Positive autoregulation of the transcription factor Pax6 in response to increased levels of either of its major isoforms, Pax6 or Pax6(5a), in cultured cells

<p>Abstract</p> <p>Background</p> <p>Pax6 is a transcription factor essential for normal development of the eyes and nervous system. It has two major isoforms, Pax6 and Pax6(5a), and the ratios between their expression levels vary within narrow limits. We tested the effects of overexpressing either one or other isoform on endogenous Pax6 expression levels in Neuro2A and NIH3T3 cells.</p> <p>Results</p> <p>We found that both isoforms caused an up-regulation of endogenous Pax6 expression in cells with (Neuro2A) or without (NIH3T3) constitutive Pax6 expression. Western blots showed that cells stably transfected with constructs expressing either Pax6 or Pax6(5a) contained raised levels of both Pax6 and Pax6(5a). Quantitative RT-PCR confirmed an increase in levels of <it>Pax6(5a) </it>mRNA in cells containing Pax6-expressing constructs and an increase in levels of <it>Pax6 </it>mRNA in cells containing Pax6(5a)-expressing constructs. The fact that the introduction of constructs expressing only one isoform increased the cellular levels of not only that isoform but also the other indicates that activation of the endogenous <it>Pax6 </it>locus occurred. The ratio between the levels of the two isoforms was maintained close to physiological values. The overexpression of either isoform in neuroblastoma (Neuro2A) cell lines also promoted morphological change and an increase in β-III-tubulin expression, indicating an increase in neurogenesis.</p> <p>Conclusion</p> <p>Our results demonstrate that Pax6 can up-regulate production of Pax6 protein from an entire intact endogenous <it>Pax6 </it>locus in its genomic environment. This adds to previous studies showing that Pax6 can up-regulate reporter expression driven by isolated <it>Pax6 </it>regulatory elements. Furthermore, our results suggest that an important function of positive feedback might be to stabilise the relative levels of Pax6 and Pax6(5a).</p

Regulation of the Pax6 : Pax6(5a) mRNA ratio in the developing mammalian brain

BACKGROUND: Early in mammalian brain development cell proliferation generates a population of progenitor cells whose subsequent divisions produce increasing numbers of postmitotic neurons. Pax6 affects both processes and it has been suggested that this changing role is due at least in part to changes in the relative concentrations of its two main isoforms, (i) Pax6 and (ii) Pax6(5a), created by insertion of a 42 bp exon (exon 5a) into one of the two DNA-binding domains. Crucially, however, no previous study has determined whether the ratio between Pax6 and Pax6(5a) transcripts alters during mammalian neurogenesis in vivo. RESULTS: Using RNase protection assays, we show that Pax6 transcripts are 6–10 times more prevalent than Pax6(5a) transcripts early in neurogenesis in the murine telencephalon, diencephalon and hindbrain and that the ratio later falls significantly to about 3:1 in these regions. CONCLUSION: These changes in vivo are similar in magnitude to those shown previously to alter target gene activity in vitro and might, therefore, allow the single mammalian Pax6 gene to carry out different functions at different times in mammalian brain development

Controlled overexpression of Pax6 in vivo negatively autoregulates the Pax6 locus, causing cell-autonomous defects of late cortical progenitor proliferation with little effect on cortical arealization

Levels of expression of the transcription factor Pax6 vary throughout corticogenesis in a rostro-lateral(high) to caudo-medial(low) gradient across the cortical proliferative zone. Previous loss-of-function studies have indicated that Pax6 is required for normal cortical progenitor proliferation, neuronal differentiation, cortical lamination and cortical arealization, but whether and how its level of expression affects its function is unclear. We studied the developing cortex of PAX77 YAC transgenic mice carrying several copies of the human PAX6 locus with its full complement of regulatory regions. We found that PAX77 embryos express Pax6 in a normal spatial pattern, with levels up to three times higher than wild type. By crossing PAX77 mice with a new YAC transgenic line that reports Pax6 expression (DTy54), we showed that increased expression is limited by negative autoregulation. Increased expression reduces proliferation of late cortical progenitors specifically, and analysis of PAX77↔wild-type chimeras indicates that the defect is cell autonomous. We analyzed cortical arealization in PAX77 mice and found that, whereas the loss of Pax6 shifts caudal cortical areas rostrally, Pax6 overexpression at levels predicted to shift rostral areas caudally has very little effect. These findings indicate that Pax6 levels are stabilized by autoregulation, that the proliferation of cortical progenitors is sensitive to altered Pax6 levels and that cortical arealization is not

SNP genotyping on pooled DNAs: comparison of genotyping technologies and a semi automated method for data storage and analysis

We have compared the accuracy, efficiency and robustness of three methods of genotyping single nucleotide polymorphisms on pooled DNAs. We conclude that (i) the frequencies of the two alleles in pools should be corrected with a factor for unequal allelic amplification, which should be estimated from the mean ratio of a set of heterozygotes (k); (ii) the repeatability of an assay is more important than pinpoint accuracy when estimating allele frequencies, and assays should therefore be optimised to increase the repeatability; and (iii) the size of a pool has a relatively small effect on the accuracy of allele frequency estimation. We therefore recommend that large pools are genotyped and replicated a minimum of four times. In addition, we describe statistical approaches to allow rigorous comparison of DNA pool results. Finally, we describe an extension to our ACeDB database that facilitates management and analysis of the data generated by association studies