A dominant modifier screen in the Drosophila eye was performed using an activated form of the calcineurin catalytic subunit to identify new targets, regulators, and functions of calcineurin. The canAact.gl rough eye phenotype is modifiable; i.e., it is sensitive to transgene dose and is specifically modified by CanB, which is essential for CanA function. An isogenic wild-type stock was prepared and canAact.gl was inserted onto the chromosome 3 balancer TM3, which carries the dominant visible marker Sb. 70,000 progeny of TM3-canAact.gl (TCAG) females and EMS- or X-ray-treated males were screened. Each individual F1 with an enhanced or suppressed TCAG rough eye was backcrossed to TCAG to confirm the modification and to determine the chromosomal location. About 21% of the modifiers initially isolated bred true, and stable lines of the confirmed modifiers were established over either TCAG or the chromosome 2 balancer CCAG (CyO-canAact.gl). Because chromosome 2 balancers, including CyO, harbor one or more suppressors of the TCAG phenotype, chromosome 2 suppressors were difficult to balance and are thus underrepresented in the final tally. Modifiers on chromosome 1 were also underrepresented, in part because the balancers carry a dominant eye mutation, Bar, that significantly interfered with scoring TCAG modification. A total of 5 viable and 123 lethal modifiers were isolated in the screen (Sullivan, 2002).
Modifier groups that act directly on the glass enhancer do not specifically modify canAact.gl. Nonspecific groups were identified by testing whether any of the complementation groups modified rough eyes caused by unrelated, glass-dependent transgenes. Two of the enhancer groups, CE3-1 and CE2-1, modified rough eye phenotypes caused by other glass-dependent transgenes, such as sinagl, a Ras pathway component, and Rho1gl. All complementation groups were additionally tested with Rasv12, phyllopod, yanact, and reaper glass-dependent transgenes, but only CE3-1 and CE2-1 modified the rough eye phenotypes caused by these transgenes (Sullivan, 2002).
The specific modifier groups were further separated into two classes by determining whether they modified the canAact.gl rough eye phenotype caused by TCAGB (TM3-canAact.gl,canBgl). Class I genes, such as Ca2+ signaling components or dephosphorylation targets, act downstream of calcineurin and will modify the rough eye phenotype of TCAG and TCAGB. However, class II groups, which act at the level of canB, such as canB or factors that regulate its expression, will modify TCAG but not TCAGB. Only two groups, CE3-3 and CS2-1, failed to modify TCAGB. Class I and class II modifier groups were mapped by meiotic recombination and by failure to complement deficiencies. The results from both methods were used to estimate the cytological map position of each group (Sullivan, 2002).
Meiotic mapping localized the class II group CS2-1 to 44A;50B, and deficiencies refined the region to 42B3;43E18. Polytene chromosome analysis revealed a large deficiency in CS2-1128 that uncovered 43E6;44B1. CS2-187 was an inversion with breakpoints at 43A1-2 and 43E13-18. The left breakpoint of CS2-187 fails to complement two independent alleles of prickle (pk), a gene in 43A1 that is required for tissue polarity in the wing, haltere, and notum. However, the other CS2-1 alleles complement pk alleles, the pk mutation in CS2-187 is viable, and pk does not modify TCAG. The right inversion breakpoint, 43E13-18, is lethal when uncovered by deficiencies in the 43E region, and these deficiencies also fail to complement other CS2-1 alleles. Thus, the right breakpoint of the CS2-187 inversion corresponds to the CS2-1 TCAG suppressor (Sullivan, 2002).
One of the canB genes, CanB2, maps to 43E16 and was a strong candidate for CS2-1. The CS2-1128 deficiency uncovered canB2, since CS2-1128/CyO,cn flies were cn, indicating that the deficiency breaks to the left of cn. PCR on genomic DNA from homozygous CS2-187 flies revealed that the right breakpoint of the insertion occurred between base pair positions -452 and +60, relative to the canB2 start of transcription. The gene on the left side of the breakpoint, cn, was not disrupted in CS2-187, because CS2-187/CyO,cn flies are cn+. Additionally, Western blots of homozygous CS2-187, CS2-1128, and CS2-1180 larvae that were probed with canB antibodies revealed that, compared to similarly staged controls, total canB protein levels are reduced in CS2-1 alleles (Sullivan, 2002).
Rescue was carried out using the UAS-GAL4 system on CS2-1180, which has no detectable lesions aside from canB2 and has a late larval/pupal lethal stage. CS2-1180/CyO;GAL4hs flies were crossed to CS2-1180/CyO;canB-4FUAS, and the GAL4hs/canB-4FUAS progeny were screened for CS2-1180 (i.e., Cy+) animals. Heat shock was not used because basal GAL4hs activity at 25° induces UAS transgenes at a low level. In three independent crosses, the percentage of CS2-1180 adults was increased from <0.2% to an average of 11%. Thus, ectopic canB-4F successfully rescues the lethality associated with CS2-1180 (Sullivan, 2002).
Deficiency mapping localized CS3-3 to 63C6;63E, and the X-ray allele CS3-3154 had a deletion spanning 63C2-5;63E1-4. The EMS allele CS3-3518 failed to complement styDelta5 and styDelta64, which are hypomorphic alleles of sprouty, a gene in 63D1. In addition, the sty alleles were able to suppress TCAG. Sequencing the sty ORF from CS3-3518 revealed that the codon corresponding to glutamine residue 250 was mutated into a stop codon. This mutation removes one of the two sty homology domains and the cysteine-rich region and is predicted to be nonfunctional. Sprouty is a negative regulator of RTK signaling in Drosophila, including Fgf receptor and Egf receptor signaling. Sprouty protein can bind to the E3 ligase Cbl, the adaptor protein Drk, and Gap1 and has been proposed to facilitate Gap1 inactivation of Ras. A single gene in flies, sprouty, has at least five homologs in mammalian genomes (Sullivan, 2002).
Increased signaling through the Egf receptor, caused by either the presence of ectopic Egfr/Ras signaling components or hypomorphic mutations in negative regulators, results in the development of extra photoreceptors and wing vein material. Conversely, a decrease in Egf receptor signaling reduces both wing vein formation and the number of photoreceptor cells. Ectopic canAact causes defects in eye and wing vein development consistent with repression of Egfr/Ras signaling. Misexpression of canAact in the posterior compartment of the developing wing by using GAL4en results in a truncation of wing veins and a decrease in compartment size. GAL4sev drives expression in the presumptive R3, R4, and R7 photoreceptor cells, as well as in cone cells. Sections of GAL4sev/canAact.UAS eyes reveal a decrease in the number of photoreceptor cells per ommatidium. Usually the missing cell is R7, but occasionally R3 or R4 are also absent. In addition, TCAG and TCAGB discs were examined for a decrease in active MAP kinase levels by using an antibody specific for the diphosphorylated, active form of MAP kinase (Sigma, M8159). However, posterior to the furrow the levels of active MAP kinase were too low to reliably detect any effect of activated calcineurin on signaling output from the Ras pathway (Sullivan, 2002).
To examine the interaction between calcineurin and individual components of the Egfr pathway, the ability of mutations in these components to modify the activated calcineurin phenotype was tested. Hypomorphic mutations in Egfr, Ras, pnt, sty, Gap1, and small wing modified activated calcineurin, although this was not the case for most downstream components of the Egfr pathway. TCAGB is enhanced by removing one copy of Egfr, Ras, or pnt and was suppressed by Gap1 and small wing. Both TCAGB and TCAG suppress the rough eye caused by hypermorphic Egfr alleles: flies that have one copy of EgfrE1 and TCAGB have a rough eye that closely resembles that of TCAGB alone. TCAG is not detectably modified by hypomorphic Egfr, Ras, or pnt alleles. Aside from CS3-3, none of the modifier groups corresponded to Egf receptor/Ras signaling components that genetically interact with TCAG. However, it is possible that these genes are present among the 61 single hits, which have not been characterized (Sullivan, 2002).
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date revised: 20 March 2007
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