Supplementary Figure 1 Expression of escargot (esg) and genetic approach for achieving IPC-specific knockdown. (a) esg MH766 -Gal4 UAS-cd8GFP (green) and esg-lacz B7-2-22 (red) show similar expression patterns. Both transgenes label cells at the p- IPC neuroepithelial margins (arrowheads), as well as migratory progenitors within cell streams (arrows) of late third instar larvae (3L). (b,c) fasciclin 3 (fas3) NP1233 -Gal4 drives expression of UAS-cd8GFP (green) in the IPC and its progeny, while ey 3.5 -Gal80 blocks Gal4 activity in R-cells (arrows, b). This approach efficiently drives expression of UAS-RNAi transgenes, as revealed by the knockdown of fas3 in p-ipc neuroepithlial cells and their progeny (asterisks, red, c). (d) In a EdU pulse-chase experiment, brains of mid third instar larvae (mid 3L) were assessed 4 hours after 2.5 hours EdU feeding. At this stage, p-ipc neuroepithelial cells extensively incorporate EdU (red) and thus are proliferative. dc, distal cells; ln, lamina neurons; lopn, lobula plate neurons; mn, medulla neurons. For genotypes and sample numbers, see Supplementary Table 2. Scale bars represent 50 µm.
Supplementary Figure 2 Validation of lethal of scute (l sc) RNAi-mediated knockdown and characterization of phenotypes in the developing IPC. (a) In control animals, maintained at 18 C, L sc is expressed in neuroepithelial cells of the OPC and p-ipc (red, arrows). (b,c) Experimental animals were grown at 29 C during the third instar larval stage. IPC-specific expression of two different l sc RNAi transgenes - l sc IR JF02399 and l sc IR KK104691 - using fasciclin 3 (fas3) NP1233 -Gal4 results in efficient knockdown of L sc expression in the p- IPC (asterisks) without affecting expression in the OPC (arrows). (d-g) IPC-specific expression of l sc IR KK104691 results in identical phenotypes as l sc IR JF02399 (cf. Fig. 7). Compared to controls, Deadpan-positive (Dpn + ) neuroblasts (red, arrows, d,e) and Dachshundpositive (Dac + ) progeny (red, arrows, f,g), including lobula plate neurons (lopn), are reduced. (h-k) Knockdown of l sc does not affect esg-lacz expression (red, h,i), p-ipc morphology (arrow, h,i), and Dichaete expression in cell streams (red, arrows, j,k). Although the d-ipc is reduced in size, Dichaete continues to be expressed (j,k). For genotypes and sample numbers, see Supplementary Table 2. Scale bars represent 50 µm.
Supplementary Figure 3 Validation of Dichaete RNAi-mediated knockdown in the IPC. (a) In controls, Dichaete (red) is expressed in cell streams (arrow), the d-ipc and medulla neurons (mn). (b,c) IPC-specific expression IR KK107194 IR GD49549 NP1233 of two different RNAi transgenes - Dichaete and Dichaete using fasciclin 3 (fas3) -Gal4 results in efficient knockdown of Dichaete expression in progenitors in cell streams and neuroblasts in the d-ipc (asterisks), without affecting expression in medulla neurons. (d-f) Asense (Ase, red) is not expressed in cell streams (arrows) in control animals (d) and upon Dichaete knockdown (e,f). Both Dichaete RNAi transgenes show identical phenotypes in impairing d-ipc neurogenesis. Discs large (Dlg) immunolabeling is shown in blue. For genotypes and sample numbers, see Supplementary Table 2. Scale bars represent 50 µm.
Supplementary Figure 4 Knockdown of tailless (tll) affects early p-ipc development. (a,b) Compared to controls (a), IPC-specific expression of a tll RNAi transgene using fasciclin 3 (fas3) NP1233 -Gal4 strongly impairs early p-ipc neuroepithelial development, visualized by apkc labeling at the third instar larval stage (red, b). The p-ipc is absent (asterisk) and the d-ipc is reduced (arrow). (c,d) neuralized (neur)-gal4 mediated tll RNAi transgene expression results in knockdown of Tll (red) in the d-ipc (asterisk, d) without affecting expression in the lamina (La) and p-ipc (arrows). Low levels of Tll expression remain in the d-ipc. Discs large (Dlg) immunolabeling is shown in blue. For genotypes and sample numbers, see Supplementary Table 2. Scale bars represent 50 µm.
Supplementary Figure 5 Model illustrating two neuroblast competence stages in the d-ipc. Migratory progenitors mature into neuroblasts (Nb) in the d-ipc, where they transition through two competence stages that are defined by the sequential expression of Asense (Ase) and Atonal (Ato)/Dachshund (Dac). These give rise to Twin of Eyeless-positive (Toy + ) distal cells (dc) and Dachshund-positive (Dac + ) lobula plate neurons (lopn), respectively. Dichaete (D) acts upstream of tailless (tll) to promote the transition between the two neuroblast stages. Genetic manipulations indicate that Dichaete is required to induce tll. tll is required to suppress Dichaete and ase, and to induce ato and dac.
Supplementary Table 1 Full genotypes and numbers of samples shown in main figure panels. Figure Panel Genotype n = a Fig. 1 c esg MH766 -Gal4/CyO or Pin YT ; UAS-cd8GFP/UAS-cd8GFP 15 d esg MH766 -Gal4/CyO or Pin YT ; UAS-cd8GFP/UAS-cd8GFP 19 e esg MH766 -Gal4/CyO or Pin YT ; UAS-cd8GFP/UAS-cd8GFP 9 f esg MH766 -Gal4/CyO or Pin YT ; UAS-cd8GFP/UAS-cd8GFP 20 g-k esg MH766 -Gal4/CyO or Pin YT ; UAS-cd8GFP/UAS-cd8GFP 11 Fig. 2 a esg MH766 -Gal4/CyO or Pin YT ; UAS-cd8GFP/UAS-cd8GFP 11 b esg MH766 -Gal4/CyO or Pin YT ; UAS-cd8GFP/UAS-cd8GFP 19 c, e esg MH766 -Gal4/CyO or Pin YT ; UAS-cd8GFP/UAS-cd8GFP 14 d esg MH766 -Gal4/CyO or Pin YT ; UAS-cd8GFP/UAS-cd8GFP 8 f esg MH766 -Gal4/CyO or Pin YT ; UAS-cd8GFP/UAS-cd8GFP 13 g esg MH766 -Gal4/CyO or Pin YT ; UAS-cd8GFP/UAS-cd8GFP 7 h w hs-flp 122 elav-gal4 c155 UAS-cd8GFP/+ or Y; FRT42D tubp-gal80/frt42d; tubp-gal4/+ i ey 3.5 -Gal80/w 1118 or Y; esg MH766 -Gal4/+; UAS-Dcr2 UAS-cd8GFP/+ 4 j w hs-flp 122 elav-gal4 c155 UAS-cd8GFP/+ or Y; FRT42D tubp-gal80/frt42d Pcl 3-78*38 ; tubp-gal4/+ Fig. 3 a esg MH766 -Gal4/CyO or Pin YT ; UAS-cd8GFP/UAS-cd8GFP 10 b ey 3.5 -Gal80/w 1118 or Y; fas3 NP1233 -Gal4/+; UAS-Dcr2 UAS-cd8GFP/+ 4 c ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/dpp-lacZ Exel.2 ; UAS-Dcr2 UAS-cd8GFP/UASesg IR GD9794 16/30 e esg MH766 -Gal4/CyO or Pin YT ; UAS-cd8GFP/UAS-cd8GFP 5 f esg MH766 -Gal4/CyO or Pin YT ; UAS-cd8GFP/UAS-cd8GFP 12 i w 1118 4 j w 1118 8 k w 1118 7 l w 1118 9 Fig. 4 a-c +/CyO or Pin YT ; dpp-gal4/uas-cd8gfp 10 d brk x47 /+ or Y; UAS-cd8GFP/+; dpp-gal4/+ 4 e omb P1 /+ or Y; UAS-cd8GFP/+; dpp-gal4/+ 3 g (wt) (tkv strii ) h (wt) (tkv strii ) i (wt) (tkv strii ) ey 3.5 -Gal80/+ or Y; ubi-gfp cyce AR95 FRT40A/FRT40A; lama-gal4 UAS-FLP Exel.2 13 mδ/dpp-lacz ey 3.5 -Gal80/+ or Y; ubi-gfp cyce AR95 FRT40A/tkv strii FRT40A; lama-gal4 UAS- Exel.2 29/33 FLP mδ/dpp-lacz ey 3.5 -Gal80/brk x47 ; ubi-gfp cyce AR95 FRT40A/FRT40A; lama-gal4 UAS-FLP mδ/tm2 ey 3.5 -Gal80/brk x47 ; ubi-gfp cyce AR95 FRT40A/tkv strii FRT40A; lama-gal4 UAS- FLP mδ/tm2 ey 3.5 -Gal80/omb P1 ; ubi-gfp cyce AR95 FRT40A/FRT40A; lama-gal4 UAS-FLP mδ/tm2 ey 3.5 -Gal80/omb P1 ; ubi-gfp cyce AR95 FRT40A/tkv strii FRT40A; lama-gal4 UAS- FLP mδ/+ Fig. 5 a acj6-gal4 UAS-cd8GFP/+; UAS-cd8GFP/+ 9 b esg MH766 -Gal4/CyO or Pin YT ; UAS-cd8GFP/UAS-cd8GFP 3 c w 1118 9 e, f, h, i w hs-flp 122 elav-gal4 c155 UAS-cd8GFP/+ or Y; FRT42D tubp-gal80/frt42d; tubp-gal4/+ g, j, k w hs-flp 122 elav-gal4 c155 UAS-cd8GFP/+ or Y; FRT42D tubp-gal80/frt42d 17 Fig. 6 a ey 3.5 -Gal80/w 1118 or Y; esg MH766 -Gal4/+; UAS-Dcr2 UAS-cd8GFP/+ 6 16 5/8 3 18 8 40 16
b w 1118 7 c w 1118 17 d w 1118 10 e w 1118 11 f w 1118 6 g h w hs-flp 122 elav-gal4 c155 UAS-cd8GFP/+ or Y; FRT42D tubp-gal80/frt42d; tubp-gal4/+ w hs-flp 122 elav-gal4 c155 UAS-cd8GFP/+ or Y; FRT42D tubp-gal80/frt42d; tubp-gal4/ + i brk x47 /+ or Y; UAS-cd8GFP/+; dpp-gal4/+ 4 Fig. 7 a ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/+; UAS-Dcr2 UAS-cd8GFP/UAS-l sc IR JF02399 10 b ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/+; UAS-Dcr2 UAS-cd8GFP/UAS-l sc IR JF02399 17 c ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/+; UAS-Dcr2 UAS-cd8GFP/UAS-l sc IR JF02399 7 d ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/+; UAS-Dcr2 UAS-cd8GFP/UAS-l sc IR JF02399 12 e ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/+; UAS-Dcr2 UAS-cd8GFP/UAS-l sc IR JF02399 6 f ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/+; UAS-Dcr2 UAS-cd8GFP/UAS-l sc IR JF02399 7 g ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/+; UAS-Dcr2 UAS-cd8GFP/UAS-l sc IR JF02399 9 h ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/+; UAS-Dcr2 UAS-cd8GFP/UAS-l sc IR JF02399 9 j w 1118 17 k Df(1)ase-1, sc ase-1 pn 1 /Y 11 l w 1118 11 m Df(1)ase-1, sc ase-1 pn 1 /Y 10 n w 1118 6 o Df(1)ase-1, sc ase-1 pn 1 /Y 7 Fig. 8 a ey 3.5 -Gal80/w 1118 or Y; fas3 NP1233 -Gal4/+; UAS-Dcr2 UAS-cd8GFP/+ 4 b ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/UAS-D IR KK107194 ; UAS-Dcr2 UAS-cd8GFP/+ 8 c ey 3.5 -Gal80/w 1118 or Y; fas3 NP1233 -Gal4/+; UAS-Dcr2 UAS-cd8GFP/+ 7 d ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/UAS-D IR KK107194 ; UAS-Dcr2 UAS-cd8GFP/+ 6 e ey 3.5 -Gal80/w 1118 or Y; fas3 NP1233 -Gal4/+; UAS-Dcr2 UAS-cd8GFP/+ 3 f ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/UAS-D IR KK107194 ; UAS-Dcr2 UAS-cd8GFP/+ 8 g ey 3.5 -Gal80/w 1118 or Y; fas3 NP1233 -Gal4/+; UAS-Dcr2 UAS-cd8GFP/+ 3 h ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/UAS-D IR KK107194 ; UAS-Dcr2 UAS-cd8GFP/+ 7 i +/w 1118 or Y; neur P72 -Gal4 UAS-ponGFP/+ 6 j +/w 1118 or Y; UAS-tll IR GD6236 /+; neur P72 -Gal4 UAS-ponGFP/UAS-Dcr2 11 k +/w 1118 or Y; neur P72 -Gal4 UAS-ponGFP/+ 7 l +/w 1118 or Y; UAS-tll IR GD6236 /+; neur P72 -Gal4 UAS-ponGFP/UAS-Dcr2 9 m +/w 1118 or Y; neur P72 -Gal4 UAS-ponGFP/+ 8 n +/w 1118 or Y; UAS-tll IR GD6236 /+; neur P72 -Gal4 UAS-ponGFP/UAS-Dcr2 12 o w 1118 7 p ato 1 7 q ey 3.5 -Gal80/+ or Y; ubi-gfp cyce AR95 FRT40A/dac 1 FRT40A; lama-gal4 UAS- FLP mδ/+ a If not otherwise indicated, in loss-of-function and knockdown experiments, all examined control samples were normal, while all experimental samples showed defects (100% penetrance). 2 6 8
Supplementary Table 2 Full genotypes and numbers of samples shown in Supplementary figure panels. Figure Panel Genotype n = a Suppl. a esg MH766 -Gal4/esg-lacZ B7-2-22 ; UAS-cd8GFP/TM2 7 Fig. 1 b ey 3.5 -Gal80/w 1118 or Y; fas3 NP1233 -Gal4/+; UAS-Dcr2 UAS-cd8GFP/+ 16 c ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/UAS-fas3 IR KK100642 ; UAS-Dcr2 UAScd8GFP/+ d w 1118 4 Suppl. a ey 3.5 -Gal80/w 1118 or Y; fas3 NP1233 -Gal4/+; UAS-Dcr2 UAS-cd8GFP/+ 10 Fig. 2 b ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/+; UAS-Dcr2 UAS-cd8GFP/UAS-l sc IR JF02399 6 c ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/UAS-l sc IR KK104691 ; UAS-Dcr2 UAS-cd8GFP/+ 4 d ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/UAS-l sc IR KK104691 ; UAS-Dcr2 UAS-cd8GFP/+ 9 e ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/UAS-l sc IR KK104691 ; UAS-Dcr2 UAS-cd8GFP/+ 7 f ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/UAS-l sc IR KK104691 ; UAS-Dcr2 UAS-cd8GFP/+ 6 g ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/UAS-l sc IR KK104691 ; UAS-Dcr2 UAS-cd8GFP/+ 5 h i ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/ esg-lacz B7-2-22 ; UAS-Dcr2 UAScd8GFP/UAS-l sc IR JF02399 14 ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/ esg-lacz B7-2-22 ; UAS-Dcr2 UAScd8GFP/UAS-l sc IR JF02399 7 j ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/+; UAS-Dcr2 UAS-cd8GFP/UAS-l sc IR JF02399 4 k ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/+; UAS-Dcr2 UAS-cd8GFP/UAS-l sc IR JF02399 6 Suppl. a ey 3.5 -Gal80/w 1118 or Y; fas3 NP1233 Gal4/+; UAS-Dcr2 UAS-cd8GFP/+ 10 Fig. 3 b ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/UAS-D IR KK107194 ; UAS-Dcr2 UAS-cd8GFP/+ 3 c ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/+; UAS-Dcr2 UAS-cd8GFP/UAS-D IR GD49549 3 d ey 3.5 -Gal80/w 1118 or Y; fas3 NP1233 Gal4/+; UAS-Dcr2 UAS-cd8GFP/+ 3 e ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/UAS-D IR KK107194 ; UAS-Dcr2 UAS-cd8GFP/+ 5 f ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/+; UAS-Dcr2 UAS-cd8GFP/UAS-D IR GD49549 6 Suppl. a ey 3.5 -Gal80/w 1118 or Y; fas3 NP1233 Gal4/+; UAS-Dcr2 UAS-cd8GFP/+ 5 Fig. 4 b ey 3.5 -Gal80/+ or Y; fas3 NP1233 -Gal4/UAS-tll IR GD6236 ; UAS-Dcr2 UAS-cd8GFP/+ 4 c +/w 1118 or Y; neur P72 -Gal4 UAS-ponGFP/+ 5 d +/w 1118 or Y; UAS-tll IR GD6236 /+; neur P72 -Gal4 UAS-ponGFP/UAS-Dcr2 4 a If not otherwise indicated, in loss-of-function and knockdown experiments, all examined control samples were normal, while all experimental samples showed defects (100% penetrance). 5