Identification of genes differentially expressed in rat brain during postnatal development

During neuronal development CNS neurons extend axons over long distances. This high growth potential is lost during postnatal development resulting in very poor axonal outgrowth and regeneration in the adult CNS. This pronounced decline of axon growth potential and regenerative capability might be related to alterations in the expression level of growth-associated genes during postnatal development. The aim of the present study was the identification of candidate molecules that might be associated with axon growth, i.e. which are strongly expressed during axonal outgrowth and are downregulated as neuronal maturation proceeds. As the time periods of developmental axonal outgrowth and decrease in growth potential are well studied in rat cerebellum and entorhinal cortex, these two brain regions were chosen as model systems for analysis of gene expression patterns during axonal extension and after completion of pathway formation. In a first approach the study focused on the identification of transcription factors, because they are known to be involved in the regulation of cellular identity and differentiation and hence might also determine the intrinsic growth state of a neuron. In order to identify transcription factors from rat cerebellum and entorhinal cortex at the time of maximal axonal outgrowth, PCR with degenerate oligonucleotides, specific for the conserved DNA-binding domains of distinct transcription factor classes, was performed with cDNA from cerebellum at E18 and entorhinal cortex at P0, respectively. A limited number of PCR products could be isolated from the above brain regions by the use of primers for the POU and zinc finger family of transcription factors. Because of the small number of candidate molecules and considerable difficulties in constructing cDNA probes for further analysis this approach was not further pursued. A second approach aimed at the comparison of the transcriptional activity of young differentiating CNS neurons, which extend axons, with that of more mature neurons, which have lost growth competence. The method of suppression subtractive hybridisation (SSH) was performed in two distinct CNS tissues, rat cerebellum and entorhinal cortex, at two developmental stages (E18 and P35 for cerebellum and P0 an P10 for entorhinal cortex, respectively) in order to enrich for genes, which are downregulated during postnatal development. Several differentially expressed genes were identified, and the temporal and spatial expression pattern of some of these genes was further examined in rat brain by Northern- and in situ-hybridisation analysis at different developmental stages. One of the identified genes, rMMS2, was not known in the rat before and was characterised in this study for the first time. In addition, CRHSP-24, whose expression pattern had not previously been examined in the developing brain, was identified as a differentially expressed gene. Further analysis showed that rMMS2 and CRHSP-24 were strongly expressed in many brain regions during late embryonic and early postnatal development. Expression of both genes was significantly downregulated during the first postnatal weeks and was only weak or absent in the adult brain. As this regulated distribution correlates well with the time period of establishment of axonal connections in the developing brain, these molecules might play a role in neuronal differentiation processes. However, their function in neuronal development is not yet clear and remains to be elucidated. Because only a fraction of the enriched genes has been analysed by now the pool of subtracted genes might serve as a valuable source for the identification of further candidate genes, which might be associated with neuronal differentiation and axonal outgrowth