Pax6 Controls Cell Migration in the Brainstem construct as indicated in E. After 3DIV several hundred neurons have left the transfected rhombic lip (RL) to migrate along the mms. At 5DIV all neurons have reached their target positions. 25 similar cultures were used to outline the territories of the LRNc and ECN in respect to surrounding ‘‘landmarks’’: the superior olivary nuclei (open arrows), the inferior olivary nuclei, the midline (white arrowhead), and the rhombic lip (RL). Double- immunolabeling of cultures transfected with a hemagglutinin (HA)-tagged Pax6 construct (M, M9, M’’) confirm that over-expression results in Pax6 levels comparable to endogenous Pax6 levels. Immunolabeling with HA-tag antibodies (M) mark transfected cells (black arrows) which express endogenous and exogenous Pax6. Note, that Pax6 levels in some untransfected cells (only endogenous Pax6) are as high as Pax6 levels in transfected cells (endogenous plus exogenous Pax6). (The scale bar represents 0.8 mm in [B]; 1 mm in [C]; 150 mm in [F]; 0.44mm in [G, J]; 0.9 mm in [H]; 70 mm in [I]; 1.8 mm in [K, K9, L]; 50 mm in [M, M9]). doi:10.1371/journal.pgen.1002099.g001 simultaneous visualization and manipulation of neurons we designed reporter constructs containing two TRE elements (Figure 1J). Control constructs co-expressed a cytoplasmic green fluorescent protein (EGFPm) and a nuclear red fluorescent protein (DsRed2nls) in 99% (61; n = 10) of labeled neurons demonstrating that our reporter constructs enable the co-expression of two genes in the same neurons (Figure 1J). To enable statistical analysis of the cultures, the territories of the LRN and the ECN were delineated using visible landmarks (Figure 1K, 1K9, 1L; see also the Materials and Methods section). Immunolabeling of cultures expressing a HA-tagged Pax6 construct demonstrate that over-expression of TRE constructs in Tg(PAX6-tTA) transgenic cultures result in moderate levels of protein expression that are in the range of physiological Pax6 concentrations (Figure 1M, 1M9, 1M’’). Pax6 plays multiple roles in patterning and guiding migrating ECN and LRN neurons Pax6 mutant Pax6Sey/Sey mice display multiple neuronal pattern- ing and migration defects. We therefore wished to determine whether Pax6 also regulates the mms. At the anatomical level, several features of the mms are severely disturbed in Pax6 mutant Pax6Sey/Sey embryos. Most noticeable, the initiation of migration and the midline crossing was delayed by 0.5 days (asterisks in Figure 2A, 2A9 and data not shown; see also Figure S2 and Figure 4A, 4A9 The expression patterns of Pax2, Dcx, NK1R, and DopH was unaltered indicating that there is no general developmental delay in the mutant brainstem (data not shown). In Pax6Sey/Sey embryos some migrating mms neurons used a sub- marginal instead of a marginal migration path (black arrowhead in Figure 2A9; see also Figure S2 and Figure 4A9) and at E14.5 a large number of mutant neurons accumulated around the midline suggesting a reduced pace in midline crossing (black arrow in Figure 2B, 2B9). Furthermore, a subset of Pax6 positive neurons migrated along the midline into the parenchyma of the hindbrain (white arrowheads in Figure 2C, 2C9). We used a variety of markers, e.g. antibodies against the potassium channel Kcnj6, and DiI tracing of mossy fiber projections to discriminate between mms neurons (generating the ECN and LRN) and neurons of the sms (generating the IO). These experiments all indicated a complete loss of neurons in the ECN and a disorganized settling of neurons in the LRN (Figure 2D, 2D9, 2E, 2E9; Figure S2, and data not shown). Many Kcnj6 positive neurons were even observed within the inferior olivary territory (black arrows in Figure 2E9) and dorsally to the IO at the midline (open arrow in Figure 2E9). In agreement with previous reports [16], we found a slight enlargement of the IO at E14.5 when we used the Ets transcription factor Etv1 as a IO specific marker [33] (Figure S3 and data not shown). However, by labeling for the axon-guidance-molecule B (RgmB) no alterations in the general architecture of the IO were seen [34] (Figure S3). This is consistent with our observation that misguided Pax6Sey/Sey mms neurons make only a negligible contribution to the IO or settle in the periphery of the IO. In summary, these data demonstrate that migration of Pax6Sey/Sey mms neurons is severely disrupted. Mutant neurons of the mms are initially delayed. Later, a number of neurons use a sub-marginal migration path, migration is disturbed at the midline and several neurons migrate to ectopic positions along the midline. Lastly, the normal structure of the LRN is lost, the ECN is completely missing, and the IO is enlarged. Figure 2. Migration defects of Pax6Sey/Sey pre-cerebellar neu- rons: histology. Coronal vibratome Pax6 in situ hybridizations of wt (A - C) and mutant (A9–C9) embryos. The Pax6Sey/Sey mutation is a result of single base pair substitution in the Pax6 gene that leads to a shortened non-functional protein product [4]. Pax6 transcript levels, however, are largely unaffected in mutants therefore allowing the detection of Pax6 expressing cells in Pax6Sey/Sey embryos. At E12.5 the mms has not yet formed (see Figure. 1B), but, at E13.5 first neurons of the mms have already crossed the midline (asterisk in A). In the mutant, migration of mms neurons is delayed, some neurons use a sub-marginal migration (arrowhead in A9), and by E13.5 neurons have not reached the midline (asterisk in A9). At E14.5 Pax6 positive neurons accumulate around the ventral midline (arrow in B9) and at E15.5 some neurons migrate dorsally along the ventral midline (white arrowheads in C9). In wt embryos all mms neurons migrate along the marginal migration route (A,B) and no Pax6 positive cells are seen at the ventral midline (C). Immunohisto- chemical labeling with an a-Kcnj6 antibody on coronal E18.5 wt (D,E) and Pax6Sey/Sey (D9,E9) sections. The a-Kcnj6 antibody strongly labels the wt ECN (D) and the LRN (E). a-Kcnj6 is completely absent from the ECN territory in Pax6Sey/Sey embryos (D9) and mutant LRN neurons (E9) are scattered at the LRN territory, within the inferior olivary complex (black arrows in E9), and at both sides of the midline (open arrow in E9). The dotted line in E and E9 indicates the midline. (Scale bar is 1 mm in [A, A9]; 1.2 mm in [B, B9]; 380 mm in [C]; 0.55 mm in [D, D9, E, E9].) doi:10.1371/journal.pgen.1002099.g002 PLoS Genetics | www.plosgenetics.org 4 June 2011 | Volume 7 | Issue 6 | e1002099