Forsøksdyr: function of cerebrospinal fluid flow in brain function and development

Godkjenningsdato 19.07.2018

The cerebrospinal fluid (CSF), which fills the cavities surrounding the central nervous system, provides nutrients and chemical homoestasis to the brain. Defective production, removal or transport of CSF across the brain cavities is associated to neurological disorders, including hydrocephalus and scoliosis. Yet, the molecular and cellular basis of the CSF circulating system remains poorly understood.
The purpose of this application is to study the function of motile cilia-mediated flow of CSF by generating new mutant lines. We aim to identify how the CSF flow in the brain ventricular system is established and regulated in order to ultimately understand its function in brain development, physiology and disease. To this end, we will use zebrafish as model system, since its external development and transparency allow us to monitor and manipulate CSF flow and neural activity while recording the physiology of the brain.
We will ablate cilia function upon genetic manipulation in order to identify the function of motile cilia in brain development. Based on the literature and our current work, we identified candidate genes (e.g. Foxj1a and Foxj1b) that we will mutate. We may generate additional mutants over the course of this project.
As we do not know the effect of these mutations on the animal well being, we are here applying for a pilot project. We want to familiarize ourselves with these mutant lines in order to optimize our breeding strategy and reduce the impact on the wellbeing of our animals. Homozygous mutant animals may not be viable or develop harmful phenotypes. In this case, only heterozygous animals will be bred and maintained in our colony. Alternatively, homozygous mutant animal may be viable. In which case, we will raise these animals and monitor their development by daily inspection of their body size, shape and monitor any developmental defects. As ciliary defects have been associated with the development of hydrocephalus and scoliosis, we will monitor these parameters closely. We will also monitor any abnormal behavior. Monitoring will be done daily by experienced staff using non-invasive methods. We will also carry post-mortem evaluation of dead or euthanized sick animals, which will be genotyped in order to evaluate the viability of the mutant lines. Based on the litterature, we do not expect heterozygous mutants to display any developmental defects. We plan to raise around 300 animals/line until the genetically altered line is characterized at the F4 generation, as indicated by the EU directives.
Using zebrafish, we replace mammalian animal models. All our experiments consider studying the function of the vertebrate brain in health and disease; hence these experiments require working with living animals.
We will put a strong emphasis on studying the function of motile cilia in larval zebrafish (less than 5days old), which are considered as a replacement to animal experimentation. This implies that we will maintain an actively breeding colony with heterozygous or homozygous mutants.
We anticipate that our results will go beyond zebrafish brain and inspire novel knowledge about the function of motile ciliated flow in the brain of mammals.