Molecular control of corpus callosum development by Gli3

Lead Research Organisation: University of Edinburgh
Department Name: Centre for Integrative Physiology

Abstract

The cerebral cortex confers humans with their unique cognitive capabilities. Thereby, it relies on a huge number of different types of nerve cells which need to be connected in a correct manner to allow different parts of the cortex to communicate with each other and with other parts of the brain. One of these connections is the corpus callosum, the largest fibre tract in the brain. It connects the two cerebral hemispheres and allows the exchange of information between nerve cells located in opposite hemispheres. Malformation of the corpus callosum is a birth defect and is a major cause of mental retardation having a wide range of cognitive, behavioural and neurological consequences.
To form the corpus callosum, nerve fibres (axons) from callosal nerve cells have to migrate from one cerebral hemisphere to the other. This important step is regulated by several "guidepost" cells which guide callosal axons towards the opposite hemisphere. Thereby, guidepost cells have to acquire specific positions at the boundary between the cortex and another brain structure, the septum (corticoseptal boundary; CSB) where callosal axons cross the midline. Failure to do so results in severe malformation of the corpus callosum. How these cells acquire their correct position is largely unknown but involves the function of the Gli3 gene. Our characterization of mice in which the Gli3 gene is altered (mutated) showed that the formation of the corticoseptal boundary is defective in these mutants leading to the absence of the corpus callosum. Moroever, human patients carrying mutations in the human GLI3 gene often show malformations of the corpus callosum.
This proposal addresses the molecular mechanisms by which Gli3 controls formation of the CSB and hence of the corpus callosum. Gli3 gene encodes a transcription factor, a molecule which can bind to DNA and can regulate the activity of other genes. The genes which are regulated by Gli3 during CSB formation are largely unknown. In the first part of the proposal, we will investigate how Gli3 and two classes of signalling molecules which allow the communication of cortical and septal cells interact to control CSB formation. In the second part of the proposal, we aim at identifying Gli3 regulated genes. We will use a mutant mouse line in which the Gli3 gene has been specifically inactivated on the cortical side of the CSB. We will compare the activity of genes between control and mutant cortex and using computational methods will identify genes with abnormal expression in mutant tissue. These analyses will be a vital step towards gaining a comprehensive understanding of the molecular functions of a key transcription factor during cortical development. It will also provide important insights into the mechanisms underlying malformation of the corpus callosum in genetic diseases.

Technical Summary

Aim 1: Regulatory relationship between Gli3, Fgf8 and Wnts
Our previous analyses showed that Gli3 interacts with Fgf8 and several Wnt genes during corticoseptal boundary (CSB) formation but the molecular basis for these interactions are unknown. We will investigate the regulatory relationships between Gli3, Fgf8 and Wnts during this process. Using electromobility shift and chromatin assays, we will determine whether the Ets transcription factors Etv4/5 bind to Gli3 and Wnt8b forebrain enhancers. We will use transgenic reporter gene assays to determine the functionality of these binding sites. Finally, we will incubate forebrain tissue in the presence of Fgf8 or of the Fgf signalling inhibitor SU 5402 to determine the effect of activating or inhibiting Fgf signalling, respectively, on Gli3 and Wnt8b expression. These analyses will test our hypothesis that Fgf signalling directly regulates Gli3 and Wnt8b expression in the forebrain.

Aim 2. Identification of Gli3 regulated genes
Gli3 controls development of the corpus callosum by regulating CSB formation. To identify Gli3 regulated genes during this process, we will conditionally inactivate Gli3 using an Emx1Cre driver line. At different time points after the start of Cre expression we will harvest cortical cells from either control or conditional mutant embryos. We will compare gene expression profiles between the two groups by RNA sequencing analyses. Bioinformatic analyses will group genes according to their temporal expression profiles (cluster analyses) and to their biological function (gene ontology analyses). We will determine the expression of candidate genes using in situ hybridization. In this way, we will identify genes which (i) are rapidly down-regulated after loss of Gli3 expression, (ii) are expressed in cortical progenitor cells and (iii) encode transcription factors, cell adhesion molecules or components of signalling pathways which likely play important roles in CSB formation.

Planned Impact

This proposal addresses a fundamental question in biomedical sciences, the answers to which will be complex but will have a long-term effect on our understanding of human disease and will contribute to the development of a rationale for treatment. In the short-term, we expect to improve our understanding of the neurological and psychiatric conditions of patients. Although we do not test new treatment for relevant diseases, this knowledge represents an important step towards the development of new therapies for currently incurable neurological and neuropsychiatric diseases.

Most of the immediate impact of the research in this proposal will be on other members of the scientific community who are interested in transcriptional regulation, cell adhesion, cell signalling and brain development. All the data obtained from work described in this proposal will be made available through peer-reviewed publication in respected journals. The proposal will also provide high quality data sets for use with bioinformatics to identify regulatory connections, molecular mechanisms of developmental processes and downstream effectors of signalling pathways. The data sets will be made publicly available by depositing in public repositories. In addition, clinicians who are interested in human syndromes with mutations in GLI3, in ciliopathies, in callosal malformations and/or in mental retardation will benefit from this research. This research will also have an early societal impact especially on patients suffering from GCPS and Acrocallosal Syndrome in which GLI3 is mutated or from other syndromes with agenesis of the corpus callosum such as ciliopathies as it provides these patients and their families with a deeper understanding of the biological causes of their disease. Since little is known about the pathogenesis underlying agenesis of the corpus callosum in these patients, our findings in the Gli3 mouse model might prompt clinicians to search for mutations in genes we identified as Gli3 targets in syndromes with callosal malformation. From meetings with patients, it is also very clear that understanding their condition and its implications for their and their families' livelihoods and prospects is of paramount importance. At future such meetings, we hope to be able to provide patients with continuously improving explanations. Although this proposal does not aim at a curative treatment for patients, the identification of alterations in transcriptional regulation and in signalling pathways underlying callosal malformations might lead to a rationale for treatment. Therefore, we consider that the results of this proposal will have an impact on society as well as economically by improving the potential for new treatments.

Publications


10 25 50
 
Title in utero electroporatoin 
Description in utero electroporation is a method to analyze gene function in the development of the cerebral cortex. In brief, this method involves surgical procedure on narcotized, pregnant mice whereby plasmid DNA is injected into the IVth ventricle of embryos with a fine glass capillary. Subsequently short electronic pulses are applied to transfect the DNA into cortical progenitor cells. Embryos are harvested 24 or 48 hours after electroporation and analysed on the effects of the electroporated DNA constructs. 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact Training of another research group in this technique 
 
Title RNAseq data from Gli3 mutants 
Description RNAseq data sets from the telencephalon of E11.5 and E12.5 Emx1Cre;Gli3 conditional mouse mutants 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact The data sets will be published and made available to research groups who are interested in the action of transcription factors or signalling pathways during the development of the brain. 
 
Description . The molecular and cellular signatures of the mouse eminentia thalami support its role as a signalling centre in the developing forebrain 
Organisation University of Edinburgh
Department Centre for Integrative Physiology
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Theil lab: provided assistence in performing the ex vivo slice culture experiments; contributed to writing the manuscript
Collaborator Contribution Vassiliki Fotaki, David Price: planning and execution of experiments, writing manuscript
Impact Adutwum-Ofosu, K.K., Magnani, D., Theil, T., Price, D.J., Fotaki, V., 2015. The molecular and cellular signatures of the mouse eminentia thalami support its role as a signalling centre in the developing forebrain. Brain Struct Funct. PMID: 26459142
Start Year 2012
 
Description Analysing the origin of mGluR1/Lot Cells. 
Organisation University of Alicante
Department Institute of Neuroscience
Country Spain, Kingdom of 
Sector Academic/University 
PI Contribution Nuria Ruiz Reig visited our lab for half a year; we provided mouse mutants, contributed to the design and write-up of the study.
Collaborator Contribution Initiated project, designed and performed most of the experiments, writing of manuscript
Impact Ruiz-Reig, N., Andrés, B., Huilgol, D., Grove, E.A., Tissir, F., Tole, S., Theil, T., Herrera, E. and Fairen, A. (2016). Lateral thalamic eminence - a novel origin for mGluR1/lot cells. Cerebral Cortex, PMID: 27178193
Start Year 2014
 
Description Cadmium effects on retinogenesis 
Organisation Technical University of Darmstadt
Country Germany, Federal Republic of 
Sector Academic/University 
PI Contribution PALMA SIMONIELLO, the first author on the paper coming from this collaboration, stayed for 3 months in the lab to learn and to perform in situ hybridisations on lizard embryos.
Collaborator Contribution The partners planned and performed the experiments and wrote the manuscript.
Impact Simoniello, P., Trinchella, F., Filosa, S., Scudiero, R., Magnani, D., Theil, T., Motta, C.M., 2014. Cadmium contaminated soil affects retinogenesis in lizard embryos. J Exp Zool A Ecol Genet Physiol 321, 207-219.
Start Year 2008
 
Description Differential requirements for Gli2 and Gli3 in the regional specification of the mouse hypothalamus. 
Organisation University of Heidelberg
Department Institute of Anatomy and Cell Biology
Country Germany, Federal Republic of 
Sector Academic/University 
PI Contribution Theil lab: provided Gli3 mutant mouse embryos; contributed to writing the manuscript
Collaborator Contribution Gonzalo Alvarez-Bolado: planning and executing experiments, writing of manuscript
Impact Haddad-Tóvolli, R., Paul, F., Zhang, Y., Zhou, X., Theil, T., Puelles, L., Blaess, S. and Alvarez-Bolado, G. (2015). Differential requirements for Gli2 and Gli3 in the regional specification of the mouse hypothalamus. Front. Neuroanatomy 9, DOI 10.3389/fnana.2015.00034.
Start Year 2011
 
Description Gli3 controls corpus callosum formation by positioning midline guideposts during telencephalic patterning 
Organisation King's College London (KCL)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Theil lab: planning and execution of most experiments, writing the manuscript
Collaborator Contribution Price: planning of experiments, contributed to writing the manuscript Basson: provided sprouty1/2 mutant mice Lebrand: provided some marker analysis
Impact Magnani D, Hasenpusch-Theil K, Benadiba C, Yu T, Basson MA, Price DJ, Lebrand C and Theil T. (2014). Gli3 controls corpus callosum formation by positioning midline guideposts during telencephalic patterning. Cerebral Cortex 24,186-98.
Start Year 2009
 
Description Gli3 controls corpus callosum formation by positioning midline guideposts during telencephalic patterning 
Organisation University of Edinburgh
Department Centre for Integrative Physiology
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Theil lab: planning and execution of most experiments, writing the manuscript
Collaborator Contribution Price: planning of experiments, contributed to writing the manuscript Basson: provided sprouty1/2 mutant mice Lebrand: provided some marker analysis
Impact Magnani D, Hasenpusch-Theil K, Benadiba C, Yu T, Basson MA, Price DJ, Lebrand C and Theil T. (2014). Gli3 controls corpus callosum formation by positioning midline guideposts during telencephalic patterning. Cerebral Cortex 24,186-98.
Start Year 2009
 
Description Gli3 controls corpus callosum formation by positioning midline guideposts during telencephalic patterning 
Organisation University of Lausanne
Department Department of Cell Biology and Morphology
Country Switzerland, Swiss Confederation 
Sector Academic/University 
PI Contribution Theil lab: planning and execution of most experiments, writing the manuscript
Collaborator Contribution Price: planning of experiments, contributed to writing the manuscript Basson: provided sprouty1/2 mutant mice Lebrand: provided some marker analysis
Impact Magnani D, Hasenpusch-Theil K, Benadiba C, Yu T, Basson MA, Price DJ, Lebrand C and Theil T. (2014). Gli3 controls corpus callosum formation by positioning midline guideposts during telencephalic patterning. Cerebral Cortex 24,186-98.
Start Year 2009
 
Description Gli3 controls corticothalamic pathfinding by regulating development of the piriform cortex 
Organisation University of Edinburgh
Department Centre for Integrative Physiology
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Theil lab: planning experiments, performing experiments, writing the manuscript
Collaborator Contribution John Mason: planning experiments, contributed to writing the manuscript
Impact Amaniti, E. M., Fu, C., Lewis, S., Saisani, M., Magnani, D., Mason, J. O. and Theil, T. (2015). Gli3 controls corticothalamic pathfinding by regulating development of the piriform cortex. Cerebral Cortex 25, 460-71.
Start Year 2011
 
Description Gli3 is required in Emx1+ progenitors for the development of the corpus callosum. 
Organisation University College London (UCL)
Department Wolfson Institute for Biomedical Research
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Theil lab: planning and execution of experiments, writing the manuscript
Collaborator Contribution Mason: planning and contributed to writing the manuscript Kessaris: provided Zic4Cre transgenic mice
Impact Amaniti E-A., Hasenpusch-Theil K, Magnani M, Kessaris N, Mason JO and Theil T. (2013). Gli3 is required in Emx1+ progenitors for the development of the corpus callosum. Dev Biol., 376, 113-24.
Start Year 2010
 
Description Gli3 is required in Emx1+ progenitors for the development of the corpus callosum. 
Organisation University of Edinburgh
Department Centre for Integrative Physiology
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Theil lab: planning and execution of experiments, writing the manuscript
Collaborator Contribution Mason: planning and contributed to writing the manuscript Kessaris: provided Zic4Cre transgenic mice
Impact Amaniti E-A., Hasenpusch-Theil K, Magnani M, Kessaris N, Mason JO and Theil T. (2013). Gli3 is required in Emx1+ progenitors for the development of the corpus callosum. Dev Biol., 376, 113-24.
Start Year 2010
 
Description Pax6 exerts regional control of cortical progenitor proliferation via direct repression of Cdk6 and hypophosphorylation of pRb. 
Organisation University of Edinburgh
Department Centre for Integrative Physiology
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution My lab helped with the DNA binding assays and the reporter gene assays in the paper and we contributed to writing the manuscript.
Collaborator Contribution Planning and execution of most of the experiments. Writing the manuscript
Impact This collaboration has resulted in a paper (PMID: 23622063) and has prompted us to start to analyse a role of the cell cycle in the Gli3 mutants we are working on.
Start Year 2011
 
Description Role of Gli3 in the development of the lateral olfactory tract 
Organisation University of Alicante
Country Spain, Kingdom of 
Sector Academic/University 
PI Contribution Nuria Ruiz Reig, a PhD student from our collaborator Alfonso Fairen, stayed in our lab for three months to characterize the lateral olfactory tract in Gli3 mutants. We provided lab space and the mouse mutants.
Collaborator Contribution Our partner planned and performed the experiments
Impact There have been no papers published yet but a manuscript is in preparation. The collaboration is not multi-disciplinary.
Start Year 2013
 
Description The ciliogenic transcription factor Rfx3 is required for the formation of the thalamocortical tract by regulating patterning of prethalamus and ventral telencephalon 
Organisation Claude Bernard University Lyon 1 (UCBL)
Country France, French Republic 
Sector Academic/University 
PI Contribution We have made the vast majority of the analysis and wrote the manuscript.
Collaborator Contribution Benedicte Durand at the University of Lyon 1 provided the Rfx3 mutant mice, made some quantification experiments and contributed to writing the manuscript. Stephane Schurmans provided the Inpp5e mutant mice.
Impact Magnani, D., Morle, L., Hasenpusch-Theil, K., Paschaki, M., Jacobi, M., Schurmans, S., Durand, B. and Theil, T. (2015). The ciliogenic transcription factor Rfx3 is required for the formation of the thalamocortical tract by regulating patterning of prethalamus and ventral telencephalon. Hum. Mol. Genet. 24, 2578-93
Start Year 2011
 
Description The ciliogenic transcription factor Rfx3 is required for the formation of the thalamocortical tract by regulating patterning of prethalamus and ventral telencephalon 
Organisation University of Liege
Department Interdisciplinary Cluster for Applied Genoproteomics (GIGA)
Country Belgium, Kingdom of 
Sector Academic/University 
PI Contribution We have made the vast majority of the analysis and wrote the manuscript.
Collaborator Contribution Benedicte Durand at the University of Lyon 1 provided the Rfx3 mutant mice, made some quantification experiments and contributed to writing the manuscript. Stephane Schurmans provided the Inpp5e mutant mice.
Impact Magnani, D., Morle, L., Hasenpusch-Theil, K., Paschaki, M., Jacobi, M., Schurmans, S., Durand, B. and Theil, T. (2015). The ciliogenic transcription factor Rfx3 is required for the formation of the thalamocortical tract by regulating patterning of prethalamus and ventral telencephalon. Hum. Mol. Genet. 24, 2578-93
Start Year 2011