Supplementary Material for: Identification and Analysis of Two Novel Sites of Rat GnRH Receptor Gene Promoter Activity: The Pineal Gland and Retina

<b><i>Background and Aims:</i></b> In mammals, activation of pituitary GnRH receptor (GnRHR) by hypothalamic GnRH increases the synthesis and secretion of LH and FSH, which, in turn, regulate gonadal functions. However, GnRHR gene <i>(Gnrhr)</i> expression is not restricted to the pituitary. <b><i>Methods:</i></b> To gain insight into the extrapituitary expression of <i>Gnrhr</i>, a transgenic mouse model that expresses the human placental alkaline phosphatase reporter gene driven by the rat <i>Gnrhr</i> promoter was created. <b><i>Results:</i></b> This study shows that the rat <i>Gnrhr</i> promoter is operative in two functionally related organs, the pineal gland, as early as embryonic day (E) 13.5, and the retina where activity was only detected at E17.5. Accordingly, <i>Gnrhr</i> mRNA were present in both tissues. Transcription factors known to regulate <i>Gnrhr</i> promoter activity such as the LIM homeodomain factors LHX3 and ISL1 were also detected in the retina. Furthermore, transient transfection studies in CHO and gonadotrope cells revealed that OTX2, a major transcription factor in both pineal and retina cell differentiation, is able to activate the <i>Gnrhr</i> promoter together with either CREB or PROP1, depending on the cell context. <b><i>Conclusion:</i></b> Rather than using alternate promoters, <i>Gnrhr</i> expression is directed to diverse cell lineages through specific associations of transcription factors acting on distinct response elements along the same promoter. These data open new avenues regarding GnRH-mediated control of seasonal and circadian rhythms in reproductive physiology.