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First published online September 30, 2004
doi: 10.1242/10.1242/dev.01398

Development 131, 5127-5138 (2004)
Published by The Company of Biologists 2004
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Time-lapse and cell ablation reveal the role of cell interactions in fly glia migration and proliferation

Benoît Aigouy, Véronique Van de Bor*, Marcel Boeglin and Angela Giangrande{dagger}

Institut de Génétique et Biologie Moléculaire et Cellulaire, IGBMC/CNRS/ULP/INSERM – BP 10142, ILLKIRCH, C. U. de Strasbourg, 67404, France



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  		Fig. 1. Outline of glial development in the pupal wing. (A) Adult wing schematic drawing indicating the position of neurones belonging to gliogenic (squares) and non-gliogenic (triangles) sensory organs. L1 and L3 indicate L1 and L3 veins, respectively. P, proximal; D, distal. (B,C) Wild-type wings labelled with glial (anti-Repo, green) and neuronal (anti-22c10, red) markers at 20 hours after puparium formation (APF; B) and at 24 hours APF (C). GSR indicates the giant sensillum of the radius. L1, L1 nerve; L3, L3 nerve, r, radial nerve; c, costal nerve. Neurones issued from the two neurogenic (L3.2 and E2, ACV and E1) and the three gliogenic (L3.3, L3.1, L3.v) sensory organs (Van De Bor et al., 2000Go) are indicated. Brackets include the L3.3-derived Repo-positive cells. On this and following panels anterior is to the top, distal to the right. (D) Model for cell division in wing gliogenic sensory organs. SOP, sensory organ precursor; IIa and IIb, second order precursors; IIIb, third order precursor; To, tormogen cell; Tr, trichogen cell [also called dome/cap cell in the case of campaniform sensilla (reviewed by Keil, 1997Go)]; Th, thecogen cells; n, neurone; GPI, II and III, first, second and third order glial precursors, respectively; g, glial cells. The variable number of glial cells is indicated by the dashed lines. Scale bars: 70 µm (B,C).

 


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  		Fig. 2. Glial development followed by time-lapse. (A) Pupa preparation for time-lapse. Red lines indicate the path of puparium case dissection. The operculum is opened (left panel, straight line), and the puparium case over the wing is removed (mid and right panel, dashed lines). Left and mid panels are dorsal views; right panel is a lateral view. Anterior is to the top. W, wing. (B) repo-actinGFP: time-lapse images of a GPI showing dynamic cell shape and filopodia reorganisation (arrowheads). (C) Time-lapse images showing repo-ncGFP (nuclear and cytoplasmic GFP) labelling (see Movie 1 in supplementary material). Symbols as in Fig. 1B. Brackets indicate the glial cells originating from L3 gliogenic sensory organs. Arrows show the L1 front of migration. Arrowheads indicate the positions of L3.3 glial nuclei. Scale bars: 20 µm (B); 70 µm (C).

 


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  		Fig. 3. Pioneer and follower L1 glial cells. (A) Schematic representation of a wing. Boxes show regions of interest on L1 (B-D) that have been analysed in different repo-ncGFP animals. (B,C) Individually labelled (repo-ncGFP MARCM clones) follower cells. (B) Distal glial cell at 17 hours APF. (C) Time-lapse sequence (17-19 hours APF) on two glial cells. (D) Time-lapse sequence (17-19 hours APF) on the whole glial population at the front of migration (see also Movie 3 in supplementary material). Pioneer glial cells display more numerous and elaborate filopodia (arrowheads) when compared with follower cells, which are more distal (see asterisk). Scale bars: 30 µm (B-D).

 


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  		Fig. 8. Establishment of glial cell contact does not inhibit proliferation. Time-lapse images from a repo-ncGFP wing showing L3.3 (artificially coloured in red) and L3.1 (green) glial lineages. Nuclei are indicated by small, coloured arrowheads. (A) L3.3 and L3.1 GPIIIs are not in contact at 25 hours and 30 minutes APF. (B) L3.3 and L3.1 GPIIIs contact each other (white arrowheads) 28 minutes later. (C-E) All L3.3 GPIIIs divide and produce eight cells; notice that cell division entails a transient loss of contact between L3.3 and L3.1 glia (asterisk in C). Scale bar: 50 µm.

 


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  		Fig. 4. Confocal-assisted cell ablation. (A) Ablation of all L3 glial lineages (L3.3, L3.1 and L3.v) at 17 hours APF on a repo-ncGFP wing. GFP labelling before (upper panel) and just after (lower panel) UV laser irradiation. Asterisks indicate the position of the ablated cells. (B) Immunolabelling on the same wing five hours after ablation. Notice the absence of specific Repo (upper and lower panels) and GFP (upper panel) labelling on L3 (bracket). (C) A wild-type wing at a comparable stage, labelled with anti-Repo. + indicates unspecific labelling corresponding to the wing cuticle. Scale bars: 40 µm.

 


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  		Fig. 5. Glia-glia contact inhibit migration. In all schematic drawings (A, panels a-c; B, panels a-d), distal glia are shown in light green and proximal/medial glia in dark green. Glial cells are represented by circles, neurones by red squares, the laser beam is indicated in purple. (A) Glia ablations on 17 hours APF repo-ncGFP wings. (a-c) Ablation of proximal/medial (L3.1 and L3.v; P+M), bilateral (L3.3 and L3.v; P+D) and distal (L3.3; D) glial cells, respectively. (d-i) GFP labelling before (d-f) and just after (g-i) ablation. Asterisks indicate the positions of the ablated cells. (B) Same wings as in A, 15 hours after cell ablation. (a-d) Schematic representations of the results. (e-l) Immunolabelling with anti-GFP (green), anti-22c10 (red; neuronal somata and axons) and anti-Elav (blue; neuronal nuclei). Symbols are as above. (e-h) anti-GFP. (i-l) Multiple labelling. Scale bars: in A, panels d-i, 40 µm; in B, panels e-l, 70 µm.

 


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  		Fig. 6. Glia migration in the absence of neurones. (A-C,E,F) Nts1: Nts1; repo-ncGFP wings after complete transformation of the L3.3 gliogenic lineage into glia immunolabelled at 13 hours (A,B), 20 hours (C) and 24 hours (F) APF. (B) Triple labelling with anti-Repo (red), anti-Elav (blue) and anti-Cut (all SOP progeny, green). (C,D) Wings labelled with anti-Elav (red) and anti-GFP (green). (E-G) Wings immunolabelled with anti-22c10 (red) and anti-GFP (green). A corresponds to the blue channel of B, and E corresponds to the red channel of F. Notice the absence of the L3.3 neurone (arrows) in the Nts1 wings, as shown by lack of neuronal labelling (anti-Elav in A-C; anti-22c10 in E,F). (D,G) Wild-type (WT): (repo-ncGFP) wings at 20 hours (D) and 24 hours (G) APF. In all panels, brackets indicate L3.3 glia. Symbols are as above. Scale bars: 40 µm (A-D); 70 µm (E-G).

 


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  		Fig. 7. Neurone-glia interactions affect directional migration. (A) Time-lapse analysis in an Hw49c; repo-ncGFP wing posterior margin. Ectopic glia are indicated in the upper panel by an asterisk (proximal cell) and a square (distal cell). Lower panel: a proximal cell has divided, arrows show the direction of migration. (B) Schematic drawing of the same wing after immunolabelling. The posterior margin region analysed by time-lapse (A) and immunolabelling (C) is shown in the box and contains neurones (red) and glial cells (green). (C) Anti-Repo, anti-22c10 and anti-Elav labelling performed after time-lapse. Arrowheads show neuronal cell bodies. Scale bars: 40 µm (A); 35 µm (C).

 


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  		Fig. 9. Glia-glia interactions do not control proliferation. Glial cell number at 32 hours APF, after proximal/medial (L3.v and L3.1) and bilateral (L3.v and L3.3) glial cell ablation, revealing the contribution of the L3.3 and L3.1 glia, respectively. Values obtained upon ablation (grey bar) or in wild-type wings (white bar). Wild-type values were obtained by following L3.3 and L3.1 lineages in seven time-lapses. Bars indicate the highest and lowest values; n indicates the number of samples analysed.

 


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  		Fig. 10. Ablation within a glial lineage. (A,B) Examples of GPIId (A) and GPIIp (B) ablations performed at 22 hours APF. Symbols are as in Fig. 5. (a) Schematic representation of the ablation. (b-d) GFP labelling before (b), just after (c) and several hours (d) after ablation. Asterisks in panel c show fading of the GFP labelling in the targeted cell. Notice (d) that the spared GPII remains labelled by GFP and undergoes division. (e) Immunolabelling at 32 hours and 30 minutes APF, using anti-GFP and anti-Elav. Brackets indicate the remaining glia progeny of L3.3 and L3.1 GPII. L3.2 indicates the L3.2 neurone. (C) Average number of glial cells at 32 hours and 30 minutes APF in a wild-type (WT) wing, and in GPIIp or GPIId ablated wings. Glial cells from each L3.3 sublineage were followed by time-lapse; n indicates the number of samples analysed. Bars indicate highest and lowest values. Scale bars: 40 µm.

 


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  		Fig. 11. Time-lapse analysis of glial cell division. (A) Time-lapse analysis of a repo-tauGFP GPI: (a-f) proximodistal extensions keep elongating (arrowheads), whereas the others prune back (asterisks). (B) Time-lapse sequence of a GPI division using repo-ncGFP. (a-d) Glial extensions rapidly prune back so that the cell adopts an almost round shape (d). Upon cell division (e), the GPIIs rapidly send out novel extensions (f-h). (C) Microtubule reorganisation at division. Confocal projections, illustrating a GPI division (repo-tauGFP). (a-c) Colour coding allows labelling quantification and thereby the identification of centrosomes as red spots (highest GFP levels). (b,c) Centrosomes migrate and position themselves perpendicular to the orientation of division. (d-f) GFP reveals the mitotic spindle, which undergoes rotation, so that division takes place along the proximodistal axis (g,h). (D) Time-lapse sequence showing that GPIIs of the same sensory organ divide synchronously. Glial nuclei are indicated by asterisks. Scale bars: 10 µm (A,C); 20 µm (B); 30 µm (D).

 

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© The Company of Biologists Ltd 2004