Molecular control of corpus callosum development by Gli3

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.

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.

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.