Pax6 Controls Cell Migration in the Brainstem Figure 1. A novel mouse model to visualize and manipulate Pax6 positive neurons. (A) Schematic diagram of tangentially migrating neurons in the mouse brainstem. Pax6 positive neurons of the marginal migration stream (mms) and of the pontine migration (pm) are derived from the rhombic lip (RL) and generate the lateral reticular (LRN), the external cuneate nuclei (ECN), the pontine nuclei and the pontine reticulotegmental nuclei (not shown), respectively. Pax6 positive external granule cells (EGL) initially migrate rostrally on the surface of the cerebellar primordium (Cb). Pax6 negative neurons of the submarginal migration stream (sms) generate the inferior olive (IO). Distribution of Pax6 positive cells in the wt hindbrain at E12.5 (B) and at E14.5 (C) as shown by in situ hybridization on coronal vibratome sections. Pax6 is expressed in precursor cells at the caudal RL (open arrows in B,C), in ventral precursor cells at the basal plate, in groups of ventrally migrating neurons and in the mms (filled arrow in C). The dorsal alar plate (brackets in B,C), the hypoglossal nuclei (nXII), and the floor plate are negative for Pax6. (D) Diagram of the targeting construct to replace the coding sequence of human PAX6 in YAC Y593 with an IRES-tau-tTA-LYS2 cassette. For a more detailed description of the construct see the Materials and Methods section. (E) Schematic drawing of the electroporation and brainstem culture procedures. Responder constructs consisting of tetracycline responsive elements (TRE), a minimal promoter (pmin), and a reporter gene (e.g. EGFP) are injected into the fourth ventricle of E12.5 Tg(PAX6-tTA) embryos and then transfected into one RL by electroporation. Subsequently, the hindbrain is dissected and cultured with the ventricular face onto MilliporeCM filters. After prolonged cultures mms neurons settle either in the ipsi- or the contralateral LRN (LRNi, LRNc), or in the ECN. All neurons project to the contralateral cerebellar hemisphere (Cb). (F and G) Migrating mms neurons visualized by EGFP fluorescence in transfected brainstems. The enlarged areas shown are indicated by white boxes in (E). After 1 day in vitro culture (1 DIV) migrating neurons with long leading and short trailing processes emerge from the transfected RL (F). After 4DIV the majority of mms neurons have settled in contralateral LRN (LRNc) (G). Genotyping of transfected cultures confirms that responder constructs are exclusively activated in Tg(PAX6-tTA) and not in non-transgenic embryos (H). Double-immunolabeling of mms neurons with Pax6 and VP16 (to recognize tTA) antibodies confirm a complete overlap of Pax6 and tTA expression (I). A responder vector containing two TRE elements drives expression of two genes simultaneously: a cytoplasmic green fluorescent protein (EGFPm) and a nuclear red fluorescent protein (DsRed2nls) (J). The left and right images are green and red fluorescent images, respectively, of migrating mms neurons after 1.5DIV. K, K9, and L are low magnification fluorescence (K, L) and phase contrast (K9) images of cultures transfected with an EGFP PLoS Genetics | www.plosgenetics.org 3 June 2011 | Volume 7 | Issue 6 | e1002099