GnRH neurogenesis depends on embryonic pheromone receptor expression

Highlights

• Male pheromones rescue hypogondal hypogonadism in Nhlh2-deficient mice.

• Inactivation of β2-m prevents pheromone-mediated rescue of infertility.

• β2-m is required for the embryonic development of Nhlh2-deficient GnRH neurons.

• Nhlh1 controls expression of V2R vomeronasal receptors.

• GnRH neurogenesis depends on VN4/V2R vomeronasal receptors.

Abstract

Gonadotropin-releasing hormone (GnRH) neurons control mammalian reproduction and migrate from their birthplace in the nasal placode to the hypothalamus during development. Despite much work on the origin and migration of GnRH neurons, the processes that control GnRH lineage formation are not fully understood. Here, we demonstrate that Nhlh genes control vomeronasal receptor expression in the developing murine olfactory placode associated with the generation of the first GnRH neurons at embryonic days (E)10–12. Inactivation of ß2-microglobulin (ß2-m), which selectively affects surface expression of V2Rs, dramatically decreased the number of GnRH neurons in the Nhlh2 mutant background, preventing rescue of fertility in female Nhlh2 mutant mice by male pheromones. In addition, we show that GnRH neurons generated after E12 fail to establish synaptic connections to the vomeronasal amygdala, suggesting the existence of functionally specialized subpopulations of GnRH neurons, which process pheromonal information.

Introduction

At the center of hypothalamic control of reproduction is a small group of neurons which regulate the reproductive endocrine status in mammals by secreting gonadotropin releasing hormone (GnRH; (Gore, 2002; Herbison, 2006)). GnRH triggers the release of gonadotropins from the pituitary, which in turn regulates puberty onset, gametogenesis, and estrus cycling. GnRH neurons do not develop inside the central nervous system but migrate from the olfactory placode (OP) to the forebrain during embryogenesis (Schwanzel-Fukuda and Pfaff, 1989; Wray et al., 1989). Despite intense research, the embryonic origin of the GnRH precursor cells is still debated (Wray, 2010). It has been hypothesized that GnRH precursors are derived from the olfactory pits, a structure that forms as the olfactory placode (OP) invaginates and/or neural crest cells.

Normal development of GnRH neurons depends on the basic helix-loop-helix (bHLH) transcription factor Nhlh2. Nhlh2 mutants lack ~30% of GnRH neurons and show signs of hypogonadal hypogonadism (Cogliati et al., 2007; Good et al., 1997; Kruger et al., 2004). Concomitant inactivation of Nhlh2 and the closely related Nhlh1 leads to a virtually complete absence of GnRH neurons in posterior parts of the brain at E18.5 indicating that both genes act in parallel to determine the fate of GnRH neurons (Kruger et al., 2004). Interestingly, conditional inactivation of Nhlh2 restricted to GnRH neurons results only in a 20% reduction of GnRH neurons and has no effects on the reproductive system (Schmid et al., 2013). Furthermore, infertile and hypogonadal female Nhlh2 mutant mice show a normal phenotype and are fertile when exposed to conspecific males (Cogliati et al., 2007; Good et al., 1997; Kruger et al., 2004), suggesting rescue of the Nhlh2 mutant phenotype by male pheromones.

Chemoreception of pheromones in mice is initiated in sensory neurons of the epithelium of the vomeronasal organ (VNO), which are equipped with a unique molecular repertoire of over 300 G-protein coupled vomeronasal receptors (VRs) encoded by two large gene families (V1Rs and V2Rs, respectively (Dulac and Torello, 2003)). To date, only few chemicals carrying pheromonal information have been identified (Liberles, 2014; Mohrhardt et al., 2018; Stowers and Kuo, 2015). These are structurally diverse and include proteins and fragments thereof, small volatile molecules found in urine and specific peptides secreted by exocrine glands (Leinders-Zufall et al., 2000; Leinders-Zufall et al., 2004; Kimoto et al., 2007; Chamero et al., 2007; Haga et al., 2010). Although more pheromones might exist to account for the large number of mouse VR genes, it is tempting to speculate that VRs not only serve in the recognition of these chemical signals but also play roles independent of their chemosensory function.

Signals generated by vomeronasal sensory neurons are relayed through the accessory olfactory bulb (AOB) to the vomeronasal amygdala. From there they proceed to the hypothalamus, which mediates the behavioural effects and neuroendocrine alterations triggered by pheromones (Scalia and Winans, 1975; Alheid et al., 1995; Newman, 1999; Halpern and Martinez-Marcos, 2003). Transsynaptic tracing studies have provided experimental evidence that pheromone signals affecting reproductive physiology and behavior are ultimately relayed to hypothalamic GnRH neurons (Boehm et al., 2005).

To determine whether members of the V1R or V2R family of vomeronasal receptors mediate pheromonal rescue of the Nhlh2 mutant phenotype we generated compound mutant mice deficient for both Nhlh2 and β2-microglobulin (β2-m), which selectively affects surface expression of V2Rs, but not V1Rs (Loconto et al., 2003). We found that infertility and hypogonadism in female β2-m/Nhlh2 compound mutant mice cannot be rescued by male pheromones, suggesting functional contribution of V2Rs to the pheromone-mediated rescue of the Nhlh2 phenotype. Interestingly, β2-m/Nhlh2 mutant mice have drastically reduced numbers of GnRH neurons at day 10–12 of gestation (E10-12). This resulted in a nearly complete absence of GnRH neurons in the posterior part of the hypothalamus similar to the Nhlh1/Nhlh2 double mutants. We found that Nhlh1 or β2-m control expression of the VN4 subfamily of V2Rs and that concomitant knockdown of VN4/V2Rs and Nhlh2 in explant cultures prevent GnRH neurogenesis. Using genetic transsynaptic tracing in the β2-m/Nhlh2 compound mutant mice, we also show that the remaining, non-E10-12 GnRH neurons fail to establish synaptic connections to the vomeronasal amygdala, suggesting the existence of functionally specialized subpopulations of GnRH neurons.