The possibility that ependymal cells might harbor stem cells brought them into the limelight. The IE still a contended issue. The NOVOcans are a novel family of developmentally regulated (putative) herparan sulfate proteiglycans, highly consevered across species. They were isolated from an oligodendrocyte (OLD) DNA library. Preliminary double immunohistochemical studies on the localization of NOVOcans in rat brains, using monoclinal antibodies (mass) against NOVOCAIN and either a polyclonal anti-GFAP (glial fibrillary acidic protein), revealed (mostly) co-localization with M.P. but not GFAP (but see below). No staining of astrocytes (protoplasmic or fibrous) or neurons was detected. Their mass specifically recognized ependymal cells. This finding established an intriguing link between OLDS ans ependymal cells. This pilot project has two objectives: first, to confirm and extend the preliminary observations concerning ependymal cells; and second, to define the temporal and spacial regulation of NOVOcans with particular emphasis on identifying spliced products and ascertaining weather the are selectively expressed in ependyma cells. The proposal has three Specific Aims.
In Aim 1, it is proposed to investigate NOVOcans' developmental pattern of expression in ependymal cells by Northern blots, immunohistochemistry and in situ hybridization. For Northern, mRNA will be extract from A. through adults and probed with DNA fragments obtained from restriction enzyme cuts of novocain DNA. Coronal sections of the forebrain of rats will be obtained and double stained with selected panels of abs.. to discriminate subventricular astrocytes and ependymal cells. Abs. that recognize progenitor cells and immature OLDS will also be used. Equivalent sections will be employed for in sity hybridization to facilitate the comparison between mRNA and protein expression.
In Aim 2, the immunohistochemistry data will be further analyze by confocal microcopy and electron micropsy in order to obtain the cellular localization of NOVOcans.
In Aims 3, rat and OLD DNA libraries will be utilized as templates to fish out novocain Informs by PER. These will be used to generate specific probes (e.g., Abs.) to investigate whether these isoforms are differentially expressed. The proposed research should provide significant parameters for assessing the importance of NOVOcans. This, in turn, would open up new avenues of research concerning the biology of ependymal cells and their role in subventricular zone neurogenesis.
