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First published online 23 October 2008
doi: 10.1242/dev.025189

Development 135, 3859-3869 (2008)
Published by The Company of Biologists 2008
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Neuroblast entry into quiescence is regulated intrinsically by the combined action of spatial Hox proteins and temporal identity factors

Takuya Tsuji1, Eri Hasegawa1,* and Takako Isshiki1,2,{dagger}

1 Center for Frontier Research, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan.
2 Department of Genetics, SOKENDAI, 1111 Yata, Mishima, Shizuoka 411-8540, Japan.


Figure 1
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  		Fig. 1. Quiescence of NB3-3T and proliferation of NB3-3. NB3-3 (arrowhead) and its progeny were visualized by eagle-GAL4>UAS-mCD8GFP (eg>GFP; green) or eagle-GAL4>UAS-LacZ (eg>LacZ; green). Drosophila NBs were labeled using anti-Miranda (Mir; white). (A-C) Embryos were pulse-labeled with BrdU (magenta) at the indicated time (AEL, after egg laying). (A) Thoracic NB3-3 (NB3-3T) and (B) abdominal NB3-3 (NB3-3A). (C) BrdU-positive NB3-3 cells were counted. n, number of NB3-3 scored. (D) Wild-type larvae were continuously labeled with BrdU (magenta) from hatching until the indicated time (ALH, after larval hatching). (E) NB3-3T (left) and NB2-4T (right) from the same hemisegment of embryo pulse-labeled with BrdU at stage 15. When NB3-3T became elongated, its neighboring NB2-4T remained round and incorporated BrdU (magenta). The shape and BrdU incorporation of NB3-3 are represented diagrammatically beneath A,B,D.

 

Figure 2
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  		Fig. 2. Sequential expression of temporal transcription factors in NB3-3T and NB3-3A. The Drosophila NB3-3T and NB3-3A lineages were visualized using Eg, eg>GFP or eg>LacZ (green). White arrowheads indicate NB3-3. (A,B) Kr, Pdm, Cas, Svp and Grh expression (magenta) in NB3-3T (A) and NB3-3A (B) during embryogenesis. (C) NB3-3T lineage from wild-type larva at 20-25 hours ALH (0 progeny, left) and 25-30 hours ALH (1 progeny, right) continuously labeled with BrdU from hatching. Cas expression and BrdU incorporation are shown in magenta. Yellow arrowhead indicates the first NB3-3T larval progeny. (D) Cas and Svp expression and BrdU incorporation (magenta) in larval NB3-3T at the indicated time (ALH). (E) Schematic of the quiescence period in NB3-3T and expression of the temporal transcription factors in NB3-3T (upper) and NB3-3A (lower). (F) Schematic model of NB3-3 lineage development. Expression patterns of the temporal factors are shown by the indicated color. EL neurons are enclosed by gray rectangles. The late Cas expression was downregulated without Svp function (data not shown).

 

Figure 3
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  		Fig. 3. Hox genes spatially regulate NB quiescence. Drosophila embryos were labeled for markers indicated at the top. (A-H) NB3-3T (A,C,E; stage 16) and thoracic EL neurons (B,D,F; stage 17) of wild-type (A,B), Antp (C,D) and wor>abd-A (E,F) and NB3-3A (G, stage 15), and abdominal EL neurons (H, stage 17) of wild type. White arrowhead, NB3-3; yellow arrowhead, DmLin29-positive EL neuron. (I) Schematics of DmLin29 expression in EL neurons. The seventh to 11th late-born abdominal EL neurons are DmLin29 positive, although the expression in seventh- and 11th-born EL neurons is hard to detect at stage 17 owing to the decay and late expression of DmLin29. (J,K) BrdU incorporation in wild-type Antp (J) and wor>abd-A (K) NB3-3T (arrowhead) pulse-labeled at the indicated times. In K, embryos were grown at 29°C. n, number of NB3-3T scored.

 

Figure 4
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  		Fig. 4. Temporal transcription factors regulate the timing of entry to quiescence. (A-X) Drosophila embryos were labeled for the indicated markers. Genotypes are shown at the top. NB3-3T was identified by eg>GFP or Eg (green). (A-H) Thoracic NBs (A-D) and NB3-3T (E-H) at stage 16. In wild-type (A,E), pdm (C,G) and pdm cas (D,H) embryos, NBs become elongated, whereas cas mutant NBs remain round (B,F). (I-L') NB3-3T (white arrowhead) in embryos pulse labeled with BrdU (I-L, magenta, I'-L',white) at the indicated stages. In pdm (K,K') and pdm cas (L,L') embryos, BrdU incorporation is only occasionally observed in NB3-3T at stage 13. In cas embryos (J,J'), BrdU incorporation is still observed at stage 16. (M-T) Nab expression (magenta) in NB3-3T. (U-X) Thoracic EL neurons at stage 16 or 17. In cas embryos (V), the number of EL neurons increases and Nab expression is not detected, whereas in pdm (W) and pdm cas (X) embryos, the number of EL neurons decreases and Nab-positive EL neurons are observed precociously. Bottom: phenotype summaries. (Y) The number of BrdU-positive NB3-3T in pdm embryos. n, number of NB3-3T scored. (Z) Schematic model of formation of thoracic EL neurons.

 

Figure 5
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  		Fig. 5. Expression of Nab and Sqz in the Drosophila NB3-3 lineage. (A,B) Nab (green, upper row in the upper box) and Sqz (green, upper row in the lower box) expression in thoracic (A) and abdominal (B) NB3-3 (white arrowhead), compared with Cas expression (green, middle row in each box) at the indicated stage. NB3-3 lineage is visualized by Eg (magenta in embryonic stage) or eg>LacZ (magenta in larval stage). Yellow arrowhead, Nab-positive NB3-3 progeny. (C,D) Nab, Sqz, Kr and DmLin29 expression (green) in thoracic (C) and abdominal (D) EL neurons (identified by Eve, magenta) at the point when the indicated number of EL neurons exist. Schematic diagrams of the expression patterns are shown at the bottom row in each box. Nab expression in abdominal sixth-born EL neuron was often reduced at late stages. (E) Schematic diagrams of gene expression in EL neurons.

 

Figure 6
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  		Fig. 6. Nab and Sqz regulate temporal state and NB quiescence. (A) BrdU incorporation (magenta) in NB3-3T (white, visualized by Mir) of nab and sqz02102 Drosophila embryos pulse-labeled at the indicated times. NB3-3 lineage is visualized by Eg (green). (B) BrdU incorporation (magenta) in the thoracic hemisegment of wild-type, nab and sqz02102 newly hatched larva (0-6 hours ALH). (C,D) Cas expression (magenta) in NB3-3T (C) and Cas and Svp expression (magenta) in NB3-3A (D) from embryos. Svp expression is not detected in NB3-3T (data not shown). Genotypes are shown on the left. The NB3-3 lineage is visualized by eg>GFP or Eg (green). Arrowhead indicates NB3-3. (E) Phenotype summaries. In sqzie nab double mutants, NB quiescence was inhibited as in nab mutants (data not shown). (F) NB3-3T (visualized by Mir), BrdU incorporation, Pdm expression and Grh expression in thoracic NBs of wild-type, cas and wor>nab sqz in cas embryos at stage 16. The co-expression of Nab and Sqz rescues the defect in Grh expression, indicating that NBs maintain stem cell characteristics and undergo temporal change.

 

Figure 7
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  		Fig. 7. Nab and Sqz regulate temporal cell fates of NB3-3 progeny. (A-D) Thoracic and abdominal EL neurons (Eve, magenta) of wild-type (A), nab (B), wor>nab (C) and sqz02102 (D) stage 17 Drosophila embryos. Kr and DmLin29 expression is shown in green. In nab (B) and sqz02102 (D) embryos, the number of thoracic EL neurons increased. In nab embryos (B), DmLin29 expression is not detected. In wor>nab embryos (C), although the number of EL neurons is mostly unchanged, Kr-positive EL neurons are missing and DmLin29-positive EL neurons are observed in the thorax. (E) Phenotype summaries.

 

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