Establishing the roles of oestrogen receptor 1 (ESR1) in olfactory development and function using novel CRISPR/Cas9-based knockouts in the zebrafish
Lead Research Organisation:
UNIVERSITY OF EXETER
Department Name: Biosciences
Abstract
In all animals, the sense of smell (olfaction) is fundamental for sensing the outside world with roles including for feeding, avoiding predators, social interactions, and reproduction. Much of the information for these smell associated behaviours is imprinted during early-life and in humans smell dysfunction is an early indicator of various behavioural disorders, including autism. The ways (mechanisms) through which smell develops in early life to influence subsequent animal behaviours, however, are largely unknown. Recently, we discovered that oestrogens (which are steroid hormones) regulate olfactory development in the embryo brain via a novel cell type which we have named oestrogen responsive olfactory bulb (EROB). In this project we will apply highly novel ways (so called CRISPR-Cas9 methods), developed by our industry partner (AstraZeneca), to remove (knock out) the key oestrogen receptor (called esr 1) in a highly controlled cell-specific and precisely-timed manner. This will help us to identify the role of esr1 in the development of smell and smell-mediated behaviour. The CRISPR-Cas9 methods we will develop will also allow other researchers to study other genes with much greater precision in the zebrafish model.
In this work we will first knock out esr1 in zebrafish in specific brain cells (called glia, which include EROB) in a highly controlled and timed manner to provide the required zebrafish study models. We will then use these zebrafish models to establish what happens to the anatomy of the brain and the neural circuits in a key region of the brain involved in smell (the olfactory bulb) when esr-1 is knocked out. We will do this by analysing brain sections and measuring the different brain cell types, their structural arrangements and the neural circuits they form. We will then cross breed our esr1 knock out zebrafish with another genetically modified zebrafish in which brain neural activity can be visualised via imaging. With this new zebrafish model we will assess the effects of the glial-specific knock out of esr-1 during embryo development on brain activity in response to selected smells using imaging, and in subsequent juveniles and adults through studies on sections of the brain. Finally, we will use behavioural assessments to determine the consequences of knocking out esr1 in EROB cells on smell-mediated behaviours in larval stages, and on social-interaction in both larval and adult animals. We provide significant pilot data supporting our approach that includes showing that esr1 specifically affects the number of EROB cells during development. As a major step in creating a brain cell- specific conditional esr 1 knock out we have also already incorporated key genetic elements into a zebrafish line to facilitate this. Furthermore, we have established an imaging system which allows us to image neural activity in the whole brain, in real time.
Our research will be of significant interest to a diverse audience including academic and industry researchers, and the medical profession, by providing new models to study smell and the roles of oestrogens in brain development and function. Our project will advance genomic editing tools for the research community relevant to anyone studying genes and their function in the zebrafish model. It will also be of great interest to industry and government regulatory bodies, as the models developed, for example, could be applied for advancing the risk assessment of chemicals with oestrogenic activity, supporting evidence-based decision-making for those chemicals. The wider public will benefit also from this research from improved understanding of basic life processes associated with smell, a sense fundamental to animal (including human) life.
In this work we will first knock out esr1 in zebrafish in specific brain cells (called glia, which include EROB) in a highly controlled and timed manner to provide the required zebrafish study models. We will then use these zebrafish models to establish what happens to the anatomy of the brain and the neural circuits in a key region of the brain involved in smell (the olfactory bulb) when esr-1 is knocked out. We will do this by analysing brain sections and measuring the different brain cell types, their structural arrangements and the neural circuits they form. We will then cross breed our esr1 knock out zebrafish with another genetically modified zebrafish in which brain neural activity can be visualised via imaging. With this new zebrafish model we will assess the effects of the glial-specific knock out of esr-1 during embryo development on brain activity in response to selected smells using imaging, and in subsequent juveniles and adults through studies on sections of the brain. Finally, we will use behavioural assessments to determine the consequences of knocking out esr1 in EROB cells on smell-mediated behaviours in larval stages, and on social-interaction in both larval and adult animals. We provide significant pilot data supporting our approach that includes showing that esr1 specifically affects the number of EROB cells during development. As a major step in creating a brain cell- specific conditional esr 1 knock out we have also already incorporated key genetic elements into a zebrafish line to facilitate this. Furthermore, we have established an imaging system which allows us to image neural activity in the whole brain, in real time.
Our research will be of significant interest to a diverse audience including academic and industry researchers, and the medical profession, by providing new models to study smell and the roles of oestrogens in brain development and function. Our project will advance genomic editing tools for the research community relevant to anyone studying genes and their function in the zebrafish model. It will also be of great interest to industry and government regulatory bodies, as the models developed, for example, could be applied for advancing the risk assessment of chemicals with oestrogenic activity, supporting evidence-based decision-making for those chemicals. The wider public will benefit also from this research from improved understanding of basic life processes associated with smell, a sense fundamental to animal (including human) life.
Technical Summary
In this project, new CRISPR Cas9 genome editing methods, combined with advanced imaging methods and studies on behaviour, will be applied to understand the role of oestrogen in the development and function of the olfactory system. The work will advance cell-type specific conditional gene KO in zebrafish, with huge benefits for the study of gene function generally in zebrafish.
We will first knock out esr1 in zebrafish using 3 different CRISPR-Cas9 mediated methods, specifically: non-conditional KO (esr1-KO1); oestrogen receptor olfactory bulb (EROB)-specific esr1-KO by driving esr1 CRISPR guide RNA under control of the glia specific promoter, gfap (esr1-KO2); and, glia- and developmental-stage specific esr1-KO using the inducible UbiDInCas9 system (esr1 -KO3) knocking out esr-1 in brain glia cells during embryonic, juvenile and adult life stages through exposure of the esr1 -KO3 line to doxycycline.
We will then use the esr1-KO3 line to assess the impacts of glia- and life stage-specific esr1-KO on the anatomy and circuitries of the olfactory bulb (OB) using brain immunohistochemistry (cryosection and/or whole mount) with antibodies to study synapses and cell types, in combination with LSM880 airyscan confocal microscopy.
We will then cross the esr1 KO lines (esr1-KO-1, KO-2 and KO-3) with a pan neuronal Ca2+reporter line, elavl3:GCaMP6s and use live-Ca2+ imaging to visualise whole brain neuronal activity in response to odourants in the embryos via light sheet microscopy, and in the subsequent juvenile and adult life stages via phospho-ERK staining on brain cryosections, to establish the functional consequences of esr1-KO in EROB cells on odour-evoked brain activity.
Finally, we will determine the functional consequences of esr1-KO in EROB cells on olfaction-mediated behaviour by exposing the fish at the different life stages to alarm and attractant odourants and quantify behaviourmeasures for anxiety and social interaction via automated video tracking
We will first knock out esr1 in zebrafish using 3 different CRISPR-Cas9 mediated methods, specifically: non-conditional KO (esr1-KO1); oestrogen receptor olfactory bulb (EROB)-specific esr1-KO by driving esr1 CRISPR guide RNA under control of the glia specific promoter, gfap (esr1-KO2); and, glia- and developmental-stage specific esr1-KO using the inducible UbiDInCas9 system (esr1 -KO3) knocking out esr-1 in brain glia cells during embryonic, juvenile and adult life stages through exposure of the esr1 -KO3 line to doxycycline.
We will then use the esr1-KO3 line to assess the impacts of glia- and life stage-specific esr1-KO on the anatomy and circuitries of the olfactory bulb (OB) using brain immunohistochemistry (cryosection and/or whole mount) with antibodies to study synapses and cell types, in combination with LSM880 airyscan confocal microscopy.
We will then cross the esr1 KO lines (esr1-KO-1, KO-2 and KO-3) with a pan neuronal Ca2+reporter line, elavl3:GCaMP6s and use live-Ca2+ imaging to visualise whole brain neuronal activity in response to odourants in the embryos via light sheet microscopy, and in the subsequent juvenile and adult life stages via phospho-ERK staining on brain cryosections, to establish the functional consequences of esr1-KO in EROB cells on odour-evoked brain activity.
Finally, we will determine the functional consequences of esr1-KO in EROB cells on olfaction-mediated behaviour by exposing the fish at the different life stages to alarm and attractant odourants and quantify behaviourmeasures for anxiety and social interaction via automated video tracking
