QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Movies Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Gilthorpe, J. D. Articles by Wingate, R. J. T. Search for Related Content PubMed PubMed Citation Articles by Gilthorpe, J. D. Articles by Wingate, R. J. T.

The migration of cerebellar rhombic lip derivatives

¹ MRC Centre for Developmental Neurobiology, King’s College London, New Hunt’s House, Guy’s Campus, London SE1 1UL, UK
² INSERM U106, Bâtiment de Pédiatrie, Hôpital de la Salpétrière, 47 boulevard de l’ Hôpital, F-75651 Paris cedex 13, France

View larger version (104K): [in a new window]   Fig. 1. Rhombic lip derivatives at E4 and E5 project to the ventral midline. The schematic diagram (bottom left) shows the preparation of flatmount explants with the rhombic lip of r1 (rl) highlighted in dark grey. Dorsal r1 is the presumptive cerebellum (cb), while ventral r1 and r2-8 comprise the hindbrain (hb). Rhombic lip derivatives were labelled, either acutely by DiI application to the rhombic lip, or cumulatively by isochronic grafting of GFP-electroporated dorsal r1 into host neural tube at E2. Acute labelling reveals that at both (A) E4 and (B) E5, the rhombic lip generates unipolar migratory cells that project leading processes directly to the ventral midline. (C) In an E6 chimaera, GFP-labelled leading processes of cells that have migrated into ventral r1 turn either rostrally or caudally close to the ventral midline (vml). An arrow indicates a neurone with rudimentary dendrites. The rhombic lip (to the left) lies outside the field of view. (D) At higher magnification, turning axons display increased growth cone complexity and short interstitial branches, but do not seem to bifurcate. (E) Immunohistochemical staining of a quail/chick chimaera constructed in a similar manner and with the same field of view shown to that in C. Cell bodies were labelled using the Q'PN antibody (green). The monoclonal antibody QNTAN (red) reveals the presence of an unspecified neuronal membrane-bound epitope (Tanaka et al., 1990) and identifies longitudinal processes as definitive axons [for experimental details, see Wingate and Hatten (Wingate and Hatten, 1999)]. In these and all other flatmount micrographs, rostral is towards the top and the dorsoventral axis runs from left (the rhombic lip) to right (the ventral midline). Plates show the pialward surface of flatmounted brains. Scale bars: in A, 100 µm for A,B,C,E; in D, 100 µm for D. mb, midbrain.

View larger version (98K): [in a new window]   Fig. 2. Rhombic lip derivatives generated after E6 are restricted to the cerebellum. (A) GFP label in dorsal r1 at E6 shows the cumulative distribution of migratory cells generated from the rhombic lip. Close to their origin, cells that are presumably younger exhibit short processes. Cell bodies have accumulated at the interface between dorsal and ventral territory (arrows) where leading processes are aligned longitudinally. The field of view does not include the ventral midline. The rhombic lip (rl) is indicated towards the left. (B) Acute labelling at E6 demonstrates that the leading processes of all later born cells are deflected rostrally at the boundary between dorsal and ventral r1 (arrow). The bright fluorescence to the left of the panel is a site of DiI application at the rhombic lip (rl). (C) Cells generated at E7 within dorsal r1 do not migrate as far as the boundary within 24 hours. The rhombic lip lies outside the panel towards the left. (D,E) At E6, in situ hybridisation for Pax6 and erbB, respectively, reveals that granule cell markers are concentrated at the ventrolateral edge of the cerebellum (arrows). (F) This corresponds with the boundary encountered by acutely labelled rhombic lip derivatives at E6 (arrow). The edge of the explant is highlighted (dotted line). Scale bars: in A, 100 µm for A,D,E,F; in B, 100 µm for B,C.

View larger version (61K): [in a new window]   Fig. 3. Time-lapse confocal microscopy in GFP-chimaeras reveals the mode of cell migration. (A) Opening frame of a time-lapse movie showing cell movement over 24 hours (see Movie 1 at http://dev.biologists.org/supplemental/). Green dots identify cell bodies (a-e) whose progress was monitored from frame to frame (composite). For reference, the cell highlighted in pink is a static neurone (n) just ventral to the cerebellum. The ventral midline is outside the field of view (towards the bottom right). The boxed area highlights the boundary between dorsal and ventral r1 (see C). (B) Superimposing the positions of cells a-e in successive frames reveals the path and speed of migrating cells. The lighter the colour of the trail, the lower the rate of cell body movement (white indicates stationary). The position of the rhombic lip is indicated by a white line (top left). (C) Opening frame of a higher magnification movie (see Movie 3 at http://dev.biologists.org/supplemental/) of the region bounded by the red box in A. An arrow indicates a single leading process approaching the boundary of the cerebellum which fasciculates with rostrally projecting processes. Scale bar: 100 µm.

View larger version (54K): [in a new window]   Fig. 4. The behaviour of rhombic lip derivatives in r1. GFP-chimaeras demonstrate that cells born at E4 and E5 (pink) migrate into ventral r1. Their leading processes turn at the ventral midline (b), while later born migrants (green) turn rostrally at the edge of the cerebellar anlage (a).

View larger version (77K): [in a new window]   Fig. 5. Co-cultures reveal migration cues within r1. (A) Cell migration can be induced from rhombic lip fragments cultured directly on the pialward surface of flatmounted explants. Cells from E4 rhombic lip fragments (green) align themselves along the same axis as endogenous migratory cells labelled with DiI (red) in the underlying E4 explant. The full bilateral dorsoventral axis is shown in the confocal montage with the ventral midline (vml) at its centre. (B) E4 fragment on E6 cerebellum. (C) E6 fragment on E6 cerebellum. (D) E4 fragment on E6 hindbrain. (E) E6 fragment on E6 hindbrain. (F) Radial plots compare the orientation of the leading process of 1683 clearly identified cells from E4 or E6 rhombic lip fragments (n=110) with respect to an axis perpendicular to the ventral midline. For each plot, orientations were scored in 15° bins and plotted as a percentage of the mode. The orientation on E6 cerebellar substrates (top) is contrasted with that on E6 hindbrain (bottom), as indicated in the accompanying schematic diagrams (left). In light grey (middle), the orientation on cerebellar territory is re-plotted with respect to an axis perpendicular to the closest segment of rhombic lip. (G) Cells only exit a fragment of rhombic lip where it contacts the explant substrate (the boundary of the explanted hindbrain is denoted by a broken line). (H) An isolated rhombic lip fragment will produce processes but few cells emerge. As no migrating cells were observed, these fragments were excluded from analysis. Scale bars: in A, 100 µm for A; in H, 100 µm for B-E,G,H.

View larger version (54K): [in a new window]   Fig. 6. Later born cells lose their response to an exogenous source of netrin 1. DiI-labelled rhombic lip cells were challenged with ectopically positioned netrin 1-expressing cells or floorplate (indicated by green dotted lines). (A) Leading processes of E4 migrants extend directly to the midline. (B) Leading processes at E4 stall beneath an ectopic floorplate (fp) fragment placed on the surface of the explant. (C) An exogenous source of netrin 1 can induce both rostral and caudal (arrow) turning in E4 labelled cells. (D) At E6, cell processes turn rostrally at the cerebellar boundary (first seen in caudally labelled migrants). (E) The normal extension and turning of leading processes at E6 cannot be deflected by an exogenous source of netrin 1. Scale bars: in E, 100 µm for A,B,D,E; in C, 100 µm for C.

View larger version (117K): [in a new window]   Fig. 7. Expression patterns and co-culture assays suggest a role for slit-Robo signalling at the rhombic lip. At E7, the expression patterns of Slit2 and Robo2 are complementary in the rhombic lip and EGL, respectively. (A) Whole-mount brain showing Slit2 expression in rhombic lip of the hindbrain (hb) and cerebellum (cb) and at the ventral midline. (B) Robo2 expression in the EGL (egl) of a flatmounted cerebellum. (C) Complementary expression of Slit2 in the rhombic lip (rl) at the same stage. (D) For E4 rhombic lip fragments, cell migration along the dorsoventral axis (left to right) is unaffected by the close proximity of an overlying pellet of control 293-T cells (whose boundary is indicated by green dotted lines). (E) Similarly, migration is unaffected by a pellet of Neuro2a cells placed at a distance to the labelled rhombic lip explant. (F) Migration is attenuated only in close proximity to a source of Slit2. (G) For E6 rhombic lip fragments, migration of labelled cells is again unaffected by control 293-T cells. (H) As at E4, Slit2-secreting Neuro2a cells are unable to affect migration at distance. (I) However, close proximity of Neuro2a cells polarises migration from rhombic lip explants such that cells only exit from the side of the fragment opposed to the source of Slit2. Scale bar: in I, 100 µm for D-I.