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Fig. S1. Isolated NBs cultured in vitro show quiescent NB-like features. NB shape was visualized by Miranda (green). Nab (magenta) is a temporal identity factor expressed in aged NBs (see Fig. 2F and sections below for detail). NB culture was initiated from 5-7 hours AEL embryos. (A-C′) BrdU (1 mg/ml in medium) was added 0 (A,A′) or 7 hours (B-C′) after culture was initiated, and pulse-labeled for 30 minutes. At 0 hours of culture, all NBs were Nab negative and round. After 7 hours of culture, isolated NBs often showed elongated shape. All the elongated NBs examined were Nab positive and BrdU negative (B,B′), whereas spherical NBs usually incorporated BrdU and more than half of them were Nab negative (C,C′).
Fig. S2. Svp expression in thoracic and abdominal NBs. (A,B) Svp expression (magenta) in NB layer in thoracic (A) and abdominal (B) hemisegments at embryonic stage 14. NBs are indicated by Miranda (green). (C) Svp expression (magenta) in NB layer in a thoracic hemisegment (second instar). NBs are indicated by grh-LacZ (grh06850 from the Bloomington Drosophila Stock Center). Ventral view; midline is on the left.
Fig. S3. Expression of temporal transcription factors in EL neurons. (A-H) Thoracic (A-D) and abdominal (E-H) EL neurons (visualized by Eve, magenta) of wild-type embryos at the point when the indicated number of EL neurons exist. Expression of the following transcription factors (green) is shown: (A,E) Cas (top row), Nab (middle row), (B,F) Cas (top row), Kr (middle row), (C,G) Sqz (top row), Kr (middle row), (D,H) Svp (top row), DmLin29 (middle row). Schematics of the expression patterns are shown at the bottom panel in each box. (I,J) Schematic diagrams of the expressions of the temporal factors in thoracic (I) and abdominal (J) EL neurons. Pale blue indicates weak Nab expression in abdominal sixth EL neuron. Pale pink indicates DmLin29 expression weak and often hard to be detected in seventh and 11th abdominal EL neurons.
Fig. S4. Svp and DmLin29 expression in larval NB3-3T lineage. (A) Svp (magenta) expression in NB3-3T lineage at 30-35 hours AEL (early second instar). NB3-3 lineage is visualized by eg>LacZ (green). (B) DmLin29 (magenta) expression in NB3-3T lineage at 50-60 hours AEL (early third instar). NB3-3 lineage is visualized by MZ360 eg>GFP (green). Arrowheads indicate NB3-3T.
Fig. S5. Expression of temporal identity factors in Hox mutant NB3-3 lineages. (A,C) Pdm, Cas, Sqz and Nab expression (magenta) in NB3-3T of the Antp mutant (A) and wor>abd-A (C) embryos. Sqz and Nab are late temporal identity factors (see Fig. 2F and sections below). NB3-3T lineage is visualized by Eg or eg>GFP (green). (B,D) Pdm, Cas, Sqz and Nab expression (magenta) in thoracic EL neurons in the Antp mutant (B) and wor>abd-A (D) embryos. EL neurons are visualized by Eve (green). Arrowhead indiactes NB3-3. (E) Phenotype summaries. In Antp mutant, NB3-3T or ectopically abd-A-expressing NB3-3T, the expression of Pdm, Cas, Sqz and Nab were unchanged until entry into quiescence. Failure of mutant NB3-3T to enter quiescence led to precocious downregulation of both Sqz and the late Cas expression during embryogenesis.
Fig. S6. Prolonged Pdm expression delays NB quiescence and the switching of temporal identity factors. Embryos at the indicated stages were labeled for the indicated markers. NB3-3 (arrowhead) was identified by Eg or eg>GFP (green). Genotypes are shown at the top. We expressed Pdm in all neuroblasts using the neuroblast-specific worniu-GAL4 line or scabrous-GAL4 line crossed with UAS-pdm (referred to as wor>pdm and sca>pdm). wor>pdm NBs express Pdm throughout from stage 11, whereas sca>pdm NBs express Pdm from early to mid stages. (A-C) NB3-3T was visualized by Miranda (white). Wild-type NB3-3T (A) becomes elongated by early stage 15, whereas in wor>pdm embryos (B) NB3-3T remains round. In sca>pdm embryos (C), NB3-3T eventually becomes elongated at stage 16, later than in wild-type embryos. (D-F) NB3-3T pulse-labeled with BrdU (magenta). In wild-type NB3-3T (D), BrdU incorporation is rarely observed at stage 15, whereas in wor>pdm NB3-3T (E), BrdU incorporation is still observed at stage 16. In sca>pdm NB3-3T (F), BrdU incorporation is observed at stage 15, but rarely at stage 16. (G-I) Cas expression (magenta) in NB3-3T. wor>pdm (H) and sca>pdm (I) NB3-3T expresses Cas continuously. (J-L) Sqz expression (magenta) in NB3-3T. In wild-type NB3-3T (J), Sqz expression starts at stage 13. In wor>pdm NB3-3T (K), Sqz expression is never observed. (M-O) Nab expression (magenta) in NB3-3T. In wild-type NB3-3T (M), Nab expression starts at stage 14. In wor>pdm NB3-3T (N), Nab expression is never observed. In sca>pdm NB3-3T (O), Nab expression is occasionally observed at stage 16. (P-R) Svp expression (magenta) in NB3-3A. In wild-type NB3-3A (P), Svp expression is observed at stage 15, whereas in wor>pdm NB3-3A (Q) Svp expression is never observed. Continuous Pdm expression in wor>pdm embryos inhibits quiescence and temporal changes completely. By contrast, sca>pdm NBs display retarded quiescence and temporal changes owing to the eventual downregulation of Pdm.
Fig. S7. Expression of temporal identity factors in pdm, cas and pdm cas double mutant NB3-3. (A-D) Embryos were labeled for the indicated markers. Genotypes are shown at the top. Pdm, Cas and Sqz expression (magenta) in NB3-3T and Svp expression (magenta) in NB3-3A are shown. NB3-3 is visualized by Eg (green). (E) Phenotype summaries regarding temporal identity factor switching. Loss of cas inhibits temporal factor switching: Sqz and abdominal Svp expression are absent, and Pdm expression is prolonged. Loss of pdm and loss of both pdm and cas cause precocious switching of temporal factor expression; Cas, Sqz and abdominal Svp expression occur precociously in the absence of pdm or both pdm and cas.
Fig. S8. Nab and Sqz cooperate to rescue the failure to trigger quiescence and temporal specification in the cas mutant. Embryos were labeled for the indicated markers. Genotypes are shown at the top. We expressed both Nab and Sqz, or Nab or Sqz alone in all NBs in the cas mutant using the NB-specific worniu-GAL4 line (referred to as wor>nab sqz in cas, wor>nab in cas or wor>sqz in cas). (A-R) NB3-3 is visualized by Eg or eg>GFP (green). (A-E) NB3-3T (white, visualized by Mir) at stage 16. cas mutant phenotype (NBs remain round) is rescued by co-expression of Nab and Sqz but not by expression of Nab or Sqz alone. (F-O,K′-O′) Pdm expression in thoracic NBs (F-J, white) and NB3-3T (arrowhead; K-O, magenta, K′-O′, white) at stage 16. Prolonged Pdm expression in cas mutant NBs is terminated by the co-expression of Nab and Sqz. (P-R,P′-R′) BrdU incorporation (P-R, magenta, P′-R′, white) in NB3-3T (arrowhead) at stage 16. BrdU incorporation into NBs stops in wor>nab sqz in cas mutant. (S-W) DmLin29 expression (green) in abdominal EL neurons (labeled for Eve, magenta) at stage 17. Loss of cas leads to the absence of DmLin29 in abdominal EL neurons. The defect in DmLin29 expression is rescued by co-expression of Nab and Sqz.
Fig. S9. Expression of temporal factors in nab or sqz mutant NB3-3. Embryos at the indicated stages were labeled for the indicated markers. Genotypes are shown at the top. NB3-3 was identified by Eg (green). (A) Pdm, Sqz and Nab expression (magenta) in NB3-3T, and Sqz expression (magenta) in NB3-3A, of wild-type embryos. (B) Pdm expression (magenta) in NB3-3T and Sqz expression (magenta) in NB3-3T and NB3-3A in nab mutant embryos. (C) Pdm and Nab expression (magenta) in sqz mutant NB3-3T. Nab and Sqz are dispensable for downregulation of Pdm. Nab is necessary for downregulation of Sqz. (D) Phenotype summaries regarding temporal identity factor switching.
Fig. S10. A model of neuroblast entry into quiescence controlled by temporal and spatial regulators. Tentative regulatory relationships among temporal identity factors are also shown. See the text for detail.
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