(mm) / % rescue / Wing cell
density(a) / Expression in 3rd instar larvae(b)
Isogenic wild-type / - / 3.22 (0.08) / - / 6.8 (0.1) / -
NF1E2/UAS-NF1; NF1E1 / - / 2.71 (0.07) / - / 7.4 (0.2) / -
P{Act5C-GAL4} / [1] / 3.26 (0.09) / 108 / ND / Ubiquitous
P{GawB}69B / B1774 / 3.26 (0.10) / 108 / 6.4 (0.2) / CNS: scattered cells in CB and VG
prospero-GAL4 / [2] / 3.19 (0.08) / 94 / 6.8 (0.1) / CNS: ganglion cells in CB and VG
P{GawB}c768 / [3] / 3.16 (0.11) / 88 / 6.7 (0.2) / CNS: widely in CB and VG
elav-GAL4 / [4] / 3.11 (0.09) / 78 / 6.7 (0.2) / CNS: pan-neuronal
P{GawB}c23 / B2703 / 3.06 (0.07) / 69 / 6.8 (0.2) / CNS: MB; few cells in CB and VG
72Y / [5] / 3.01 (0.09) / 59 / ND / CNS: MB, widely in CB and VG
P{GAL4-Hsp70.PB}31-1 / B1822 / 3.00 (0.11) / 57 / ND / CNS: pan-neuronal
P{GawB}OK307 / B6488 / 2.99 (0.12) / 55 / ND / CNS: giant descending neuron circuit; RG
GAL4-386Y / [6] / 2.99 (0.10) / 55 / 7.1 (0.2) / CNS: peptidergic neurons
P{GawB}drl[PGAL8] / B4669 / 2.97 (0.09) / 51 / 6.9 (0.1) / CNS: MB, OPC, IPC; few cells in CB/VG
156Y / [3] / 2.95 (0.12) / 47 / 7.0 (0.1) / PNS, CNS: scattered cells in CB and VG
P{GawB}C1003 / B2735 / 2.92 (0.06) / 41 / ND / CNS: brain hemispheres
Feb 211 / [7] / 2.72 (0.13) / NR / ND / CNS: PG innervating NSNs
amnc651 / [8] / 2.71 (0.11) / NR / ND / CNS: amnesiac expressing neurons
c929 / [9] / 2.71 (0.09) / NR / ND / CNS: peptidergic neurons
Ddc-GAL4HL-8 / [10] / 2.71 (0.08) / NR / ND / CNS: dopaminergic/serotonergic neurons
Mai 301 / [7] / 2.71 (0.06) / NR / ND / CNS: 6 NSNs in PI
Mai 369 / [7] / 2.71 (0.09) / NR / ND / CNS: 7 lateral NSNs innervating the CC
P{lsp2-GAL4.H}3 / B6357 / 2.70 (0.09) / NR / 7.4 (0.2) / Fat body
repo-GAL4 / [11] / 2.70 (0.07) / NR / 7.3 (0.1) / CNS glial cells
phm-GAL4 / [13} / 2.70 (0.08) / NR / ND / RG: strong in PG
c302 / [5] / 2.69 (0.09) / NR / ND / Fore-, mid- and hind-gut
P{Cg-GAL4.A}2 / B7011 / 2.67 (0.06) / NR / ND / Fat body, lymph gland
P{GawB}V55 / B2683 / 2.66 (0.08) / NR / ND / Epidermis, PNS, gut, SG
P{GawB}5015 / B2721 / 2.64 (0.06) / NR / ND / Lymph gland
201Y / [5] / 2.64 (0.04) / NR / 7.5 (0.1) / Epidermis, CNS, MB, SG
dILP2-GAL4 / [12] / 2.64 (0.09) / NR / ND / Insulin producing cells in CNS
P{GawB}c747 / [3] / 2.64 (0.09) / NR / 7.4 (0.2) / CNS, MB, VG, NBs in CB
P{GAL4-Eh.2.4}C21 / B6301 / 2.61 (0.09) / NR / ND / Eclosion hormone producing neurons
P{Cha-GAL4.7.4}19B / B6798 / 2.61 (0.05) / NR / 7.3 (0.1) / CNS: cholinergic neurons
238Y / [5] / 2.61 (0.09) / NR / ND / SG, MB, imaginal discs
PO206-GAL4 / [13] / 2.61 (0.08) / NR / ND / RG: strong in CA, moderate in PG
Aug 21 / [7] / 2.60 (0.08) / NR / 7.5 (0.1) / RG: CA
P{GawB}167Y / [3] / 2.57 (0.10) / NR / ND / NBs in CB and VG; Bolwig's nerve
Table S1. Rescue of NF1 pupal size and adult wing cell size defects upon driving UAS-NF1 with the indicated GAL4 drivers. Each cross was set up using an equal number of females per vial. At least 15 female pupae of each genotype were measured for each GAL4 driver. Pupal size is shown as the mean length, with standard deviation in parentheses. Percentage rescue is given for those GAL4 drivers that significantly (p<0.0001) rescued pupal size. NR - no significant rescue. a Calculated by counting the number of wing hairs in a 0.01 mm2 area between the L2 and L3 veins. Standard deviation in parentheses (n=6). b Expression not in all cases limited to the indicated tissues. Determined by crossing GAL4 drivers to UAS-GFP and dissecting third instar larvae, or from published data. Other Gal4 drivers that rescue significantly and express in parts of the CNS include c698a, c81 [3], and T80 (B1878). Gal4 drivers that express in parts of the CNS but do not significantly rescue include: Okt 30, Mai 179, Kurs 21, Kurs 58 [7], P{GawB}389 (B1874), Nrv2 (B6800), 82Y [14], c701b, 91Y, and c315a [3], Eip71CD (B6871), and ddc-GAL44-36 [10]. Additional mushroom body drivers that do not rescue include OK107 (B854), c217, c284a, c445 and c681 [3], c35, c772, 30Y [5] and MB247 [15]. Numbers in brackets are Bloomington stock numbers. Abbreviations: CA: Corpora allata; CB: central brain; CC: corpora cardiaca; CNS: central nervous system; IPC: inner proliferative center; MB: mushroom bodies; NB: neuroblast; NSN: neurosecretory neuron; OPC: outer proliferative center; PG: prothoracic gland; PI: pars intercerebralis; PNS: peripheral nervous system; RG: ring gland SG: salivary glands; VG: ventral ganglion.
References to Table S1
1. Ito, K., Awano, W., Suzuki, K., Hiromi, Y., and Yamamoto, D. 1997. The Drosophila mushroom body is a quadruple structure of clonal units each of which contains a virtually identical set of neurones and glial cells. Development 124: 761-771.
2. Ohshiro, T., Yagami, T., Zhang, C., and Matsuzaki, F. 2000. Role of cortical tumour-suppressor proteins in asymmetric division of Drosophila neuroblast. Nature 408: 593-596.
3. Manseau, L., Baradaran, A., Brower, D., Budhu, A., Elefant, F., Phan, H., Philp, A.V., Yang, M., Glover, D., Kaiser, K., Palter, K., and Selleck, S. 1997. GAL4 enhancer traps expressed in the embryo, larval brain, imaginal discs, and ovary of Drosophila. Dev Dyn 209: 310-322.
4. Robinow, S. and White, K. 1988. The locus elav of Drosophila melanogaster is expressed in neurons at all developmental stages. Dev Biol 126: 294-303.
5. Yang, M.Y., Armstrong, J.D., Vilinsky, I., Strausfeld, N.J., and Kaiser, K. 1995. Subdivision of the Drosophila mushroom bodies by enhancer-trap expression patterns. Neuron 15: 45-54.
6. Taghert, P.H., Hewes, R.S., Park, J.H., O'Brien, M.A., Han, M., and Peck, M.E. 2001. Multiple amidated neuropeptides are required for normal circadian locomotor rhythms in Drosophila. J Neurosci 21: 6673-6686.
7. Siegmund, T. and Korge, G. 2001. Innervation of the ring gland of Drosophila melanogaster. J Comp Neurol 431: 481-491.
8. Waddell, S., Armstrong, J.D., Kitamoto, T., Kaiser, K., and Quinn, W.G. 2000. The amnesiac gene product is expressed in two neurons in the Drosophila brain that are critical for memory. Cell 103: 805-813.
9. Hewes, R.S., Schaefer, A.M., and Taghert, P.H. 2000. The cryptocephal gene (ATF4) encodes multiple basic-leucine zipper proteins controlling molting and metamorphosis in Drosophila. Genetics 155: 1711-1723.
10. Li, H., Chaney, S., Roberts, I.J., Forte, M., and Hirsh, J. 2000. Ectopic G-protein expression in dopamine and serotonin neurons blocks cocaine sensitization in Drosophila melanogaster. Curr Biol 10: 211-214.
11. Sepp, K.J., Schulte, J., and Auld, V.J. 2001. Peripheral glia direct axon guidance across the CNS/PNS transition zone. Dev Biol 238: 47-63.
12. Rulifson, E.J., Kim, S.K., and Nusse, R. 2002. Ablation of insulin-producing neurons in flies: growth and diabetic phenotypes. Science 296: 1118-1120.
13. Warren, J.T., Petryk, A., Marques, G., Parvy, J.P., Shinoda, T., Itoyama, K., Kobayashi, J., Jarcho, M., Li, Y., O'Connor, M.B., Dauphin-Villemant, C., and Gilbert, L.I. 2004. Phantom encodes the 25-hydroxylase of Drosophila melanogaster and Bombyx mori: a P450 enzyme critical in ecdysone biosynthesis. Insect Biochem Mol Biol 34: 991-1010.
14. Armstrong, J.D., de Belle, J.S., Wang, Z., and Kaiser, K. 1998. Metamorphosis of the mushroom bodies; large-scale rearrangements of the neural substrates for associative learning and memory in Drosophila. Learn Mem 5: 102-114.
15. Schulz, R.A., Chromey, C., Lu, M.F., Zhao, B., and Olson, E.N. 1996. Expression of the D-MEF2 transcription in the Drosophila brain suggests a role in neuronal cell differentiation. Oncogene 12: 1827-1831.
Genotype: / Pupal length(mm) / Wing area
(mm2) / Cell density
(x 10-3/mm2)
w1118 / 3.26 (0.08) / 1.78 (0.05) / 5.1 (0.1)
NF1E2 / 2.73 (0.09) / 1.51 (0.08) / 6.0 (0.1)
Ras2-Gal4(12)/+; UAS-Ras1V12/+ / 2.75* (0.1) / 1.44 (0.05) / 6.5 (0.2)
Ras2-Gal4(12)/UAS-Ras2V14 / 2.72* (0.08) / 1.45 (0.09) / 6.6 (0.2)
Ras2-Gal4(12)/+; UAS-Rafgof/+ / 2.69* (0.1) / 1.43 (0.03) / 6.7 (0.2)
UAS-Ras1V12/+ / 3.34 (0.08) / ND / ND
UAS-Ras2V14/+ / 3.20 (0.07) / ND / ND
UAS-Rafgof/+ / 3.27 (0.09) / ND / ND
Table S2. Expression of constitutively active Ras1V12, Ras2V14, or Rafgof mutants in Ras2-expressing cells phenocopies the NF1 size defect. All crosses and cultures were maintained at 16˚ C. Females of each genotype were measured for each of the criteria. Pupal size is shown as the mean length with standard deviation in parentheses (n=15). Measurements of wing areas were made using NIH Image 1.62 (n=5). Cell density was calculated by counting the number of wing hairs in a 0.01 mm2 area of the wing either between L2 and L3 (n=5). Transgenic lines used in this experiment were: P[UAS-Ras1V12 ] (Go, M. and Artavanis-Tsakonas, S., unpublished), P[UAS-Ras2V14] (Brand and Perrimon, 1993) and P[UAS-Rafgof ] (F179 from Bloomington Stock Center). Asterisks indicate statistical significance (p<0.0001 for w1118 vs Ras2-Gal4(12) driving UAS-Ras1v12, UAS-Ras2v14, or UAS-Rafgof)
Legends to Supplemental Figures:
Figure S1. NF1 regulates Drosophila growth non cell-autonomously. A-D. Confocal micrographs of anti-NF1 stained 3rd instar CNS from the following genotypes: (A) w1118 isogenic control, (B) UAS-NF1, NF1E1/NF1E2, (C) en-GAL4/+; UAS-NF1, NF1E1/NF1E2, and (D) elav-GAL4, NF1E2/UAS-NF1, NF1E1. The scale bar represents 200 mm. E-H. Anti-NF1 stained 3rd instar larval wing discs from the same genotypes. Scale bar represents 100 mm. I-L. Wings from adult female flies representing the above genotypes. Scale bar is 500 mm. Percentage indicates wing area size relative to wild-type.
Figure S2. NF1 expression in 3rd instar CNS overlaps substantially with p-ERK staining, and with Ras1 or Ras2 expression. Confocal images of wild-type 3rd instar larval CNS showing NF1 staining in red (A), p-ERK staining in green (B), or a merge of both images (C). Panels D-F show higher magnification images of cells in the central brain region. p-ERK staining in NF1 mutant CNS is moderately more intense, but no obvious changes in the staining pattern are apparent (not shown). The rabbit p-ERK antiserum used is #9101 from Cell Signaling. Confocal images of the central brain (G-L) or thoracic ganglion (M-O) from the CNS of third instar larvae driving UASGFP expression (shown in green) with either Ras1-GAL4 (G-I) or Ras2-GAL2(41). Dissected larval CNS was fixed and stained with anti-NF1, shown in red. Scale bars represent 50 mm.
Figure S3. Mutations in canonical Ras pathway components do not modify NF1 pupal size or larval p-ERK phenotypes. A. Pupal size of the indicated genotypes. B. p-ERK levels observed in dissected 3rd instar CNS of the indicated genotypes. See Materials and Methods for a list of mutants used.