Supplemental Data SUPPLEMENTAL EXPERIMENTAL PROCEDURES

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1 Temporal transcription factors and their targets schedule the end of neural proliferation in Drosophila. Cédric Maurange, Louise Cheng and Alex P. Gould Supplemental Data SUPPLEMENTAL EXPERIMENTAL PROCEDURES Drosophila genetics Most genetic elements are described in Flybase (Drysdale and Crosby, 2005) or in the appropriate references provided below. The genotype of grh 370 mutants is b pr cn grh 370 bw/df(2r)pcl 7B. Genetic elements used in this study are elav-gal4 c155 ; tubp-gal80 ts ; en- GAL4; wor-gal4; UAS-CD8::GFP; UAS-GFP; UAS-YFP-pros (Choksi et al., 2006); His2AvDGFP (Clarkson and Saint, 1999); FRT19A npr 1 ; FRT40A chinmo 1 ; FRT2A Df(3L)H99; FRT82B pros 17 ; FRTG13 grh B37 ; cas 24 (Cui and Doe, 1992); UAS-cas; UAShb; UAS-Kr; UAS-pdm1; UAS-otd; UAS-kni; UAS-msh; UAS-D; svp e22 (Hiromi et al., 1993) and tubp-gal80 ts 7. nab-gal4 (NP1316 from GETDB) is a GAL4 trap inserted into nab (CG33545) that is active in late embryonic and larval neuroblast lineages (Clements et al., 2003). The clonemaker stocks used for mosaic analysis with a repressible cell marker (MARCM, Lee and Luo, 1999) were: for chromosome X: w hsflp 1, FRT19A, tubp-gal80 LL1 ; UAS-nlsLacZ 20b, UAS-mCD8::GFP LL5 ; tub-gal4 LL7. For chromosome II: w, elav-gal4 C155, hsflp 1 ; FRTG13, tubp-gal80 LL2 ; UASnlsLacZ J312, UAS-mCD8::GFP LL6 and w, elav-gal4 C155, hsflp 1 ; tubp-gal80 LL10 ; FRT40A, UAS-nlsLacZ J312, UAS-mCD8::GFP LL6. For chromosome III: w, tub-gal4, hsflp 122, UAS-GFP::6xMyc::NLS; tub-gal80 LL9, FRT2A and w, tubp-gal4, hsflp 122, UAS-GFP::6xMyc::NLS; FRT82B, tubp-gal80 LL3 and w, elav-gal4 C155, hsflp 1 ; UASnlsLacZ 20b, UAS-mCD8::GFP LL5 ; FRT82B, tubp-gal80 LL3 and w, elav-gal4 C155, hsflp 1 ; UAS-nlsLacZ 20b, UAS-mCD8::GFP LL5 ; tubp-gal80 LL9, FRT2A. Control (wildtype) genotypes were generated by crossing GAL4 drivers to yw 1118 or MARCM clonemakers to FRT2A, + or FRT82B, +. Five recombinant chromosomes were constructed for this study: UAS-cas, FRT2A; UAS-cas, UAS-abdA; FRT82B, svp e22 and FRT82B, cas 24. Due to the close proximity of FRT82B and cas, we scored recombinants between cas 24 and a

2 P{neoFRT}82B P{w +mc =lacw}l(3)l2100 L2100 chromosome (Chen et al., 2005). All 5 white - recombinants obtained from 3700 progeny raised in the presence of 625μg/ml of Geneticin were verified by failure to complement cas j1c2. Embryonic MARCM clones were induced using hsflp 122 at 37 C for 45 min, following either an 8 hr (for D analysis) or a 16 hr (for Chinmo and Br-C analyses) egg collection. Postembryonic MARCM clones were induced at 0-24 hr after larval hatching for 80 min at 37 C unless otherwise stated. Neural expression of UAS transgenes was temporally restricted to postembryonic stages by combining elav-gal4 C155 or nab-gal4 with tubp-gal80 ts (McGuire et al., 2003) and shifting from 18 C to 30 C from ~4 hr for induction during all larval stages. elav-gal4 C155 /+; UAS-cas/tub-GAL80 ts larvae do not detectably express UAS-cas when raised at 18 C but the switch to 30 C induces robust expression. For transient induction of Pros, UAS-YFP-pros/tub-GAL80 ts ; nab-gal4/+ larvae were raised at 18ºC and shifted to 30ºC during the or hr time windows. Antibodies The primary antibodies used for immunohistochemistry were: 1:2000 rabbit anti-cas (gift of W. Odenwald), 1:1000 rabbit anti-chinmo (gift of T. Lee), 1:100 rabbit anti-grh (gift of B. Bello), 1:2000 rabbit anti-corebr-c (gift of J-A. Lepesant), 1:1000 mouse anti-svp (gift of Y. Hiromi), 1:400 guinea-pig anti-eve (gift of D. Kosman and J. Reinitz), 1:10 mouse anti-pdm1/nubbin (gift of S. Cohen), 1:20 rat anti-brdu (Oxford Biotechnology), 1:100 mouse anti-core Br-C (Developmental Studies Hybridoma Bank, University of Iowa), 1:20 rat anti-pros (gift of F. Matsuzaki), 1:1000 rabbit anti-cnn (gift of T. Kaufman), 1:1000 rabbit anti-d (gift of J. Nambu), 1:400 rabbit anti-ph3 (Upstate Biotechnology), 1:300 rat anti-ph3 (Abcam), 1:50 mouse anti-mira (gift of F. Matsuzaki), 1:7000 rabbit anti- -galactosidase (Cappel), 1:100 mouse anti- galactosidase (Promega), 1:1000 rabbit anti-gfp (Molecular Probes), 1:200 mouse anti- GFP (Molecular Probes) and 1:100 rat anti-cd8 (Caltag). Secondary goat antibodies were conjugated to Alexa Fluor 488 or 568 (Molecular Probes) or to Cy5 (Jackson ImmunoResearch).

3 SUPPLEMENTAL REFERENCES Choksi, S. P., Southall, T. D., Bossing, T., Edoff, K., de Wit, E., Fischer, B. E., van Steensel, B., Micklem, G., and Brand, A. H. (2006). Prospero acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells. Dev Cell 11, Clarkson, M., and Saint, R. (1999). A His2AvDGFP fusion gene complements a lethal His2AvD mutant allele and provides an in vivo marker for Drosophila chromosome behavior. DNA Cell Biol 18, Clements, M., Duncan, D., and Milbrandt, J. (2003). Drosophila NAB (dnab) is an orphan transcriptional co-repressor required for correct CNS and eye development. Dev Dyn 226, Cui, X., and Doe, C. Q. (1992). ming is expressed in neuroblast sublineages and regulates gene expression in the Drosophila central nervous system. Development 116, Drysdale, R. A., and Crosby, M. A. (2005). FlyBase: genes and gene models. Nucleic Acids Res 33, D Grosskortenhaus, R., Robinson, K. J., and Doe, C. Q. (2006). Pdm and Castor specify late-born motor neuron identity in the NB7-1 lineage. Genes Dev 20, Hiromi, Y., Mlodzik, M., West, S. R., Rubin, G. M., and Goodman, C. S. (1993). Ectopic expression of seven-up causes cell fate changes during ommatidial assembly. Development 118, Isshiki, T., Pearson, B., Holbrook, S., and Doe, C. Q. (2001). Drosophila neuroblasts sequentially express transcription factors which specify the temporal identity of their neuronal progeny. Cell 106, Kambadur, R., Koizumi, K., Stivers, C., Nagle, J., Poole, S. J., and Odenwald, W. F. (1998). Regulation of POU genes by castor and hunchback establishes layered compartments in the Drosophila CNS. Genes Dev 12, Lee, T., and Luo, L. (1999). Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22, Ma, Y., Niemitz, E. L., Nambu, P. A., Shan, X., Sackerson, C., Fujioka, M., Goto, T., and Nambu, J. R. (1998). Gene regulatory functions of Drosophila fish-hook, a high mobility group domain Sox protein. Mech Dev 73, McGuire, S. E., Le, P. T., Osborn, A. J., Matsumoto, K., and Davis, R. L. (2003). Spatiotemporal rescue of memory dysfunction in Drosophila. Science 302, Zhu, S., Lin, S., Kao, C. F., Awasaki, T., Chiang, A. S., and Lee, T. (2006). Gradients of the Drosophila Chinmo BTB-zinc finger protein govern neuronal temporal identity. Cell 127,

4 Supplemental Figure 1

5 Figure S1. Early- and late-born neurons express Chinmo and Br-C respectively Panels in A-D show single confocal sections. (A) Confocal sections of the embryonic CNS (anterior to the left) showing most, if not all, neurons are Chinmo + at stage 17. Neuroblast 7-1 expresses Hb, Kr, Pdm1 and Cas in sequence to generate the U1-U5 Eve + motor neurons (Isshiki et al., 2001) and all five of these are Chinmo +. Dashed box is enlarged in triptych showing U2, U3 and U4 (dotted circles). (B) Confocal sections of the ventral thoracic CNS showing postembryonic neuroblasts (large cells) and their last-born progeny (small cells) marked with nab>mcd8::gfp (mgfp). At 48 hr, thoracic neuroblasts, postembryonic-born (mgfp + ) and embryonicborn (mgfp - ) cells express Chinmo and Br-C is absent from all postembryonic-born neural cells. This contrasts with mushroom-body neuroblasts which are reported not to express Chinmo (Zhu et al., 2006). At 55 hr, most but not all (dotted circle) neuroblasts weakly express Chinmo and some also begin to express Br-C (dotted circle). By 65 hr, most neuroblasts express Br-C but not Chinmo and begin to generate Br-C + neurons (data not shown). At 96 hr, Br-C (but not Chinmo) is expressed in neuroblasts and late-born neurons. Note that early-born Chinmo + progeny are located below the plane of this section (see Figure 1C) and that immature neurons do not express Br-C immediately after birth. (C-D) npr 1 (C) and chinmo 1 (D) MARCM clones, induced at 0-24 hr and marked with elav>mgfp, express the normal layer-specific patterns of Chinmo and Br-C respectively at 96 hr.

6 Supplemental Figure 2 Figure S2. Thoracic postembryonic neuroblasts express Cas and Svp sequentially Single confocal sections of the ventral thoracic CNS with postembryonic neuroblasts (large cells) and their progeny (small cells) marked with nab>mcd8::gfp. At 30 hr, some larval neuroblasts leaving quiescence express Cas in a similar pattern in each segment (T1, T2 and T3) but no neuroblasts express Svp. At 40 hr, some neuroblasts (arrowheads and enlargement of dotted box) express Svp in a pattern resembling Cas at 30 hr. At 60 hr and 96 hr, neuroblasts no longer express Cas or Svp but new-born progeny continue to express Cas strongly, even though they must have been generated from Cas - neuroblasts. This contrasts with the temporally-linked Cas expression pattern observed in embryonic neuroblasts and their progeny (Isshiki et al., 2001).

7 Supplemental Figure 3

8 Figure S3. Loss of Pros but not RHG activity prolongs thoracic and central brain neuroblast divisions Panels in A-C show ventral views of the CNS. (A) A thoracic MARCM clone lacking the activity of RHG proapoptotic genes (Df(3L)H99), induced at 0-24 hr and marked with elav>mgfp, does not contain PH3 + or Mira + progenitors at 128 hr. (B) Thoracic neuromeres contain numerous Mira + neuroblasts (some of which are PH3 + ) at 96 hr but lose most of these by 128 hr. Inset shows an enlargement of one of the last remaining Mira + neuroblasts to undergo cell-cycle exit (arrow). (C) Ventral view of 7-day adult central brain with several pros 17 MARCM clones, induced at 0-24 hr and marked with tub>nlacz, each containing numerous Mira + adult progenitors, many of which are PH3 +. Such progenitors are absent from control tissue surrounding the clones.

9 Supplemental Figure 4

10 Figure S4. Loss or mis-expression of Cas leads to neuroblast divisions and Chinmo + neurons in the adult Ventral views of the central brain and thoracic neuromeres of 3-day old adults. MARCM UAS-cas clones, induced at 0-24 hr (A-B) and cas 24 clones, induced during embryogenesis (C-D), were marked with tub>ngfp. Both types of clone often retain a single Mira + neuroblast that is mitotically active (some are PH3 + ) in the adult and continues to generate superficial (late-born) Chinmo + cells. Deep (early-born) neurons in surrounding control tissue (GFP - ) have down regulated Chinmo before adult eclosion.

11 Supplemental Figure 5 Figure S5. Svp is required for nuclear Pros in neuroblasts at 120 hr A svp e22 MARCM clone, induced at 0-24 hr, marked with tub>ngfp and analyzed at 120 hr, lacks Br-C + neurons, indicating that the postembryonic temporal series is stalled. Although Pros is absent from the nucleus of the interphase Mira + neuroblast (arrow), it remains normally expressed in the nucleus of the GMC (arrowhead).

12 Supplemental Figure 6

13 Figure S6. Dichaete is down regulated in most wild-type neuroblasts during development (A) Single confocal sections of the embryonic CNS at stages At stage 9, D is expressed in most medial (m) and intermediate (i) but not lateral (l) Mira + neuroblasts. From stage 10 onwards, many lateral neuroblasts express D. As previously reported, D is expressed in both the nucleus and cytoplasm (Ma et al., 1998). (B) At 55 hr, a small subset of thoracic Mira + neuroblasts (and other cells) expresses D. (C) At 65 hr, D is not expressed in wild-type Mira + abdominal neuroblasts. Three dl lineages (marked with nab>mgfp) in segments A3-A5 are shown. (D) Histogram showing that the percentage of thoracic Mira + neuroblasts expressing D progressively decreases. It is 47% in stage 12 embryos (n=12 hemisegments, sd=11.3), 50.7% in stage 14 embryos (n=10, sd=11.2), 41.5% in stage 15 embryos (n=8,sd=10.5), 23.6% in 55 hr larvae (n=18, sd=3.5) and only 14.6% in 96 hr larvae (n=18, sd=3). Asterisk, P < for stage 14 embryos versus 96 hr larvae.

14 Supplemental Figure 7 Figure S7. Embryonic Cas activates Grh and down regulates D in neuroblasts (A) Ventral views of T2 and T3 neuromeres at embryonic stage 14 showing that early continuous expression of Hb within the Engrailed stripes (brackets) of en>hb embryos or loss-of-function of Cas in cas 24 embryos prevents Grh expression in Mira + neuroblasts. (B) Ventral views of T2 and T3 neuromeres at embryonic stage 12. In most Mira + neuroblasts, Grh has yet to be expressed in wild-type (wt) embryos but within the Engrailed stripes (brackets) of en>cas embryos it is prematurely activated. Consistent with previous reports, we also observe that Cas represses Pdm (Grosskortenhaus et al., 2006; Kambadur et al., 1998). (C-D) Ventral views of T2-A1 neuromeres at embryonic stages 14 and 15. (C) At embryonic stage 15, D down regulation in Mira + neuroblasts, particularly in lateral

15 progenitors (arrows) is prevented by early and continuous expression of Hb within the Engrailed stripes (brackets) of en>hb embryos, or by loss-of-function of Cas in cas 24 embryos. More neuroblasts with M-phase Mira crescents are found in en>hb and cas 24 than in wild-type embryos at this stage, suggesting that entry into quiescence is delayed. (D) At embryonic stage 14, early and persistent expression of Cas in wor>cas embryos leads to precocious down regulation of D in lateral Mira + neuroblasts (arrows).

16 Supplemental Figure 8 Figure S8. Persistent adult neuroblasts in svp e22 clones retain Grh expression Postembryonic svp e22 MARCM clones were induced at 0-24 hr, marked with tub>ngfp, and analyzed in the central brain of 3-day old adults. Inset shows enlargement of a clone (arrow) containing a Mira + Grh + neuroblast. Supplemental Figure 9 Figure S9. The Chinmo->Br-C switch is not blocked in grh B37 clones Single confocal section of a grh B37 MARCM lineage in a thoracic neuromere, induced at 0-24hr and marked with elav>mgfp, showing that although these postembryonic lineages are smaller-than-normal they nevertheless contain both Chinmo + and Br-C + cells at 96 hr (n=34/34 clones).

17 Supplemental Figure 10

18 Figure S10. dl neuroblasts express Cas and Svp sequentially and generate Chinmo + and Br-C + progeny (A) Single confocal sections of two wild-type dl lineages in the central abdomen at 96 hr. The postembryonic component of these lineages is labeled with BrdU (the most anterior dl lineage is dotted) and contains Br-C + cells and somewhat deeper Chinmo + cells. (B) Single confocal sections of a wild-type dl lineage, labeled with nab>mgfp. Both the dl neuroblast and its progeny express Cas at 55 hr but only the progeny expresses Cas at 62 hr and 65 hr. Svp is expressed in dl neuroblasts at 62 hr but not at 55 hr or 65 hr. (C) Schematic representation of Cas and Svp expression in the dl neuroblast (large circle) and its progeny (small circles). For clarity, Cas expression is omitted from postmitotic cells and Chinmo and Br-C are only shown at 96 hr.

19 Supplemental Figure 11

20 Figure S11. Cas and Svp are required for timely apoptosis of dl neuroblasts (A) Abdominal neuromeres containing several svp e22 clones, induced at 0-24 hr and marked with elav>mgfp or elav>nlslacz. At 96 hr, dl clones retain a single Mira + neuroblast (arrow), PH3 + progenitors (inset) and supernumerary mgfp + cells. (B) Abdominal neuromeres containing a single cas 24 clone, induced during embryogenesis and marked with tub>ngfp. At 96 hr, this dl clone retains a single Mira + PH3 + neuroblast, a PH3 + GMC (inset) and supernumerary mgfp + cells. (C) Abdominal neuromeres continuously expressing neural Cas during postembryonic stages (UAS-Cas, referring to elav-gal4 C155 ; UAS-cas/tub-GAL80 ts ). At 96 hr, dl and other abdominal lineages contain more postembryonic cells (BrdU + ) than normal and retain Mira + neuroblasts (some of which are PH3 + ). (D) Quantification of supernumerary (GFP + ) cells generated in dl MARCM clones marked with tub>ngfp that are wild-type (0 or 1 cell only) or svp e22 (m=22.5 cells, sd=5.5, n=12). Asterisk indicates P < (E) Numbers of postembryonic-born (BrdU + ) cells in dl lineages for wt, UAS-cas (elav- GAL4 C155 ; UAS-cas/tub-GAL80 ts ) and UAS-D (elav-gal4 C155 ; UAS-D; tub-gal80 ts ) larvae at 96 hr. Asterisks indicate P < for wt (m=9.5, sd=2, n=17) versus UAS-cas (m=16.6, sd=2.4 and n=18) or UAS-D (m=23.8, sd=5.6 and n=10). (F) Single confocal sections of Df(3L)H99, svp e22 and UAS-cas clones, all induced at 0-24hr, and cas 24 clones, induced during embryogenesis. All four types of MARCM clone generate larger-than-normal dl lineages by 96 hr but the supernumerary (tub>ngfp labeled) cells in Df(3L)H99 clones are Br-C + but do not express Chinmo, whereas those in svp e22, UAS-cas and cas 24 clones are Chinmo + but do not express Br-C. This indicates that the Chinmo->Br-C transition is blocked in svp e22, UAS-cas and cas 24 clones but not in Df(3L)H99 clones.

21 Supplemental Figure 12 Figure S12. Postembryonic mis-expression of Hb, Kr or Pdm1 blocks neuroblast apoptosis and the Chinmo->Br-C switch in dl lineages (A-E) Abdominal neuromeres at 96 hr from elav C155 -GAL4; tub-gal80 ts larvae carrying either UAS-otd (A), UAS-hb (B,C), UAS-kr (D) or UAS-pdm1 (E) transgenes. Continuous postembryonic neural expression of Hb, Kr, Pdm1 but not Otd produces supernumerary cells, some of which are PH3 +, in the vm, vl and dl lineages (BrdU + ). Control elav C155 - GAL4; tub-gal80 ts ; UAS-hb larvae raised at the non-induced temperature (18 C) display no such over proliferation (B). UAS-knirps and UAS-msh gave similar negative results to UAS-otd (data not shown). (F) A superficial confocal section of a dl lineage (BrdU + ) from an elav C155 -GAL4; tub- GAL80 ts ; UAS-hb larva at 96hr. Many of the supernumerary late-born neurons generated after continuous postembryonic neural expression of Hb are Chinmo + but none are Br-C +.

22 Supplemental Figure 13 Figure S13. Postembryonic mis-expression of D blocks dl neuroblast apoptosis but not Grh expression nor the Chinmo->Br-C switch (A) Postembryonic mis-expression of D (UAS-D refers to elav-gal4 C155 ; UAS-D; tub- GAL80 ts larvae) leads to persistent dl neuroblasts that are Grh + (and PH3 + in some cases) at 96 hr. Postembryonic cells are labeled with BrdU. (B) Postembryonic mis-expression of D (UAS-D refers to UAS-D/tub-GAL80 ts ; nab- GAL4, UAS-mGFP larvae) leads to persistent dl neuroblasts that generate supernumerary Chinmo - Br-C + neurons at 96 hr.

23 Supplemental Figure 14 Figure S14. Postembryonic blockade of the temporal series does not prevent dl neuroblasts from expressing AbdA At 96 hr, AbdA expression is retained by the persistent dl neuroblasts in UAS-cas (elav C155 -GAL4; tub-gal80ts/uas-cas) larvae (A) and also in svp e22 MARCM clones (B), induced at 0-24 hr and marked with elav>mgfp.

24 Supplemental Movie 1. Self-renewing asymmetric division of a neuroblast at 96 hr Time-lapse movie of a 96 hr thoracic neuroblast (large mitotic cell, ~10μm diameter) and smaller surrounding cells expressing the chromatin label His2AvDGFP. The interval between frames (single confocal sections) is 30 seconds and anaphase and telophase of this type-i neuroblast take ~3 min to complete. Supplemental Movie 2. Atypical division of a neuroblast at 120 hr Time-lapse movie of a 120 hr thoracic neuroblast (intermediate-size mitotic cell, ~7μm diameter) and smaller surrounding cells expressing the chromatin label His2AvDGFP. The interval between frames (single confocal sections) is 30 seconds and anaphase and telophase of this type-i neuroblast take ~16 min to complete.