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

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.

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.

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.