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First published online 17 September 2008
doi: 10.1242/dev.023986

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Antagonistic roles for Ultrabithorax and Antennapedia in regulating segment-specific apoptosis of differentiated motoneurons in the Drosophila embryonic central nervous system

Institute of Genetics, University of Mainz, 55099 Mainz, Germany.

View larger version (104K): [in this window] [in a new window]   Fig. 1. Two differentiated motoneurons undergo segment-specific rpr-dependent cell death. An NB7-3 lineage (A-C) and an NB2-4t lineage (D-F) of wild-type (A-D,F) and eg-Gal4,UAS-mCD8::GFP (E) Drosophila embryos at late stage 16. Dashed lines mark the midline. Dashed circles indicate NB7-3 (B) and NB2-4t (E). B',B'' and E',E'' show magnified single scans of one lineage as boxed in B and E, respectively. Anterior is up in all images. (A) A DiI-labeled NB7-3 clone. The GW motoneuron (arrow) lies at the posterior end of the cluster and exhibits an ipsilateral outward projection. (B) GWs (arrows) undergo apoptosis in segments T3 and A1, but not in T2 (arrowheads). Asterisk indicates that the GW has already been removed. (B') A magnified single scan of the A1 NB7-3 lineage. The apoptotic GW is outlined. (B'') Same image as in B', showing only the activated Caspase 3 (act Casp3) staining. (C) A magnified single scan of an A1 NB7-3 lineage. (C') rpr is transcriptionally activated in apoptotic GWs (outlined). (D) A DiI-labeled NB2-4t clone. The motoneurons (arrows) are the most medial and most dorsal cells of the cluster and show contralateral outward projections. (E) The anterior NB2-4t motoneuron in segment T3 undergoes apoptosis (arrows). In A1, only one motoneuron lies in the same position (curved arrows). (E') A magnified single scan of the T3 NB2-4t. The apoptotic MNa is outlined. (E'') Same image as in E', showing only the activated Caspase 3 staining. (F) A magnified single scan of a T3 NB2-4t lineage. (F') rpr is transcriptionally activated also in apoptotic MNas (outlined).

View larger version (19K): [in this window] [in a new window]   Fig. 2. Summary of Ubx expression in the NB7-3 and NB2-4 lineages. The segmental pattern of Ubx expression is depicted at the indicated developmental stages. The intensity of the blue shading reflects the Ubx expression level; white indicates no expression. (A) The neuroblast and progeny of NB7-3. (B) The neuroblast and the two motoneurons of NB2-4t.

View larger version (98K): [in this window] [in a new window]   Fig. 3. Apoptosis of both motoneurons requires Ubx function at a late developmental stage. NB7-3 neurons in late stage 16 (A,B,G,H) and stage 15 (C) Drosophila embryos and NB2-4 (D-F) neurons in late stage 16 embryos. Dashed circles, NB7-3 and NB2-4; dashed lines mark the midline. (B',C',G',H') Magnified single scans of NB7-3 with GW outlined. (B'',C'',G'',H'') Only the activated is shown; Caspase 3 staining (act Casp3) is shown. (E',F') Magnified single scans of NB2-4t with MNa outlined. (E'',F'') Only the activated Caspase 3 staining (act Casp3) is shown. Anterior is up in all images. (A) Ubx expression levels are high in GWs that undergo apoptosis (arrows) and are barely, if at all, detectable in GWs that survive (arrowheads). (B-B'') Apoptosis of GWs is abolished in a Ubx loss-of-function mutant (arrows). The apoptotic cell in T2 (curved arrow) is not the GW (arrowhead). (C-C'') Ectopic Ubx expression induces apoptosis of GWs in anterior thoracic segments (arrowheads). These neurons now die prematurely in T3 and in abdomen as well (arrows). (D) Ubx is strongly expressed in the anterior, apoptotic NB2-4t motoneuron (arrows). Expression levels are low in the posterior motoneuron. In T2, Ubx is not expressed in these neurons (arrowheads). (E-E'') In a Ubx loss-of-function mutant, apoptosis of the MNa in T3 is abolished (arrows). In A1, these neurons are transformed towards a thoracic phenotype (arrows), and do not undergo apoptosis. T2 is unaffected (arrowheads). (F-F'') Ectopic Ubx expression induces apoptosis of MNa in anterior thoracic segments (arrowheads), and increases MNa apoptosis in T3 (arrow). Asterisks indicate that MNa has already been removed. (G-G'') Early ectopic Ubx expression cannot induce GW apoptosis in anterior thoracic segments (arrowheads). (H-H'') Late ectopic Ubx expression induces GW apoptosis in anterior thoracic segments (arrowheads), and increases it in T3 and abdomen. Genotypes: (A,D) wild type, (B) Ubx1, (C) en-Gal4/+;UAS-Ubx/+, (E) UAS-CD8::GFP/+;eg-al4,Ubx1/Ubx1, (F) poxN-Gal4,UAS-GFP/UAS-Ubx, (G,H) hs-Ubx.

View larger version (130K): [in this window] [in a new window]   Fig. 4. Antp is necessary and sufficient for survival of the GW motoneuron. (A-B') NB7-3 neurons (dashed circles) in late stage 16 Drosophila AntpW10 (A) and en-Gal4/UAS-Antp (B) embryos. Dashed lines mark the midline. (A',B') Magnified single scans of NB7-3 with GW outlined. (A'',B'') Only the activated Caspase 3 staining (act Casp3) is shown. Anterior is up in all images. (A) GWs undergo apoptosis in anterior thoracic segments of Antp loss-of-function mutants (arrowheads). Apoptosis in T3 and the abdomen is unchanged (arrows). (B) Ectopic Antp expression reduces apoptosis of GWs in T3 and abdomen (arrows). T2 is unaffected (arrowheads).

View larger version (82K): [in this window] [in a new window]   Fig. 5. Ubx induces apoptosis by preventing Antp from promoting survival of the GW motoneuron. NB7-3 neurons in late stage 16 (A-F,J) or stage 15 (G-I) Drosophila embryos. Dashed lines mark the midline. Insets show magnified single scans of NB7-3 as follows: (B,C) the indicated segments from A; (E,F) the indicated segments from D; (H,I) the indicated segments from G. (B',C',E',F',H',I') Only the Antp staining from respective insets is shown. GWs are indicated by arrowheads. Anterior is up in all images. (A) GWs show high Antp expression levels in segments in which they do not undergo apoptosis (arrowheads). In segment T3, in which they undergo apoptosis, they express Antp at low levels (arrows). (B,B') In T1, all NB7-3 cells show strong Antp expression. (C,C') In T2, Antp expression is strong in GW (arrowhead) and barely detectable in the EW interneurons. (D) In Ubx mutants, Antp expression is strongly upregulated in all thoracic segments. (E,E') In T1, Antp expression is unchanged. (F,F') In T2, Antp expression is equally strong in all NB7-3 cells. (G) Ectopic Ubx expression suppresses Antp in the anterior thoracic segments. (H,H') In T1, ectopic Ubx strongly suppresses Antp expression in all cells of NB7-3. (I,I') In T2, GW (arrowhead) also shows reduced Antp levels upon ectopic Ubx expression. (J) In Antp,Ubx double mutants, GW apoptosis is restored (arrow). Asterisks indicate that the GW has already been removed. (J') A magnified single scan of the A6 NB7-3. The apoptotic GW is outlined. (J'') Same image as in J', showing only the activated Caspase 3 (act Casp3) staining.

View larger version (17K): [in this window] [in a new window]   Fig. 6. Summary of GW and NB2-4t motoneuron apoptosis. Expression of Antp and Ubx as observed at late stage 16. Antp expression is indicated in red (strong) or pink (weak) and Ubx expression in blue. Crosses indicate apoptotic cell death, with the different line widths reflecting its frequency. Dashed crosses indicate only observed (not evaluated) MNa apoptosis. ND, no data.

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