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Gliogenesis in Drosophila: genome-wide analysis of downstream genes of glial cells missing in the embryonic nervous system

¹ Biozentrum/Pharmazentrum, University of Basel, CH-4056 Basel, Switzerland
² Roche Bioinformatics, F. Hoffmann-La Roche, Ltd, CH-4070 Basel, Switzerland
³ Genetics Pharmaceuticals Division, F. Hoffmann-La Roche, Ltd, CH-4070 Basel, Switzerland

View larger version (85K): [in a new window]   Fig. 1. Targeted misexpression of gcm results in gain of glial cells at the expense of neuronal cells. (A,B) In situ hybridization of stage 10 embryos shows gcm expression in wild-type (A) and in sca-gcm embryos (B); lateral views, anterior towards the left. In the wild type, small clusters of cells in the neuroectoderm express gcm; in sca-gcm embryos, all cells of the neuroectoderm express gcm. (C,D) Immunostaining with anti-REPO in wild-type (C) and in sca-gcm (D) embryos; laser confocal microscopy of stage 11, ventral views of the VNC, anterior is towards the left. In the wild-type, single gcm-expressing glial precursors in each hemisegment express the repo gene. In sca-gcm embryos, virtually all of the neuronal and glial precursor cells are REPO positive. (E,F) Double immunostaining with anti-REPO (green) and anti-HRP (red) in wild-type (E) and in sca-gcm (F) embryos; laser confocal microscopy of stage 15/16 embryos. In the wild type, neurons and glial cells are differentiated and correctly positioned, and all lateral glial cells express repo. In sca-gcm embryos, 80%-90% of the cells in the CNS express repo.

View larger version (41K): [in a new window]   Fig. 2. Changes in transcript levels of the genes encoding transcription factors after gcm misexpression. Bars represent the fold changes in gene expression levels between wild-type embryos and sca-gcm embryos. Positive values indicate that the relative expression level of a gene is increased (upregulation) and negative values indicate a decrease (downregulation). Normalized average difference values are given for the wild-type condition as follows: yellow bars represent <100; orange bars represent 100-1000; red bars represent >1000.

View larger version (34K): [in a new window]   Fig. 3. Changes in transcript levels for the genes encoding protein kinases and phosphatases following gcm misexpression. Data are presented as in Fig. 2.

View larger version (26K): [in a new window]   Fig. 4. Changes in transcript levels for the genes encoding DNA/chromatin-binding proteins (A), cell cycle regulators (B) or cell adhesion molecules (C) after gcm misexpression. Data are presented as in Fig. 2.

View larger version (55K): [in a new window]   Fig. 5. Spatial expression of selected candidate gcm downstream genes by in situ hybridization and immunocytochemistry. Whole-mount in situ hybridization (A-J,Q,R) and immunostaining (K-T) show expression of differentially regulated genes in wild-type, sca-gcm and gcm mutant embryos. Ventral views of stage 11 (A-D) and stage 15/16 (E,F,I,J,K-P) embryos, and lateral views of stage 15/16 embryos (G,H,Q-T), anterior is towards the left. Fold changes and P values are indicated on the right. (A,B) Expression of htl in stage 11 wild-type embryos is visible in a distinct set of neural precursors; in sca-gcm embryos, htl is expressed throughout the neurogenic region. (C,D) In stage 11 embryos, the scrt gene is expressed in neural precursors; in stage 11 sca-gcm embryos, the expression of scrt is diminished in most of the neural precursors, but is still apparent in a subset of these cells. (E-H) In stage 15/16 wild-type embryos, bnb gene is expressed in lateral glial cells; in stage 15/16 sca-gcm embryos, the expression of bnb increases markedly and appears virtually in all of the cells of the nervous system. (I,J) In stage 15/16 wild-type embryos, the elav gene is expressed in all neurons; in stage 15/16 in sca-gcm embryos, expression of elav is strongly reduced in most of the neurons. (K,L) In stage 15/16 wild-type embryos, the EY protein is expressed in a segmentally reiterated subset of neurons in the CNS; in stage 15/16 sca-gcm embryos, the number of EY-expressing cells in the CNS is dramatically reduced. (M,N) In stage 15/16 wild-type embryos, the TEN-M protein is expressed on the axons that make up the longitudinal and commissural tracts of the CNS; this axonal expression of TEN-M is virtually abolished in stage 15/16 sca-gcm embryos. (O,P) In stage 15/16 wild-type embryos, the WRAPPER protein is expressed in midline glial cells, in some lateral glial cells and in glial cells supporting the chordotonal sensory organs; this expression has spread to the complete CNS region in stage 15/16 sca-gcm embryos. (Q,R) In late stage embryos REPO (brown) is expressed in all and bnb (blue) is expressed in a subset of lateral glial cells; in gcm mutants REPO expression is reduced to a few cells, and bnb expression is completely absent in the CNS. (S,T) In late stage embryos, WRAPPER is expressed in midline glial cells, in some lateral glial cells and in glial cells supporting chordotonal sensory organs (arrowheads); in gcm mutant embryos WRAPPER expression in lateral glia (CNS) and in chordotonal sensory organs (PNS) is absent, whereas expression in midline glial cells remains.

View larger version (26K): [in a new window]   Fig. 6. Changes in transcript levels of the genes with differential expression in both early and late embryonic stages after gcm misexpression. Ninety-three genes show significant changes in expression levels in response to gcm misexpression at stage 11 (yellow), as well as at stage 15/16 (red). Bars represent the fold changes in gene expression levels between wild-type embryos and sca-gcm embryos. Positive values indicate that the relative expression level of a gene is increased (upregulation) and negative values indicate a decrease (downregulation).