Antibodies directed against epitopes located between residues 1472 and 1673 in DAKAP550/Rugose were produced in rabbits and purified by affinity chromatography. In adult Drosophila, DAKAP550 accumulates preferentially in anterior tissues. The concentration of DAKAP550 in cytosol derived from fly heads is ~10-fold higher than the level of anchor protein in extracts of fly bodies. Binding assays also detected a 550-kDa protein, which accounts for a very high proportion of total RII tethering activity in adult Drosophila. Like the immunoreactive DAKAP550 polypeptide, the 550-kDa RIIß/RIIalpha-binding protein is isolated in cytosol and enriched ~8-12-fold in anterior tissues (head). Thus, DAKAP550 appears to be the principal PKA anchoring protein in mature flies (Han, 1997).
Affinity-purified IgGs directed against DAKAP550 were employed to monitor the expression and location of the anchor protein during Drosophila development. During early embryogenesis DAKAP550 was evident in most cells. From gastrulation onward the anchor protein seemed to be ubiquitous, but DAKAP550 levels differ markedly in distinct tissues. For example, at gastrulation anchor protein content is elevated in the ventral furrow and adjacent mesectodermal cells. DAKAP550 immunoreactivity remains high in mesoderm for several hours, and was also elevated in neuroblasts delaminating from the ectoderm during stages 8-9. Some DAKAP550 appeared to be in the nuclei of several neuroblasts. Late in embryogenesis DAKAP550 is reduced in epidermis and skeletal muscle and becomes maximal in ventral nerve cord. Peripheral neurons, subsets of central neurons, hindgut, the tracheal system and salivary gland, also have substantial levels of DAKAP550 at this stage. Postembryonically, DAKAP550 is detected in imaginal discs and primordia for various adult tissues. The anchor protein is especially-enriched in neural cells such as photoreceptor neurons of the developing eye. In embryonic hindgut, trachea, and salivary gland, DAKAP550 is selectively concentrated in the apical portion of highly-polarized cells. In the photoreceptor neurons DAKAP550 is also concentrated apically; in addition, a basal localization is noted for the anchor protein in cytoplasmic granules in the same cells. Granular staining was also seen in cells of the larval brain (Han, 1997).
Synthesized antisense single-strand probes from the RgcD10 cDNA were used to follow the expression of rugose mRNA. The expression of rugose mRNA is dynamic during development and was detected in all the embryonic stages. The early expression at stage 5 showed enrichment anteriorly and expression was also seen ventrally in the posterior regions. The late stage 5 showed strong expression in the region of the cephalic furrow formation. During stage 6, the anterior localization of the message is strong including the cephalic furrow region. At stage 7, rg expression becomes maximized in the region of the anterior midgut and persists through stage 8. During the later stages of development (stages 13–17), the expression is pronounced in the developing nervous system. The RgcD10 expression is also seen in the antenna-maxillary complex. In the developing eye imaginal disc, RgcD10 is expressed throughout the disc and in the region of the morphogenetic furrow. The expression of the RgcD10 transcript is highly reduced or absent in the rg mutant eye discs. The rg transcript expression patterns seen in these experiments are similar to the rg expression pattern reported by Schreiber (2002) and consistent with the DAKAP expression patterns reported by Han in 1997 (Shamloula, 2002).
Hairless was identified as antagonist in the Notch signaling pathway based on genetic interactions. Molecularly, Hairless inhibits Notch target gene activation by directly binding to the Notch signal transducer Su(H). Additional functional domains apart from the Su(H) binding domain, however, suggest additional roles for the Hairless protein. To further understanding of Hairless functions, a genetic screen was performed for modifiers of a rough eye phenotype caused by overexpression of Hairless during eye development. A number of enhancers were identified that comprise mutations in components of Notch- and Egfr-signaling pathways, some unknown genes and the gene rugose. Mutant alleles of rugose display manifold genetic interactions with mutants in Notch and Egfr signaling pathway components. Accordingly, the rugose eye phenotype is rescued by Hairless and enhanced by Delta. Molecularly, interactions might occur at the protein level because rugose appears not to be a direct transcriptional target of Notch (Schreiber, 2002).
Although several alleles of the rg mutation have been described in the literature, all but rg1 are extinct. rg1 is a hypomorphic allele and the rough eye phenotype is temperature sensitive. At 17° the eyes are almost normal with a very slight rough appearance, whereas at 25° the eyes are moderately rough. When flies are grown at 29° the eyes become severely rough. A number of rugose mutant alleles have been isolated using EMS, gamma-rays and P elements as mutagenic agents. New rg alleles were isolated on the basis of their failure to complement the rg1 allele. The severity of the eye phenotype varies in the different alleles. On the basis of the degree of roughness of the eye the mutant eyes have been classified into mild, moderate, and severe classes. From mutagenic screens, several alleles have been isolated that have severe eye phenotypes and behave as genetic nulls. Hypomorphic alleles or partial loss-of-function mutations show mild or moderate roughness of the eye. In the gamma-ray and EMS mutagenesis, 60,000 and 35,000 F1 progeny, respectively, have been screened. Six P-element-induced alleles of rg were isolated from screening 30,000 F2 progeny (Shamloula, 2002).
P-element- induced mutations can be due to either insertions or deletions caused by imprecise excision of an element. Remobilization of the P element, resulting in the reversion of the mutant phenotype, can identify insertions. Reversion analysis of the two P-induced alleles, rgP2 and rgP5, was carried out. The revertants show restoration of the cone cell number as well as the smooth eye characteristic of the wild-type eye. The remobilization of the P element also yielded several partial revertants consistent with imprecise excision of the inserted P element. These results indicate that rgP2 and rgP5 are insertion mutants (Shamloula, 2002).
Genetic interactions uncover interrelationships between components of a cellular pathway or interactions between different cellular pathways. Genetic modifier screens offer a simple and powerful method of identifying genetic interactions of the gene of interest. The rough eye phenotype of the rg mutants has been used to identify the genetic interactions of rugose. A dominant modifier F1 screen was conducted by using autosomal deficiencies from the Bloomington Stock Center deficiency kit. The hemizygous rg males were placed in double mutant combination with a single copy of the autosomal deficiency and scored for either suppression or enhancement of the rough eye phenotype. A total of 118 autosomal deficiency stocks (51 on chromosome II and 68 on chromosome III), covering ~60% of the autosomes, were screened. Genes that mapped within the deficiency breakpoints were identified and mutant alleles were obtained and tested for their interactions with various rugose mutant alleles. Effects of a 50% reduction in the dosage of the interacting locus were sought. For some of the interacting genetic loci, the effect of one copy of the gain-of-function allele was tested on rugose phenotype. To do this, transgenic lines were used carrying heat-shock promoter constructs ectopically expressing the wild-type gene product. The results suggest that rugose interacts with the components of the Drosophila EGFR- and Notch-activated signal transduction cascades (Shamloula, 2002).
Argos (aos) is a secreted protein having an EGF motif. Partial loss-of-function mutations in argos result in rough eyes with supernumerary cone and R cells and extra-wing-vein phenotypes. Complete loss-of-function mutants of argos are embryonic lethals. While loss-of-function argos mutants have increased numbers of cone cells, heat-shock promoter-driven overexpression of Argos leads to a reduction in the number of cone cells. Cell culture experiments have shown that Argos is a negative regulator of the Egfr. argos mutations act as strong suppressors of the rugose mutation. The rough eye phenotype of rg is completely suppressed by a single copy of the argos loss-of-function mutation. rg/Y; argos/+ double mutants have smooth eyes and normal complement of R cells as well as cone cells. Consistent with this result is the finding that heat-shock promoter-driven overexpression of Argos acts as a dominant enhancer of the rg mutant phenotype. In a genetic screen for second-site modifiers of the argos phenotype two interacting genes have been identified and it has been suggested that they may function in the Argos-mediated signaling pathway. Mutations in bulge and soba act as dominant suppressors of the rough eye phenotype of an argos amorphic allele as well as the rough eye phenotype caused by the heat-shock-induced overexpression of the Argos protein. A single copy of bulge and soba dominantly enhance the rough eye phenotype of the rg mutants. These results are consistent with the finding that argos mutations act as strong suppressors of rugose (Shamloula, 2002).
Star, rhomboid, and spitz belong to the 'spitz' group of genes and encode an essential function necessary for ventral midline development. In addition to the recessive lethal embryonic phenotype, S mutations are haplo-insufficient and show a dominant, rough eye phenotype. During development, S is required in a wide variety of tissues and S mutations show genetic interactions with genes from multiple signaling pathways. S encodes a putative membrane protein that, in combination with Rhomboid (rho), participates in the processing of the EGFR ligand, Spitz. In the modifier screen an S deficiency was identified as a strong enhancer of the rugose rough phenotype. A number of S alleles have been tested for their interactions with multiple alleles of rugose. Mutations at the S locus act as strong enhancers of the rugose eye phenotype and conversely heat-shock promoter-driven overexpression of the wild-type Star protein acts as a dominant suppressor of the rugose mutant phenotype. Consistent with these results, rhomboid (rho) mutations act as dominant enhancers of the rough eye phenotype of rugose mutants. rho encodes a novel intramembrane serine protease and is involved in the proteolytic processing of the EGFR ligand Spitz. A single copy of the rho mutation acts as an enhancer of the rg eye phenotype and a single copy of the hs-rho acts as a weak suppressor (Shamloula, 2002).
The Drosophila homolog of Egfr is an RTK that activates a highly conserved signal transduction cascade in a variety of tissue and cell types during Drosophila development. The activation of the Egfr is dependent on the tissue/cell type-specific ligands at the specific developmental stage. In the developing eye, Egfr function is required for the determination of all retinal cell types. A single copy of the mutations in Egfr acts as a mild enhancer of the rugose eye phenotype. In addition, Ellipse, a dominant mutation in Egfr (EgfrE), acts as a suppressor suggesting that rugose interacts with the Egfr-mediated signal cascade (Shamloula, 2002).
ras1 is a Drosophila homolog of the human ras genes (H-ras, Ki-ras, and N-ras). Ras1 is a GTPase, which functions as the key transducer in several of the receptor tyrosine kinase-activated cellular signal transduction pathways. In the developing eye, ras1 is required for the specification of photoreceptors as well as cone cells. Reduction or loss of Ras1 activity results in the failure of photoreceptor cell determination. A constitutively active form of Ras1 (Rasv12) results in the overrecruitment of retinal cells. The effects were tested of the ras1 mutations on the rg eye phenotype. A 50% reduction in ras1 activity acts as a dominant enhancer of the rg rough eye phenotype. In addition, a single copy of the dominant negative mutant form of RasN17 acts as a strong enhancer of the rg eye phenotype. In these experiments, a single copy of the constitutively active Rasv12 was a weak suppressor of the rough eye phenotype of rg. These data suggest that Ras1 and rugose interact in a dose-dependent manner and may function synergistically in retinal pattern formation (Shamloula, 2002).
Rolled is a mitogen-activated protein kinase that functions at the last step in the Ras-MAPK phosphorylation cascade. Activated Rolled activates downstream transcription factors and thus plays a key role in the Egfr-mediated signaling required for cell determination and pattern formation. rll/+; rg/Y double mutants were constructed to test for genetic interactions with rg, and a single copy of the rolled loss-of-function mutation enhances the rough eye phenotype of rg. A single copy of the dominant gain-of-function rolled mutation acts a suppressor of the rough eye phenotype. Taken together these results suggest that rugose interacts with the components of the signal cascade activated by Egfr (Shamloula, 2002).
Sparkling is a Drosophila homolog of the vertebrate pax-2 gene and is involved in cone cell specification. The runt family transcription factor, Lozenge, has been shown to directly regulate spa and Lozenge is a key downstream mediator of the Notch and Egfr pathways. A single copy of the spa mutation acts as dominant enhancer of the rg eye phenotype. These results are consistent with the data showing that rg interacts with the Egfr and Notch signaling pathways (Shamloula, 2002).
The Delta-Notch pathway is involved in a variety of cell fate decisions during development. A single copy of the Delta mutation acts as strong dominant enhancer of the rough eye phenotype of rugose. Similarly, a single copy of the Suppressor of Hairless [Su(H)] mutation also acts as an enhancer of the rugose eye phenotype. Conversely, a single copy of the Notch pathway antagonist, Hairless (H), acts as dominant suppressor of the rg phenotype. The results suggest that rugose interacts with the components of the Notch signaling pathway (Shamloula, 2002).
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date revised: 3 August 2002
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