Abstract
Drosophila adult leg development provides an ideal model system for characterizing the molecular mechanisms of hormone-triggered morphogenesis. A pulse of the steroid hormone ecdysone at the onset of metamorphosis triggers the rapid transformation of a flat leg imaginal disc into an immature adult leg, largely through coordinated changes in cell shape. In an effort to identify links between the ecdysone signal and the cytoskeletal changes required for leg morphogenesis, we performed two large-scale genetic screens for dominant enhancers of the malformed leg phenotype associated with a mutation in the ecdysone-inducible broad early gene (br1). From a screen of >750 independent deficiency and candidate mutation stocks, we identified 17 loci on the autosomes that interact strongly with br1. In a complementary screen of approximately 112,000 F1 progeny of EMS-treated br1 animals, we recovered 26 mutations that enhance the br1 leg phenotype [E(br) mutations]. Rho1, stubbloid, blistered (DSRF), and cytoplasmic Tropomyosin were identified from these screens as br1-interacting genes. Our findings suggest that ecdysone exerts its effects on leg morphogenesis through a Rho1 signaling cascade, a proposal that is supported by genetic interaction studies between the E(br) mutations and mutations in the Rho1 signaling pathway. In addition, several E(br) mutations produce unexpected defects in midembryonic morphogenetic movements. Coupled with recent evidence implicating ecdysone signaling in these embryonic morphogenetic events, our results suggest that a common ecdysone-dependent, Rho1-mediated regulatory pathway controls morphogenesis during the two major transitions in the life cycle, embryogenesis and metamorphosis.
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- Affolter M., Montagne J., Walldorf U., Groppe J., Kloter U., LaRosa M., Gehring W. J. The Drosophila SRF homolog is expressed in a subset of tracheal cells and maps within a genomic region required for tracheal development. Development. 1994 Apr;120(4):743–753. doi: 10.1242/dev.120.4.743. [DOI] [PubMed] [Google Scholar]
- Andres A. J., Thummel C. S. Methods for quantitative analysis of transcription in larvae and prepupae. Methods Cell Biol. 1994;44:565–573. doi: 10.1016/s0091-679x(08)60932-2. [DOI] [PubMed] [Google Scholar]
- Appel L. F., Prout M., Abu-Shumays R., Hammonds A., Garbe J. C., Fristrom D., Fristrom J. The Drosophila Stubble-stubbloid gene encodes an apparent transmembrane serine protease required for epithelial morphogenesis. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):4937–4941. doi: 10.1073/pnas.90.11.4937. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ashburner M., Chihara C., Meltzer P., Richards G. Temporal control of puffing activity in polytene chromosomes. Cold Spring Harb Symp Quant Biol. 1974;38:655–662. doi: 10.1101/sqb.1974.038.01.070. [DOI] [PubMed] [Google Scholar]
- Baker B. S., Hoff G., Kaufman T. C., Wolfner M. F., Hazelrigg T. The doublesex locus of Drosophila melanogaster and its flanking regions: a cytogenetic analysis. Genetics. 1991 Jan;127(1):125–138. doi: 10.1093/genetics/127.1.125. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bayer C. A., Holley B., Fristrom J. W. A switch in broad-complex zinc-finger isoform expression is regulated posttranscriptionally during the metamorphosis of Drosophila imaginal discs. Dev Biol. 1996 Jul 10;177(1):1–14. doi: 10.1006/dbio.1996.0140. [DOI] [PubMed] [Google Scholar]
- Bayer Cynthia A., Halsell Susan R., Fristrom James W., Kiehart Daniel P., von Kalm Laurence. Genetic interactions between the RhoA and Stubble-stubbloid loci suggest a role for a type II transmembrane serine protease in intracellular signaling during Drosophila imaginal disc morphogenesis. Genetics. 2003 Nov;165(3):1417–1432. doi: 10.1093/genetics/165.3.1417. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Belyaeva E. S., Aizenzon M. G., Semeshin V. F., Kiss I. I., Koczka K., Baritcheva E. M., Gorelova T. D., Zhimulev I. F. Cytogenetic analysis of the 2B3-4--2B11 region of the X-chromosome of Drosophila melanogaster. I. Cytology of the region and mutant complementation groups. Chromosoma. 1980;81(2):281–306. doi: 10.1007/BF00285954. [DOI] [PubMed] [Google Scholar]
- Birr C. A., Fristrom D., King D. S., Fristrom J. W. Ecdysone-dependent proteolysis of an apical surface glycoprotein may play a role in imaginal disc morphogenesis in Drosophila. Development. 1990 Sep;110(1):239–248. doi: 10.1242/dev.110.1.239. [DOI] [PubMed] [Google Scholar]
- Bocchinfuso W. P., Lindzey J. K., Hewitt S. C., Clark J. A., Myers P. H., Cooper R., Korach K. S. Induction of mammary gland development in estrogen receptor-alpha knockout mice. Endocrinology. 2000 Aug;141(8):2982–2994. doi: 10.1210/endo.141.8.7609. [DOI] [PubMed] [Google Scholar]
- Brouns M. R., Matheson S. F., Hu K. Q., Delalle I., Caviness V. S., Silver J., Bronson R. T., Settleman J. The adhesion signaling molecule p190 RhoGAP is required for morphogenetic processes in neural development. Development. 2000 Nov;127(22):4891–4903. doi: 10.1242/dev.127.22.4891. [DOI] [PubMed] [Google Scholar]
- Chávez V. M., Marqués G., Delbecque J. P., Kobayashi K., Hollingsworth M., Burr J., Natzle J. E., O'Connor M. B. The Drosophila disembodied gene controls late embryonic morphogenesis and codes for a cytochrome P450 enzyme that regulates embryonic ecdysone levels. Development. 2000 Oct;127(19):4115–4126. doi: 10.1242/dev.127.19.4115. [DOI] [PubMed] [Google Scholar]
- Condic M. L., Fristrom D., Fristrom J. W. Apical cell shape changes during Drosophila imaginal leg disc elongation: a novel morphogenetic mechanism. Development. 1991 Jan;111(1):23–33. doi: 10.1242/dev.111.1.23. [DOI] [PubMed] [Google Scholar]
- DiBello P. R., Withers D. A., Bayer C. A., Fristrom J. W., Guild G. M. The Drosophila Broad-Complex encodes a family of related proteins containing zinc fingers. Genetics. 1991 Oct;129(2):385–397. doi: 10.1093/genetics/129.2.385. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Edwards K. A., Kiehart D. P. Drosophila nonmuscle myosin II has multiple essential roles in imaginal disc and egg chamber morphogenesis. Development. 1996 May;122(5):1499–1511. doi: 10.1242/dev.122.5.1499. [DOI] [PubMed] [Google Scholar]
- Erdélyi M., Michon A. M., Guichet A., Glotzer J. B., Ephrussi A. Requirement for Drosophila cytoplasmic tropomyosin in oskar mRNA localization. Nature. 1995 Oct 12;377(6549):524–527. doi: 10.1038/377524a0. [DOI] [PubMed] [Google Scholar]
- Fehon R. G., Dawson I. A., Artavanis-Tsakonas S. A Drosophila homologue of membrane-skeleton protein 4.1 is associated with septate junctions and is encoded by the coracle gene. Development. 1994 Mar;120(3):545–557. doi: 10.1242/dev.120.3.545. [DOI] [PubMed] [Google Scholar]
- Fehon R. G., Johansen K., Rebay I., Artavanis-Tsakonas S. Complex cellular and subcellular regulation of notch expression during embryonic and imaginal development of Drosophila: implications for notch function. J Cell Biol. 1991 May;113(3):657–669. doi: 10.1083/jcb.113.3.657. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fessler L. I., Condic M. L., Nelson R. E., Fessler J. H., Fristrom J. W. Site-specific cleavage of basement membrane collagen IV during Drosophila metamorphosis. Development. 1993 Mar;117(3):1061–1069. doi: 10.1242/dev.117.3.1061. [DOI] [PubMed] [Google Scholar]
- Geneste Olivier, Copeland John W., Treisman Richard. LIM kinase and Diaphanous cooperate to regulate serum response factor and actin dynamics. J Cell Biol. 2002 May 28;157(5):831–838. doi: 10.1083/jcb.200203126. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gineitis D., Treisman R. Differential usage of signal transduction pathways defines two types of serum response factor target gene. J Biol Chem. 2001 May 7;276(27):24531–24539. doi: 10.1074/jbc.M102678200. [DOI] [PubMed] [Google Scholar]
- Gotwals P. J., Fristrom J. W. Three neighboring genes interact with the Broad-Complex and the Stubble-stubbloid locus to affect imaginal disc morphogenesis in Drosophila. Genetics. 1991 Apr;127(4):747–759. doi: 10.1093/genetics/127.4.747. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Graves B. J., Schubiger G. Cell cycle changes during growth and differentiation of imaginal leg discs in Drosophila melanogaster. Dev Biol. 1982 Sep;93(1):104–110. doi: 10.1016/0012-1606(82)90243-3. [DOI] [PubMed] [Google Scholar]
- Guillemin K., Groppe J., Ducker K., Treisman R., Hafen E., Affolter M., Krasnow M. A. The pruned gene encodes the Drosophila serum response factor and regulates cytoplasmic outgrowth during terminal branching of the tracheal system. Development. 1996 May;122(5):1353–1362. doi: 10.1242/dev.122.5.1353. [DOI] [PubMed] [Google Scholar]
- Halsell S. R., Chu B. I., Kiehart D. P. Genetic analysis demonstrates a direct link between rho signaling and nonmuscle myosin function during Drosophila morphogenesis. Genetics. 2000 Jul;155(3):1253–1265. doi: 10.1093/genetics/155.3.1253. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Halsell S. R., Kiehart D. P. Second-site noncomplementation identifies genomic regions required for Drosophila nonmuscle myosin function during morphogenesis. Genetics. 1998 Apr;148(4):1845–1863. doi: 10.1093/genetics/148.4.1845. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Karim F. D., Thummel C. S. Ecdysone coordinates the timing and amounts of E74A and E74B transcription in Drosophila. Genes Dev. 1991 Jun;5(6):1067–1079. doi: 10.1101/gad.5.6.1067. [DOI] [PubMed] [Google Scholar]
- Kiss I., Beaton A. H., Tardiff J., Fristrom D., Fristrom J. W. Interactions and developmental effects of mutations in the Broad-Complex of Drosophila melanogaster. Genetics. 1988 Feb;118(2):247–259. doi: 10.1093/genetics/118.2.247. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kraminsky G. P., Clark W. C., Estelle M. A., Gietz R. D., Sage B. A., O'Connor J. D., Hodgetts R. B. Induction of translatable mRNA for dopa decarboxylase in Drosophila: an early response to ecdysterone. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4175–4179. doi: 10.1073/pnas.77.7.4175. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mack C. P., Somlyo A. V., Hautmann M., Somlyo A. P., Owens G. K. Smooth muscle differentiation marker gene expression is regulated by RhoA-mediated actin polymerization. J Biol Chem. 2001 Jan 5;276(1):341–347. doi: 10.1074/jbc.M005505200. [DOI] [PubMed] [Google Scholar]
- Magie C. R., Meyer M. R., Gorsuch M. S., Parkhurst S. M. Mutations in the Rho1 small GTPase disrupt morphogenesis and segmentation during early Drosophila development. Development. 1999 Dec;126(23):5353–5364. doi: 10.1242/dev.126.23.5353. [DOI] [PubMed] [Google Scholar]
- Mandaron P. Développement in vitro des disques imaginaux de la drosophile. Aspects morphologiques et histologiques. Dev Biol. 1970 Jun;22(2):298–320. doi: 10.1016/0012-1606(70)90156-9. [DOI] [PubMed] [Google Scholar]
- Miralles Francesc, Posern Guido, Zaromytidou Alexia-Ileana, Treisman Richard. Actin dynamics control SRF activity by regulation of its coactivator MAL. Cell. 2003 May 2;113(3):329–342. doi: 10.1016/s0092-8674(03)00278-2. [DOI] [PubMed] [Google Scholar]
- Mizuno Tomoaki, Tsutsui Kyoko, Nishida Yasuyoshi. Drosophila myosin phosphatase and its role in dorsal closure. Development. 2002 Mar;129(5):1215–1223. doi: 10.1242/dev.129.5.1215. [DOI] [PubMed] [Google Scholar]
- Montagne J., Groppe J., Guillemin K., Krasnow M. A., Gehring W. J., Affolter M. The Drosophila Serum Response Factor gene is required for the formation of intervein tissue of the wing and is allelic to blistered. Development. 1996 Sep;122(9):2589–2597. doi: 10.1242/dev.122.9.2589. [DOI] [PubMed] [Google Scholar]
- Moore J. T., Fristrom D., Hammonds A. S., Fristrom J. W. Characterization of IMP-E3, a gene active during imaginal disc morphogenesis in Drosophila melanogaster. Dev Genet. 1990;11(4):299–309. doi: 10.1002/dvg.1020110409. [DOI] [PubMed] [Google Scholar]
- Nakamura M., Nishida W., Mori S., Hiwada K., Hayashi K., Sobue K. Transcriptional activation of beta-tropomyosin mediated by serum response factor and a novel Barx homologue, Barx1b, in smooth muscle cells. J Biol Chem. 2001 May 25;276(21):18313–18320. doi: 10.1074/jbc.m101127200. [DOI] [PubMed] [Google Scholar]
- Natzle J. E., Hammonds A. S., Fristrom J. W. Isolation of genes active during hormone-induced morphogenesis in Drosophila imaginal discs. J Biol Chem. 1986 Apr 25;261(12):5575–5583. [PubMed] [Google Scholar]
- Pino-Heiss S., Schubiger G. Extracellular protease production by Drosophila imaginal discs. Dev Biol. 1989 Apr;132(2):282–291. doi: 10.1016/0012-1606(89)90225-x. [DOI] [PubMed] [Google Scholar]
- Riddiford L. M., Cherbas P., Truman J. W. Ecdysone receptors and their biological actions. Vitam Horm. 2000;60:1–73. doi: 10.1016/s0083-6729(00)60016-x. [DOI] [PubMed] [Google Scholar]
- Settleman J. Rac 'n Rho: the music that shapes a developing embryo. Dev Cell. 2001 Sep;1(3):321–331. doi: 10.1016/s1534-5807(01)00053-3. [DOI] [PubMed] [Google Scholar]
- Sotiropoulos A., Gineitis D., Copeland J., Treisman R. Signal-regulated activation of serum response factor is mediated by changes in actin dynamics. Cell. 1999 Jul 23;98(2):159–169. doi: 10.1016/s0092-8674(00)81011-9. [DOI] [PubMed] [Google Scholar]
- Symons M., Settleman J. Rho family GTPases: more than simple switches. Trends Cell Biol. 2000 Oct;10(10):415–419. doi: 10.1016/s0962-8924(00)01832-8. [DOI] [PubMed] [Google Scholar]
- Tetzlaff M. T., Jäckle H., Pankratz M. J. Lack of Drosophila cytoskeletal tropomyosin affects head morphogenesis and the accumulation of oskar mRNA required for germ cell formation. EMBO J. 1996 Mar 15;15(6):1247–1254. [PMC free article] [PubMed] [Google Scholar]
- Thummel C. S. Flies on steroids--Drosophila metamorphosis and the mechanisms of steroid hormone action. Trends Genet. 1996 Aug;12(8):306–310. doi: 10.1016/0168-9525(96)10032-9. [DOI] [PubMed] [Google Scholar]
- Van Aelst L., D'Souza-Schorey C. Rho GTPases and signaling networks. Genes Dev. 1997 Sep 15;11(18):2295–2322. doi: 10.1101/gad.11.18.2295. [DOI] [PubMed] [Google Scholar]
- Ward Robert E., Reid Pamela, Bashirullah Arash, D'Avino Pier Paolo, Thummel Carl S. GFP in living animals reveals dynamic developmental responses to ecdysone during Drosophila metamorphosis. Dev Biol. 2003 Apr 15;256(2):389–402. doi: 10.1016/s0012-1606(02)00100-8. [DOI] [PubMed] [Google Scholar]
- Wei L., Roberts W., Wang L., Yamada M., Zhang S., Zhao Z., Rivkees S. A., Schwartz R. J., Imanaka-Yoshida K. Rho kinases play an obligatory role in vertebrate embryonic organogenesis. Development. 2001 Aug;128(15):2953–2962. doi: 10.1242/dev.128.15.2953. [DOI] [PubMed] [Google Scholar]
- Wissmann A., Ingles J., Mains P. E. The Caenorhabditis elegans mel-11 myosin phosphatase regulatory subunit affects tissue contraction in the somatic gonad and the embryonic epidermis and genetically interacts with the Rac signaling pathway. Dev Biol. 1999 May 1;209(1):111–127. doi: 10.1006/dbio.1999.9242. [DOI] [PubMed] [Google Scholar]
- Wissmann A., Ingles J., McGhee J. D., Mains P. E. Caenorhabditis elegans LET-502 is related to Rho-binding kinases and human myotonic dystrophy kinase and interacts genetically with a homolog of the regulatory subunit of smooth muscle myosin phosphatase to affect cell shape. Genes Dev. 1997 Feb 15;11(4):409–422. doi: 10.1101/gad.11.4.409. [DOI] [PubMed] [Google Scholar]
- von Kalm L., Fristrom D., Fristrom J. The making of a fly leg: a model for epithelial morphogenesis. Bioessays. 1995 Aug;17(8):693–702. doi: 10.1002/bies.950170806. [DOI] [PubMed] [Google Scholar]