PAR polarity proteins and Neurogenesis

Par polarity proteins in building the nervous system The brain develops from a thin sheet of cells in the embryo. During development to the adult, it becomes an extraordinarily complex structure with many layers and many different cell types. One of the ways of generating different cell types is by cells dividing asymmetrically, that is a cell becoming two daughter cells that are different from one another. Typically, one daughter cell is like its mother (i.e. a stem cell that can divide in the same way again) while the other is a more specialised cell such as a neuron. Such divisions are known to take place in the developing mammalian brain at many stages, and so how they are regulated is very important in understanding brain development in general. How do asymmetrical cell divisions happen in the developing brain? We have been studying a group of proteins that in flies and worms are needed for certain asymmetric cell divisions at several stages of development in non-neural tissues. To do this, we have used the Xenopus frog as an experimental system because, like humans, it is a vertebrate, but unlike humans or laboratory mice, its embryos are large and can be watched easily from spawning to tadpole stages, a period of four days. We decided to look at these proteins in the neural tissue. We found that when we deplete either of two of these proteins, known as Par-1 and Par-4, early neurons are no longer formed. When we introduce extra Par-1 protein in the wrong place, we can get extra neurons. This tells us that these Par proteins are important and so we want to discover how they work. We believe that both the loss and the gain of neurons we see happen because the earliest asymmetrical cell divisions in the thin sheet of cells that will make the brain are now not sufficiently asymmetrical. To test this idea, we will observe directly whether the daughter cells of the early divisions go on to become the same type of cell when Par-1 or Par-4 is altered. This involves either manually separating the cells early or tracking them in place and then using specific cell staining reagents to see what cell types they are. In a separate set of experiments, we will also find out whether the role of Par-1 and Par-4 is similar in other asymmetric divisions, specifically in the early skin and in the later cell divisions that give rise to neurons. We will also look at cells from mouse brains that are known to divide asymmetrically, but for which the role of Par-1 and Par-4 is completely unknown. Finally, in a third project, we will examine the biochemistry of what Par-1 and Par-4 are doing. They are both enzymes ('kinases') that add phosphate groups to other proteins. This is a common way that changes are transmitted from one part of a cell to another and there are hundreds of different kinases that each relays a different signal. We know some of the proteins that the Par kinases can regulate. Whether they do this during neuron development, we do not know / but can find out. We have special reagents ('phospho-specific-antibodies') that can be used to stain the specific candidate proteins only when they are phosphorylated. Sophisticated microscopes and image processing will show us exactly where and when the phosphorylation evens take place. Seeing where and when these phosphorylation events take place in the developing nervous system will enable us to draw a few more important linkages in the vast circuit diagram that drives development. Ultimately, all of the above studies will not only help us understand how brains are made, but it will also contribute to the knowledge that is needed to make cells that can repair the brains and spinal cords of patients.

The Par (partition defective) proteins were identified in C. elegans and Drosophila as essential for embryo polarity. The proteins are conserved and known to play roles in epithelial apicobasal polarity and neuron polarity (neurite-to-axon establishment) in mammalian cells. Their role in vertebrate development is less well understood. We have used the advantages of Xenopus, especially the rapid assays for Organiser and convergent extension functions, to demonstrate interaction between the Par proteins Par-1 and Par-4/LKB1 and branches of the Wnt signalling pathway and are critical for primary axis establishment. New data (including both gain- and loss-of-function with our well-validated, published reagents) indicate that Par-1 and Par-4/LKB1 proteins are also critical in primary neurogenesis in the Xenopus neural plate. Our protein localisation studies show that the basolateral localisation of Par-1 protein in particular are critical for its neurogenic functions. Previous detailed lineage studies and dissectable cell layers of the Xenopus ectoderm have shown that there is a link between neurogenesis and apicobasal cell polarity. We therefore propose to test experimentally the link between cell polarity and primary neurogenesis, and to define the role of Par-1 and Par-4/LKB1 in primary and secondary neurogenesis in Xenopus and mouse. We will first determine whether Par-1 and Par-4/LKB1 control only the early neurogenic programme or instead layer specification as such. We will define the layer origins of supernumary neurons by dissection and cell-labelling methods. We will establish whether layer specification in the non-neural ectoderm is also affected.. To establish the stages at which Par-1 and Par-4/LKB1 are important, and whether they are important in secondary neurogenesis and mammalian cortical neurogenesis, we will make transgenic embryos with inducible expression of activated Par-1 to identify the stages of competence for induction of extra neurons. Endogenous Par-1 will be tracked in Xenopus and mammalian cortical cells to identify its role in asymmetrical cell divisions in relation to the better-described polarised determinant, Numb. Finally, we shall analyse the mechanism of regulation and action of Par-1 by defining functional interactions with Par-4/LKB1 and Wnt signalling. Published data indicates a possible pathway linking Par-4/LKB1 via Par-1 to Wnt signaling. We shall conduct cross-rescue experiments on Par-1 and Par-4/LKB1 depletants and determine in situ the effects of each depletion and rescue on phosphorylation and localisation of Dishevelled, GSK3 beta and beta-catenin proteins.