Pax6 Controls Cell Migration in the Brainstem Figure 6. Summary of phenotypes observed in Pax6Sey/Sey RL derived neurons. (A) In Pax6Sey/Sey embryos the mms migration is delayed, some neurons migrate sub-marginally, neurons are arrested at the midline, the ECN is lost, the LRN is disrupted, and the IO is enlarged [16]. Additionally, the aes is reduced resulting in an absence of the pontine nuclei and granule cells of the cerebellum migrate ectopically into the inferior culliculus [14]. To search for genes expressed in mms neurons a large scale in situ hybridization screen was performed and a summary of the expression data of selected genes is shown in (B). The color code indicates expression of individual genes during migration of mms neurons: dark grey marks expression at the RL, beige before midline crossing, red during midline crossing, blue after crossing the midline and light grey marks expression during settlement in the ECN and LRN. Genes, which show altered expression levels in Pax6Sey/Sey embryos are marked in red. (C) A gene cascade in which Pax6 and Math1 positively regulate Pou4f2 expression leads to a repression of Pou4f1 expression. mms: marginal migration stream forming the external cuneate (ECN) and lateral reticular (LRN) nuclei; sms: sub-marginal migration stream forming the inferior olive (IO); aes: anterior extramural migration stream forming the pontine and pontine reticulotegmental nuclei; egl: external granule cell layer of the cerebellum; RL: rhombic lip; FP: floor plate. doi:10.1371/journal.pgen.1002099.g006 study axonal pathfinding and neuronal cell migration processes. Migrating mms neurons are easily accessible as they navigate along the pial surface. Our model takes advantage of the superficial migration of these neurons and provides a straightforward assay to specifically label and manipulate these cells without affecting their surroundings. Members of most families of guidance receptors (Netrin receptors, Slit receptors, Semaphorins, Eph receptors, and Ephrins) are expressed in migrating mms neurons [48,53,54] (Engelkamp, unpublished) and at least two of these pathways are essential for the correct guidance of mms neurons: the Slit/ Robo - [48] and the Netrin/ Dcc- pathways [53,56,57] (see also Table 1). Our system should therefore also have important implications for the study of the signal cascades entailed in these pathways. In summary, we have established a novel model system which allows the simultaneous visualization and manipulation of neuronal subpopulations. As a prototypical model we have focused on the role of Pax6 in migrating brainstem neurons. Yet, our results imply that our model system is applicable to a range of other cells in the developing brain and may therefore serve as a general tool to quickly study axonal pathfinding, neuronal cell migration or patterning processes. Materials and Methods Animals The Small Eye allele [4] was maintained on a CD1 background. Embryos were obtained from matings of heterozygote (Pax6Sey/+) mice. 0.5 denotes the morning when the vaginal plug was found. Experiments were always performed on matching pairs of control (wt) and Pax6Sey/Sey embryos that were carefully staged. All phenotypes described were confirmed on at least six individual Pax6Sey/Sey embryos obtained from different crossings. There was no noticeable phenotypic difference between Pax6Sey/+ and wt embryos and therefore, in our experiments wt designates wt and Pax6Sey/+ embryos. For brainstem cultures, matings between heterozygote Tg(PAX6-tTA) and wt CD1 mice or between heterozygote Pax6Sey/+/ Tg(PAX6-tTA) and heterozygote Pax6Sey/+ mice (to generate Pax6Sey/Sey cultures) were used. Genotyping was performed by PCR with primers directed against the Tet repressor (upper: GCG- CTGTGGGGCATTTTACTTTAG; lower: CCGCCAGCCCC- GCCTCTTC). All animal procedures were carried out in accordance to the guideline approved by institutional protocols. Generation of Tg(PAX6-tTA) mice YAC Y593 [30] was modified such that exons 8 to 11 of the Pax6 gene were replaced by homologous recombination with a construct containing the following elements in 59 to 39 order: Pax6k30 – IRES – tTA – loxP – LYS2 – loxP – Pax6k32. Pax6k30 and Pax6k32 corresponded to the sequences 29.792 to 30.296 and 31.587 to 32.095 of the Pax6 cosmid cFAT5 (NCBI accession no. Z95332), respectively, and were generated via PCR. The IRES (internal ribosomal entry site) was derived from pIRES-EGFP (Invitrogen), however, the original ATG-11 start codon was reconstituted to enhance translational initiation. tTA (Tet-On- system), a fusion of the tetracycline repressor and the activation domain of VP16, was derived from pUHD15-1neo (Clontech). The LYS2 gene from S. cerevisiae was derived from pAF107, which was obtained from B. Dujon, Institute Pasteur, Paris, France [59]. LoxP sequences were generated via PCR. All constructs were sequence verified. Homologous recombination in yeast was performed using standard techniques. The integrity of the recombined YAC was then verified by PCR and southern blotting. Preparation of the YAC DNA and the generation of transgenic mice were as described [30]. In situ hybridisation In situ hybridization was performed on free floating vibratome sections as previously described [14]. Probes for Math1 [60], Neurod1 and Neurod2 [61], Pax6 [47], Unc5h3 [62] and Slit1, Slit2 [63] were PLoS Genetics | www.plosgenetics.org 10 June 2011 | Volume 7 | Issue 6 | e1002099