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First published online July 21, 2003
doi: 10.1242/10.1242/dev.00593

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The regulation of apoptosis by Numb/Notch signaling in the serotonin lineage of Drosophila

Martha J. Lundell*, Hyung-Kook Lee, Ernesto Pérez and Linda Chadwell

Department of Biology, University of Texas at San Antonio, San Antonio, TX 78249, USA



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  		Fig. 1. Molecular markers uniquely identify all NB7-3 progeny neurons. (A) Schematic representation of the NB7-3 lineage showing molecular markers of the four neuronal progeny, axonal projections and positions relative to the midline (M), anterior commissure (A) and posterior commissure (P). Ddc and corazonin are cytoplasmic antigens; all others are nuclear. The yellow numbers 1, 2 and 3 label the EW1, EW2 and EW3 neurons, respectively. M indicates the midline. (B,C) One abdominal segment from the ventral cord of stage 15 eg289/+ embryos, shows the four Eg-lacZ positive neurons of the NB7-3 lineage. The EW2 and EW3 neurons express Zfh-2 (orange cells). (B) The GW and EW1 neurons express Hb (purple cells). (C) The GW neuron expresses Zfh-1 (purple cell). (D-F) One abdominal segment from the ventral cord of stage 17 eg289/+ embryos. (D) The two serotonergic neurons (1, 2) express Ddc and can be distinguished by their differential expression of Hb and Zfh-2. (E) The EW3 neuron expresses corazonin. (F) The EW2 neuron expresses Pdm1. The GW neuron is usually not detectable at stage 17. Scale bars: in C, 5 µm for B,C; in F, 5 µm for D-F.

 


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  		Fig. 2. Numb is required for development of EW2 and EW3. All images are ventral cord abdominal segments from stage 17 embryos. The yellow numbers 1, 2 and 3 label the EW1, EW2 and EW3 neurons, respectively. M indicates the midline. (A,B) Immunostaining with Ddc (red), Zfh-2 (green) and Hb (blue) shows a preferential loss of EW2 cells in embryos homozygous for numb1. The remaining Ddc-positive cells in numb1 are Hb positive, indicating they are EW1 cells. (A'-B') Same images as (A,B), but showing only Ddc immunoreactivity. (C,D) Immunostaining with Ddc (red), Eg-lacZ (green) and corazonin (blue), showing a reduction of Eg-lacZ in numb1 relative to wild type. Corazonin expression is completely absent in numb1. Midline dopaminergic cells (M) are visible in wild type but are undetectable in numb1. Scale bar in D: 5 µm for A-D.

 


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  		Fig. 4. Comparison of serotonergic cells in segment A8 from wild-type and numb1 mutant embryos. (A,D) Ddc immunoreactivity of stage 17 embryos showing the entire ventral cord and demonstrating that A8 cells are not absent in numb1 embryos. T1-T3 are thoracic segments. A1-A8 are abdominal segments. E is the embryonic posterior Ddc cluster that is only visible during embryogenesis. (B,E) A magnification of A8 from the same CNS as in A,D, showing immunoreactivity of Ddc (red), Zfh-2 (green) and Hb (blue). The single Ddc-expressing cell in A8 shows Zfh-2 expression in wild-type and Hb expression in numb1 embryos. (C,F) Stage 15 embryos immunostained with Eg (red), Zfh-2 (green) and Hb (blue). (C) A wild-type A8 hemisegment shows three Eg cells, two are Hb positive and one is Zfh-2 positive. (D) A numb1 A8 hemisegment shows that Eg and Hb expression are unaltered, but Zfh-2 expression is reduced. Scale bars: in D, 15µm for A,D; in E, 10 µm for B,E; in F, 5 µm for C,F.

 


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  		Fig. 3. CNS phenotypes of stage 15 embryos. (A-G) Embryos were immunostained for Eg (red), Zfh-2 (green) and Hb (blue). (A) A wild-type segment showing the four NB7-3 progeny. 1, 2, 3 and GW label the EW1, EW2, EW3 and GW neurons, respectively. M indicates the midline. (B) Segments from numb1 mutant embryos show an absence of EW2 and EW3 neurons, only Hb-positive neurons remain (EW1 and GW). (C) Twenty-two percent of numb1 hemisegments have three Eg-positive cells. The third cell expresses Zfh-2 at reduced levels relative to wild type. (D) UAS-NotchACT: eggal4 embryos shows a variable pattern, some hemisegments are identical to the wild-type pattern and some hemisegments having a reduced number of Eg-positive cells. (E-G) Hemisegments from spdoG104, UAS-numb:eg-gal4 and eg-gal4/UAS-p35 embryos show a similar phenotype of five to six Eg-positive cells. The ectopic cells express Zfh-2. (H-K) Embryos were immunostained with Eg (red) and Zfh-1 (green). (H) A wild-type segment showing the four NB7-3 progeny and expression of Zfh-1 in the GW neuron. (I,J) Hemisegments from numb1 mutant embryos show that the expression of Zfh-1 in GW is unaltered. J shows an example of a numb1 hemisegment were both remaining cells express Zfh-1, indicating a transformation of EW1 to a GW cell fate. (K) UAS-Notch: eg-gal4 embryos show a variable number of cells similar to D and two Zfh-1 staining cells indicating a transformation of EW1 to a GW cell fate. Scale bar in J: 5 µm.

 


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  		Fig. 5. Numb/Notch regulation of NB7-3 development. All panels with the exception of F show segments of the ventral cord immunostained for Ddc (red) and corazonin (green). (A) The wild-type larval pattern shows that corazonin is not expressed in segments A7 and A8. (B) numb1 mutant embryos show the absence of EW2 and EW3. (C) The UAS-Notch: eg-gal4 larval pattern shows the absence of EW1, EW2 and EW3. (D-F) spdoG104 mutant embryos show ectopic Ddc-expressing cells, D is a thoracic hemisegment and E shows abdominal segments. (F) A spdoG104 mutant embryo immunostained for Ddc (red), Zfh-2 (green) and Hb (blue), shows two Ddc cells immunoreactive for Hb and three Ddc cells immunoreactive for Zfh-2. (G) A spdoG104 heterozygous embryo shows a pattern that is wild type but expression of markers in EW2 and EW3 is incomplete at this stage. (H) numb1/numb1:spdoG104/+ embryos show rescue of the numb1 mutant phenotype. Arrows indicate hemisegments that have two dc-expressing cells, arrowheads indicate hemisegments with no Ddc-expressing cells (indicative of the numb phenotype) and asterisks mark the midline dopamine cells. E and L indicate whether the image is from a stage 17 embryo or third instar larva, respectively. M indicates the midline. Scale bar: in B, 15 µm for B,G,H; in C, 15 µm for A,C; in D, 10 µm for D; in F is 3 µm for F; in E, 10 µm for E.

 


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  		Fig. 6. TUNEL analysis of apoptosis in the NB7-3 lineage. Stage 15 embryos were immunostained for TUNEL (green), Eg (red) and Hb (blue). All panels are arranged with anterior at the top and the midline towards the right. (A-A') A wild-type hemisegment showing an extra NB7-3 Eg cell that is apoptotic. (B) A numb1 hemisegment showing three Eg cells; the two GMC-1 progeny and an apoptotic cell. (C) A numb1 hemisegment showing apoptosis of the two GMC-1 progeny, these cells are immunostained for all three labels. (D-D') A UAS-NotchACT:eg-gal4 hemisegment showing four Eg cells; the two GMC-1 progeny and two apoptotic cells. (E-E') A UAS-NotchACT:eg-gal4 hemisegment showing six cells; the two GMC-1 progeny and four closely associated cells that are apoptotic and appear as mitotic pairs. Only one of the apoptotic cells maintains Eg expression. (F-F') A UAS-numb:eggal4 hemisegment showing six Eg cells; the two GMC-1 progeny and paired GMC-2 and GMC-3 progeny (outlined). Only one cell in this hemisegment is apoptotic. Scale bar: 5 µm.

 


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  		Fig. 7. Notch-mediated apoptosis in the NB7-3 lineage. (A-D) Abdominal segments of the ventral cord showing immunoreactivity of Ddc (red) and corazonin (green). E and L indicate whether the image is from a stage 17 embryo or a larva, respectively. M indicates the midline. (A) A eg-gal4/UAS-p35 third instar larval nerve cord shows a wild-type pattern of cells plus ectopic corazonin-containing cells marked with arrowheads. (B) A numb1/numb1:eg-gal4/UAS-p35 embryos show evidence that p35 can rescue the numb1 mutant phenotype. Arrows indicate hemisegments that have two Ddc-expressing cells, and the arrowheads marks a hemisegment with ectopic corazonin-containing cells. A8 shows two Ddc-expressing cells. (C) A UAS-NotchACT/+:eggal4/UAS-p35 third instar larval nerve cord has a pattern identical to wild type. (D) A en-gal4/+:UAS-p35/+ third instar larval CNS shows two abdominal hemisegments with triplets of Ddc-expressing cells. (E-E') An entire spdo/+ first instar larval CNS showing overproliferation of Ddc expressing cells. E' is a DIC image of the same CNS as in E, showing the top surface and a protrusion that matches the largest area of cell proliferation in E. Scale bars: in C, 15 µm for A-C; in D, 5 µm for D; in E', 30 µm for E,E'.

 


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  		Fig. 8. Specification of NB7-3 cell fates by Numb/Notch signaling. GMC terminal divisions in the NB7-3 lineage are asymmetric, with only one daughter cell receiving Numb protein. Daughter cells that receive Numb inactivate Notch signaling, which leads to a neuronal cell fate. The broken green line in EW1 indicates that Numb is not an absolute requirement for this cell fate. In the progeny that do not receive Numb, Notch signaling is maintained. In GMC-1 progeny, Notch signaling induces the differentiation of the GW motoneuron. In GMC-2 and GMC-3 progeny, Notch signaling activates apoptosis. Previous work has shown that Hb is necessary for development of the GMC-1 lineage and that Kr is necessary for development of the GMC-2 lineage (Isshiki et al., 2001Go; Novotny et al., 2002Go).

 

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© The Company of Biologists Ltd 2003