Current Biology, Volume 30

Supplemental Information

Egfr Signaling Is a Major Regulator of Ecdysone

Biosynthesis in the Drosophila Prothoracic Gland

Josefa Cruz, David Martín, and Xavier Franch-Marro

Figure S1. Disruption of downstream members of the RTK signaling downregulates enzyme-encoding genes independently of cell survival and polyploidy of PG cells, related to Figure 1. (A) In all experiments phmGal4 and amnc651 Gal4 were used as specific PG drivers. Compared with the control (phmGal4), overexpression of rasDN, rafRNAi and ERKRNAi in the PG induces the arrested development of L3 larvae. Representative model of RTK signaling pathway showing the main components of the MAPK pathway. (B) PG image of 120h control, EgfrRNAi and pntRNAi larva under control of phm>CD8GFP. Nuclei are visualized by DAPI staining (in blue). Scale bar represents 100 μm. (C) Tunel staining of DNAse treated (positive control), EgfrRNAi and pntRNAi PG cells. Scale bar represents 50 μm. (D) Graph showing the number of cells of PGs in control, EgfrRNAi and pntRNAi animals. (E) Graph showing the C value of PG cells of EgfrRNAi and EgfrRNAi; Cyc-E larvae at 120 h AEL. (F) DAPI staining of PG cells nuclei of 120h control, EgfrRNAi and EgfrRNAi; Cyc-E larva under control of phm>. Lower panels show a high magnification of the corresponding nuclei of PG cells (outlined). Scale bars represents 50 μm in the upper panels and 10 μm (lower panels). (G) Percentages of pupariated control, EgfrRNAi, and EgfrRNAi; Cyc-E animals shown at indicated stages. Numbers of animals analysed are indicated in parenthesis. (H) Expression of ecdysone biosynthetic genes in control, EgfrRNAi and EgfrRNAi; Cyc-E larvae at 120 hours AEL measured using RT-PCR. Average values of three independent data sets are shown with standard errors. Asterisks indicate differences statistically significant at p≤0.05 (*), and p≤0.005 (**) (t-test).

Figure S2. Vesicle aggregation upon inactivation of Egfr signaling in the PG at 120h AEL, related to Figure 2. (A) PGs of wandering control, EgfrRNAi and pntRNAi larva co-overexpressing either Syt-GFP (phm22>Syt-GFP) or YPet-Atet (phm22>YPet-Atet). Magnified view of the PG cells at 120h shows a more diffused aggregation of small vesicle-like structures nearby the membrane (arrowheads). Note that in contrast, in EgfrRNAi and pntRNAi PG cells vesicles accumulate in bigger aggregates. Scale bar represent 100 μm in upper panels and 25 μm in magnified views. (B) Expression of a list of genes involved in vesicle-mediated release of ecdysone of dissected ring glands, at 120 hours AEL, in animals depleted of either Egfr or pnt in the PG. Relative expression level of Broad-Complex (Br-C), torso, synaptobrevin (Syb), Secretory 6 (Sec6), CG30054, Phospholipase C at 21C (Plc21C), Inositol 1,4,5,-tris-phosphate receptor (IP3R), Staccato (Stac), Rab3, Rab3 interacting molecule (Rim), Syt and Atet was measured by qRT-PCR. Dashed line represents control relative expression for each gene. Br-C and torso are used as positive and negative controls respectively.

Figure S3. Expression of the different members of Egfr signalling in PG cells, related to Figure 3. (A-B) Expression of of grk and krn is undetected by RNA in situ hybridization in dissected PGs (C-D) mRNA level of grk and krn of control larvae at 72, 96 and 120 hours AEL, measured by qRT-PCR. (E) Egfrlacz expression visualized by ßGal staining. (F-G) PntP2 expression in the PG detected by either PntP2 antibody (F) or ßGal staining (G), respectively. In all panels DAPI (in blue) was used to visualize the cell nucleus. Scale bars represent 50 μm.

Figure S4. Progressive activation of MAPK cascade induces vesicle trafficking and endoreplication in PG cells, related to Figure 4. (A) Expression of Ptth and torso in animals with inactivated Egfr signaling in the PG. mRNA level of Ptth and torso in control and animals depleted of EgfrRNAi and pntRNAi specifically in the PG, measured by qRT-PCR at 72, 96 and 120 hours AEL. Average values of three independent data sets are shown with standard errors. t-test analysis reveals no statistically significant differences. (B) Vesicle aggregation upon inactivation of Egfr and Ptth/torso signaling in the PG at 120h AEL. PGs of wandering control, torsoRNAi and EgfrRNAi co-overexpressing YPet-Atet (phm22>YPet-Atet). Note that in torsoRNAi and EgfrRNAi PG cells vesicles accumulation in the cytoplasm correlates with the level of activation of MAPK shown in Figure 4. Scale bar represent 100 μm in upper panels and 25 μm in magnified views. (C) Gradual activation of MAPKs regulates nuclear size. Nuclear staining with DAPI (in blue) of control PG cells compared to PG cells overexpressing either torsoRNAi, EgfrRNAi or rasV12. Note that the nucleus size correlates with the level of activation of MAPK. Scale bar represent 100 μm.

Oligonucleotides DmSyt1F: 5’-AGTTGTTGGGATCGGCGTACAA-3’ Sigma-Aldrich N/A

DmSyt1R: 5’-TCACGGCCAAACTGTTGGAGTT-3’ Sigma-Aldrich N/A DmCG30054F: 5’-CAAGCGTGGATACATCAAGCT-3’ Sigma-Aldrich N/A

DmCG30054R: 5’-GGTAACGGTCTCGTAATCGAT-3’ Sigma-Aldrich N/A

DmRIMF: 5’-AAGAAATCGAACTCCACGTCG-3’ Sigma-Aldrich N/A

DmRIMR: 5’-TTCGCAGTTCCCGCGTTATAT-3’ Sigma-Aldrich N/A

DmRab3F: 5’-AAGGCTGAGTCGAGTCCTTTT-3’ Sigma-Aldrich N/A

DmRab3R: 5’-CCTGCTGTCACAAGAATATCCT-3’ Sigma-Aldrich N/A

DmIP3F: 5’-GATGTCTGCCACTTCTGCATCA-3’ Sigma-Aldrich N/A

DmIP3R: 5’-GCAATGTCCGCAGAATACGGAA-3’ Sigma-Aldrich N/A

DmPlc21CF: 5’-CAGAGATTTCAGCTTTGGTCAA-3’ Sigma-Aldrich N/A

DmPlc21CR: 5’-CTTCAATAATGTTGTCGCCTGT-3’ Sigma-Aldrich N/A

DmStacF: 5’-AAGAGATTGGATGTGCCAGACG-3’ Sigma-Aldrich N/A

DmStacR: 5’-TCGGAGCAAAGAGATTCTCCTA-3’ Sigma-Aldrich N/A

DmSybF: 5’-GGAGTGAGGTAAACACATGGAT-3’ Sigma-Aldrich N/A

DmSybR: 5’-TCTTCATCTCCCCTTCTCTCAT-3’ Sigma-Aldrich N/A

DmSec6F: 5’-TCTACGACGCTTTGGAAGTTGT-3’ Sigma-Aldrich N/A

DmSec6R: 5’-CTGATGGGCATTAAGCAGCTTA-3’ Sigma-Aldrich N/A

DmsadF: 5’-GTTCGTATCGTCCGCAAATCTCAT-3’ Sigma-Aldrich N/A

DmsadR: 5’-CAGTCGATTAAGTATGCGTCGGTT-3’ Sigma-Aldrich N/A

DmnvdF: 5’-AACTTGGGTATTGGTGGAAGCGTT-3’ Sigma-Aldrich N/A

DmnvdR: 5’-TCCACTTCCTGCGAAATCCACTTT-3’ Sigma-Aldrich N/A

DmspoF: 5’-CACACTCGCTGCATAGTGGTAAAT-3’ Sigma-Aldrich N/A

DmspoR: 5’-GTTGTAATTGAGACCAGTCGCACA-3’ Sigma-Aldrich N/A

DmtorF: 5’-CTCTCACGATAAGACGCTTCTCAA-3’ Sigma-Aldrich N/A

DmtorR: 5’-CATCTGGTAGTTTTCCTGCACCTT-3’ Sigma-Aldrich N/A

DmHr4F: 5’-CGTTTGCATGATCTGCGAGGACA-3’ Sigma-Aldrich N/A

DmHr4R: 5’-TTTGGTTATCTCGCAGGTGCCGT-3’ Sigma-Aldrich N/A

DmBrCcomF: 5’-CATCTGGCTCAGATACAGAACCT-3’ Sigma-Aldrich N/A

DmBrCRcomR:5’-CTTCAGCAGCTGGTTGTTGATGT-3’ Sigma-Aldrich N/A

DmshdF: 5’-CAAGCGGATATTTGTAGACTTGG-3’ Sigma-Aldrich N/A

DmshdR: 5’-AAGCCCACTGACTGCTGAAT-3’ Sigma-Aldrich N/A

DmdibF: 5’-CCAAAGAGTGTGCGGAACTTTGTT-3’ Sigma-Aldrich N/A

DmdibR: 5’-GCTCCAAAGGTAAGCAAACAGGTT-3’ Sigma-Aldrich N/A

DmphmF: 5’-CTGGTGATTCTCTACCTGTGCGA-3’ Sigma-Aldrich N/A

DmphmR: 5’-CTCGAAAATTCCACCATACCGCTT-3’ Sigma-Aldrich N/A

DmptthF: 5’-AATGCTCAGTGCCCTATTCGGATT-3’ Sigma-Aldrich N/A

DmptthR; 5’-GGAATGGACATCATTGCGTTTGGT-3’ Sigma-Aldrich N/A

DmHR3F: 5’-GAGGCTTTTCAATCTGAGCATGAA-3’ Sigma-Aldrich N/A

DmHR3R: 5’-CGATTCCATGTGCAAGATGGAAAT-3’ Sigma-Aldrich N/A

DmsroF: 5’-CGAATCGCTGCACATGAC-3’ Sigma-Aldrich N/A

DmsroR: 5’-TAGGCCCTGCAGCAGTTTAG-3’ Sigma-Aldrich N/A

DmEgfrF: 5’-TTATCCGTGGAGGAGGAGAAGTA-3’ Sigma-Aldrich N/A

DmEgfrR: 5’-AGGTGGCACCATAGGCATACTTT-3’ Sigma-Aldrich N/A

DmvnF: 5’-ATGAAGGTGGAGAAGGTGTACAA-3’ Sigma-Aldrich N/A

DmvnR: 5’-TTGATGGACTTTTCGTCCTTGAA-3’ Sigma-Aldrich N/A

DmspiF: 5’-CAAATGTCCGGAAACCTTCGAT-3’ Sigma-Aldrich N/A

DmspiR: 5’-GTCGATCTCCTTGTATTCGCAT-3’ Sigma-Aldrich N/A

DmrhoF: 5’-AGGCGAGCACATCTACATG-3’ Sigma-Aldrich N/A

DmrhoR: 5’-AATGATGGAGATCACTAGGATGAA-3’ Sigma-Aldrich N/A

DmRpL32F: 5’-CAAGAAGTTCCTGGTGCACAA-3’ Sigma-Aldrich N/A

DmRpL33R: 5’-AAACGCGGTTCTGCATGAG-3’ Sigma-Aldrich N/A

DmGrkF: 5’-CCAATGATGCAAATCCCATT-3’ Sigma-Aldrich N/A

DmGrkR: 5’-ACTTCTCCGAGAGCATACGC-3’ Sigma-Aldrich N/A

DmKrnF: 5’-GCCAGCCAAAGCCAAGAATTC-3’ Sigma-Aldrich N/A

DmKrnR: 5’-GCAAGCAGCAGCAGATCCTG-3’ Sigma-Aldrich N/A

Table S1. List of oligonucleotides used in this study.