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First published online 5 January 2006
doi: 10.1242/dev.02229

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Timing of identity: spatiotemporal regulation of hunchback in neuroblast lineages of Drosophila by Seven-up and Prospero

Institut für Genetik, Universität Mainz, 55099 Mainz, Saarstraße 21, Germany.

View larger version (54K): [in a new window]   Fig. 1. pros and svp mutants show an opposite phenotype with respect to neural Hunchback expression. Dorsal views of wild-type (A), prosC7 (B), svpe22 mutant (C) and prosC7, svpe22 double mutant (D) embryos at stage 14, stained for Hb protein (green). Thin dashed lines indicate lateral borders of the ventral nerve cord; thick dashed line indicates the midline. (B) In the absence of pros function there are fewer Hb-expressing cells within the ventral nerve cord than in wild type (A), whereas svpe22 mutant (C) and prosC7, svpe22 double mutant embryos (D) show more Hb-expressing cells. Scale bar: 20 µm.

View larger version (93K): [in a new window]   Fig. 2. Regulation of hb within the NB7-3 lineage. Embryos double labelled for the NB7-3 lineage marker Eg (magenta) and Hb (green). Midline is towards the left. Insets show Eg expression separately. (A-C) Hb expression during normal NB7-3 lineage development. At late stage 11 the NB has divided once to generate the Hb+ GMC7-3a (A). After hb downregulation the NB has divided again, and Hb is only detectable in GMC7-3a (B). At stage 14 the progeny of GMC7-3a (GW and EW1) express Hb, whereas the progeny of GMC7-3b (EW2) and GMC7-3c (EW3) are Hb- (C). (D-F) Hb expression in svpe22. Even after the generation of the second GMC, the NB has not yet downregulated Hb (E). At stage 14, three instead of two Hb+ neurons can be detected in many cases (F). Note that not all NB7-3-derived cells express Hb. (G-I) Hb expression in prosC7. In prosC7, the NB generates a Hb+ GMCa as normal (G) but the expression is not maintained (H,I). (J-L) Hb expression in svpe22, prosC7. After the first division Hb has not been downregulated in the NB (K), which results in a svp-like phenotype at stage 14 (L). (M) Scheme of normal NB7-3 lineage development, with markers used in our analysis. (N) Continuous expression of Pros within the NB inhibits the downregulation of Hb. All cells of the NB7-3 cluster are Hb+. (O) prosC7/Df(3L)H99 double mutant. There are no NB7-3-derived Hb+ cells present in the absence of apoptosis. Scale bar: 20 µm.

View larger version (95K): [in a new window]   Fig. 3. Regulation of hb within the NB6-4T lineage. Embryos double labelled with the lineage marker Eg (magenta), and Hb (green) or the glial marker Repo (green), as indicated. Midline is towards left (A-F) or marked by a dashed line (G,G',H, H'). Insets show Eg expression separately. (A,B,G,G') Wild type. At stage 11 there are four cells generated by this lineage: two Hb+ glial cells (arrowheads), one Hb- GMC and the Hb- neuroblast (A). At stage 14 the lateral neuronal cluster consists mostly of six cells (B). At stage 13, one of the two glial cells in A has divided again and the three resulting cells have migrated towards the ventral midline: two (MMM3 and MML3) near the midline and one (ML2) left behind (outlined in G'). (C,D,H,H') svpe22. At stage 11, all four cells (inset in C) are often Hb+, indicating a prolonged expression of Hb (C). Concomitantly, the size of the lateral cluster is reduced (D). In many hemineuromeres there is one additional NB6-4T-derived glial cell at the ventral midline (H,H'). (E,F) prosC7. At stage 11, all four cells are Hb- (E), showing that Hb expression could not be maintained. The lateral neuronal cluster is enlarged (F), probably due to the deregulation of dacapo. (I) Scheme of NB6-4T lineage development, with markers used in our analysis. Scale bar: 20 µm.

View larger version (91K): [in a new window]   Fig. 4. Regulation of hb within the thoracic NB2-4T lineage. Embryos are double labelled for the NB2-4T lineage marker Eg (magenta) and Hb (green). NB2-4T-derived cells are outlined; midline is towards the left. Insets show Eg expression separately. (A-C) Wild type. At early stage 11 NB2-4T has divided once to give rise to the Hb+ GMC2-4Ta (A). At early stage 12 the NB has generated three GMCs, two of them are the first-born Hb+ GMC2-4Ta and GMC2-4Tb (B, arrowheads). At stage 14, four neurons are Hb+, which are the progeny of the two first born GMCs (C, arrowheads). (D-F) svpe22. Hb expression is not downregulated after the second mitosis (E), resulting in up to eight Hb+ neurons (F, arrowheads). (G-I) prosC7. Hb is initially expressed in the first and second born GMCs (H, arrowheads), but the expression is often not maintained in one GMC (most likely GMC2-4Tb), resulting in only two Hb+ neurons at stage 14 (I, arrowheads). (J) Scheme of early NB2-4T lineage development with markers used in our analysis. Scale bar: 20 µm.

View larger version (142K): [in a new window]   Fig. 5. svp mRNA and protein expression in early NB7-3 lineage development. Embryos are double labelled for the NB7-3 lineage marker Eg (magenta), and svp mRNA (green) or Svp protein (green), as indicated. NB7-3-derived cells are outlined; midline is towards left. (A-D) svp mRNA expression in wild type. NB7-3 initiates svp expression before its first division (A). After this division, svp mRNA can be detected only in the NB (B) or in both cells (C). After the second NB division there is no detectable mRNA in most cases (D). (E-G) svp mRNA expression in prosC7. As in wild type, NB7-3 expresses svp mRNA before the first division takes place (E). After this division, both cells are usually positive for svp mRNA, although the amount of transcripts in GMCa seems to be generally lower than in the NB (F). After the second NB division there is still detectable svp mRNA in GMCa in most cases (G). (H-J) Svp protein expression in wild type. There is only a little or no protein in NB7-3 prior to the first mitosis (H). After division, both cells are Svp+ (I). After the birth of GMCb, all cells are Svp+ but the highest amount of Svp protein is found in GMCa (J). (K-M) Svp protein expression in prosC7 is similar to wild type. Scale bar: 20 µm.

View larger version (117K): [in a new window]   Fig. 6. Temporal regulation of svp mRNA expression. In situ hybridisation for svp mRNA (magenta, A-C',E-G') combined with anti-Eg (green, A-C) and anti-Hb (green, E-G), and double staining with anti-Eg (green, D) and anti-Svp (magenta, D,D'). NBs and their GMCs are outlined. (A,B) Wild type. NB6-4T expresses svp before it undergoes the first mitosis (arrowhead in A,A'). After mitosis, svp expression stops immediately (6-4T cells in B,B'). Likewise, NB7-3 expresses svp before its first divison (B,B', arrow). NB2-4T generates one GMC (small 2-4T cell in A,A') before it expresses svp (curved arrow in B,B'). There is no detectable svp mRNA within GMC2-4Ta (B,B'). NB3-3 and its progeny do not express svp mRNA at these early stages. (C) stg4. At late stage 11, NB6-4T, 2-4T and 7-3 still express svp mRNA in cell cycle-arrested embryos, indicating that svp expression without cell division is not sufficient to switch off hb expression. (D) Even at early stage 12 there is hardly any Svp protein detectable in NB2-4T, 6-4T and 7-3 in stg4 mutant NBs. (E,F) Wild type. NB7-1 (identified by position and En expression, not shown) is initially negative for svp mRNA (E,E'). svp expression starts after the generation of its first GMC (F,F'). (G) stg4. Like NB2-4T, NB7-1 also starts to express svp mRNA at normal time, even without the generation of its first GMC. Scale bar: 20 µm.

View larger version (125K): [in a new window]   Fig. 7. Nuclear svp mRNA localisation and attenuation of hb expression in stg mutant embryos. Dashed line indicates the midline. Cells expressing high levels of Hb protein are outlined. (A) Double labelling for Hb protein (green) and svp mRNA (magenta). Cells that express high levels of Hb, do not express svp mRNA. In contrast to that, svp-expressing cells show low levels of Hb protein. Note the complete colocalisation of the nuclear Hb protein with svp mRNA. (B) Double labelling for Hb (green) and Svp protein (magenta). There is no Svp protein detectable in cells that show high expression of Hb protein. Those cells that are weakly Hb+ show a concomitant low level of Svp protein. (C) Double labelling for hb mRNA (green) and svp mRNA (magenta). Although hb mRNA is enriched in the cytoplasm of the NBs, the svp mRNA is found mainly in the nucleus. (D) Double labelling for Hb protein (green) and hb mRNA (magenta). Low Hb protein levels correlate with low hb mRNA levels, suggesting a transcriptional downregulation of hb by Svp. Scale bar: 20 µm.

View larger version (16K): [in a new window]   Fig. 8. Model of hb regulation in NB lineage development. Expression of svp mRNA starts in the NB in a lineage-specific time window, which is dependent on an unknown timing mechanism. In the case of two Hb-dependent GMCs with different fates, svp expression starts after the birth of the first GMC (GMCa). Before the next mitosis, most of the svp mRNA is kept in the nucleus, allowing only a limited translation of Svp protein. The resulting Svp activity leads to a reduction of hb expression, which is necessary to specify the fate of GMCb. Subsequently, as a result of the next mitotic division, the translation of svp mRNA is enhanced and hb expression is switched off in the NB. Within GMCb, Svp is inhibited by Pros, which has segregated into the GMC during mitosis, thereby maintaining hb expression in this cell.