A genetic screen for modifiers of a kinase suppressor of Ras-dependent rough eye phenotype in Drosophila

kinase suppressor of Ras (ksr) encodes a putative protein kinase that by genetic criteria appears to function downstream of RAS in multiple receptor tyrosine kinase (RTK) pathways. While biochemical evidence suggests that the role of KSR is closely linked to the signal transduction mechanism of the MAPK cascade, the precise molecular function of KSR remains unresolved. To further elucidate the role of KSR and to identify proteins that may be required for KSR function, we conducted a dominant modifier screen in Drosophila based on a KSR-dependent phenotype. Overexpression of the KSR kinase domain in a subset of cells during Drosophila eye development blocks photoreceptor cell differentiation and results in the external roughening of the adult eye. Therefore, mutations in genes functioning with KSR might modify the KSR-dependent phenotype. We screened approximately 185,000 mutagenized progeny for dominant modifiers of the KSR-dependent rough eye phenotype. A total of 15 complementation groups of Enhancers and four complementation groups of Suppressors were derived. Ten of these complementation groups correspond to mutations in known components of the Ras1 pathway, demonstrating the ability of the screen to specifically identify loci critical for Ras1 signaling and further confirming a role for KSR in Ras1 signaling. In addition, we have identified 4 additional complementation groups. One of them corresponds to the kismet locus, which encodes a putative chromatin remodeling factor. The relevance of these loci with respect to the function of KSR and the Ras1 pathway in general is discussed.     

A genetic screen for synaptic transmission mutants mapping to the right arm of chromosome 3 in Drosophila

Neuronal function depends upon the proper formation of synaptic connections and rapid communication at these sites, primarily through the regulated exocytosis of chemical neurotransmitters. Recent biochemical and genomic studies have identified a large number of candidate molecules that may function in these processes. To complement these studies, we are pursuing a genetic approach to identify genes affecting synaptic transmission in the Drosophila visual system. Our screening approach involves a recently described genetic method allowing efficient production of mosaic flies whose eyes are entirely homozygous for a mutagenized chromosome arm. From a screen of 42,500 mutagenized flies, 32 mutations on chromosome 3R that confer synaptic transmission defects in the visual system were recovered. These mutations represent 14 complementation groups, of which at least 9 also appear to perform functional roles outside of the eye. Three of these complementation groups disrupt photoreceptor axonal projection, whereas the remaining complementation groups confer presynaptic defects in synaptic transmission without detectably altering photoreceptor structure. Mapping and complementation testing with candidate mutations revealed new alleles of the neuronal fate determinant svp and the synaptic vesicle trafficking component lap among the collection of mutants recovered in this screen. Given the tools available for investigation of synaptic function in Drosophila, these mutants represent a valuable resource for future analysis of synapse development and function.     

The Doublesex Locus of Drosophila Melanogaster and Its Flanking Regions: A Cytogenetic Analysis

The region of the third chromosome (84D-F) of Drosophila melanogaster that contains the doublesex (dsx) locus has been cytogenetically analyzed. Twenty nine newly induced, and 42 preexisting rearrangements broken in dsx and the regions flanking dsx have been cytologically and genetically characterized. These studies established that the dsx locus is in salivary chromosome band 84E1-2. In addition, these observations provide strong evidence that the dsx locus functions only to regulate sexual differentiation and does not encode a vital function. To obtain new alleles at the dsx locus and to begin to analyze the genes flanking dsx, 59 lethal and visible mutations in a region encompassing dsx were induced. These mutations together with preexisting mutations in the region were deficiency mapped and placed into complementation groups. Among the mutations we isolated, four new mutations affecting sexual differentiation were identified. All proved to be alleles of dsx, suggesting that dsx is the only gene in this region involved in regulating sexual differentiation. All but one of the new dsx alleles have equivalent effects in males and females. The exception, dsxEFH55, strongly affects female sexual differentiation, but only weakly affects male sexual differentiation. The interactions of dsxEFH55 with mutations in other genes affecting sexual differentiation are described. These results are discussed in terms of the recent molecular findings that the dsx locus encodes sex-specific proteins that share in common their amino termini but have different carboxyl termini. The 72 mutations in this region that do not affect sexual differentiation identify 25 complementation groups. A translocation, T(2;3)Es that is associated with a lethal allele in one of these complementation groups is also broken at the engrailed (en) locus on the second chromosome and has a dominant phenotype that may be due to the expression of en in the anterior portion of the abdominal tergites where en is not normally expressed. The essential genes found in the 84D-F region are not evenly distributed throughout this region; most strikingly the 84D1-11 region appears to be devoid of essential genes. It is suggested that the lack of essential genes in this region is due to the region (1) containing genes with nonessential functions and (2) being duplicated, possibly both internally and elsewhere in the genome.     

A Screen for Genes That Function Downstream of Ras1 during Drosophila Eye Development

Cell-fate specification of the R7 photoreceptor cell is controlled by the sevenless receptor tyrosine kinase (SevRTK) and Ras1, the Drosophila homologue of mammalian H-ras, K-ras and N-ras oncogenes. An activated form of Ras1 expressed under control of the sevenless enhancer/promoter (sev-Ras1(V12)) induces production of supernumerary R7 photoreceptor cells, which causes the eye to become rough in appearance. To isolate mutations in genes functioning downstream of Ras1, we carried out a screen for dominant suppressors and enhancers of this rough eye phenotype. Approximately 850,000 mutagenized flies were screened, and 282 dominant suppressors and 577 dominant enhancers were isolated. Mutations in the Drosophila homologues of Raf, MEK, MAPK, type I Geranylgeranyl Transferase and Protein Phosphatase 2A were isolated, as were mutations in several novel signaling genes. Some of these mutant genes appear to be general signaling factors that function in other Ras1 pathways, while one seems to be more specific for photoreceptor development. At least two suppressors appear to function either between Ras1 and Raf or in parallele to Raf.     

The splicing factor Prp31 is essential for photoreceptor development in Drosophila

Retinitis pigmentosa is a leading cause of blindness and a progressive retinal disorder, affecting millions of people worldwide. This disease is characterized by photoreceptor degeneration, eventually leading to complete blindness. Autosomal dominant (adRP) has been associated with mutations in at least four ubiquitously expressed genes encoding pre-mRNA splicing factors—Prp3, Prp8, Prp31 and PAP1. Biological function of adRP-associated splicing factor genes and molecular mechanisms by which mutations in these genes cause cell-type specific photoreceptor degeneration in humans remain to be elucidated. To investigate the in vivo function of these adRP-associated splicing factor genes, we examined Drosophila in which expression of fly Prp31 homolog was down-regulated. Sequence analyses show that CG6876 is the likely candidate of Drosophila melanogaster Prp31 homolog (DmPrp31). Predicted peptide sequence for CG6876 shows 57% similarity to the Homo sapiens Prp31 protein (HsPrp31). Reduction of the endogenous Prp31 by RNAi-mediated knockdown specifically in the eye leads to reduction of eye size or complete absence of eyes with remarkable features of photoreceptor degeneration and recapitulates the bimodal expressivity of human Prp31 mutations in adRP patients. Such transgenic DmPrp31RNAi flies provide a useful tool for identifying genetic modifiers or interacting genes for Prp31. Expression of the human Prp31 in these animals leads to a partial rescue of the eye phenotype. Our results indicate that the Drosophila CG6876 is the fly ortholog of mammalian Prp31 gene.     

Characterization of New Punch Mutations: Identification of Two Additional Mutant Classes

The Punch locus of Drosophila melanogaster which encodes the pteridine biosynthetic enzyme, GTP cyclohydrolase, is genetically complex. Lethal alleles of the locus resolve into an array of interallelic complementation groups, and at least one class of mutations is developmentally specific, affecting GTP cyclohydrolase activity only in the heads of adults. All previously isolated Punch alleles were identified on the basis of a mutant eye color phenotype. By screening mutagenized chromosomes over Punch region deficiencies, we have now isolated new alleles on the basis of lethal and visible phenotypes. Most of these alleles fall into previously identified genetic classes, but two new classes of mutations were also found. One class contains two alleles that behave as dominant lethal mutations in some genetic backgrounds. The other class represents a second developmentally specific set of alleles that affect the function of the Punch locus only during embryogenesis.     

Function for Hedgehog genes in zebrafish retinal development

The hedgehog (hh) genes encode secreted signaling proteins that have important developmental functions in vertebrates and invertebrates. In Drosophila, expression of hh coordinates retinal development by propagating a wave of photoreceptor differentiation across the eye primordium. Here we report that two vertebrate hh genes, sonic hedgehog (shh) and tiggy-winkle hedgehog (twhh), may perform similar functions in the developing zebrafish. Both shh and twhh are expressed in the embryonic zebrafish retinal pigmented epithelium (RPE), initially in a discrete ventral patch which then expands outward in advance of an expanding wave of photoreceptor recruitment in the subjacent neural retina. A gene encoding a receptor for the hedgehog protein, ptc-2, is expressed by retinal neuroepithelial cells. Injection of a cocktail of antisense (alphashh/alphatwhh) oligonucleotides reduces expression of both hh genes in the RPE and slows or arrests the progression of rod and cone photoreceptor differentiation. Zebrafish strains known to have mutations in Hh signaling pathway genes similarly exhibit retardation of photoreceptor differentiation. We propose that hedgehog genes may play a role in propagating photoreceptor differentiation across the developing eye of the zebrafish.     

A role for the deep orange and carnation eye color genes in lysosomal delivery in Drosophila.

Deep orange and carnation are two of the classic eye color genes in Drosophila. Here, we demonstrate that Deep orange is part of a protein complex that localizes to endosomal compartments. A second component of this complex is Carnation, a homolog of Sec1p-like regulators of membrane fusion. Because complete loss of deep orange function is lethal, the role of this complex in intracellular trafficking was analyzed in deep orange mutant clones. Retinal cells devoid of deep orange function completely lacked pigmentation and exhibited exaggerated multivesicular structures. Furthermore, a defect in endocytic trafficking was visualized in developing photoreceptor cells. These results provide direct evidence that eye color mutations of the granule group also disrupt vesicular trafficking to lysosomes.     

Star is required in a subset of photoreceptor cells in the developing Drosophila retina and displays dosage sensitive interactions with rough.

We report that mutations at the Star locus act as dominant enhancers of the eye phenotype displayed by flies carrying a null allele of rough. Our analysis of double mutants at different stages of eye development suggests that this phenotype results from defects in the early stages of photoreceptor cell differentiation in the eye imaginal disc. Complete loss of Star function during retinal development, analyzed in mosaic animals, results in cell death, visible as scars in the adult eye. The requirement for wild-type Star function, however, is confined to only a subset of photoreceptor cells, R8, R2, and R5, which are the first three cells to differentiate neurally in the developing retina. These results suggest an essential role for the Star gene in the initial events of ommatidial cluster formation during the development of the Drosophila compound eye.     

Genetic analysis of mutations at the Glued locus and interacting loci in Drosophila melanogaster

A genetic analysis of the dominant mutation Glued that perturbs the development of the normal axonal architecture of the fly's visual system was undertaken. Ten new alleles at this locus were identified and characterized. Two complementation groups that were identified failed to complement the original allele, suggesting that it is a double mutant or that it resides at a complex locus. Several of the new alleles display visual-system abnormalities similar to those of the original mutation. Seven of the eight members of one complementation group are embryonic/early larval lethals, like the original mutation. The other allele in this group is temperature sensitive. Homozygous mutant adults exhibit a temperature-sensitive female sterile phenotype. Unsuccessful attempts to recover genetic mosaics carrying clones of cells homozygous for some of these mutations revealed that they are either essential for the viability of individual cells or that they affect some other fundamental cellular function, such as mitosis or the ability to participate in tissue level organization, which prevents them from being recovered in adult mosaics. This also indicates that these mutations do not specifically affect neural cells. A number of X-ray- and EMS-induced partial and complete phenotypic "revertants" of the original allele have also been isolated as material for a comparative analysis of visual system development. All "revertants" that alter the abnormal eye phenotype towards the wild type have similar impact on the organization of the optic lobe.     

The Drosophila roughened mutation: activation of a rap homolog disrupts eye development and interferes with cell determination

Roughened is a dominant mutation of D. melanogaster that disrupts eye development. The majority of the ommatidia in the adult eye lack a single photoreceptor cell, which is most commonly the R7 cell. The Roughened mutation disrupts the early stages of photoreceptor cell determination. Roughened is a dominant gain-of-function mutation that results from a single amino acid change (Phe157 to Leu) in the Drosophila Rap1 protein. Loss of function Rap1 mutations are lethal. Drosophila Rap1 protein is 88% identical to human rap1A/K-rev1 protein, a putative antagonist of ras action.     

Drosophila JAB1/CSN5 acts in photoreceptor cells to induce glial cells.

Different classes of photoreceptor neurons (R cells) in the Drosophila compound eye form connections in different optic ganglia. The R1-R6 subclass connects to the first optic ganglion, the lamina, and relies upon glial cells as intermediate targets. Conversely, R cells promote glial cell development including migration of glial cells into the target region. Here, we show that the JAB1/CSN5 subunit of the COP9 signalosome complex is expressed in R cells, accumulates in the developing optic lobe neuropil, and through the analysis of a unique set of missense mutations, is required in R cells to induce lamina glial cell migration. In these CSN5 alleles, R1-R6 targeting is disrupted. Genetic analysis of protein null alleles further revealed that the COP9 signalosome is required at an earlier stage of development for R cell differentiation.     

A Screen for Modifiers of Deformed Function in Drosophila

Proteins produced by the homeotic genes of the Hox family assign different identities to cells on the anterior/posterior axis. Relatively little is known about the signalling pathways that modulate their activities or the factors with which they interact to assign specific segmental identities. To identify genes that might encode such functions, we performed a screen for second site mutations that reduce the viability of animals carrying hypomorphic mutant alleles of the Drosophila homeotic locus, Deformed. Genes mapping to six complementation groups on the third chromosome were isolated as modifiers of Deformed function. Products of two of these genes, sallimus and moira, have been previously proposed as homeotic activators since they suppress the dominant adult phenotype of Polycomb mutants. Mutations in hedgehog, which encodes secreted signalling proteins, were also isolated as Deformed loss-of-function enhancers. Hedgehog mutant alleles also suppress the Polycomb phenotype. Mutations were also isolated in a few genes that interact with Deformed but not with Polycomb, indicating that the screen identified genes that are not general homeotic activators. Two of these genes, cap `n' collar and defaced, have defects in embryonic head development that are similar to defects seen in loss of function Deformed mutants.     

A Genetic Analysis of the Drosophila Closely Linked Interacting Genes Bulge, Argos and Soba

The Drosophila gene argos encodes a diffusible protein that acts as a negative regulator of cell fate decisions. To define interacting gene products, we performed a genetic analysis of argos, which suggests the presence of several partially redundant gene functions in its immediate vicinity at the chromosomal position 73A. Dose titration experiments have identified two of these loci. One of them corresponds to the gene bulge. Loss of function bulge alleles suppress the rough eye phenotype associated with overexpression of argos; conversely, amorphic argos mutations suppress the eye phenotype seen in flies bearing a single dominant bulge allele. Recombination mapping localized bulge 0.15 cM distal to argos. A second gene, suppressor of bulge and argos (soba), corresponds to the recently described lethal complementation group 73Aj. soba alleles suppress the eye phenotypes seen in flies expressing either the dominant bulge allele or the hs-argos construct. soba resides 120 kb proximal to argos. In addition, we have identified one allele of a new gene, clown, which like soba suppresses the eye phenotypes associated with hs-argos and bulge(Dominant). clown maps on chromosome 3 at the cytological position 68CD.     

pipsqueak encodes a novel nuclear protein required downstream of seven-up for the development of photoreceptors R3 and R4.

Photoreceptor induction in the Drosophila eye is mediated by activation of the Ras signal transduction cascade. Although this process is well understood, little is known about how the diversity of photoreceptor subtypes is generated. The pipsqueak (psq) gene is expressed at high levels in the R3/R4 precursors during eye development and this expression depends on seven-up (svp) gene function. Moreover, strong psq alleles are dominant suppressors of a svp-induced cone cell transformation phenotype. Although the gene was previously identified and described as a member of the maternal posterior group of genes, the strong semilethal alleles isolated here demonstrate a specific requirement for psq function downstream of svp for the development of photoreceptors R3/R4. The gene has three independent 5' ends and codes for several nuclear protein isoforms, some containing the POZ domain which has been implicated in protein-protein interactions. Interestingly, all viable alleles with a maternal posterior group phenotype cluster around one specific 5' exon, while all semilethal alleles have lesions which map to a different alternative 5' exon.     

A novel strategy for identifying mutations that sensitize Drosophila eye development to caffeine and hydroxyurea.

This report describes a novel strategy for isolating Drosophila mutants with conditional eye phenotypes that should be generally applicable for identifying genes required for cellular responses to specific drugs. To test the strategy, we screened 3 of the 5 major chromosome arms for hydroxyurea- and (or) caffeine-sensitive (huc) mutants, and isolated mutations affecting 5 different complementation groups. Most of these were represented by single alleles; however, we also isolated multiple alleles of huc(29DE) gene, an essential gene that is also associated with a nonconditional pupal lethal phenotype. We also identified huc(95E) mutants, which are extremely sensitive to caffeine. Although huc(95E) is a nonessential gene, mutant imaginal disc cells undergo caffeine-dependent apoptosis, and huc(95E) gene function is required for the viability of the organism when mutant larvae are exposed to levels of caffeine that controls can easily tolerate. We have mapped the cytological positions of huc(29D) and huc(95E) as a first step toward molecularly characterizing the relevant genes.     

Multiple regulatory genes control expression of a gene family during development of Dictyostelium discoideum.

Mutant strains of Dictyostelium discoideum carrying dis mutations fail to transcribe specifically the family of developmentally regulated discoidin lectin genes during morphogenesis. The phenotypes of these mutants strongly suggested that the mutations reside in regulatory genes. Using these mutant strains, we showed that multiple regulatory genes are required for the expression of the lectin structural genes and that these regulatory genes (the dis+ alleles) act in trans to regulate this gene family. These regulatory genes fall into two complementation groups (disA and disB) and map to linkage groups II and III, respectively. A further regulatory locus was defined by the identification of an unlinked supressor gene, drsA (discoidin restoring), which is epistatic to disB, but not disA, and results in the restoration of lectin expression in cells carrying the disB mutation. Mutant cells carrying the drsA allele express the discoidin lectin gene family during growth and development, in contrast to wild-type cells which express it only during development. Therefore, the suppressor activity of the drsA allele appears to function by making the expression of the discoidin lectins constitutive and no longer strictly developmentally regulated. The data indicate that normal expression of the discoidin lectins is dependent on the sequential action of the disB+, drsA+, and disA+ gene products. Thus, we described an interacting network of regulatory genes which in turn controls the developmental expression of a family of genes during the morphogenesis of D. discoideum.