obtained from H. Zoghbi, A. Bartholoma¨, R. Hill, S. Ackerman, and M. Little, respectively. Probes for Dcc and RgmB were as published [34,64]; other probes were obtained by RT-PCR. The PCR products were subcloned and their identities were confirmed by sequencing. The general staining patterns of all probes matched published expression patterns. Probes were as follows: Etv1 (bp 853– 1820 of NM_007960); Fgfr2 (bp 343–1192 of NM_201601); Pou4f1 (bp 1321–2199 of NM_011143), Pou4f2 (bp 216 – 1762 of S68377); Robo3 (bp 3648–4673 of NM_011248). Several genes which are down- or up-regulated in Pax6Sey/Sey embryos were identified with the help of a large scale in situ screen using .300 putative candidates. Individual probes are available on request. Recombinant constructs The vector for the co-expression of two constructs in Tg(PAX6-tTA) mice contained the following elements in 59 to 39 order: MCSI – TRE – PminCMV – IntronA - BGHPolyA – MCSII – TRE - SV40PolyA; MCS = multiple cloning sites; TRE = 7 repeats of the tetracycline responsive element, PminCMV = minimal CMV promoter, and IntronA were from ptetOi-MCS (obtained from Martin Spiegel, T u¨bingen); SV40polyA and BGHPolyA = polyadenylation signals (derived from pTetOi-MCS and pRc/ CMV, Invitrogen, respectively). Fluorescent markers to label migrating cells were a modified EGFP or DsRed2 (Clontech). Full length clones for Gfi1, Math1, Math5, Ngn1, and Ngn2 were obtained from the German Resource Center for Genome Research (RZPD) and sequence verified; clones for all other genes were obtained by RT-PCR and confirmed by sequencing. Fusions with a triple HA- tag or the engrailed repressor domain (EnR) were generated by PCR. shRNA constructs were generated in the psiSTRIKE vector (Promega) using the Promega Web tool for designing the hairpin oligonucleotides. In the psiSTRIKE vector shRNAs are expressed under control of the U6 RNA polymerase promoter. Efficiency of shRNA knockdown was demonstrated in HEK293 cells using the psiCHECK/ Dual Luciferase system according to the manufactur- ers protocol (Promega). All constructs were sequence verified. Brainstem cultures Responder constructs (2-4 ml at 0.5 mg/ml in GBSS/ 0.01% Methyl Fast, Sigma) were injected into the fourth ventricle of E12.5 wt and Tg(PAX6-tTA) mouse embryos by using glass needles. Electroporation was then performed with forceps-like electrodes with platinum ending (Ø = 0.5 mm) (one Electrode above the right RL and the other under the left jaw). Conditions were 8 pulses at 50V, 50msec with a pulse interval of 1sec. We used the square pulse generator EPI2500 (L. Fischer, Heidelberg). After electro- poration, the hindbrain (rhombomeres 1–8 including the cerebel- lar anlage) was dissected, opened at the roof plate and cultivated with the ventricular site onto MillicellCM filters (Millipore) in culture medium (DMEM/F12 (1:1); 0.6% Glucose; 0.02 mM Glutamine; 5 mM HEPES; 5% Fetal Calf Serum; 5% Horse Serum) at 37uC and 5%CO2. Immunohistochemistry Depending on the antibodies used, brainstem preparations were fixed with 4% or 0.2% PFA in PBS for 12 hours at 4uC. Cryosections were cut at 14 mm. Primary and secondary antibodies used for staining were as follows: mouse monoclonal (mAb) a-Pax6 ([10], 1:1000, DSHB); rabbit pAb a-Pou4f2 (also Brn3b, 1:300, Covance); rabbit pAb a-Kcnj6 (also Girk2, 1:300, Chemicon); rabbit a-Wt1 (Santa Cruz); rabbit a-VP16 (Clontech); a-HA-tag (1:100, Roche) and a-mouse and a-rabbit secondary antibodies conjugated with Alexa488 or Alexa596 (Molecular Pax6 Controls Cell Migration in the Brainstem Probes). Quantification of Pou4f2 positive mms neurons was done on every 3rd of serial sections double stained for Pax6 and Pou4f2. Image analysis Images were taken at a Zeiss Axiophot microscope equipped with a Spot camera, at a confocal Zeiss LSM microscope, or at a Leica MZ12 equipped with a camera device. Images were processed using the MetaView software (Universal Imaging Corporation) and Adobe Photoshop. To perform statistical analysis the position of the ECN and the LRN were determined in wt un-manipulated cultures by in situ RNA staining of Pax6 and Kcnj6. The resulting territories were then overlaid onto the electroporated cultures with the help of three landmarks: a) the position of the rhombic lip; b) the position of the floor plate; and c) the position of the superior and inferior olivary complexes, which both are visible in phase contrast images of the cultures. This procedure allowed classifying 97% of labeled neurons on the contralateral side and 90% on the ipsilateral side as either ECN or LRN neurons. The remaining 3% (or 10% for the ipsilateral side) of labeled cells were scattered neurons mainly in between the ECN and the LRN. Quantification of growth cones arrested at the midline in wt cultures was done by counting all growth cones in a 25 mm wide territory at the midline. Continuous observations of cultures implied that mms growth cones traveled at an average speed of at least 500 mm/day, suggesting that within any 25 mm interval only 5% of growth cones should be detected if migration would not pause. Quantification of the volume of the inferior olive was done with AxioVision (Zeiss). Supporting Information Figure S1 Pax6, tTA and EGFP expression in tg(PAX6-tTA) embryos. (A,B,E,F,I,J) In situ hybridizations of coronal vibratome sections of tg(PAX6-tTA) transgenic embryos. Alternating sections were stained for either Pax6 (A,E,I) or tTA (B,F,J). In the developing eye, Pax6 and tTA are co-expressed in the neural retina (nr), the lens, and the surface ectoderm (s.e.) which generates the future cornea. In the forebrain, both genes are co-expressed in the cortex (ctx), the ventral diencephalon (di), and the epithalamus (epi). In the cerebellum, Pax6 and tTA are co-expressed in granule cells of the external granule cell layer (egl). (C,D,G,H,K,L) Electroporation of EGFP reporter constructs into tg(PAX6-tTA) transgenic embryos. After electroporation tissues were cultured for one or two days on MilliporeCM filters. Reporter gene expression is activated in the developing retina, but not in the surrounding tissue (C); (D) Higher magnification of retinal precursor cells. (G) Electroporation of the telencephalon at E13.5 results in a specific labeling of the Pax6 positive cortex (ctx, blue dotted line), but not of the Pax6 negative striatum (red dots) or other surrounding tissues. (H) Electroporation of the E18.5 anterior telencephalon reveals precursor cells with the typical appearance of radial glial cells; vz, svz, iz and cp denote the ventricular, subventricular, and intermediate zones, and the cortical plate, respectively. (K) In the developing cerebellum EGFP expression is seen in granule cells which leave the transfected region and start to migrate parallel to the cerebellar surface. (L) Higher magnification of (K). Scale bar: 0.6 mm in [A,B]; 1.9 mm in [E,F]; 0.8 mm in [I,J]; 400 mm in [C,H, K]; 1.7 mm in [G]; and 150 mm in [D,L]. (TIF) Figure S2 Pax6Sey/Sey Brainstem defects. The hypoglossal nucleus (black arrows) is lost in Pax6Sey/Sey embryos as shown by Slit2, Isl1, L1Cam, and Unc5h3 labelling (A–D). Migrating mms neurons (white arrows) take a submarginal route in Pax6Sey/Sey embryos as shown by Robo3, Dcc, and Cntn2 (also: Tag1) staining (E–I). Zic1 marks mms neurons which ectopically settle in the IO territory of PLoS Genetics | www.plosgenetics.org 11 June 2011 | Volume 7 | Issue 6 | e1002099