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First published online 14 June 2006
doi: 10.1242/dev.02429

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The brain tumor gene negatively regulates neural progenitor cell proliferation in the larval central brain of Drosophila

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

View larger version (121K): [in a new window]   Fig. 1. brat mutation perturbs development of the larval central brain. GAL4NP3262 driven UAS-mCD8::GFPexpression in heterozygous (A,C,E) and homozygous (B,D,F) zygotic brat mutant larval CNS reveals all cell membranes of secondary lineages in central brain (CB) and optic lobe (OL). (A,B) In late third instar brat mutant larvae, brain hemispheres (Br) are markedly enlarged and characterized by cellular overgrowth whereas ventral ganglia (VG) appear normal (compare B with A). (C,D) In early to mid third instar larvae, brain hemispheres of brat mutants appear of same size as heterozygous brain hemispheres when viewed as whole mounts (D, compare with C). The cellular architecture of optic lobes is unaltered but is pushed aside by overgrowth of the central brain area that displays abnormal cells types. (E) Higher magnification of heterozygous central brain area in C reveals a regular pattern of large superficial neuroblasts (asterisks), in association with smaller numerous progeny cells. (F) By contrast, higher magnification of central brain area in D reveals that brat mutant brain tissue is characterized by dense cellular masses of numerous small, pleiomorphic cells in addition to larger cells, presumably neuroblasts (asterisks). Scale bar, 70 µm. Genotypes: (A,C,E) brat11, GAL4NP3262/FRT40A; UAS-mCD8::GFPLL6, UAS-nlslacZJ312/+. (B,D,F) brat11, GAL4NP3262/FRT40A, brat11; UAS-mCD8::GFPLL6, UAS-nlslacZJ312/+.

View larger version (117K): [in a new window]   Fig. 2. The brat mutation affects larval central brain proliferation in a cell-autonomous manner. Wild-type (A,B) and brat11 (C,D) MARCM clones labelled with CD8::GFP in third instar larval brain hemispheres (A,C) and ventral ganglia (B,D), counterstained with the DNA dye TOTO-3. When induced at low frequency in newly hatched larvae, wild-type clones contain progeny of a single neuroblast occupying a small area of third instar larval brain (single clone shown in A). Similar heat shock conditions generate brat11 mutant clones of large size, which appear difficult to resolve as single neuroblast lineages (two or more merged clones are shown in C). In ventral ganglia, brat11 mutant clones (D) are recovered at similar frequency as in wild-type clones (B) and appear indistinguishable in size and shape. Scale bars, 50 µm. Genotypes: (A,B) hsFLP/+; FRT40A, UAS-mCD8::GFPLL5, UAS-nlslacZ20b/FRT40A, tubP-GAL80LL10; tubP-GAL4LL7/+; (C,D) hsFLP/+; FRT40A, brat11, UAS-mCD8::GFPLL5, UAS-nlslacZ20b/FRT40A, tubP-GAL80LL10; tubP-GAL4LL7/+.

View larger version (128K): [in a new window]   Fig. 3. Cell markers and proliferation of wild-type neural lineages in the larval central brain. Wild-type MARCM clones labelled with membrane-tethered CD8::GFP (A-I; white) and centrosomin-GFP for centrosomes (D,E; white dots). Neuroblasts are indicated by asterisks and closely associated GMCs by arrowheads. (A) GFP-labelling of an entire clone shows a single large neuroblast and its associated progeny of adult-specific neurons (ganglion cells; GCs) which send their neurite in a common bundle (cell body fibre tract; CBT). (B-I) Neuroblast and late born cells are shown, immunostained as indicated in each panel. Mitosis, detected by PH3 immunostaining, is always restricted to neuroblasts (B: metaphase; D: telophase) and GMCs (E: anaphase/telophase; F,G: prophase/metaphase). CycE is detectable in neuroblast from interphase (E) to telophase (D) and in terminally dividing GMCs at interphase (D), but not during terminal division (E, inset shows CycE only). Mira is polarised at the neuroblast cortex during mitosis forming a crescent at metaphase (B) that segregates in budding GMC at telophase (C). Uniform cortical Mira is detected in neuroblasts at interphase (F,I) or in GMCs during mitosis (F, inset shows Mira only). Both neuroblasts and GMCs show Grh in their nuclei (H), whereas Pros is detectable in all nuclei of GMCs and GCs but not in the nucleus of neuroblasts (G,H, inset shows Pros staining only). Elav is expressed exclusively in nuclei of GCs that do not express Mira (I). Genotypes: (A-C,F-G) hsFLP/+; FRT40A/FRT40A, tubP-GAL80LL10; UAS-mCD8::GFPLL6, UAS-nlslacZJ312/tubP-GAL4LL7. (D,E) hsFLP/UAS-cnn::GFP; FRT40A, UAS-mCD8::GFPLL5/FRT40A, tubP-GAL80LL10; tubP-GAL4LL7/+.

View larger version (129K): [in a new window]   Fig. 4. brat mutant clones comprise an excessive number of neural progenitor cells. brat11 MARCM clones positively labelled with CD8::GFP (white) and immunostained as indicated for each panel. The outline of GFP-labelled cellular masses of mutant clones are indicated with dots when GFP label is omitted for clarity. GFP-labelled mutant clones appear heterogeneous in size. (A,B) Most cells express cyclin E (A) and numerous cells are engaged in mitosis (B, magenta, E, yellow). (C-E) In contrast to heterozygous wild-type tissues that surround clones, the majority of brat mutant cells show expression of markers characteristic of neural progenitors: nuclear Grh (C,C'; blue) and cortical Mira (E, magenta). Only very few nuclei express Pros (C', arrowheads) and Elav (D, arrowheads). This apparent deficit in neural differentiation of brat mutant cells is also revealed by the absence of axonal processes. Scale bar, 50 µm; genotype as in Fig. 2.

View larger version (100K): [in a new window]   Fig. 5. larval brat mutant central brain clones continue to proliferate into adulthood. (A-D) Dissected whole-mount 3-week-old adult brains showing MARCM clones which had been induced in early first instar larvae. (A) X-gal detection of nuclear lacZ marker (blue) reveals typical sizes of wild-type MARCM clones in central brain (arrowhead) and optic lobe (arrow). (B) Close up view of wild-type clones in central brain immunostained with anti-ß-galactosidase (nuclei, white). Wild-type clones lack PH3 immunoreactivity (magenta) consistent with proliferation arrest during puparium formation and metamorphosis. (C) X-gal labelled brat11 MARCM clones in central brain (arrowheads) are dramatically enlarged in size, whereas mutant clones in optic lobe (arrows) appear wild-type-like. [The brown axon-like structures on the specimen (asterisks) are the remains of the adult head cuticule.] (D) Double-immunolabelling of nuclear ß-gal (white) and PH3 (magenta) reveals numerous cells of the enormously enlarged brat mutant clones mitotically active even 3 weeks after adult eclosion (compare with B). Scale bars, 50 µm; genotypes as in Fig. 2.

View larger version (132K): [in a new window]   Fig. 6. Pros mutant clones phenocopy brat mutant clones in the larval central brain. Wild type (A) and pros17 MARCM clones (B-G') labelled with nuclear lacZ (A-C, white) or CD8::GFP (D,E,F,G, white) and immunostained as indicated for each panel. pros17 clones in central brain are enlarged and can cover most of the brain hemisphere (compare B with A) with multiple pros mutant clones occasionally merged into a labelled cell mass. (C-G') Close up views of labelled mutant cells reveal the presence of pleiomorphic cell types including large neuroblast-like cells (arrows) and many smaller cells (arrowheads). Brat expression is ubiquitous in larval CNS (A,B) and detected at comparable levels in cytoplasm of all mutant pros cells labelled with nuclear lacZ, or in heterozygous wild-type cells that surround labelled mutant cells (C,C'). Many pros mutant cells dispersed throughout clones are engaged in mitosis as indicated by PH3 immunoreactivity (D,D"; magenta) and most cells express CycE (D,D'; blue) and CycB (E, magenta), irrespective of their size and location within clones. Only a few nuclei are positive for the postmitotic marker Elav (F,F'; blue), whereas most cell express Mira at the cortex (F-G'; magenta) and Grh in nuclei (G,G'; blue). Cortical expression of Mira is uniform in vast majority of small cells (F-G', arrowheads), whereas polarized Mira forming crescents can be observed in large neuroblast-like cells (F-G', arrows). Scale bar, 100 µm (A,B) or 20 µm (C-G). Genotypes: (A) hsFLP/+; UAS-mCD8::GFPLL5, UAS-nlslacZ20b/tub-Gal4; FRT82B/FRT82B, tubP-GAL80LL10; (B-G') hsFLP/+; UAS-mCD8::GFPLL5, UAS-nlslacZ20b/tub-Gal4; FRT82B, pros17/FRT82B, tubP-GAL80LL10.

View larger version (84K): [in a new window]   Fig. 7. pros can promote cell cycle exit and differentiation of brat mutant cells. Targeted expression of UAS-brat (A,B) or UAS-pros (C,D) in brat11 mutant clones restores wild type-like features of larval central brain lineages. Clones labelled with CD8::GFP (white) were immunostained in late third instar larvae as indicated for each panel. Outline of clones is indicated with dots when GFP label is omitted for clarity. Clones display a limited number of neuroblast-like cells (asterisks) that specifically stain for Grh (A,B,D) or Mira (C). Mitosis is restricted to large nuclei (C,C'; asterisks) or to smaller, closely associated GMC-like cells (C,C'; arrowhead). Most cells in clones project axons contributing to fibre tracts (A-D; arrows) and show nuclear Elav (A,A') as well as nuclear Pros (B,B',D,D'). Scale bar, 40 µm. Genotypes: (A,B) hsFLP/+; FRT40A, brat11, UAS-mCD8::GFPLL5, UAS-nlslacZ20b/FRT40A, tubP-GAL80LL10; UAS-bratFL1B/tubP-GAL4LL7. (C,D) hsFLP/+; FRT40A, brat11, UAS-mCD8::GFPLL5, UAS-nlslacZ20b/FRT40A, tubP-GAL80LL10; UAS-prosL(wt)9D/tubP-GAL4LL7.

View larger version (15K): [in a new window]   Fig. 8. Neural lineage formation in the larval central brain of wild-type, brat and pros mutant clones. (A) In wild-type Drosophila, neural progenitor cells are required for lineage formation: neuroblasts (NB) divide asymmetrically to self-renew and to produce a series of ganglion mother cells (GMC); GMCs instead differentiate by undergoing a single terminal division that produces two postmitotic ganglion cells (GCs). GCs send out axons contributing to fibre tracts. (B) Somatic mutation of the tumour suppressor brat, and loss of function of the cell fate determinant pros impede differentiation of progenitor cells into GCs. Instead, these mutant cells retain progenitor cell-like characteristics and indefinitely self-renew, thereby generating clonally derived brain tumours. Targeted misexpression of either brat or pros in brat mutant cells restores differentiation which abrogates brain tumour formation.