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.