Pax6 Controls Cell Migration in the Brainstem Figure 5. Pou4f2 induces a midline arrest of neurons in the mms. Immunohistochemical labeling of Pou4f2 on coronal wt (A) and Pax6Sey/Sey (A9) E14.5 sections counterstained with DAPI (B and B9, respectively); high magnifications of the ventral mms are shown as indicated in (L). Pou4f2 is expressed in E14.5 wt embryos in neurons of the inferior olive (IO) and in a fraction of migrating mms neurons (white arrows in A and B). In Pax6Sey/Sey embryos Pou4f2 is expressed in the IO but not in neurons of the mms (A9,B9). In situ hybridization of coronal wt (C) and Pax6Sey/Sey (C9) E15.5 sections reveals that Pou4f1 expression is massively up-regulated in Pax6Sey/Sey embryos (arrow in C, C9). Over-expression of Pou4f2 (F) and of Math1 (H), but not of control constructs (D), result in a complete arrest of growth cones at the midline (black arrows in F and H) after 2DIV. In control cultures after 5DIV few cells settle in the ipsilateral LRN (LRNi) (E), whereas, in cultures over-expressing Pou4f2 or Math1 a significant higher proportion of cells settles in the LRNi (G, I). (J, K) Distribution of Wt1, Pax6 and Math1 on coronal wt E14.5 sections as indicated in (L). (J) Immunohistochemical labeling of Pax6 (green) and Wt1 (red) indicates that Wt1 is expressed in the roof plate dorsally to the rhombic lip and that Pax6 and Wt1 are non-overlapping. (K) In situ hybridization with Math1 indicates that Math1 is expressed at the rhombic lip and in a subset of cells emanating from the rhombic lip (arrowhead in K). (M) Quantification of mms neurons settling in the LRN; values are given for the proportion of cells settling in the LRNc or LRNi. The significance of changes is indicated by a p-value of ,0.001; values are 6SEM. Over-expression of Pou4f2, Math1, and Gfi1 resulted in a higher ratio of LRNi cells and down-regulation of Gap43 led to a lower ratio of LRNi cells. (N-P) In situ hybridization of sectioned cultures after over-expression of Pou4f2 or Math1. Pou4f1 expression is reduced in Pou4f2 and in Math1 transfected rhombic lips (open arrows in N,P), but not in the control rhombic lip (open arrowheads in N,P). Pou4f2 is up-regulated in Math1 transfected (open arrow in O) but not in control (open arrowhead in O) rhombic lips. (Scale bar is 110 mm in [A, A9, B, B9, J, K]; 1,4mm in [C, C9, N, O, P].); 440 mm in [D–I].) doi:10.1371/journal.pgen.1002099.g005 (Figure 5H, 5I, 5M). Mis-expression of Math1 resulted in an up- regulation of Pou4f2 and a down-regulation of Pou4f1 (Figure 5O, 5P). Together, these data suggest, that Pou4f2 expression in rhombic lip derived mms neurons depends on Pax6 and Math1 and that Pou4f2 may regulate Pou4f1 and Gap43 in mms neurons. In summary, our work led to the identification of a gene cascade acting in tangentially migrating neurons of the brainstem, in which Pou4f2 plays a central role to induce a previously unknown mechanism that controls midline crossing behavior. Furthermore, our results imply that our model system is applicable to quickly analyze genetic hierarchies in Pax6 positive cells and may therefore serve as a general tool. Discussion Tg(PAX6-tTA) mice as a model for Pax6 function, cell migration, and axonal pathfinding processes The extraordinary complexity of cell determination, migration and wiring processes in the developing mammalian brain creates a major challenge for developmental neurobiologists. Here, we introduced a simple yet powerful technology to quickly analyze any gene potentially involved in these processes. Our model is of threefold use: first to study the function of Pax6 and of Pax6 downstream genes in their genuine environment, second to investigate genes involved in general patterning, axonal pathfind- ing and cell migration processes, and third to enable the analysis of tissue-specific gene functions. The Tg(PAX6-tTA) model complements and improves existing approaches and has certain benefits: it combines cell specific transfection protocols and organotypic culture assays, thus, facilitating the quick analysis of genes in a natural tissue environment. The experimental design and the binary nature of the Tg(PAX6-tTA) model is fundamentally simple and has several advantages over systems that are based purely on transgenic animals. First, the electroporation and subsequent culture of embryonic tissues allows the screening of large number of genes without the need of generating new transgenic animals for each construct. In fact, less than 10% of the constructs we have tested revealed phenotypes. Thus, only those genes showing PLoS Genetics | www.plosgenetics.org 8 June 2011 | Volume 7 | Issue 6 | e1002099