A Novel Approach to Selectively Target Neuronal Subpopulations Reveals Genetic Pathways That Regulate Tangential Migration in the Vertebrate Hindbrain Karsten Benzing1, Stefanie Flunkert1, Andreas Schedl2,3, Dieter Engelkamp1,4* 1 Max Planck Institute for Brain Research, Department of Neuroanatomy, Frankfurt, Germany, 2 INSERM UMR636, Centre de Biochimie, Nice, France, 3 University of Nice Sophia Antipolis, Nice, France, 4 Department of Biology, Friedrich-Alexander-University of Erlangen-Nu¨ rnberg, Erlangen, Germany Abstract Vertebrate genes often play functionally distinct roles in different subsets of cells; however, tools to study the cell-specific function of gene products are poorly developed. Therefore, we have established a novel mouse model that enables the visualization and manipulation of defined subpopulations of neurons. To demonstrate the power of our system, we dissected genetic cascades in which Pax6 is central to control tangentially migrating neurons of the mouse brainstem. Several Pax6 downstream genes were identified and their function was analyzed by over-expression and knock-down experiments. One of these, Pou4f2, induces a prolonged midline arrest of growth cones to influence the proportion of ipsilaterally versus contralaterally settling neurons. These results demonstrate that our approach serves as a versatile tool to study the function of genes involved in cell migration, axonal pathfinding, and patterning processes. Our model will also serve as a general tool to specifically over-express any gene in a defined subpopulation of neurons and should easily be adapted to a wide range of applications. Citation: Benzing K, Flunkert S, Schedl A, Engelkamp D (2011) A Novel Approach to Selectively Target Neuronal Subpopulations Reveals Genetic Pathways That Regulate Tangential Migration in the Vertebrate Hindbrain. PLoS Genet 7(6): e1002099. doi:10.1371/journal.pgen.1002099 Editor: Sabine Cordes, University of Toronto and Samuel Lunenfeld Research Institute at Mount Sinai Hospital, Canada Received August 9, 2010; Accepted April 8, 2011; Published June 16, 2011 Copyright: 2011 Benzing et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Max Planck Society. KB was supported by the Graduiertenkolleg Neuronale Plastizita¨t. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: dengelka@biologie.uni-erlangen.de Introduction Understanding cell-specific regulatory mechanisms is a major challenge in the post-genome era. Particularly in mammals, the reiterated usage of the same transcription factor in distinct subsets of cells or during distinct developmental time points provides the basis to generate thousands of individual cell types with a relatively small number of genes. A single transcription factor may therefore elicit variable downstream effects depending on the context of its expression. Tissue-specific knockout strategies based e.g. on the Cre-lox-system, or promoter-driven transgenic models allow a cell- specific manipulation of genes. However, as these techniques rely on the generation of new transgenic animals for each gene- combination analyzed they are laborious and time-consuming. Here, we combined a transgenic model 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 applied our system to decode molecular pathways initiated by the transcription factor Pax6 which is involved in neuronal cell migration and axonal pathfinding processes. Pax6, a homeodomain and paired domain containing transcrip- tion factor, is a major determinant of visual and olfactory sensory structures and is essential for a variety of patterning and pathfinding processes throughout the nervous system [1–3]. Depending on the context and area of expression Pax6 initiates varying downstream effects. Homozygous small eye (Pax6Sey/Sey) mouse and rat embryos, which lack functional Pax6, do neither generate eye nor nasal structures and are deficient in ventral diencephalic structures [4–9]. In the ventral hindbrain and spinal cord, Pax6 controls the dorso-ventral patterning of motorneurons and of interneurons [5,10]. In the cerebral cortex Pax6 determines the neurogenic potential of radial glial cells [11,12]. Throughout the developing nervous system, with the exception of the midbrain, Pax6 is expressed in a ventral and a dorsal pool of progenitor cells. Although the dorsal Pax6 expression domain has achieved much less attention than the ventral domain there is evidence that Pax6 plays a pivotal role in the specification and migration of neurons derived from this domain [13–16]. The dorsal domain of Pax6 positive neuronal precursors of the hindbrain includes the rhombic lip (RL) [14,16] which comprises the interface between the dorsal neuroepithelium and the roof plate. The RL is the source of several tangentially migrating neurons (see also Figure 1A) [14,17–24]. The most notable are the neurons of the marginal migratory stream (mms; also pes) which migrate from the rhombic lip circumferentially around the medulla towards their contralateral destinations to settle in the ECN (external cuneate nuclei) and the LRN (lateral reticular nuclei) [17,25]. Owing to the superficial nature of the mms migration these neurons serve as paradigm to study neuronal migration and axonal pathfinding processes. The highly complex neuronal circuits of the vertebrate nervous system are established during development when growing axons travel considerable distances towards their targets to generate the PLoS Genetics | www.plosgenetics.org 1 June 2011 | Volume 7 | Issue 6 | e1002099