positive results in culture assays may be used subsequently to generate transgenic lines. Of note however, some of the phenotypes reported here, for example, the midline arrest or the altered ipsi- to contra-lateral ratio, would have been missed in purely transgenic systems. Second, variation of the electroporation protocol allows transfections ranging from just a few cells to a complete Pax6 expression domain with thousands of cells. Hence, our approach allows adjustment according to the needs: either to monitor single migrating cells or to determine global patterning effects. In addition, neighboring cells and non-electroporated contra-lateral sides serve as internal controls. The usefulness of our system critically depends on the tightness of the TRE based promoter and on the ability of the constructs to express two genes simultaneously. To ascertain the tightness of our system we used repeated electroporations and high DNA concentrations (up to 5 mg/ml). Even under these extreme conditions we were never able to detect any reporter gene expression in Pax6 negative cells at any developmental stage. Thus, under the conditions used in this report the combination of Tg(PAX6-tTA) mice and TRE based promoters allow expression of reporter gene constructs only in Pax6 positive cells. It is also important to note, that our strategy to use a YAC based technology combined with an internal ribosomal entry site (IRES) resulted in moderate levels of reporter gene expression which were in the range of physiological concentra- tions. To ensure the simultaneous expression of two reporter genes we tested several types of TRE constructs. Only our approach, to use two consecutive TRE based promoters led to the activation of nearly equal amounts of two genes at the same time in the same cell. A bidirectional TRE element that previously had been shown to work in transgenic animals failed in our system [45]. One obvious difference is that in transgenic animals typically multiple copies of constructs are stably integrated into the genome, whereas, in our assay transfections were transient. Pax6 loss of function phenotypes are often highly complex involving massive malformations in the affected organs. Pax6 is expressed in neuronal precursors of the telencephalon, commis- sural neurons in the dorsal spinal cord, in adult neuronal stem cells, the early eye cup, in the pancreas, in precursors and in migrating cells of several tangential and radial migration streams of the rhombencephalon and of the forebrain [5,6,10,11,14, 28,46,47]. In addition to its technical advances, the Tg(PAX6-tTA) model represents a novel, highly versatile technology to study the function of Pax6 or any other gene in these tissues. As a paradigm, we have dissected the role of Pax6 in tangentially migrating cells of the brainstem. In principle, however, this system shall be applicable to any Pax6 positive tissue and we have initial evidence that our model allows to specifically target Pax6 positive telencephalic precursor cells, cerebellar granule cells, the devel- oping retina, the rostral migratory stream, the pontine migration and ventral precursor cells of the brainstem and spinal cord (Figure S1 and data not shown). With the help of this model it should therefore be possible to systematically analyze cell fate decisions and the migratory behavior of Pax6 expressing cells at any developmental stage. Several studies have revealed that Pax6 is required for hindbrain and spinal cord development [5,7,10,14,15]. Our work adds that Pax6 also controls the determination and migration of rhombic lip derived neurons (for a summary see Table 1 and Figure 6). Pax6 functions twofold: first, Pax6 controls guidance cues which push migrating mms neurons to the marginal path and which control the settling pattern of LRN neurons. The most likely sources of these cues are the hypoglossal nuclei which are located close to the midline and in proximity to the LRN. Slit1 and Slit2 are expressed in the hypoglossal nuclei and the Slit receptor Robo3 is expressed in Pax6 Controls Cell Migration in the Brainstem migrating mms neurons [48]. Slit expression provided by the hypoglossal nuclei may therefore act as repellent to push mms neurons to a marginal migration route and may also specify the settlement of neurons in the LRN. The loss of Slit-expressing hypoglossal nuclei in Pax6Sey/Sey embryos [5,10] causes a major reduction of the repellent (a minor source of Slit is still present in midline cells). Consequently, migrating mms neurons would use a more sub-marginal migration route and settle less organized in Pax6Sey/Sey embryos. Furthermore, Slit expression at the RL may be involved during the initial phase of mms migration. Secondly, Pax6 functions cell-autonomously in migrating mms neurons to control the determination, the timing of migration, and midline crossing. Several genes show altered expression in Pax6Sey/Sey mms neurons (Table 1: Pou4f1, Pou4f2, Unc5h1, Mafb, Chordin) and may convey individual aspects of migration. We and others find that several transcription factors relay Pax6 downstream effects in dorsal brainstem neurons: Ngn1 in precursor cells ventral to the RL [16], and Pou4f1, and Pou4f2 in migrating neurons (this report). Mis-expression of Ngn1 or Ngn2 in Pax6Sey/Sey embryos failed to rescue the migration defects observed in the Pax6 mutant (Table 1). Neither did the mis-expression or down- regulation of these genes generate small eye - like migration defects in wt embryos (Table 1). On the other hand, Pou4f2, which is lost in the mms of Pax6Sey/Sey embryos (Figure 5B and also in the pontine migration and in the cerebellum, data not shown), alters migration behavior of mms neurons. Together these data suggest that Pou4f2 may regulate genes involved in pathfinding processes, whereas, Ngn1 acts earlier in the cell determination process. There are striking similarities in gene expression pattern between sensory neurons and RL derived neurons. We found that at least two thirds of the genes which are co-expressed with Pax6 and Pou4f2 in retinal ganglion cells are also co-expressed with these genes in mms neurons. Furthermore, genetic hierarchies seem to be analogous: in the retina Math5 controls Pou4f2, which then acts upstream of Pou4f1 [38,41–43], whereas, in RL derived neurons Math1, a close homologue of Math5, initiates related pathways. General genetic pathways are conserved between retinal and RL derived neurons and our model may therefore help to elucidate some of the phenotypes observed in Pou4f1-/- and Pou4f2-/- mice. Both mouse models have revealed distinct axonal pathfinding errors [39,49–52]. Mis-expression of Pou4f2 (or Math1) in RL derived neurons stalls growth cones at the midline for several hours. To our knowledge, this is the first report of such a midline arrest and it may thereby be a paradigm for a novel mechanism controlling midline crossing. The arrest does neither induce a growth cone collapse nor does it inhibit midline crossing per se as all neurons generate axons that cross the midline after a ‘‘waiting period’’. These axons all migrated into the cerebellum like those of control cultures. Gfi1 mis-expression and Gap43 knockdown were able to partially mimic the Pou4f2 induced phenotype, however, additional unknown targets or a combinatory code may be needed to elicit the full phenotype. As Pou4f2 was only expressed in about 1/4 of wt mms neurons, the loss of Pou4f2 in Pax6Sey/Sey embryos mimics only minor aspects of the Pax6Sey/Sey phenotype. The down regulation of Pou4f2 by shRNA constructs resulted in a severe midline disturbance of neuronal processes at similar to the phenotype observed in Pax6Sey/Sey cultures, whereas, in control cultures neuronal processes crossed the midline instantly (data not shown). Comparable phenotypes were also observed in Pax6Sey/Sey cultures, in cultures transfected with Pax6 shRNA constructs, and in Pax6Sey/Sey brainstem sections. Tangentially migrating neurons follow similar navigational cues as developing axons [14,19,48,53–58]. Hence, tangentially migrating neurons of the mms provide an excellent system to PLoS Genetics | www.plosgenetics.org 9 June 2011 | Volume 7 | Issue 6 | e1002099