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First published online 7 February 2007
doi: 10.1242/dev.02793

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Polycomb group genes are required for neural stem cell survival in postembryonic neurogenesis of Drosophila

Biozentrum, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland.

View larger version (78K): [in this window] [in a new window]   Fig. 1. PcG mutant neuroblast lineages fail to proliferate. Confocal images of late third-instar nervous system immunostained for Cyclin E (CYC E), phosphohistone H3 (PH3) and nuclear ß-galactosidase (nLACZ) in positively labeled MARCM clones. All panels show z projections of image stacks recorded on the ventral side. (A) Ventral view of a whole-mount nervous system showing a large number of mitotic cells in the optic lobes (OL) and in discrete foci evenly distributed in the central brain (CB) and in the thoracic part (T) of the ventral ganglia (VG), but not in the abdominal region (A). (B-E) Mitosis in isolated wild-type neuroblast lineages is revealed by PH3 staining in a maximum of two cells: the large neuroblast (asterisk) and/or the closely associated GMC (arrowhead) at the frequency indicated for each panel (n=148). In B, mitotic activity is seen outside of the clone. In C, mitotic activity is seen in the neuroblast. In D, mitotic activity is seen in the GMC. In E, mitotic activity is seen in neuroblast and GMC. (F-I) Compared with wild-type MARCM neuroblast clones in the central brain (F) or the ventral ganglia (H), Sce mutant clones in the same areas (G,I) contain a much smaller number of nuclei and do not stain for PH3. The dotted lines in F,G demarcate optic lobe (OL) and central brain (CB) by the density of mitotic cells. (J) The percentage of mitotic Sce and other PcG mutant clones is plotted with the number of clones examined indicated in parentheses. For genotypes, see Materials and methods. Scale bars: A, 50 µm; B-E, 5 µm, F-I, 30 µm.

View larger version (53K): [in this window] [in a new window]   Fig. 2. PcG mutant lineages lack neuronal precursor cells. Confocal images of wild-type control (wt) and Pc mutant MARCM clones labeled with membrane-tethered GFP (CD8::GFP, white) and immunostained as indicated. (A-D) Grh is detectable in the large neuroblasts (arrowheads) of the central brain (CB), but not in the precursor cells of the optic lobes (OL). Elav is expressed in all adult-specific neurons in both areas of the brain hemispheres. A,B are ventral views; C,D are optical cross-sections. Unlike wild-type clones which contain a single neuroblast and a large postmitotic progeny expressing Elav (A,C), the small labeled Pc mutant clones lack Grh-positive nuclei (B,D). Optical cross-sections (C,D) further show that neuronal precursors lie in the outer-most layer (top), and a wild-type neuroblast lineage forms a column spanning the cellular cortex. Pc mutant clones comprise a few neurons loosely associated and located away from the neuroblast layer. (E,F) Mira is evenly detected at the cellular cortex of the large neuroblasts at interphase (PH3-negative, arrowhead). During mitosis, Mira transiently accumulates on one side of the neuroblast forming crescents (PH3-positive, asterisk). The small, labeled mutant Pc clones in F contain only postmitotic cells lacking Mira. (G) The percentage of wt and PcG mutant clones containing Grh- and/or Mira-positive neuronal precursor cells is plotted with the number of clones examined indicated in parentheses. For genotypes see Materials and methods. Scale bars: A,B, 25 µm; C-F, 10 µm.

View larger version (52K): [in this window] [in a new window]   Fig. 3. Pc mutant neuroblasts undergo programmed cell death. (A) Time course analysis of programmed cell death. Clones were induced at 24 hours, 48 hours and 72 hours before examination in late third-instar larvae (top). The percentage of mutant Pc clones containing a neuroblast is plotted for each of the three temporal induction programs (bottom, light gray bars). The number of clones examined is indicated in parentheses. The percentage of clones containing anti-caspase immunoreactive neuroblasts is also plotted (dark gray bar). Caspase activation and disappearance of the progenitor marker was observed predominantly 48 hours after clonal induction. (B-B") Confocal image of a CD8::GFP-labeled Pc mutant clone stained for the progenitor marker Mira (blue) showing a dying neuroblast (arrowhead). Programmed cell death is specifically detected in the neuroblast by anti-activated Caspase-3 antibody (magenta).

View larger version (126K): [in this window] [in a new window]   Fig. 4. Abberant expression of homeotic genes in Pc mutant clones. Confocal images of wild-type control and Pc mutant MARCM clones labeled with membrane-tethered GFP (CD8::GFP, white) and immunostained as indicated. Shown are late third-instar ventral ganglia (A-F, outlined with dots) or close-up views on the central brain (G-I), anterior at the top. (A-C) Large wild-type clones are visible in the thorax which contains most of the proliferative neural precursors (labeled for Mira or Grh, blue). Hox genes of the Bithorax complex are expressed in restricted domains along the anteroposterior axis. (D-F). Small Pc clones show ectopic anterior expression of the homeotic genes (white and magenta). Ectopic expression of UBX (F) is not as widespread as Abd-B (D) or Abd-A (E). (G-I) Variability in Hox gene derepression is also evident on the close-up views of isolated small clones in the brain. Scale bars: A-F, 25 µm; G-I, 20 µm.

View larger version (82K): [in this window] [in a new window]   Fig. 5. Targeted misexpression of Abd-A in neuroblast clones mimics the PcG mutant phenotype. (A) Schematic of the larval CNS showing the expression domain of Abd-A (see also Fig. 4B,E). (B-F') Confocal images of late third-instar ventral ganglia immunostained for Abd-A (magenta) and the clonal markers CD8::GFP (B, green) or nuclear ß-galactosidase (C-F, green). Shown are ventral views at the junction between the endogenous domain of abdA expression and the more anterior thoracic region carrying clones. Anterior is at the top. Coexpression of Abd-A and the clonal marker is visible in white (e.g. see Fig. 4B). Close-up views in D-F are shown as split channels for clarity. Targeted misexpression of Abd-A under tubulinGAL4 control in wild-type MARCM clones generates small neuroblast lineages (UAS-abd-A). (B) A large-field view of the ventral ganglia (outlined with dashed lines) shows small clusters of nuclei expressing Abd-A, anterior to the endogenous expression domain in the abdomen. (C) In a similar field, most of the small Sce mutant clones also show ectopic expression of Abd-A. (D-F) Close-up views of the thoracic area immediately anterior to the endogenous Abd-A domain and carrying a clone of the genotype indicated. (D'-F') Abd-A expression in the same field without the clonal marker. A wild-type clone in the thorax contains a large progeny around the neuroblast and does not express Abd-A (D,D', neuroblast outlined with dots). Targeted misexpression of Abd-A results in small clones lacking a neuroblast (E,E'). Coexpression of P35 with Abd-A restores to a wild-type-like clone (F,F'), in spite of the elevated level of Abd-A in the neuroblast (dotted circle). Scale bars: B,C, 25 µm; D-F, 10 µm.

View larger version (75K): [in this window] [in a new window]   Fig. 6. Blocking apoptosis in PcG mutant clones restores wild-type-like neurogenesis. Confocal images of Pc+P35 clones labeled with CD8::GFP in the late third-instar central brain. A,C,E show the region around the junction of the central brain and optic lobe (dotted line); B,D,F,F' show high magnification of isolated clones. (A-D) Targeted expression of P35 in Pc mutant clones results in clones that contain a single large neuroblast cell (asterisk) expressing Mira (A,B) and Grh (C,D), whereas the adjacent GMCs (arrowheads) express low levels of Mira at the cortex (B and inset) or coexpress Grh and Pros in the nucleus (D shows an isolated clone adjacent to wild-type cells and the inset is a close-up view on the neuroblast and GMC). Nonneural stem cells within the clones express Elav (A,B) and Pros (C,D). (E-F') Neural progenitors survive in spite of the expression of Abd-A and are engaged in mitosis as indicated by PH3 immunoreactivity (G) at a frequency similar to wild-type controls (see Fig. 1J). (G) Quantification of the presence of neural progenitors and mitosis indicates a large degree of rescue of the three mutants by P35 (F).