Targeted mutation of a novel gene with developmentally down-regulated expression in the mouse CNS

By means of subtractive hybridization and differential screening of mRNAs expressed at different developmental stages in the mouse brain, a novel gene was identified which we termed 1A13. Its open reading frame comprises 1453 nucleotides encoding a protein of 477 amino acids with a predicted molecular weight of 53.3 kDa. Using Northern blot analysis, we have followed 1A13 expression from gestational day 10 to adulthood, demonstrating that 1A13 mRNA levels decline markedly towards birth. In the adult brain, 1A13 expression is no longer detectable on Northern blots. Our results from in-situ hybridization have revealed that 1A13 is mainly expressed in the developing brain and the spinal cord. Interestingly, a distinct signal was also observed in the retina and the interdigital areas. In the developing neocortex, 1A13 appears restricted to the migratory neurons, while the marginal zone is clearly devoid of 1A13 expression. In order to elucidate the biological significance of 1 Al 3 in the developing mouse brain, we are planning to generate 1A13 loss-of-f unction mutants by homologous recombination, transformation of embryonic stem cells and generation of chimeric animals. The phenotype of homozygous offspring will be analyzed by histochemical/immunohistochemical methods and, if necessary, in vitro analysis.

Aim of our study was to elucidate the expression pattern and the function of a novel neuroanl gene termed 1A13 which we had previously identified in the murine brain by the means of subtractive and differential hybridization. With these techniques it is possible to analyze the expression of developmentally regulated genes in the growing cerebral hemispheres in the second half of brain development, when major steps of neurogenesis take place. We found that 1A13 (GenBank accession number:X83587) is the murine homologue of the human transcriptional co- repressor CoREST and thus will be refered to as 1A13/M-CoREST. The gene comprises 2037 base pairs and encodes a protein of 479amino acids wich an apparent molecular weiht of 53.3. kDa. It localizes to the nucleus and the existence of two so called SANT domains indicates a role in transcriptional regulation. 1A13/M-CoREST is expressed in the mouse embryo from early on (gestational periode of E7) in nearly every rudimentary organ. As development proceeds it is nearly exclusively expressed in the nervous system and in a few non-neural cells like the interdigitary cells or in the tooth-anlage. In the adult mouse only few neuronal cells express the gene weakly. Co-REST was originally described as a corepressor to the transcriptional repressor REST, a master negative regulator of neurogenesis. However it has also been detected in undifferentiated neural cells. Thus it is believed that, apart from its repressive action on neuronal gene transcription in extraneural tissue, REST may act as a silencer of neuron specific gene expression in neuronal precursors. Whether human CoREST respectively 1A13/M-CoREST shares this function with REST needs to be determined. This will be an important question to ask in light of largely unknown mechanisms of neuronal differentiation.