Estrogen receptor regulation of brain sexual differentiation

项目摘要/摘要 在有性繁殖物种中，雄性和雌性表现出不同的社会和生殖行为，例如 交配和攻击性。这些行为通常不需要训练，这表明它们处于发育阶段。 在大脑中进行编程。在包括灵长类和啮齿动物在内的许多哺乳动物物种中， 大脑受核受体转录因子的调节，核受体转录因子与性腺类固醇激素结合，如 作为睾丸素和雌激素。在小鼠中，围产期睾丸激素的激增永久地使关键大脑男性化。 控制性典型行为的区域，称为终纹床核(BNST)。在围产期内 BNST，睾酮转化为雌二醇，雌二醇激活雌激素受体α(ERα)。以前的基因 基因敲除和药理学研究表明，围产期ERα信号是必要和充分的 BNST电路和行为的男性化。具体地说，ERα的激活导致BNST细胞中的男性偏见 存活和轴突指导，特别是对前腹侧脑室周围核(AVPV)的指导 第4天(P4)和第10天。该项目旨在确定和表征ERα基因组结合位点和靶基因 参与BNST的性别分化。最近，我们的研究小组发现，雌二醇能调节 在成体BNST ERα细胞中表达Netrin受体Unc5b。Unc5b在新生儿BNST和 此前已被证明可调节神经元存活和轴突引导。此项目的目标1调查 在缺乏Unc5b的小鼠中，雌激素对Unc5b的调节是否有助于BNST的性别分化 在ERα细胞中表达(Esr1cre/；Unc5blx/Lx)。Aim 1中的实验验证了雌二醇的假设 Unc5b的调节有助于偏向男性的BNST细胞存活和/或AVPV神经支配。因为ERα很可能 通过多个生物途径使BNST男性化，本项目的目标2寻求识别完整的 发育中的BNST中的ERα基因组结合位点和靶基因的谱系。之前，我发现了 使用最近发表的一种低输入转铁蛋白分析方法，在成人大脑中首次发现ERα基因组结合位点，该方法称为 切割和运行(在目标下切割，在核酸酶下释放)。这一方法揭示了大脑特异的ERα 结合位点在参与神经发育过程的基因附近丰富。此应用程序的目标2用于 切割和运行以测量围产期BNST/下丘脑中雌激素调节的ERα结合位点。AIM 2也 检测围产期雌二醇对P4 BNST ERα细胞基因表达的影响。使用CRISPR中介的 在初级神经元的激活(CRISPRA)系统中，远端的ERα结合位点将与 围产期雌二醇调节基因的表达。总体而言，该项目将揭示核受体如何调节 参与大脑性别分化的基因，通过这样做，将提供对分子基础的新见解 有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.