Estrogen receptor regulation of brain sexual differentiation

项目概要/摘要 在有性繁殖的物种中，雄性和雌性表现出不同的社会和生殖行为，例如 交配和攻击这些行为通常不需要训练，表明它们是发育性的。 在大脑中编程。在许多哺乳动物物种中，包括灵长类和啮齿类， 大脑受核受体转录因子（TF）的调节，TF与性腺类固醇激素结合，如 睾丸激素和雌激素。在老鼠中，围产期睾丸激素的激增使一个关键的大脑永久男性化 控制性典型行为的区域，称为终纹床核（BNST）。围产期内 BNST，睾酮转化为雌二醇，激活雌激素受体α（ERα）。既往遗传 基因敲除和药理学研究表明，围产期ERα信号传导是必要的，也是足够的， BNST电路和行为的男性化。ERα激活导致BNST细胞的雄性偏好 存活和轴突引导，特别是前腹侧室周核（AVPV），出生后 第4天（P4）和P10。该项目旨在鉴定和表征ERα基因组结合位点和靶基因 参与BNST性分化。最近，我们的研究小组发现，雌二醇调节 在成年BNST ERα+细胞中netrin受体Unc 5 b的表达。Unc 5 b在新生儿BNST中稳健表达， 先前已经显示出调节神经元存活和轴突引导。本项目的目标1调查 雌二醇对Unc 5 b的调节是否有助于BNST的性分化，使用缺乏Unc 5 b的小鼠 ERα+细胞表达（Esr 1cre/+; Unc 5 blx/lx）。目标1中的实验检验了雌二醇 Unc 5 b的调节有助于雄性偏向的BNST细胞存活和/或AVPV神经支配。因为ERα可能 通过多种生物途径使BNST雄性化，本项目的目标2旨在确定完整的 ERα基因组结合位点和靶基因在发育中的BNST的库。之前，我发现 第一个ERα基因组结合位点在成人大脑中，使用最近发表的低输入TF分析方法，称为 CUT&RUN（切割下的目标和释放下的核酸酶）。这种方法揭示了脑特异性ERα 结合位点富集在参与神经发育过程的基因附近。本申请的目标2使用 CUT&RUN测量围产期BNST/下丘脑中雌二醇调节的ERα结合位点。目标2 测量P4 BNST ERα+细胞中围产期雌二醇调节的基因表达。使用CRISPR介导的 在原代神经元中的CRISPRa激活（CRISPRa）系统中，远端ERα结合位点将与CRISPRa激活系统的激活有因果关系。 围产期雌二醇调节基因的表达。总的来说，该项目将揭示核受体如何调节 基因参与大脑性别分化，并在这样做，将提供新的见解的分子基础 性别偏见的心理健康状况和神经发育疾病。

PROJECT SUMMARY/ABSTRACT In sexually reproducing species, males and females display different social and reproductive behaviors, such as mating and aggression. These behaviors typically require no training, indicating they are developmentally programmed in the brain. In many mammalian species, including primates and rodents, sexual differentiation of the brain is regulated by nuclear receptor transcription factors (TFs), which bind gonadal steroid hormones, such as testosterone and estrogens. In mice, a perinatal surge of testosterone permanently masculinizes a key brain region controlling sex-typical behaviors, called the bed nucleus of the stria terminalis (BNST). Within the perinatal BNST, testosterone is converted to estradiol, which activates estrogen receptor α (ERα). Previous genetic knockout and pharmacological studies demonstrate perinatal ERα signaling is necessary and sufficient for masculinization of BNST circuitry and behavior. Specifically, ERα activation leads to a male-bias in BNST cell survival and axon guidance, particularly to the anteroventral periventricular nucleus (AVPV), between postnatal day 4 (P4) and P10. This project seeks to identify and characterize ERα genomic binding sites and target genes involved in BNST sexual differentiation. Recently our group discovered that estradiol regulates the expression of the netrin receptor, Unc5b, in adult BNST ERα+ cells. Unc5b is robustly expressed in the neonatal BNST and has previously been shown to regulate neuron survival and axon guidance. Aim 1 of this project investigates whether estradiol regulation of Unc5b contributes to BNST sexual differentiation, using mice lacking Unc5b expression in ERα+ cells (Esr1cre/+;Unc5blx/lx). The experiments in Aim 1 test the hypothesis that estradiol regulation of Unc5b contributes to male-biased BNST cell survival and/or AVPV innervation. Because ERα likely masculinizes the BNST through multiple biological pathways, Aim 2 of this project seeks to identify the complete repertoire of ERα genomic binding sites and target genes in the developing BNST. Previously, I discovered the first ERα genomic binding sites in the adult brain, using a recently published low-input TF profiling method, called CUT&RUN (Cleavage Under Targets & Release Under Nuclease). This approach revealed brain-specific ERα binding sites are enriched near genes involved in neurodevelopmental processes. Aim 2 of this application uses CUT&RUN to measure estradiol-regulated ERα binding sites in the perinatal BNST/hypothalamus. Aim 2 also measures perinatal estradiol-regulated gene expression in P4 BNST ERα+ cells. Using a CRISPR-mediated activation (CRISPRa) system in primary neurons, distal ERα binding sites will be causally linked to the expression of perinatal estradiol-regulated genes. Overall, the project will reveal how nuclear receptors regulate genes involved in brain sexual differentiation and, in doing so, will provide novel insight into the molecular basis of sex-biased mental health conditions and neurodevelopmental diseases.