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First published online 27 July 2005
doi: 10.1242/dev.01932

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Drosophila Grainyhead specifies late programmes of neural proliferation by regulating the mitotic activity and Hox-dependent apoptosis of neuroblasts

Medical Research Council, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK

View larger version (109K): [in a new window]   Fig. 1. Neural Grh expression is restricted to progenitors and its distribution is cell-cycle dependent. (A-C) Wild-type thoracic MARCM clones marked in green with nuclear ß-galactosidase (n-ßgal) or membrane-tethered GFP (mGFP) under tubulin-GAL4 control. Expression of Grh (red) is observed in neuroblasts (large dotted circles) and GMCs (arrowhead), but not in adult-specific neurons. (A) In interphase (I-phase) neuroblasts, Grh is excluded from the nuclelus and co-localizes with DNA (blue) in a crescent. (B,C) In mitotic (M-phase) neuroblasts, Grh is evenly distributed throughout the cell and does not specifically co-localise with DNA (blue in B) or phosphorylated histone H3 (H3p, blue in C), and is evenly distributed throughout the cell. In this and all subsequent figures, the neuroblast is indicated by a dotted circle.

View larger version (55K): [in a new window]   Fig. 2. Grh regulates neural proliferation in a segment-specific manner. (A) Cartoon showing the distribution of neuroblasts (circles) in the postembryonic CNS; the thorax and central abdomen are boxed. The major domains of expression of the Hox genes Antennapedia (Antp, blue), Ultrabithorax (Ubx, cyan), Abdominal-A (AbdA, green) and Abdominal-B (AbdB, yellow) are shown. Anterior is to the top in panels A-E. (B-E) Single confocal sections of the thoracic (above dotted line) and abdominal (below dotted line) regions of the CNS isolated from 78-hour larvae. (B,C) H3p/AbdA labelling of balanced control (B) and grh370 hemizygous (C) larvae showing that loss-of-grh activity reduces the mitotic index in the thorax but leads to ectopic mitoses in the abdomen. (D,E) 72-78-hour BrdU-pulse labelling of wild-type control (D) and grh370 hemizygous (E) larvae showing that removing neural grh function leads to fewer thoracic cells progressing through S-phase but that numerous ectopic S-phases occur in the abdomen. (F) Histogram showing the total thoracic number of Miranda-positive cells at 72 hours (Mira+ neuroblasts), or lineages incorporating BrdU from 72-78 hours (BrdU+ lineages). In both cases, a reduction (star indicates significance at P=0.002) is observed in grh370 hemizygotes (for Mira: n=9 larvae, mean=114.6, s.d.=8.6; for BrdU: n=7, mean=100.7, s.d.=18) relative to balanced siblings (for Mira: n=9, mean=138.1, s.d.=16.8; for BrdU: n=6, mean=140.1, s.d.=16.9). (G) Histogram showing that the number of cells per thoracic lineage incorporating BrdU during the 72- to 78-hour window is higher for wild-type control larvae (n=52 lineages, mean=7.1, s.d.=2.1) than for grh370 hemizygotes (n=57, mean=5.3, s.d.=2.4).

View larger version (58K): [in a new window]   Fig. 3. Abdominal neuroblast lineages lacking Grh activity proliferate for longer than normal. (A,B) Four to five abdominal segments labelled by continuous BrdU treatment from 0-96 hours, showing rows of vm and vl clusters from wild-type control larvae (A) and corresponding but enlarged clusters in grh370 hemizygotes (B). The hemizygotes also show BrdU-positive clusters in ectopic abdominal locations (outlined). (C,D) Confocal projections of one vl cluster containing six cells from a balanced control larva (C) and part of one vm cluster, where nine out of a total of 14 cells are shown from a grh370 hemizygous larva (D; see also Fig. S1 in the supplementary material). BrdU-labelled nuclei are outlined in white. (E,F) Histograms comparing the number of cells generated per vm/vl lineage (from 0 hours onwards) in wild-type larvae and grh370 hemizygous larvae at 72 hours (E) and 96 hours (F). Average lineage size remains at four cells from 72-96 hours in wild-type larvae (72 hours: n=19 lineages, mean=3.9, s.d.=1.5; 96 hours: n=18, mean=4.2, s.d.=1.5) but in grh370 hemizygotes it increases from 7.5 to 14 cells (72 hours: n=14, mean=7.4, s.d.=2.4; 96 hours: n=18, mean=13.7, s.d.=2.4).

View larger version (43K): [in a new window]   Fig. 4. Grh is required to maintain a large mitotically active neuroblast at late stages. (A-C) Single thoracic MARCM clones marked with n-ßgal driven by elav-GAL4 and labelled for M-phase with H3p. Wild-type clones contain many neurons and the large neuroblast frequently undergoes mitosis at 96 hours (A), whereas grhB37 homozygous clones contain less neurons and the neuroblast ceases mitosis by 72 hours (B) and is no longer evident by 96 hours (C). (D) Histogram showing that the average number of adult-specific thoracic neurons at 96 hours in grhB37 clones (n=43 clones, mean=28.6, s.d.=12.7) is approximately half the wild-type value (n=61, mean=57.3, s.d.=15.7).

View larger version (63K): [in a new window]   Fig. 5. Repression of Ubx in postembryonic neuroblasts does not require larval Grh activity. All panels show the pattern of Ubx expression in MARCM neuroblast clones in the posterior thorax (n-ßgal driven by elav-GAL4) at the stages indicated on the left. (A-D) Wild-type clones do not express Ubx in the neuroblast or GMCs at any of the stages shown, but it is progressively upregulated within adult-specific neurons. At 72 hours, few if any cells are Ubx positive (in A, projected sections produce false overlap), by 96 hours early-born adult-specific neurons become Ubx positive (B), and, by 104 hours, the majority of neurons in many of the lineages become Ubx positive, but not the GMCs (arrowheads) or neuroblasts (C,D). (E-G) grhB37 mutant clones do not express Ubx in the neuroblast or adult-specifc neurons at 72 hours (E). By 96 (F) and 104 hours (G), although clone size is small and the large neuroblast is absent, Ubx is upregulated in a subset of adult-specific neurons in some but not all clones (in G, two clones are shown).

View larger version (37K): [in a new window]   Fig. 6. Grh may prevent apoptosis of thoracic neuroblasts at late stages. (A,B) Single confocal sections of grhB37 (A) and grhB37; UAS-P35 (B) MARCM clones, showing that inhibiting cell-death caspases rescues a large neuroblast at 96 hours. (C) Histogram showing MARCM clone size for wild type (with elav-GAL4, mean=57.3, n=61, s.d.=12.7), grhB37 (with elav-GAL4, n=43, mean=28.6, s.d.=15.7) and grhB37; UAS-P35 (with elav-GAL4, n=31, mean=32, s.d.=8.6). Although elav-GAL4 driving the UAS-P35 transgene rescues the neuroblast in grhB37 clones at 96 hours, it does not significantly increase clone size. (D) Histogram showing the percentage of MARCM clones containing a large neuroblast at 96 hours (sample sizes and genotypes are as in C). elav-GAL4 driving the UAS-p35 transgene rescues the presence of a large neuroblast in >40% grhB37 clones.

View larger version (87K): [in a new window]   Fig. 7. Grh is required for maintaining but not initiating the late phase of AbdA expression in neuroblasts. (A) At 96 hours, a neuroblast in an abdominal H99 MARCM clone expresses Grh. (B,C) The abdominal region of a balanced sibling control (B) and grh370 hemizygous CNS (C) at 96 hours, showing that Mira-positive neuroblasts are normally absent by this stage but persist when Grh activity is removed. (D) At 72 hours, a wild-type neuroblast maintains AbdA expression but displays weak and punctate Mira labelling, suggestive of the early stages of apoptosis. (E) At 96 hours, a neuroblast and its last-born progeny in an abdominal H99 MARCM clone maintain AbdA expression. (F,G) At 63 hours, Mira-positive interphase (F) and H3p-positive M-phase (G) neuroblasts in grh370 hemizygotes express AbdA as normal. (H) At 96 hours, a Mira-positive neuroblast from a grh370 hemizygous CNS fails to maintain AbdA expression.

View larger version (66K): [in a new window]   Fig. 8. grh mutant neuroblasts are insensitive to AbdA-induced apoptosis. (A-D) Abdominal region of balanced control (A), hs-abdA (B) and grh370, hs-abdA (C) larvae that received a heat shock at 48 hours and were labelled for Mira and AbdA at 72 hours. Ectopic AbdA expression from the hs-abdA transgene persists for at least 24 hours after heat shock and is observed posterior to the endogenous AbdA domain. The exogenous AbdA activity is sufficient to eliminate all Mira-positive neuroblasts from the abdomen of hs-abdA but not grh370, hs-abdA larvae. (D) A single confocal section of a grh370, hs-abdA larva showing three central abdominal neuroblasts that have expressed AbdA for 24 hours after heat-shock induction but have remained resistant to apoptosis.