Due to the genomic organization, the transcription of discs lost and α-spectrin must be regulated by the same promoter elements. To determine whether splicing of the two transcripts is temporally and spatially regulated, whole-mount in situ hybridization experiments were performed using digoxygenin-labeled exon-specific RNA probes. During embryonic development both genes are ubiquitously expressed, no specific differences could be detected between the expression patterns of α-spectrin and CG32315, suggesting that differential splicing is not involved in regulating the expression pattern of the two genes. Similarly, a ubiquitous expression is observed in imaginal discs, salivary glands, and fat body, and low levels of expression in the gut (Pielage, 2003).
The predicted molecular weight corresponds well to the size determined by Western blot analyses (140 kDa) using antisera generated against a bacterial expressed Discs lost GST-fusion protein. High levels of Discs lost protein were detected only following ectopic expression of an UAS-dlt construct in the daughterless or rhomboid pattern. Due to the addition of an 8× Myc tag, the Discs lost-Myc protein is slightly larger than the wild-type protein on the Western blot (rho > dltmyc). The identity of this protein band was verified by using an anti-Myc antibody. The Myc-tagged version of the protein is still able to rescue the discs lost phenotype to full viability when expressed ubiquitously during development. The specificity of the antisera was further confirmed by RNA interference (RNAi) experiments (Pielage, 2003).
Unfortunately, antisera generated against different domains of the Discs lost protein did not allow the detection of the protein in wild-type embryos. However, when discs lost was ectopically expressed using the engrailed GAL4 driver, the protein was detected in the engrailed expression domain. In these cells, the Discs lost protein is localized in the cytoplasm. Similar results were obtained when the expression of the Myc-tagged Discs lost protein was analyzed in salivary gland cells or ectodermal cells. Discs lost protein never colocalized with α-Spectrin, which demarcates the cell membrane (Pielage, 2003).
Mutant discs lost animals are characterized by a variable loss of imaginal discs. In addition, it was reported that discs lost mutations led to a disruption of cell polarity (Bhat, 1999). The variable loss of imaginal discs has been confirmed and it is noted that eye imaginal discs preferentially lack their antennal part. Because cell polarity markers such as Armadillo or the Patj protein previously assigned to the discs lost locus are still expressed in their normal apical domain in mutant epithelial cells, polarity does not appear to be disrupted. Since the discs lost alleles may not be null alleles, the phenotype of the My10 deficiency, which fails to complement discs lost mutations, was determined. Because Df(3L)My10 removes cdc37 (Bhat, 1999) as well as α-spectrin (Tanentzapf, 2000), these genes were complimented by crossing the corresponding rescue construct (P[ubi-α-spec] and P[cdc37]) into the background of the Df(3L)My10. Again, larvae with small eye discs were recovered that preferentially lacked the antenna part, suggesting that the EMS alleles are strong hypomorphic or null mutations in discs lost. These eye discs lacked expression of the Patj protein, and the severity of the imaginal disc phenotype was comparable to the phenotype of homozygous mutant discs lost larvae. α-Spectrin expression outlines regular-shaped cell clusters, suggesting that some patterning occurred in these rudimentary discs. These analyses did not indicate any function of discs lost in cell polarity, and further support the finding that discs lost does not encode the Patj protein (Pielage, 2003).
To further address whether discs lost affects cell polarity, mutant clones in the follicular epithelium were analyzed. Mutant cell clones were detected by the lack of GFP expression. α-Spectrin expression was used to assess the cellular phenotype. As observed in the homozygous mutant condition, the loss of discs lost in epithelial cells does not alter PDZ protein expression or localization, indicating that cell polarity is not affected. This result was confirmed by assaying several other markers. In discs lost mutant cell clones, the apical localization of Crumbs is unchanged. Similarly, the distribution of the cell polarity markers SAS and Armadillo was not affected (Pielage, 2003).
To assess the consequences of the loss of discs lost in the eye discs, the Minute FRT approach was used to generate large mutant cell clones. When homozygous mutant discs lost clones were generated, the mutant eyes were significantly smaller than wild-type eyes. However, this phenotype was extremely variable. Some flies were found with almost normal sized mutant eyes and in the most extreme situation a small and a large eye developed in the same fly. These phenotypes indicated that discs lost function is required to a variable extent for cell proliferation or survival (Pielage, 2003).
The reduction of eye size suggests that discs lost mutant cell clones had a growth disadvantage during larval development. To directly examine the growth behavior, discs lost mutant cell clones were generated in the absence of a Minute mutation. Heat shock-induced expression of the Flp recombinase was generated and cell proliferation was tested by BrdU labeling. discs lost mutant cells were able to incorporate BrdU normally, and the size of the mutant cell clone was comparable to the wild-type twin spot. Thus, discs lost mutant cells are at least initially able to divide at rates similar to wild-type cells. However, during pupal development, these cells must have a growth disadvantage, as mutant cell clones never appear in the adult eye (Pielage, 2003).
As observed in the follicular epithelium, discs lost mutant imaginal disc cells did not show any cell polarity defects. No alterations in the apical localization of the Patj protein were detected. Using different cellular markers, no alterations in cell polarity or shape could be detected. The DNA content of mutant cells was similar to wild-type cells (Pielage, 2003).
In the eye imaginal discs, neuronal cells differentiate following a series of inductive signaling events in the wake of the morphogenetic furrow. The induction and later the differentiation of these neuronal cells require the formation and maintenance of polarized cells. Both processes proceed normally in mutant discs lost cells. Although mutant cell clones of considerable size in the eye imaginal disc could be detected, no discs lost mutant cell clones in the adult compound eye could be found, suggesting that discs lost affects survival of differentiated cells. Only when neighboring cells carried a Minute mutation, which affects ribosomal function and thus leads to a growth disadvantage of these cells, were mutant discs lost cells able to survive to adulthood (Pielage, 2003).
The above-mentioned phenotypes tended to be relatively variable. This may be due to a perdurance of maternal Discs lost protein or RNA. To overcome the problems of RNA perdurance, discs lost function was inhibited by RNA interference (RNAi). Here, in vivo expression of double-stranded RNA (UAS-dlt RNAi) is able to interfere efficiently with endogenous gene expression. To control the efficiency of the RNAi treatment, discs lost was expressed ubiquitously using the P[ubi-discs lost] transgene to allow the detection of the Discs lost protein by polyclonal antisera. Expression of the discs lost RNAi construct in the posterior compartment of the wing imaginal disc by an engrailed GAL4 driver led to an efficient elimination of discs lost expression. Because the discs lost gene shares its first exon with α-spectrin, RNAi directed against discs lost might also interfere with α-spectrin function. However, α-Spectrin expression levels are not reduced in the discs lost RNAi-treated compartment. Furthermore, expression of discs lost RNAi during eye imaginal disc development was able to induce a phenocopy of the discs lost mutant phenotype (Pielage, 2003).
Two possible scenarios may lead to the reduction in cell number observed in all discs lost mutant animals. The proliferation of cells may be disturbed due to defects in cell cycle progression, or the loss of discs lost could lead to an increase in apoptosis. To discriminate between these possibilities, discs lost function was inhibited by RNAi in a stripe across the wing disc epithelium using a patched-GAL4 driver. Reduction of discs lost function led to a reduction in cell number in the corresponding wing domain. Whereas in wild-type wings about 18 cells separate the longitudinal veins L3 and L4, only 16 cells were found following discs lost RNAi expression. Concurrently, a slight increase in cell size was noted. This phenotype was subsequently used to quantify the effects of different genetic backgrounds (Pielage, 2003).
When one copy of discs lost was removed in this genetic background, the number of cells surviving dlt RNAi was further reduced. This result again demonstrates that the gene located in the first intron of α-spectrin corresponds to the discs lost gene. As expected, the dlt RNAi phenotype is dose dependent. When higher levels of discs lost RNAi were expressed, the number of cells was further reduced to ten and the crossvein 1 was missing. In extreme cases, necrotic cells were observed in the patched expression domain. Acridine orange staining, which labels dying cells, showed that reduction of discs lost function in the patched domain leads to cell death (Pielage, 2003).
If loss of discs lost function leads to apoptosis, the mutant discs lost phenotype may be rescued by expression of the caspase inhibitor baculovirus p35, which counteracts apoptosis. However, coexpression of discs lost RNAi and p35 did not alter the number of surviving cells. Instead, it was frequently observed that the dorsal and ventral wing blades detached and that the L3 vein appeared broader. Similar, although slightly weaker phenotypes were obtained following coexpression of discs lost RNAi and Drosophila myc (dmyc), which affects cell growth. Thus, Discs lost might be part of a pathway required for cell survival because mutant cells cannot be rescued by blocking caspase activity or increasing cellular growth (Pielage, 2003).
Alternatively, the discs lost phenotype could be explained by a failure in cell cycle progression. Thus, a test was performed to see whether expression of cyclin E, which prompts additional mitoses by forcing cells into S phase, might rescue the discs lost mutant phenotype. Expression of cyclin E in the patched domain led to a slightly enlarged wing compartment. However, coexpression of both cyclin E and discs lost RNAi resulted in a dramatic loss of wing cells, which was more than twice as severe as compared to expression of discs lost RNAi alone. When discs lost RNAi was expressed in a heterozygous cycE mutant background, the number of wing cells was decreased only very moderately. Because Cyclin E prompts transition from G1 to S phase, these data may indicate that a reduction of discs lost function results in an arrest in G1 phase. Reduction of the level of Cyclin E does not overcome this arrest. However, when cells are forced into S phase by ectopic Cyclin E expression they die. Coexpression of discs lost RNAi and M phase promoting genes (cyclin A or string) had little or no effect. This suggests that discs lost is required for both G1 and S phase and that in the absence of discs lost, S phase cannot be completed and thus mitosis cannot be initiated (Pielage, 2003).
Most animals depend on olfaction for survival and procreation. Odor-guided behavior is a quantitative trait, with phenotypic variation due to multiple segregating quantitative trait loci (QTL). Despite its profound biological importance, the genetic basis of naturally occurring variation in olfactory behavior remains unexplored. Here, a single Drosophila QTL affecting variation in avoidance response to benzaldehyde has been mapped using a population of recombinant inbred lines. Deficiency complementation mapping resolved this region into one female- and one male-specific QTL. Subsequent quantitative complementation tests to all available mutations of positional candidate genes showed that the female-specific QTL failed to complement a P-element insertional mutation, l(3)04276. The P-element insertion was in the intron of a novel gene, Vanaso, which contains a putative guanylate binding protein domain, is highly polymorphic, and is expressed in the third antennal segment, the major olfactory organ of Drosophila. No expression was detected in the fly brain, suggesting that Vanaso plays a role in peripheral chemosensory processes rather than in central integration of olfactory information. QTL mapping followed by quantitative complementation tests to deficiencies and mutations is an effective strategy for gene discovery that allows characterization of effects of recessive lethal genes on adult phenotypes and in this study enabled identification of a candidate gene that contributes to sex-specific quantitative variation in olfactory behavior (Fanara, 2002).
Reference names in red indicate recommended papers.
Bhat, M. A., et al. (1999). Discs Lost, a novel multi-PDZ domain protein, establishes and maintains epithelial polarity. Cell 96: 833-845.
Dgany, O., Avidan, N., Delaunay, J., Krasnov, T., Shalmon, L., Shalev, H., Eidelitz-Markus, T., Kapelushnik, J., Cattan, D. and Pariente, A. et al. (2002). Congenital dyserythropoietic anemia type I is caused by mutations in codanin-1. Am. J. Hum. Genet. 71: 1467-1474. 12434312
Fanara, J. J., Robinson, K. O., Rollmann, S. M., Anholt, R. R. and Mackay, T. F. (2002). Vanaso is a candidate quantitative trait gene for Drosophila olfactory behavior. Genetics 162: 1321-1328. 12454076
Lemmers, C., et al. (2002). hINADl/PATJ, a homolog of Discs lost, interacts with Crumbs and localizes to tight junctions in human epithelial cells. J. Biol. Chem. 277: 25408-25415. 11964389
Pielage, J., Stork, T., Bunse, I. and Klämbt, C. (2003). The Drosophila cell survival Gene discs lost encodes a cytoplasmic Codanin-1-like protein, not a homolog of tight junction PDZ protein Patj. Dev. Cell 5: 841-851. 14667407
Roh, M. H., et al. (2002a). The Maguk protein, Pals1, functions as an adapter, linking mammalian homologues of Crumbs and Discs Lost. J. Cell Biol. 157(1): 161-72. 11927608
Roh, M. H., et al. (2002b). The carboxyl terminus of Zona occludens-3 binds and recruits a mammalian homologue of Discs lost to tight junctions. J. Biol. Chem. 277: 27501-27509. 12021270
Sliter, T. J., Henrich, V. C., Tucker, R. L., and Gilbert, L. I. (1989). The genetics of the Dras3-Roughened-ecdysoneless chromosomal region (62B3-4 to 62D3-4) in Drosophila melanogaster: analysis of recessive lethal mutations. Genetics 123: 327-336. 2511069
Tanentzapf, G., et al. (2000). Apical, lateral, and basal polarization cues contribute to the development of the follicular epithelium during Drosophila oogenesis. J. of Cell Bio. 151: 891-904. 11076972
Wickramasinghe, S. N. (1997). Dyserythropoiesis and congenital dyserythropoietic anaemias. Br. J. Haematol. 98: 785-797. 9326170
Wickramasinghe, S. N. (2000). Congenital dyserythropoietic anemias. Curr. Opin. Hematol. 7: 71-78. 10698292
date revised: 10 May 2004
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