Identification of transcriptional regulatory elements for Ntng1 and Ntng2 genes in mice.

BACKGROUND: Higher brain function is supported by the precise temporal and spatial regulation of thousands of genes. The mechanisms that underlie transcriptional regulation in the brain, however, remain unclear. The Ntng1 and Ntng2 genes, encoding axonal membrane adhesion proteins netrin-G1 and netrin-G2, respectively, are paralogs that have evolved in vertebrates and are expressed in distinct neuronal subsets in a complementary manner. The characteristic expression patterns of these genes provide a part of the foundation of the cortical layer structure in mammals. RESULTS: We used gene-targeting techniques, bacterial artificial chromosome (BAC)-aided transgenesis techniques, and in vivo enhancer assays to examine transcriptional mechanisms in vivo to gain insight into how the characteristic expression patterns of these genes are acquired. Analysis of the gene expression patterns in the presence or absence of netrin-G1 and netrin-G2 functional proteins allowed us to exclude the possibility that a feedback or feedforward mechanism mediates their characteristic expression patterns. Findings from the BAC deletion series revealed that widely distributed combinations of cis-regulatory elements determine the differential gene expression patterns of these genes and that major cis-regulatory elements are located in the 85-45 kb upstream region of Ntng2 and in the 75-60 kb upstream region and intronic region of Ntng1. In vivo enhancer assays using 2-kb evolutionarily conserved regions detected enhancer activity in the distal upstream regions of both genes. CONCLUSIONS: The complementary expression patterns of Ntng1 and Ntng2 are determined by transcriptional cis-regulatory elements widely scattered in these loci. The cis-regulatory elements characterized in this study will facilitate the development of novel genetic tools for functionally dissecting neural circuits to better understand vertebrate brain function.

Monoclonal antibodies discriminating netrin-G1 and netrin-G2 neuronal pathways.

Netrin-G1 and netrin-G2, belonging to a vertebrate-specific subfamily of the netrin family, distribute on axons of distinct neuronal pathways. To add to the array of molecular probes available for labeling unique neuronal circuits, we generated monoclonal antibodies against the netrin-G1 and netrin-G2 proteins. The monoclonal antibody clones 171A18 and 30B15 differentially labeled specific neuronal circuits, the so-called netrin-G1 or netrin-G2 circuits in mice, respectively. Epitope mapping revealed linear epitopes for these monoclonal antibodies, which are common among splicing variants, and suggested that the anti-netrin-G1 monoclonal antibodies are applicable to various species including humans.

Axonal netrin-Gs transneuronally determine lamina-specific subdendritic segments.

Axons from a distinct group of neurons make contact with dendritic trees of target neurons in clearly segregated and laminated patterns, thereby forming functional units for processing multiple inputs of information in the vertebrate central nervous system. Whether and how dendrites acquire lamina-specific properties corresponding to each pathway is not known. We show here that vertebrate-specific membrane-anchored members of the UNC-6/netrin family, netrin-G1 and -G2, organize the lamina/pathway-specific differentiation of dendrites. Netrin-G1 and -G2 distribute on axons of different pathways and specifically interact with receptors NGL-1 and -2, respectively. In the hippocampus, parietal cortex, and piriform cortex, NGL-1 is concentrated in the dendritic segments corresponding to the lamina-specific termination of netrin-G1-positive axons, and NGL-2 is concentrated in distinct dendritic segments corresponding to the termination of netrin-G2-positive axons. In netrin-G1- and -G2-deficient mice, in which axonal path-finding is normal, the segmental distribution of NGL-1 and -2 is selectively disrupted, and the individual receptors are diffused along the dendrites. These findings indicate that transneuronal interactions of netrin-Gs and their specific receptors provide a molecular basis for the axonal innervation-dependent mechanism of postsynaptic membrane organization, and provide insight into the formation of the laminar structure within the dendrites.