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Identification of Novel Factors That Regulate GnRH Gene Expression and Neuronal Migration

Research Service, Veterans Affairs Medical Center and Department of Medicine, University of Colorado School of Medicine, Denver, Colorado 80220

Address all correspondence and requests for reprints to: Margaret E. Wierman, University of Colorado, Endocrinology (III H), VAMC Room 9C104, 1055 Clermont, Denver, Colorado 80220-3808.

We used differential display PCR on two GnRH producing cell lines to identify genes involved in GnRH gene expression and neuronal migration. RNA from Gn10 cells (derived from a tumor in the olfactory area when GnRH neruons are migrating and make low levels of GnRH) and from GT1-7 cells (derived from a tumor in forebrain when GnRH neurons are postmigratory and make high levels of GnRH) was reverse transcribed into cDNA. The cDNA was amplified using three anchored primers and eight random primers from each cell line and products from duplicate reactions electrophoresed in parallel in a denaturing acrylamide gel. Differentially expressed cDNAs were excised, reamplified and used as probes in Northern analysis of total RNA from each cell line to confirm differentially expressed RNA. The cDNAs were sequenced and compared to the Genbank database. Four of five clones isolated from GT1-7 GnRH neurons are novel, while four of five clones isolated from Gn10 cells have homology to known DNA sequences. One clone, Gn8-01 encodes adhesion related kinase (Ark), a molecule that has an N-terminal domain characteristic of cell adhesion molecules and whose kinase domain may play a role in protection from apoptosis. Together these data support the usefulness of the technique to identify novel genes that play a role in the control of GnRH expression and neuronal migration.

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