Functional role(s) of oestrogen signalling on neuronal progenitor cell development and fate in the brain

Lead Research Organisation: University of Exeter
Department Name: Biosciences

Abstract

Nerve cells (called neurons) form in the brain principally during embryo development but they can also be synthesised in the adult brain. Defects in the process of neuron synthesis (neurogenesis) have been linked with various brain diseases, psychiatric illnesses and addictions. Neurogenesis is also critically involved in the repair of an injured brain. One type of cell, called radial glial cells (RGCs) can give rise to all types of neurons in the cerebral cortex of the brain but how this is controlled is not well understood. Recently, hormones, called oestrogens, have been indicated to play important roles in RGCs and in neurogenesis.

In this project we will apply genetically engineered zebrafish models to investigate the roles of oestrogen in neurogenesis and brain development, investigating also for sex related differences. We are using zebrafish for this work because of the relative ease to create transgenic animals and the fact that there is a high neurogenic activity in the brain throughout life.

We have already successfully developed one genetically engineered (transgenic) zebrafish in which cells responsive to oestrogen produce a green fluorescent protein (GFP) that can be detected using imaging methods in living animals and in real time. Using this model we will establish the role of oestrogens in the RGCs in the telecephalon and identify the different cells in the brain responsive to oestrogens and how they develop. We will also apply a method called two-photon microscopy to selectively remove the oestrogen responsive RGCs in the telecephalon and investigate subsequent effects on brain development to help establish their function.

We will then develop a new transgenic zebrafish model with a different genetic insert that prolongs the green fluorescent signal generated in response to oestrogen exposure. This will enable us to follow the fate of the oestrogen responsive RGCs and other neural cells of early life in the brain of juvenile and adult fish. We will also use this model to investigate for differences that occur between males and females.

We will develop a further, more complex (triple), transgenic zebrafish that will enable us to identify activity in oestrogen responding neurons. This model includes two fluorescent colours, red identifying oestrogen responsive neurons and green identifying when these cells are active (produced in response to increased levels of calcium in the cell). Using this fish we will investigate the roles of oestrogen responding neurons in smell, including assessing for sex related differences. We will also apply a technique to block oestrogen action responsive neurons in the telecephalon during early life to establish how this affects their subsequent development and function in smell.

Applying these transgenic models, finally we will initiate studies into the effects of exposure to so called endocrine disrupting chemicals (EDCs), for which there is widespread health concern, on oestrogen responding neurons and their subsequent fate and function.

The research will be significant interest to a diverse audience including academic and industry researchers, and the medical profession, by provision of new models to study neurogenesis and the roles of an important group of environmental chemicals on human health. It will also be of strong interest to industry and government regulatory bodies, as the models develop could be applied for advancing 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 neurological conditions and better evidence of chemical effects that may affect their own health. The models developed will provide more integrative systems for chemical effects analysis with great potential for reducing the numbers of vertebrate animals used in testing.

Technical Summary

We will apply an oestrogen responsive transgenic zebrafish model (developed at Exeter) and develop new transgenic models to investigate the roles of oestrogen in neurogenesis and brain development, investigating for sex related differences, including in intact animals in real time using advanced imaging methods.

Specifically we will:

1.) Apply our current transgenic zebrafish model (ERE- GFP) to establish the role of oestrogens in telencephalic radial glial cell (RGC) differentiation and function, identifying the different cell types in the brain responsive to oestrogens, their ontogeny, sensitivity and behaviours. The work will include ablation of oestrogen responsive neurones during early life using two-photon microscopy.

2.) Develop a new transgenic zebrafish model [ERE_Gal4ff x UAS_GFP_KalTA4] and use this to follow the fate and behaviour of oestrogen responsive RGCs and other neural cells in juvenile and adult fish brains, investigating also for differences between the sexes.

3.) Create a further transgenic zebrafish [ERE_Gal4ff x UAS_ mCherry x UAS _GCaMP3] to identify neuronal activity in oestrogen responsive neurons. We will investigate the spatio-temporal activation of individual oestrogen responding neurons in the telecephalon associated with olfaction, including assessing for sex related differences. Oestrogen receptor morpholino knockdown will be applied to assess how oestrogen signal interference during early life affects subsequent telecephalon oestrogen responsive cell development and neuronal activity for olfactory stimuli.

4.) Apply our ERE_Gal4ff x UAS_GFP_KalTA4 transgenic model to investigate effects of exposure to a series of endocrine disrupting chemicals (EDCs) on oestrogen responding neuronal progenitors and their subsequent fate. Also examine for effects of these EDCs on the neuronal activity of oestrogen sensitive telencephalic cells using the ERE_Gal4ff x UAS_mCherry x UAS _GCaMP3 transgenic line.

Planned Impact

We expect our research findings and the new zebrafish models we develop will be of significant interest to academic and medical researchers working in similar or related fields by providing new zebrafish models for studies on understanding brain development and function and the roles of an important group of environmental (oestrogenic) chemicals on human health.The work will aso benefit industry researchers by providing new models for advancing risk assessment of chemicals with oestrogenic activity. Industry, government and government regulatory bodies may benefit from the exploitation of these models through better evidence-based decision-making for chemicals with oestrogenic action. The wider public will benefit from improved understanding of neurological conditions and better evidence of chemical effects that may affect their own health.

Major impact activities will be centred on delivering high quality science, training, engagement with potential industry (and other) partners, and informing the wider public.

Results of this work are expected to make a significant scientific impact and major routes for dissemination will be via relevant ISI scientific journals with open access, and national and international conferences and workshops. This will include a workshop we will run through the trans-channel LARC-neurosciences network (larc.neurosciences.org) and dedicated to supporting uptake of zebrafish models into other laboratories. The project team has a very strong track record in international research collaboration.

Tyler has research associations with large organizations that include AstraZeneca, Syngenta, UK water companies, the Chemical Manufacturers Association, and various SMEs, who may seek to exploit the models we develop in this project. This is evidenced through a recently awarded BBSRC FLIP to Tyler and Kudoh with AstraZeneca to apply our oestrogen sensitive transgenic zebrafish to screen for oestrogenic activity of pharmaceuticals and facilitate molecular technology transfer. We will gain direct support on commercialisation opportunities for our new models, and be provided with advice on the protection of IP, through the University's Research & Knowledge Transfer section. CRT/TK has partnered KEP awards and has 5 current CASE (2 BBSRC) studentships, including for the development of new transgenic fish models. We will balance any possible commercial exploitation opportunities with our desire to make the new models widely available to academic researchers.

We have a strong record in public engagement activities. The PI, Co-I and research co-I and other lab members will contribute to primary school visits and public University open days during the study, continuing current activity. These presentations will include how fundamental bioscience can be translated into applications that benefit us all and affects our daily lives. We will, with support from our Press Office, ensure that results are disseminated from our work in order to maximise publicity opportunities, in agreement with the policies of the BBSRC. Our work on endocrine disruption and zebrafish models has received considerable media attention and has featured in the national and international press, on television, and has been presented to The Royal Society, the House of Commons, the European Commission, and more globally via web bases (e.g. Webinar, Harvard University) and u-tube presentations. In 2011, Tyler's team's research won the University Exeter's Outstanding Achievement Award for its contribution to Policy and Public Services.

The PI will be responsible for ensuring that impact activities are conducted, but Dr Takesono (Research co-I) will be expected to contribute substantially to these activities. Dr Takesono will receive training in a wide range of science disciplines, including interaction with industry, policy makers and the wider public, and in communicating science, enhancing her employment prospects.

Publications

10 25 50

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Lee O (2015) Transgenic fish systems and their application in ecotoxicology. in Critical reviews in toxicology

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MOURABIT S A novel transgenic biosensor zebrafish for detecting oxidative stress in Environmental Health Perspectives

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Ogino Y (2018) Functionalization of the sex steroid hormone receptors ESR and AR in the regulation of diverse characteristics. in Journal of Steroid Biochemistry and Molecular Biology

 
Description Work package 1: Investigate the role of oestrogens in neuronal progenitor cells differentiation in the telencephalon.
Using our ERE-GFP zebrafish and imaging methods, we have established detailed ontogenic profiles of oestrogen responsive neuronal progenitors (ER-forebrain cells) for developmental stages of embryos/larvae up to 30 days post fertilisation. Via immune-staining we have confirmed that the ER-forebrain cells are SOX2 (a neuronal progenitor marker), aromatase B (a brain specific estrogen synthesising enzyme expressing in radial glia cells) and GFAP (a glia marker) positive. We have shown that the earliest ER-forebrain cells occur from 28 hours post fertilisation (28 hpf) when they are located in the most dorsal layer of the olfactory bulbs. They subsequently increase in number and extend towards the posterior part of telencephalon up until 14 days post fertilisation (14 dpf). After this life stage they become reduced in number (by 21 dpf). We have shown that at 10-14dpf there are two distinct groups of ERE-GFP larvae, that have either high or low numbers of ER-forebrain cells, and we are currently testing if this difference in the numbers of ER-forebrain cells relates to sex. Through histological analysis of the gonads of adults resulting from the larvae with either high or low numbers of ER-forebrain cells at 10-14dpf we will be able to establish whether these differences are encoded for (encode for) sex.

We planned originally to develop a laser cell ablation system using multiphoton microscope to investigate the roles of ER-forebrain cells in forebrain development. In this project however, we have adopted a more refined approach to ablate ER-forebrain cells, choosing instead to use a chemical/genetic approach, which utilises nitroreductase-mediated in vivo cell ablation, and sought to optimise the conditions for cell ablation (currently, we are able ablate around 50% of the ER-forebrain cells with no other obvious adverse animal effects). Our findings to date in this work indicate that the ablation of the ER-forebrain cells affects the spatial localisation and volume of olfactory glomeruli as well as axonal pathfinding of olfactory sensory neurons. These data indicate that ER-forebrain cells in the olfactory bulb play an important role in the olfactory bulb development and development/ differentiation of associated olfactory sensory neurons.

Work package 2: Develop a new transgenic zebrafish model (ERE_Gal4ff x UAS_GFP_KalTA4) to study the fate of oestrogen responsive neuronal progenitors throughout life and establish sex related differences.

We generated ERE_Gal4ff x UAS_GFP_KalTA4 line as set out in our original proposal. However, in this line we found that the original tissue specificities for oestrogen responses in the liver, muscle, heart and forebrain were not consistent with the original ERE-GFP line. Rather, the ERE_Gal4ff x UAS_GFP_KalTA4 line showed oestrogen independent spontaneous expression of GFP in other parts of the brain including the habenula, retina, and hindbrain. This was an unexpected result and may have resulted from a compatibility issue between the ERE and KalTA4. We have overcome this problem through generating a completely new ERE_Gal4ff x UAS_Cre x Ubi_Zebrabow line which allows for permanent multicolour single cell labelling of oestrogen responding cells. Using the ERE_Gal4f x Ubi_Zebrabow we are currently analysing the cell fate of oestrogen responding cells. To explore the possibility if ER-forebrain cells are involved with/contribute to sex differentiation, we are testing for associations between the numbers of ER-forebrain cells (at 10-14 dpf) and sex differentiation, as described in work package 1.

Work package 3: Develop a second new, triple, transgenic zebrafish model (ERE_Gal4ff x UAS_mCherry x UAS _GCaMP3) to identify neuronal activity in oestrogen responsive neurons and investigate sex related differences.
To visualise neuronal activity of ER-forebrain cells in real-time, we have successfully established an ERE_Gal4ff x UAS_mCherry x UAS_GCaMP6s line (a modified/latest version of UAS_GCaMP line) which will allows us to detect neuronal responses in the target cells of interest. We are examining the neuronal activities of the identified ER-forebrain cells in response to amino acid mixture using light-sheet microscopy. We have recently established and optimised the imaging system for detection and quantification of GCaMP6 signals in zebrafish larvae and the ImageJ macro/ python pipelines for quantification of these for responses (Winter et al., 2017; 4-Dimensional functional profiling of in the convulsant treated larval zebrafish brain. Scientific Reports. DOI:10.1038/s41598-017-06646-6 ).


Work package 4: Initiate studies into the effects of exposure to oestrogen-mimicking chemicals on neuronal progenitor cell development and function.
We have tested the effects of two EDCs, bisphenol A (BPA) and nonlyphenol (NP) on our transgenic zebrafsh models and found that ER-forebrain cells are responsive to these environmental oestrogens for early life exposures. NP, however, unlike for BPA, induce alterations in ER-forebrain cell development, markedly inhibiting the establishment of their projection networks, with also ectopic distributions of the cell bodies. We have shown that these effects for NP are dose-dependent, with effects at exposure concentrations from 11.4mg NP/L in the water. NP did not alter the spatiotemporal expression of olfactory output neuron marker, lhx2 or tbx21, but impaired the innervation of ER-forebrain cells to lhx2 and tbx21 positive cells. We also found that NP markedly reduced the size of axonal termini of hemocyanin positive olfactory input neurons at glomeruli, as well as the size of the
olfactory bulb/forebrain. To further investigate the adverse effect of NP in olfactory-mediated neuronal activation and behaviours, we are currently testing if NP exposure in early life could affect olfactory-mediated neuronal activity (pERK staining) and odour-evoked behaviour, i.e. Kin, skin extracts, food extracts, cadaverine-mediated preference test in later life (juvenile and adult). These findings highlight that NP exposure during early life has the potential to alter forebrain development, adding further concern about exposure to this EDC for wildlife (and human) health.
Exploitation Route Provision of new transgenic fish models and tools or studies into brain development and function.
Sectors Chemicals,Environment,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Using new transgenic zebrafish lines generated in this project, we have discovered a novel estrogen-mediated mechanism modulating spontaneous neuronal activity in the olfactory bulbs (OBs) during embryo development. We demonstrated that, in the zebrafish embryo, estrogen inhibits spontaneous neuronal activity specifically in the olfactory bulbs (OB) through novel estrogen responding glial progenitor cells (namely, Estrogen Responding Olfactory Bulb cells: EROB cells) which are found exclusively in the OBs during the early onset of brain development. We show that EROB cells are critically involved in OB development by regulating the axonal pathfinding of olfactory sensory neurons in the OBs. We also show altering estrogen signalling pathways at an early embryonic stage causes a significant defect in olfaction-mediated fear response. These observations emphasize a fundamental importance of estrogen in olfactory development and functions, and highlight potential influences of Neuronal endocrine disrupting chemicals in olfactory-mediated brain functions and behaviour in animals. The information generated from this project has been disseminated widely at international meetings. Furthermore, we are at an advanced stage in preparing two research papers from this work for publication in prominent journals (Neuron, eLife) with submissions due imminently.
Sector Chemicals,Environment,Pharmaceuticals and Medical Biotechnology
 
Description DEFRA BBSRC FLIP
Geographic Reach Europe 
Policy Influence Type Participation in a national consultation
 
Description EU Commission
Geographic Reach Europe 
Policy Influence Type Influenced training of practitioners or researchers
Impact This research project has contributed to the validation process for use of transgenic zebrafish in a new OECD guideline test protocol, in a collaboration developed with Dr Francois Brion , INERIS, France..
 
Description Ministry of Environment for Japan
Geographic Reach Asia 
Policy Influence Type Participation in a advisory committee
Impact This research project has contributed to the validation process for use of transgenic zebrafish in a new OECD guideline test protocol.
 
Description 2014-17. BBSRC Industrial CASE studentship with AstraZeneca(PhD - Dylan Windell). Tracing nanodrugs in aquatic organisms using light sheet microscopy.
Amount £80,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start  
 
Description AstraZeneca Contract Research: zebrafish development assays
Amount £80,000 (GBP)
Organisation AstraZeneca 
Sector Private
Country United Kingdom
Start 01/2018 
End 12/2018
 
Description BBSRC China - UK Partnering Award: Breeding and health for improving robustness in aquaculture.
Amount £42,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2019 
End 01/2020
 
Description BBSRC Industry CASE studentship - Molly Payne
Amount £80,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start  
 
Description BBSRC Japan Partnering Award - Engineering novel transgenic zebrafish with CRISPR/Cas9 technology
Amount £42,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2017 
End 08/2020
 
Description Developing Alternative Models to Evaluate the Impact of Nanomaterials on Neutrophils during the Stimulation and Resolution of Inflammation.
Amount £344,251 (GBP)
Funding ID NC/P001149/1 
Organisation National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) 
Sector Private
Country United Kingdom
Start 11/2016 
End 10/2026
 
Description Effects of water chemistry on the exposure risk for pharmaceutical drugs, including antibiotics
Amount £80,000 (GBP)
Organisation AstraZeneca 
Sector Private
Country United Kingdom
Start 10/2018 
End 09/2022
 
Description Industry CASE (PhD) - Advancing transgenic zebrafish bioassays for drug analysis using imaging
Amount £100,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2018 
End 12/2021
 
Description Merck - Integrated health effects analysis of SSRIs
Amount £75,000 (GBP)
Organisation Merck 
Department Merck UK
Sector Private
Country United Kingdom
Start 10/2017 
End 09/2020
 
Description NC3Rs/Simonics The human iDART framework
Amount £43,000 (GBP)
Organisation National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) 
Sector Private
Country United Kingdom
Start 01/2018 
End 06/2018
 
Description PhD studentship - An alternative approach for assessing drug-induced seizures, using non-protected larval zebrafish
Amount £100,000 (GBP)
Funding ID NC/R001421/1 
Organisation National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) 
Sector Private
Country United Kingdom
Start 05/2018 
End 04/2021
 
Description Single Molecule Detection Platform for a Leica SP8 TCS
Amount £275,399 (GBP)
Funding ID BB/R013764/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2018 
End 01/2018
 
Title Applied our current transgenic zebrafish models to establish the role of oestrogens in differentiation and function of ER-forebrain cells 
Description We applied our current transgenic zebrafish models (ERE- GFP, ERE-mCherry and ERE-Kaede and ERE-mCherry x Cyp19-GFP) to establish the role of oestrogens in differentiation and function of ER-forebrain cells. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact Identified the different cell types in the brain responsive to oestrogens, their ontogeny, sensitivity and behaviours. The work includes ablation of oestrogen responsive neurones during early life usingnitroreductase-mediated cell specific ablation.. we have discovered a new and earliest target of oestrogen in developing zebrafish; glial progenitor cells in the forebrain that exclusively respond to oestrogen (named ER-forebrain cells) and revealed the roles of these ER-forebrain cells in forebrain development and the associated neuronal networks 
 
Title Currently developing a [ERE:Gal4ff] x [UAS:Cre] x [Ubi:zebrabow] transgenic line, which enables permanent multispectral cell labelling in response to estrogens, to trace the fate of estrogen responsive forebrain cells throughout the life. 
Description Currently developing a [ERE:Gal4ff] x [UAS:Cre] x [Ubi:zebrabow] transgenic line, which enables permanent multispectral cell labelling in response to estrogens, to trace the fate of estrogen responsive forebrain cells throughout the life. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
Impact n/a 
 
Title Developed ImageJ macros which allow us to measure the fluorescent intensity in a variety of tissues in our transgenic fish in response to stressors that induce oxidative stress via the EpRE. 
Description Developed ImageJ macros which allow us to measure the fluorescent intensity in a variety of tissues in our transgenic fish in response to stressors that induce oxidative stress via the EpRE. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Developed ImageJ macros which allow us to measure the fluorescent intensity in a variety of tissues in our transgenic fish in response to stressors that induce oxidative stress via the EpRE. 
 
Title Establish odour-evoked behaviour assay (olfaction preference tests) using a video tracking system (ViewPoint) 
Description Establish odour-evoked behaviour assay (olfaction preference tests) using a video tracking system (ViewPoint) 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact Examined the consequences of altering oestrogen signalling pathway (i.e. by ablation of ER-forebrain cells or by an oestrogen antagonist ICI182780 ) in olfactory-mediated behaviours. 
 
Title Established ERE-mCherry transgenic line in casper mutant zebrafish background, which possesses a better photostability compared to GFP, for studies into the roles of oestrogen responsive cells in brain development. 
Description Established ERE-mCherry transgenic line in casper mutant zebrafish background, which possesses a better photostability compared to GFP, for studies into the roles of oestrogen responsive cells in brain development. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
Impact A new tool for studying brain development and function. 
 
Title Established [ERE:mCherry] x [Cyp19a1b:GFP] double transgenic line which responds to estrogen and expresses two fluorescent colours derived from two estrogen responsive promoters (synthetic ERE promoter with mCherry reporter and cyp19a1b promoter with GFP reporter). 
Description Established [ERE:mCherry] x [Cyp19a1b:GFP] double transgenic line which responds to estrogen and expresses two fluorescent colours derived from two estrogen responsive promoters (synthetic ERE promoter with mCherry reporter and cyp19a1b promoter with GFP reporter). 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
Impact n/a 
 
Title Established an appropriate live imaging system to monitor the development of estrogen responsive forebrain cells in live zebrafish larvae (<5day post fertilization) using confocal microscopy, that includes optimization of sample mounting, anaethetic treatment and the conditions of the laser settings. These skills are essential to investigate the details in the processes and dynamics and ontogeny of estrogen responsive brain cells. 
Description Established an appropriate live imaging system to monitor the development of estrogen responsive forebrain cells in live zebrafish larvae (<5day post fertilization) using confocal microscopy, that includes optimization of sample mounting, anaethetic treatment and the conditions of the laser settings. These skills are essential to investigate the details in the processes and dynamics and ontogeny of estrogen responsive brain cells. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact None as yet 
 
Title Established appropriate conditions for whole mount antibody stainings using PFA fixed zebrafish larvae, which includes conditions for PFA fixation, permeabilisation with Proteinase K and incubation of the samples with various antibodies. 
Description Established appropriate conditions for whole mount antibody stainings using PFA fixed zebrafish larvae, which includes conditions for PFA fixation, permeabilisation with Proteinase K and incubation of the samples with various antibodies. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
Impact n/a 
 
Title Established imaging based screening system for homozygous ERE-mCherry/GFP fish. 
Description Established imaging based screening system for homozygous ERE-mCherry/GFP fish. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
Impact n/a 
 
Title Established the live imaging of spontaneous and odour-mediated neuronal activity in elav3-GCamp6s transgenic zebrafish and ERE_Gal4ff x UAS_NRT_mCherry x UAS_GCaMP6m 
Description Established the live imaging of spontaneous and odour-mediated neuronal activity in elav3-GCamp6s transgenic zebrafish and ERE_Gal4ff x UAS_NRT_mCherry x UAS_GCaMP6m using a light sheet microscope 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact . These systems have allowed us to examine how the alteration of oestrogen signalling during early embryo development, i.e., +/- oestrogen (EE2), an oestrogen antagonist (ICI182780) and ER-forebrain cell ablation, can affect the neuronal activities in different brain regions. 
 
Title Immuno-staining of the markers for olfactory sensory neurons and olfactory glomeruli 
Description Applied immuno-staining of the markers for olfactory sensory neurons and olfactory glomeruli (synaptic termini of the olfactory sensory neurons) 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact Applied immuno-staining of the markers for olfactory sensory neurons and olfactory glomeruli (synaptic termini of the olfactory sensory neurons) to investigate the oestrogen functions in olfactory development and olfactory neuronal circuitry. 
 
Title Immunohistochemical analyses using pERK specific antibody 
Description conducted immunohistochemical analyses for neuronal activation in zebrafish brain using pERK specific antibody 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact To examine the oestrogen functions in odour-mediated neuronal activation. 
 
Title To examine neuronal activity of estrogen responsive brain cells, we aim to generate [ERE:Gal4ff] x [UAS:mCherry]x[UAS:GCamp6f,m,s] by natural crossing 
Description To examine neuronal activity of estrogen responsive brain cells, we aim to generate [ERE:Gal4ff] x [UAS:mCherry]x[UAS:GCamp6f,m,s] by natural crossing 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
Impact n/a 
 
Title Transgenic Zebrafish models (x3) 
Description 1..) Developed a new transgenic zebrafish model [ERE_Gal4ff x UAS_Cre x Ubi_zebrabow] 2..) created a further transgenic zebrafish [ERE_Gal4ff x UAS_ mCherry x UAS _GCaMP6m] 3..) applied our ERE_Gal4ff x UAS_GFP transgenic model 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact 1) Follows the fate and behaviour of oestrogen responsive RGCs in single cell level in larvae and juvenile zebrafish fish brains, investigating also for differences between the sexes. 2) Identified neuronal activity in oestrogen responsive neuronal cells. 3) Investigated effects of exposure to a series of endocrine disrupting chemicals (EDCs) on oestrogen responding neuronal progenitors. 
 
Title Validated proof of concept for a transgenic zebrafish model (i.e. oestrogen responsive ERE-GFP zebrafish) as a reliable, rapid and integrated tool for screening chemical products for endocrine activity in human and environmental safety assessment. 
Description Validated proof of concept for a transgenic zebrafish model (i.e. oestrogen responsive ERE-GFP zebrafish) as a reliable, rapid and integrated tool for screening chemical products for endocrine activity in human and environmental safety assessment. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2015 
Provided To Others? Yes  
Impact Validated proof of concept for a transgenic zebrafish model (i.e. oestrogen responsive ERE-GFP zebrafish) as a reliable, rapid and integrated tool for screening chemical products for endocrine activity in human and environmental safety assessment. 
 
Title oestrogen responsive transgenic zebrafish model 
Description At Exeter, we have applied an oestrogen responsive transgenic zebrafish model and also developed new transgenic models to investigate the roles of oestrogen in neurogenesis and brain development, investigating for sex related differences, including in intact animals in real time using advanced imaging methods. Specifically we have: 1.) applied our current transgenic zebrafish models (ERE- GFP, ERE-mCherry and ERE-Kaede and ERE-mCherry x Cyp19-GFP) to establish the role of oestrogens in differentiation and function of ER-forebrain cells, identifying the different cell types in the brain responsive to oestrogens, their ontogeny, sensitivity and behaviours. The work includes ablation of oestrogen responsive neurones during early life using nitroreductase-mediated cell specific ablation.. 2.) developed a new transgenic zebrafish model [ERE_Gal4ff x UAS_Cre x Ubi_zebrabow] to follow the fate and behaviour of oestrogen responsive forebrain cells in single cell level in larvae and juvenile zebrafish fish brains, investigating also for differences between the sexes. 3.) created a further transgenic zebrafish [ERE_Gal4ff x UAS_ mCherry x UAS _GCaMP6m] to identify neuronal activity in oestrogen responsive neuronal cells. 4.) applied our ERE_Gal4ff x UAS_GFP transgenic model to investigate effects of exposure to a series of endocrine disrupting chemicals (EDCs) on oestrogen responding neuronal progenitors. 5) conducted immunohistochemical analyses for neuronal activation in zebrafish brain using pERK specific antibody to examine the oestrogen functions in odour-mediated neuronal activation. 6) Applied immuno-staining of the markers for olfactory sensory neurons and olfactory glomeruli (synaptic termini of the olfactory sensory neurons) to investigate the oestrogen functions in olfactory development and olfactory neuronal circuitry. 7) Established the live imaging of spontaneous and odour-mediated neuronal activity in elav3-GCamp6s transgenic zebrafish and ERE_Gal4ff x UAS_NRT_mCherry x UAS_GCaMP6m using a light sheet microscope. These systems have allowed us to examine how the alteration of oestrogen signalling during early embryo development, i.e., +/- oestrogen (EE2), an oestrogen antagonist (ICI182780) and ER-forebrain cell ablation, can affect the neuronal activities in different brain regions. 8) Establish odour-evoked behaviour assay (olfaction preference tests) using a video tracking system (ViewPoint) to examine the consequences of altering oestrogen signalling pathway (i.e. by ablation of ER-forebrain cells or by an oestrogen antagonist ICI182780) in olfactory-mediated behaviours. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2019 
Provided To Others? Yes  
Impact ?? 
 
Description Collaborations with University college London and obtaining UAS-GCamp6f,m and s lines. 
Organisation University College London
Department Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Sharing of knowledge on transgenic zebrafish systems
Collaborator Contribution provision of UAS-GCamp6f, m and s zebrafish lines.
Impact Joint publication in preparation
Start Year 2014
 
Description Japan Partnering Award 
Organisation University of Yamanashi
Country Japan 
Sector Academic/University 
PI Contribution This project has helped to obtain a Japan Partnering Award: Engineering novel transgenic zebrafish with CRISPR/Cas9 technology. At Exeter, we have applied an oestrogen responsive transgenic zebrafish model and also developed new transgenic models to investigate the roles of oestrogen in neurogenesis and brain development, investigating for sex related differences, including in intact animals in real time using advanced imaging methods. Aya Takesono visited our Japanese partner, Professor Atsuo Kawahara (Medical School, Yamanashi University) from 30/July/2017 to 11/Aug/2017. Tetsu Kudoh also visited Kawahara lab from 21st to 31st August 2017. During the visit, Takesono and Kudoh applied novel CRISPR/Cas9 genome editing technology to zebrafish embryo
Collaborator Contribution Through UK-Japan partnerships we are applying CRISPR/Cas9 to produce novel biosensor transgenic fish and gene knock-outs for studies on hormone receptors in zebrafish. This has lead to a new international research partnership with Professor Atsuo Kawahara (Medical School, Yamanashi University).
Impact We have established new transgenic zebrafish lines to elucidate a novel mechanism of action of oestrogen on the forebrain development, in particular, olfactory development. Through this work, we have discovered a new and earliest target of oestrogen in developing zebrafish; glial progenitor cells in the forebrain that exclusively respond to oestrogen (named ER-forebrain cells) and revealed the roles of these ER-forebrain cells in forebrain development and the associated neuronal networks. Further, we have developed several functional assays to assess the developmental roles of ER-forebrain cells, including immunohistochemical analyses for neuronal activation markers, a live imaging for neuronal activation using a genetically encoded calcium indicator GCaMP system and odour-mediated behaviour assays.
Start Year 2018
 
Description UEMS Dr John Chilton/Prof Andrew Randall 
Organisation University of Exeter
Department Medical School
Country United Kingdom 
Sector Academic/University 
PI Contribution We have undertaken collaborations with Dr. John Chilton (University of Exeter, Medical School) to test the transient expression of GFP derived from estrogen responsive promotor (ERE) activation in the live chick embryo. The aim of this approach was to examine whether tissue specific estrogen responses seen in our ERE-TG line (e.g. brain, heart, liver and heart) are conserved in a higher vertebrate. Collaboration with Prof. Andrew Randall's lab (Medical School, University of Exeter) for Ca++ imaging of estrogen responsive neuronal cells.
Collaborator Contribution Expertise in neural development and function. Collaborators conducted supporting experiments in neural electrophysiology. Joint research paper in preparation.
Impact Collaborators conducted supporting experiments in neural electrophysiology. Joint research paper in preparation. Joint collaborative research grant application based on this collaborative work submitted to BBSRC.
Start Year 2014
 
Description 50th Anniversary of the Fisheries Society of the British Isles 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The Jack Jones Lecture, 50th Anniversary of the Fisheries Society of the British Isles. Feminisation of Nature - An Unnatural History, Exeter, July 2017. C.R.Tyler
Assessing the oestrogenic potency and health impacts of wastewater treatment work effluents using ERE-GFP transgenic zebrafish.
FSBI Annual Symposium, Exeter, UK 3-7 July 2017
Oral presentation
Cooper, R.; Kudoh, T.; Tyler, C.R.; David, A.; Hill, E.
Year(s) Of Engagement Activity 2017
 
Description Annual Scientific Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact The 20th UK-Japan Annual Scientific Workshop on Research into Environmental Endocrine Disrupting Chemicals, Downing College, Cambridge
Year(s) Of Engagement Activity 2019
 
Description Government report: The 20th UK-Japan Annual Scientific Workshop on Research into Environmental Endocrine Disrupting Chemicals 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact GOVERNMENT REPORT (DEFRA)
Year(s) Of Engagement Activity 2018,2019
 
Description International Congress on Comparative Endocrinology, Canada 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Endocrine Disruption in Aquatic Systems. International Congress on Comparative Endocrinology, Canada. June 2017. Keynote speech.
Year(s) Of Engagement Activity 2017
 
Description PRESENTATION: Endocrine Disruption in Aquatic Wildlife - Reproductive Toxicity an Endocrine Disruption. CRU Symposium, Uppsala University, Sweden October 18-19 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact CRU Symposium, Uppsala University, Sweden October 18-19 2018
Year(s) Of Engagement Activity 2018
 
Description SETAC Barcelona 2015 - seminar/workshop 'The use of transgenic zebrafish in ecotoxocology' 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact We also coordinated and ran an international seminar/workshop on "The use of transgenic fish in ecotoxicology" held at the Society for Ecotoxicology And Chemistry (SETAC) European Meeting in Barcelona.
Year(s) Of Engagement Activity 2015
 
Description kEYNOTE SPEECH: The Feminisation of Freshwater Fish - From Individuals to Population Consequences 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The Feminisation of Freshwater Fish - From Individuals to Population Consequences. International Symposium on the Reproductive Physiology of Fish, Manaus Brazil. Keynote 3-8th June 2018
Year(s) Of Engagement Activity 2018