Microarray investigation of the role of Pax6 at the PSPB using a novel tauGFP-Pax6 reporter mouse

Pax6 encodes a highly conserved transcriptional regulator that is widely expressed during development of the eye, olfactory bulbs and central nervous system. Pax6 mice exhibit severe brain defects, lack eyes and nasal structures, and die at birth. Included among the functions of Pax6 are cell adhesion, cell cycle progression, axon guidance and boundary formation. The pallial-subpallial boundary (PSPB) is both a physical and gene expression boundary separating dorsal and ventral telencephalon. Pax6 is required for this boundary to develop. In Pax6 embryos, genes which normally have a sharp border of expression at the PSPB become ectopically expressed and the radial glial fasicles that make up the physical component of the boundary fail to form. There is also an increase in the number of interneurons migrating dorsally across the boundary to enter the cortex while corticofugal axons struggle to cross the PSPB and enter the ventral telencephalon. Here a novel tauGFP-Pax6 reporter mouse, DTy54, is described in which cells capable of expressing Pax6 are tauGFP positive. In general the expression pattern of tauGFP corresponds well with the known Pax6 expression pattern in the eye and forebrain and the gradient of cortical Pax6 expression from high rostro-laterally to low caudomedially is also recapitulated by tauGFP. The cytoskeletal localisation of the tauGFP also labels cellular processes and the axons projecting from Pax6 positive cells such as those forming the optic nerve can be clearly seen. At E10.5 the forebrain expression patterns of tauGFP and Pax6 correspond exactly, but at later stages tauGFP expression can be seen in areas negative for Pax6. This can be seen at E12.5 in the ventral telencephalon and in both the dorsal and ventral telencephalon at E15.5. Pax6 and tauGFP expression colocalise more closely in the diencephalon. In situ hybridization analysis of Pax6 and tauGFP transcripts suggests that many of the discrepancies in expression seen at the protein level are due to a longer protein halflife for tauGFP than for Pax6. The expression of tauGFP allows the PSPB to be accurately dissected. The cells from this region can then be sorted by FACS to isolate cells expressing high levels of tauGFP and enrich for the Pax6 positive population. Microarray analysis of gene expression is this population of cells in Pax6.DTy54 and Pax6.DTy54 embryos is described here. This analysis identified many genes that show a significant change in expression at the PSPB in the absence of Pax6 expression including Ngn2, Lhx6, Neurod6 and CyclinD1 and 2. The biological processes and molecular functions in which these genes are involved were examined to provide insight into the role of Pax6 in this population of cells. Several processes previously reported to be regulated by Pax6 were identified together with a number of novel processes with which Pax6 has not formerly been associated. Some of these include cell cycle, neurogenesis, transcription and metabolic and signalling pathways. This study has also identified many novel downstream targets of Pax6, such as Sema3G and PlexinA4, which will help to elucidate the genetic basis for the Pax6 phenotype at the PSPB. The changes in expression levels of Ngn2, Lhx6 and Gsh2, identified by microarray, were validated by in situ hybridization, which showed a good correspondence with the microarray results.