but a disorganized LRN (Figure 3E9), whereas, the Pax6(+5a) variant was ineffective (not shown). Thus, in the RL Pax6 splice variants differ in their biological activity, similar to the embryonic cortex [35]. Lastly, we diminished the endogenous Pax6 mRNA by using siRNAs. Transfection of siRNAs or over expression of shRNA constructs directed against Pax6 (Figure S4) resulted in a massive reduction of ECN cells in wt explants (Figure 3F’’), whereas, control constructs had no effect (not shown). The effect was quantified by counting labeled cells that had settled in the ECN (Figure 3L). Taken together, these experiments demonstrate that our model system enables the simultaneous visualization and manipulation of tangentially migrating cells in the mouse brainstem. In addition, we have shown that Pax6 plays numerous distinct roles in the formation and migration of mossy fiber producing neurons. Moreover, the combination of a binary model and organotypic culture assays facilitates a quick discrimination between cell-autonomous and non-autonomous effects. Axonal pathfinding receptors guide mms neurons We identified several genes whose expression was altered in Pax6Sey/Sey mms neurons (Figure S5; see also Materials and Methods). To gain more insights into the function of these putative Pax6 downstream targets all genes were over-expressed or their Table 1. Summary of phenotypes obtained in organotypic cultures. Pax6 Controls Cell Migration in the Brainstem expression level was diminished with shRNAs. Those genes which showed the most noticeable effects are summarized in Table 1. The altered migration and settling behavior of Pax6Sey/Sey ECN/ LRN neurons suggested that migration cues were changed in Pax6Sey/ Sey embryos. The most prominent candidates are ligand/ receptor couples of the Slit/ Robo- and Netrin/ Dcc- pathways [36,37]. Expression of Netrin1, Dcc, and Robo1,2 and 3 was unaltered in migrating Pax6Sey/Sey mms neurons (Figure 4A, 4A9, Figure S2, and data not shown). However, Slit1 and Slit2 which were expressed in the hypoglossal nuclei were both lost in Pax6Sey/Sey embryos (Figure 4B, 4B9 and Figure S2) [5,10]. Motorneurons of the hypoglossal nuclei are in close proximity to the LRN settling territories suggesting that Slit1 and Slit2 expression provided from these neurons may determine the place of LRN settlement. To test this hypothesis we performed transplantation experiments and shRNA driven knock- down of the Slit-receptor Robo3 in migrating mms neurons. Both types of experiments resulted in a disorganized LRN similar to the phenotype observed in Pax6Sey/Sey mice (Figure 4C-4H). The above results indicate that factors provided from the hypoglossal nucleus, (most likely Slit1 and Slit2) determine the place of LRN settlement. These data also explain the cell non-autonomous role of Pax6 during this process. Hypoglossal neurons are Pax6 negative, but are completely lost in Pax6Sey/Sey embryos (Figure S2) [5,10]; hence, Slit1 gene Pax6 Slit1,2 Robo3 Dcc Unc5h1 Unc5h3 Neurod1 Neurod2 Pou4f2 Pou4f1 Math1 Gap43 Shh Chordin Mafb Pax6Sey/Sey expression (Q) (nXII:Q) Q(RL only) < < < < < Q q < Q < Q Q phenotype : unaltered migration : unaltered migration : (shRNA or siRNA or EnR) migration delay, ECN loss, midline arrest : (ablation of the Slit1-positive hypoglossal nuclei): sub-marginal migration, LRN disruption : (shRNA) sub-marginal migration, LRN disruption : enlarged ECN : (shRNA or siRNA) migration delay, ECN loss, altered migration path : reduced cell migration and cell death : migration delayed, altered migration path : (shRNA) unaltered migration : migration delay, altered migration path (EnR): no migration : migration delay, altered migration path (EnR): no migration : (shRNA) midline arrest of cells : unaltered migration : (shRNA) unaltered migration : growth cone stop at midline, altered ipsi-/contra-lateral settlement : growth cone stop at midline, altered ipsi-/contra-lateral settlement : normal migration : (shRNA) altered ipsi-/contra-lateral settlement : no migration : unaltered migration : unaltered migration Q: gene downregulated in the Pax6Sey/Sey mms; <: gene expressed similarly in the wt and the Pax6Sey/Sey mms; q: gene upregulated in the Pax6Sey/Sey mms; ?: overexpression via TRE responder constructs; : knockdown via siRNAs or shRNA constructs, ablation of expression domain, or functional knockdown by fusion to the repressor domain of engrailed (EnR); nXII: hypoglossal nucleus; Genes showing no or only minor mms migration phenotypes after over-expression: Apbb2, Calpactin LC, Diras2, Gata3, Gli1, Hermes, Hip1, Internexin, Irx2, Irx6, Isl2, L1Cam, Math5, Nell2, Neurod6, Nfl, Ngn1, Ngn2, Nrp1, Olf1, Persyn, Pou6f1, RgmA, RgmB, Sema 7a, Stathmin, Syt4, Syt13. doi:10.1371/journal.pgen.1002099.t001 PLoS Genetics | www.plosgenetics.org 6 June 2011 | Volume 7 | Issue 6 | e1002099