In order to dissect the complex neuronal cell migration defects observed in Pax6Sey/Sey mice, Pax6Sey/Sey mice were crossed to the Tg(PAX6-tTA) transgenic line. Comparison of cultures obtained from wt and from Pax6Sey/Sey embryos confirmed the anatomical observations described above. Pax6Sey/Sey mms cells showed an initial delay in the onset of the migration (Figure 3A, 3B) and a disturbance at midline crossing later on. After 5 DIV (5 days in vitro) Pax6Sey/Sey mms neurons settled randomly in the LRN (Figure 3B9) but failed to form any ECN structures (Figure 3B’’). In contrast, wt cultures formed a well organized LRN in which cells settled in a dorsal and ventral sub-nucleus of the LRN (Figure 3A9) and in a distinguished ECN (Figure 3A’’). To quantify the effect we counted labeled cells in cultures from wt (LRNi = ipsilateral LRN = 11267; LRNc = contralateral LRN = 266616; ECN = 86614; total number of cells = 610634; n = 50) and Pax6Sey/Sey embryos (LRNi = 313623; LRNc = 426646; ECN = 862; total number of cells = 670671; n = 8) (Figure 3K). These data suggested that there were no alterations in the gross number of migrating neurons between wt and Pax6Sey/Sey embryos and confirmed the complete absence of an ECN in Pax6Sey/Sey embryos. In both, mutant and wt tissues, a proportion of LRN neurons settled ipsilaterally (Figure 3K). All LRN neurons, however, projected to the contralateral cerebellum in Pax6 Controls Cell Migration in the Brainstem respect to their origin from one rhombic lip, explaining the observations by Bourrat and Sotelo of an ipsilateral and contralateral contribution of mossy fibers [17]. The Pax6Sey/Sey migration defect could be caused either by a direct cell autonomous action of Pax6 in migrating RL precursors or via an indirect non-autonomous effect, for example in the ventral domain of Pax6expression (e.g. by altering migration cues at the midline). We performed three types of experiments to discriminate between these alternatives. First, we transplanted transfected Pax6Sey/Sey rhombic lips onto wt brainstems and vice versa (Figure 3C, 3D). Unexpectedly, migrating Pax6Sey/Sey neurons (in a wt host) formed a well organized LRN (Figure 3C9), but no ECN (Figure 3C’’). In contrast, wt neurons (in a mutant host) failed to form a correctly organized LRN (Figure 3D9), but were able to generate a normal ECN (Figure 3D’’). These data suggested, that Pax6 may act cell autonomously in generating ECN neurons, but non-autonomously in specifying the correct sub-organization of LRN neurons. To further validate this assumption we rescued the Pax6Sey/Sey migration defect by re- expression of Pax6. We tested the two major splice variants and of these, the expression of the Pax6(-5a) isoform in PaxSey/Sey6 rhombic lips resulted in a full recovery of the ECN (Figure 3E’’), Figure 3. Migration defects of Pax6Sey/Sey pre-cerebellar neurons: cultures. (A–F) Fluorescence images of transfected brainstem cultures. The enlargements shown are indicated by dotted boxes in (G). Dotted lines in A9–F9 and A’’–F’’ indicate the LRN and ECN territories, respectively. The respective positions of the LRN and ECN are placed according to landmarks derived from wt cultures (see Figure 1). 1.2DIV wt mms neurons have left the rhombic lip (RL) and have almost reached the midline (A). Neurons settle after 5DIV in two sub-nuclei of the contralateral LRN (A9, LRNc) and in the ECN (A’’). In contrast, Pax6Sey/Sey mms neurons are initially delayed (B), and then settle in a scattered LRNc (B9) but not in the ECN (B’’). After transplantation of a Pax6Sey/Sey RL (beige area in C) onto a wt brainstem (grey area in C), two LRN sub-nuclei are formed (C9), but no ECN (C’’), whereas, transplantation of a wt RL (grey area in D) onto a Pax6Sey/Sey brainstem (beige area in D), results in a scattered LRNc (D9) and a normal ECN (D’’). Transfection of the Pax6Sey/Sey RL with Pax6(-5a) results in a rescue of the ECN (E’’), but not of the LRNc phenotype (E9). shRNA down regulation of Pax6 causes a loss of the ECN (F’’), however, the LRNc remains unaffected (F9). Responder constructs used are: (H) for (A–D), (I) for (E), and (J) for (F), respectively. A9, B9, and E9 are red fluorescence images of the nuclear DsRednls protein; all other images are green fluorescence images of the cytoplasmic EGFP. (K) Quantification of settled LRN and ECN cells after 5DIV. There are no significant differences between the total number of migrating cells in wt and Pax6Sey/Sey cultures. (L) Quantification of neurons settling in the ECN territory after 5DIV in cultures. Standard Student’s t test was used to assess the significance of changes as indicated by a p-value of ,0.001; values are 6SEM. (Scale bar is 0.44 mm in [D–I’’].) doi:10.1371/journal.pgen.1002099.g003 PLoS Genetics | www.plosgenetics.org 5 June 2011 | Volume 7 | Issue 6 | e1002099