Genetic modifier screens in Drosophila demonstrate a role for Rho1 signaling in ecdysone-triggered imaginal disc morphogenesis

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.     

DHR3 is required for the prepupal-pupal transition and differentiation of adult structures during Drosophila metamorphosis.

Pulses of the steroid hormone ecdysone activate genetic regulatory hierarchies that coordinate the developmental changes associated with Drosophila metamorphosis. A high-titer ecdysone pulse at the end of larval development triggers puparium formation and induces expression of the DHR3 orphan nuclear receptor. Here we use both a heat-inducible DHR3 rescue construct and clonal analysis to define DHR3 functions during metamorphosis. Clonal analysis reveals requirements for DHR3 in the development of adult bristles, wings, and cuticle, and no apparent function in eye or leg development. DHR3 mutants rescued to the third larval instar also reveal essential functions during the onset of metamorphosis, leading to lethality during prepupal and early pupal stages. The phenotypes associated with these lethal phases are consistent with the effects of DHR3 mutations on ecdysone-regulated gene expression. Although DHR3 has been shown to be sufficient for early gene repression at puparium formation, it is not necessary for this response, indicating that other negative regulators may contribute to this pathway. In contrast, DHR3 is required for maximal expression of the midprepupal regulatory genes, EcR, E74B, and betaFTZ-1. Reductions in EcR and betaFTZ-F1 expression, in turn, lead to submaximal early gene induction in response to the prepupal ecdysone pulse and corresponding defects in adult head eversion and salivary gland cell death. These studies demonstrate that DHR3 is an essential regulator of the betaFTZ-F1 midprepupal competence factor, providing a functional link between the late larval and prepupal responses to ecdysone. Induction of DHR3 in early prepupae ensures that responses to the prepupal ecdysone pulse will be distinct from responses to the late larval pulse and thus that the animal progresses in an appropriate manner through the early stages of metamorphosis.     

Inducible expression of double-stranded RNA directs specific genetic interference in Drosophila

BACKGROUND: The introduction of double-stranded RNA (dsRNA) can selectively interfere with gene expression in a wide variety of organisms, providing an ideal approach for functional genomics. Although this method has been used in Drosophila, it has been limited to studies of embryonic gene function. Only inefficient effects have been seen at later stages of development. RESULTS: When expressed under the control of a heat-inducible promoter, dsRNA interfered efficiently and specifically with gene expression during larval and prepupal development in Drosophila. Expression of dsRNA corresponding to the EcR ecdysone receptor gene generated defects in larval molting and metamorphosis, resulting in animals that failed to pupariate or prepupae that died with defects in larval tissue cell death and adult leg formation. In contrast, expression of dsRNA corresponding to the coding region of the betaFTZ-F1 orphan nuclear receptor had no effect on puparium formation, but led to an arrest of prepupal development, generating more severe lethal phenotypes than those seen with a weak betaFTZ-F1 loss-of-function allele. Animals that expressed either EcR or betaFTZ-F1 dsRNA showed defects in the expression of corresponding target genes, indicating that the observed developmental defects are caused by disruption of the genetic cascades that control the onset of metamorphosis. CONCLUSIONS: These results confirm and extend our understanding of EcR and betaFTZ-F1 function. They also demonstrate that dsRNA expression can inactivate Drosophila gene function at later stages of development, providing a new tool for functional genomic studies in Drosophila.     

A conditional rescue system reveals essential functions for the ecdysone receptor (EcR) gene during molting and metamorphosis in Drosophila

In Drosophila, pulses of the steroid hormone ecdysone trigger larval molting and metamorphosis and coordinate aspects of embryonic development and adult reproduction. At each of these developmental stages, the ecdysone signal is thought to act through a heteromeric receptor composed of the EcR and USP nuclear receptor proteins. Mutations that inactivate all EcR protein isoforms (EcR-A, EcR-B1, and EcR-B2) are embryonic lethal, hindering analysis of EcR function during later development. Using transgenes in which a heat shock promoter drives expression of an EcR cDNA, we have employed temperature-dependent rescue of EcR null mutants to determine EcR requirements at later stages of development. Our results show that EcR is required for hatching, at each larval molt, and for the initiation of metamorphosis. In EcR mutants arrested prior to metamorphosis, expression of ecdysone-responsive genes is blocked and normal ecdysone responses of both imaginal and larval tissues are blocked at an early stage. These results show that EcR mediates ecdysone signaling at multiple developmental stages and implicate EcR in the reorganization of imaginal and larval tissues at the onset of metamorphosis.     

The ecdysone-induced DHR4 orphan nuclear receptor coordinates growth and maturation in Drosophila

A critical determinant of insect body size is the time at which the larva stops feeding and initiates wandering in preparation for metamorphosis. No genes have been identified that regulate growth by contributing to this key developmental decision to terminate feeding. We show here that mutations in the DHR4 orphan nuclear receptor result in larvae that precociously leave the food to form premature prepupae, resulting in abbreviated larval development that translates directly into smaller and lighter animals. In addition, we show that DHR4 plays a central role in the genetic cascades triggered by the steroid hormone ecdysone at the onset of metamorphosis, acting as both a repressor of the early ecdysone-induced regulatory genes and an inducer of the betaFTZ-F1 midprepupal competence factor. We propose that DHR4 coordinates growth and maturation in Drosophila by mediating endocrine responses to the attainment of critical weight during larval development.     

Temporal regulation of metamorphic processes in Drosophila by the let-7 and miR-125 heterochronic microRNAs

BACKGROUND: The let-7 and lin-4 microRNAs belong to a class of temporally expressed, noncoding regulatory RNAs that function as heterochronic switch genes in the nematode C. elegans. Heterochronic genes control the relative timing of events during development and are considered a major force in the rapid evolution of new morphologies. let-7 is highly conserved and in Drosophila is temporally coregulated with the lin-4 homolog, miR-125. Little is known, however, about their requirement outside the nematode or whether they universally control the timing of developmental processes. RESULTS: We report the generation of a Drosophila mutant that lacks let-7 and miR-125 activities and that leads to a pleiotropic phenotype arising during metamorphosis. We focus on two defects and demonstrate that loss of let-7 and miR-125 results in temporal delays in two distinct metamorphic processes: the terminal cell-cycle exit in the wing and maturation of neuromuscular junctions (NMJs) at adult abdominal muscles. We identify the abrupt (ab) gene, encoding a nuclear protein, as a bona fide let-7 target and provide evidence that let-7 governs the maturation rate of abdominal NMJs during metamorphosis by regulating ab expression. CONCLUSIONS: Drosophila Iet-7 and miR-125 mutants exhibit temporal misregulation of specific metamorphic processes. As in C. elegans, Drosophila let-7 is both necessary and sufficient for the appropriate timing of a specific cell-cycle exit, indicating that its function as a heterochronic microRNA is conserved. The ab gene is a target of let-7, and its repression in muscle is essential for the timing of NMJ maturation during metamorphosis. Our results suggest that let-7 and miR-125 serve as conserved regulators of events necessary for the transition from juvenile to adult life stages.     

Temporally restricted expression of transcription factor betaFTZ-F1: significance for embryogenesis, molting and metamorphosis in Drosophila melanogaster

FTZ-F1, a member of the nuclear receptor superfamily, has been implicated in the activation of the segmentation gene fushi tarazu during early embryogenesis of Drosophila melanogaster. We found that an isoform of FTZ-F1, betaFTZ-F1, is expressed in the nuclei of almost all tissues slightly before the first and second larval ecdysis and before pupation. Severely affected ftz-f1 mutants display an embryonic lethal phenotype, but can be rescued by ectopic expression of betaFTZ-F1 during the period of endogenous betaFTZ-F1 expression in the wild type. The resulting larvae are not able to molt, but this activity is rescued again by forced expression of betaFTZ-F1, allowing progression to the next larval instar stage. On the other hand, premature expression of betaFTZ-F1 in wild-type larvae at mid-first instar or mid-second instar stages causes defects in the molting process. Sensitive periods were found to be around the time of peak ecdysteroid levels and slightly before the start of endogenous betaFTZ-F1 expression. A hypomorphic ftz-f1 mutant that arrests in the prepupal stage can also be rescued by ectopic, time-specific expression of betaFTZ-F1. Failure of salivary gland histolysis, one of the phenotypes of the ftz-f1 mutant, is rescued by forced expression of the ftz-f1 downstream gene BR-C during the late prepupal period. These results suggest that betaFTZ-F1 regulates genes associated with ecdysis and metamorphosis, and that the exact timing of its action in the ecdysone-induced gene cascade is important for proper development.     

Cellular and molecular processes leading to embryo formation in sponges: evidences for high conservation of processes throughout animal evolution

The emergence of multicellularity is regarded as one of the major evolutionary events of life. This transition unicellularity/pluricellularity was acquired independently several times (King 2004). The acquisition of multicellularity implies the emergence of cellular cohesion and means of communication, as well as molecular mechanisms enabling the control of morphogenesis and body plan patterning. Some of these molecular tools seem to have predated the acquisition of multicellularity while others are regarded as the acquisition of specific lineages. Morphogenesis consists in the spatial migration of cells or cell layers during embryonic development, metamorphosis, asexual reproduction, growth, and regeneration, resulting in the formation and patterning of a body. In this paper, our aim is to review what is currently known concerning basal metazoans—sponges’ morphogenesis from the tissular, cellular, and molecular points of view—and what remains to elucidate. Our review attempts to show that morphogenetic processes found in sponges are as diverse and complex as those found in other animals. In true epithelial sponges (Homoscleromorpha), as well as in others, we find similar cell/layer movements, cellular shape changes involved in major morphogenetic processes such as embryogenesis or larval metamorphosis. Thus, sponges can provide information enabling us to better understand early animal evolution at the molecular level but also at the cell/cell layer level. Indeed, comparison of molecular tools will only be of value if accompanied by functional data and expression studies during morphogenetic processes.