The upper rhombic lip (URL), a germinal zone in the dorsoanterior hindbrain, has long been known to be a source for neurons of the vertebrate cerebellum [1]. It was thought to give rise to dorsally migrating granule cell precursors [2](Figure 1e)Figure 1Migration of neuronal precursor cells from the cerebellar rhombic lip. (a) Lateral view of the head of a zebrafish wild-type embryo at 28 hpf. The region surrounding the MHB, including the posterior mesencephalon, the entire cerebellar anlage, and the anterior rhombencephalon, is marked by a red rectangle. The schematic drawings in (e) and (f) as well as all of the time-lapse movies in the following figures were generated for this area of the brain. In all cases, the figures and schematics were oriented with anterior to the left. (b) Lateral view of the head of a 40 hpf zebrafish embryo simultaneously stained with TO-PRO-3 to label nuclei and BodipyCeramide to visualize cytoplasmic membranes. At this stage, no clear subdivision in the cerebellar anlage between primitive neuroepithelium and differentiation zones can be observed. The apical side of the neuroepithelium along the IVth ventricle is outlined by a yellow line (note the bent nature of this ventricluar zone).Gene expression indicates that the rhombic lip becomes established at 18 hpf in zebrafish embryos and is well set up at 28 hpf, the starting time point of our time-lapse analysis. (c) Lateral view of the hindbrain region of a 20 hpf zebrafish embryo stained for atonal1 expression (blue), a marker for the rhombic lip and its migratory derivatives, and fgf8 expression (red) to demarcate the MHB. First, atonal1 expression can be detected at 18 hpf along the ventricular zone (see inset), just shortly after the opening of the IVth ventricle (our unpublished data). Initially, atonal1 expression does not reach the fgf8 expression domain, which demarcates the posterior part of the isthmus, showing that the rhombic lip is not part of the isthmus but localized posterior to it. Subsequently, expression of atonal1 covers the entire dorsal aspect of the cerebellar anlage and later (d) also the lateral regions of the cerebellar anlage (as example, 26 hpf prior to imaging, inset shows dorsal view). This is in accordance with atonal1 being expressed in migratoy rhombic lip-derived cells. The dynamics of the atonal1 expression pattern already suggest a migratory behavior for URL-derived cells in zebrafish embryos and indicates that at the start point of imaging the rhombic lip is well established. opl, another rhombic lip marker gene, is expressed similarly (data not shown), indicating that the rhombic lip is well established at 28 hpf, the start point for our subsequent time-lapse analysis. (e) Schematic representation of the neuronal migratory pathways previously characterized. Upper rhombic lip derivatives at the ventricular surface in the cerebellar anlage (blue arrows) are thought to migrate dorsally inside the rhombic lip to eventually migrate over the cerebellar anlage to settle in dorsal positions. Ventrally migrating lower rhombic lip derivatives (green arrows) give rise to precerebellar nuclei in the ventral brain stem underneath the cerebellum. (f) In vivo time-lapse analysis reveals that most upper rhombic lip derivatives instead turn to migrate ventrally (blue arrows) at the MHB and eventually settle in the ventral brainstem region underneath the cerebellum. Only URL descendants in very dorsomedial position migrate dorsally over the cerebellar anlage. Schematic drawings according to [5, 25, 26].; however, recent fate mapping studies have questioned the exclusive contributions of the URL to granule cells [3–5]. By taking advantage of the clarity of the zebrafish embryo during the stages of …