The functions of the transcription factor Pax6 in thalamic neuronal specification and axonogenesis

In this research we are trying to discover why a single gene called Pax6 is so important for the normal growth of a critical part of the brain called the thalamus. The thalamus is essential for our ability to perceive the world around us since it transmits information about touch, temperature, pain, vision, hearing and so on through the brain to the cerebral cortex. The Pax6 gene codes for a protein that exerts high-level control of development by affecting the activity of other genes by binding to DNA. It is identical in mice and humans and when it is prevented from working the thalamus becomes seriously abnormal: it is too small, its cells contain some of the wrong molecules and it fails to make normal connections with the cerebral cortex. We still do not know if these are all of the defects and we do not know why they develop. Obtaining detailed answers particularly to the second of these questions will take a long time and here we plan to take a first step. We have designed experiments to compare all the major events that occur as the thalamus forms in normal mice and in Pax6 mutants of various types, to look for defects when Pax6 levels are lowered or raised. We shall examine thalamic cell production, the exchange of cells with adjacent tissue, molecules produced by thalamic cells and thalamic connections with the cerebral cortex. Where we show that Pax6 is needed for a particular process, we shall test whether the need is because Pax6 normally acts within the cells that show abnormalities. It is possible that some defects might be knock-on effects of changing Pax6 levels in other cells that then interact incorrectly with the cells showing abnormalities. To home in on the source of the defects we shall alter expression of Pax6 in specific sets of cells only, leaving others intact. Future work will build on results obtained here: we expect that by finding out which thalamic cells need Pax6 for which events we will be able to design further experiments to discover the biochemistry of Pax6?s actions within those cells.

Our long-term goal is to understand the molecular mechanisms of action of the highly conserved transcription factor Pax6 in thalamic and prethalamic neuronal development and function. In both mice and humans, loss of Pax6 causes congenital defects of the eyes and brain. The thalamus and prethalamus are complex brain structures involved in the transmission and processing of most sensory information. Pax6 is expressed in a dynamic pattern in thalamic and prethalamic progenitors and some neurons from early embryonic stages through to adulthood. Studies of mutant mice have shown that Pax6 is required for normal thalamic and prethalamic neuronal development and axonogenesis, but the full extent of its actions and why it is needed remain unknown. This proposal describes a first step towards our long-term goal. We shall exploit new transgenic tools that we have made successfully, including a conditional allele for Pax6 and a YAC transgenic Pax6-reporter mouse (both made with MRC funding) to discover what aspects of the cellular and molecular development of thalamus and prethalamus are affected by Pax6 and to test whether effects are cell autonomous. Current evidence from the analysis of Pax6-/- embryos suggests that Pax6?s major functions are to ensure that thalamus and prethalamus acquire the correct numbers of cells and to regulate those cells? subsequent gene expression and axonal connections. Here, our specific aims will be as follows. (A) To complete a set of mouse models for manipulating Pax6 expression levels in vivo. We have most of the strains we require, but need additional Cre recombinase-expressing strains that allow production of conditional loss-of-function mutations of Pax6. (B) To study the effects of altered Pax6 expression on cell proliferation in embryonic thalamus and prethalamus. (C) To study whether loss of Pax6 affects cell movement across embryonic thalamic and prethalamic boundaries, as a potential explanation for altered gene expression patterns. (D) To study the effects of altered Pax6 expression on the morphological and molecular development of thalamic and prethalamic nuclei from embryo to adult. (E) To study the effects of loss-of-function of Pax6 in thalamus or prethalamus on the development of thalamic axonal projections. (F) To investigate cell autonomous actions of Pax6 in the morphological and molecular development of thalamic and prethalamic nuclei and their axonal connections using chimaeras. Knowledge of the cell autonomous actions of Pax6 will pave the way for future strategies to find the molecular pathways that it regulates.