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First published online October 12, 2006
doi: 10.1242/10.1242/dev.02606

Development 133, 4331-4339 (2006)
Published by The Company of Biologists 2006
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The columnar gene vnd is required for tritocerebral neuromere formation during embryonic brain development of Drosophila

Simon G. Sprecher1,2,*, Rolf Urbach3, Gerhard M. Technau3, Filippo M. Rijli2, Heinrich Reichert1 and Frank Hirth1,{dagger},{ddagger}

1 Biozentrum, University of Basel, CH-4056 Basel, Switzerland.
2 Institut de Génétique et de Biologie Moléculaire et Cellulaire, UMR 7104, CNRS/INSERM/ULP, BP 10142, F-67404 Illkirch Cedex, CU de Strasbourg, France.
3 Institute of Genetics, University of Mainz, D-55099 Mainz, Germany.


Figure 1
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  		Fig. 1. Spatiotemporal expression of vnd during embryonic brain development. Laser confocal microscopy, reconstructions of optical sections, lateral views (A,C) and 3D reconstructed models of confocal microscopic stacks, covering corresponding optical sections (B,D) are shown for embryonic late stage 12 (A,B), and stage 15 (C,D). (A) Embryo double-immunolabelled with anti-Neurotactin (NRT) antibody (red) and anti-VND antibody (green/yellow). (C) Double-immunolabelling with anti-HRP antibody (red) and anti-VND antibody (green, yellow). (A,C) At stage late 12, three vnd expression domains become apparent that are still observable at stage 15. (B,D) 3D reconstructed models show relative location of domains within developing brain (vnd expression domains: blue, protocerebral; green, deuterocerebral; red, tritocerebral).

 

Figure 2
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  		Fig. 2. Mutant brain phenotype observed in vnd-null mutant embryos at embryonic stage 15. Laser confocal microscopy reconstructions of optical sections, lateral views. (A,B) Double-immunolabelling with anti-HRP antibody (red) and anti-ELAV antibody (yellow/green). (C,D) Double-immunolabelling with anti-HRP antibody (red) and glial-specific anti-REPO antibody (yellow/green). Arrows indicate general trito-deutocerebral region. (E,F) Double immunolabelling with anti-HRP antibody (red) and an anti-EN antibody (yellow/green). (A) In wild type, neuron-specific marker ELAV reveals all neural cell bodies. (B) By contrast, in vnd-null mutants a large gap is seen in the tritocerebral/deuterocerebral region (arrow). (C) The glia-specific marker REPO reveals localization of glial cell bodies in embryonic wild-type brain. (D) In vnd mutant embryos, REPO-expressing cells in residual tritocerebral/deuterocerebral region appear to be present but are severely misplaced (arrow). (E) In wild type, the protocerebral b1 en-stripe (b1), deuterocerebral b2 en-stripe (b2), tritocerebral b3 en-stripe and anteriormost en expressing secondary head spot (shs) are visible (arrowheads). (F) By contrast, in vnd-null mutant embryos only b1 en-stripe and en expressing secondary head spot are present (arrowheads), and neuron-specific HRP marker reveals a cellular gap in deuto- and tritocerebral region (arrow).

 

Figure 3
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  		Fig. 3. Defective neuroblast formation in lab-expressing tritocerebral domain of vnd mutants. (A-D) Double-immunolabelling with neuroblast-specific anti-DPN (blue) and anti-LAB (brown), at embryonic stage 11, in wild type (WT) (A,B) and vnd-null mutants (C,D). (A-D) Ventral views of flat preparations. (B,D) Higher magnification of regions indicated in A,C by black frames, at level of brain neuroblasts. (A) In wild-type embryos, all brain neuroblasts have developed by stage 11. (B) Two deutocerebral and complete set of tritocerebral neuroblasts developing from LAB domain are indicated [according to nomenclature of Urbach et al. (Urbach et al., 2003Go)]: Tv1-5, ventral tritocerebral neuroblasts; Td1-8, dorsal tritocerebral neuroblasts; Dv2, Dv4 ventral deutocerebral neuroblasts. Broken line encircles group of dorsal neuroblasts that are assumed to be retained in vnd-null mutants (compare with D). (C) In vnd-null mutants, overall expansion of LAB domain appears to be reduced when compared with wild type (A), and invagination of the foregut (Fg) is affected (compare lateral extension of foregut invagination as marked by red arrowheads in A and C). (D) Number of DPN-positive neuroblasts (white asterisks) is diminished, when compared with B. One neuroblast (white dot) does not express DPN at detectable levels; similarly, in wild type a neuroblast expressing DPN at significantly lower levels is found in same relative position (see Td5 in cluster of neuroblasts encircled by broken line, B). Fg, foregut; Lr, labrum; Md and Mx, mandibular and maxillary segment, respectively.

 

Figure 4
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  		Fig. 4. vnd and the anterior Hox gene labial act independently in tritocerebral neuromere formation. Laser confocal microscopy of stage 15 embryos, reconstructions of optical sections, lateral views. Arrows indicate tritocerebral region. (A) Wild-type embryonic brain immunolabelled with anti-HRP antibody (blue). (B) Wild-type embryonic brain triple-immunolabelled with anti-HRP (blue), anti-LAB (green) and anti-VND (red); co-expression of vnd and lab is seen in part of lab-expressing tritocerebral domain (arrow); A and B are from same section. (C) vnd mutant embryonic brain immunolabelled with anti-HRP (blue). (D) vnd mutant embryonic brain double-immunolabelled anti-HRP (blue) and anti-LAB (green); only a few cells remain in the tritocerebrum and express lab; C and D are from same section. (E) P{ry+ 7.31 lab-LacZ};;labvd1/labvd1: null mutant embryonic brain immunolabelled with anti-HRP (blue); no anti-HRP immunoreactivity is detected in tritocerebral domain. (F) P{ry+ 7.31 lab-LacZ};;labvd1/labvd1: null mutant embryonic brain triple-immunolabelled with anti-HRP (blue), anti-VND (red) and anti-ßGAL, revealing 7.31 lab-LacZ reporter (green). vnd expression is seen in a part of tritocerebral domain mutant for lab and expression overlaps with lab-lacZ specific reporter gene expression; E and F are from same section.

 

Figure 5
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  		Fig. 5. Increased apoptosis in vnd-null mutant embryos at embryonic stage 12. Laser confocal microscopy, reconstructions of optical sections, lateral views. (A-C,G,H) Embryos at early stage 12; average maximal extent of lab-expressing domain in wild-type embryos is outlined (white line, arrow) and projected onto each figure in top row. (D-F,I,J) Embryos at late stage 12; average maximal extent of lab-expressing domain in wild-type embryos is outlined (white line, arrow) and projected onto each figure in bottom row. (A,D) Wild type double-immunolabelled with anti-NRT (green) and anti-LAB (red) showing lab-expression domain (arrow). (B,E) P{ry+ 7.31 lab-LacZ} in wild-type background. Double-immunolabelling using anti-NRT (green) and anti-ßGAL shows that 7.31 lab-LacZ reporter construct mimics endogenous lab expression. (C,F) P{ry+ 7.31 lab-lacZ} in vnd-null background. Double-immunolabelling using anti-NRT (green) and anti-ßGAL reveals extent of lab expression domain, as assayed by 7.31 lab-lacZ reporter construct. (G,I) Wild-type double-immunolabelled with anti-LAB (red) and TUNEL staining (green) showing low level of apoptotic activity in lab domain. (H,J) vnd-null mutant; anti-LAB immunolabelling (red) and TUNEL staining (green) showing increased level of apoptotic activity in lab expression domain at early stage 12. (K) Quantitation of TUNEL-positive cells and of ß-galpositive 7.31 lab-lacZ-expressing cells detectable in wild-type and vnd mutant background. Values are means of n=17 preparations counted in each case: wt TUNEL=10, vnd TUNEL=20; wt cells=43, vnd cells=27. Standard deviations are indicated as bars (P<0.000005 each) of Student's t-test are indicated as stars (**). By early stage 12, the number of TUNEL-positive apoptotic cells in vnd mutant tritocerebrum are significantly increased; by late stage 12, number of lab-lacZ expressing cells in vnd mutant tritocerebrum are significantly reduced.

 

Figure 6
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  		Fig. 6. Partial restoration of brain structures and lab expression in vnd mutants by blocking apoptosis. Laser confocal microscopy of stage 15 embryos, reconstructions of optical sections, lateral views. Arrows indicate tritocerebral region. (A) Wild type; (B) vnd mutant; (C,D) sca::Gal4/UAS::p35 in vnd null mutant background. (A-C) Embryonic brain immunolabelled with anti-HRP (red). (D) Embryonic brain double-immunolabelled with anti-HRP (red) and anti-LAB (green). When compared with wild-type (A) and vnd mutant brain (B), p35-mediated block of apoptosis partially restores HRP-immunoreactive tissue in vnd mutant tritocerebrum, and descending longitudinal connectives that transverse the tritocerebrum are detectable (C). Restoration of neural tissue also results in wild-type-like lab expression domain in vnd mutant tritocerebrum (D, compare with Fig. 4B).

 

Figure 7
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  		Fig. 7. Partial restoration of brain tracts in cell death prevented vnd mutant. Laser confocal microscopy of stage 15 embryos, reconstructions of optical sections, lateral views. Arrows indicate tritocerebral region. (A,B) Wild-type; (C,D) vnd mutant; (E,F) sca::Gal4/UAS::p35 in vnd-null mutant background. Embryos in B,D,F are not the same as in A,C,E, respectively. (A,C,E) Embryonic brain immunolabelled with anti-FAS2 (red). (B,D,F) Embryonic brain double-immunolabelled with anti-FAS2 (red) and anti-ELAV (green). (A,B) In wild type, FAS2 immunostaining reveals a number of early differentiating neurons as well as axon tracts, and ELAV expression is apparent within postmitotic neurons of all brain neuromeres, including the tritocerebrum. (C,D) By contrast, in vnd-null mutants, a gap is seen in the area of the tritocerebral region and the majority of ELAV-expressing cells are lacking in this domain. (E,F) Ubiquitous block of apoptosis partially restores the gap-like defects observed in vnd loss-of-function mutants: FAS2-immunoreactive longitudinal connectives are detectable, although neural fibres display fasciculation defects, and a significant number of ELAV-expressing cells are detectable in the cell death prevented vnd mutant tritocerebrum (F, arrow; compare with B).

 

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© The Company of Biologists Ltd 2006