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Binary cell death decision regulated by unequal partitioning of Numb at mitosis

Ecole Normale Supérieure, CNRS UMR 8542, 46 rue d’Ulm, 75230 Paris Cedex 05, France

View larger version (50K): [in a new window]   Fig. 1. Analysis of cell divisions in the vmd1a lineage. (A) Diagram of the ventral Cut-positive md neurons and es organs in segments A1-A7. Each vp1-4a organ is composed of an es neuron (large pink circular cell), a sheath cell (green) and a socket/shaft cell pair (yellow). The vmd1a and vmd4a neurons (dark purple diamond-shaped cells) can be distinguished from the vmd1-4 neurons (pink diamond-shaped cells) as they express the B6-2-25 enhancer-trap marker. (B) Md-es lineage (Orgogozo et al., 2001). Numb-inheriting cells are in red. (C-K) Confocal images showing the vmd1a-vp1 region. (C-D) In stage 12 CutA3-lacZ embryos (Jack and DeLotto, 1995) stained for ß-galactosidase in red, Numb in green and DNA in blue, the vmd1a pI cell divides asymmetrically soon after the vp1 pI cell has divided. (E) Stage 12 wild-type embryo stained for Cut in red, Numb in green and DNA in blue. (F-H) Stage 13 wild-type embryo stained for Cut in red, Pros in green and DNA in blue. A small Cut-positive dot is detectable at the pIIa position (arrowhead in F). (I) Late stage 13 wild-type embryo stained for Cut in red, Pros in green and Elav in blue. (J) Late stage 13 B6-2-25 embryo stained for Cut in red and ß-galactosidase in green. (I-J) Cells of the vp1 lineage are indicated by a bracket. (K-K') Same confocal section of a stage 13 CutA3-lacZ embryo stained for Cut (K,K') in red and ß-galactosidase (K') in green. A small Cut-positive and some ß-galactosidase-positive dots are detectable at the position of the pIIa cell (arrowhead in K, K'). In all figures, anterior is left and dorsal is up

View larger version (19K): [in a new window]   Fig. 2. Genetic evidence for cell death in the vmd1a lineage. (A-C) Ventral Cut-positive md neurons and es organs in segments A1-A7 of wild-type (A), Df(1)H99 (B) and arm-Gal4 UAS-p35 (C) embryos stained for Cut (red), Elav (blue) and Pros (green). (D) Diagram of the ventral-most Cut-positive md neurons and es organs in segments A1-A7 of Df(1)H99 or arm-Gal4 UAS-p35 embryos. The Cut-positive md neurons (diamond-shaped cells) are either highly Cut-positive (pink) or weakly Cut-positive (light purple). Es neurons, sheath cells and socket/shaft cell pairs are as in Fig. 1A. One ectopic es organ (bold outline in D) is observed near the vmd1a neuron in Df(1)H99 (B) and arm-Gal4 UAS-p35 (C) embryos. This es organ is composed of one Elav-positive es neuron (arrow), one Pros-positive sheath cell (arrow) and two Cut-positive cells identified as the socket and shaft cells (arrowheads; one of these two cells accumulates Suppressor of Hairless [Su(H)], a socket cell marker; data not shown (Gho et al., 1996)). This phenotype is observed in 40% of the segments of arm-Gal4 UAS-p35 embryos (n=52). In other segments, we observed three Cut-positive cells (12%), two Cut-positive cells (12%) or no additional Cut-positive cell (36%). Therefore, in these cases, the level of p35 accumulation may not be high enough to fully inhibit apoptosis in the vmd1a lineage. (E) Model proposed for the vmd1a lineage. The pIIa and pIIIb cells die.

View larger version (48K): [in a new window]   Fig. 3. Additional apoptotic lineages similar to the vmd1a lineage in other embryonic regions. (A-D') Cut-positive md neurons and es organs in the dorsal region of segments A1-A7 (A,B) and in the ventral region of segment A8 (C,D) of wild-type (A,C) and Df(1)H99 (B,D) embryos stained for Cut (red), Elav (blue) and Pros (green). The schematic representations of the Cut-positive md neurons and es organs (A',B',C',D') are as in Fig. 2. Two or three ectopic es organs are observed in the dorsal region of segments A1-A7, as well as five ectopic es organs in the ventral region of segment A8. Arrowheads indicate the ectopic socket and shaft cells. Cells shown into brackets in A',B' are present in 58% of the segments of wild-type embryos (A'; n=33) and in 90% of the segments of Df(1)H99 embryos (B'; n=20). Ectopic cells in B' and D' have a bold outline. (E) Ventral region of segments A7-A8 of a wild-type stage 12 embryo stained for Cut (red) and the sensory organ marker Senseless (Sens; blue (Nolo et al., 2000)). (E') Schematic representation of the Cut-positive and Sens-positive sensory organ precursor cells (yellow) and of the chordotonal (ch) organ precursor cells (blue) which are Cut-negative and Sens-positive. At this stage, six pIIa-pIIb cell clusters are seen in the ventral region of segment A7. In the ventral region of segment A8, five pIIa-pIIb cell clusters are observed at positions corresponding to the vmd1a, vp1, vp2, vp4 and vp4a positions in segment A7.

View larger version (40K): [in a new window]   Fig. 4. Numb inhibits the Notch-mediated expression of rpr and grim in the vmd1a lineage. (A-D) Wild-type embryos stained for Cut in red, Pros in blue and rpr transcripts (A,C) or grim transcripts (B,D) in green. The rpr and grim genes are specifically expressed in the pIIa and pIIIb cells of the vmd1a lineage. No expression is seen in the vp1 lineage cells. (E-F) In numb796 mutant embryos stained for Cut in red and rpr in green, rpr transcripts accumulate in both vmd1a pI daughter cells (E) or in the only pI daughter cell remaining (F). No expression is seen in the vp1 lineage cells. This indicates that both vmd1a pI daughter cells undergo cell death in the absence of numb (F'). (G) In arm-Gal4 UAS-numb embryo stained for Cut in red, Pros in green and Elav in blue, four Cut-positive cells accumulating various levels of Pros and Elav can be observed at the vmd1a position, suggesting that the pIIa cell did not die and was transformed into a pIIb cell (G'). Note that in G, the pIIa cell of the vp1 lineage was not completely transformed into a pIIb cell since two Pros-negative cells accumulating high levels of Cut, corresponding to socket-like and shaft-like cells, are observed. (H-I') B6-2-25 (H) and B6-2-25 insc22 mutant embryos (I) stained for Cut in red, ß-galactosidase in green and Elav in blue. In the ventral md cluster of insc22 mutant embryos (I), six md neurons instead of the five seen in wild-type (H) are found. Two of them express the vmd1a-specific enhancer-trap marker B6-2-25. This indicates that the pIIIb cell did not die and was transformed into a vmd1a neuron (I'). (J,K) Heat-shocked hs-Nintra embryos stained for Cut in red and rpr (J) or grim (K) in green. rpr (J) or grim (K) transcripts accumulate in both pI (J) and pIIb (K) daughter cells. This indicates that in these conditions, both pI (J') or pIIb (K') daughter cells undergo cell death. Staged hs-Nintra and control yw embryos (15.5-16.5 or 17.5-18.5 hours after egg laying at 19°C) were heat-shocked 30 minutes at 37°C, to induce the expression of Nintra, and allowed to develop for another 45 minutes at 19°C prior to fixation. We only analyzed embryos in which at least one segment shows a dividing vp1 pIIb cell or a dividing vp1 pIIIb cell. In each panel, the vp1 lineage cells are indicated by a bracket.

View larger version (10K): [in a new window]   Fig. 5. Regulation of cell death in the vmd1a lineage. Alternative cell death decision is regulated by the unequal partitioning of Numb. Numb inhibits the pro-apoptotic function of Notch in one of the two daughter cells, while activation of Notch induces the expression of rpr and grim in the other cell.