The proteins encoded by polar-localized mRNAs play an important role in cell fate specification along the anteroposterior axis of the Drosophila embryo. The only maternally synthesized mRNA known previously to be localized to the anterior cortex of both the oocyte and the early embryo is the bicoid mRNA whose localization is required to generate a homeodomain protein gradient that specifies position along the anteroposterior embryonic axis. A second mRNA has been identified and characterized that is localized to the anterior pole of the oocyte and early embryo. This mRNA encodes a Drosophila homolog of mammalian adducin, a membrane-cytoskeleton-associated protein that promotes the assembly of the spectrin-actin network. A comparison of the spatial distribution of bicoid and Adducin-like transcripts in the maternal-effect RNA-localization mutants exuperantia, swallow, and staufen indicates different genetic requirements for proper localization of these two mRNAs to the anterior pole of the oocyte and early embryo (Ding, 1993; full text of article).
Adducin is a cytoskeletal protein that can function in vitro to bundle F-actin and to control the assembly of the F-actin/spectrin cytoskeletal network. The Drosophila Adducin-like (Add) locus, also referred to as hu-li tai shao encodes two adducin-related protein isoforms: a 95 x 10(3) Mr form (ADD-95) and an 87 x 10(3) Mr form (ADD-87). ADD-87 protein is present throughout the oocyte cortex at stages 9 and 10 of oogenesis but is restricted to its anterior pole from stage 11 onward. This ADD-87 protein localization is preceded by localization of Add-hts mRNA first to the cortex and then to the anterior pole of the oocyte. Mutation of the swallow gene results in delocalization of Add-hts mRNA and ADD-87 protein from the cortex of stage 9 and 10 oocytes, and from the anterior pole of later stage oocytes. Early embryos produced by swallow or Add-hts mutant females have severe defects in the distribution of F-actin and spectrin as well as abnormalities in nuclear division, nuclear migration, and cellularization. In addition to their cytoskeletal defects, embryos produced by swallow females have an abnormal anterior pattern because bicoid mRNA is delocalized from the anterior pole. In contrast, bicoid mRNA is still found at the anterior of embryos produced by Add-hts mothers. Thus swallow functions to restrict bicoid mRNA and Add-hts mRNA to the cortex of the oocyte. Cortical restriction of Add-hts mRNA and protein is required for the normal structure and function of the early embryonic F-actin/spectrin cytoskeleton. A defective embryonic cytoskeleton can be induced in either of two ways: (1) by delocalization of functional ADD from the oocyte cortex (as in swallow mutants), or (2) by reduction of ADD function while retaining its normal cortical localization during oogenesis (as in Add-hts mutants) (Zaccai, 1996b).
In Drosophila oogenesis, the development of a mature oocyte depends on having properly developed ring canals that allow cytoplasm transport from the nurse cells to the oocyte. Ring canal assembly is a step-wise process that transforms an arrested cleavage furrow into a stable intercellular bridge by the addition of several proteins. A gene is described, cheerio, that provides a critical function for ring canal assembly. Mutants in cheerio fail to localize ring canal inner rim proteins including filamentous actin, the ring canal-associated products from the hu-li tai shao (hts) gene, and kelch. Since Hts and Kelch are present but unlocalized in cheerio mutant cells, cheerio is likely to function upstream from each of them. Examination of mutants in cheerio places it in the pathway of ring canal assembly between cleavage furrow arrest and localization of hts and actin filaments. Furthermore, this mutant reveals that the inner rim cytoskeleton is required for expansion of the ring canal opening and for plasma membrane stabilization (Robinson, 1997; full text of article).
Cytoplasmic mRNA localization is one method by which protein production is restricted to a particular intracellular site. A novel mechanism is described for localization of transcripts encoding distinct protein isoforms to different destinations. Alternative processing of transcripts produced in the Drosophila ovary by the hu-li tai shao locus introduces distinct 3' untranslated regions (3'UTRs) that differentially localize the mRNAs. Three classes of hts mRNA (R2, N32 and N4) are synthesized in the germ line nurse cells and encode proteins with adducin-homologous amino-terminal regions but divergent carboxy-terminal domains. The R2 and N32 classes of mRNA remain in the nurse cells and are not transported into the oocyte. In contrast, the N4 class of transcripts is transported from the nurse cells into the oocyte starting at stage 1, is subsequently localized to the oocyte cortex at stage 8 and then to the anterior pole from stage 9 on. All aspects of N4 transcript transport and localization are directed by the 345-nucleotide(nt)-long 3' untranslated region (3'UTR). The organization of localization elements in the N4 3'UTR is modular: a 150 nt core is sufficient to direct transport and localization throughout oogenesis. Additional 3'UTR elements function additively together with this core region at later stages of oogenesis to maintain or enhance anterior transcript anchoring. The swallow locus is required to maintain hts transcripts at the anterior pole of the oocyte and functions through the N4 3'UTR. In addition to the three classes of germ line-expressed hts transcripts, a fourth class (R1) is expressed in the somatic follicle cells that surround the germ line cells. This transcript class encodes the Drosophila orthologue of mammalian adducin (Whittaker, 1999; full text of article).
Elimination of maternal expression of the Drosophila RNA-binding protein Lark results in female sterility. This is due to a requirement during oogenesis. Developing oocytes from lark mutatn germline clones (GLCs) are often smaller than normal due to defects in nurse cell cytoplasmic 'dumping.' Late-stage egg chambers from lark mutant GLCs contain low levels of cortical and ring canal associated actin and completely lack nurse cell cytoplasmic F-actin bundles, suggesting the 'dumping' phenotype is due to a defect in the actin cytoskeleton. Localization of Hu-li tai shao (Hts) protein, a component of ring canals, is also disrupted in these mutants. In addition to the dumpless phenotype, a buildup of late-stage egg chambers is observed, a phenotype that correlates with the decrease in egg-laying observed in the mutants. It is postulated that this phenotype is due to defects in the cytoskeletal integrity of eggs since retained and oviposited eggs are fragile and often deflated. These mutant phenotypes are likely due to disruption of an RNA-binding function of Lark since similar phenotypes were observed in flies carrying specific RNA-binding domain mutations. It is proposed that Lark functions during oogenesis as an RNA-binding protein, regulating mRNAs required for nurse cell transport or apoptosis (McNeil, 2004).
The Rho-kinases are widely utilized downstream targets of the activated Rho GTPase that have been directly implicated in many aspects of Rho-dependent effects on F-actin assembly, acto-myosin contractility, and microtubule stability, and consequently play an essential role in regulating cell shape, migration, polarity, and division. The single closely related Drosophila Rho-kinase ortholog, DRok, is required for several aspects of oogenesis, including maintaining the integrity of the oocyte cortex, actin-mediated tethering of nurse cell nuclei, 'dumping' of nurse cell contents into the oocyte, establishment of oocyte polarity, and the trafficking of oocyte yolk granules. These defects are associated with abnormalities in DRok-dependent actin dynamics and appear to be mediated by multiple downstream effectors of activated DRok that have previously been implicated in oogenesis. DRok regulates at least one of these targets, the membrane cytoskeletal cross-linker DMoesin, via a direct phosphorylation that is required to promote localization of DMoesin to the oocyte cortex. The collective oogenesis defects associated with DRok deficiency reveal its essential role in multiple aspects of proper oocyte formation and suggest that DRok defines a novel class of oogenesis determinants that function as key regulators of several distinct actin-dependent processes required for proper tissue morphogenesis (Verdier, 2006a).
The observation of dumpless-like oversized nurse cells in most of Drok2 GLCs supports a role for DRok in the rapid phase of cytoplasmic transport at stages 10B–11 of oogenesis. Unlike other classes of dumpless mutants including chickadee, singed or quail, failure of rapid cytoplasmic transport from the Drok2 mutant nurse cells to the oocyte does not result from the obstruction of the ring canals by unanchored nurse cell nuclei, suggesting that Drok constitutes a distinct class of dumpless-like mutants. In addition, in sqhAX3 GLCs, dumpless nurse cells are associated with a lack of acto-myosin contractility by nurse cells, as revealed by mislocalization of myosin II and by absence of the perinuclear organization of actin filaments bundles in the nurse cells. Therefore, sqhAX3 mutant nurse cells cannot contract properly to expulse their cytoplasm through otherwise weakly damaged ring canals. Drok2 and sqhAX3 mutant nurse cells do not share the same actin filament phenotype; Drok2 mutant nurse cells exhibit a more dramatic phenotype associated with absence of radial filaments and disorganization of cortical actin. It is, however, likely that DRok and Sqh are part of the same signaling pathway that regulates acto-myosin contractility in nurse cells; it has already been shown that DRok phosphorylates Sqh in Drosophila development. Moreover, the severity of the Drok2 mutant F-actin phenotypes may reflect DRok's potential to engage multiple distinct downstream substrates, of which Sqh is only one. Significantly, the actin-binding protein, adducin, is also reportedly a direct substrate for mammalian Rho-kinases, and the Drosophila Adducin ortholog, Hts, is a major component of ring canals. Thus, it is possible that the observed defects in ring canal morphology in Drok2 GLCs involve abnormal regulation of adducin by DRok. However, it is difficult to determine whether this ring canal phenotype contributes to the dumpless-like nurse cell phenotype observed in Drok2 GLCs (Verdier, 2006a).
The observation that nurse cell nuclei are substantially increased in size in Drok2 GLCs suggests a possible involvement of DRok in increased endoreplication of the nurse cells. The Rho-related Rac and Cdc42 GTPases have previously been associated with endoreplication in porcine aortic endothelial (PAE) cells, although Rho has not been implicated thus far. Interestingly, this nurse cell nuclei phenotype has not been observed in other previously described GLC mutants of other actin cytoskeleton-regulating signaling components that exhibit oogenesis defects. Thus, chic as well as sqhAX3 GLCs reveal cytokinesis defects associated with the presence of multinucleated nurse cells. In addition, the majority of sqhAX3 mutant egg chambers harbor less than 15 nurse cells (64% of sqhAX3 mutant egg chambers have less than 7 nurse cells), a phenotype that is not shared by Drok2 mutant nurse cells. These findings also suggest that Drok2 defines a new category of oogenesis mutants that affect the actin cytoskeleton (Verdier, 2006a).
Both Dmoe and Drok2 GLCs exhibit similar actin defects in the oocyte, associated with a loose uneven cortical actin distribution and the presence of actin clumps in the ooplasm and near the cortex. Moreover, phospho-DMoesin levels are decreased at the cortex or mislocalized within the ooplasm of Drok2 GLCs and the conserved kinase domain of Rho-kinase phosphorylates DMoesin on threonine 559 in vitro. A potential mechanism for the DRok-DMoesin signal in this setting is that DRok controls actin reorganization through phosphorylation of DMoesin, which has been previously shown to cross-link actin to the plasma membrane when phosphorylated on T559 at the oocyte cortex. However, the detection of some phospho-DMoesin in the Drok2 GLCs indicates that the critical T559 residue can be phosphorylated by other kinases in the oocyte. Indeed, direct phosphorylation of T559 of mammalian Moesin by protein kinase C (PKC)-θ has been shown in vitro. In addition, mammalian Rho-kinase and PAK have been reported to both phosphorylate the very conserved T508 residue of LIM-kinase in vitro. Therefore, phosphorylation of the conserved T559 residue of Moesin by additional kinases might also occur in Drosophila, highlighting the complexity of cross-talk within developmental signaling pathways (Verdier, 2006a).
The observation that Drok2 mutant oocytes are morphologically more affected than Dmoe mutant oocytes with regard to the deformed plasma membrane also suggests that to exert its functions at the oocyte cortex, DRok is not only signaling to DMoesin but probably also to additional downstream targets that cooperate with DMoesin in the maintenance of the cortical actin cytoskeleton. The strong phenotype associated with the deformed oocyte plasma membrane, which separates dramatically from the apical plasma membranes of the follicle cell layer in most Drok2 GLCs, raises an intriguing question about DRok's apparent role in an adhesive process. That specific phenotype has not been previously reported in studies of other oogenesis mutants associated with defective adhesion between the oocyte and the surrounding follicle cells. Previous reports regarding such adhesion largely address cross-signaling between the apical Notch receptor and the germline-derived putative secreted and transmembrane proteins, Brainiac and Egghead, respectively, in which germline loss of either Brainiac or Egghead results in loss of epithelial apico-basal polarity and accumulation of follicular epithelial cells in multiple layers around the oocyte, but does not lead to a physical separation between the oocyte and the follicle cells membranes. The unique phenotype of Drok2 GLCs could reflect a role for DRok in mediating a distinct signaling pathway from the oocyte to regulate its shape and its adherence to the surrounding follicle cells. Alternatively, the aberrant morphology of the nurse cells, which appear to 'push' against the oocyte without contracting, might produce a mechanical stress on the oocyte itself that prevents it from remaining apposed to the follicle cell layer. Notably, it was found that the follicle cells themselves also appear to require DRok function for the maintenance of their shape, and it is possible that their ability to signal to the oocyte is also affected by DRok deficiency (Verdier, 2006a).
In summary, the single closely related Drosophila Rho-kinase ortholog, DRok, is required for several aspects of oogenesis, including maintaining the integrity of the oocyte cortex, actin-dependent tethering of nurse cell nuclei, 'dumping' of nurse cell contents into the oocyte, establishment of oocyte polarity, and the trafficking of oocyte yolk granules. It is likely that several previously identified direct phosphorylation targets of DRok, including DMoesin, Sqh (myosin light chain), and Hts (adducin), which have each been implicated in various aspects of oogenesis, mediate at least some of the functions of DRok in developing egg chambers. These findings indicate an essential role for Rho-DRok signaling via multiple DRok effectors in several distinct aspects of oogenesis (Verdier, 2006a).
The Rho-kinases (ROCKs) are major effector targets of the activated Rho GTPase that have been implicated in many of the Rho-mediated effects on cell shape and movement via their ability to affect acto-myosin contractility. The role of ROCKs in cell shape change and motility suggests a potentially important role for Rho-ROCK signaling in tissue morphogenesis during development. Indeed, in Drosophila, a single ROCK ortholog, DRok, has been identified and has been found to be required for establishing planar cell polarity. A potential role for DRok in additional aspects of tissue morphogenesis was examined using an activated form of the protein in transgenic flies. The findings demonstrate that DRok activity can influence multiple morphogenetic processes, including eye and wing development. Furthermore, genetic studies reveal that Drok interacts with multiple downstream effectors of the Rho GTPase signaling pathway, including non-muscle myosin heavy chain, adducin, and Diaphanous in those developmental processes. Finally, in overexpression studies, it was determined that Drok and Drosophila Lim-kinase interact in the developing nervous system. These findings indicate widespread diverse roles for DRok in tissue morphogenesis during Drosophila development, in which multiple DRok substrates appear to be required (Verdier, 2006b; full text of article).
The ability to rescue RCs with Ovhts transgenes provided an opportunity to investigate the function of Ovhts-RC. To determine when full-length Ovhts needs to be expressed for RC localization, stage-specific induction of Ovhts expression was done. Wild-type flies carrying a P{UASH-Ovhts::GFP} transgene were crossed to two different Gal4 lines: MTD-Gal4 that induces strong expression throughout oogenesis or P{bam-Gal4} that induces expression only in Region 1 of the germarium. In ovaries from P{UASH-Ovhts::GFP}, MTD-Gal4 flies, Ovhts::GFP was on RCs in all stages of oogenesis. However, in ovaries from P{UASH-Ovhts::GFP}; P{bam-Gal4}, Ovhts::GFP was only in the germaria, and on rare occasions on RCs in stage 2 egg chambers. Thus, continuous expression of Ovhts-RC protein is needed to maintain localization to RCs (Petrella, 2007).
The continual expression and localization of Ovhts-RC throughout oogenesis indicates that it may be important for actin maintenance at the RC. To test this possibility, P{UASH-Ovhts::GFP} was expressed in htsDeltaG flies using the P{nos-GAL4} driver whose expression is high in the germarium, low during stages 2-6, and then high again starting approximately at stage 7. This allowed rescue of RCs when they form in the germaria of mutants, followed by about a day where little or no new Ovhts protein is produced. As expected Ovhts::GFP was present on RCs in the germaria, absent in mid-stage egg chambers, and present again in later egg chambers within the same ovariole. Both the amount of F-actin and its organization at RCs mirrored the presence of Ovhts::GFP. When Ovhts::GFP was present, RCs appeared wild type. In egg chambers lacking Ovhts::GFP, there were no clear F-actin-containing RC rims. There were, however, actin-rich areas that may be disintegrating RC rims. Thus, continued expression of Ovhts is needed for the recruitment and/or maintenance of F-actin on RCs (Petrella, 2007).
Drosophila females bearing mutations in a previously undescribed gene, hu-li tai shao [(hts) too little nursing], produced egg chambers that contained fewer than the normal 15 nurse cells and that usually lacked an oocyte. The cytoplasmic bridges (ring canals) interconnecting nurse cells and the oocyte appeared abnormal, and lacked associated actin rings. The hts locus was found to encode a homolog of the mammalian membrane skeletal protein adducin. During oogenesis, hts mRNA became localized at the anterior of the oocyte and was subsequently expressed in a variety of embryonic tissues. These studies suggested that Drosophila adducin is needed to assemble actin at specialized regions of cell-cell contact in developing egg chambers and may also function at other times during the Drosophila life cycle (Yue, 1992; full text of article).
The structure of cytoplasmic bridges called ring canals were analyzed in Drosophila egg chambers. Two mutations, hu-li tai shao (hts) and kelch, disrupt normal ring canal development. Antibodies were raised against the carboxy-terminal tail of hts and it was found that they recognize a protein that localizes specifically to ring canals very early in ring canal assembly. Accumulation of filamentous actin on ring canals coincides with the appearance of the Hts protein. Kelch, which is localized to the ring canals hours after Hts and actin, is necessary for maintaining a highly ordered ring canal rim since kelch mutant egg chambers have ring canals that are obstructed by disordered actin and hts. Anti-phosphotyrosine antibodies immunostain ring canals beginning early in the germarium before Hts and actin and throughout egg chamber development. The use of antibody reagents to analyze the structure of wild-type and mutant ring canals has shown that ring canal development is a dynamic process of cytoskeletal protein assembly, possibly regulated by tyrosine phosphorylation of some ring canal components (Robinson, 1994; full text of article).
Adducin is a cytoskeletal protein that can function in vitro to bundle F-actin and to control the assembly of the F-actin/spectrin cytoskeletal network. The Drosophila Adducin-like (Add) locus (also referred to as hu-li tai shao) encodes a family of proteins of which several are homologous to mammalian adducin. Two novel adducin isoforms have been identified: a 95 x 10(3) Mr form (ADD-95) and an 87 x 10(3) Mr form (ADD-87). A detailed analysis of the distribution patterns of ADD-95 and ADD-87 during oogenesis and embryogenesis is presented. The isoforms are co-expressed in several cell- and tissue-types; however, only ADD-87 is present in mid- to late-stage oocytes. ADD-87 is present throughout the oocyte cortex at stages 9 and 10 of oogenesis but is detectable only at the anterior pole from stage 11 onward, correlated with localisation of Add-hts mRNA first to the cortex and then to the anterior pole of the oocyte. ADD-87 co-localises with F-actin and spectrin in the cortex of the oocyte through stage 10 of oogenesis, consistent with a possible role in cytoskeletal assembly or function predicted by mammalian studies (Zaccai, 1996a).
Because previous examination of Hts protein localization was done before there was a complete understanding of the hts locus, Hts antibody localization was characterized in more detail. Four antibodies directed against different Hts protein domains were used. htsF (Lin, 1994; Robinson, 1997) recognizes ShAdd, Add1 and Add2 (Add1/2) and Ovhts. In germaria, htsF antibody labels the fusome in the germline and plasma membranes in follicle cells. 1B1 antibody (Zaccai, 1996b), which recognizes Ovhts and Add1/2, has an identical germarium labeling pattern to htsF. htsM antibody, which recognizes only Add1/2, labeled follicle cell membranes and shows no labeling of the germline. In later-stage egg chambers, htsF, 1B1 and htsM antibodies continued to show specific labeling of lateral follicle cell membranes but no germline labeling. Consistent with RNA in-situ data (Whittaker, 1999), Ovhts is germline-specific and Add1/2 are follicle-cell-specific. As shadd mRNA is also exclusively found in the germline, and the htsF antibody only labels the fusome in the germline, ShAdd is likely a fusome component. Although both shadd and ovhts mRNAs are expressed in the germline throughout oogenesis (Whittaker, 1999), their protein products detected with 1B1 and htsF antibodies are only present in the germarium. This suggests that the proteins are either not translated or are not stable once egg chambers are formed (Petrella, 2007).
Because it was not possible able to make a useful peptide antibody specific for ShAdd, a ShAdd transgene was produced expressing ShAdd fused to Venus, a modified fluorescent protein (EYFP). When ShAdd::Ven was expressed with the ovarian tumor (otu) promoter in the germline of wild-type flies, it localized specifically to spectrosomes and fusomes. Since the fusome began to degrade in Region 2, the localization of ShAdd::Ven also became more dispersed. Thus, ShAdd::Ven provided additional evidence that ShAdd is a fusome protein (Petrella, 2007).
HtsRC antibody (Robinson, 1994), which recognizes the C-terminus of Ovhts, has a completely different localization pattern. Starting in Region 2a of germaria, htsRC labeled discrete puncta, which resolve into RCs in Region 2. htsRC antibody labels RCs throughout the rest of oogenesis (Petrella, 2007).
In order to verify the different labeling patterns of antibodies against the N- and C-termini of Ovhts, Ovhts transgenes were made that were expressed specifically in the germline. In separate constructs containing the native ovhts UTRs, the N-terminus of Ovhts was tagged with Cerulean, a modified ECFP, and the C-terminus was tagged with GFP. The Cer::Ovhts transgene did not produce a fluorescent product; however, upon labeling with anti-GFP antibodies, Cer::Ovhts was detected on the fusomes in germaria. Like the N-terminus of Ovhts as seen by antibody labeling, Cer::Ovhts localizes to both spectrosomes and branched fusomes. Co-staining with 1B1 showed that as the fusome begins to break down in Region 2, Cer::Ovhts becomes dispersed and looses colocalization with 1B1 (Petrella, 2007).
GFP fluorescence from Ovhts::GFP localizes specifically to RCs. As with htsRC antibody, protein is first detected in Region 2a as puncta that appear to be near, although not within the fusome. By Region 2b Ovhts::GFP is in rings. Ovhts::GFP is seen on RCs in all subsequent stages until stage 13. Thus, localization of tagged Ovhts transgenes recapitulates antibody labeling, with the N-terminus present on fusomes and the C-terminus localizing to RCs (Petrella, 2007).
Oogenesis in Drosophila takes place within germline cysts that support polarized transport through ring canals interconnecting their 15 nurse cells and single oocyte. Developing cystocytes are spanned by a large cytoplasmic structure known as the fusome that has been postulated to help form ring canals and determine the pattern of nurse cell-oocyte interconnections. The adducin-like hts product and alpha-spectrin have been identified as molecular components of fusomes, a related structure has been identified in germline stem cells, and regular associations between fusomes and cystocyte centrosomes have been documented. hts mutations completely eliminated fusomes, causing abnormal cysts containing a reduced number of cells to form. These results imply that Drosophila fusomes are required for ovarian cyst formation and suggest that membrane skeletal proteins regulate cystocyte divisions (Lin, 1994; full text of article).
Cytokinesis partitions a centrosome to each daughter cell at cell division that will duplicate and assemble a bipolar spindle in the subsequent M phase. Cytokinesis is incomplete in proliferating germ cells in Drosophila and cytoplasmic channels connect sibling germ cells. Although centrosomes are essential to male fertility, the molecular mechanism that retains centrosomes in parental germ cells is not known. Cortical cytoplasmic structures known as fusomes extend through ring canals and connect cells within the cyst. Fusome assembly in males requires function of hu-li tai-shao (hts), an adducin like protein found in fusomes and in the cortical membrane cytoskeleton of somatic cells. This work used immunological and cytological methods to place hts mutants in an allelic series. Male fertile hts mutants express Hts protein and generate apparently normal or fragmented fusomes. A male sterile allele does not express Hts protein or show fusome structures. Gonial cells in all hts mutants showed 2 centrosomes and mitotic spindles were bipolar. Yet, primary spermatocytes, with and without fusome structures, frequently contained too many or too few centrosomes. Although spindle structures were not found in spermatocytes without centrosomes, meiotic spermatocytes with centrosomes generated bipolar, monopolar, and multipolar spindles. Collectively, these results indicate that hts function is necessary for centrosome inheritance in spermatocytes as well as for male fertility (Wilson, 2005).
In order to elucidate the functions of the individual hts proteins, new alleles of hts were characterized. All previously described alleles of hts were P-element insertions or imprecise excisions that reduce expression of all hts transcripts. Two new EMS-induced hts alleles were examined. htsW532X contains a single nonsense mutation, W532X, in the tail domain. DNA sequencing of htsDeltaG showed a deletion of a single G in the last part of the Tail domain (G2346 of the ovhts transcript). This results in a frame shift followed by six novel amino acids and a stop codon. Conceptual translation of htsDeltaG results in a truncated protein that does not contain any of the normal C-terminal domains. These mutations are downstream of the entire ShAdd coding sequence (Petrella, 2007).
The phenotypes of these truncation alleles are indistinguishable from the P-element alleles. Both are female sterile and show a loss of oocyte specification, too few nurse cells, and no actin on RCs. However, labeling of htsDeltaG and htsW532X with Hts antibodies and western analysis showed a distinct difference between the alleles. Even though both truncation alleles should encode the epitope for the htsF antibody, protein was detected only in htsDeltaG. Western analysis showed that whereas htsDeltaG expressed a single truncation product, htsW532X produced no detectable protein and is therefore a null allele. Additionally in htsDeltaG, antibodies 1B1 and htsF labeled a cytoplasmic protein that persisted in egg chambers after they emerged from the germarium, which is never seen in wild type. Therefore, the mutant truncated protein is aberrantly stable in germline cells that normally do not have Ovhts-Fus. Mutant follicle cells labeled with 1B1 antibody show a significant, but not complete loss of Add1/2 localization to lateral membranes (Petrella, 2007).
To determine the functional requirements of the different Hts proteins in the germline, tagged hts transgenes expressed from the otu promoter were crossed into both htsDeltaG and htsW532X mutant backgrounds for rescue experiments. Both P{Ovhts::GFP} and P{Cer::Ovhts} rescued recruitment of Ovhts-RC and actin on RCs. However, other hts phenotypes were not rescued. Labeling with htsF, 1B1 or alpha-spectrin antibodies showed no fusome-like structure. Anti-GFP labeling in mutants expressing P{Cer::Ovhts} showed only cytoplasmic labeling. Additionally, the egg chambers still have too few cells and degenerate. Whether the addition of ShAdd would improve rescuing activity was tested. When P{ShAdd-Ven} is expressed alone in htsDeltaG, Venus fluorescence was diffuse in the cytoplasm, and hts phenotypes were not rescued. Expression of P{ShAdd::Ven} with either P{Cer::Ovhts} or P{Ovhts::GFP} in a htsDeltaG background showed the same phenotype as the single rescue: only RCs were rescued, but not the fusome or any of the phenotypes resulting from the loss of the fusome (Petrella, 2007).
Recent work has shown that the fusome precursor, the spectrosome, first begins to form during stage 11 of embryogenesis (Wawersik, 2005). Since the fusome develops from the spectrosome, it is possible that the otu promoter does not provide Ovhts at an early enough stage. However, earlier expression of Ovhts by driving P{UASH-ovhts::GFP} with either P{nos-GAL4} or P{tub-GAL4} produces the same result as the otu promoter (Petrella, 2007).
To determine whether there is a somatic contribution to the hts phenotype that results in the inability to rescue the fusome, clonal analysis was performed with htsDeltaG. Germline clones showed the same phenotype as homozygous mutants, whereas egg chambers that had only follicle cell clones were normal. Therefore, the loss of the fusome and RCs is caused solely by the loss of full-length Ovhts in the germline (Petrella, 2007).
During the rescue experiments with P{Ovhts::GFP}, it was noticed that localization of Ovhts-RC::GFP to RCs was delayed. In wild-type flies expressing this transgene, GFP is always present in Region 2 of germaria. In contrast, in either htsDeltaG or htsW532X flies expressing P{Ovhts::GFP}, GFP is often absent from germaria and only appears later. Quantitation of this phenotype revealed that in htsW532X there is a delay in 30% of germaria, and in htsDeltaG there is a delay in 91% of germaria. Thus, in hts mutants the recruitment of Ovhts-RC and actin, and therefore the establishment of RCs, does not occur at the correct developmental stage, suggesting that the fusome is necessary for the timing of RC development (Petrella, 2007).
Advantage of the ability of S2 cells to cleave Ovhts to identify amino acids necessary for its cleavage. A series of small in-frame deletion mutations were made in the region of the predicted cleavage site, and expressed in S2 cells. The Delta1 deletion, which removes 20 amino acids in the Tail domain, is cleaved at a wild-type level. Deletion Delta2 removed the last 20 amino acids of the Tail domain, and Delta3 removed the last nine amino acids of the Tail and the first 11 amino acids of the RC domain. The Delta2 protein was cleaved, although less efficiently than wild-type protein, whereas the Delta3 protein was not cleaved. This result demonstrates that the first 11 amino acids (ALVSQLAQKYA) of the RC domain are required for cleavage (Petrella, 2007).
To study the effect of uncleavable Ovhts in the ovary, a P{Ovhts-Delta3::GFP} transgene was made that was expressed from the otu promoter. Except for the 20 amino acid deletion, this transgene was identical to the P{Ovhts::GFP} transgene. Western analysis of Ovhts-Delta3::GFP from ovary extracts demonstrated that, as in S2 cells, this protein is not cleaved. When expressed in wild-type flies, Ovhts-Delta3::GFP was present not only on RCs, but also on the fusome. Additionally, the 1B1 antibody, which normally only labels the fusome, now also labeled RCs. Therefore, the uncleaved protein localized to both structures where the cleavage products are normally found (Petrella, 2007).
Although wild-type flies expressing Ovhts-Delta3::GFP are fertile and produce apparently normal egg chambers, uncleaved Ovhts does cause a subtle, but completely penetrant dominant defect in the disappearance of the fusome. In wild-type germaria, the fusome begins to disappear where the RCs are starting to form. This results in unobstructed RCs with fragmented fusome material between them, but not through them. In flies expressing P{Ovhts-Delta3::GFP}, fusome material is present within the RCs as late as stage 2 and 3 egg chambers. In some cases, GFP-positive rings are occluded with GFP-negative fusome material that can be visualized with 1B1 antibody. Therefore, at least some of the aberrant fusome contains only wild-type Ovhts-Fus protein and not the N-terminal portion of P{Ovhts- Delta3::GFP}. Additionally, RC rims were thicker than normal, less organized and misshapen. These results suggest that the cleavage and proper maintenance of the Ovhts domains may play a role in the transition from a fusome to RCs (Petrella, 2007).
Whether the P{Ovhts-Delta3::GFP} transgene could rescue hts mutants, htsDeltaG and htsW532X, was also tested. As with expression of P{Ovhts::GFP}, P{Ovhts-Delta3::GFP} rescues RCs but not the fusome. The rescued RCs were also labeled with 1B1 antibody indicating that the N-terminus of Ovhts was present (Petrella, 2007).
Reference names in red indicate recommended papers.
Search PubMed for articles about Drosophila Hts
Aoshima, R., Hiraoka, R., Shimada, N. and Kawata, T. (2006). Analysis of a homologue of the adducin head gene which is a potential target for the Dictyostelium STAT protein Dd-STATa. Int. J. Dev. Biol. 50(6): 523-32. Medline abstract: 16741867
Barkalow, K. L., et al. (2003). Alpha-adducin dissociates from F-actin and spectrin during platelet activation. J. Cell Biol. 161(3): 557-70. Medline abstract: 12743105
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date revised: 2 July 2007
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