Pilot grant to determine the transcription factor code that specifies CiA interneurons

Lead Research Organisation: University of Cambridge
Department Name: Physiology

Abstract

In this one-year research grant we will determine which genes are involved in making a specific population of spinal cord nerve cells, called CiAs, in zebrafish embryos. CiAs are made, and function in very similar ways to V1 nerve cells in mammals, so our findings should also be relevant to humans.

This research is important because the more we learn about how different neurons are made, the more likely we are to be able to develop treatments for neurological diseases or spinal cord injuries. For example, once we know which genes specify particular neurons we can use this information to instruct stem cells to grow into those neurons.

We will exploit the cell biology and genetics of zebrafish embryos to identify CiAs in live embryos and determine which genes control their particular characteristics (e.g. shape of the nerve cell, what other nerve cells it communicates with and the chemicals that it uses to communicate).

We have already identified two genes that are important for instructing CiAs to acquire their particular characteristics. These genes encode transcription factors (proteins that are responsible for turning other genes on and off). I

In this one-year project, we aim to identify all of the other transcription factors that are present in CiAs, so that we can start to analyse their individual and combined functions in making these nerve cells.

Technical Summary

This one-year pilot grant aims to determine the complete post-mitotic transcription factor code that specifies zebrafish spinal cord Circumferential Ascending (CiA) interneurons. CiAs are a multifunctional class of interneurons that provide all of the ipsilateral glycinergic-inhibition in zebrafish spinal cord and hence are essential for both motor-pattern generation and sensory gating. CiAs also share many characteristics and are probably functionally homologous with mammalian V1 interneurons. Currently, we do not understand how the unique characteristics of CiAs/V1 interneurons are specified in any vertebrate. The current model is that the functional characteristics of specific spinal cord interneurons are specified, at least in part, by the combination of transcription factors that they express post-mitotically. We already know that CiAs and V1 interneurons express the transcription factors Engrailed1 and Pax2, but it is likely that these are not the only post-mitotic transcription factors involved in specifying these cells. This pilot grant will determine the full complement of transcription factors expressed by CiAs by RNA profiling FAC-sorted GFP-labelled CiAs using microarrays. This will provide a basis for future work to establish the functions of these genes, singly and in combination, in specifying the unique characteristics of CiAs. Most of these findings should also be applicable to mammalian V1 interneurons. This grant will also provide a proof-of-principle for future experiments to RNA profile other FAC-sorted GFP-labelled zebrafish interneurons.

We will use existing stable transgenic lines Tg(pax2a:GFP), that drives Green Fluorescent Protein (GFP) expression in CiA cell bodies and axons, and Tg(huC:GFP), that drives GFP expression in most differentiated neurons. We will dissect out spinal cords and then dissociate and FAC sort spinal cord cells from Tg(pax2a:GFP) and Tg(huC:GFP) embryos. We will then RNA profile GFP +ve spinal cord cells from both of these lines and GFP ?ve cells from the Tg(huC:GFP) line using microarray analysis. By comparing the RNA profiles of these three populations of cells we will be able to identify transcription factors that are specifically upregulated in CiAs and transcription factors that are upregulated in both CiAs and other post-mitotic neurons. We will then confirm that these transcription factors are expressed by CiAs using double labels. As CiAs are probably homologous to mammalian V1 cells our results should also identify transcription factors important for V1 cell specification and function and hence should be relevant to studies of human neural development and disease.

Publications

10 25 50
 
Description HFSP
Amount $300,000 (USD)
Organisation Human Frontier Science Program (HFSP) 
Sector Charity/Non Profit
Country France, French Republic
Start 06/2014 
End 06/2017
 
Description MRC Project Grant
Amount £360,707 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 02/2009 
End 02/2012
 
Description NIH R01
Amount $1,619,750 (USD)
Organisation National Institutes of Health (NIH) 
Sector Public
Country United States of America
Start 09/2014 
End 05/2019
 
Description R21
Amount $270,500 (USD)
Organisation National Institutes of Health (NIH) 
Sector Public
Country United States of America
Start 04/2011 
End 04/2013
 
Title FAC sorting protocol 
Description We have developed a successful protocol for FAC-sorting zebrafish neurons. We have written a paper describing this technique in detail which is now published. 
Type Of Material Technology assay or reagent 
Year Produced 2008 
Provided To Others? Yes  
Impact We published a paper and have supplied our protocol to several groups 
 
Description Department Open Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Primary Audience Public/other audiences
Results and Impact We had an exhibit at the department's open day as part of Cambridge Science week

We successfully interacted with the public and told them about our research.
Year(s) Of Engagement Activity 2007,2008,2009
 
Description departmental open day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Primary Audience Public/other audiences
Results and Impact Ran a stall describing our research to the public

Interacted successfully with members of the public
Year(s) Of Engagement Activity 2009