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 ? (ER?). Previous genetic knockout and pharmacological studies demonstrate perinatal ER? signaling is necessary and sufficient for masculinization of BNST circuitry and behavior. Specifically, ER? 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 ER? 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 ER?+ 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 ER?+ 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 ER? likely masculinizes the BNST through multiple biological pathways, Aim 2 of this project seeks to identify the complete repertoire of ER? genomic binding sites and target genes in the developing BNST. Previously, I discovered the first ER? 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 ER? binding sites are enriched near genes involved in neurodevelopmental processes.
Aim 2 of this application uses CUT&RUN to measure estradiol-regulated ER? binding sites in the perinatal BNST/hypothalamus.
Aim 2 also measures perinatal estradiol-regulated gene expression in P4 BNST ER?+ cells. Using a CRISPR-mediated activation (CRISPRa) system in primary neurons, distal ER? 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.

Public Health Relevance

While steroid hormones, such as estrogens, play a conserved role in sexual differentiation of mammalian brain and behavior, little is known about their influence on molecular processes in the developing brain. This study aims to identify molecular mechanisms by which estrogen receptor ? (ER?) regulates sexual differentiation of the mouse bed nucleus of the stria terminalis (BNST) ? a region controlling sex-typical social behaviors. By identifying sex differences in circuit wiring and ER? gene regulation during a critical developmental window, this study will provide novel insight into the pathophysiology of sex-biased mental health conditions and neurodevelopmental diseases, such as anxiety and autism spectrum disorder.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
1F31MH124365-01
Application #
10067180
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Driscoll, Jamie
Project Start
2020-09-01
Project End
2023-08-31
Budget Start
2020-09-01
Budget End
2021-08-31
Support Year
1
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Cold Spring Harbor Laboratory
Department
Type
DUNS #
065968786
City
Cold Spring Harbor
State
NY
Country
United States
Zip Code
11724