Author Summary In mammals, many genes execute a unique set of distinctive and common functions in different cell types. Strategies to address these individual roles often involve the generation of series of transgenic animals. Here, we present a novel approach that combines a single transgenic mouse line with tissue-specific transfection protocols and organotypic cultures to enable the quick analysis of numerous genes in a cell-specific manner. As a proof of principle, we analyzed the function of transcrip- tion factors in tangentially migrating neurons of the developing vertebrate hindbrain. We identified a tempo- rary halt in migration as a novel mechanism for neurons to decide whether to cross or not cross the midline. Our model may serve as a general tool to quickly study axonal pathfinding, neuronal cell migration, or patterning pro- cesses in a well-defined population of neurons. appropriate connections. This wiring process depends on attrac- tive and repulsive factors which emanate from final or interme- diate cellular targets and which are interpreted by cell surface receptors located on axonal growth cones [26,27]. Although the general principles were uncovered during the past years our understanding of axonal pathfinding processes is far from being complete. Current methods to analyze candidates regulating neuronal migration and axonal navigation processes are laborious and often involve the generation of transgenic animals for each gene analyzed. Non-transgenic methods, as DiI labeling of neurons or vector-driven mis-expression of gene constructs, are suitable for use with certain applications, however, they are neither cell specific nor can they be targeted to distinct neuronal subpopulations. Here we describe a novel transgenic mouse model, which allows the specific and exclusive visualization and manipulation of subsets of neurons in the developing brain. To demonstrate the power of this system we have analyzed the role of Pax6 in migrating neurons of the brainstem. In Pax6 mutant mice migration of these neurons is distorted and some neurons differentiate at ectopic positions. Using transplantation, knock-down and over-expression experi- ments we show that distinct migratory features are controlled by discrete sets of Pax6 downstream genes. These results demonstrate the potential of our transgenic mouse model as a tool to study the role of Pax6 in individual neurons. Moreover, our system should be widely applicable to study virtually any gene that acts during cell determination, axonal pathfinding and/ or cell migration processes. Results A novel mouse model to visualize and manipulate subsets of neurons The functional analysis of genes in restricted tissues often involves the generation of inducible knockout mice or mice over expressing transgenic constructs. To simplify this time-consuming process we developed an in vitro model that enables the visualization and manipulation of defined populations of neurons. To label neurons in a largely unlabelled background we searched for genes that were expressed in only a subset of neuronal precursors and in migrating neurons. Pax6 meets these criteria ideally. Pax6 is expressed in several groups of tangentially migrating neurons and their precursors as well as in a small population of radially migrating neurons and their precursors (Figure 1A–1C) [5,10,12,14,16,28]. Pax6 Controls Cell Migration in the Brainstem We adopted the Tet binary system [29] and generated YAC (yeast artificial chromosome) transgenic mice which expressed the tetracycline dependent transactivator (tTA) in all Pax6 positive cells. A 420 kb YAC spanning the human PAX6 locus (Y593) [30] was modified such that the PAX6 coding region was replaced with a cassette containing an IRES (internal ribosomal entry site) and the tTA (Figure 1D). Previously, we and others had shown that the unmodified YAC Y593 contains all elements driving full functional PAX6 expression [30–32] and, in agreement with this, Tg(PAX6-tTA) mice showed a wide overlap of tTA and endogenous murine Pax6 expression (Figure S1). Tg(PAX6-tTA) mice were entirely normal and control experiments insured that neuronal patterning and migration was unaltered. tTA is a transcriptional activator that at moderate levels of expression is completely inert in vertebrates, yet, enables the activation of artificial constructs containing a tTA-DNA-binding element (TRE = tetracyline responsive element). To examine whether our transgenic model specifically allows the labeling of only Pax6 positive cells we introduced by electroporation reporter constructs driving the green fluorescent protein into transgenic embryos. In all instances only Pax6 positive cells, e.g. retinal precursor cells, cortical precursors, or cerebellar granule cells, expressed the reporter genes (Figure S1). Non-transgenic embryos or Pax6 negative tissues did not induce reporter gene expression (Figure 1H, Figure S1). Together these results demonstrate that Tg(PAX6-tTA) mice enable the targeting of reporter gene constructs specifically to Pax6 positive cells and tissues during development. As Tg(PAX6-tTA) mice allow any gene to be targeted to Pax6 expressing cells, they are of potential value to study neuronal migration and axonal pathfinding processes and for the analysis of Pax6 downstream effects. As a proof of principle, we chose to focus on the marginal migration stream (mms). Like other tangentially migrating neurons, mms neurons use the same or similar navigational cues as do growing axons, and migration of mms neurons is severely disturbed in Pax6 mutant Pax6Sey/Sey mice (see below). Neurons of the mms are generated at the rhombic lip and migrate circumferentially around the embryonic brainstem to generate the contralateral lateral reticular (LRN) and the external cuneate (ECN) nuclei (Figure 1A) [16,21–23,25]. Migration starts at E13.0 and is completed by E16.5. Pax6 is expressed in precursors at the rhombic lip, in all migrating neurons of the mms and during the initial period of settling in the target nuclei (Figure 1B, 1C and data not shown). Antibody staining and in situ hybridization (not shown) of Tg(PAX6-tTA) mice confirmed a complete overlap of Pax6 and tTA expression in these neurons (Figure 1I). To visualize migrating mms neurons in Tg(PAX6-tTA) mice, reporter constructs were introduced into neuronal precursor cells in the left rhombic lip by electroporation at E12.5 before migration had begun (Figure 1E). Whole brainstems including the cerebellar primordium were then sustained in organotypic filter cultures for up to 14 days as an open book preparation which allowed the observation of migrating neurons with a fluorescence microscope from above (Figure 1F, 1G). Our approach to use a binary system ensured that only Pax6 positive neurons containing tTA and a TRE reporter construct expressed the desired reporter genes. This procedure resulted in the specific labeling of mms neurons originating only from one rhombic lip. Pax6 positive neurons originating from the opposite rhombic lip remained unlabelled as were Pax6 negative (and therefore tTA negative) neurons originating from regions close to the rhombic lip. Unlabelled neurons included neurons of the submarginal migra- tion stream (sms) which generate the inferior olive (IO) thus demonstrating the specificity of our model. To allow the PLoS Genetics | www.plosgenetics.org 2 June 2011 | Volume 7 | Issue 6 | e1002099