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Hunchback is required for the specification of the early sublineage of neuroblast 7-3 in the Drosophila central nervous system

Tanja Novotny, Regina Eiselt and Joachim Urban*

Institut für Genetik, Universität Mainz, Saarstrasse 21, D-55122 Mainz, Germany



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  		Fig. 1. All neurons of the NB7-3 lineage can be identified individually. (A) The NB7-3 lineage as revealed by DiI labeling (Bossing et al., 1996Go): three interneurons (arrows) with contralateral projections across the posterior commissure (thick arrow) and one motorneuron (arrowheads). Anterior is upwards; broken line indicates the midline. (B) Schematic representation of the NB7-3-derived neurons with their designations. Anterior is upwards; P, posterior commissure; A, anterior commissure. (C,D) Dorsal view of the VNC of an egGal4::GFP (C) and a wild-type (D) L1 larva. Anterior is upwards. Crz is stained in red, while GFP (C) and Serotonin (D) are stained in green. One Eg-positive NB7-3 derived neuron co-expresses Crz in the segments T2 to A6 (yellow in C; arrows). This neuron lies in most cases lateral to the serotonergic neurons (D, arrows) in the EW3 position. (E) NB7-3 lineage development showing markers and genealogy of the lineage. The markers listed are expressed in the postmitotic neurons as indicated by each bracket. 5-HT, Serotonin; Crz, Corazonin; DDC, Dopa-decarboxylase; dSERT, Drosophila Serotonin transporter; Eg, Eagle; En, Engrailed; Hb, Hunchback; Hkb, Huckebein; Isl, Islet; Pdm-1, POU-domain protein 1; TrpH, Tryptophan hydroxylase; Zfh-1 and Zfh-2: Zinc-finger homeodomain 1 and zinc-finger homeodomain 2; PCD, programmed cell death; NB, neuroblast; GMC, ganglion mother cell.

 


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  		Fig. 2. BrdU incorporation reveals that the NB7-3 lineage generates three GMCs. Dorsal views; anterior is towards the left; the broken line indicates the midline; arrowheads, GW neuron. (A-E) Labeling obtained in NB7-3-derived neurons at stage 15 after BrdU injections between 6.5 and 7.5 hours AEL. The corresponding images are shown underneath (A'-E'). Green, BrdU; red, Eg; yellow, doublestaining. We found all (A,A'), three (B,B'), two (C,C'), one (D,D') and none (E,E') of the neurons double labeled (yellow). The EW3 neuron is always labeled last (D,D'). GW and EW1 are always either both labeled together (A,B) or not labeled (C,D) indicating that they are siblings. (A''-E'') Diagram to explain how BrdU incorporation at different times during NB7-3 lineage development affects labelling. Vertical axis is the time axis; green, BrdU incorporation; green horizontal line, timepoint of BrdU application. (F,G) An Eg-positive NB7-3-derived cell cluster in a stage 14 Df(L3)H99 mutant embryo consisting of eight cells (G), compared with four in wild type (F). (H) In many hemisegments of CycA mutant embryos at stage 14, we find two cells in NB7-3 position where the most dorsal cell is smaller and Hb positive (yellow), indicating that this is the first born GMC. Eg is shown in red.

 


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  		Fig. 3. hb and pdm1, but not cas, are expressed in the NB7-3 lineage. Flat preparations of wild-type embryos. Dorsal views, anterior is leftwards; broken line represents the midline; arrowheads indicate GW. (A) Three hemineuromeres of a stage 12 embryo stained for Hb (green) and Eg (red) showing different phases of NB7-3 lineage development in each hemisegment. Left position: Hb is expressed in NB7-3 (curved arrow) before the generation of progeny. Right position: two Hb-positive cells are detected, i.e. NB7-3 and GMC7-3a (long arrows). Medial position: three Hb-positive cells are seen corresponding to NB7-3 plus the early progeny (short arrows). At stage 15 (B-D), two of the four Eg positive neurons (green) in the NB7-3 lineage are Hb positive (yellow, B). These are always GW (arrowhead) and EW1 (arrow). Hb is shown in red. (C) Pdm1 (green) in the NB7-3 lineage is very variable in stage 15 embryos and is detectable in up to all four Eg-positive neurons (yellow) of the lineage. (D) Cas (green) is not detected in the NB7-3 progeny neurons (red).

 


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  		Fig. 4. Hb specifies the identity of the first two GMCs. (A-L) Flat preparations of embryos at stage 15 (except E', which is stage 13); one hemineuromere; dorsal view; anterior is towards the left; arrowheads indicate GW neuron. (A,D,G,J,M) Wild type, (B,E,H,K,N) hb12 and (C,F,I,L,O) egGal4::hb. Eg is red; double stained neurons are yellow. (A-C) NB7-3 lineage as revealed by anti-Eg staining. In hb mutant embryos at stage 15 (B), only two Eg-positive neurons are detected when compared with wild type (A). (C) Ectopic hb expression in the late sublineage leads to additional Eg-positive neurons. Shown is an NB 7-3 lineage consisting of seven Eg-positive cells (arrows). (D-F) Zfh1 (green) within the NB7-3 lineage. (D) In wild type, only the motorneuron (GW) of the NB7-3 lineage is Zfh1 positive (arrowhead). (E) In hb mutant embryos at stage 13, this Zfh1-positive neuron can still be detected in those hemisegments that show three Eg positive neurons (arrowhead, E'). At a later stage (stage 15, E''), this Eg-positive neuron has disappeared. (F) In egGal4::hb embryos, often two or more Zfh1-positive neurons can be found in the NB 7-3 lineage (arrowheads). (G-I) Zfh2 (green) within the NB7-3 lineage. (G) In wild type, Zfh2 labels exclusively the late born neurons EW2 and EW3 (arrows). (H) In hb mutant embryos, the remaining two neurons express Zfh2. (I) Ectopic expression of hb in the late part of the lineage results in absence of zfh2 expression in all neurons of the NB7-3 lineage. (J-L) dSerT mRNA expression. (J) In wild type, each abdominal hemisegment except the last shows two dSerT-positive neurons (EW1 and EW2). (K) In hb mutant embryos, only one dSerT-positive neuron (EW2) is left, whereas after ectopic hb expression, up to four dSerT positive neurons (EW1) are obtained (L). (M-O) Summaries of the result obtained in A,D,G,J, B,E',E'',H,K and C,F,I,L, respectively.

 


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  		Fig. 5. hb affects Serotonin and Corazonin synthesis in the NB7-3 lineage. Ventral nerve cords of L1 larval stage; dorsal view; anterior is upwards. (A-C) anti-Crz; (D-F) anti-Serotonin. (A) In wild-type hemisegments, T2 to A6 show one corazonergic EW3 neuron each (arrows). (B) In the hb9 allele, Crz (arrows) is missing in a significant number of hemisegments (asterisks). (C) Overexpression of hb leads to a complete loss of Crz. (D) In wild-type segments, T2 to A7 show two serotonergic neurons in each hemisegment (arrows), whereas the last segment shows only one serotonergic neuron. (E) In the hb9 mutant a significant number of hemisegments shows only one serotonergic neuron (arrows). (F) Overexpression of hb yields often three to four serotonergic neurons per hemisegment (arrows).

 


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  		Fig. 6. hb affects the number of eve-expressing neurons. Flat preparations of embryos at stage 15, dorsal view, anterior is leftwards. Horizontal square brackets, aCC and pCC; vertical square brackets, NB 7-1 derived neurons; thin arrows, RP2; curled brackets, EL neurons; arrowheads, CQ neurons; curved arrows, fpCC. Sections of the confocal images were combined, but for the purpose of clarity presented in two layers: the left images (') are dorsal planes, the right images are ventral planes (''). Eve is red; Hb (A) and Sal (B-E) are green; double stained neurons are yellow. (A) In wild type, hb/eve co-expression is detected in aCC, pCC and RP2 in the most dorsal layer (A'). In the ventral layer (A'') fpCC and the first innermost CQ neuron show hb co-expression. (B) In wild type, co-expression with sal is shown only in aCC, pCC, RP2 (B'), occasionally in fpCC (not shown) and in one of the EL neurons (B''). (C',C'') In the hb mutant embryo, RP2 (arrow) and either aCC and/or pCC are missing in several hemisegments (C', broken square bracket). In addition, the number of NB 7-1-derived neurons is decreased (C'', arrowheads). (D',D'') Ectopic expression of hb using scaGal4 as a driver leads to ectopic neurons in medial position (D'', square brackets) and occasionally to additional Sal-positive neurons in aCC/pCC/fpCC positions (D', thick arrow). (E',E'') Ectopic expression of hb using enGal 4 as a driver gives rise to ectopic neurons in the position of the NB 7-1-derived neurons (large square brackets, E''). One hemisegment shows additional Sal staining in fpCC (thick arrow).

 


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  		Fig. 7. A model explaining the loss-of-function and gain-of-function phenotypes of hb with respect to the NB7-3 lineage. (A) The development of the wild-type NB7-3 showing the relevant markers in the postmitotic neurons. (B) Lack of Hb leads to a complete loss of EW1 and only a transient presence of GW. By stage 15, the missing cells might have died by apoptosis (PCD). Although EW3 is detectable by markers, it does not express the neuropeptide Crz. (C) Overexpression of hb might transform the second GMC completely into a GMC7-3a fate, while the third GMC might often generate only EW1 type cells.

 

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© The Company of Biologists Ltd 2002