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Gene Regulation of Cell Adhesion Molecules During Neural Morphogenesis

F.S. Jones, P. Kallunki, R. Meech, G.M. Edelman

Cell adhesion molecules (CAMs) play important roles in the development of the nervous system by regulating neural differentiation, axonal guidance and fasciculation, and synapse formation. We seek to understand how certain CAMs are expressed in defined spatiotemporal patterns during neural morphogenesis. Our approach has been to determine cis regulatory sequences and transcription factors that regulate expression of the genes for CAMs of the immunoglobulin superfamily, particularly those related to the neural CAM (N-CAM).

We found that several CAMs of the N-CAM family are regulated by transcription factors encoded by homeobox-containing genes, including those of the Hox gene network, and by the paired box (Pax) gene family. For instance, the N-CAM promoter contains binding sites for Hox and Pax proteins, and both of these elements are necessary for the correct anteroposterior and dorsoventral expression pattern of N-CAM in the nervous system of transgenic mice.

Recently, we focused on dissecting the regulatory mechanisms responsible for the tissue-specific expression of the CAM L1. Mutations in the gene for L1 are responsible for a variety of neurologic disorders, including hydrocephalus, mental retardation, and agenesis of the corpus callosum. These observations suggest that understanding how expression of the gene for L1 is regulated would be a significant step in relating the place-dependent appearance of L1 with its role in morphogenesis.

We determined the promoter for the mouse gene for L1 and found that a segment of the gene spanning the region from the promoter to the fourth exon (approximately 18 kb) was required to give a neurally restricted expression pattern in transgenic mice. Deletion of a single 21-bp regulatory element called the neural restrictive silencer element (NRSE) within the second intron resulted in extraneural expression of the gene for L1, primarily in mesenchymal derivatives of the neural crest. These experiments indicated that tissue-specific expression of L1 is modulated by the NRSE.

Although the gene for L1 is first expressed at neural differentiation, it is expressed most abundantly during postnatal development when extensive neural outgrowth and formation of synaptic connections occur. These features motivated us to examine how the NRSE controls expression of the gene for L1 during postnatal development. We found that the NRSE can function as both a silencer and an enhancer during postnatal development and in adults. Expression of ß-galactosidase in neurons within the cortex, striatum, thalamus, and hippocampus and in the peripheral glia that ensheathe olfactory and cochlear nerves was greater in newborn mice with the NRSE-mutated L1lacZ reporter gene than in mice with the wild-type L1lacZ transgene. Later, during postnatal development and in adulthood, mice with the NRSE-mutant transgene had a loss of expression of ß-galactosidase in several neural structures, whereas mice with the wild-type transgene did not. Collectively, these data indicate that the NRSE plays an active role in modulating expression of the gene for L1 in the nervous system, where the NRSE plays a dual role as a silencer and an enhancer.

To screen for additional elements and transcription factors that control the neural pattern of expression of the L1 gene, we examined sequences in the gene that were responsive to signals from bone morphogenetic proteins (BMPs), an important family of extracellular factors that regulate cellular differentiation. A composite 30-bp element called the HPD was detected within the first intron. This element contains binding sequences for both homeodomain and Pax proteins and can bind these 2 different classes of transcription factors independently. One part of the HPD bound to Barx2, a new member of the Bar family of homeodomain proteins that we discovered in an earlier study. A different part of the HPD bound to Pax-6, a transcription factor known to regulate the development of the forebrain, eye, and olfactory system. In cellular transfection experiments, the HPD was necessary for high levels of expression of the L1 gene in neural cells, essential for induction of expression of the gene by BMP2 and BMP4, and required for activation of expression of the gene by Pax-6 and Barx2.

Currently, we are examining the role of the HPD in the pattern of expression of the gene for L1 in transgenic mice. Our working hypothesis is that the HPD is part of a developmental mechanism that controls L1 gene expression mediated by homeobox and Pax proteins and integrates signals from BMPs. We plan to expand these studies on gene regulation by using invertebrate systems such as Drosophila.