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First published online 22 October 2003
doi: 10.1242/dev.00822

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The potential to induce glial differentiation is conserved between Drosophila and mammalian glial cells missing genes

Yasuno Iwasaki1, Toshihiko Hosoya2, Hirohide Takebayashi1, Yasuhiro Ogawa1, Yoshiki Hotta2 and Kazuhiro Ikenaka1,*

1 Division of Molecular Neurobiology, National Institute for Physiological Sciences, Okazaki, Aichi 444-8585, Japan
2 Department of Developmental Genetics, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan



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  		Fig. 1. Developmental profiles of Gcm gene expression in the mouse brain. (A) Messenger RNA was prepared from E16 placenta and brain, and used for RT-PCR analysis. Primers specific for mouse Gcm1, mouse Gcm2 and ß-actin were designed to recognize positions on different exons. (B) Total RNA was prepared from different developmental stages of the mouse brain, and used for quantification of mouse Gcm gene expression by real-time PCR analysis. Bars represent the relative mouse Gcm transcript levels normalized against ß-actin transcript levels.

 


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  		Fig. 2. Effects of the Gcm genes in fibroblast cells. (A,B) Staining of mouse Gcm1 (A) and mouse Gcm2 (B) proteins expressed in each stable transformant. The proteins were localized in nuclei. The top right panels show negative controls in which the first antibodies were omitted. (C-F) Gcm gene expression leads to morphological changes in fibroblast cells. Cells were transfected with control (C), mouse Gcm1 (D), mouse Gcm2 (E) or truncated mouse Gcm2 (F) retroviral vectors and cultured for 3 days. Truncated mouse Gcm2 contains the DNA-binding domain but not the transactivating domain. The cells were subsequently stained with X-gal. (G-K) Fibroblast cells were transduced with control (G), mouse Gcm1 (H), mouse Gcm2 (I) or Drosophila gcm (J) viruses at low titers and cultured for 6 days. The cells were stained with X-gal (G-J) and the X-gal+ cell number in a cluster was counted. More than 100 colonies for each viral transduction were examined and the distribution of colony size was plotted (K). (L) Total RNA was prepared from stable transformants of control (CT), mouse Gcm1 (a) and mouse Gcm2 (b), and used for RT-PCR analysis of S100ß and ß-actin gene expression. Induction of S100ß expression by mouse Gcm1 and mouse Gcm2 was observed in fibroblast cells. Scale bars: 25 µm in A,B; 50 µm in C-F; 50 µm in G-J.

 


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  		Fig. 3. Effect of the Gcm genes in cultured brain cells. (A-G) Primary cultured cells from E12 mouse hemispheres were transduced with control (A,D), mouse Gcm1 (B,E,G) or mouse Gcm2 (C,F) retrovirus and cultured in chemically defined medium for 3 days (A-F) or 2 days (G). The cells were stained with X-Gal, followed by staining with anti-GFAP (A-C,G) or anti-MAP2 (D-F) antibodies. GFAP+ (H) or MAP2+ (I) cells in X-gal+ cells after 3 days culture were counted. (J-N) After control (J,K) or mouse Gcm1 (L,M) transduction, brain cells were stained with both ß-galactosidase (red) and S100ß (green) antibodies. J and L are merged images. Arrowheads show overlapped signals in mouse Gcm1 transduction. The percentage of overlap was plotted in N. (O,P) Brain cells were infected with control lacZ or mouse Gcm1-lacZ virus together with virus harboring alkaline phosphatase (ALK). lacZ+ and ALK+ cells were visualized by X-gal-staining (arrows) and NBT-staining (arrowheads), respectively. They were further stained with anti-GFAP antibodies (brown). Cells double-stained for X-gal and anti-GFAP Ab or ALK and anti-GFAP Ab were counted (P). Scale bars: 30 µm in A-G; 40 µm in J-M,O.

 


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  		Fig. 4. Effect of mouse Gcm1 expression during gliogenesis in vivo. Vibratome sections of P24 mouse forebrain that was injected with ß-gal-harboring retrovirus at E13 were stained with X-gal (A). B is a higher magnification of a box in A. The observed morphologies of infected cells were suggestive of neurons (arrows) and astrocytes (arrowheads). Neuron-like cells have a small clear cell body while astrocyte-like cells have a large obscure cell body. To confirm this classification, the cells were further stained (brown) with a neuronal marker, NeuN (C,D), or an astrocyte marker, GFAP (E,F). Using these criteria, the lineage of cells infected with CT or mouse Gcm1 retrovirus was examined (G). For mouse Gcm1-transduced cells, 69.9±7.2% of cells (n=2, 725 cells scored in total) were astrocytes while 33.6±10.2% (n=2, 1742 cells scored in total) of the cells were astrocytes for CT transduction. Scale bars: in A, 150 µm for A and 60 µm for B; in C, 25 µm for C,D; in E, 50 µm for E,F.

 


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  		Fig. 5. Cultures from mouse Gcm1-deficient mice. Heads were dissected from E9.5 mouse Gcm1-heterozygote or homozygote mice and cultured according to Kitani et al. (Kitani et al., 1991Go). The cells were stained with anti-MAP2 or S100ß antibody after 3 days or with anti-GFAP antibody after 5 days. There were no significant differences in the appearance of GFAP+ cells among the cultures from mouse Gcm1-heterozygote or homozygote mice. Scale bar: 30 µm.

 


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  		Fig. 6. In situ hybridization analysis of mouse Gcm1. Coronal sections of E14.5 mouse forebrains were hybridized with a digoxigenin-labeled mouse Gcm1 antisense riboprobe (A,B). (B) Higher magnification of the boxed area in A; inset shows higher magnification of the boxed area in B. Dispersed distribution of mouse Gcm1 mRNA was observed in the ganglionic eminence and in the thalamus. Only a few signals lining the ventricular zone were observed in the cerebral cortex. Sections were counterstained with Methyl Green after in situ hybridization. As controls, sections of E11.5 placenta were hybridized with a digoxigenin-labeled mouse Gcm1 antisense (C) and sense (D) riboprobes, respectively. mouse Gcm1 expression was observed only in labyrinth layer of placenta using antisense riboprobe. No signal was observed in embryonic brain sections (not shown), or in placenta sections (D). Scale bars: 150 µm in A-D; 50 µm inset of B.

 

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© The Company of Biologists Ltd 2003