Drosophila gene discs lost (dlt) has been reported to encode a homolog of the vertebrate tight junction PDZ protein Patj, and was thought to play a role in cell polarity (Bhat, 1999). Using rescue experiments and sequence analyses, dlt mutations have been shown to disrupt the Drosophila Codanin-1 homolog, a cytoplasmic protein, and not the PDZ protein. Mutations in human Codanin-1 are associated with congenital dyserythropoietic anemia type I (CDA I). In Drosophila, the genomic organization of dlt is unusual. dlt shares its first untranslated exon with α-spectrin, and both genes are coexpressed throughout development. dlt is not required for cell polarity but is needed for cell survival and cell cycle progression. Finally, evidence suggests that the PDZ protein previously thought to be encoded by dlt is not required for viability. It is proposed that PDZ protein be named after its vertebrate homolog, Patj (Pals-associated tight junction protein) (Pielage, 2003).

In the course of generating P element-induced alleles of an unrelated gene, three independent, lethal P element-induced discs lost mutations were identified, that all failed to complement the original dlt^dre1 mutation. A previous analysis of the discs lost locus had suggested a complex genetic scenario (Bhat, 1999) and only a single EMS-induced discs lost mutation was available (dlt^dre1). Thus, an F2 EMS-mutagenesis was conducted to isolate four EMS-induced discs lost alleles (dlt^A1, dlt^C1, dlt^N1, and dlt^O2). The original dlt^dre1 mutation (Sliter, 1989) as well as the dlt^N1 allele led to larval lethality. In contrast, homozygous dlt^A1, dlt^C1, or dlt^O2 animals die as pupae. To discriminate whether the differences in the lethal phase are due to different allelic strengths or to background effects, the lethal period of all discs lost alleles was determined in trans to deficiencies. In this paradigm, all dlt alleles led to pupal lethality (Bhat, 1999; Sliter, 1989). This indicates that additional background mutations are responsible for the larval lethality that was observed for dlt^dre1 and dlt^N1 mutant animals (Pielage, 2003).

The discs lost locus was previously shown to encode a protein with four PDZ domains (Bhat, 1999). To determine the molecular nature of the newly induced discs lost EMS alleles, the genomic DNA encompassing the PDZ domain protein-encoding gene was sequenced in two EMS-induced dlt mutations. Only third base changes were found, none of which resulted in an amino acid exchange, indicating that discs lost may not correspond to the PDZ domain protein that is now called Patj (Pielage, 2003).

In contrast to the EMS-induced mutations, all P element-induced discs lost alleles result in late embryonic, early larval lethality. Sequence analyses demonstrate that all three P element insertions occur within the first intron of α-spectrin. The analysis of a large number of cDNA clones identified by the Berkeley Drosophila Genome Project (BDGP) was demonstrated a rather unusual organization of the α-spectrin locus. Within its first intron, 3' to the P element insertion sites, resides a 1240 amino acid large open reading frame called CG32315 or vanaso (Fanara, 2002). Interestingly, this transcript shares the first noncoding exon with α-spectrin (Pielage, 2003).

Given the genetic organization of the α-spectrin locus, it was not surprising that the genetic complementation analyses demonstrate that all P element-induced discs lost mutations do not complement mutations in α-spectrin. Thus, the P element-induced discs lost alleles are indeed double mutations, and the loss of α-spectrin function is likely to be responsible for the earlier lethal phase of the P element-induced dlt alleles compared to the EMS-induced dlt alleles. On reexamining the previously identified P element-induced discs lost allele (Bhat, 1999); it too failed to complement α-spectrin mutations (Pielage, 2003).

Because none of the EMS-induced discs lost alleles could be linked to sequence alterations in the genomic region encompassing the patj gene, it was assumed that discs lost might correspond to the open reading frame CG32315 located within the first α-spectrin intron. To test this assumption, rescue experiments were performed using different CG32315 transgenes. To facilitate these experiments, the CG32315 coding region was placed under the control of the ubiquitin promoter. As observed for GAL4-UAS-activated expression, ubiquitous expression of CG32315 in otherwise wild-type animals did not cause any dominant phenotype. Using the ubi-CG32315 transgene, it was not possible to rescue EMS-induced discs lost mutations (dre1, N1, A1, and C1) in trans to the deficiencies Df(3L)Aprt32 or Df(3L)My10. In addition, it was not possible to rescue any allelic combinations of discs lost alleles tested. Homozygous dlt^A1 mutants could be rescued, which indicates that no other lethal mutations are present on the background of this chromosome. In all cases, lethality was completely rescued and fertile flies with normal appendages successfully eclosed. Although it was not possible to rescue homozygous dlt^dre1 animals, the rescue of dlt^dre1 in trans to the deficiencies clearly demonstrates that discs lost corresponds to CG32315 and not to the previously published PDZ domain protein (Pielage, 2003).

To further verify that discs lost is encoded by the transcription unit CG32315, the corresponding genomic sequence was examined in two mutant alleles. In dlt^dre1, which is likely to represent a strong allele, a CAA -> TAA exchange was determined at nucleotide 865 of the open reading frame, which leads to an early premature translational stop leaving only a small protein with 288 amino acids. In dlt^N1 mutants, a CAG -> TAG exchange was detected at position 1807 resulting in a truncated protein of 602 amino acids. In summary, these data clearly demonstrate that discs lost corresponds to CG32315 and not the previously published PDZ protein (Pielage, 2003).

Recently, the CG32315 transcription unit has been identified as the quantitative trait locus vanaso that affects olfaction (Fanara, 2002). Since the correlation to CG32315 is based upon a P element insertion affecting both α-spectrin and CG32315, it is suggested that the phenotype-based name discs lost be kept (Pielage, 2003).

Therefore, it is proposed that discs lost does not correspond to the PDZ protein as previously reported but to a conserved protein related to the human Codanin-1 protein encoded by CG32315. First, the discs lost mutant phenotype could be rescued to full viability by expression of the CG32315 transcript. Second, no sequence alterations were detected in the patj gene encoding the PDZ domain protein, but nucleotide changes leading to a premature translational stop were detected in the transcription unit CG32315 of discs lost mutant animals. Third, RNAi-mediated inhibition of CG32315 function is sensitive to the discs lost gene dose. Fourth, the discs lost mutant phenotype could be phenocopied by RNA interference of CG32315. Finally, by using a rescue approach, the Patj protein was shown not to be essential for viability (Pielage, 2003).

The decision between proliferation and survival is an important aspect of cellular regulation during the formation of a well-sized animal organ. The mutant phenotype of discs lost is characterized by a dramatic reduction in the number of imaginal disc cells and consequently in the size of the respective adult tissues. The imaginal discs of Drosophila larvae are simple epithelia that within 4 days grow from 50 to 50,000 cells. During imaginal disc development, cell growth can be uncoupled from cell division. When cell division is increased, cell size is reduced, whereas when cell division is blocked, cells continue to grow to a larger size and multiple trichomes are observed on the surface of wings. These phenotypes can be detected following discs lost inactivation, suggesting that discs lost function is required for the control of cell cycle progression (Pielage, 2003).

In addition, prominent cell death was noted in epithelial cells expressing a discs lost RNAi construct. A similar phenotype can be induced by the deregulation of the cell cycle in imaginal discs. In these experiments, programmed cell death can be efficiently blocked by the caspase inhibitor baculovirus p35. However, coexpression of p35 did not rescue the defects caused by the inhibition of discs lost function. The coexpression of p35 even slightly enhanced the severity of the discs lost RNAi phenotype. Crossvein 1 was generally missing and the L3 vein increased in size. Thus, discs lost regulates cell survival independent of the caspase-induced apoptosis pathway (Pielage, 2003).

To analyze the requirement of Discs lost for cell proliferation, attempts were made to modulate the discs lost mutant phenotype by coexpressing CycE or Dmyc. Expression of CycE (and to a weaker extent Dmyc) is sufficient to activate cell proliferation. Dmyc, unlike its vertebrate homolog, functions primarily as a regulator of cell growth, and overexpression of dmyc in the wing disc leads to an increase in cell size. When discs lost RNAi and dmyc are coexpressed no alterations in the phenotypic strength are observed. In contrast, coexpression of cycE, which promotes G1/S phase transition, leads to an enhancement of the phenotype that now resembles the discs lost loss-of-function phenotype. A possible explanation for these results is that a reduction of Discs lost protein levels leads to a prolonged G1 phase and to a lower survival rate once the cells enter S phase. If these cells are now prematurely forced to enter S phase by ectopic expression of Cyclin E, they eventually die. To test whether discs lost mutant cells can complete S phase, cyclin A and string, which are both regulators of M phase initiation, were coexpressed. Because coexpression of the M phase promoting factors had little or no effect, discs lost seems to be required for the progression through S phase during the cell cycle (Pielage, 2003).

The analysis of discs lost mutant cell clones suggested that discs lost is also required for cell survival of differentiated cells independent of cell division. Although discs lost mutant cell clones could be detected during larval development, they were absent in the resulting adult compound eyes (Pielage, 2003).

Mutations in the human Discs lost homolog Codanin-1 cause an inherited disorder associated with morphological and functional abnormalities of erythropoiesis called congenital dyserythropoietic anemia type I (CDA I; Dgany, 2002). Patients with CDA I present with moderate to severe macrocytic anemia. Cell division of the erythroblasts appears to be arrested in late S phase (Wickramasinghe, 1997 and 2000). Interestingly, these phenotypes are only detectable in about 60% of the erythroblasts, whereas the remaining cells have a rather normal appearance (Wickramasinghe, 2000). This resembles the discs lost phenotype, which is primarily manifested in the fast dividing imaginal cells (Pielage, 2003).
To test whether discs lost affects Drosophila hematopoiesis, the number of blood cells (hemocytes) were analyzed in discs lost mutant larvae. Drosophila hematopoiesis occurs in two waves. At first, presumptive hemocytes are specified in the anterior mesoderm and colonize the entire embryo during midembryogenesis. Later, the lymph gland, which is the larval hematopoietic organ, generates hemocytes that are found in the larval hemolymph. In mutant discs lost third instar larvae, a 30% decrease in the number of blood cells was noted, opening the possibility to set up a Drosophila model system to study CDA I (Pielage, 2003).

Previously, the discs lost mutation was assigned to a PDZ protein, which localizes to the subapical domain of the cell membrane in epithelial cells (Bhat, 1999). Interaction of this PDZ domain protein with cell polarity proteins was shown in flies and vertebrates (Bhat, 1999; Lemmers, 2002; Roh, 2002a and 2002b). Because discs lost does not encode the PDZ protein, the function of this protein remained unclear. To generate animals lacking the patj gene, the deficiency Df(3L)My10 (Bhat, 1999) was used and the different gene functions were restored using a set of transgenes (Pielage, 2003).
Homozygous Df(3L)My10 animals carrying both the P[ubi-spec] and the P[cdc37] transgenes are pupal lethal and develop a discs lost phenocopy indicating that no functional Discs lost protein is expressed. When the functions of α-spectrin, discs lost, and cdc37 were complemented in the background of Df(3L)My10, viable and fertile females were generated. In these females, the patj coding region is absent, as determined by PCR and antibody staining of ovaries. Similarly, Tanentzapf (2000) failed to detect Patj expression in Df(3L)My10 mutant cell clones. Flies were then mated to Df(3L)My10/TM6 males and normal homozygous Df(3L)My10 flies emerged, indicating that recombination had occurred between the P[ubi-discs lost] and the P[cdc37] rescue constructs. These flies very likely develop without any maternal Patj protein, which may have contributed to the rescue in the first generation. All rescued males were sterile. Whether this phenotype is linked to the loss of Patj expression or due to the loss of additional genes that are affected by the deficiency remains to be determined. These results argue against the notion that the Patj protein exerts important functions during the establishment or maintenance of apico-basal polarity (Pielage, 2003).