1. Direct imaging of in vivo neuronal migration in the developing cerebellum 
Reinhard W. Köster, Scott E. Fraser 
Current Biology 
Volume 11, Issue 23, Pages (November 2001)
DOI: /S (01)

2. Figure 2
URL descendants follow a ventral migratory stream. (a–f) Lateral view of cerebellar anlage of an EF-GVP-UG-injected embryo that has been studied by time-lapse analysis. Cells marked with a colored dot (f) have been traced back to their cell clusters of origin inside the URL marked with the same color (a). Cells from different dorsoventral positions in the upper rhombic lip (blue and pink dots) migrate toward the MHB to turn into a ventral pathway. Eventually, they settle in the ventral brainstem close to cells that are derived from the anterior lower rhombic lip (green dots). Insets in (b)–(e) display a higher magnification of the area indicated, to show the morphology of individual migrating URL cells. The process connecting the migrating cell with the URL can be seen clearly (e.g., [d], upper process is 15 μm long) until it gets pulled in (see Movie 1 and Figure 3b–d). Each frame in the figure represents a merged projection of the image stacks collected at each time point. In the first (a) and last (f) frame of the time-lapse analysis, overlays with transmitted light images are shown. (a,f) The MHB is outlined as dashed yellow line; (a) the upper rhombic lip and (f) the posterior border of the cerebellum are outlined as yellow lines. See Movie 1 for full time-lapse presentation. cb, cerebellar anlage; LRL, lower rhombic lip; mes, mesencephalon; MHB, midhindbrain boundary; rh, rhombencephalon; URL, upper rhombic lip.
Current Biology  , DOI: ( /S (01) )

3. Figure 3
Migrating URL-derived neuronal precursors change their morphology and behavior at the MHB. (a–d) Lateral view of cerebellar anlage of a tub-GFP-Uunc-injected embryo. (a) With reaching the MHB, the leading neuronal precursor (blue star) halts in forward movement and extends a longer process running ventrally along the MHB ([b,c], arrowhead; [b,c] are higher magnification to better demonstrate the changes of the processes of the individual neuron marked with a blue star in [a] and [d]). At the same time, the trailing process gets loosened ([b], blue arrow) and retracted ([c], blue arrow), eventually resulting in a change of cell polarity from bipolar (b) to unipolar (d). (e–g) Frames taken from different time-lapse studies in which process elongation along the MHB is particularly pronounced. (e) Neuronal precursor cell projects process ventrally along the MHB. (f) Higher magnification of cell shown in (e) (yellow arrowhead); note that process is thickened at the end, reminiscent to a growth cone (blue arrowhead). (f) At later stages, when more neuronal precursors have reached the MHB, process length can reach between 30 μm (e) and 50 μm (f), three to five cell diameters, in length. Each frame in the figure represents a merged projection of the image stack collected at each time point. The first and last sections were overlaid with a transmitted light image (a,d,e) to allow localization of the observed neurons inside the cerebellar anlage. The MHB is outlined as yellow dashed line; the URL is marked as a continuous yellow line (a,d). Times in (b)–(d) are given as minutes passed since frame (a). See Movie 2 for full time-lapse presentation. cb, cerebellar anlage; mes, mesencephalon; MHB, midhindbrain boundary; URL, upper rhombic lip.
Current Biology  , DOI: ( /S (01) )

4. Figure 1
Migration 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].
Current Biology  , DOI: ( /S (01) )