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

Abstract

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 neurons 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.

Estrogen regulation of gene expression in GnRH neurons

“…The GN11 cells were derived from an olfactory tumor from a mouse containing a transgene consisting of Tag under the regulatory control of 1131bp of the human GnRH promoter (Radovick et al, 1991b; Zhen et al, 1997). They are thought to be derived from GnRH neurons transformed early in development (Pimpinelli et al, 2003; Fang et al, 1998; Maggi et al, 2000). The GT1-7 cells were derived from an hypothalamic tumor from a mouse containing a transgene consisting of Tag under the regulatory control of a rat promoter fragment (Mellon et al, 1990).…”

“…The GT1-7 cells were derived from an hypothalamic tumor from a mouse containing a transgene consisting of Tag under the regulatory control of a rat promoter fragment (Mellon et al, 1990). These cells are thought to represent a more mature cell as they were transformed at a later developmental age (Pimpinelli et al, 2003; Fang et al, 1998; Maggi et al, 2000). This is the first report documenting the relative expression of the estrogen receptors in the different GnRH expressing cell lines and their contributions to GnRH gene expression and secretion.…”

Gonadotropin-releasing hormone (GnRH) neuron migration: Initiation, maintenance and cessation as critical steps to ensure normal reproductive function

“…We showed that Axl and Tyro3 were expressed in NLT cells as models for early migrating GnRH neurons while Tyro3 and Mer were expressed in GT1-7 cells as models for postmigratory GnRH neurons (87). In NLT GnRH neuronal cells, Growth arrest specific gene 6 (Gas6), the ligand for the TAM family, induced GnRH neuronal migration via a novel p38MAP kinase pathway(88) and protection from programmed cell death via ERK MAP kinase and PI3 kinase to Akt signaling(89), suggesting a potential role for Axl and Tyro3 in GnRH neuron development.…”

Hypothalamic Dysregulation and Infertility in Mice Lacking the Homeodomain Protein Six6

“…In contrast, GN11 cells represent a developmentally earlier, migratory GnRH neuron that expresses low levels of GnRH and responds to migratory cues (Radovick et al, 1991). Using these cell lines, enhancer and promoter elements have been characterized (Kim et al, 2002, 2007; Lawson et al, 2002; Givens et al, 2004; Iyer et al, 2010) and several transcription factors that regulate GnRH gene expression have been elucidated (Clark and Mellon, 1995; Lawson et al, 1996; Wierman et al, 1997; Fang et al, 1998; Kelley et al, 2000; Wolfe et al, 2002; Rave-Harel et al, 2004, 2005; Pierce et al, 2008; Larder and Mellon, 2009). …”