Germline Clones Research Articles

The R-SNARE Ykt6 is required for multiple events during oogenesis in Drosophila.

Ykt6 has emerged as a key protein involved in a wide array of trafficking events, and has also been implicated in a number of human pathologies, including the progression of several cancers. It is a complex protein that simultaneously exhibits a high degree of structural and functional homology, and yet adopts differing roles in different cellular contexts. Because Ykt6 has been implicated in a variety of vesicle fusion events, we characterized the role of Ykt6 in oogenesis by observing the phenotype of Ykt6 germline clones. Immunofluorescence was used to visualize the expression of membrane proteins, organelles, and vesicular trafficking markers in mutant egg chambers. We find that Ykt6 germline clones have morphological and actin defects affecting both the nurse cells and oocyte, consistent with a role in regulating membrane growth during mid-oogenesis. Additionally, these egg chambers exhibit defects in bicoid and oskar RNA localization, and in the trafficking of Gurken during mid-to-late oogenesis. Finally, we show that Ykt6 mutations result in defects in late endosomal pathways, including endo- and exocytosis. These findings suggest a role for Ykt6 in endosome maturation and in the movement of membranes to and from the cell surface.

Machine Learning to Quantify In Situ Humoral Selection in Human Lupus Tubulointerstitial Inflammation.

In human lupus nephritis, tubulointerstitial inflammation (TII) is associated with in situ expansion of B cells expressing anti-vimentin antibodies (AVAs). The mechanism by which AVAs are selected is unclear. Herein, we demonstrate that AVA somatic hypermutation (SHM) and selection increase affinity for vimentin. Indeed, germline reversion of several antibodies demonstrated that higher affinity AVAs can be selected from both low affinity B cell germline clones and even those that are strongly reactive with other autoantigens. While we demonstrated affinity maturation, enzyme-linked immunosorbent assays (ELISAs) suggested that affinity maturation might be a consequence of increasing polyreactivity or even non-specific binding. Therefore, it was unclear if there was also selection for increased specificity. Subsequent multi-color confocal microscopy studies indicated that while TII AVAs often appeared polyreactive by ELISA, they bound selectively to vimentin fibrils in whole cells or inflamed renal tissue. Using a novel machine learning pipeline (CytoSkaler) to quantify the cellular distribution of antibody staining, we demonstrated that TII AVAs were selected for both enhanced binding and specificity in situ. Furthermore, reversion of single predicted amino acids in antibody variable regions indicated that we could use CytoSkaler to capture both negative and positive selection events. More broadly, our data suggest a new approach to assess and define antibody polyreactivity based on quantifying the distribution of binding to native and contextually relevant antigens.

Multiple Functions of the Essential Gene PpV in Drosophila Early Development.

Protein phosphatase V (PpV) encodes the Drosophila homolog of the evolutionarily conserved Protein Phosphatase 6 (PP6). The physiological and developmental functions of PpV/PP6 have not been well characterized due to lack of a genetically defined mutant. Here, we identified a PpV non-sense mutation and describe multiple mutant phenotypes in oogenesis and early embryogenesis. Specifically, we found that the defects in chromosome segregation during nuclear cycles are related to AuroraA function, which is consistent with the interaction of PP6 and AuroraA in mammalian cells. Surprisingly, we also identified a PpV function specifically in blastoderm cell cycle but not in cell proliferation in the follicle epithelium or larval wing imaginal discs. Embryos from PpV germline clones frequently undergo an extra nuclear division cycle. By epistasis analysis, we found that PpV functions in parallel with tribbles, but independently of auroraA for the remodeling of the nuclear cycles. Taken together, this study reports novel developmental functions of PpV and provides a framework for further genetic analysis under physiological conditions.

Selection of potential cytokeratin-18 monoclonal antibodies following IGH repertoire evaluation in mice

Cytokeratin 18 (CK18), the main scaffold protein of keratinocyte, is distributed in epithelial cells. This structural protein maintains the integrity and continuity of epithelial tissue. Cytokeratin is also frequently used as an immunohistochemical marker of tumor growth. In recent years, immune repertoire (IR) evaluation using next-generation sequencing (NGS) have become increasingly efficient. Here we deep sequenced the mouse IR of the immunoglobulin heavy chain (IGH) after CK18 immunization. We comprehensively analyzed the IR based on complementarity determining region 3 (CDR3) abundance, germline gene usage polarization, clone diversity, and lineage. We found many convergence characteristics after CK18 immunization. Convergence represents a phenomenon that antigen stimulation or pathogen exposure induces the antigen specific clone expansion and enrichment. The convergence could be used for the immune evaluation and antibody screen. After immunization, the IGHV5 gene clusters became preponderant. The abundance and length of the most frequent CDR3 both increased, nevertheless the IR diversity level decreased. From the convergent IGH repertoires, we selected and expressed six antibodies with the most frequent CDR3s and IGH V-J combinations. The ELISA results suggested all screened six antibodies bound CK18 specifically. The most potential antibody had 9.424E-10M M affinity for the interaction with the CK18. Therefore, this is the NGS platform has been first used for anti-CK18 monoclonal antibodies (MAbs) discovery. These analyses methods could also be used for vaccine evaluation.

Ribosomal protein L10A and signaling pathway

Ribosome: machinery in control of messenger RNA's (mRNAs) and several ribosomal proteins are in the small and large subunit of the ribosome. Various aspects of ribosomal proteins have related to cell growth, cell cycle, and diseases. Ribosomal protein L10A (RpL10A) in shrimp and fruit fly has been demonstrated to play a role in oogenesis. Interestingly, deletion RpL10A gene (RpL10Ab−/−) germ line clone of the fruit fly showed a loss of follicle cells surrounding the egg chamber, but nurse cells appeared normal. This phenotype is reminiscent of insulin receptor mutants (InR−/−). In contrast, over-expression of RpL10A in the eyes of the fruit flies resulted in abnormal ommatidia with a loss of red pigment in the center of the eyes. In this study, the abnormal rearrangements of nuclei and lack of cell membranes in those eyes were demonstrated. Furthermore, the expression of InR gene and the InR protein were extensively increased as determined by real-time PCR and immunohistochemistry, respectively. In addition, some insulin signaling mediators were also detected. The Akt and FOXO proteins were highly phosphorylated in the RpL10A over-expressed mutant. The results revealed that RpL10A induced over-expression of the insulin receptor and consequently activated in insulin signaling pathway which affects cell proliferation and we suggest RpL10A stimulates cell proliferation via the insulin signaling pathway.

The retromer subunit Vps26 mediates Notch signaling during Drosophila oogenesis

During endocytosis, molecules are internalized by the cell through the invagination of the plasma membrane. Endocytosis is required for proper cell function and for normal development in Drosophila. One component of the endocytic pathway is the retromer complex, which recycles transmembrane proteins to other parts of the cell such as the plasma membrane and the trans-Golgi network. Previous studies have shown that mutations to the retromer complex result in developmental defects in Drosophila. In humans, retromer dysfunction has been implicated in Alzheimer's and Parkinson's disease, but little is known about the role of the retromer complex in Drosophila oogenesis. In the current project, we examined the role of the retromer protein Vps26 in oogenesis by characterizing the phenotype of vps26 germline clones. Immunofluorescence was used to visualize the expression of membrane proteins and vesicular trafficking markers in mutant egg chambers. We find that vps26 germline clones exhibit a signaling defect between the germline cells and follicle cells indicated by an increase in LysoTracker staining of the border cells in the mutants. We show that this signaling defect in vps26 mutants may be the result of impaired Notch signaling based on the misexpression of multiple proteins in the Notch signaling pathway in vps26 mutants.

Essential Function of the Serine Hydroxymethyl Transferase (SHMT) Gene During Rapid Syncytial Cell Cycles in Drosophila.

Many metabolic enzymes are evolutionarily highly conserved and serve a central function in the catabolism and anabolism of cells. The serine hydroxymethyl transferase (SHMT) catalyzing the conversion of serine and glycine and vice versa feeds into tetrahydrofolate (THF)-mediated C1 metabolism. We identified a Drosophila mutation in SHMT (CG3011) in a screen for blastoderm mutants. Embryos from SHMT mutant germline clones specifically arrest the cell cycle in interphase 13 at the time of the midblastula transition (MBT) and prior to cellularization. The phenotype is due to a loss of enzymatic activity as it cannot be rescued by an allele with a point mutation in the catalytic center but by an allele based on the SHMT coding sequence from Escherichia coli. The onset of zygotic gene expression and degradation of maternal RNAs in SHMT mutant embryos are largely similar to that in wild-type embryos. The specific timing of the defects in SHMT mutants indicates that at least one of the SHMT-dependent metabolites becomes limiting in interphase 13, if it is not produced by the embryo. Our data suggest that mutant eggs contain maternally-provided and SHMT-dependent metabolites in amounts that suffice for early development until interphase 13.

Drosophila chem mutations disrupt epithelial polarity in Drosophila embryos.

Drosophila embryogenesis has proven to be an extremely powerful system for developmental gene discovery and characterization. We isolated five new EMS-induced alleles that do not complement the l(3R)5G83 lethal line isolated in the Nüsslein-Volhard and Wieschaus screens. We have named this locus chem. Lethality of the new alleles as homozygous zygotic mutants is not completely penetrant, and they have an extended phenocritical period. Like the original allele, a fraction of mutant embryos die with cuticular defects, notably head involution and dorsal closure defects. Embryonic defects are much more extreme in germline clones, where the majority of mutant embryos die during embryogenesis and do not form cuticle, implying a strong chem maternal contribution. chem mutations genetically interact with mutations in cytoskeletal genes (arm) and with mutations in the epithelial polarity genes coracle, crumbs, and yurt. chem mutants dorsal open defects are similar to those present in yurt mutants, and, likewise, they have epithelial polarity defects. chem1 and chem3 mutations suppress yurt3, and chem3 mutants suppress crumbs1 mutations. In contrast, chem1 and coracle2 mutations enhance each other. Compared to controls, in chem mutants in embryonic lateral epithelia Crumbs expression is mislocalized and reduced, Coracle is increased and mislocalized basally at embryonic stages 13–14, then reduced at stage 16. Arm expression has a similar pattern but levels are reduced.

Analysis of novel alleles of brother of tout-velu, the drosophila ortholog of human EXTL3 using a newly developed FRT42D ovoD chromosome.

The FLP/FRT system permits rapid phenotypic screening of homozygous lethal mutations in the context of a viable mosaic fly. Combining this system with ovoD dominant female-sterile transgenes enables efficient production of embryos derived from mutant germline clones lacking maternal contribution from a gene of interest. Two distinct sets of FRT chromosomes, carrying either the mini-white (w + mW.hs ), or rosy (ry+ ) and neomycin (neoR ) transgenes are in common use. Parallel ovoD lines were developed using w + mW.hs FRT insertions on the X and chromosomes 2R and 3L, as well as ry+ , neoR FRT insertions on 2L and 3R. Consequently, mutations isolated on the X, 2R and 3L chromosomes in a ry+ , neoR FRT background, are not amenable to germline clonal analysis without labor-intensive recombination onto chromosome arms containing a w + mW.hs FRT. Here we report the creation of a new ovoD line for the ry+ , neoR FRT insertion at position FRT42D on chromosome 2R, through induced recombination in males. To establish the developmental relevance of this reagent we characterized the maternal-effect phenotypes of novel brother of tout-velu alleles generated on an FRT42D chromosome in a somatic mosaic screen. We find that an apparent null mutation that causes severe defects in somatic tissues has a much milder effect on embryonic patterning, emphasizing the necessity of analyzing mutant phenotypes at multiple developmental stages.

Drosophila glob1 is required for the maintenance of cytoskeletal integrity during oogenesis.

Hemoglobins (Hbs) are evolutionarily conserved heme-containing metallo-proteins of the Globin protein family that harbour the characteristic "globin fold." Hemoglobins have been functionally diversified during evolution and their usual property of oxygen transport is rather a recent adaptation. Drosophila genome possesses three globin genes (glob1, glob2, and glob3), and we have reported earlier that adequate expression of glob1 is required for various aspects of development, as well as to regulate the cellular level of reactive oxygen species (ROS). The present study illustrates the explicit role of Drosophila globin1 in progression of oogenesis. We demonstrate a dynamic expression pattern of glob1 in somatic and germ cell derivatives of developing egg chambers during various stages of oogenesis, which largely confines around the F-actin-rich cellular components. Reduced expression of glob1 leads to various types of abnormalities during oogenesis, which were primarily mediated by the inappropriately formed F-actin-based cytoskeleton. Our subsequent analysis in the somatic and germ line clones shows cell autonomous role of glob1 in the maintenance of the integrity of F-actin-based cytoskeleton components in the somatic and germ cell derivatives. Our study establishes a novel role of glob1 in maintenance of F-actin-based cytoskeleton during progression of oogenesis in Drosophila. Developmental Dynamics 245:1048-1065, 2016. © 2016 Wiley Periodicals, Inc.

Ultrastructural study of spermatogenesis and sperm in the African medicinal leech Hirudo troctina Johnson, 1816 (Annelida, Hirudinida).

This paper presents the process of spermatogenesis in the leech Hirudo troctina Johnson, 1816 using light, fluorescent and transmission electron microscopy. At the onset of spermatogenesis in testes, the pear-shaped spermatogonia divide mitotically without full cytokinesis and as a result isogenic groups are formed (clusters, clones) with 2, 4, 8, 16, 32, 64, 128 spermatogonia and, finally, 256 primary spermatocytes occur. The final meiotic divisions of spermatocytes give rise to clones with 1024 spermatids. There are hundreds of developing germ-line clones in each testis. In each clone, the male germ cells divide in full synchrony and they are in the same phase of spermatogenesis. During complex spermiogenesis each spermatid becomes a filiform spermatozoon with a helicoid nucleus, which is characterized by the presence of a long acrosome with two regions - anterior and posterior, which are followed by a helicoid nucleus, a midpiece with only one mitochondrion and a long flagellum. Our results were compared to those on other clitellate annelids that have been studied to date, especially to sperm formation in Hirudo medicinalis Linnaeus, 1785. Only minor differences were found in the length and the diameter of different organelles and the number of spermatids in germ-line clones.

Slmb antagonises the aPKC/Par-6 complex to control oocyte and epithelial polarity.

The Drosophila anterior-posterior axis is specified when the posterior follicle cells signal to polarise the oocyte, leading to the anterior/lateral localisation of the Par-6/aPKC complex and the posterior recruitment of Par-1, which induces a microtubule reorganisation that localises bicoid and oskar mRNAs. Here we show that oocyte polarity requires Slmb, the substrate specificity subunit of the SCF E3 ubiquitin ligase that targets proteins for degradation. The Par-6/aPKC complex is ectopically localised to the posterior of slmb mutant oocytes, and Par-1 and oskar mRNA are mislocalised. Slmb appears to play a related role in epithelial follicle cells, as large slmb mutant clones disrupt epithelial organisation, whereas small clones show an expansion of the apical domain, with increased accumulation of apical polarity factors at the apical cortex. The levels of aPKC and Par-6 are significantly increased in slmb mutants, whereas Baz is slightly reduced. Thus, Slmb may induce the polarisation of the anterior-posterior axis of the oocyte by targeting the Par-6/aPKC complex for degradation at the oocyte posterior. Consistent with this, overexpression of the aPKC antagonist Lgl strongly rescues the polarity defects of slmb mutant germline clones. The role of Slmb in oocyte polarity raises an intriguing parallel with C. elegans axis formation, in which PAR-2 excludes the anterior PAR complex from the posterior cortex to induce polarity, but its function can be substituted by overexpressing Lgl.

Drosophila Hephaestus/Polypyrimidine Tract Binding Protein Is Required for Dorso-Ventral Patterning and Regulation of Signalling between the Germline and Soma

In the Drosophila oocyte, gurken (grk) mRNA encodes a secreted TGF-α signal that specifies the future embryonic dorso-ventral axes by altering the fate of the surrounding epithelial follicle cells. We previously identified a number of RNA binding proteins that associate specifically with the 64 nucleotide grk localization signal, including the Drosophila orthologue of polypyrimidine tract-binding protein (PTB), Hephaestus (Heph). To test whether Heph is required for correct grk mRNA or protein function, we used immunoprecipitation to validate the association of Heph with grk mRNA and characterized the heph mutant phenotype. We found that Heph is a component of grk mRNP complexes but heph germline clones show that Heph is not required for grk mRNA localization. Instead, we identify a novel function for Heph in the germline and show that it is required for proper Grk protein localization. Furthermore, we show that Heph is required in the oocyte for the correct organization of the actin cytoskeleton and dorsal appendage morphogenesis. Our results highlight a requirement for an mRNA binding protein in the localization of Grk protein, which is independent of mRNA localization, and we propose that Heph is required in the germline for efficient Grk signalling to the somatic follicle cells during dorso-ventral patterning.

Depleting Gene Activities in Early Drosophila Embryos with the “Maternal-Gal4–shRNA” System

In a developing Drosophila melanogaster embryo, mRNAs have a maternal origin, a zygotic origin, or both. During the maternal-zygotic transition, maternal products are degraded and gene expression comes under the control of the zygotic genome. To interrogate the function of mRNAs that are both maternally and zygotically expressed, it is common to examine the embryonic phenotypes derived from female germline mosaics. Recently, the development of RNAi vectors based on short hairpin RNAs (shRNAs) effective during oogenesis has provided an alternative to producing germline clones. Here, we evaluate the efficacies of: (1) maternally loaded shRNAs to knockdown zygotic transcripts and (2) maternally loaded Gal4 protein to drive zygotic shRNA expression. We show that, while Gal4-driven shRNAs in the female germline very effectively generate phenotypes for genes expressed maternally, maternally loaded shRNAs are not very effective at generating phenotypes for early zygotic genes. However, maternally loaded Gal4 protein is very efficient at generating phenotypes for zygotic genes expressed during mid-embryogenesis. We apply this powerful and simple method to unravel the embryonic functions of a number of pleiotropic genes.

A Germline Clone Screen on theXChromosome Reveals Novel Meiotic Mutants inDrosophila melanogaster

In an effort to isolate novel meiotic mutants that are severely defective in chromosome segregation and/or exchange, we employed a germline clone screen of the X chromosome of Drosophila melanogaster. We screened over 120,000 EMS-mutagenized chromosomes and isolated 19 mutants, which comprised nine complementation groups. Four of these complementation groups mapped to known meiotic genes, including mei-217, mei-218, mei-9, and nod. Importantly, we have identified two novel complementation groups with strong meiotic phenotypes, as assayed by X chromosome nondisjunction. One complementation group is defined by three alleles, and the second novel complementation group is defined by a single allele. All 19 mutants are homozygous viable, fertile, and fully recessive. Of the 9 mutants that have been molecularly characterized, 5 are canonical EMS-induced transitions, and the remaining 4 are transversions. In sum, we have identified two new genes that are defined by novel meiotic mutants, in addition to isolating new alleles of mei-217, mei-218, mei-9, and nod.

The role of atg18 in signal transduction pathways during Drosophila development

8J16 and 9E6 are allelic mutations that disrupt Wnt/Wingless (Wg) or Hedgehog (Hh) signaling based upon their embryonic ‘lawn of denticles’ phenotype. Goals of this project were to identify the disrupted gene and determine its role in Drosophila developmental signaling. Complementation analysis revealed 8J16/9E6 are alleles of autophagy‐specific gene 18, atg18, which plays a role in autophagosome‐lysosome fusion. In yeast, atg18 negatively regulates endosome‐lysosome targeting. In Drosophila, endocytic machinery mutations that block lysosome targeting cause continuous signaling. Thus, atg18 mutants should show a buildup of lysosomes at the expense of endosomes and decrease signaling. To the contrary, Distalless, a long‐range Wg target, was enhanced in 8J16/9E6 mutant tissue in Drosophila wing discs. Analysis of 8J16/9E6 germline clone (glc) mutant embryos confirmed atg188J16, atg189E6, and atg18KG03090 are allelic. Immunofluorescent staining of glc embryos showed the segmentation defect is due to loss of Wg expression at embryonic stage 9. 8J16/9E6 glc embryos also display severe CNS defects not strictly the result of Wg loss and thus, are pleiotropic effects of these mutations. 8J16/9E6 are intronic point mutations not predicted to disrupt mRNA splicing and are temperature sensitive, yielding lethality at 25ºC, but are viable at 29ºC. Molecular explanations for this phenotype are explored. HHMI.

The role of N‐linked glycosylation during Drosophila embryonic development

N‐linked glycosylation is a key post‐translational modification for many secretory proteins. It has roles in cell recognition and adhesion, protein folding, and cellular signaling. Endoplasmic reticulum glycosyltranferases construct a 14‐sugar precursor on a dolichol carrier, which is then transferred en mass to proteins on consensus sites. The function of N‐glycosylation during Drosophila embryogenesis was investigated in alg9 and alg10 germ line clone mutant embryos. alg10 encodes the enzyme catalyzing terminal glucose addition to the sugar‐substrate prior to protein transfer, while the alg9 encoded enzyme acts five steps earlier in sugar synthesis. alg9 and alg10 displayed severe and pleotrophic defects in cuticle secretion. Expression of Wingless (Wg), a model secreted glycoprotein of the Wnt pathway, was examined to identify earlier effects of these mutations. Normal Wg secretion was disrupted in alg9 and alg10 glc embryos. However, defects in secretion did not block downstream Wg signaling, as Engrailed (En) was expressed fairly normally in both backgrounds. The central nervous system (CNS) develops later and is highly dependent on glycosylation, alg10 loss caused severe defects in CNS development, but alg9 glc embryos lack CNS. Hence, alg10 may cause global underglycosylation or is only needed for some glycoproteins, while alg9 may be required for N‐glycosylation of most or all protein substrates.

Shaggy/glycogen synthase kinase 3β and phosphorylation of Sarah/regulator of calcineurin are essential for completion of Drosophila female meiosis

The Ca 2+ /Calmodulin-dependent phosphatase calcineurin is essential for exit from meiotic arrest at metaphases I and II in Drosophila and Xenopus oocytes. We previously found that Sarah, the Drosophila homolog of regulator of calcineurin, acts as a positive regulator of calcineurin and is required to complete anaphase I of female meiosis. Here, we undertook biochemical approaches, including MS and posttranslational modification analyses, to better understand the mechanism by which Sarah regulates calcineurin. A search for phosphorylated residues revealed that Sarah is highly phosphorylated at Ser100, Thr102, and Ser219 in both ovaries and activated eggs and that Ser215 is phosphorylated only in activated eggs. Functional analyses using mutant forms of Sarah showed that phosphorylation at Ser215, a consensus phosphorylation site for glycogen synthase kinase 3β (GSK-3β) and its priming kinase site Ser219, are essential for Sarah function. Furthermore, germ-line clones homozygous for a null allele of shaggy ( Drosophila GSK-3β) both fail to complete meiosis and lack phosphorylation of Sarah at Ser215, suggesting that the phosphorylation of Sarah by Shaggy/GSK-3β is required to complete meiosis. Our findings suggest a mechanism in which Shaggy/GSK-3β activates calcineurin through Sarah phosphorylation on egg activation in Drosophila .

Conserved requirement for DEAD-box RNA helicase Gemin3 in Drosophila oogenesis

BackgroundDEAD-box RNA helicase Gemin3 is an essential protein since its deficiency is lethal in both vertebrates and invertebrates. In addition to playing a role in transcriptional regulation and RNA silencing, as a core member of the SMN (survival of motor neurons) complex, Gemin3 is required for the biogenesis of spliceosomal snRNPs (small nuclear ribonucleoproteins), and axonal mRNA metabolism. Studies in the mouse and C. elegans revealed that loss of Gemin3 function has a negative impact on ovarian physiology and development.FindingsThis work reports on the generation and characterisation of gemin3 mutant germline clones in Drosophila adult females. Gemin3 was found to be required for the completion of oogenesis and its loss led to egg polarity defects, oocyte mislocalisation, and abnormal chromosome morphology. Canonical Cajal bodies were absent in the majority of gemin3 mutant egg chambers and histone locus bodies displayed an atypical morphology. snRNP distribution was perturbed so that on gemin3 loss, snRNP cytoplasmic aggregates (U bodies) were only visible in wild type.ConclusionsThese findings establish a conserved requirement for Gemin3 in Drosophila oogenesis. Furthermore, in view of the similarity to the phenotypes described previously in smn mutant germ cells, the present results confirm the close functional relationship between SMN and Gemin3 on a cellular level.

The Drosophila TRPP Cation Channel, PKD2 and Dmel/Ced-12 Act in Genetically Distinct Pathways during Apoptotic Cell Clearance

Apoptosis, a genetically programmed cell death, allows for homeostasis and tissue remodelling during development of all multi-cellular organisms. Phagocytes swiftly recognize, engulf and digest apoptotic cells. Yet, to date the molecular mechanisms underlying this phagocytic process are still poorly understood. To delineate the molecular mechanisms of apoptotic cell clearance in Drosophila, we have carried out a deficiency screen and have identified three overlapping phagocytosis-defective mutants, which all delete the fly homologue of the ced-12 gene, known as Dmel\\ced12. As anticipated, we have found that Dmel\\ced-12 is required for apoptotic cell clearance, as for its C. elegans and mammalian homologues, ced-12 and elmo, respectively. However, the loss of Dmel\\ced-12 did not solely account for the phenotypes of all three deficiencies, as zygotic mutations and germ line clones of Dmel\\ced-12 exhibited weaker phenotypes. Using a nearby genetically interacting deficiency, we have found that the polycystic kidney disease 2 gene, pkd2, which encodes a member of the TRPP channel family, is also required for phagocytosis of apoptotic cells, thereby demonstrating a novel role for PKD2 in this process. We have also observed genetic interactions between pkd2, simu, drpr, rya-r44F, and retinophilin (rtp), also known as undertaker (uta), a gene encoding a MORN-repeat containing molecule, which we have recently found to be implicated in calcium homeostasis during phagocytosis. However, we have not found any genetic interaction between Dmel\\ced-12 and simu. Based on these genetic interactions and recent reports demonstrating a role for the mammalian pkd-2 gene product in ER calcium release during store-operated calcium entry, we propose that PKD2 functions in the DRPR/RTP pathway to regulate calcium homeostasis during this process. Similarly to its C. elegans homologue, Dmel\\Ced-12 appears to function in a genetically distinct pathway.