rhomboid and Star interact synergistically to promote EGFR/MAPK signaling during Drosophila wing vein development.

Genes of the ventrolateral group in Drosophila are dedicated to developmental regulation of Egfr signaling in multiple processes including wing vein development. Among these genes, Egfr encodes the Drosophila EGF-Receptor, spitz (spi) and vein (vn) encode EGF-related ligands, and rhomboid (rho) and Star (S) encode membrane proteins. In this study, we show that rho-mediated hyperactivation of the EGFR/MAPK pathway is required for vein formation throughout late larval and early pupal development. Consistent with this observation, rho activity is necessary and sufficient to activate MAPK in vein primordium during late larval and early pupal stages. Epistasis studies using a dominant negative version of Egfr and a ligand-independent activated form of Egfr suggest that rho acts upstream of the receptor. We show that rho and S function in a common aspect of vein development since loss-of-function clones of rho or S result in nearly identical non-autonomous loss-of-vein phenotypes. Furthermore, mis-expression of rho and S in wild-type and mutant backgrounds reveals that these genes function in a synergistic and co-dependent manner. In contrast, spi does not play an essential role in the wing. These data indicate that rho and S act in concert, but independently of spi, to promote vein development through the EGFR/MAPK signaling pathway.

Boundaries in the Drosophila wing imaginal disc organize vein-specific genetic programs.

Previous studies have suggested that vein primordia in Drosophila form at boundaries along the A/P axis between discrete sectors of the larval wing imaginal disc. Genes involved in initiating vein development during the third larval instar are expressed either in narrow stripes corresponding to vein primordia or in broader 'provein' domains consisting of cells competent to become veins. In addition, genes specifying the alternative intervein cell fate are expressed in complementary intervein regions. The regulatory relationships between genes expressed in narrow vein primordia, in broad provein stripes and in interveins remains unknown, however. In this manuscript, we provide additional evidence for veins forming in narrow stripes at borders of A/P sectors. These experiments further suggest that narrow vein primordia produce secondary short-range signal(s), which activate expression of provein genes in a broad pattern in neighboring cells. We also show that crossregulatory interactions among genes expressed in veins, proveins and interveins contribute to establishing the vein-versus-intervein pattern, and that control of gene expression in vein and intervein regions must be considered on a stripe-by-stripe basis. Finally, we present evidence for a second set of vein-inducing boundaries lying between veins, which we refer to as paravein boundaries. We propose that veins develop at both vein and paravein boundaries in more 'primitive' insects, which have up to twice the number of veins present in Drosophila. We present a model in which different A/P boundaries organize vein-specific genetic programs to govern the development of individual veins.

The spalt gene links the A/P compartment boundary to a linear adult structure in the Drosophila wing.

During Drosophila embryogenesis, each segment is subdivided into an anterior and a posterior compartment through the action of the engrailed gene. Compartmental boundaries bisect imaginal disc primordia which give rise to adult appendages. In early larval development, a short-range Hedgehog signal originating from the posterior compartment of the imaginal wing disc activates expression of genes including decapentaplegic (dpp) in a stripe running along the anterior-posterior compartment boundary. Secreted Dpp emanating from the A/P boundary of wing discs then acts as a secondary signal to organize the wing over large distances. The transcription factor encoded by spalt major (salm) gene, which is expressed in a broad wedge centered over the dpp stripe, is one target of Dpp signaling. In this manuscript, we show that the anterior edge of the salm expression domain abuts a narrow stripe of rhomboid (rho)-expressing cells corresponding to the L2 longitudinal vein primordium. hh mis-expression along the anterior wing margin induces a surrounding domain of salm expression, the anterior edge of which abuts a displaced rho L2 stripe. salm plays a key role in defining the position of the L2 vein since loss of salm function in mosaic patches induces the formation of ectopic L2 branches, which comprise salm- cells running along clone borders where salm- cells confront salm+ cells. These data suggest that salm determines the position of the L2 vein primordium by activating rho expression in neighboring cells through a locally non-autonomous mechanism. rho then functions to initiate and maintain vein differentiation. We discuss how these data provide the final link connecting the formation of a linear adult structure to the establishment of a boundary by the maternal Bicoid morphogen gradient in the blastoderm embryo.

The Drosophila decapentaplegic and short gastrulation genes function antagonistically during adult wing vein development.

TGF-beta-related signaling pathways play diverse roles during vertebrate and invertebrate development. A common mechanism for regulating the activity of TGF-beta family members is inhibition by extracellular antagonists. Recently, the Drosophila short gastrulation (sog) gene was shown to encode a predicted diffusible factor which antagonizes signaling mediated by the TGF-beta-like Decapentaplegic (Dpp) pathway in the early blastoderm embryo. sog and dpp, which are among the earliest zygotic genes to be activated, are expressed in complementary dorsal-ventral domains. The opposing actions of sog and dpp in the early embryo have been highly conserved during evolution as their vertebrate counterparts, chordin and BMP-4, function homologously to define neural versus non-neural ectoderm in Xenopus. Here we exploit the genetically sensitive adult wing vein pattern to investigate the generality of the antagonistic relationship between sog and dpp. We show that dpp is expressed in vein primordia during pupal wing development and functions to promote vein formation. In contrast, sog is expressed in complementary intervein cells and suppresses vein formation. sog and dpp function during the same phenocritical periods (i.e. 16-28 hours after pupariation) to influence the vein versus intervein cell fate choice. The conflicting activities of dpp and sog are also revealed by antagonistic dosage-sensitive interactions between these two genes during vein development. Analysis of vein and intervein marker expression in dpp and sog mutant wings suggests that dpp promotes vein fates indirectly by activating the vein gene rhomboid (rho), and that sog functions by blocking an autoactivating Dpp feedback loop. These data support the view that Sog is a dedicated Dpp antagonist.

The Drosophila rhomboid protein is concentrated in patches at the apical cell surface.

Patterned expression of the Drosophila rhomboid (rho) gene is thought to promote signaling by the EGF receptor (EGFR) in specific cell types. In this report we examine the subcellular localization of the Rhomboid protein (Rho) which is predicted to be an integral membrane protein. At the light level, immunocytochemical staining for Rho reveals a small number of large patches (or plaques) at or near the apical cell surface. In some cells Rho plaques colocalize with Armadillo at adherens junctions, while in other cells plaques are only found basal to the adherens junction. Immunoelectron microscopy reveals that Rho plaques are composed of a highly localized patch of plasma membrane and a densely staining underlying structure. Concentration of Rho in distinct plaques depends on a balance of synthesis and membrane recycling since increasing the amount of rho expression or blocking membrane recycling leads to more uniform cell surface labeling. A limiting cellular component also appears to be required for concentrating Rho in plaques. We discuss clustering of Rho in plasma membrane patches with respect to the proposed role of Rho in promoting EGF-R signaling.

scratch, a pan-neural gene encoding a zinc finger protein related to snail, promotes neuronal development.

The Drosophila scratch (scrt) gene is expressed in most or all neuronal precursor cells and encodes a predicted zinc finger transcription factor closely related to the product of the mesoderm determination gene snail (sna). Adult flies homozygous for scrt null alleles have a reduced number of photoreceptors in the eye, and embryos lacking the function of both scrt and the pan-neural gene deadpan (dpn), which encodes a basic helix-loop-helix (bHLH) protein, exhibit a significant loss of neurons. Conversely, ectopic expression of a scrt transgene during embryonic and adult development leads to the production of supernumerary neurons. Consistent with scrt functioning as a transcription factor, various genes are more broadly expressed than normal in scrt null mutants. Reciprocally, these same genes are expressed at reduced levels in response to ectopic scrt expression. We propose that scrt promotes neuronal cell fates by suppressing expression of genes promoting non-neuronal cell fates. We discuss the similarities between the roles of the ancestrally related scrt, sna, and escargot (esc) genes in regulating cell fate choices.

Analysis of the genetic hierarchy guiding wing vein development in Drosophila.

The Drosophila rhomboid (rho) and Egf-r genes are members of a small group of genes required for the differentiation of various specific embryonic and adult structures. During larval and early pupal development expression of rho in longitudinal vein primordia mediates the localized formation of wing veins. In this paper we investigate the genetic hierarchy guiding vein development, by testing for genetic interactions between rho alleles and a wide variety of wing vein mutations and by examining the pattern of rho expression in mutant developing wing primordia. We identify a small group of wing vein mutants that interact strongly with rho. Examination of rho expression in these and other key vein mutants reveals when vein development first becomes abnormal. Based on these data and on previous genetic analyses of vein formation we present a sequential model for establishment and differentiation of wing veins.

Down-regulation of Drosophila Egf-r mRNA levels following hyperactivated receptor signaling.

Internalization of ligand-receptor complexes is a well-documented mechanism for limiting the duration and magnitude of a signaling event. In the case of the EGF-Receptor (EGF-R), exposure to EGF or TGF-alpha results in internalization of up to 95% of the surface receptor pool within 5 minutes of exposure to ligand. In this report, we show that levels of Drosophila Egf-r mRNA are strongly down-regulated in epidermal cells likely to have recently undergone high levels of EGF-R signaling. The cells in which Egf-r mRNA levels are down-regulated express the rhomboid gene, which is thought to locally amplify EGF-R signaling. Widespread Egf-r mRNA down-regulation can be induced by ubiquitous expression of rhomboid or by eliminating the Gap1 gene. These results suggest that cells engaged in intense EGF-R/RAS signaling limit the duration of the signal through a combination of short-acting negative feedback mechanisms such as receptor internalization followed by a longer lasting reduction in receptor transcript levels. Control of Egf-r mRNA levels by altering transcription or mRNA stability is a new tier of regulation to be considered in analysis of EGF-R signaling during development.

Blistered: a gene required for vein/intervein formation in wings of Drosophila.

We have characterized the blistered (bs) locus phenotypically, genetically and developmentally using a set of new bs alleles. Mutant defects range from wings with ectopic veins and intervein blisters to completely ballooned wings where the distinction between vein and intervein is lost. Mosaic analyses show that severe bs alleles behave largely autonomously; homozygous patches having vein-like properties. Developmental analyses were undertaken using light and electron microscopy of wild-type and bs wings as well as confocal microscopy of phalloidin- and laminin-stained preparations. bs defects were first seen early in the prepupal period with the failure of apposition of dorsal and ventral wing epithelia. Correspondingly, during definitive vein/intervein differentiation in the pupal period (18-36 hours after puparium formation), the extent of dorsal/ventral reapposition is reduced in bs wings. Regions of the wing that fail to become apposed differentiate properties of vein cells; i.e. become constricted apically and acquire a laminin-containing matrix basally. To further understand bs function, we examined genetic interactions between various bs alleles and mutants of two genes whose products have known functions in wing development. (i) rhomboid, a component of the EGF-R signalling pathway, is expressed in vein cells and is required for specification of vein cell fate. rhove mutations (lacking rhomboid in wings) suppress the excess vein formation and associated with bs. Conversely, rho expression in prepupal and pupal bs wings is expanded in the regions of increased vein formation. (ii) The integrin genes, inflated and myospheroid, are expressed in intervein cells and are required for adhesion between the dorsal and ventral wing surfaces. Loss of integrin function results in intervein blisters. Integrin mutants interact with bs mutants to increase the frequency of intervein blisters but do not typically enhance vein defects. Both developmental and genetic analyses suggest that the bs product is required during metamorphosis for the initiation of intervein development and the concomitant inhibition of vein development.

New functions of the Drosophila rhomboid gene during embryonic and adult development are revealed by a novel genetic method, enhancer piracy.

Localized expression of the Drosophila rhomboid (rho) gene has been proposed to hyperactivate EGF-Receptor signaling in specific cells during development of the embryo and adult. In this report we use a novel transposon based genetic method, enhancer piracy, to drive ectopic expression of a rho cDNA transgene by endogenous genomic enhancers. Many enhancer piracy transposon-rho insertions cause dominant phenotypes, over half of which cannot be duplicated by ubiquitous expression of rho. Genetic interactions between various dominant enhancer piracy alleles and mutations in the EGF-R/RAS signaling pathway indicate that many of these novel phenotypes result from ectopic activation of EGF-R signaling. Patterned mis-expression of the rho cDNA transgene correlates in several cases with localized dominant enhancer piracy phenotypes. Enhancer piracy lines reveal an unanticipated role for rho in imaginal disc formation and provide the first evidence that mis-expression of rho is sufficient for converting entire intervein sectors into veins. Enhancer piracy may prove to be a general strategy for obtaining dominant alleles of a gene of interest in diverse insects, worms, plants, and potentially in vertebrates such as mice and fish.

The Drosophila rhomboid gene mediates the localized formation of wing veins and interacts genetically with components of the EGF-R signaling pathway.

The rhomboid (rho) gene, which encodes a transmembrane protein, is a member of a small group of genes (ventrolateral genes) required for the differentiation of ventral epidermis in the Drosophila embryo. The ventrolateral genes include spitz, which encodes an EGF-like ligand, and Star. The receptor for spitz may be the gene encoding the Drosophila epidermal growth factor-receptor (Egf-r) because the phenotype resulting from partial loss of function of Egf-r is similar to that of ventrolateral group mutants. Among ventrolateral genes encoding cell-surface or secreted proteins, rho is the only member expressed in a localized pattern corresponding to cells requiring the activity of the ventrolateral pathway. In this paper we provide evidence that spatial localization of rho plays an analogous role in establishing vein pattern in the adult wing. rho is expressed in early wing disc cells likely to be wing vein primordia and later is sharply restricted to developing veins. Flies homozygous for the viable rho(ve) allele have missing veins and rho fails to be expressed in rho(ve) mutant wing discs. Ectopic expression of rho during wing development leads to the formation of extra veins. Gene dosage studies among ventrolateral genes suggest that the rho product (Rho) may facilitate Spi-EGF-R signaling, resulting in activation of RAS. We discuss models for how localized expression of Rho may amplify signaling mediated by ubiquitously distributed ligand and receptor components.

Premenstrual tension and functional infertility. Aetiology and treatment.

17 women with premenstrual symptoms received bromocriptine (CB 154) and placebo in a double-blind crossover manner. 5 because pregnant and 10 who completed 2 cycles showed significant improvement in breast symptoms, oedema, weight gain, and mood with bromocriptine. Prolactin concentrations were suppressed. In 34 women with premenstrual symptoms, who had been warned of possible increased fertility, bromocriptine 2-5 mg twice daily from the 10th day of the menstrual cycle for 1--11 months gave marked or complete relief. 45 women attending the infertility clinic took 2-5 mg bromocriptine twice daily for 186 cycles; 23 became pregnant, 2 had marked relief and 20 complete relief from premenstrual symptoms. The relief of premenstrual symptoms by bromocriptine may be due to suppression of prolactin concentrations, which may be a major factor in premenstrual syndrome.