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

The subcommissural organ (SCO) secretes specific glycoproteins into th e cerebrospinal fluid that aggregate to constitute Reissner's fiber (R F), a thread-like structure running along the central canal of the spi nal cord. For further identification of the gene(s) encoding these sec retions, we have prepared a cDNA library in the vector IGT11 from bovi ne embryonic SCO. The screening of this library was performed using a polyclonal antibody raised against bovine RE Three positive clones wer e isolated and purified and one of these lambda RF101 comprising an in sert 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 subcommi ssural organ of the embryonic bovine by in situ hybridization. A label ing signal was observed in the embryonic SCO both in the secretory epe ndymal and hypendymal cells. This labeling is specific since the epend ymal layer bordering the ventricular cavity as well as the surrounding nervous tissue remained negative. Thus, the embryonic SCO contains sp ecific transcripts that are colocalized with the specific glycoprotein s 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 subsequ ent set of experiments the expression pattern was compared in embryos at two different stages of development (4-month-old and 8-month-old em bryos). No difference in the intensity of labeling could be detected i n the SCO of both stages suggesting that the level of expression remai ns stable at least during the second part of gestation. The identifica tion of the complete cDNA sequence is now required to find out homolog ies with known factors and to provide information about the role of th ese proteins in the developing nervous system.