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doi: 10.1242/10.1242/dev.00377

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The dead ringer/retained transcriptional regulatory gene is required for positioning of the longitudinal glia in the Drosophila embryonic CNS

Tetyana Shandala1,2, Kazunaga Takizawa3,* and Robert Saint1,4,{dagger}

1 Centre for the Molecular Genetics of Development, Adelaide University, Adelaide SA 5005, Australia
2 Department of Molecular Biosciences, Adelaide University, Adelaide SA 5005, Australia
3 Department of Developmental Genetics National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan
4 Research School of Biological Sciences, Australian National University, Canberra, ACT 2601, Australia
* Present address: Laboratory for Neural Network Development RIKEN Center for Developmental Biology 2-2-3 Chuo Kobe 650-0047, Japan



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  		Fig. 1. dri is not expressed in midline-derived cells of the Drosophila embryonic CNS. Confocal micrographs of stage 15 whole-mount embryos carrying specific enhancer trap lines directing ß-galactosidase expression in midline cells of the CNS. Embryos were immunochemically stained with polyclonal rabbit anti-ß-gal (Alexa488-conjugated secondary antibody, green) and rat anti-DRI (Lissamine-Rhodamine-conjugated secondary antibody, red). Anterior is towards the left. Some dri-positive cells are located in the vicinity of the embryonic midline: (A) Posterior to the posterior commissures (arrow) as revealed by the mAb 22C10 axonal marker; (B) posterior to X55-positive cells (the VUM neurones, the MNB and its support cells and the posterior pair of the midline glia) (arrow); (C) posterior to Star-positive (P 2333) midline glia (long arrow); and (D) half way between neighbouring segmental clusters of AA142-positive midline glia (MGA, MGM and MGP) (long arrow). There is one dri-positive cell per hemineurome located at the very edge of the VNC (A-D, notched arrowhead), posterior to the intersegmental nerve root as revealed by the mAb 22C10 (A). Other dri-positive CNS cells are labelled with short arrows in C-D.

 


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  		Fig. 2. dri is expressed in longitudinal glial cells of the Drosophila embryonic CNS. Confocal micrographs of stage 15 whole-mount embryos. Embryos are immunochemically stained with polyclonal rabbit anti-ß-galactosidase (Alexa488-conjugated secondary antibody, green) and rat anti-DRI (Lissamine-Rhodamine-conjugated secondary antibody, red). Anterior is towards the left. (A,B) Focal planes showing that dri is expressed in the medialmost cell body glia (MM CBG, arrow in A), the subperineural glia (SPG, double arrowheads in B) and longitudinal glial (LG, short arrow in B) that express ß-galactosidase in the rA87 enhancer trap line. (C) dri is expressed in the A and B SPG (double arrowhead) and LG (short arrow) that express ß-galactosidase in the pntrm254 enhancer trap line. (D) dri is expressed in the A and B SPG (double arrowheads) and LG (short arrow) that express ß-galactosidase in the rC56 enhancer trap line. (E) dri is expressed in the A and B SPG (double arrowhead) and LG (short arrow) that express repo. (F) dri is expressed in all six prospero-expressing LG (short arrows, only five of these cells are in focus). (G) ct and dri are co-expressed in the LG (short arrow). (H) CNS stained for DRI alone showing the assignments of cells derived from the data shown in (A-F): LG (short arrow), A and B SPG (double arrowheads), MM CBG (long arrow) and N (neurone, notched arrowhead).

 


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  		Fig. 3. DRI is not involved in initiation of the glial cell fate or repression of the neural cell fate decision in lateral glioblasts. (A) dri expression in longitudinal glia of a stage 12 embryo (arrow). (B) Whole-mount stage 12 dri1/dri2 embryo, derived from a maternal dri1 homozygous germline clone, stained with anti-REPO antibody. Loss of maternal and zygotic DRI does not interfere with the early behaviour of the LG. (C) Wild-type stage 15 embryo stained with anti-REPO antibody. (D) Ectopic expression of dri in the mesectoderm using sim-GAL4 does not induce longitudinal glial cell fate specification, as detected by anti-REPO staining (compare with C). (E) Ectopic expression of dri in parasegments T2-A4 using Kr-GAL4 (between arrowheads) does not inhibit neural differentiation, as detected by anti-ELAV antibody. (F) 22C10 antigen is correctly localized in a single somatic muscle fibre in dri1/dri2 homozygotes (arrowheads).

 


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  		Fig. 4. Loss of DRI results in axon defasciculation in the Drosophila embryonic CNS. Dorsal view of dissected ventral nerve cord preparations. Anterior is towards the left. (A) The first axon bundle in control dri heterozygotes is revealed by anti-GFP staining of ftz-GAL4-driven UAS-tauGFP. (B) ftz-GAL4-driven UAS-tauGFP staining of a dri homozygous mutant embryo shows that the first axon bundle is correctly established. (C) Immunochemical staining with mAB 1D4, which recognizes the Fasciclin 2 protein, reveals the three axon bundles either side of the midline in a wild-type stage 16 embryo. (D) In dri1/dri2 mutant embryos, the first axon tract occupies the correct position relative to the midline, while the spacing between other tracts is slightly increased. Moreover, a moderate axon defasciculation is evident. In addition, axons occasionally cross between fascicles (arrow). (E) Another example of a dri1/dri2 mutant embryos, showing defasciculation of the three axon bundles. (F) pntrm254 mutants exhibit a mild defasciculation of axons, similar to those observed in dri mutant embryos. (G) Strong pathfinding defects are observed in gcmP1 mutant embryos.

 


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  		Fig. 5. The longitudinal glial cells expressing repo and cut are misplaced in dri mutants. Dorsal view of dissected ventral nerve cord preparations of dri/CyO wgß gal (A,C,E) and dri1/dri2 homozygous (B,D,F) stage 16 embryos, immunochemically stained with anti-CT (A-B) or anti-REPO (C-F) antibodies and AP-conjugated (for REPO or CT in A-F) and HRP-conjugated (for ß-gal in CyO wgß-gal, A, C) secondary antibody. Anterior is towards the left. (A,C,E) dri heterozygotes exhibit the proper positions of two rows of glial cells either side of the midline. (B,D,F) Loss of DRI results in the misplacement of nuclei marked with CT (compare heterozygotes in A with dri homozygotes in B) or REPO (compare C with D). Higher magnification (E,F) more clearly shows that repo-positive nuclei of the longitudinal glial cells in dri homozygotes do not occupy a flat layer underlying the longitudinal connectives (F; arrowhead, nucleus in focus; arrows, mispositioned nuclei out of the focal plane) as in the wild type (E).

 


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  		Fig. 6. Loss of DRI results in downregulation of Neuroglian in subsets of longitudinal glial cells. Whole-mount immunochemical staining of wild-type (A-C) and dri1/dri2 (D-F) embryos. Dorsal glial cells are labelled with Pß-gal (driven by a gcm enhancer trap, green) and mAb 1B7 against NRG CAM (red). In all panels anterior is upwards. LG are slightly misplaced in some hemisegments. Some of those cells do not express Neuroglian and show a rounded cell shape (arrows in D,F indicate cells shown at higher magnification in inset), although the penetrance is low, the phenotype being detected on average in three hemisegments from T1 to A8 in an embryo.

 


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  		Fig. 7. dri expression in the longitudinal glial cells of the Drosophila embryonic CNS depends on GCM and REPO. Dorsal view of dissected embryonic ventral nerve cords immunochemically stained with anti-DRI and detected with AP-conjugated secondary antibody. Anterior is towards the left. (A) The distribution of dri-expressing cells in wild-type embryos. (B) dri expression is completely abolished in all glial (arrow) but not neural (arrowhead) cells in the gcmP1 mutants. (C) dri expression is lost from most longitudinal glial cells but not from the neurone (arrowhead) nor, perhaps, from A/B SPG (thin arrows) in a repo3 mutant embryo. (D) Expression of dri in the dorsal glia (arrow) seems to be unaffected in a stage 15 embryo mutant for pntD88. (E,F) Expression of dri in the dorsal glia is also unaffected in stage 15 embryos mutant for flb. (E) One focal plane, showing MM CBG, long arrow with asterisk. (F) A different focal plane showing the LG (short arrow) and the dri-positive neural cells (arrowhead).

 


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  		Fig. 8. The number of cells expressing loco and pros is reduced in dri mutants. Dorsal view of dissected ventral nerve cord preparations of dri/CyO wgß gal (A,C,E) and dri1/dri2 (B,D,F) stage 16 embryos, immunochemically stained with anti-PROS and anti-ß-galactosidase (A-D), anti-ß-galactosidase and anti-DRI antibodies (E,F) and detected with AP-conjugated (for PROS and rC56 ß-gal; A-F) and, in some cases, HRP-conjugated (for ß-gal: A,C; and DRI in E) secondary antibody. Anterior is towards the left. (A) In dri/CyO embryos there are five pros-positive cells in the focal plane shown here. (B) In dri mutant embryos, pros expression is absent in one cell per hemineuromere, depicted by an arrow. (C,D) Higher magnifications of A,B, respectively, showing that the four remaining pros-positive cells are not positioned in a flat sheet as they are in wild type. (E,F) rC56-directed ß-galactosidase expression is reduced or absent in the longitudinal glia of dri mutant embryos (arrows in F).

 

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