Drosophila Tribbles (Trbl) encodes the founding member of the Trib family of kinase-like proteins that regulate cell migration, proliferation, growth and homeostasis. Trbl was identified in a misexpression screen in the ovary as an antagonist of border cell migration and acts in part by directing turnover of the C/EBP protein encoded by the gene slow border cells (slbo). The ability of mammalian Trib isoforms to promote C/EBP turnover during tissue differentiation indicates that this function is highly conserved. To better understand the role of Trbl in cell migration, specific Trbl antisera, a trbl null allele and Trbl transgenes bearing site-directed mutations were tested. Trbl is expressed at high levels in the nuclei of follicle cell epithelia and is downregulated in delaminating epithelia as expression of Slbo (C/EBP) is upregulated. This complementary pattern of expression during subsequent cell migration is achieved by negative feedback whereby slbo represses Trbl expression and trbl is necessary and sufficient to promote Slbo protein turnover. A series of point mutations that scan the conserved kinase domain of Trbl reveal that the conserved DLK catalytic loop is required for Trbl-Slbo binding and turnover, as well as for interactions between Trbl subunits, suggesting a mechanism of Trbl function (Masoner, 2013).
Mammalian Trib proteins have diverse functions (reviewed in Dobens, 2012), as transcriptional co-activators and repressors in the nucleus, and as MAP kinase kinase inhibitors and proteosome adapters in the cytoplasm. Tribs bind and direct the degradation of key regulatory proteins, in particular members of the C/EBP family, notably (1) C/EBP α, which is degraded by Trib1 to promote the formation of acute myelogenous leukemia (AML) tumors and by Trib2 during myeloid differentiation (Keeshan, 2006) and (2)C/EBPβ, which is degraded by Trib2 during differentiation of 3T3-L1 preadipocytes (Naiki, 2007). As well, C/EBPβ levels increase in Trib1 knockout mice (Yamamoto, 2007; Keeshan, 2008; Masoner, 2013 and references therein).
For Trib2, it has been demonstrated recently that a point mutation in the DLK catalytic loop motif disrupts its ability to direct turnover of C/EBP α and promote acute myelogenous leukemia (Keeshan, 2010), and this study demonstrates that the same DLK/R mutation in fly Trbl compromises its ability to direct Slbo turnover and block BC migration. It was also shown that an intact DLK catalytic motif is required for Trbl interactions both with Slbo and with Trbl, itself. These data support the notion that the conserved catalytic domain is critical for Trbl function, however it remains unclear if this mutation disrupts ATP binding-dependent protein folding or whether Tribs are functional kinases, a distinction which will be resolved ultimately by identifying bonafide Trib substrates (Masoner, 2013).
The D/NLK Trbl mutant surprisingly retains WT activity when misexpressed in the posterior wing compartment, resulting in larger cells as measured by more widely spaced bristles when compared to the anterior compartment. It is noted that a similar mutation in Trbl located just C-terminal to DLK (DLKLK/R) also retains WT activity to block cell proliferation when injected into the blastoderm epithelium. Because WT Trbl is thought to block cell division by directing String/cdc25 turnover, the data suggest another domain outside the Trbl catalytic loop must be required for String degradation (Masoner, 2013).
Several observations are worth noting regarding the trbl protein null allele and specific antisera that were analyzed. The trblD13 deletion allele described in this study is a stronger allele than those used previously, but still yields a few escaper adults in combination with a deficiency of the locus. Though trbl is not required for viability, it was found that rare escaper animals hatch late and are infertile with vestigial ovaries, suggesting trbl has unexplored roles both in larval tissue growth and cell proliferation during early oogenesis. Antisera to Trbl reveal dynamic changes in subcellular localization during late oogenesis: Trbl accumulates in FC nuclei up to stage 11 after which nuclear levels drop and Trbl accumulates at low levels in the cytoplasm. The notion that Trib localization is regulated and has functional importance is supported by observations that (1) GFP-tagged versions of Tribs are localized variously to the cytoplasm, nucleus and even the mitotic spindle (Saka, 2004), and (2) during BMP/TGF-β-signaling Tribs bind BMP receptors at the cell cortex, upon signaling are released into the cytoplasm to bind and degrade SMURFS, and subsequently translocate to the nucleus to serve as SMAD co-activators (Hegedus, 2006; Hegedus, 2007; Chan, 2007; Hua, 2011; Masoner, 2013 and references therein).
Antisera detect dynamic changes in Trbl levels during BC migration: Trbl is strongly expressed prior to delamination from the anterior epithelium and Trbl levels decrease during posteriorwards BC migration. In slbo mutant egg chambers Trbl levels are significantly higher compared to WT at all stages examined, indicating that slbo is necessary to repress Trbl. However Slbo misexpression alone is not sufficient to repress Trib expression, suggesting that cofactors mediate Slbo repression of Trbl. The observation that slbo represses Trbl stands at odds with previous work showing that Slbo turnover is reduced in slbo mutant egg chambers. While further work must be done to reconcile these observations, it is possible that slbo both represses Trbl expression and activates an unidentified Trbl activator, whose existence is implied by work in embryos. In this way, while Trbl levels increase in a slbo mutant, its ability to direct Slbo turnover could be compromised (Masoner, 2013).
Together these data indicate that Slbo and Trbl are at the core of a negative feedback loop in which Slbo represses Trbl expression and conversely Trbl (via its catalytic loop) binds and degrades Slbo. A yeast two-hybrid interaction assay was used to test the strength of Trbl interactions in the absence of Trbl-directed Slbo turnover and it was shown (1) that weak interactions occur between either Trbl-Trbl or Slbo-Slbo, while (2) comparatively stronger Trbl-Slbo interactions occur. These observations adds a key feature to the model: the proposition that weak homomeric complexes of Trbl multimers and Slbo dimers exchange preferentially for strong heteromeric Trbl-Slbo complexes to direct Slbo turnover. Such a model is consistent with observations that in some instances protein kinase dimerization leads to autoinhibited complexes and is supported by work showing that (1) purified Trbl protein expressed in E. coli can form dimers and tetramers and (2) the strength of Trbl-Trbl interaction impacts the activity of target pathways (Masoner, 2013).
Snail-related transcription factors, which direct E-cadherin-dependent cell migration in a wide range of normal and diseased tissues, are also regulated by protein turnover. Several modifications affect Snail stability, including phosphorylation by PAK and GSK3β, dephosphorylation by the small C-terminal domain phosphatase (SCP), and lysine oxidation is promoted by NFkappaB. In the latter case, NFkappaB prevents Snail phosphorylation by GSK-3 and subsequent degradation, whereas formation of a ternary complex between wild-type p53, the ubiquitin ligase Mdm2, and Snail2 promotes degradation. It is likely that during BC migration a similar level of complexity underlies Trbl effects on Slbo stability, and the proposed feedback between these genes results in oscillating levels of Slbo and corresponding fluctuating expression of Slbo target genes, notably DE-cadherin, whose turnover at the cell membrane has been demonstrated to promote proper BC migration (Masoner, 2013).
In mammals, Trb family members have been identified as tumor suppressors or oncogenes, depending on tissue context and sorting out these conflicting data may be aided from a simpler Drosophila model. Conserved interactions between Trbl and C/EBP during cell differentiation in flies and mammals suggest the possibility that interactions between Trbl and the cdc25 phosphatase String observed during cell division in fly tissue might be conserved in mammals as well. Conversely, mammalian work holds the promise to illuminate and direct further tests of Trbl function in Drosophila, notably documented interactions between mouse Trib3 and ATF4, another member of the B-Zip class of transcription factors active during pancreas β-cell differentiation, and human Trib3 with Atk kinase during insulin-target cell metabolism. Synergies between these parallel lines of investigation will shed light on the diverse roles of Trb family members in cell growth, proliferation and differentiation (Masoner, 2013 and references therein).
Drosophila Tribbles (Trbl) is the founding member of the Trib family of kinase-like docking proteins that modulate cell signaling during proliferation, migration and growth. In a wing misexpression screen for Trbl interacting proteins, the Ser/Thr protein kinase Akt1. Given the central role of Akt1 in insulin signaling, the function of Trbl was tested in larval fat body, a tissue where rapid increases in size are exquisitely sensitive to insulin/insulin-like growth factor levels. Consistent with a role in antagonizing insulin-mediated growth, trbl RNAi knockdown in the fat body increased cell size, advanced the timing of pupation and increased levels of circulating triglyceride. Complementarily, overexpression of Trbl reduced fat body cell size, decreased overall larval size, delayed maturation and lowered levels of triglycerides, while circulating glucose levels increased. The conserved Trbl kinase domain is required for function in vivo and for interaction with Akt in a yeast two-hybrid assay. Consistent with direct regulation of Akt, overexpression of Trbl in the fat body decreased levels of activated Akt (pSer505-Akt) while misexpression of trbl RNAi increased phospho-Akt levels, and neither treatment affected total Akt levels. Trbl misexpression effectively suppressed Akt-mediated wing and muscle cell size increases and reduced phosphorylation of the Akt target FoxO (pSer256-FoxO). Taken together, these data show that Drosophila Trbl has a conserved role to bind Akt and block Akt-mediated insulin signaling, and implicate Trib proteins as novel sites of signaling pathway integration that link nutrient availability with cell growth and proliferation (Das, 2014: 25329475).
tribbles is expressed in stage 5 embryos. Tribbles mRNA is expressed in the germline and provided maternally. During cellularization this ubiquitous maternal mRNA disappears and zygotic expression is observed in the ventral region. The expression domain includes the presumptive mesoderm but extends beyond it both anteriorly and posteriorly. Older embryos show very little expression of tribbles (Mata, 2000).
Tribbles can be localized both in the nucleus and the cytoplasm, and preliminary results suggest that this localization may be cell cycle dependent (Mata, 2000).
trbl transcript is absent from early cleavage embryos and accumulates to high levels at the beginning of cycle 14. During cellularization, expression levels decline, but transcripts are present throughout gastrulation, and in fact persist until late embryogenesis. At the beginning of gastrulation, the RNA distribution is not uniform. Highest levels are seen in ventral cells; the extent of this expression, however, is wider than the ventral furrow itself and thus wider than the domain in which trbl-dependent mitotic delays are observed (Großhans, 2000).
Tribbles, mesoderm formation and the timing of mitosis
The final shape and size of an organism is determined by both morphogenetic processes and cell proliferation and it is essential that these processes be properly coordinated. In particular, cell division is incompatible with certain types of morphogenetic cell behaviour, such as migration, adhesion and changes in cell shape. Mechanisms must therefore exist to ensure that one does not interfere with the other. This study addresses the coordination of proliferation and morphogenesis during the development of the mesoderm in Drosophila. It is essential that mitosis be blocked in the mesoderm during early gastrulation, and the putative serine/threonine kinase Tribbles is identified as controlling this block. In its absence, the mitotic block is lifted, resulting in severe defects during early gastrulation. Tribbles, a homolog of a group of vertebrate proteins of unknown function, acts in concert with another, as yet unidentified, factor to counteract the activity of the protein phosphatase Cdc25/String. It has been concluded that in a finely tuned balance with Cdc25/String, Tribbles controls the timing of mitosis in the prospective mesoderm, allowing cell-shape changes to be completed. This mechanism for coordinating cell division and cell-shape changes may have helped Drosophila to evolve its mode of rapid early development (Seher, 2000).
The mitoses in each of the mitotic domains are preceded by the expression of the protein phosphatase Cdc25/String. Initially, cells in the blastoderm-stage embryo are arrested in G2, with the cyclin-dependent kinase Cdk1 in the cyclin-Cdk1 complex in its inactive, phosphorylated form. As soon as String is expressed in a domain, it removes the inhibitory phosphates and allows the cells to enter mitosis. The prospective mesoderm represents an exception, being the first domain to express String, but the tenth domain to divide. The reason for this delay in cell division is not known, although the level of String in this domain is initially low and rises until the point at which mesodermal cells eventually divide (Seher, 2000).
The developmental relevance of the precise sequence and timing of cell division in the Drosophila embryo is not understood for most domains. There are, however, instances when cell division would interfere with other developmental processes. One such case is the mesoderm, which invaginates by a series of precisely orchestrated cell-shape changes immediately after the cellular blastoderm has formed. During invagination, the cell sheet representing the mesodermal primordium retains its epithelial integrity and no cell divisions occur until the mesoderm is fully internalized. Prematurely induced cell division has been suggested to disrupt mesoderm morphogenesis (Seher, 2000).
Even the initially low level of String present in the early mesoderm is shown to be sufficient to induce mitosis. Mitosis is blocked, however, by the concerted activity of at least two factors until the completion of ventral furrow formation. One of these factors is Tribbles, an atypical member of the protein kinase superfamily (Seher, 2000).
In a screen for mutations that disrupt gastrulation four regions of the genome were identified that are necessary for proper invagination of the mesoderm primordium, two of which will be discussed here. One (chromosomal region 77B7-77D1) is represented by the overlap of the deficiencies Df(3L)rdgCco2 and Df(3L)ri79c; the other (region 71C3-71E5) by Df(3L)BK10. In embryos homozygous for any one of these deficiencies, or trans-heterozygous for Df(3L)rdgCco2 and Df(3L)ri79c, the ventral furrow is not formed properly. Whereas most of the mesoderm has been internalized in a wild-type embryo by the time germ-band extension begins, the entire mesoderm primordium is still on the surface of the mutant embryos. The mesoderm does, however, eventually move into the interior and in many embryos no lasting defects due to the early failure in morphogenesis are seen. The successful completion of mesoderm invagination could be due to redundancy of genes in these two regions of the genome. If this were the case, simultaneous deletion of both gene functions should exacerbate the phenotype, perhaps leading to a complete failure to internalize the mesoderm. The gastrulation phenotype of double mutants of Df(3L)BK10 and Df(3L)ri79c is, however, identical to that of the single mutants (Seher, 2000).
A striking aspect of the phenotype of the mutants is that, while the mesoderm is still on the surface of the embryo, many mesodermal cells appear to have already undergone mitosis. Cell division in mutant embryos was analyzed by staining them with an antibody against tubulin and an antibody specific for the phosphorylated form of histone 3 (PH3) to visualize mitotic cells. In wild-type embryos in which mitotic domains 1-3 are stained, the mesodermal cells mostly remain in interphase. By contrast, mesodermal cells in the mutants are undergoing or have completed mitosis, as judged by mitotic spindles visualized using the antitubulin antibody and condensed chromosomes at various stages of mitosis visualized using anti-PH3 antibodies. Analysis of younger mutant embryos shows that the mesodermal domain is in fact the first to begin mitosis. Thus the mesodermal cells in the mutant embryos show two defects: they fail to undergo the normal cell-shape changes associated with ventral furrow formation and they divide much too early (Seher, 2000).
An additional defect in cell-cycle control has been observed in the primordial germ cells. The pole cells normally divide twice after they have budded from the early syncytial embryo, and then remain mitotically silent until larval life. By contrast, dividing pole cells are found in approximately 75% of mutant embryos between stage 6 (ventral furrow formation) and stage 10 (extended germ band). Only the individual cells are seen in mitosis, never are many seen, nor are all the pole cells seen to divide simultaneously (Seher, 2000).
The causal relationships between the morphogenetic and the mitotic defects in the mesoderm have been investigated. Normally, no cell divisions occur while mesodermal cells are changing their shapes to create the ventral furrow. To test whether it is important that mitosis be prevented during ventral furrow formation, String was specifically overexpressed in the mesoderm primordium. In these embryos, the first cells to divide are those of the mesodermal primordium. The resulting premature mitoses disrupt gastrulation severely, showing that suppression of mitosis is essential to allow early gastrulation to proceed properly. Conversely, there is no evidence from embryos that fail to undergo normal mesodermal cell-shape changes that this failure causes premature mitoses (Seher, 2000).
To investigate whether premature mitoses are indeed the cause of the gastrulation defects that are observed in embryos homozygous for Df(3L)ri79c, mitosis in the mutant embryos was blocked genetically by making double mutants of Df(3L)ri79c and string. In string mutant embryos, which form a ventral furrow indistinguishable from wild-type embryos, none of the post-blastoderm mitoses occurs. In Df(3L)ri79c string double-mutant embryos no cell divisions occurs and gastrulation is no longer abnormal. Thus the premature divisions are the cause of the morphogenetic defect, showing that Df(3L)ri79c deletes a gene that is responsible for blocking mitosis in the mesoderm until the formation of the ventral furrow has been completed. The loss of this gene does not affect mitosis by causing abnormally early or high levels of String expression in the mesoderm. Furthermore, there are no obvious effects on the accumulation or stability of String protein, since the level of staining in the mutant mesoderm primordium seems no higher than in the wild type. Thus, this gene must affect cell division either by affecting regulation of String activity at the post-translational level or by interfering with a process downstream of or in parallel to String (Seher, 2000).
To identify the gene responsible for the observed phenotype, mutations were screened that map to the overlap of the two deficiencies. Two P-element insertions, EP(3)3519 and EP(3)1119, when homozygous, cause the same phenotype as the deficiencies. Trans-heterozygotes for the two mutations and each mutation over Df(3L)ri79c also show the same phenotype, indicating that they are alleles of the same gene. By excising the inserted P element EP(3)3519 the mutant phenotype can be reverted. The P elements are inserted 8 bp upstream and 186 bp downstream of the presumed transcription start site of an expressed sequence tag (EST) identified by the Drosophila Genome Project. The RNA for this gene is initially found throughout the newly laid egg but fades by the end of cellularization. Zygotic expression is first seen in the region of the prospective mesoderm, shortly afterwards in the ectoderm, and later in a dynamic pattern throughout embryogenesis. Injection of double-stranded RNA of this EST phenocopies the defects seen in the mutants. Together, these findings indicate that the transcript corresponds to the tribbles gene (Seher, 2000).
Animals homozygous for tribbles can survive to adulthood. Loss of homozygotes does not appear to occur at any specific stage but throughout all developmental stages. Homozygous adults appear normal, except that females are infertile, laying very few eggs; these look normal but are rarely fertilized. No gross abnormalities are seen in ovaries and the cause of infertility was not investigated further (Seher, 2000).
The loss of tribbles homozygotes throughout development suggests that Tribbles may modulate cell proliferation at many developmental stages. To test whether it could affect division in cells outside the mesoderm, tribbles was overexpressed in a wide range of cells and organs during embryogenesis and larval stages. No obvious defects were seen in any of these situations. For example, as judged by staining for PH3, no pair-rule segmental aberrations were seen in the patterns of mitotic domains in embryos in which tribbles was expressed in the paired domain under the control of a paired-GAL4 driver line. Defects such as slowing down of the cell cycle, for example, by extension of the G2 phase, or a change in the number of cells in mitosis should have been easily detectable as changes in the regularity of the mitotic domains. Moreover, embryos overexpressing tribbles in the paired domain were viable and larvae overexpressing tribbles in the eye discs or wing discs produced flies with normal eyes and wings. These results show that Tribbles cannot generally override normal cell-cycle control mechanisms (Seher, 2000).
It is concluded that in at least two situations Tribbles acts as a mitotic inhibitor. In the primordial germ cells of tribbles mutants a low level of mitotic activity is seen that is not detectable in wild-type germ cells at similar stages. In contrast to the situation in the mutant mesoderm, however, only a few of these cells divide. This shows that germ cells at this stage are able to divide (as has also been found in nanos mutants) and must therefore contain all the necessary components to drive cells through mitosis. However, not all cells divide, suggesting that there must either be other inhibitors apart from Tribbles, or that at least one of the mitotic activators in primordial germ cells must be present at threshold concentration (Seher, 2000).
Mitosis in other cells could not be blocked by supplying high levels of Tribbles. This might mean that to block mitosis Tribbles needs a cofactor that is present in the mesoderm but absent in other tissues, or that the cofactor is present in limiting amounts sufficient only to interact with naturally occurring levels of Tribbles. A potential candidate for such a partner is the product of the gene in the second genomic region, uncovered by Df(3L)BK10, since the gastrulation phenotype of this deficiency resembles that of tribbles. This gene does not act as a transcriptional regulator of tribbles, as demonstrated by the finding that the expression pattern of tribbles is not affected in Df(3L)BK10 mutant embryos. Its product might instead modify Tribbles post-transcriptionally, form a complex with Tribbles or act in a parallel pathway that converges with the Tribbles pathway further downstream to block cell division. Further elucidation of this problem will have to await the molecular identification of the gene in Df(3L)BK10 (Seher, 2000).
It is possible that Tribbles can block only mitosis driven by low levels of String, as found in the early mesoderm primordium. The precise level of String in the mesoderm should then determine whether Tribbles is able to block mitosis. This is consistent with the findings that overexpression of String in the mesoderm of wild-type embryos overcomes the block Tribbles imposes on mitosis and produces the same phenotype as that seen in homozygous tribbles mutants. Only transgenes expressing high levels of String have this effect, however, with others showing weaker or no mutant phenotypes. This demonstrates that the ability of Tribbles to block mitosis depends on the balance between String and Tribbles (Seher, 2000).
These results explain why the very early expression of String in the mesoderm does not lead to correspondingly early mitosis. Furthermore, they show that even the low level of String present in the mesoderm is sufficient to trigger mitosis. A further reduction of String by 50% (in string heterozygotes) does not compromise its ability to trigger mitosis in the mesoderm. And in the absence of Tribbles, such reduced levels of String still cause premature mitosis and disruption of the ventral furrow (Seher, 2000).
A large number of Drosophila and Caenorhabditis elegans genes identified by sequencing are not essential under laboratory conditions and may not have easily detectable mutant phenotypes even though they may have highly conserved homologs in other species. tribbles is an example of such a gene. While not essential, it is required for the fine tuning and optimization of developmental processes, especially during gastrulation. In this context, it is interesting to note that the appearance of the process of mesoderm invagination in tribbles mutants resembles that in the beetle Tribolium. Tribolium is a short-germ insect whose development proceeds much more slowly than that of the long-germ Drosophila embryo, and in which the mesoderm, as in tribbles mutants, invaginates slowly as a cell mass rather than rapidly as an epithelial tube. The speed and regularity with which the mesoderm invaginates in Drosophila is not essential, but may have helped Drosophila to evolve its mode of rapid early development (Seher, 2000).
The opposing phenotypes of tribbles overexpression and loss-of-function mutant in the ovary, as well as the similarity between the latter and string GOF indicates that tribbles does downregulate CDC25 in vivo. To establish the importance of this regulation, the loss-of-function phenotype of tribbles was investigated in more detail. The fact that tribbles1119 homozygotes show poor viability which can be rescued by the tribbles transgene, suggests that tribbles plays an important role during development. Control of CDC25 activity is particularly important at the transition between maternally and zygotically controlled cell cycles in the embryo. To ask whether Tribbles might regulate String at these critical stages, patterns of mitosis were analyzed in tribbles mutant embryos (Mata, 2000).
tribbles mutant embryos from tribbles1119/Deficiency mothers have distorted patterns of mitosis. A very small percentage of embryos undergo an extra round of division before cellularization, similar to the phenotype observed for embryos with extra copies of string and twine. However, the most clear and penetrant defect is seen in the majority of embryos of late stage 5 or stage 6. These embryos show precocious mitosis occurring throughout a broad band in the ventral region. Mitosis is either observed in this domain by itself, or in this domain plus several smaller patches in the head region. The smaller patches correspond to the normal mitotic domains 1-3 (the earliest mitotic domains). At stage 5/6, wild-type cells are arrested in G2 of cycle 14 due to degradation of maternal CDC25 protein and mRNA. Zygotic reexpression of string spatially and temporally controls cycle 14 mitoses. The broad ventral band of mitosis seen in stage 5/6 tribbles mutant embryos corresponds to the earliest zygotic expression of string, in domain 10, the mesoderm anlage. In wild-type embryos, the mesoderm anlage is one of the few domains in which string mRNA does not temporally predict mitosis. Mitosis normally occurs with some delay, after the cells have undergone gastrulation. In tribbles mutant embryos, the early patterns of mitosis appear to accurately reflect the pattern of string mRNA accumulation (Mata, 2000).
It has been assumed that the delay of mitosis in the mesoderm anlage is required because mitosis would interfere with the morphogenetic movement of gastrulation. The progression of gastrulation was examined in both control and tribbles mutant embryos by examining expression of single-minded (sim), which marks CNS midline precursors at the transition between mesoderm and neurectoderm. In control embryos, sim-expressing cells become aligned at the midline. In contrast, tribbles mutant embryos show serious gastrulation defects. None of the mutant embryos show normal early alignment of sim-expressing cells. Since tribbles1119/Deficiency produces no detectable tribbles mRNA, tribbles1119 is a null or close to a null mutant. Nevertheless, the mutation is not completely lethal. Many (45%) tribbles mutant embryos hatch but look abnormal and die during larval stages. Overall 14% of tribbles mutant flies survive to adulthood. This may be explained by the fact that although the gastrulation defects in tribbles embryos are severe, some embryos appear to recover after being delayed from normally entering gastrulation, or after being deformed. Finally, this is consistent with the observation that inducing a synchronous 14th mitosis by driving string with a heat shock promoter is lethal to only 60% of the embryos (Mata, 2000 and references therein)
The phenotype of premature ventral mitosis and the resulting gastrulation phenotype are primarily due to lack of zygotic tribbles expression. This conclusion was reached because the phenotype is rescued by crossing tribbles1119/Deficiency females to wild-type males but not to tribbles1119/tribbles1119 males. In addition, the phenotypes are observed at the expected frequency in embryos from tribbles1119/TM3 stocks (Mata, 2000).
If the phenotypic effects of zygotic tribbles were strictly due to upregulation of zygotic string, then removing string should rescue the tribbles phenotype. This prediction was tested by analyzing gastrulation of embryos that were mutant for both string and tribbles. Double-mutant embryos do not show the tribbles phenotype. This confirms genetically that the role of tribbles is to negatively regulate string (Mata, 2000).
To determine whether endogenous tribbles acts by affecting CDC25/String turnover as proposed, String mRNA and protein accumulation were examined in tribbles mutant embryos. While String mRNA appears to accumulate normally, tribbles mutant embryos show a moderate, but reproducible, increase in String protein level at 2-4 hr of development. string mRNA and high level of tribbles mRNA coincide primarily in the ventral region in late stage 5 embryos. Since Western blots average the effect over the total String protein in the embryo, it is probable that the magnitude of the effect is higher in the presumptive mesodermal cells. This suggests that the main role of Tribbles is to delay accumulation of zygotic String protein. This view is supported by the observation that removing string suppresses the zygotic requirement for tribbles (Mata, 2000).
Tribbles did not appear to be required for the maternally controlled turnover of the bulk of String protein following cycle 10, although there may be a small increase in early String levels in mutant embryos. Unfortunately, no CDC25/Twine antibody is available. Since an additional division before cellularization is occasionally observed in tribbles mutant embryos, it is therefore possible that tribbles also plays a minor role in the turnover of maternal CDC25 (Mata, 2000).
If string expression is already regulated at the transcriptional level, why is this additional post-transcriptional control required? string transcriptional control 'piggybacks' on the cell fate patterning genes and events. In the mesoderm anlage, string transcription is regulated by the mesoderm determining factors twist, snail, and other genes. As these factors are already active before gastrulation, another mechanism is needed to ensure that mitosis is delayed. tribbles is also transcriptionally upregulated in the ventral region, including the mesoderm anlage where it is required to downregulate String. tribbles may be under direct transcriptional control of twist and snail or their upstream regulators. Using the same transcription factors to upregulate string and tribbles provides a simple mechanism to delay mitosis in this critical region. This mechanism is sufficient to explain the mitotic delay in the mesoderm anlage (Mata, 2000).
Tribbles and regulation of oocytic cell division
tribbles mutation produces an extra division within oocytes with very high frequency (>90%). The mutant egg chambers have one oocyte (31+1) and, apart from their slightly larger than usual size, they look normal. The 31+1 arrangement appears to be functional because 40% of embryos from such females hatched. The 60% lethality may be due to maternally provided tribbles overexpression in the embryo (Mata, 2000).
Interestingly, oocyte determination is also affected in mutant lines. Females overexpressing the tribbles cDNA using UASp vector and nanosGAL4:VP16 drivers have smaller ovaries with a reduced number of egg chambers per ovariole. Most cysts exhibited the 31+1 phenotype, but a few percent of them had 30 nurse cells and two oocytes. Each of the two oocytes contained five ring canals, one ring canal was between them, and they appeared equal in terms of morphology and yolk accumulation. Direct insertion of a cDNA in a UAS vector usually gives higher level of GAL4-induced expression than an EP insertion in the gene. Thus, moderate overexpression of tribbles induces an extra cystocyte division, and higher level expression can in addition convert an extra cell into an oocyte (Mata, 2000).
In the case of string GOF, half of the 8-cell cysts had a normal oocyte, with very condensed DNA and were located at the posterior of the egg chamber. The rest the DNA of the putative oocyte showed different degrees of polyploidization, and the cell often failed to reach the posterior. It is not known why some cysts have an oocyte and others do not. It may reflect different levels of string overexpression in different cysts, with higher level of expression resulting in loss of oocyte. The 16-cell cysts were mostly normal, but 2% of them failed to develop a normal oocyte (Mata, 2000).
Additional EP insertions have been identified in the tribbles locus. EP1119 was inserted in the 5'UTR and it is a strong loss-of-function allele of tribbles, producing no detectable tribbles mRNA. EP1119 homozygous or EP1119 heterozygous with a deletion of the region has low viability (14% of mutant flies can survive to adulthood). The surviving females showed frequent (average 20%) 8-cell germline cysts. Half of such cysts have an oocyte, half do not. Thus, reduced tribbles activity has an effect opposite that of tribbles overexpression. This indicates that tribbles is normally involved in controlling cystocyte divisions and oocyte determination, and does so in a dose-dependent manner. The oogenesis phenotype (as well as the reduced viability) of tribbles1119 is rescued by one copy of a transgene containing the tribbles locus (Mata, 2000).
The control of cystocyte divisions is not well understood. Both germline-specific factors, such as bag-of-marbles (bam), and general cell cycle factors appear to play important roles. Removing one copy of bam can suppress the phenotype of encore mutant females, which contain 31+1 egg chambers. However, removing one copy of bam has no effect on the tribbles GOF phenotype (Mata, 2000).
What are the targets of Tribbles in the germline? Overexpression of string and mutations in tribbles have similar phenotypes, and simultaneous overexpression of string and tribbles suppresses the phenotype of the other. These data, together with tissue culture results, suggest that String is downregulated by Tribbles in the germline. Twine is also a likely target, since it is present in the germline and Tribbles can promote its degradation. String and Twine appear to perform largely redundant functions in cystocyte divisions and in early blastoderm divisions. Thus, the phenotypes observed may be due to an effect on String, on Twine or on both of these CDC25 proteins (Mata, 2000).
The fact that overexpression of String in the germline decreases the number of cystocyte divisions is unexpected. Overexpression of other positive regulators of mitosis (Cyclin A and Cyclin B) has the opposite effect (Lilly, 2000). One possibility is that the effect of overexpression of String or mutations in Tribbles is caused by reducing the length of the G2 phase of the cell cycle. For instance, divisions can be counted by producing a positive regulator during the G2 phase of the first cycle, the supply of which would be exhausted after four cycles. Shortening G2 would result in less regulator being accumulated and thus one division less, while elongating G2 by Tribbles-induced downregulation of String/Twine would have the opposite effect. However, there are many possible explanations. Understanding the role of specific regulators such as Tribbles and CDC25 in the germline will require a careful description of the cystocyte cell cycles. It will be necessary to know at which transitions these cell cycles are regulated, and which molecules are rate limiting (Mata, 2000).
The effects of string overexpression or tribbles mutations on oocyte differentiation could also be explained by a shortened G2 phase. The fusome, which is required for oocyte formation, appears to undergo distinct morphological changes during each phase of the cell cycle. It is possible that a completely functional fusome cannot be assembled in a shortened G2 period, leading to impaired oocyte development. It is also possible that the levels of CDC25 proteins have a more direct effect on the decision to become oocyte (meiotic cycle) or nurse cell (endocycle). The formation of a second oocyte upon increased expression of tribbles suggests a more direct effect of Tribbles and CDC25 on oocyte determination. Hypomorphic mutations in cyclin E give rise to a second (or third) cell having some, but not all, oocyte features. This cell has a typical oocyte nucleus, but does not accumulate actin or yolk. In contrast, the two oocytes seen in tribbles GOF are equivalent and appear to have all oocyte characteristics. This may reflect that the latter egg chambers have a total of 32 cells and thus can 'support' two oocytes. It may also reflect a more fundamental difference in the process leading to an extra oocyte (Mata, 2000).
Tribbles and regulation of Slbo degradation
The C/EBP transcription factor, Slbo, is required for migration of border cells during Drosophila oogenesis. Neither increase nor decrease of Slbo activity is tolerated in border cells. Correct protein level is in part ensured by cell type-specific regulated turnover of Slbo protein. Through genetic screening, two genes that are involved in this regulation have been identified. The Ubp64 ubiquitin hydrolase acts as a stabilizer of Slbo protein. A novel gene, tribbles, is a negative regulator of slbo in vivo. Tribbles acts by specifically targeting Slbo for rapid degradation via ubiquitination (Rørth, 2000).
Border cells are a group of six to ten cells that delaminate from the follicular epithelium and migrate as a cluster to the oocyte at a specific time during Drosophila oogenesis, called stage 9. slbo is expressed in border cells and is absolutely required for their migration. If border cell migration is perturbed, the resulting eggs cannot be fertilized; thus, slbo mutant females are sterile. Slbo protein is detected in border cells before and as they migrate, both in the centrally located anterior polar cells (APCs) and the remaining 'outer border cells.' After migration, the level of Slbo protein in border cells decreases. Another group of cells, the centripetal cells, migrate over the anterior part of the oocyte during stage 10. slbo is also expressed in centripetal cells but is not required for their migration (Rørth, 2000).
To determine which cells require slbo activity as well as the fate of individual cells with no slbo activity, a clonal analysis was carried out using a slbo null mutant. A total of 185 border cell clusters with one or more mutant cells were analyzed. If all border cells are mutant, then the cells do not move from the anterior tip. Similarly, no migration was seen in over 200 egg chambers from slbonull females (slbonull females were rescued from embryonic lethality with a transgene). If the only wild-type cell in the cluster is one (or both) of the APCs, migration is also not initiated. However, if one or two outer border cells are wild type, these initiate movement but remain associated with the mutant border cells and the cluster moves very little. Conversely, if one or two outer border cells are mutant, these mostly remain associated with the wild-type border cells and are 'dragged along' at the rear of the cluster. If one or both APCs is mutant, this does not affect migration or position of the cell. The first conclusion from these experiments is that APCs are not, by themselves, migratory cells. The APCs have separate lineage and different morphology than the remaining border cells. Also posterior polar cells (PPC) express Slbo protein but do not migrate. The second conclusion is that slbo is required in outer border cells for active migration, but not for the selective adhesion of border cells (Rørth, 2000).
Although slbo is autonomously required for each outer border cell to be actively migratory, the efficiency of cluster movement reflects the fraction of cells that are migration competent (slbo+). Similarly, altering the overall level of Slbo in the cluster using different hypomorphic slbo mutants does not result in complete cessation of movement, but in quantitative defects. Less Slbo protein results in longer delay and smaller percentage of clusters initiating migration. Complete block of border cell migration is only observed in the slbo null mutant. In a screen for gain-of-function suppressors of the slbo mutant phenotype, a slbo mutant, slbo1310, was used that allows a low level of slbo expression. This should allow for identification of suppressors that act by affecting the level of slbo activity (Rørth, 2000).
One line isolated in the suppressor screen, EP3584, was found to have an EP insertion upstream of the Ubp64 gene. To verify the activity of the gene, the Ubp64 cDNA was expressed directly using the UAS-GAL4 system and the slboGAL4 driver. Overexpression of Ubp64 suppresses the slbo phenotype significantly. Ubp64 encodes a putative ubiquitin hydrolase (Henchoz, 1996). Based on the similarity to specific ubiquitin hydrolases in yeast, it is likely that Ubp64 removes ubiquitin moieties from ubiquitinated proteins, thereby stabilizing them. The suppressor activity of EP3584 suggested that Slbo might be a target for Ubp64 deubiquitination, since stabilization of residual Slbo protein should suppress the slbo mutant phenotype. Whether Slbo protein levels are affected by Ubp64 overexpression was tested. Slbo protein levels are very low in the slbo1310 mutant but markedly increase when Ubp64 is induced. slbo transcription, reflected by the slbo-lacZ reporter gene, is unaltered by Ubp64 expression. These results suggest that increased expression of Ubp64 stabilizes Slbo protein (Rørth, 2000).
The suppressor activity of the ubiquitin hydrolase indicates that Slbo protein is targeted for degradation by ubiquitination and furthermore that Slbo might have a short half-life in vivo. To look at Slbo protein turnover in vivo, but in a situation where its transcription is under temporal control, slbo mRNA was ectopically induced by a heat shock pulse to flies carrying a HS-slbo construct. Abundant ectopic protein was detected 1 hr after HS in all follicle cells of stage 10 egg chambers, including border cells. Most follicle cells retain a high level of Slbo protein 1 hr later. However, Slbo protein is almost undetectable in border cells. By 4 hr, Slbo protein has disappear from almost all follicle cells. The difference between border cells and other follicle cells is also observed in stage 9 egg chambers. As expected, slbo RNA is detected in all follicle cells 30 min after heat shock in these experiments and declines in all cells thereafter. Since Slbo protein initially accumulates equally in all follicle cells, the difference in protein levels is best explained by differential turnover. Thus, Slbo protein appears to be selectively unstable in border cells, the cells that endogenously express the protein (Rørth, 2000).
When the same experiment was performed in a slbo mutant background, Slbo protein levels were identical in border cells and other follicle cells at different time points. This indicates that the selective instability of Slbo protein in border cells is dependent on the activity of Slbo itself. In both wild-type and slbo mutant chambers, Slbo protein has almost disappeared from all follicle cells 4 hr after heat shock. This time coincides with the time when the first border cell clusters have initiated migration in the slbo mutant background as a result of rescue by HS-slbo. Note that HS-slbo does not induce significantly precocious migration, nor migration of more than the normal number of border cells. Thus, when expressed in cells with no prior exposure to Slbo protein, the decline in Slbo levels coincides with the time when downstream events are triggered (migration of border cells). This further supports that Slbo directly or indirectly stimulates its own degradation (Rørth, 2000).
Given that Slbo turnover is specifically regulated in border cells, it might be possible to genetically identify factors important for this regulation. A screen was performed to identify genes that when overexpressed using the slboGAL4 driver, would stop border cell migration. slboGAL4 drives efficient expression in both border cells and centripetal cells. Out of 2000 EP lines, one line with this phenotype was identified: EP3519. This gene corresponds to tribbles (Rørth, 2000).
The specificity of the tribbles phenotype for border cell migration raised the possibility that tribbles might affect Slbo expression or activity, since slbo is required for border cell, but not centripetal cell migration. Slbo protein expression was examined by antibody staining. Overexpression of tribbles in border cells causes a dramatic decrease in Slbo protein levels. Slbo protein is reduced to a level similar to that seen in the slbo1310 mutant border cells, thus explaining the phenotype of tribbles overexpression. To see whether tribbles normally controls Slbo protein levels, loss-of-function mutant clones of tribbles were analyzed. Cells mutant for tribbles show slightly higher levels of Slbo protein, indicating that tribbles normally contributes to downregulating Slbo. The effect is detectable from early stage 9 to stage 10. The role of tribbles was addressed by looking at genetic interactions between tribbles and slbo. Removing one copy of tribbles suppresses the slbo phenotype 2-fold (from 5% migration to 10%). Flies homozygous mutant for tribbles are poorly viable, due to defects unrelated to slbo, but the slbo mutant phenotype is suppressed about 4-fold. Thus, both gain-of-function and loss-of-function results show that tribbles is a negative regulator of Slbo expression in migrating border cells (Rørth, 2000).
Overexpression of tribbles in border cells does not affect beta-galactosidase expression from the slbo enhancer trap, indicating that tribbles is affecting slbo at a posttranscriptional level. This was confirmed by looking at the effect of tribbles on slbo, which was expressed under control of a heterologous promoter. For example, ectopic expression of slbo in wings using vestigialGAL4 and UAS-slbo results in small wings, and this effect is suppressed by coexpressing tribbles. Thus, the effect of tribbles on slbo in vivo is independent of cell context and is posttranscriptional (Rørth, 2000).
To further analyze the effect of tribbles, Schneider cell transfections were carried out. Induced expression of tribbles decreases the level of cotransfected epitope-tagged Slbo protein. This is observed without any effect on Slbo mRNA level. Addition of lactacystin, a potent inhibitor of proteosome-mediated degradation, increases Slbo levels and partially blocks the effect of tribbles. Since lactacystin has a more pronounced effect in the presence of tribbles (7-fold versus 3.5-fold increase in Slbo level), it has been concluded that Slbo is normally degraded via the proteosome pathway fairly rapidly and that this turnover is stimulated by Tribbles. Support for this idea came from looking at addition of His-tagged ubiquitin moieties to Slbo protein. Expression of Tribbles causes an increase in higher molecular weight bands, which most likely correspond to Slbo-ubiquitin conjugates, suggesting that Tribbles stimulates Slbo ubiquitination. Finally, epitope-tagged Tribbles and Slbo proteins could be coimmunoprecipitated from cells, indicating that the proteins can physically associate, directly or indirectly. In conjunction with the in vivo data, these experiments indicate that Tribbles affects Slbo protein levels by targeting Slbo for degradation via the ubiquitin-proteosome pathway (Rørth, 2000).
One conclusion from these experiments is that Slbo protein level is under very tight control in vivo. In addition to transcriptional regulation of the slbo gene, Slbo protein is targeted for degradation via the proteosome pathway by Tribbles. Slbo protein turnover is particularly high in border cells, possibly as a negative feedback regulation. To address the importance of this tight control of protein level, attempts were made to override it by forcing overexpression of Slbo. Ectopic expression of Slbo is deleterious in most tissues. More importantly, increased Slbo level is deleterious even in cells where it is normally expressed. Overexpression of Slbo in border cells delays their migration. This effect is observed when Slbo protein levels are about 5-fold over wild-type level (shown by quantitation of immunoflouresence staining). Thus, border cells fail to migrate properly if the level of Slbo protein is too low or too high. The effect of overexpressing slbo was alleviated by coexpressing tribbles. Thus, cooverexpression of slbo and tribbles reciprocally rescues the gain-of-function phenotypes. This confirms that Tribbles stops border cells by removing Slbo. Conversely, delay in border cell migration due to moderate overexpression of Slbo is worsened by coexpressing Ubp64, confirming its role as a positive regulator of Slbo protein levels (Rørth, 2000).
The deleterious effects of Slbo overexpression could either be due to increased activity of this transcription factor or to effects such as squelching or other abnormal interactions. To see if Slbo activity is responsible for the effect, an inactive version of Slbo with a leucine to proline mutation in the leucine zipper (Slbo-LZ) was tested. Slbo-LZ protein cannot dimerize and therefore cannot bind DNA and is not able to rescue border cell migration when expressed in vivo. Because the mutant cannot dimerize, it also does not act as a dominant-negative. However, the mutant still goes to the nucleus and has functional activation domains. When overexpressed at a level similar to that of the control Slbo protein, the mutant Slbo protein does not affect border cell migration. This result suggests that the problems caused by high levels of Slbo are due to excessive activity of the transcription factor (Rørth, 2000).
It is concluded that the role of APCs may be to signal to and/or recruit the adjacent cells to become migratory outer border cells. slbo is not required for selective border cell adhesion, indicating that slbo null mutant border cells retain some border cell characteristics. Also ectopic expression of slbo by HS-slbo or the GAL4/UAS system does not convert other follicle cells into migratory border cells. Thus, slbo is not a master regulator of border cell fate. In addition, although HS-slbo can induce migration later than normal in slbo mutant egg chambers, precocious expression of slbo cannot force much earlier migration. Thus, specification of border cells must be dependent upon temporal and spatial control of one or more factor(s) in addition to slbo (Rørth, 2000).
Expression of slbo in the ovary is under tight transcriptional control, spatially and temporally. In addition, Slbo protein is rapidly degraded in a regulated fashion. Why might the organism impose this extra control on Slbo protein accumulation? One rationale is the need for efficient induction of Slbo to certain levels for border cell differentiation (inducing migration), coupled with the need to keep Slbo from overaccumulating. That the increased turnover of Slbo seen in border cells may be induced by Slbo itself (autoregulation) fits with this rationale; it may prevent Slbo levels from becoming too high. Rapid turnover may also contribute to timing the initial effect of Slbo. Although there is obvious upregulation of slbo transcription at stage 8/9, transcriptional activity of slbo reporter genes can be seen earlier in polar cells and adjacent cells. The cues regulating slbo transcription may be present at earlier stages, whereas functional activity of Slbo requires protein level above a certain threshold. Rapid turnover, in part imposed by tribbles, may keep Slbo below this threshold by preventing its accumulation early. In any case, the Slbo overexpression experiments indicate that control by proteolysis can be overridden if Slbo is sufficiently overexpressed (Rørth, 2000).
The observations raise two further questions. The first is why overaccumulation of Slbo protein is deleterious. Sustained overexpression of Slbo is deleterious to all tissues tested, including where it is normally expressed. The latter shows that Slbo levels are critical. It is assumed that the problem is elevated Slbo transcriptional activity, as indicated by the Slbo-LZ mutant result, although it is formally possible that Slbo (and not the Slbo-LZ mutant) engages in some other aberrant activity. The increased transcriptional activity could, in turn, reflect increased expression of normal target genes, or activation of inappropriate, suboptimal 'target genes'. Slbo, like other proteins of the C/EBP family, has a reasonable affinity for suboptimal target sites in vitro. Slbo may bind to low affinity sites if present at higher than normal concentration and influence transcription of genes not intended to be target genes (Rørth, 2000).
It is possible that Tribbles is part of a ubiquitin E3 complex for Slbo. E3 complexes are defined by their activity, to stimulate transfer of ubiquitin from specific ubiquitin-charged E2 complexes to substrates, and can be unrelated in sequence. It is also possible that the effect on Slbo protein levels is indirect and, for example, reflects an effect on subcellular localization. Tribbles can be located both in the nucleus and the cytoplasm; when overexpressed in border cells, it is primarily nuclear. In vivo, the GOF phenotype of tribbles was very specific, and in cell transfections, the effect of Tribbles was specific to Slbo (as well as the mammalian homolog C/EBPalpha). In conclusion, these studies of Slbo regulation have revealed an unexpected importance of transcription factor levels and precise control thereof, even for a transcription factor that acts as an apparent on/off switch and is itself transcriptionally regulated. These observations stress the importance of looking at transcription factors and targets in their natural context, with physiological levels of protein present (Rørth, 2000).
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date revised: 21 November 2016
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