Identification of genes differentially expressed in rat brain during postnatal development

Field: Neuroscience (Life Sciences)

During neuronal development CNS neurons extend axons over long distances. This high \ngrowth potential is lost during postnatal development resulting in very poor axonal \noutgrowth and regeneration in the adult CNS. This pronounced decline of axon growth \npotential and regenerative capability might be related to alterations in the expression level \nof growth-associated genes during postnatal development. The aim of the present study \nwas the identification of candidate molecules that might be associated with axon growth, \ni.e. which are strongly expressed during axonal outgrowth and are downregulated as \nneuronal maturation proceeds. As the time periods of developmental axonal outgrowth and \ndecrease in growth potential are well studied in rat cerebellum and entorhinal cortex, these \ntwo brain regions were chosen as model systems for analysis of gene expression patterns \nduring axonal extension and after completion of pathway formation. In a first approach the \nstudy focused on the identification of transcription factors, because they are known to be \ninvolved in the regulation of cellular identity and differentiation and hence might also \ndetermine the intrinsic growth state of a neuron. In order to identify transcription factors \nfrom rat cerebellum and entorhinal cortex at the time of maximal axonal outgrowth, PCR \nwith degenerate oligonucleotides, specific for the conserved DNA-binding domains of \ndistinct transcription factor classes, was performed with cDNA from cerebellum at E18 \nand entorhinal cortex at P0, respectively. A limited number of PCR products could be \nisolated from the above brain regions by the use of primers for the POU and zinc finger \nfamily of transcription factors. Because of the small number of candidate molecules and \nconsiderable difficulties in constructing cDNA probes for further analysis this approach \nwas not further pursued. A second approach aimed at the comparison of the transcriptional \nactivity of young differentiating CNS neurons, which extend axons, with that of more \nmature neurons, which have lost growth competence. The method of suppression \nsubtractive hybridisation (SSH) was performed in two distinct CNS tissues, rat cerebellum \nand entorhinal cortex, at two developmental stages (E18 and P35 for cerebellum and P0 an \nP10 for entorhinal cortex, respectively) in order to enrich for genes, which are \ndownregulated during postnatal development. Several differentially expressed genes were \nidentified, and the temporal and spatial expression pattern of some of these genes was \nfurther examined in rat brain by Northern- and in situ-hybridisation analysis at different \ndevelopmental stages. One of the identified genes, rMMS2, was not known in the rat \nbefore and was characterised in this study for the first time. In addition, CRHSP-24, whose \nexpression pattern had not previously been examined in the developing brain, was \nidentified as a differentially expressed gene. Further analysis showed that rMMS2 and \nCRHSP-24 were strongly expressed in many brain regions during late embryonic and early \npostnatal development. Expression of both genes was significantly downregulated during \nthe 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 \nthe developing brain, these molecules might play a role in neuronal differentiation \nprocesses. However, their function in neuronal development is not yet clear and remains to \nbe elucidated. Because only a fraction of the enriched genes has been analysed by now the \npool of subtracted genes might serve as a valuable source for the identification of further \ncandidate genes, which might be associated with neuronal differentiation and axonal \noutgrowth.