3T3 cell lines stably expressing Pax6 or Pax6(5a)--a new tool used for identification of common and isoform specific target genes

Pax6 and Pax6(5a) are two isoforms of the evolutionary conserved Pax6 gene often co-expressed in specific stochiometric relationship in the brain and the eye during development. The Pax6(5a) protein differs from Pax6 by having a 14 amino acid insert in the paired domain, causing the two proteins to have different DNA binding specificities. Difference in functions during development is proven by the fact that mutations in the 14 amino acid insertion for Pax6(5a) give a slightly different eye phenotype than the one described for Pax6. Whereas quite many Pax6 target genes have been published during the last years, few Pax6(5a) specific target genes have been reported on. However, target genes identified by Pax6 knockout studies can probably be Pax6(5a) targets as well, since this isoform also will be affected by the knockout. In order to identify new Pax6 target genes, and to try to distinguish between genes regulated by Pax6 and Pax6(5a), we generated FlpIn-3T3 cell lines stably expressing Pax6 or Pax6(5a). RNA was harvested from these cell lines and used in gene expression microarrays where we identified a number of genes differentially regulated by Pax6 and Pax6(5a). A majority of these were associated with the extracellular region. By qPCR we verified that Ncam1, Ngef, Sphk1, Dkk3 and Crtap are Pax6(5a) specific target genes, while Tgfbi, Vegfa, EphB2, Klk8 and Edn1 were confirmed as Pax6 specific target genes. Nbl1, Ngfb and seven genes encoding different glycosyl transferases appeared to be regulated by both. Direct binding to the promoters of Crtap, Ctgf, Edn1, Dkk3, Pdgfb and Ngef was verified by ChIP. Furthermore, a change in morphology of the stably transfected Pax6 and Pax6(5a) cells was observed, and the Pax6 expressing cells were shown to have increased proliferation and migration capacities.     

The Trichoplax PaxB gene: a putative Proto-PaxA/B/C gene predating the origin of nerve and sensory cells

Pax genes play key regulatory roles in embryonic and sensory organ development in metazoans but their evolution and ancestral functions remain widely unresolved. We have isolated a Pax gene from Placozoa, beside Porifera the only metazoan phylum that completely lacks nerve and sensory cells or organs. These simplest known metazoans also lack any kind of symmetry, organs, extracellular matrix, basal lamina, muscle cells, and main body axis. The isolated Pax gene from Trichoplax adhaerens harbors a paired domain, an octapeptide, and a full-length homeodomain. It displays structural features not only of PaxB and Pax2/5/8-like genes but also of PaxC and Pax6 genes. Conserved splice sites between Placozoa, Cnidaria, and triploblasts, mark the ancient origin of intron structures. Phylogenetic analyses demonstrate that the Trichoplax PaxB gene, TriPaxB, is basal not only to all other known PaxB genes but also to PaxA and PaxC genes and their relatives in triploblasts (namely Pax2/5/8, Pax4/6, and Poxneuro). TriPaxB is expressed in distinct cell patches near the outer edge of the animal body, where undifferentiated and possibly multipotent cells are found. This expression pattern indicates a developmental role in cell-type specification and/or differentiation, probably in specifying-determining fiber cells, which are regarded as proto-neural/muscle cells in Trichoplax. While PaxB, Pax2/5/8, and Pax6 genes have been linked to nerve cell and sensory system/organ development in virtually all animals investigated so far, our study suggests that Pax genes predate the origin of nerve and sensory cells.