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