Molecular mechanisms regulating subventricular zone progenitor migration

The large majority of our nerve cells are generated during fetal development. However, two regions of the brain contain stem cells and continuously make hundreds of thousands of new nerve cells throughout life. My laboratory has actively studied these stem cells and the nerve cells they make for over twenty years. One of the regions, the subependymal zone (SEZ), makes nerve cells that migrate long distances. They move from the SEZ to a brain region called the olfactory bulb which has to do with the sense of smell. When we experimentally inhibit the migration, the newborn nerve cells whither and die and olfaction diminshes.
There are many unsolved questions surrounding SEZ nerve cell migration. The answers are vital to discover as proper migration is fundamental for brain function. When migration goes wrong, brain malformations can arise in the embryo and lead to neurological problems such as mental retardation and epilepsy. Migration is a dynamic process best visualized and understood in real-time; we developed a powerful 2-photon time-lapse microscope that detects nerve cell migration deep in tissue without damaging it. Our microscope employs lasers that pulse incredibly rapidly to generate three-dimensional images of fluorescently labeled cells, which we then reconstruct into movies. Using advanced software, we track cell positions and thus generate rich and detailed information on cell movement.
We recently discovered that a protein called galectin-3 (Gal-3) specifically regulates SEZ nerve cell migration. Our novel finding was fascinating because Gal-3 was known to affect cancer and inflammation but not normal brain processes. Although Gal-3 was known in brain injury, our work proved that it also works in the healthy brain. In fact, Gal-3 was found only in the SEZ, and no other brain region. Gal-3 frequently "multi-tasks": in addition to migration, it can also regulate cell division and survival. We found such effects of Gal-3 do not occur in the SEZ; it was only important for migration. Although we showed Gal-3 regulates migration, the specific way in which it does so is unclear. In this project we seek to discover the ways Gal-3 regulates nerve cell movement.
1) We will test how much it promotes adhesion of SEZ cells. We believe Gal-3 interacts with other proteins to maintain the right level of adhesion between cells. Too much adhesion causes cells to get stuck and not move; too little adhesion and they will not have the traction needed for motility. We will also test how much Gal-3 mediated adhesion causes shifts from individual cell migration to group migration.
2) We will test whether Gal-3 regulates motile cilia and the establishment of these road maps. Gal-3 was expressed on SEZ cells with motile cilia, small whip-like protrusions that cause brain fluid (cerebrospinal fluid) flow. The fluid flow creates molecular gradients, which form a road map for nerve cells to migrate in the right direction.
3) We will next test if Gal-3 regulates migration by interacting with another protein called epidermal growth factor receptor (EGFr). We showed EGFr is important for SEZ nerve cell migration and that it is controlled by Gal-3.
4) Finally, we will test if Gal-3's cousin, Gal-1, interacts with it to regulate nerve cell migration. This last experiment is important since Gal-1 regulates SEZ proliferation but its function in migration remains untested.
By defining the molecular regulation of nerve cell migration, this project will fill a big gap in the understanding of the overall process of adult nerve cell production. Cell migration is a general feature of embryonic development and is also relevant for the adult immune system's normal function and in cancer. Therefore the insights gleaned from these studies are predicted to be of multiple interest and applicable to biologists in research and clinicians in practice.

The SEZ is the major stem cell niche in the adult brain. We discovered that the pleiotropic lectin Gal-3 is selectively expressed in the SEZ and is necessary for migration homeostasis. In this project we will expand these exciting new findings and ask intersecting questions to determine the mechanisms Gal-3 uses to regulate SEZ neuroblast migration. Our research programme is significant because we are in a unique position to answer these important questions. We have already uncovered mechanisms and fundamental patterns in adult neurogenesis.
Aim 1. Gal-3 regulates migration via chemoattraction and adhesion. We will use 2-photon time-lapse and transwell migration to ask if neuroblasts move up or down Gal-3 concentration gradients. We will next examine shifts from chain to individual migration in vivo and in slices in the presence of blocking Gal-3 antibodies and in Gal-3-/- mice. We will finally explore the rate of isolated SEZ cell heterotopic and homotopic adhesion using FACsorted Dcx-GFP+ cells.
Aim 2. The role of ependymal cilia. Using a live whole mount assay we will determine if ciliary flow is altered in Gal-3-/- mice, after knockdown and upon administration of blocking antibodies. We will then ask if rGal-3 is sufficient to rescue ependymal cliary motility.
Aim 3. Gal-3 regulates migration via EGFr. We will use EGFr inhibitors to ask if it is necessary for Gal-3's effects. Then we will determine EGFr expression in Gal-3-/- mice. We will finally test if Gal-3 loss is permissive for stem and progenitor cell motility.
Aim 4. Gal-1 and Gal-3 interact to regulate SEZ migration. We have evidence that Gal-1/Gal-3 double knockouts have altered rates of neurogenesis. We will first determine if loss of Gal-1 function alters SEZ migration and then test double knockouts. We will then test the notion that Gal-1 and Gal-3 regulate each other's expression levels in the SEZ. Finally we will determine if Gal-3 works via interactions with a6b1 integrin, as does Gal-1.

This project is designed to solidify my laboratory, the University of Oxford and therefore the United Kingdom as a pre-eminent center for the investigation of adult neurogenesis and cell migration. We have already pushed forward the boundaries of knowledge in this important field and have carved out a unique niche. Our laboratory uncovered several mechanisms regulating adult neurogenesis. We recently showed that Gal-3 is necessary for SEZ neuroblast migration. This intersects with our published work showing a novel role for epidermal growth factor receptor (EGFr) in the SEZ. It reduces migration, thus contributing to balancing migration and proliferation in the system. Interestingly Gal-3-/- mice had increased levels of activated pEGFr. In this project we will determine if EGFr and other molecules are necessary for Gal-3's actions.

We view adult neurogenesis within the broader context of stem cell biology. The UK has outstanding resources and legislative frameworks for research in human embryonic stem (hESC) cells. We believe it is important to complement this portfolio with a robust research stream in adult endogenous tissue stem cells. The intersection of the hESC and adult stem cells will inform both fundamental biological questions and translational approaches. With regards to this I have a firm standing within the Oxford Stem Cell Institute (OSCI). I am the departmental OSCI representative on committee; I interview students, invite lecturers, and present data at our annual retreat. I represented OSCI at the St. Edwards School event "New body parts for old: stem cells and regenerative medicine". Every year I teach in the OSCI outreach course "Stem Cells a Pathway through the Maze". Individual investigators like myself combined with healthy institutional and intellectual support from organizations like OSCI will result in increasing the importance of stem cell research in the UK.

We have developed one of the most effective 2-photon time-lapse microscopy systems in the world and integrated it into investigations of molecular actions and models of disease. In this project we will use this powerful tool as well as a suite of related techniques to address the fundamental problem of SEZ cell migration. A large part of work will use the advanced software, Volocity, developed by the company Improvision, who are based in the UK (Coventry). I have already spoken at International Meetings (Society for Neuroscience, Chicago, 2009) on their behalf and transparently discuss the centrality of Volocity software in our studies. I am certain that the experiments in this project will stimulate further purchases of Improvision's software.

A significant portion of the project will entail training and dissemination of our techniques and data. I and others in the lab will train the research assistants funded by this project as well as undergraduates doing shorter stints allied with the project. Our laboratory is always open to those in, or outside of, our field interested in learning our techniques. We have recently hosted PhD and Masters students from Oxford, the University of Nottingham, Paris, Amsterdam and Qatar. We have also become interested in biological modeling and interact with colleagues (Drs. Sarah Waters and James Oliver) in Oxford's Mathematical Institute and Centre for Mathematical Biology.

The work in this project is predicted to enhance our ability to acquire grants from the European Union, the USA National Institutes of Health and other international funding sources. For example we have recently been awarded a Dana Foundation grant to examine the human SEZ.