Deletion of Six3 in post-proliferative neurons produces weakened SCN circadian output, improved metabolic function, and dwarfism in male mice.

OBJECTIVE: The increasing prevalence of obesity makes it important to increase the understanding of the maturation and function of the neuronal integrators and regulators of metabolic function. METHODS: Behavioral, molecular, and physiological analyses of transgenic mice with Sine oculis 3 (Six3) deleted in mature neurons using the Synapsincreallele. RESULTS: Conditional deletion of the homeodomain transcription factor Six3 in mature neurons causes dwarfism and weakens circadian wheel-running activity rhythms but increases general activity at night, and improves metabolic function, without impacting pubertal onset or fertility in males. The reduced growth in 6-week-old Six3fl/fl:Synapsincre (Six3syn) males correlates with increased somatostatin (SS) expression in the hypothalamus and reduced growth hormone (GH) in the pituitary. In contrast, 12-week-old Six3syn males have increased GH release, despite an increased number of the inhibitory SS neurons in the periventricular nucleus. GH is important in glucose metabolism, muscle function, and bone health. Interestingly, Six3syn males have improved glucose tolerance at 7, 12, and 18 weeks of age, which, in adulthood, is associated with increased % lean mass and increased metabolic rates. Further, 12-week-old Six3syn males have reduced bone mineralization and a lower bone mineral density, indicating that reduced GH levels during early life cause a long-term reduction in bone mineralization. CONCLUSION: Our study points to the novel role of Six3 in post-proliferative neurons to regulate metabolic function through SS neuron control of GH release.

The transcription factors SIX3 and VAX1 are required for suprachiasmatic nucleus circadian output and fertility in female mice.

The homeodomain transcription factors sine oculis homeobox 3 (Six3) and ventral anterior homeobox 1 (Vax1) are required for brain development. Their expression in specific brain areas is maintained in adulthood, where their functions are poorly understood. To identify the roles of Six3 and Vax1 in neurons, we conditionally deleted each gene using Synapsincre , a promoter targeting maturing neurons, and generated Six3syn and Vax1syn mice. Six3syn and Vax1syn females, but not males, had reduced fertility, due to impairment of the luteinizing hormone (LH) surge driving ovulation. In nocturnal rodents, the LH surge requires a precise timing signal from the brain's circadian pacemaker, the suprachiasmatic nucleus (SCN), near the time of activity onset. Indeed, both Six3syn and Vax1syn females had impaired rhythmic SCN output, which was associated with weakened Period 2 molecular clock function in both Six3syn and Vax1syn mice. These impairments were associated with a reduction of the SCN neuropeptide vasoactive intestinal peptide in Vax1syn mice and a modest weakening of SCN timekeeping function in both Six3syn and Vax1syn mice. Changes in SCN function were associated with mistimed peak PER2::LUC expression in the SCN and pituitary in both Six3syn and Vax1syn females. Interestingly, Six3syn ovaries presented reduced sensitivity to LH, causing reduced ovulation during superovulation. In conclusion, we have identified novel roles of the homeodomain transcription factors SIX3 and VAX1 in neurons, where they are required for proper molecular circadian clock function, SCN rhythmic output, and female fertility.

Homeodomain Proteins SIX3 and SIX6 Regulate Gonadotrope-specific Genes During Pituitary Development.

Sine oculis-related homeobox 3 (SIX3) and SIX6, 2 closely related homeodomain transcription factors, are involved in development of the mammalian neuroendocrine system and mutations of Six6 adversely affect fertility in mice. We show that both small interfering RNA knockdown in gonadotrope cell lines and knockout of Six6 in both embryonic and adult male mice (Six6 knockout) support roles for SIX3 and SIX6 in transcriptional regulation in gonadotrope gene expression and that SIX3 and SIX6 can functionally compensate for each other. Six3 and Six6 expression patterns in gonadotrope cell lines reflect the timing of the expression of pituitary markers they regulate. Six3 is expressed in an immature gonadotrope cell line and represses transcription of the early lineage-specific pituitary genes, GnRH receptor (GnRHR) and the common α-subunit (Cga), whereas Six6 is expressed in a mature gonadotrope cell line and represses the specific ß-subunits of LH and FSH (LHb and FSHb) that are expressed later in development. We show that SIX6 repression requires interaction with transducin-like enhancer of split corepressor proteins and competition for DNA-binding sites with the transcriptional activator pituitary homeobox 1. Our studies also suggest that estradiol and circadian rhythm regulate pituitary expression of Six6 and Six3 in adult females but not in males. In summary, SIX3 and SIX6 play distinct but compensatory roles in regulating transcription of gonadotrope-specific genes as gonadotrope cells differentiate.

Roles of binding elements, FOXL2 domains, and interactions with cJUN and SMADs in regulation of FSHß.

We previously identified FOXL2 as a critical component in FSHß gene transcription. Here, we show that mice deficient in FOXL2 have lower levels of gonadotropin gene expression and fewer LH- and FSH-containing cells, but the same level of other pituitary hormones compared to wild-type littermates, highlighting a role of FOXL2 in the pituitary gonadotrope. Further, we investigate the function of FOXL2 in the gonadotrope cell and determine which domains of the FOXL2 protein are necessary for induction of FSHß transcription. There is a stronger induction of FSHß reporter transcription by truncated FOXL2 proteins, but no induction with the mutant lacking the forkhead domain. Specifically, FOXL2 plays a role in activin induction of FSHß, functioning in concert with activin-induced SMAD proteins. Activin acts through multiple promoter elements to induce FSHß expression, some of which bind FOXL2. Each of these FOXL2-binding sites is either juxtaposed or overlapping with a SMAD-binding element. We determined that FOXL2 and SMAD4 proteins form a higher order complex on the most proximal FOXL2 site. Surprisingly, two other sites important for activin induction bind neither SMADs nor FOXL2, suggesting additional factors at work. Furthermore, we show that FOXL2 plays a role in synergistic induction of FSHß by GnRH and activin through interactions with the cJUN component of the AP1 complex that is necessary for GnRH responsiveness. Collectively, our results demonstrate the necessity of FOXL2 for proper FSH production in mice and implicate FOXL2 in integration of transcription factors at the level of the FSHß promoter.

Kisspeptin regulates gonadotropin genes via immediate early gene induction in pituitary gonadotropes.

Kisspeptin signaling through its receptor, Kiss1R, is crucial for many reproductive functions including puberty, sex steroid feedback, and overall fertility. Although the importance of Kiss1R in the brain is firmly established, its role in regulating reproduction at the level of the pituitary is not well understood. This study presents molecular analysis of the role of kisspeptin and Kiss1R signaling in the transcriptional regulation of the gonadotropin gene ß-subunits, LHß and FSHß, using LßT2 gonadotrope cells and murine primary pituitary cells. We show that kisspeptin induces LHß and FSHß gene expression, and this induction is protein kinase C dependent and mediated by the immediate early genes, early growth response factor 1 and cFos, respectively. Additionally, kisspeptin induces transcription of the early growth response factor 1 and cFos promoters in LßT2 cells. Kisspeptin also increases gonadotropin gene expression in mouse primary pituitary cells in culture. Furthermore, we find that Kiss1r expression is enhanced in the pituitary of female mice during the estradiol-induced LH surge, a critical component of the reproductive cycle. Overall, our findings indicate that kisspeptin regulates gonadotropin gene expression through the activation of Kiss1R signaling through protein kinase C, inducing immediate early genes in vitro, and responds to physiologically relevant cues in vivo, suggesting that kisspeptin affects pituitary gene expression to regulate reproductive function.

Msx1 homeodomain protein represses the αGSU and GnRH receptor genes during gonadotrope development.

Multiple homeodomain transcription factors are crucial for pituitary organogenesis and cellular differentiation. A homeodomain repressor, Msx1, is expressed from the ventral aspect of the developing anterior pituitary and implicated in gonadotrope differentiation. Here, we find that Msx1 represses transcription of lineage-specific pituitary genes such as the common α-glycoprotein subunit (αGSU) and GnRH receptor (GnRHR) promoters in the mouse gonadotrope-derived cell lines, αT3-1 and LßT2. Repression of the mouse GnRHR promoter by Msx1 is mediated through a consensus-binding motif in the downstream activin regulatory element (DARE). Truncation and mutation analyses of the human αGSU promoter map Msx1 repression to a site at -114, located at the junctional regulatory element (JRE). Dlx activators are closely related to the Msx repressors, acting through the same elements, and Dlx3 and Dlx2 act as transcriptional activators for GnRHR and αGSU, respectively. Small interfering RNA knockdown of Msx1 in αT3-1 cells increases endogenous αGSU and GnRHR mRNA expression. Msx1 gene expression reaches its maximal expression at the rostral edge at e13.5. The subsequent decline in Msx1 expression specifically coincides with the onset of expression of both αGSU and GnRHR. The expression levels of both αGSU and GnRHR in Msx1-null mice at e18.5 are higher compared with wild type, further confirming a role for Msx1 in the repression of αGSU and GnRHR. In summary, Msx1 functions as a negative regulator early in pituitary development by repressing the gonadotrope-specific αGSU and GnRHR genes, but a temporal decline in Msx1 expression alleviates this repression allowing induction of GnRHR and αGSU, thus serving to time the onset of gonadotrope-specific gene program.

Neurokinin B causes acute GnRH secretion and repression of GnRH transcription in GT1-7 GnRH neurons.

Genetic studies in human patients with idiopathic hypogonadotropic hypogonadism (IHH) identified mutations in the genes that encode neurokinin B (NKB) and the neurokinin 3 receptor (NK3R). However, determining the mechanism whereby NKB regulates gonadotropin secretion has been difficult because of conflicting results from in vivo studies investigating the luteinizing hormone (LH) response to senktide, a NK3R agonist. NK3R is expressed in a subset of GnRH neurons and in kisspeptin neurons that are known to regulate GnRH secretion. Thus, one potential source of inconsistency is that NKB could produce opposing direct and indirect effects on GnRH secretion. Here, we employ the GT1-7 cell model to elucidate the direct effects of NKB on GnRH neuron function. We find that GT1-7 cells express NK3R and respond to acute senktide treatment with c-Fos induction and increased GnRH secretion. In contrast, long-term senktide treatment decreased GnRH secretion. Next, we focus on the examination of the mechanism underlying the long-term decrease in secretion and determine that senktide treatment represses transcription of GnRH. We further show that this repression of GnRH transcription may involve enhanced c-Fos protein binding at novel activator protein-1 (AP-1) half-sites identified in enhancer 1 and the promoter, as well as chromatin remodeling at the promoter of the GnRH gene. These data indicate that NKB could directly regulate secretion from NK3R-expressing GnRH neurons. Furthermore, whether the response is inhibitory or stimulatory toward GnRH secretion could depend on the history or length of exposure to NKB because of a repressive effect on GnRH transcription.

Aberrant development of the suprachiasmatic nucleus and circadian rhythms in mice lacking the homeodomain protein Six6.

The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus is the central pacemaker for peripheral and organismal circadian rhythms. The development of this hypothalamic structure depends on genetic programs throughout embryogenesis. We have investigated the role of the homeodomain transcription factor Six6 in the development of the SCN. We first showed that Six6 mRNA has circadian regulation in the mouse SCN. We then characterized the behavioral activity patterns of Six6-null mice under various photoperiod manipulations and stained their hypothalami using SCN-specific markers. Six6-null mice display abnormal patterns of circadian behavior indicative of SCN abnormalities. The ability of light exposure to reset rhythms correlates with the presence or absence of optic nerves, but all Six6-null mice show irregular rhythms. In contrast, wild-type mice with crushed optic nerves maintain regular rhythms regardless of light exposure. Using immunohistochemistry for arginine vasopressin (AVP), vasoactive intestinal polypeptide (VIP), and ß-galactosidase, we demonstrated the lack of these SCN markers in all Six6-null mice regardless of the presence of optic nerve or partial circadian rhythms. Therefore, Six6 is required for the normal development of the SCN, and the Six6-null mouse can mount independent, although irregular, circadian rhythms despite the apparent absence of a histochemically defined SCN.

Prenatal exposure to low levels of androgen accelerates female puberty onset and reproductive senescence in mice.

Sex steroid hormone production and feedback mechanisms are critical components of the hypothalamic-pituitary-gonadal (HPG) axis and regulate fetal development, puberty, fertility, and menopause. In female mammals, developmental exposure to excess androgens alters the development of the HPG axis and has pathophysiological effects on adult reproductive function. This study presents an in-depth reproductive analysis of a murine model of prenatal androgenization (PNA) in which females are exposed to a low dose of dihydrotestosterone during late prenatal development on embryonic d 16.5-18.5. We determined that PNA females had advanced pubertal onset and a delay in the time to first litter, compared with vehicle-treated controls. The PNA mice also had elevated testosterone, irregular estrous cyclicity, and advanced reproductive senescence. To assess the importance of the window of androgen exposure, dihydrotestosterone was administered to a separate cohort of female mice on postnatal d 21-23 [prepubertal androgenization (PPA)]. PPA significantly advanced the timing of pubertal onset, as observed by age of the vaginal opening, yet had no effects on testosterone or estrous cycling in adulthood. The absence of kisspeptin receptor in Kiss1r-null mice did not change the acceleration of puberty by the PNA and PPA paradigms, indicating that kisspeptin signaling is not required for androgens to advance puberty. Thus, prenatal, but not prepubertal, exposure to low levels of androgens disrupts normal reproductive function throughout life from puberty to reproductive senescence.