SPECIFIC TRANSCRIPTS ANALYZED BY IN-SITU HYBRIDIZATION IN THE SUBCOMMISSURAL ORGAN OF BOVINE EMBRYOS

The subcommissural organ (SCO) secretes specific glycoproteins into the cerebrospinal fluid that aggregate to constitute Reissner's fiber (RF), a thread-like structure running along the central canal of the spinal cord. For further identification of the gene(s) encoding these secretions, we have prepared a cDNA library in the vector IGT11 from bovine embryonic SCO. The screening of this library was performed using a polyclonal antibody raised against bovine RE Three positive clones were isolated and purified and one of these lambda RF101 comprising an insert of #400 nucleotides was under-cloned into pBluescript plasmid and mapped. After labeling with S-35 (ATP) this cDNA fragment served as a probe to analyse the presence of specific transcripts in the subcommissural organ of the embryonic bovine by in situ hybridization. A labeling signal was observed in the embryonic SCO both in the secretory ependymal and hypendymal cells. This labeling is specific since the ependymal layer bordering the ventricular cavity as well as the surrounding nervous tissue remained negative. Thus, the embryonic SCO contains specific transcripts that are colocalized with the specific glycoproteins as shown after the use of a specific monoclonal antibody C(1)B(8)A(8). In addition, the pattern of labeling with the specific SCO cDNA is different from those of beta actin cDNA and tear lipocalin cDNA, which, respectively, served as positive and negative controls. In a subsequent set of experiments the expression pattern was compared in embryos at two different stages of development (4-month-old and 8-month-old embryos). No difference in the intensity of labeling could be detected in the SCO of both stages suggesting that the level of expression remains stable at least during the second part of gestation. The identification of the complete cDNA sequence is now required to find out homologies with known factors and to provide information about the role of these proteins in the developing nervous system.