Transcription Factor Activator Protein-2 Is Required for Continued Luteinizing Hormone-Releasing Hormone Expression in the Forebrain of Developing Mice

Abstract

LHRH is the neuropeptide responsible for reproductive function. Prenatally, LHRH expression begins when neurons are in the olfactory pit and continues as these cells migrate into the brain. Thus, LHRH neurons maintain neuropeptide expression through very distinct environments. The regulatory interactions that control onset and continued expression of the LHRH phenotype are unknown. To begin to address this question primary LHRH neurons were removed from nasal explants at different ages. A complementary DNA (cDNA) subtraction screen was performed comparing a 3.5-days in vitro LHRH neuron [approximately embryonic day 15 (E15) in vivo] to two 10.5-days in vitro LHRH neurons (approximately postnatal day 1 in vivo). The transcription factor activator protein-2 (AP-2␣) was differentially expressed and was present in the developmentally younger LHRH neuron. In vivo analysis revealed that LHRH neurons expressed AP-2 as they migrated across the cribriform plate and into the forebrain beginning on E13.5, but that coexpression of LHRH and AP-2 was no longer detected in postnatal day 1 animals. This suggested a regulatory role for AP-2 in LHRH neurons. Analysis of animals lacking AP-2␣ revealed a dramatic decrease in forebrain LHRH neurons between E13.5 and E14.5, correlating with normal onset of AP-2 expression in LHRH neurons as they entered the central nervous system. Nasal cells robustly expressing LHRH were still present on E14.5. The continued presence of forebrain LHRH cells is proposed based on a second marker, galanin, and lack of increased apoptotic/ necrotic cells in this region. A decrease in LHRH messenger RNA in forebrain neurons indicates regulation of LHRH occurred at the transcriptional or posttranscriptional level in mutant animals. These results indicate a developmentally restricted involvement of the transcription factor AP-2 in LHRH expression once the LHRH neurons have migrated into the forebrain, but before establishment of an adult-like distribution.

Regulation of Gonadotropin-Releasing Hormone (GnRH) Gene Expression during GnRH Neuron Migration in the Mouse

“…The spatial localization of this change in gene transcription is intriguing given suggestions that both GABAergic and glutamatergic signaling are involved in regulating the passage of GnRH neurons through the cribiform plate [5, 9]. Furthermore, Kramer et al [28]have recently reported that the transcription factor AP-2 is also expressed by GnRH neurons just as they enter the brain and demonstrated that AP-2 is required for the normal expression of GnRH mRNA. Together, these data raise the possibility that the induction of AP-2 in GnRH neurons may underlie the increase in GnRH gene transcription observed as these neurons enter the brain.…”

Developmental Changes in GABAA Receptor Subunit Composition Within the Gonadotrophin‐Releasing Hormone‐1 Neuronal System

“…Complimentary DNA libraries were created as previously described (20). Briefly, single cells or 1 µg of total brain RNA (Ambion, Inc., Austin, TX, USA) in lysis buffer were incubated at 65 °C for 1 min; ice for1 min; room temperature for 2 min.…”

“…A PCR reaction was conducted to proportionally amplify transcripts within the cells (20). PCR mix [10 µl of 10 × PCR buffer II (Applied Biosystems, Foster City, CA, USA), 10 µl of 25 m M MgCl 2 (Applied Biosystems), 0.5 ml of 20 mg/ml BSA (Roche), 1 µl each 100 m M deoxynucleotide triphosphate (Invitrogen), 1 µl 5% triton (Sigma), 5 µg AL1 primer (ATTGGATCCAGGCCGCTC‐TGCAAAAATATGAATTC[T]24), 2 µl AmpliTaq (Applied Biosystems), and 57.5 ml dH 2 O] was prepared and 90 µl added to each tube containing 10 µl template.…”

“…For fluorescent staining, the procedures for the first day were the same as described above. On the second day, tissue was rinsed and incubated in fluorescent secondary (Alexa‐555 goat anti‐guinea pig for GABA A α2 and goat anti‐rabbit Cy3 for GABA A α 6 , both of which result in a red reaction product) for 1 h. Tissue was then rinsed, blocked (anti‐rabbit Fab fragment; 80 ng/ml) (20), and fixed as described above and put into the second primary antibody (SW‐1) for 48 h at 4 °C. The tissue was then rinsed and incubated in fluorescent secondary (Alexa‐488 goat anti‐rabbit, which results in a green reaction product).…”

Disorganized olfactory bulb lamination in mice deficient for transcription factor AP-2ɛ

“…AP-2α and AP-2γ are also expressed in the developing OB (Feng and Williams, 2003), however, a detailed understanding of their importance in olfaction remains to be determined because Tcfap2a and Tcfap2c knockout mice do not survive post-natally (Auman et al, 2002; Nottoli et al, 1998; Schorle et al, 1996; Zhang et al, 1996). Nevertheless, previous studies have implicated AP-2α in Luteinizing Hormone-Releasing Hormone (LHRH) neuronal development and presumably behavior linked to olfaction (Kramer et al, 2000). Therefore, the disruption of AP-2ε in combination with AP-2α or AP-2γ may be required to uncover redundant functions for this family of transcription factors in OB development and function.…”