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First published online December 7, 2008
doi: 10.1242/10.1242/dev.025502

Development 136, 41-50 (2009)
Published by The Company of Biologists 2009
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Biased selection of leading process branches mediates chemotaxis during tangential neuronal migration

Francisco J. Martini1,*, Manuel Valiente1,*, Guillermina López Bendito1, Gábor Szabó2, Fernando Moya1, Miguel Valdeolmillos1,{dagger},{ddagger} and Oscar Marín1,{dagger},{ddagger}

1 Instituto de Neurociencias de Alicante, Consejo Superior de Investigaciones Científicas and Universidad Miguel Hernández, 03550 Sant Joan d'Alacant, Spain.
2 Institute of Experimental Medicine, 1083 Budapest, Hungary.


Figure 1
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  		Fig. 1. Stereotyped dynamic behavior of tangentially migrating interneurons. (A,B) Different morphologies of interneurons derived from the medial ganglionic eminence (MGE). (C) Experimental paradigm. NCx, neocortex. (D) Migration of MGE-derived cells in E13.5 dsRed-electroporated slice after 36 hours in culture. (E) Time-lapse sequence of a dsRed-electroporated interneuron (asterisk) migrating from the subpallium to the cortex in a slice culture. Time is depicted in hours: minutes. The bifurcation point of the leading process is marked by a broken blue line. The last frame in the sequence shows superimposed images of the frame t=0:00 (green) and t=0:52 (red). (F,F') Time-lapse sequence of a dsRed-electroporated interneuron migrating through the subpallium in a slice culture. This neuron was recorded for more than 7 hours to analyze several migratory cycles; only selected frames are displayed (see Movie 1 in the supplementary material for a complete movie version). The bifurcation points are marked by broken blue lines, and each leading process branch is coded with a colored arrowhead. Small arrows indicate small very transient processes. The drawings in F' illustrate the morphology of this cell for each of the frames shown in F. Scale bars: 20 µm in A,B,E-F'; 300 µm in D.

 

Figure 2
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  		Fig. 2. Branch dynamics during tangential migration. (A) A telencephalic slice from an E16.5 Gad65-Gfp embryo. GABAergic cells are observed in green in the different regions in which migration was studied: subpallium (lateral ganglionic eminence, LGE), cortical subventricular zone (SVZ) and cortical plate (CP). Owing to the massive accumulation of migrating neurons in the subpallium, analysis of neurons migrating through the LGE was performed in slice cultures in which the MGE was electroporated with Gfp or dsRed (as in Fig. 1). (B-D) Representative images of the morphology of cells migrating through the LGE (B), SVZ (C) or CP (D). (B'-D') Quantification of the frequency of angles form between leading process branches in cells migrating through the LGE (B'), SVZ (C') or CP (D'). Mean angles are 45.32° (LGE), 35.76° (SVZ) and 51.83° (CP), respectively. Cells inside the highly packed SVZ generated very small branching angles with high frequency (C'), whereas neurons in the CP branched at large angles with much more frequency (D'). The frequency of angles greater than 70° is significantly higher in the CP than in the LGE or SVZ ({chi}2 test, ***P<0.001). (B'',D'') Superimposed images of movie frames recreate the path followed by migrating neurons in the LGE (B'') and CP (D''). Green dots indicate the position of the nucleus in each nucleokinesis. Diagrams depict directional changes in these cells. New branches are shown in green, and the chosen branch is marked with a red arrowhead. Numbers indicate the angle formed by the branches. ac, anterior commissure; H, hippocampus; MZ, marginal zone; NCx, neocortex; PCx, piriform cortex; Str, striatum. Scale bars: 500 µm in A; 25 µm in B-D; 20 µm in B'',D''.

 

Figure 3
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  		Fig. 3. Branch dynamics during Nrg1-induced chemotaxis. (A-A'') Images of a dsRed-electroporated slice perfused with a micropipette containing recombinant EGF domain from Nrg1 (13 nM) and Alexa 488 (green channel). To induce drastic changes in direction, cortical interneurons (red channel) migrating through the LGE were confronted with the micropipette at an angle that is perpendicular to their normal trajectory. (B-B') Schematic representation of the trajectory change followed by the cell shown in C. White arrow indicates the micropipette. (C) Representative time-lapse sequences of a migrating cell that developed drastic trajectory changes in response to the chemoattractant. The cell generates a new leading process toward the pipette immediately before changing its trajectory (t=1:35). The angle generated before the most significant change in direction is the largest made by the neuron during this sequence (C). The cell chose the branch oriented towards the chemoattractant to continue migration. The gradient is also visualized in red owing to laser cross-contamination. (C'-C'') Drawings illustrate the morphology of the cell shown in C. Diagrams in C'' depict the movement of this cell. New branches are shown in green; chosen branch is tipped with a red arrowhead. The numbers indicate the angle formed by the branches. Scale bars: 100 µm in A-A'',B,B'; 25 µm in C,C'.

 

Figure 4
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  		Fig. 4. Chemotaxis in MGE-derived cells requires leading process branching. (A) Schematic diagram of experimental design. (B-D) Migration of MGE-derived cells in response to mock-transfected (B) or Nrg1-transfected (C,D) COS cells aggregates cultured in matrigel matrices for 36 hours in the presence of vehicle solution (B,C) or the ROCK inhibitor Y27632 (30 µM) (D). COS cells were also transfected with dsRed to aid their visualization. Broken lines indicate the limits of the explants before culture. (B'-D') Confocal images of cells migrating through Sector 1, as defined in the schematic shown in A, in control (B'), Nrg1 (C') and in Nrg1+Y27632 (30 µM) (D')-treated explants. Solid and open arrowheads indicate branched and non-branched interneurons, respectively. (E) Schematic view of the method used to quantify the orientation of cells ({alpha} angle). (F) Quantification of {alpha} angle in Sector 1. Bars show mean±s.e.m. 37.02±2.14° (control, n=167 cells from three independent experiments), 49.65±1.29° (Nrg1, n=520 cells from three independent experiments) and 38.10±1.41° (Nrg1 +Y27631, n=381 cells from three independent experiments). t-test, ***P<0.001. (G) Quantification of percentage of neurons located in Sector 1 with at least two leading process branches. Bars show mean±s.e.m. 54.85±7.41% (Ctrl, n=161 cells from three independent experiments), 72.21±4.49% (Nrg1, n=528 cells from three independent experiments) and 55.73±5.34% (Nrg1+Y27631, n=317 cells from three independent experiments). t-test, *P<0.05. Scale bars: 100 µm in B,C,D; 40 µm B',C',D'.

 

Figure 5
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  		Fig. 5. Physically induced turning of interneurons requires leading process branching. (A) Schematic diagram of experimental design. After dissection of an E13.5 GFP MGE into small pieces, microtransplants were placed on a host E13.5 wild-type MGE and 5 hours later the pallium was removed from slices by cutting at the pallial-subpallial boundary. After 12 hours of incubation, GFP interneurons have migrated just a short distance from the cut (B). At this time, vehicle or Y27632 (30 µM) was added to the medium and slices were incubated for another 12 hours. (C-D') Images of GFP-expressing interneurons migrating in slices in the presence of vehicle (C,C') or Y27632 (D,D'). (C',D') High-magnification images of the boxed areas shown in C,D, respectively. Note that interneurons reach the cut in a fairly delineated stream in both control and experimental slices (arrows in C,D). In control slices, many interneurons turn 90° to continue migrating parallel to the incision (C), while turning is diminished in the presence of Y27632 (D). (E) Light-gray, dark-gray and pink arrowheads indicate type 1 (leading process parallel to the cut), type 2 (leading process away from the cut) and type 3 (leading process towards the cut) cells, respectively. (F) Quantification of the relative proportion of type 1-3 cells in control and experimental slices. Histograms show averages ±s.e.m. Control: 56.56±1.79%, 18.68±1.86%, 24.76±2.09% for cell type 1, 2 and 3 respectively; 1151 cells from eight slices in three independent experiments. Y27632: 42.80±1.85%, 18.17±1.98%, 39.03±2.36% for cell type 1, 2 and 3 respectively; 743 cells from eight slices in three independent experiments. {chi}2-test: ***P<0.001. Scale bars: 100 µm in B,C,D; 25 µm in C',D'.

 

Figure 6
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  		Fig. 6. Switch from tangential to radial position during CP invasion is perturbed after addition of Y27632. (A) Schematic showing the preferential orientation of interneurons in the cortex at E15.5 and after 48 hours in culture. Interneurons invade the CP performing a rapid change in direction (Ang et al., 2003Go; Polleux et al., 2002Go). (B-D) Images of cortical slices at E15.5 (B) or after 48 hours in culture (C,D) showing the distribution of GFP/BrdU+ interneurons, treated with vehicle (C) or Y27632 (D). Solid and open arrowheads indicate tangentially and radially oriented cells, respectively. (E) Quantification of percentage of GFP/BrdU+ radially and tangentially oriented neurons in control and Y27632-treated slices after 48 hours in culture. Bars show mean±s.e.m. Control: 41.99±5.37% (radially oriented), 58.00±6.02% (tangentially oriented), n=231 cells from four slices of two independent experiments; Y27632: 23.21±2.42% (radially oriented), 76.79±2.42% (tangentially oriented), n=280 cells from four slices of two independent experiments. {chi}2-test, ***P<0.001, **P<0.01. (F) Schematic showing the criteria for the classification of the orientation of interneurons in cortical slices. (G,H) Schematic representation of the distribution of GFP/BrdU+ interneurons in C and D, respectively. Solid and open arrowheads indicate tangentially and radially oriented cells, respectively. Scale bar: 100 µm.

 

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