (OLs) in vitro. However, whether cortical OLs are derived from the cortical neuroepithelium or migrate from the ventral forebrain is under severe debate yet. This is due to the fact that OL progenitor cells (OPCs), as marked by the expression of PDGFRalpha or NG2, are generated at around embryonic day (E) 11 or 12 in the mouse ganglionic eminences, but the myelinating OLs appear during the second week postnatally in the cortex. There has been no labeling method for long-term glial cell-lineage tracing. Thus, we developed a new strategy: plasmid DNA encoding Cre recombinase was introduced into the Cre/loxP reporter forebrain in ventral- or dorsal-specific manner by in utero DNA electroporation. The reporter gfp gene is expressed permanently owing to the chromosomal DNA recombination. The GFP-labeled myelinating OLs were detected in the adult cortex when electroporation was targeted to the ventral neuroepithelium, demonstrating at least some of the myelinating OLs are derived from the ventral forebrain. However, when electroporation was targeted to the dorsal, we could not find GFP-labeled myelinating OLs. This suggests that the progenitors of cortical OPCs are absent or located at restricted regions in the dorsal forebrain (cortex) at E12.

Neuronal generation, migration, and differentiation in the mouse hippocampal primoridium as revealed by enhanced green fluorescent protein gene transfer by means of in utero electroporation.

Neuronal migration defects in the hippocampus during development are thought to be involved in various mental disorders. Studies of neural cell migration in the developing cerebrum have focused mainly on the neocortex, but those that have been performed on the developing hippocampal formation have not been adequately carried out. In the present study, the morphological differentiation of immature neurons that form the laminar structure of the hippocampus was investigated by labeling ventricular surface cells with the expression vector of the enhanced-green-fluorescent-protein (EGFP) gene. Vector DNA was transfected into spatially and temporally restricted neuroepithelium of the hippocampal primordium by in utero electroporation, and the morphology of EGFP-labeled migratory neurons and their interrelationships with the radial glial arrangement were observed. Pyramidal cells of Ammon's horn began to migrate radially along glial processes from a broad area of neuroepithelium on embryonic day (E)14. Large numbers of multipolar cells were found in the intermediate zone in the initial stage and stratified pyramidal cells appeared later. Dentate granule cells were labeled later than (E)16 and originated from a restricted area of neuroepithelium adjacent to the fimbria. Their initial migration was rapid and independent of radial glial fibers. Subsequent tangential migration in the subpial space and their ultimate settling into the forming dentate gyrus were closely associated with the radial glia. These findings indicate that distinct cellular mechanisms are involved in the development of the cortical layer of Ammon's horn and dentate gyrus.

Evidence for a second wave of oligodendrogenesis in the postnatal cerebral cortex of the mouse.

The existing view is that cortical oligodendrocytes (OLs) in rodents are born from the cortical subventricular zone (SVZ) after birth, but recent data suggest that many forebrain oligodendrocyte progenitor cells (OPCs) are specified much earlier (between E9.5 and E13.5 in the mouse) in the ventricular zone of the ventral forebrain under the control of sonic hedgehog (Shh) and migrate into the cortex afterward. We examined expression of specific early OL markers (PDGFRalpha, PLP/DM20, Olig2, and NG2) in the developing forebrain to clarify this issue. We propose that OPCs colonize the developing cortex in two temporally distinct waves. The gray matter is at least partially populated by a first wave of OPCs that arises in the medial ganglionic eminence and the entopeduncular area and spreads into the cortex via the developing cortical plate. The cerebral cortex benefits from the second wave of OPCs coming from residential SVZ. In the second wave, there might be two different types of precursor cells: PLP/DM20(+) cells populating only inner layers and PDGFRalpha(+) cells, which might eventually myelinate the outer regions as well.

[Regulation of development of oligodendrocyte].

Oligodendrocyte (OL) progenitors in the mouse spinal cord are generated from a strictly restricted region in the ventricular zone of the ventral cord as early as on embryonic day 11 (E11). We previously reported that one of the factors that restrict this ventral specific appearance of OLs is an inhibitory factor secreted from the dorsal spinal cord, in addition to well-known stimulatory ventral factors such as sonic hedgehog. We characterized the developmental change of the inhibitory activities. They were very strong at E11, gradually reduced, and disappeared by E14. This pattern seemed to be well correlated with the developmental profile of Wnt3a expression at/near the roof plate. A conditioned medium of L cells that stably express Wnt3a showed significant reduction of O4 positive OLs in the ventral spinal cord explants, indicating that Wnt3a is one of the dorsal factors that inhibit OL development. Addition of Wnt3a supernatant to CG4 cells, an OL progenitor strain, and to the dissociated primary cultured cells suggested that Wnt3a directly acts on OL lineage cells and inhibits a differentiation step from OL progenitor to O4-positive stage. Thus, Wnt3a may directly control the timing of OL differentiation and the motility of OL lineage cells. A population of myelinating OLS in the dorsal area of telencephalon was further demonstrated to be ventral origin by the newly established cell marking system using in utero DNA electroporation.