RGMb controls aggregation and migration of Neogenin-positive cells in vitro and in vivo.

The proliferation, migration and differentiation of dentate gyrus stem and precursor cells have aroused keen interest. Neogenin and RGMb are expressed in non-overlapping compartments of the developing dentate gyrus. While Neogenin is expressed in migrating and proliferating dentate precursors, RGMb is localized in structures bordering the developing dentate, such as cornus ammonis cells and Cajal-Retzius cells in the marginal zone including the hippocampal fissure. Co-immunoprecipitation and binding assays indicate a strong physical interaction. In vitro and in vivo migration of dentate neuroepithelial cells is abolished by RGMb, and cell adhesion is reduced when cells expressing Neogenin comes into contact with cells expressing RGMb. Ectopic expression of RGMb in organotypic slice cultures and after in utero electroporation in the hippocampus modifies precursor cell migration. Our results imply that Neogenin-RGMb interaction might be involved in neuronal migration in the dentate gyrus.

Regulatory pathway analysis by high-throughput in situ hybridization.

Automated in situ hybridization enables the construction of comprehensive atlases of gene expression patterns in mammals. Such atlases can become Web-searchable digital expression maps of individual genes and thus offer an entryway to elucidate genetic interactions and signaling pathways. Towards this end, an atlas housing approximately 1,000 spatial gene expression patterns of the midgestation mouse embryo was generated. Patterns were textually annotated using a controlled vocabulary comprising >90 anatomical features. Hierarchical clustering of annotations was carried out using distance scores calculated from the similarity between pairs of patterns across all anatomical structures. This process ordered hundreds of complex expression patterns into a matrix that reflects the embryonic architecture and the relatedness of patterns of expression. Clustering yielded 12 distinct groups of expression patterns. Because of the similarity of expression patterns within a group, members of each group may be components of regulatory cascades. We focused on the group containing Pax6, an evolutionary conserved transcriptional master mediator of development. Seventeen of the 82 genes in this group showed a change of expression in the developing neocortex of Pax6-deficient embryos. Electromobility shift assays were used to test for the presence of Pax6-paired domain binding sites. This led to the identification of 12 genes not previously known as potential targets of Pax6 regulation. These findings suggest that cluster analysis of annotated gene expression patterns obtained by automated in situ hybridization is a novel approach for identifying components of signaling cascades.

Emx2 in the developing hippocampal fissure region.

Mice deficient in transcription factor gene Emx2 show developmental alterations in the hippocampal dentate gyrus. Emx2, however, is also expressed in the region around the developing hippocampal fissure. The developing fissure contains a radial glial scaffolding, and is surrounded by the outer marginal zone and the dentate marginal zone, which become specifically colonized by neurons and differentiate into stratum lacunosum-moleculare and molecular layer of the dentate, respectively. In this study we show that the Emx2 mutant lacks the glial scaffolding of the fissure and has an outer marginal zone (precursor of the stratum lacunosum-moleculare), as well as a dentate marginal zone severely reduced in size while most of the reelin (Reln)-expressing cells that should occupy it fail to be generated. We have also identified a subpopulation of hippocampal Reln-expressing cells of the marginal zone, probably born in the hem, expressing a specific combination of markers, and for which Emx2 is not essentially required. Additionally, we show differential mutant phenotypes of both Emx2 and Pax6 in neocortical vs. hippocampal Reln-expressing cells, indicating differential development of both subpopulations.

bHLH gene expression in the Emx2-deficient dentate gyrus reveals defective granule cells and absence of migrating precursors.

Dentate gyrus development is uniquely characterized by the existence of migrating precursors. The production of precursors by the neuroepithelium is regulated by the proneural cascade of bHLH genes, which show distinctive expression patterns in dentate. Mice carrying a mutation in Emx2, a neuroepithelial transcription factor, lack the granule cell layer which forms most of the dentate, although the corresponding neuroepithelium is correctly specified. To understand this phenotype, we have analyzed the expression of proneural genes (bHLH gene family) and other markers in Emx2-deficient dentate. Here we show that, in the mutant dentate, expression of bHLH genes, Tenascin C and GFAP is abnormally confined to the germinal layer, as are most neuronal and astrocytic precursors. Additionally, Mash1 expression (marker of migrating precursors) is lost during development. Mutant granule cells show arrested migration and lack NeuroD2 expression. These results are evidence that in Emx2 mutants, migrating precursors (secondary matrix) and astrocytes are absent, the radial migration substrate impaired and granule cells deficiently differentiated. Our analysis gives insight into how a general defect caused by the absence of Emx2 translates into the dentate-specific phenotype.

The repulsive guidance molecule RGMa is involved in the formation of afferent connections in the dentate gyrus.

In the developing dentate gyrus, afferent fiber projections terminate in distinct laminas. This relies on an accurately regulated spatiotemporal network of guidance molecules. Here, we have analyzed the functional role of the glycosylphosphatidylinositol (GPI)-anchored repulsive guidance molecule RGMa. In situ hybridization in embryonic and postnatal brain showed expression of RGMa in the cornu ammonis and hilus of the hippocampus. In the dentate gyrus, RGM immunostaining was confined to the inner molecular layer, whereas the outer molecular layers targeted by entorhinal fibers remained free. To test the repulsive capacity of RGMa, different setups were used: the stripe and explant outgrowth assays with recombinant RGMa, and entorhino-hippocampal cocultures incubated either with a neutralizing RGMa antibody (Ab) or with the GPI anchor-digesting drug phosphatidylinositol-specific phospholipase C. Entorhinal axons were clearly repelled by RGMa in the stripe and outgrowth assays. After disrupting the RGMa function, the specific laminar termination pattern in entorhino-hippocampal cocultures was lost, and entorhinal axons entered inappropriate hippocampal areas. Our data indicate an important role of RGMa for the layer-specific termination of the perforant pathway as a repulsive signal that compels entorhinal fibers to stay in their correct target zone.

Expression pattern of the repulsive guidance molecules RGM A, B and C during mouse development.

We have characterized for the first time the mouse expression patterns of the three known members of the novel RGM gene family ('Repulsive Axonal Guidance molecules' A, B and C) by in situ hybridization. Both RGM A and B are mostly expressed in central nervous system, while RGM C is exclusively expressed in all striated muscle and in the myocardium. RGM A and B appear at every level of the developing neural axis, where they colocalize to a large extent in the mantle layer, although only RGM A appears in the neuroepithelium, and only RGM B in the peripheral nervous system. During development, both RGM A and B appear also in lung and in limb cartilage, while RGM B has additional expression domains in pancreas.

Identification of neurabin II as a novel doublecortin interacting protein.

The neuronal migration protein doublecortin (DCX) that associates with microtubules through a tandem DCX repeat, is required for the development of the complex architecture of the human cerebral cortex. Using a yeast two-hybrid screen with Dcx as bait, we have isolated neurabin II/spinophilin, an F-actin binding protein known to play a role in dendritic spine formation. The coiled-coil domain of neurabin II binds to a DCX region encompassing the C-terminal portion of the second DCX repeat and the N-terminal portion of the Ser/Pro-rich domain. Immunoprecipitation experiments with brain extracts show that neurabin II and Dcx interact in vivo. Several Dcx constructs that mimic human DCX mutant alleles failed to interact with neurabin II. Since Dcx and neurabin II colocalized in the developing and adult brain, a neurabin II-DCX heterodimer may be involved in neuronal migration and dendritic spine formation.