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First published online January 10, 2007
doi: 10.1242/10.1242/dev.02750

Development 134, 625-634 (2007)
Published by The Company of Biologists 2007
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Neurogenic role of Gcm transcription factors is conserved in chicken spinal cord

Laurent Soustelle1,*, Françoise Trousse2,*, Cécile Jacques1, Julian Ceron1,{dagger}, Philippe Cochard2, Cathy Soula2 and Angela Giangrande1,{ddagger}

1 Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, B.P.10142, 67404 Illkirch Cedex, C.U. de Strasbourg, France.
2 Centre de Biologie du Développement, UMR5547 CNRS/UPS, Université Paul Sabatier, 118 Route de Narbonne, 31062 Toulouse Cedex, France.


Figure 1
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  		Fig. 1. Expression of c-Gcm1 in developing chicken embryonic CNS. c-Gcm1 in situ hybridization on whole chick embryos (A-D) and on transverse sections of embryonic spinal cord (E-G). (A,B) Dorsal views showing that c-Gcm1 expressing cells (purple) are detected in the anterior neural plate at stage 4, in an area corresponding to the epiblast as shown on transverse section in A'. (B) Colabeling shows that c-Gcm1-expressing cells are included in the Sox2 expression domain (red). (C) During neurulation, c-Gcm1 is strongly expressed throughout neural tube and neural plate. (D,E) Starting from E1.5 (stage 11), c-Gcm1 is expressed in the neural tube (D), along the entire dorsoventral axis except in the dorsal-most region (E). (F) At E2.5 (stage 14), c-Gcm1 expression is downregulated in neural progenitors lining the lumen whereas it is maintained in cells located in the newly formed intermediate mantle layer. (G) At E8 (stage 34), c-Gcm1 RNA is no longer detected in the spinal cord. hn, Hensen's node; ps, primitive streak. Scale bars: 60 µm in E; 70 µm in F; 150 µm in G.

 

Figure 2
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  		Fig. 2. Overexpression of c-Gcm1 leads to depletion of proliferative neural progenitors. (A-F,J-P) Transverse sections after electroporation with control or c-Gcm1 expression vectors at the level of the truncal neural tube of E1.5 chicken embryos. For all images, the electroporated side is on the left; electroporated cells were detected by GFP immunolabeling or by GFP fluorescence (green in both cases). (A-B') GFP immunolabeling (A,B) and in situ hybridization using Sox2 (A') and NeuroM (B') probes. Note the decrease in Sox2 (A') and the increase in NeuroM (B') expression 6 hours after c-Gcm1 overexpression. (C-D') Ngn2 expression domain is not modified upon c-Gcm1 overexpression (C,C'), whereas Ngn2 overexpression induces c-Gcm1 expression (D,D') 24 hours after electroporation. Note that signal development was stopped before detection of endogenous c-Gcm1 mRNA in D'. (E,F) BrdU (red) and GFP colabeling in embryos subjected to a 1 hour BrdU pulse performed 30 hours after electroporation of control (E) or c-Gcm1 (F) expression vectors. Many of the cells electroporated with the control expression vector are in a proliferative state (arrows in E), whereas overexpression of c-Gcm1 markedly reduces the number of transfected BrdU-positive cells (F). Pax6 (J-M) and Pax7 (N-P) immunolabeling performed 24 hours after electroporation of control (J,J',L,O) or c-Gcm1 (K,K',M,N,N',P) expression vectors. c-Gcm1 overexpression strongly reduces the number of Pax6 (K,K',M) and Pax7 (N,N',P) expressing progenitors in the intermediate and dorsal neural tube, respectively. High magnifications of neural tube sections show GFP (L,M,O,P) and Pax6 (red in L,M) or Pax7 (red in O,P) colabeling. Neural progenitors electroporated with control vector maintain the expression of Pax6 and Pax7 (arrows in L,O), whereas neural progenitors electroporated with c-Gcm1 do not (arrows in M,P). (G-I) Percentage of transfected cells expressing Sox3 (G), incorporating BrdU (H) or expressing Pax6 (I), after electroporation of control or c-Gcm1 expression vectors. Asterisks indicate significant differences (P<0.05 in G, P<0.0001 in H; P<0.01 in I). Scale bars: 40 µm in A-F,J-K',N-O; 10 µm in L,M,O,P.

 

Figure 3
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  		Fig. 3. Overexpression of c-Gcm1 promotes premature neuronal differentiation. Electroporation with control or c-Gcm1 expression vectors in truncal neural tube (A-B',E-J') or telencephalon (C,D) of E1.5 embryos. Immunolabeling with ßIII-tubulin (Tuj1 in A-E), HuC/D (G-H') and Lim1/2 (I-J') neuronal markers were performed 24 hours after electroporation. (A-H') Electroporation of control vector (green in A,C,G,I) does not modify the expression pattern of ßIII-tubulin (red in A,A',C) or HuC/D (red in G,G'), whereas c-Gcm1 overexpression (green in B,D,E,H,J) leads to ectopic differentiation of Tuj1-positive (red in B,B',D; blue in E, arrows) and HuC/D-positive (red in H,H') neurons in the neuroepithelium. Inserts in B and H are high magnifications of c-Gcm1-overexpressing cells that coexpress ßIII-tubulin and HuC/D, respectively. (E) ßIII-tubulin (blue) and Pax7 (red) coimmunolabeling shows that c-Gcm1-overexpressing cells coexpress ßIII-tubulin but not Pax7 (arrows). (I-J') Compared with control (I,I'), c-Gcm1 overexpression induces the generation of Lim1/2-positive neurons within the ventricular zone (J,J'). (F) Percentage of transfected cells expressing ßIII-tubulin (Tuj1) and Lim1/2 after electroporation of control or c-Gcm1 expression vectors. Differences between the percentage of c-Gcm1 transfected cells expressing neuronal markers versus controls are statistically significant (asterisks, P<0.001). Scale bars: 40µm in A-D,G-J'; 10µm in insert in B,H; 20µm in E.

 

Figure 4
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  		Fig. 4. Overexpression of c-Gcm1 promotes neuronal but not glial differentiation in spinal cord. (A,B) E1.5 neural tube electroporated with control or c-Gcm1 expression vectors (green) and analysis of O4 expression (red) three days later (E5/E5.5). (A) Electroporation of control vector does not modify the pattern of O4 decorating oligodendrocyte progenitors in the ventral neuroepithelium and no ectopic expression of O4 is observed. (B) Note that the endogenous O4-positive domain is strongly reduced (arrow) and no ectopic expression of O4 is detected when the c-Gcm1-expressing vector is used. (C-I) Electroporation with control (F,H) or c-Gcm1 (D,E,G,I) expression vector was performed in E4.5/E5 embryonic spinal cord that was further dissected, opened dorsally and plated in culture with neuroepithelial precursors up, as depicted in C. Glial and neuronal differentiation was assessed three days later by immunolabeling using Glast (D, red), O4 (E,F,G, red) and Lim1/2 (H,I, red). (D,E) Transverse sections of open-book spinal cords showing that c-Gcm1 overexpression does not induce ectopic Glast-positive (D) or O4-positive (E) cells. Note in E that the O4-positive domain is strongly reduced in the ventral spinal cord. (F,G) High magnifications show that some cells electroporated with control vector have adopted an O4-positive fate (F, arrows), whereas no c-Gcm1-overexpressing cells adopt such a fate (G). (H,I) High magnifications of explants showing that most cells electroporated with control vector are located in the neuroepithelium, and only a few of them reach the mantle layer and express Lim1/2 (H). By contrast, most c-Gcm1-overexpressing cells have left the neuroepithelium and all of them express Lim1/2 (I). ne, neuroepithelium; ML, mantle layer; FP, floor plate. Scale bars: 120µm in A-E; 40µm in F-I.

 

Figure 5
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  		Fig. 5. Repression of c-Gcm1 targets inhibits neuronal differentiation without affecting cell cycle exit. (A-O') Electroporation of E1.5 embryos with control (C,C',E,G,I-K,M,N) or c-Gcm1BD-ER (BD-ER; A-B',D,D',F,H-J,L-O') expression vectors. Immunolabeling and in situ hybridization on transverse sections were performed 24 hours after electroporation using anti-ßIII-tubulin (Tuj1 in A,A') or Lim1/2 (B,B',E,F) antibodies or NeuroM probe (C-D'). (A-B') Overexpression of c-Gcm1BD-ER inhibits the generation of terminally differentiated neurons. (E,F) High magnification of electroporated neural tube showing that cells overexpressing c-Gcm1BD-ER do not express Lim1/2, whereas a fraction of cells electroporated with a control vector express Lim1/2 (arrow in E) as they reach the mantle layer. (I,J) Percentage of transfected cells expressing Lim1/2 (I) or MNR2 (J) after electroporation of control or c-Gcm1BD-ER expression vectors; asterisks indicate significant difference (P<0.01). (G,H) BrdU (red) and GFP immunolabeling on transverse sections obtained from embryos subjected to a 1 hour BrdU pulse performed 24 hours after electroporation of control (G) or c-Gcm1BD-ER (H) expression vectors. Note the presence of double-labeled cells in both cases (arrows). (M,N) Percentage of transfected cells incorporating BrdU (M) or expressing Sox3 (N) after electroporation of control or c-Gcm1BD-ER expression vectors. Note that values are not significantly different. (K,L,O,O') Similar profile of Sox3 (red in K,L) and Pax7 (red in O,O') expression upon electroporation of control (K) or c-Gcm1BD-ER (L,O,O') expression vectors. Scale bars: 50 µm in A-D',O,O'; 20 µm in G,H; 60 µm in I,J; 40 µm in K,L.

 

Figure 6
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  		Fig. 6. Context-dependent role of gcm during post-embryonic development. (A-C'') Elav (green) and Repo (red) coimmunolabeling on wing imaginal discs. Right panels (A'',B'',C'') show merge of Elav (A,B,C) and Repo (A',B',C') immunolabeling. gcm overexpression leads to neuronal and glial differentiation (compare B-C'' with A-A''). Note that some ectopic neurons and glial cells are closely associated but neuronal and glial markers never colocalize (see C''). (D-F'') Elav (green) and Acj6 (red) coimmunolabeling on wing imaginal discs. Right panels (D'',E'',F'') show merge of Elav (D,E,F) and Acj6 (D',E',F') immunolabeling. Note that some ectopic Elav-positive neurons coexpress Acj6 (white asterisks in F). C-C'' and F-F'' are magnifications of squares shown in B'' and E'', respectively. Scale bars: 50 µm in A-B'',D-E''; 200µm in C-C'',F-F''.

 

Figure 7
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  		Fig. 7. gcm and gcm2 are expressed in central brain neuronal lineages. (A-A'') gcm (A) and gcm2 (A') double in situ hybridization on wild-type Drosophila LIII brain hemispheres. (A'') Merge of gcm (red) and gcm2 (green) labeling. gcm and gcm2 RNAs are coexpressed in lamina neuronal [lamina precursor cells (LPCs)] and glial [glial precursor cells (GPCs)] progenitors as well as in a central brain cluster (area encircled by dotted line in A-A''). (B-C') GFP immunolabeling on gcm-gal4,UAS-mCD8GFP (gcm>GFP) LIII (B) or whole-mount adult brains (C,C'). Note that the GFP expression profile mimics gcm expression pattern in larvae. Anterior (C) and posterior (C') views of the same adult brain are shown. Neuronal somata of dorsolateral (dcbcs) and medial (mcbcs) clusters are encircled by white (C) and yellow (C') dotted lines, respectively. (D-D''') GFP (green), phospho-Histone H3 (red) and DAPI (blue) colabeling at late LI gcm-gal4,UAS-ncGFP larva. (D''') Merge of D-D''; note that a single GFP-positive cell undergoes mitosis as shown by phospho-Histone H3 expression. Scale bars: 40 µm in A-A''; 40 µm in B; 50 µm in C-C'';400 µm in D-D''.

 

Figure 8
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  		Fig. 8. Neuronal differentiation requires Gcm activity in central brain. GFP immunolabeling in (A) control (gcm-gal4,tub-gal80ts;UAS-ncGFP;UAS-gcmN7-4DN) and (B) gcm-gcm2 loss-of-function (LOF) (gcm-gal4,tub-gal80ts;UAS-ncGFP;UAS-gcmDN) LIII CNS. Note that dcbcs and mcbcs (encircled by white and yellow dotted lines, respectively) are missing in gcm-gcm2 LOF but not in control animals as marked by the white and yellow asterisks in B. In these conditions of a single dose of the UAS-gcmDN transgene, lamina development (neurons and glia) is less affected than with two doses of UAS-gcmDN transgene (see Fig. S5 in the supplementary material). Arrows in A,B indicate processes of glia at the interhemispheric junction. (C,D) Development of central brain atonal-positive lineage is not affected by the gcmDN construct. GFP immunolabeling in control (atonal-gal4,UAS-mCD8GFP in C) and gcm-gcm2 LOF (atonal-gal4,UAS-mCD8GFP,UAS-gcmDN in D) LIII CNS shown in dorsal view. In control (C) as well as in gcm-gcm2 LOF (D) animals, atonal lineage includes two clusters of 20-30 neurons that are connected by a commissure crossing the midline (dashed line) and which extend a bundle of ipsilateral axons (arrows) into the optic lobes. Thus, gcmDN expression does not affect the development of atonal-positive neurons. Scale bars: 100 µm in A,B; 180 µm in C,D.

 

Figure 9
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  		Fig. 9. Fly, chick and mouse gcm genes induce expression of neuronal and glial markers in HeLa cells. GFAP (A,B,C,D), GFP (A',B',C',D'), Tuj1 (A'',B'',C'',D'') and DAPI (A''',B''',C''',D''') colabeling and merges (A'''',B'''',C'''',D'''') of HeLa cells transfected with expression vectors carrying c-Gcm1 (A-A''''), gcm (d-gcm1 in B-B''''), mouse Gcm1 (m-gcm1 in C-C'''') or vector as control (D-D''''). Scale bar: 20 µm. (E) Percentage of transfected cells expressing neuronal/glial markers. n indicates the number of GFP-positive cells counted. Note that all gcm1 genes induce the expression of GFAP and Tuj1 markers and that these two markers colocalize in most transfected cells.

 

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