- THIS ARTICLE
-
Abstract
- Full Text (PDF)
- Alert me when this article is cited
- Alert me if a correction is posted
- SERVICES
- Email this article to a friend
- Similar articles in this journal
- Similar articles in PubMed
- Alert me to new issues of the journal
- Download to citation manager
- Reprints & Permissions
- CITING ARTICLES
- Citing Articles via HighWire
- Citing Articles via Google Scholar
- GOOGLE SCHOLAR
- Articles by Ward, R. E.
- Articles by Thummel, C. S.
- Search for Related Content
- PUBMED
- PubMed Citation
- Articles by Ward, R. E.
- Articles by Thummel, C. S.
Genetic Modifier Screens in Drosophila Demonstrate a Role for Rho1 Signaling in Ecdysone-Triggered Imaginal Disc Morphogenesis
Robert E. Ward1,a, Janelle Evansa, and Carl S. Thummelaa Howard Hughes Medical Institute, Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, Utah 84112-5331
Corresponding author: Carl S. Thummel, University of Utah, 15 N. 2030 East, Rm. 5100, Salt Lake City, UT 84112-5331., carl.thummel{at}genetics.utah.edu (E-mail)
Communicating editor: T. SCHÜPBACH
 | ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
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 
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.
MORPHOGENETIC movements define the body plan of metazoan animals. Gastrulation, neural tube formation, limb development, and organogenesis all depend on precisely timed, coordinated cell shape changes and cell rearrangements. In certain developmental contexts, endocrine signals provide temporal cues and also aid in the proper coordination of these morphogenetic events. For example, estrogen is required for mammary epithelial growth and ductal morphogenesis (
Development of the adult leg in Drosophila provides an ideal model system for characterizing the molecular mechanisms of hormone-triggered morphogenesis. In Drosophila, the adult legs are derived from imaginal discs that are specified during embryogenesis and set aside as discrete clusters of diploid cells that undergo extensive proliferation and patterning during larval development. At the end of the third larval instar each of the six leg imaginal discs consists of a single-layered columnar epithelium that is covered and apposed by a squamous peripodial epithelium. Transformation of this disc epithelium into an immature adult leg is triggered by a pulse of 20-hydroxyecdysone (hereafter referred to as ecdysone), the steroid hormone that directs the major developmental transitions in the Drosophila life cycle (5 hr after puparium formation, bringing them to the outside of the puparium (
Ecdysone exerts its effects primarily at the level of gene regulation (18 hr after puparium formation (
A number of molecular, biochemical, and genetic approaches have been employed to characterize leg disc morphogenesis. These studies have revealed that elongation and eversion of the leg imaginal discs occur in the absence of cell proliferation, largely in response to changes in cell shape (
A genetic approach for investigating imaginal disc morphogenesis was employed by
In this study we expand upon the original Gotwals and Fristrom screen to identify links between the ecdysone signal and the cytoskeletal machinery that drives leg morphogenesis. Two independent screens were undertaken for dominant enhancers of the malformed leg phenotype associated with the br1 mutation, using either a collection of autosomal chromosomal deficiencies or random methanesulfonic acid ethyl ester (EMS)-generated mutants. From these screens we identified 17 loci on the autosomes that interact with br1 and we isolated 26 EMS-induced br1-interacting mutations. Included in this collection of br1-interacting genes are those encoding the small GTPase Rho1, the Drosophila serum response factor (SRF) transcription factor Blistered (Bs), a cytoplasmic isoform of Tropomyosin1 (cTm), and the Sb/sbd protease. These results imply an important role for Rho1 signaling in leg disc morphogenesis, a notion that we support by genetic interaction studies between the EMS-generated mutations and previously characterized mutations in the Rho1 signaling pathway. In addition, we observe defects in midembryonic morphogenetic movements in animals bearing some of the EMS-induced mutations, suggesting that common regulatory mechanisms drive morphogenesis at different stages in the life cycle.
| MATERIALS AND METHODS |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Drosophila stocks:
All Drosophila stocks were maintained on corn meal/yeast/molasses/agar media in a room maintained at a constant temperature of 21°. The deficiency and P-element-insertion stocks used in this study were obtained from the Bloomington Drosophila Stock Center at Indiana University (Bloomington, IN). The zipE(br), RhoJ3.8, and RhoE3.10 stocks were obtained from S. Halsell (James Madison University;
EMS mutagenesis and screening:
Twenty cohorts, each consisting of 30 3- to 5-day-old br1 males, were treated with 25 mM EMS. Each cohort was mated to 30 br1 virgin females. Mutagenized males were subsequently mated to a second set of br1 virgin females to produce two broods of progeny. All progeny were maintained in bottles at 21°. In the F1 generation, all flies showing malformed legs were backcrossed to br1 males or females as appropriate. In the F2 and subsequent generations, sibling flies showing malformed legs were mated inter se in an effort to remove unlinked second-site mutations by free recombination. Starting at the F5 generation, inter se crosses producing 10% or more malformed progeny were mapped and balanced using the mapping stocks br1;Sco/Cyo and br1;T(2;3)apXa/TM6B. The br1 mutation was subsequently outcrossed leaving balanced Enhancer of br [E(br)] stocks. Complementation tests were conducted on E(br) stocks that mapped to the same chromosome.
Deficiency screen:
Dominant genetic interaction tests with br1 were performed by mating five to seven br1 virgin females to five to seven deficiency- or specific mutation-bearing heterozygous males in vials containing standard Drosophila medium. After 3 days the adults were transferred to fresh vials and, subsequently, to a third vial after two additional days. Newly eclosing F1 flies were separated by genotype and examined for malformed legs each day for a total of 10 days. Second-site noncomplementation (SSNC) tests with br5 were performed in a similar manner, except that y br5/Binsn females were used and the crosses were maintained in an incubator at 25°.
EMS screen:
Dominant genetic interaction tests between br1 and the E(br) mutations were performed at 21° as described above, using males of the genotype br1/Y;E(br)/CyO or br1/Y;E(br)/TM6B. Two vials were scored for each E(br) line in the experiment reported in Table 3. SSNC tests between br5 and the E(br) mutations were performed at 25° as described above using y br5/Binsn virgin females and w1118/Y;E(br)/CyO or w1118/Y;E(br)/TM6B males. SSNC tests between E(br) mutations and Rho1 pathway mutations were performed by mating five to seven w1118;E(br)/CyO or w1118;E(br)/TM6B virgin females to five to seven heterozygous mutant males. The crosses were maintained at 21° and three vials for each cross were examined in the manner described above. In some cases the reciprocal cross was also performed.
|
|
|
Complementation tests were performed by mating five to seven w1118;E(br)/CyO or w1118;E(br)/TM6B virgin females to five to seven heterozygous mutant males. Crosses were maintained in an incubator at 25°. The adults were transferred to fresh vials after 3 days. Newly eclosing F1 flies were separated into genotypic classes and counted each day for a total of 7 days. In some cases the reciprocal cross was also performed.
Meiotic mapping of the E(br) mutations was performed by mating br1;E(br)/CyO or br1;E(br)/TM6B virgin females to br1/Y;b pr c px sp or br1/Y;ru th st ri roe p e ca males, respectively. In the F1 generation, virgin females of the genotypes br1;E(br)/b pr c px sp or br1;E(br)/ru th st ri roe p e ca were mated to br1/Y;b pr c px sp or br1/Y;ru th st ri roe p e ca males, respectively. In the F2 generation, all animals bearing malformed legs [and therefore likely containing the E(br) mutation] were examined for the presence of recessive markers. Recombination distances were calculated between the E(br) mutation and each recessive marker, and the map position of the E(br) mutation was determined using values from the two or three closest recessive markers. From 150 to 400 informative recombination events were scored for each E(br) mutation.
Lethal-phase analysis:
Embryonic lethality was determined by collecting 0- to 2-hr embryos from E(br)/Cyo, P{w+, ActGFP} or E(br)/TM6B, P{w+, UbiGFP} stocks. The embryos were aged at 25° for 15 hr and dechorionated in 50% bleach, and homozygous E(br) embryos were identified and separated on the basis of absence of green fluorescent protein (GFP) expression. The homozygous mutant embryos were allowed to develop at 25° until 48 hr after egg laying, at which point the dead embryos were counted and mounted in Hoyer's medium (
Embryonic and adult specimen preparations:
The devitellinized embryonic cuticles shown in Fig 4 were prepared by collecting unhatched embryos from E(br)/CyO or E(br)/TM6B stocks 48 hr after egg laying at 25°. The embryos were dechorionated in 50% bleach and devitellinized in a 1:1 mixture of heptane: 90% MeOH, 50 mM EGTA, pH 8.0. The embryos were then mounted in One-Step mounting medium (2:1:1 glacial acetic acid:CMCP10:85% lactic acid) on microscope slides. Adult leg cuticles were prepared by dissecting leg pairs from the third thoracic segment of w1118, br1;E(br)/CyO or br1;E(br)/TM6B males in PBS, clearing them overnight in 10% KOH, and mounting them in Euporal on microscope slides. Images of the embryonic and adult leg cuticles were captured on either a Cool Snap or a SensiCamQE high performance digital CCD camera mounted on a Zeiss Axiophot microscope. Images of the dorsal thoraces from sbdE(br)228 and sbdE(br)228/+ animals were captured on a Cool Snap digital CCD camera mounted on a Leica stereomicroscope. Indirect immunofluorescence analysis of E(br)165 embryos was performed by collecting embryos from E(br)165/CyO parents at 25° for 2 hr, aging the embryos for an additional 19 hr, and then fixing and staining the embryos as described (
|
|
|
|
RNA isolation and Northern blot analysis:
Progeny from a cross of y br5/Binsn x Binsn/Y were staged on standard Drosophila media supplemented with 0.1% bromophenol blue as described in
| RESULTS |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Screening autosomal deficiency stocks for genetic interactions with br:
As a first step toward identifying loci that function with br to direct leg morphogenesis, we screened through an ordered collection of chromosomal deficiency stocks from the second and third chromosomes. We screened for deficiencies that exert a dominant increase in the penetrance of the malformed leg phenotype associated with the weakly hypomorphic br1 allele. In control experiments conducted prior to the screen, we found that br1 animals (hemizygous males and homozygous br1 females), maintained at 21°, display malformed legs at a low frequency of 0.4% (n = 3629). For the purpose of this screen, we arbitrarily considered an interaction to be significant if 20% or more of the br1/Y;Df/+ animals displayed at least one malformed second or third leg, representing a 50-fold increase over the br1 background.
Out of an initial collection of 154 autosomal deficiency stocks, we tested 133 stocks for genetic interactions with br1. The remaining deficiency stocks could not be tested either because of unmarked duplications that prevented unambiguous identification of progeny classes or due to the presence of Sb/sbd alleles on the deficiency chromosome that would obscure a possible genetic interaction with br1 (
Rho1:
Five overlapping deficiency stocks, Df(2R)Jp1, Df(2R)Jp4, Df(2R)Jp5, Df(2R)Jp7, and Df(2R)Jp8, enhance the br1 malformed leg phenotype to frequencies ranging from 23 to 78% (Table 1). Each of these deficiencies is predicted to remove sequences in the 52F cytological region. We tested five P-element-insertion stocks from this interval and found one, l(2)k02107, that also strongly enhances br1 (Table 2). Plasmid rescue of genomic DNA adjacent to the P-element-insertion site revealed that l(2)k02107 is an insertion into an intron of the Rho1 gene, confirming a subsequent report that this P-element mutation is a bona fide allele of Rho1 (
blistered:
Df(2R)Px2 (60C05;60D09-10) is a very strong dominant enhancer of the br1 malformed leg phenotype, producing malformed legs at a frequency of 50% (Table 1). Crosses with an overlapping deficiency, Df(2R)Px1 (60B08-10;60D01-02), produced malformed legs at a frequency of 19% (n = 112) in the br1 genetic background, mapping the br1-interacting locus to the 60C5-D1 interval. Mutations in blistered (bs), which encodes the Drosophila SRF transcription factor, fail to complement these two deficiencies, and bs2 was previously shown to enhance the malformed leg phenotype of br1, suggesting that bs might contribute to the interaction seen with these deficiencies (
cytoskeletal Tropomyosin:
Df(3R)ea (88E07-13;89A01) enhances the malformed leg phenotype of br1 with a frequency of 62% (Table 2), a particularly strong enhancement. We were unable to refine the interval containing the br1-interacting locus using available deficiencies and therefore tested 20 P-element-insertion stocks that map within the interval from 88E1 to 89A9. We found one stock, l(3)2299, that acts as a dominant enhancer of br1 and also fails to complement Df(3R)ea (Table 2;
Other interacting loci defined by deficiencies:
In addition to the genetic interactions of br1 with Rho1, bs, and cTm, we predict that at least seven loci on the second chromosome and at least seven loci on the third chromosome harbor br1-interacting genes (Table 1). This prediction is based on the finding of two or more overlapping br1-interacting deficiency stocks that were derived from different parental chromosomes (Table 1). In five cases, each of the overlapping deficiency stocks produce an interaction phenotype at a frequency of >40%, >100-fold over the br1 background. Of these, one interval is particularly noteworthy. Df(3R)D6, Df(3R)D7, and Df(3R)p712 compose a set of three overlapping deficiencies that remove genomic sequences from 84D04 to 84F02. Efforts to identify a br1-interacting gene within this interval have thus far failed, although we have tested 36 deficiency stocks, 16 P-element-insertion stocks, and 21 representative EMS alleles derived from a saturation mutagenesis screen of this region (
br5 second-site noncomplementation screen with deficiency stocks:
We conducted SSNC tests between a collection of 133 autosomal deficiency stocks and br5, an amorphic allele of br, to determine whether a stronger allele might identify novel br-interacting genes. In pilot studies we were unable to detect malformed legs in br5/+ females and therefore set an arbitrary threshold for significant interaction at 5% malformed legs in the br5/+;Df/+ genotypic class. Surprisingly, we found only one deficiency that specifically and reproducibly interacts with br5 in this SSNC assay. Df(3R)DG2 (89E01-F04;92D03-06) displays malformed legs at a frequency of 11% (n = 62) when heterozygous in a br5/+ genetic background. Df(3R)DG2 also shows a very strong dominant genetic interaction with br1 (Table 1).
br1 dominant genetic interaction screen of EMS-treated animals:
There are two significant limitations when chromosomal deficiencies are used for a genetic interaction screen: incomplete coverage of the genome and the requirement for detecting an interaction with an amorphic allele. In an effort to overcome these limitations, we conducted an F1 screen of EMS-treated animals. We mutagenized br1 males, mated them to br1 virgin females, and screened through 112,000 F1 progeny for flies displaying the malformed leg phenotype. Malformed progeny were backcrossed to br1 animals to generate stocks. These stocks were then maintained for several generations as inter se crosses, selecting for animals with malformed legs to remove unlinked second-site mutations by free recombination. We kept those stocks in which the inter se crosses produced 10% or more malformed progeny at the F5 generation. From this screen we identified 26 mutations that map to a single chromosome and reproducibly enhance the br1 leg phenotype. Two E(br) mutations map to the X chromosome, 9 map to the second chromosome, and 15 map to the third chromosome. We have analyzed 20 of these E(br) lines in detail (Table 3 and Table 4). Lethal complementation analyses revealed one complementation group consisting of six members that maps to the third chromosome, one complementation group with two members that maps to the second chromosome, and 12 E(br) mutations that complement every other E(br) mutation. Six of these map to the second chromosome and 6 map to the third chromosome.
|
Complementation analyses identify Rho1, sbd, and bs alleles as E(br) mutations:
As a first step toward identifying the br1-interacting genes disrupted by the EMS mutations, we performed a large-scale complementation analysis between each of the E(br) lines and a panel of deficiencies and specific mutations. This set of stocks included representative br1-interacting deficiencies from each of the intervals identified in the deficiency screen, as well as mutations in bs, Rho1, RhoGEF2, sbd, cTm, and zip. From these analyses we determined that the large complementation group on the third chromosome is allelic to Sb/sbd on the basis of the following four observations. First, E(br)20, E(br)48, E(br)448, E(br)536, and E(br)623 compose a single complementation group, and E(br)228 partially fails to complement these mutations for lethality. Second, E(br)20, E(br)536, and E(br)623 fail to complement sbd45. Third, complementation tests between these same three E(br) lines and sbd105 produce viable trans-heterozygous adults that show a stubble bristle phenotype. Fourth, E(br)228 is partially viable, producing homozygous mutant adults at 20% of the expected frequency, all of which show a completely penetrant stubble bristle phenotype (Fig 2B). All of the sbdE(br) mutations show normal bristle morphology when heterozygous, classifying all six alleles as sbd with respect to the bristle phenotype (Fig 2A). Similar complementation analyses demonstrated that E(br)233 and E(br)246 are allelic to Rho1. Both mutations fail to complement one another, E(br)246 fails to complement Df(2R)Jp8 (52F05-09; 52F10-53A01), and both mutations fail to complement Rho1k02107, Rho1J3.8, and Rho1E3.10. Finally, E(br)292 is allelic to bs on the basis of its failure to complement Df(2R)Px2 (60C05;60D09-10) and two bs alleles, bsk03267 and bsk07909.
Meiotic mapping of the E(br) mutations:
Our complementation studies revealed one instance of noncomplementation between an E(br) mutation and a br1-interacting deficiency. E(br)420 fails to complement Df(3R)RD31 (89E02;90D) and Df(3R)DG2 (89E01-F04;91B01-02). It does, however, fully complement Df(3R)C4 (89E03-04;90A01-07), suggesting that the E(br)420 mutation is located in 89E02-04 or 90A-D. To verify this mapping, we performed meiotic mapping experiments with br1;ru th st ri roe p e ca. We conducted these crosses in the br1 genetic background to specifically map the br1-interacting mutation rather than a lethal lesion that might be linked but not causative of the interaction (see MATERIALS AND METHODS). Recombination distance from thread (3-[43.2] on the recombination map) places E(br)420 at 59.9 on the recombination map, which approximately corresponds to 89F (
In addition to E(br)420, we used meiotic mapping to locate the br1-interacting mutations in E(br)24, E(br)65, E(br)121, E(br)155, and E(br)165 (Table 3). The mapping of E(br)121 to 3-[41-43] (corresponding to 70C-71C) is supported by the failure of E(br)121 to complement Df(3L)fz-CALS (70C02-06;70E01). We are currently attempting to confirm the meiotic mapping of the other four E(br) mutations through complementation studies and are beginning the initial mapping of the remaining five E(br) mutations.
Characterization of the E(br) mutations:
We undertook preliminary characterization of the E(br) mutations as a first step toward understanding the function of the affected genes. To this end we have: (1) determined the penetrance of the dominant genetic interaction with br1 (Table 3), (2) tested for SSNC with an amorphic allele of br (Table 3), (3) conducted thorough lethal-phase and terminal phenotypic analyses of the E(br) mutations (Table 4), (4) conducted SSNC analyses with representative mutations in the Rho1 pathway (Table 5), and (5) performed Northern blot analysis of br1-interacting genes on RNA collected from staged wild-type and br5 mutant imaginal discs (Fig 5). The results of these studies are reported below.
|
|
Phenotypic analyses of interactions between br1 and the E(br) mutations:
Individual E(br) mutations vary in their ability to enhance the br1 malformed leg phenotype, ranging from 5% for sbdE(br)228 to 100% for E(br)165 (Table 3). Eight E(br) mutations enhance br1 with a penetrance of <20%, which is the threshold used for the deficiency screen. These results are, however, significant because the E(br) mutations were generated in a uniform genetic background, unlike the wide range of genetic backgrounds found in the deficiency stocks. Therefore, for example, the 5% malformations seen in br1/Y; sbdE(br)228/+ animals represent a 12-fold increase over br1/Y that can be attributed primarily to the mutation. It is also noteworthy that the poorest interacting mutation is an allele of Sb/sbd, a known br1-interacting gene (
The malformed leg phenotypes in br1/Y;E(br)/+ males can be classified into three distinct classes, as shown in Fig 3. Fourteen of the E(br) mutant lines produce legs that have short, fat femurs and tibias that are often kinked or twisted (Fig 3B; Table 3). Included in this group of mutations are all the sbd, Rho1, and bs alleles. In two E(br) lines, the interaction phenotype is predominantly restricted to the tarsal segments, which are shorter and fatter than those of wild type (Fig 3C; Table 3). In four E(br) lines, the interaction phenotype consists entirely of a moderate to strong bend in the mid-tibia that is not associated with an ectopic joint (Fig 3D; Table 3). In all cases, the defects manifest themselves in the third pair of legs and are often unilateral. Malformations of the second pair of legs occur much less frequently and are almost always associated with extreme malformations of the third pair of legs. E(br)420 is unique in producing malformations at a high frequency in the first pair of legs. In this case, the malformed legs show defects primarily in the tibia and tarsal segments, and occasionally the first pair legs are missing middle tarsal segments (data not shown). We have not, however, quantified these phenotypes. Finally, we occasionally observe wing malformations as a br1 interaction phenotype with the E(br) mutations. We did not quantify these phenotypes because their penetrance is low and the expressivity is more varied than that of the malformed leg phenotypes.
We also outcrossed the br1 allele and examined w1118;E(br)/+ animals for the presence of malformed legs. This study revealed that five of the E(br) mutations show a semidominant malformed leg phenotype. E(br)24, E(br)121, E(br)155, E(br)333, and E(br)444 all show >5% malformed legs under these conditions (Table 3). In each case, however, the penetrance of malformed legs in the br1 background is at least threefold higher. Interestingly, all four of the E(br) mutant lines that show the bent tibia malformed leg phenotype are also semidominant (Fig 3D; Table 3).
SSNC analyses between E(br) mutations and br5:
To test the specificity of the E(br) mutations we conducted SSNC experiments with br5. Whereas only one deficiency from the deficiency screen showed >5% malformed legs in this assay (see above), 10 of the E(br) mutations display >5% malformed legs when heterozygous in a br5/w1118 genetic background (Table 3). Included in this collection are two sbd alleles, both Rho1 alleles, the bs allele and five of the unidentified E(br) mutations. The percentage of malformed legs seen with E(br)155 and E(br)444 in a br5/w1118 genetic background, however, was similar to the level of malformed legs found in w1118; E(br)/+ animals, arguing against a relevant genetic interaction.
Lethal-phase and terminal phenotypic analyses of E(br) mutants:
To characterize the function of the genes disrupted by the E(br) mutations, we conducted lethal-phase studies using balancer chromosomes that express GFP to unambiguously identify homozygous mutant embryos and larvae (Table 4). These experiments revealed that both Rho1E(br)233 and Rho1E(br)246 show completely penetrant embryonic lethality with nearly every embryo possessing a large dorsal anterior hole with the head skeleton extruded (Fig 4B and Fig C). In addition, 5–10% of these embryos possess a second cuticular hole often positioned near the posterior pole (data not shown), and many of the dead embryos display slight curvature of the ventral surface, indicating a mild defect in germband retraction (Fig 4C). These observations are consistent with phenotypic analyses describing the zygotic loss-of-function phenotypes for Rho1 mutations (
Lethal-phase analysis of bsE(br)292 indicated a requirement for bs throughout the life cycle (Table 4). The predominant lethal period occurs during larval stages, although we also detected significant embryonic lethality. All of the mutant late embryos and larvae examined lack tertiary branching of the tracheal system (data not shown), consistent with strong loss-of-function mutations at the bs locus (
Lethal-phase analyses of the sbdE(br) mutations revealed that five of the six mutants are predominantly larval lethal, whereas sbdE(br)228 shows some larval and pupal lethality with 21% viable adults (Table 4). sbdE(br)228 adults and adult escapers from trans-heterozygous combinations between sbdE(br)228 and the other sbdE(br) alleles show the short, thick, and barbed bristles characteristic of mutations at the Sb/sbd locus (Fig 2B). Most of these adult escapers also display severely malformed legs (data not shown). Unexpectedly, we found that mutant animals from all six sbdE(br) alleles show defects in larval molting characterized by two complete sets of head skeleton. Occasionally, some cuticle from an earlier molt was found attached to the mouth hooks of dead mutant larvae (Fig 2D). sbdE(br)48 and sbdE(br)623 also display a unique phenotype characterized by excessive sclerotization of the anterior-most cuticle (Fig 2D). Interestingly, 9% of the dead embryos from sbdE(br)48 and 19% of the dead embryos from sbdE(br)623 show a dorsal anterior hole similar to that seen with Rho1 mutations (Fig 4D). These embryonic and larval phenotypes have not been described previously, although
Characterization of the unidentified E(br) mutations revealed four mutant lines that display embryonic lethality characterized by defects in the midembryonic morphogenetic processes of dorsal closure and head involution (Table 4). E(br)24, E(br)65, and E(br)155 mutants show 25, 14, and 97% embryonic lethality, respectively. In all three E(br) lines at least 10% of the dead embryos display a dorsal anterior hole similar to that found in Rho1E(br) mutants (compare Fig 4E with 4, B and C). In addition, we found that E(br)155 and E(br)165 mutants show a high penetrance of dorsal holes (Table 4 and Fig 4F). Specifically, nearly 50% of the E(br)155 mutant embryos fail to complete dorsal closure (data not shown). Lethal-phase and phenotypic analyses of E(br)165 revealed completely penetrant embryonic lethality characterized by a naked cuticle (data not shown). Because it was not possible to assess the terminal phenotype of E(br)165 mutants using cuticle preparations, we resorted to indirect immunofluorescence analysis of late stage 17 embryos with an antibody directed against the septate junction marker Coracle (
SSNC analyses of E(br) mutations and mutations in Rho1 signaling genes:
The similar embryonic lethal phenotypes seen in several E(br) and Rho1 mutants raises the possibility that one or more of the genes affected by the E(br) mutations may function in a Rho1 signaling pathway. To examine this possibility, we conducted a series of SSNC experiments between the E(br) mutations and several alleles of genes known to function in Rho1 signaling, including Rho1, RhoGEF2, and zip (Table 5). The strong genetic interaction observed between the two Rho1E(br) alleles and mutations in RhoGEF2 and zip serve as a useful control for these experiments and show that the Rho1 pathway is amenable to dose-sensitive genetic interaction studies (Table 5). In general, the sbdE(br) alleles display strong SSNC with zipE(br) and weak SSNC with Rho1 alleles, although there is considerable allele-specific variation. Similar findings were observed by
Transcription profiles of br1-interacting genes and Rho1 signaling pathway genes in wild-type and br5 mutants:
The central role of ecdysone signaling and br function in imaginal disc morphogenesis raises the possibility that one or more of the genes identified through our screens might be transcriptionally regulated by ecdysone and dependent on br activity. To test these possibilities, we analyzed the expression of genes identified through the br1-interacting screens as well as additional genes in the Rho1 signaling pathway in both wild-type and br5 mutants. Total RNA was isolated from collections of 100 hand-dissected leg imaginal discs per time point from staged br5/Y mid-third instar larvae and prepupae, as well as from their Binsn/Y siblings. The expression of BR-C, an ecdysone-inducible early gene, was used as a control to follow the timing of the late larval ecdysone pulse (Fig 5). Previous studies have demonstrated that all BR-C isoforms are induced as a primary response to ecdysone in imaginal discs and that BR-C Z2, Z3, and Z4 isoforms are expressed at the beginning of the ecdysone peak, while the strongest expression of BR-C Z1 is delayed several hours coincident with a reduction in the expression of Z2, Z3, and Z4 (2-hr developmental delay described above. In similar Northern blot experiments using RNA collected from whole animals and pooled leg and wing imaginal discs, we found that Rho1, RhoGEF2, Rho kinase, and bs are expressed in imaginal discs, that the level of expression of each gene remains constant from -18 to +6 hr, and that the expression of these genes is unaffected in br5 mutant whole animals (data not shown).
We also examined the pattern of ImpE3 transcription in control and br5 leg imaginal discs in an effort to test for a possible regulatory interaction between br and ImpE3 suggested by the deficiency screen results described above. In control leg imaginal discs, ImpE3 expression begins 4 hr before puparium formation, peaks at 2–4 hr after pupariation, and begins to subside by +6 hr (Fig 5). Interestingly, this expression is substantially reduced in br5 leg imaginal discs, indicating that the ImpE3 expression is dependent upon br function in this tissue.
| DISCUSSION |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Detailed studies over the past decade have focused on understanding how the imaginal discs undergo proliferation and pattern formation during larval development. In contrast, we know little about how the mature imaginal discs are transformed into their corresponding adult structures during metamorphosis—structures that bear no physical resemblance to the imaginal discs from which they were derived. This study is aimed at addressing this topic by focusing on the ecdysone-dependent morphogenesis of the adult leg in Drosophila.
Two screens for dominant enhancers of the br1 malformed leg phenotype:
We conducted two large-scale genetic modifier screens as a first step toward identifying novel links between the ecdysone signal and the cytoskeletal components that drive Drosophila leg morphogenesis. Both approaches took advantage of a hypomorphic mutation in the ecdysone-inducible br early gene, screening for enhancement of a rare malformed leg phenotype in adult flies. Screening through >750 stocks bearing either a chromosomal deficiency or a specific mutation, we identified nine loci on the second chromosome and eight loci on the third chromosome that interact with br1. In a complementary F1 screen of EMS-treated br1 animals, we obtained 26 enhancer lines of which 20 were analyzed in detail. From these screens combined, we identified Rho1, bs, sbd, and cTm as br1-interacting genes.
Conducting both a deficiency-based screen and a random mutagenesis screen allowed us to play the strengths of one approach off the weakness of the other. While a deficiency-based screen can quickly map br1-interacting loci, it can be difficult to subsequently identify specific mutations to account for those interactions. On the other hand, an EMS screen rapidly generates specific br1-interacting mutations, but significantly more effort is required to map and clone the corresponding gene. Here, we used complementation analyses between br1-interacting deficiencies and EMS-induced mutations to identify br1-interacting genes. Both screening strategies identified mutations in Rho1 and bs. We also found an unidentified E(br) mutation that fails to complement a br1-interacting deficiency. Moreover, further complementation tests between the E(br) mutations and mutations in previously identified br1-interacting genes allowed us to identify six EMS-derived mutations as new alleles of Sb/sbd.
The results of our screens indicate that we have not yet begun to approach saturation in this pathway. Although we identified six alleles of sbd and two alleles of Rho1 from the EMS screen, we also identified 12 mutations that are each represented by a single allele. In addition, we found at least 14 br1-interacting loci by deficiency screening for which we did not recover an EMS-derived mutation. Principally, these results illustrate the complexity of imaginal disc morphogenesis and suggest that many genes are required to ensure the fidelity of this process. It is apparent that a larger screen will generate additional br1-interacting mutations, some of which might map to genomic intervals identified through the deficiency screen.
A central role for Rho1 signaling in ecdysone-triggered leg disc morphogenesis:
The identification of Rho1 mutations as dominant enhancers of br1, as well as the genetic interactions we observe between E(br) alleles and mutations in the Rho1 signaling pathway, indicate a central role for Rho1 in directing leg morphogenesis at the onset of metamorphosis (Fig 6). Signaling through the Rho1 small GTPase depends on a shift in the cellular equilibrium between inactive Rho-GDP and active Rho-GTP (reviewed in
|
Over the past several years, genetic, molecular, and pharmacological perturbations of Rho1 signaling have revealed key roles for this signaling cascade in directing a variety of morphogenetic processes, including embryonic elongation in Caenorhabditis elegans and neural tube closure in the mouse (
Genetic studies indicate that at least five components of the Drosophila Rho1 signaling cascade are required during imaginal disc morphogenesis: Rho1, RhoGEF2, myosin phosphatase, myosin regulatory light chain (encoded by spaghetti squash or sqh), and nonmuscle myosin heavy chain (encoded by zip; Fig 6). First, we found that deficiencies that uncover the Rho1 locus as well as specific mutations in Rho1 enhance the malformed leg phenotype associated with the br1 mutation (Table 2). We also recovered two new alleles of Rho1 as E(br) mutations from our EMS mutagenesis screen (Table 3). Second, we found strong SSNC between both Rho1E(br) alleles and RhoGEF211-3 (Table 5), suggesting that RhoGEF2 is playing a pivotal role in activating Rho1 during imaginal disc morphogenesis. Consistent with this observation,
In addition to direct effects on the actin cytoskeleton, Rho1 signaling can also transduce extracellular signals to the nucleus by activating SRF transcription factors. The mechanism of Rho1-dependent SRF activation is poorly understood but appears to require at least one of several Rho1-specific effector molecules, including Rho kinase, LIM kinase, and formin-homology proteins of the mDia family, in a cell-type-specific manner (
- and ß-actin, vinculin, and tropomyosin (
Roles for ecdysone in directing leg disc morphogenesis:
The identification of genetic interactions between members of the Rho1 signaling pathway and the ecdysone-inducible transcription factor encoded by br provides an intriguing tie between the steroid hormone and the cellular machinery that drives morphogenesis. To investigate whether members of the Rho1 pathway might be transcriptionally regulated by ecdysone, we examined the expression of Rho1, RhoGEF2, and Rho kinase in whole animals and imaginal discs dissected from staged late larvae and prepupae of both wild type and br5 mutants (data not shown). This study, however, revealed no changes in transcript levels in response to the late larval ecdysone pulse, and no effects of the amorphic br5 mutation on their expression (data not shown). It should be noted, however, that there are many possible targets for ecdysone regulation of Rho1 activity, including multiple RhoGEFs and RhoGAPs. Ecdysone may also be responsible for inducing the expression of one or more proteins required for the appropriate subcellular localization of the Rho1 complex, a level of regulation that is thought to be critical for its activation (reviewed in
Intriguingly, Sb/sbd represents the only known br1-interacting gene that is induced directly by ecdysone in imaginal discs as they undergo morphogenesis (Fig 5;
It is interesting to note that most sbd mutants die during larval stages with molting defects (Fig 2D). This function for sbd has not been described previously and provides an additional unexpected tie to ecdysone signaling. Ecdysone pulses during larval development trigger molting of the cuticle as the animal grows in size (
Finally, our Northern blot study provides the first observation of a br-dependent transcript expressed in imaginal discs, ImpE3 (Fig 5). This gene is induced rapidly by ecdysone, is expressed primarily in imaginal discs, and encodes a secreted protein with a potential glycosylphosphatidylinositol anchor (
Parallel ecdysone-triggered morphogenetic responses during embryogenesis and metamorphosis:
Our EMS mutagenesis screen identified 11 E(br) mutations that appear to reside in unique genes and whose identities remain unknown. Seven of these mutations show SSNC with mutations in Rho1, RhoGEF2, or zip (Table 5), suggesting that the corresponding genes may function with Rho1 to facilitate the cell shape changes that drive leg morphogenesis. Remarkably, although we originally isolated these mutations as dominant enhancers of br1 during prepupal imaginal disc morphogenesis, we found that the zygotic loss-of-function phenotype for four of them included defects in embryonic morphogenetic events—head involution and dorsal closure (Table 4; Fig 4). Zygotic loss-of-function mutations in Rho1 also show defects in head involution and dorsal closure (Fig 4B and Fig C;
Taken together, these observations raise the interesting possibility that a common ecdysone-directed, Rho1-mediated signaling cascade controls the morphogenetic movements that occur during two major developmental transitions in the life cycle, embryogenesis and metamorphosis. This idea fits with recent data that implicate a central role for ecdysone signaling in midembryonic morphogenetic events. A high titer pulse of ecdysone occurs midway through embryonic development, peaking from 6 to 12 hr after egglay, coincident with the morphogenetic movements of germband retraction, dorsal closure, and head involution (
| FOOTNOTES |
|---|
1 Present address: Department of Molecular Biosciences, University of Kansas, 1200 Sunnyside Ave., Lawrence, KS 66045-7534. 
| ACKNOWLEDGMENTS |
|---|
We thank S. Halsell, L. von Kalm, D. Kiehart, and the Bloomington Drosophila Stock Center for fly stocks and R. Fehon for the anti-Coracle antibodies used in this study. We are greatly indebted to J. Fristrom, L. von Kalm, and S. Halsell for stimulating conversations and sharing unpublished results. We thank P. Reid and J. Gallafant for technical assistance during part of this study. We also thank A. Bashirullah and T. Kozlova for critical comments on the manuscript. R.E.W. was supported as an Associate of the Howard Hughes Medical Institute and through a National Institutes of Health National Research Service Award. C.S.T. is an Investigator with the Howard Hughes Medical Institute.
Manuscript received March 13, 2003; Accepted for publication June 3, 2003.
| LITERATURE CITED |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
AFFOLTER, M., J. MONTAGNE, U. WALLDORF, J. GROPPE, and U. KLOTER et al., 1994 The Drosophila SRF homolog is expressed in a subset of tracheal cells and maps within a genomic region required for tracheal development. Development 120:743-753.[Abstract]
ANDRES, A. J., and C. S. THUMMEL, 1994 Methods for quantitative analysis of transcription in larvae and prepupae, pp. 565–573 in Drosophila melanogaster: Practical Uses in Cell and Molecular Biology, edited by L. GOLDSTEIN and E. FRYBERG. Academic Press, New York.
APPEL, L. F., M. PROUT, R. ABU-SHUMAYS, A. HAMMONDS, and J. C. GARBE et al., 1993 The Drosophila Stubble-stubbloid gene encodes an apparent transmembrane serine protease required for epithelial morphogenesis. Proc. Natl. Acad. Sci. USA 90:4937-4941.
ASHBURNER, M., 1989 Drosophila: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
ASHBURNER, M., C. CHIHARA, P. MELTZER, and G. RICHARDS, 1974 Temporal control of puffing activity in polytene chromosomes. Cold Spring Harbor Symp. Quant. Biol. 38:655-662.
BAKER, B. S., G. HOFF, T. C. KAUFMAN, M. F. WOLFNER, and T. HAZELRIGG, 1991 The doublesex locus of Drosophila melanogaster and its flanking regions: a cytogenetic analysis. Genetics 127:125-138.[Abstract]
BAYER, C. A., B. HOLLEY, and J. W. FRISTROM, 1996 A switch in Broad-Complex zinc-finger isoform expression is regulated posttranscriptionally during the metamorphosis of Drosophila imaginal discs. Dev. Biol. 177:1-14.[Medline]
BAYER, C. A., S. R. HALSELL, J. W. FRISTROM, D. P. KIEHART, and L. VON KALM, 2003 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 165:1417-1432.
BEATON, A. H., I. KISS, D. FRISTROM, and J. W. FRISTROM, 1988 Interaction of the Stubble-stubbloid locus and the Broad-Complex of Drosophila melanogaster.. Genetics 120:453-464.
BELYAEVA, E. S., M. G. AIZENZON, V. F. SEMESHIN, I. KISS, and K. KOCZKA et al., 1980 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 81:281-306.[Medline]
BIRR, C. A., D. FRISTROM, D. S. KING, and J. W. FRISTROM, 1990 Ecdysone-dependent proteolysis of an apical surface glycoprotein may play a role in imaginal disc morphogenesis in Drosophila.. Development 110:239-248.[Abstract]
BOCCHINFUSO, W. P., J. K. LINDZEY, S. C. HEWITT, J. A. CLARK, and P. H. MYERS et al., 2000 Induction of mammary gland development in estrogen receptor-alpha knockout mice. Endocrinology 141:2982-2994.
BROUNS, M. R., S. F. MATHESON, K. Q. HU, I. DELALLE, and V. S. CAVINESS et al., 2000 The adhesion signaling molecule p190 RhoGAP is required for morphogenetic processes in neural development. Development 127:4891-4903.[Abstract]
CHÂVEZ, V. M., G. MARQUÂES, J. P. DELBECQUE, K. KOBAYASHI, and M. HOLLINGSWORTH et al., 2000 The Drosophila disembodied gene controls late embryonic morphogenesis and codes for a cytochrome P450 enzyme that regulates embryonic ecdysone levels. Development 127:4115-4126.[Abstract]
CONDIC, M. L., D. FRISTROM, and J. W. FRISTROM, 1991 Apical cell shape changes during Drosophila imaginal leg disc elongation: a novel morphogenetic mechanism. Development 111:23-33.[Abstract]
D'AVINO, P. P. and C. S. THUMMEL, 1998 crooked legs encodes a family of zinc finger proteins required for leg morphogenesis and ecdysone-regulated gene expression during Drosophila metamorphosis. Development 125:1733-1745.[Abstract]
DIBELLO, P. R., D. A. WITHERS, C. A. BAYER, J. W. FRISTROM, and G. M. GUILD, 1991 The Drosophila Broad-Complex encodes a family of related proteins containing zinc fingers. Genetics 129:385-397.[Abstract]
EDWARDS, K. A. and D. P. KIEHART, 1996 Drosophila nonmuscle myosin II has multiple essential roles in imaginal disc and egg chamber morphogenesis. Development 122:1499-1511.[Abstract]
ERDELYI, M., A. M. MICHON, A. GUICHET, J. B. GLOTZER, and A. EPHRUSSI, 1995 Requirement for Drosophila cytoplasmic tropomyosin in oskar mRNA localization. Nature 377:524-527.[Medline]
FEHON, R. G., K. JOHANSEN, I. REBAY, and S. ARTAVANIS-TSAKONAS, 1991 Complex cellular and subcellular regulation of notch expression during embryonic and imaginal development of Drosophila: implications for notch function. J. Cell Biol. 113:657-669.
FEHON, R. G., I. A. DAWSON, and S. ARTAVANIS-TSAKONAS, 1994 A Drosophila homologue of membrane-skeleton protein 4.1 is associated with septate junctions and is encoded by the coracle gene. Development 120:545-557.[Abstract]
FEKETE, E., D. FRISTROM, I. KISS, and J. W. FRISTROM, 1975 The mechanism of evagination of imaginal discs of Drosophila melanogaster. II. Studies on trypsin-accelerated evagination. Wilhelm Roux's Arch. Dev. Biol. 173:123-138.
FESSLER, L. I., M. L. CONDIC, R. E. NELSON, J. H. FESSLER, and J. W. FRISTROM, 1993 Site-specific cleavage of basement membrane collagen IV during Drosophila metamorphosis. Development 117:1061-1069.[Abstract]
FLYBASE,, 1999 The FlyBase database of the Drosophila genome projects and community literature. Nucleic Acids Res. 27:85-88.
FRISTROM, D. and J. W. FRISTROM, 1975 The mechanism of evagination of imaginal discs of Drosophila melanogaster. I. General considerations. Dev. Biol. 43:1-23.[Medline]
FRISTROM, D., and J. W. FRISTROM, 1993 The metamorphic development of the adult epidermis, pp. 843–897 in The Development of Drosophila melanogaster, edited by M. BATE and A. MARTINEZ ARIAS. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
FRISTROM, J. W., W. R. LOGAN, and C. MURPHY, 1973 The synthetic and minimal culture requirements for evagination of imaginal discs of Drosophila melanogaster in vitro. Dev. Biol. 33:441-456.[Medline]
GENESTE, O., J. W. COPELAND, and R. TREISMAN, 2002 LIM kinase and Diaphanous cooperate to regulate serum response factor and actin dynamics. J. Cell Biol. 157:831-838.
GINEITIS, D. and R. TREISMAN, 2001 Differential usage of signal transduction pathways defines two types of serum response factor target gene. J. Biol. Chem. 276:24531-24539.
GOTWALS, P. J. and J. W. FRISTROM, 1991 Three neighboring genes interact with the Broad-Complex and the Stubble-stubbloid locus to affect imaginal disc morphogenesis in Drosophila. Genetics 127:747-759.[Abstract]
GRAVES, B. J. and G. SCHUBIGER, 1982 Cell cycle changes during growth and differentiation of imaginal leg discs in Drosophila melanogaster.. Dev. Biol. 93:104-110.[Medline]
GUILLEMIN, K., J. GROPPE, K. DUCKER, R. TREISMAN, and E. HAFEN et al., 1996 The pruned gene encodes the Drosophila serum response factor and regulates cytoplasmic outgrowth during terminal branching of the tracheal system. Development 122:1353-1362.[Abstract]
HALSELL, S. R. and D. P. KIEHART, 1998 Second-site noncomplementation identifies genomic regions required for Drosophila nonmuscle myosin function during morphogenesis. Genetics 148:1845-1863.
HALSELL, S. R., B. I. CHU, and D. P. KIEHART, 2000 Genetic analysis demonstrates a direct link between rho signaling and nonmuscle myosin function during Drosophila morphogenesis. Genetics 155:1253-1265.
KARIM, F. D. and C. S. THUMMEL, 1991 Ecdysone coordinates the timing and the amount of E74A and E74B transcription in Drosophila.. Genes Dev. 5:1067-1079.
KISS, I., A. H. BEATON, J. TARDIFF, D. FRISTROM, and J. W. FRISTROM, 1988 Interactions and developmental effects of mutations in the Broad-Complex of Drosophila melanogaster.. Genetics 118:247-259.
KOZLOVA, T. and C. S. THUMMEL, 2003 Essential roles for ecdysone signaling during Drosophila mid-embryonic development. Science 301:1911-1914.
KRAMINSKY, G. P., W. C. CLARK, M. A. ESTELLE, R. D. GIETZ, and B. A. SAGE et al., 1980 Induction of translatable mRNA for dopa decarboxylase in Drosophila: an early response to ecdysterone. Proc. Natl. Acad. Sci. USA 77:4175-4179.
LEFEVRE, G., 1976 A photographic representation and interpretation of Drosophila melanogaster salivary glands, pp. 31–66 in The Genetics and Biology of Drosophila, edited by M. ASHBURNER and E. NOVITSKI. Academic Press, London.
MACK, C. P., A. V. SOMLYO, M. HAUTMANN, A. P. SOMLYO, and G. K. OWENS, 2001 Smooth muscle differentiation marker gene expression is regulated by RhoA-mediated actin polymerization. J. Biol. Chem. 276:341-347.
MAGIE, C. R., M. R. MEYER, M. S. GORSUCH, and S. M. PARKHURST, 1999 Mutations in the Rho1 small GTPase disrupt morphogenesis and segmentation during early Drosophila development. Development 126:5353-5364.[Abstract]
MANDARON, P., 1970 Dâeveloppement in vitro des disques imaginaux de la drosophile. Aspects morphologiques et histologiques. Dev. Biol. 22:298-320.[Medline]
MARÓY, P., G. KAUFMANN, and A. DÜBENDORFER, 1988 Embryonic ecdysteroids of Drosophila melanogaster.. J. Insect Physiol. 34:633-637.
MARTIN, P., and I. SCHNEIDER, 1978 Drosophila organ culture, pp. 219–264 in The Genetics and Biology of Drosophila, edited by M. ASHBURNER and T. R. F. WRIGHT. Academic Press, London.
MIRALLES, F., G. POSERN, A. ZAROMYTIDOU, and R. TREISMAN, 2003 Actin dynamics control SRF activity by regulation of its coactivator MAL. Cell 113:329-342.[Medline]
MIZUNO, T., K. TSUTSUI, and Y. NISHIDA, 2002 Drosophila myosin phosphatase and its role in dorsal closure. Development 129:1215-1223.
MONTAGNE, J., J. GROPPE, K. GUILLEMIN, M. A. KRASNOW, and W. J. GEHRING et al., 1996 The Drosophila Serum Response Factor gene is required for the formation of intervein tissue of the wing and is allelic to blistered.. Development 122:2589-2597.[Abstract]
MOORE, J. T., D. FRISTROM, A. S. HAMMONDS, and J. W. FRISTROM, 1990 Characterization of IMP-E3, a gene active during imaginal disc morphogenesis in Drosophila melanogaster.. Dev. Genet. 11:299-309.[Medline]
NAKAMURA, M., W. NISHIDA, S. MORI, K. HIWADA, and K. HAYASHI et al., 2001 Transcriptional activation of beta-tropomyosin mediated by serum response factor and a novel Barx homologue, Barx1b, in smooth muscle cells. J. Biol. Chem. 276:18313-18320.
NATZLE, J. E., A. S. HAMMONDS, and J. W. FRISTROM, 1986 Isolation of genes active during hormone-induced morphogenesis in Drosophila imaginal discs. J. Biol. Chem. 261:5575-5583.
PINO-HEISS, S. and G. SCHUBIGER, 1989 Extracellular protease production by Drosophila imaginal discs. Dev. Biol. 132:282-291.[Medline]
POODRY, C. A. and H. A. SCHNEIDERMAN, 1971 Intercellular adhesivity and pupal morphogenesis in Drosophila melanogaster.. Wilhelm Roux's Arch. Dev. Biol. 168:1-9.
RIDDIFORD, L. M., 1993 Hormones and Drosophila development, pp. 899–940 in The Development of Drosophila melanogaster, edited by M. BATE and A. MARTINEZ ARIAS. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
RIDDIFORD, L. M., P. CHERBAS, and J. W. TRUMAN, 2000 Ecdysone receptors and their biological actions. Vitam. Horm. 60:1-73.[Medline]
ROBERTSON, C. W., 1936 The metamorphosis of Drosophila melanogaster, including an accurately timed account of the principal morphological changes. J. Morphol. 59:351-399.
SETTLEMAN, J., 2001 Rac 'n Rho: the music that shapes a developing embryo. Dev. Cell 1:321-331.[Medline]
SOTIROPOULOS, A., D. GINEITIS, J. COPELAND, and R. TREISMAN, 1999 Signal-regulated activation of serum response factor is mediated by changes in actin dynamics. Cell 98:159-169.[Medline]
SPILLMAN, E. and R. NOTHIGER, 1978 Cytology, genetics and lethality patterns of homozygous lethal mutations in the sbd region. Dros. Inf. Serv. 53:164-165.
SYMONS, M. and J. SETTLEMAN, 2000 Rho family GTPases: more than simple switches. Trends Cell Biol. 10:415-419.[Medline]
TATA, J. R., 1999 Amphibian metamorphosis as a model for studying the developmental actions of thyroid hormone. Biochimie 81:359-366.[Medline]
TETZLAFF, M. T., H. JÈACKLE, and M. J. PANKRATZ, 1996 Lack of Drosophila cytoskeletal tropomyosin affects head morphogenesis and the accumulation of oskar mRNA required for germ cell formation. EMBO J. 15:1247-1254.[Medline]
THUMMEL, C. S., 1996 Flies on steroids—Drosophila metamorphosis and the mechanisms of steroid hormone action. Trends Genet. 12:306-310.[Medline]
VAN AELST, L. and C. D'SOUZA-SCHOREY, 1997 Rho GTPases and signaling networks. Genes Dev. 11:2295-2322.
VON KALM, L., D. FRISTROM, and J. FRISTROM, 1995 The making of a fly leg: a model for epithelial morphogenesis. Bioessays 17:693-702.[Medline]
WARD, R., P. REID, A. BASHIRULLAH, P. P. D'AVINO, and C. THUMMEL, 2003 GFP in living animals reveals dynamic developmental responses to ecdysone during Drosophila metamorphosis. Dev. Biol. 256:389-402.[Medline]
WEI, L., W. ROBERTS, L. WANG, M. YAMADA, and S. ZHANG et al., 2001 Rho kinases play an obligatory role in vertebrate embryonic organogenesis. Development 128:2953-2962.
WISSMANN, A., J. INGLES, J. D. MCGHEE, and P. E. MAINS, 1997 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. 11:409-422.
WISSMANN, A., J. INGLES, and P. E. MAINS, 1999 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. 209:111-127.[Medline]
This article has been cited by other articles:
 |
|
 |
|
  G. E. Chalkley, Y. M. Moshkin, K. Langenberg, K. Bezstarosti, A. Blastyak, H. Gyurkovics, J. A. A. Demmers, and C. P. Verrijzer The Transcriptional Coactivator SAYP Is a Trithorax Group Signature Subunit of the PBAP Chromatin Remodeling Complex Mol. Cell. Biol., May 1, 2008; 28(9): 2920 - 2929. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Konopova and M. Jindra Broad-Complex acts downstream of Met in juvenile hormone signaling to coordinate primitive holometabolan metamorphosis Development, February 1, 2008; 135(3): 559 - 568. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. S. Hammonds and J. W. Fristrom Mutational Analysis of Stubble-stubbloid Gene Structure and Function in Drosophila Leg and Bristle Morphogenesis Genetics, March 1, 2006; 172(3): 1577 - 1593. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. G. Wilson, Y. Yerushalmi, D. M. Donnell, and L. L. Restifo Interaction Between Hormonal Signaling Pathways in Drosophila melanogaster as Revealed by Genetic Interaction Between Methoprene-tolerant and Broad-Complex Genetics, January 1, 2006; 172(1): 253 - 264. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Bayer, S. R. Halsell, J. W. Fristrom, D. P. Kiehart, and L. von Kalm 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, November 1, 2003; 165(3): 1417 - 1432. [Abstract] [Full Text] [PDF]
|
|
- THIS ARTICLE
-
Abstract
- Full Text (PDF)
- Alert me when this article is cited
- Alert me if a correction is posted
- SERVICES
- Email this article to a friend
- Similar articles in this journal
- Similar articles in PubMed
- Alert me to new issues of the journal
- Download to citation manager
- Reprints & Permissions
- CITING ARTICLES
- Citing Articles via HighWire
- Citing Articles via Google Scholar
- GOOGLE SCHOLAR
- Articles by Ward, R. E.
- Articles by Thummel, C. S.
- Search for Related Content
- PUBMED
- PubMed Citation
- Articles by Ward, R. E.
- Articles by Thummel, C. S.