The DmUbc9 protein, which localizes primarily to the nucleus in Drosophila S2 cells, is found at high levels in embryos but is also present at lower levels throughout development (Joanisse, 1998).
To investigate why lwr mutant larvae develop melanotic tumors, the number of circulating hemocytes of lwr mutants was measured. Total hemocyte counts of wild type and heterozygous controls varied from 2.1 × 106 to 4.1 × 106 per ml of hemolymph. The number of hemocytes of lwr mutant larvae was 23.0 × 106 per ml of hemolymph, which was statistically higher than that of the heterozygous control animals. This high hemocyte count was very similar to that of Tl10B mutants (20.1 × 106 per ml of hemolymph). Therefore, an excess number of circulating hemocytes probably causes the development of melanotic tumors in lwr mutant larvae (Huang, 2005).
Since three different hemocytes are known to be in the hemolymph, how hemocyte populations were affected in these mutant larvae was examined. Crystal cells were omitted from this study because they usually burst within 1 min or less after bleeding, leading to inaccurate estimates of crystal cells. The proportion of lamellocytes in the total circulating hemocytes was estimated as a parameter to represent differences in the hemocyte population. Lamellocytes were distinguished by using a lamellocyte specific marker, msn-lacZ, or by their characteristic morphology. The percentage of lamellocytes in the total hemocyte pool significantly increased in lwr and Tl10B mutant larvae. It was also noted that most aggregated hemocytes in the hemolymph consisted of lamellocytes, and that these aggregated masses were partially to fully melanized in most cases. These observations strongly suggest that lamellocytes are heavily involved in the formation of melanotic tumors in lwr mutant larvae (Huang, 2005).
It was also of interest to discover whether the lwr mutant hemocytes divide in circulation. The mitotic indexes of lwr mutant hemocytes were measured as well as those of Tl10B and Canton-S hemocytes as control. Cells in mitosis were identified using anti-phospho-Histone H3 antibodies. The mitotic index of lwr mutants was 10.4% in a total of 3109 cells from five larvae, which was slightly higher than that of Tl10B (7.1% in a total of 2515 cells from five larvae). In contrast, no dividing hemocytes were observed in Canton-S (a total of 1344 cells from ten larvae). Based on their sizes and morphologies, these dividing cells in lwr and Tl10B mutants were plasmatocytes and prohemocytes, not lamellocytes. Therefore, some hemocytes do indeed divide in circulation in lwr mutants. However, the excess number of mature lamellocytes in circulation is due to proliferation of lamellocyte precursors, presumably in the lymph gland. Taken together, it is concluded that the effects of the lwr mutation on hemocytes are similar to those of other known hematopoietic mutants in the Tl pathway such as cact and Tl10B (Huang, 2005).
To determine whether the overproduction of hemocytes can be attributed to the loss of lwr function in hematopoietic tissues, a dominant negative form of lwr (lwrDN) was expressed in the lymph gland and hemocytes. Since the lwr mutation exhibits pleiotropic effects, it is possible that the increased number of hemocytes in lwr mutant larvae is due to a secondary effect that might stimulate hemocyte production. For this purpose, the GAL4/UAS system was used. Two GAL4 drivers, CgGAL4 and e33CGAL4, were chosen for this experiment because they are known to induce expression of GAL4 in the lymph glands and hemocytes. Although these drivers induce GAL4 expression in multiple tissues, the common cell types expressing GAL4 with these two drivers are hemocytes in the lymph gland and in circulation. The e33CGAL4 driver induces a UAS-GFP transgene in most cells in the lymph gland at different expression levels, while the CgGAL4 promotes GFP expression in a subset of the cells in the anterior lobes at a relatively consistent level. The overall expression level of GFP by the e33CGAL4 is lower than that of the CgGAL4 driver. Nevertheless, these GAL4 drivers exhibited similar effects on the total hemocyte counts (Huang, 2005).
The CgGAL4 driver effectively increased the number of hemocytes to a level that was higher than those of lwr and Tl10B. The total hemocyte count was 25.0 × 106 per ml of hemolymph, which was statistically higher than that of the corresponding control. The e33CGAL4 driver also induced many hemocytes at 28°C, and this level was higher than that of the corresponding heterozygous control. Since hemocytes in the lymph gland and in circulation are the only common cell types that express GAL4 with these drivers, it is most likely that the loss of lwr function in hematopoietic tissues is responsible for high hemocyte counts in lwr mutants. This conclusion is also supported by the fact that a fat body specific driver, Lsp2GAL4, does not show any significant effect on hemocyte counts when the lwrDN allele is induced by this driver (Huang, 2005).
In addition to the total hemocyte counts, the UAS-lwrDN/CgGAL4 combination promoted lamellocyte production. Lamellocyte levels rose to 15.5%. This value was lower than that of lwr mutants, but higher than that of Tl10B mutants. Furthermore, most hemocytes expressing lwrDN showed nuclear localization of Dl protein, which is characteristic of the lwr mutant hemocytes. These observations strongly suggest that this lwrDN allele is very effective with the CgGAL4 in mimicking effects of the lwr mutation on hemocyte production in larvae. Thus, it is concluded that the increase in the total hemocyte counts in the lwr mutant background is attributed to the loss of lwr function in the hematopoietic tissues such as the lymph gland (Huang, 2005).
How does the lwr mutation affect hemocyte production? Because the hematopoietic defects of lwr mutants are similar to those of dominant Tl mutations, Tl10B and Tl3, possible interactions between lwr and the Tl signal transduction pathway were investigated. Moreover, IκBα, a human Cact homologue, is subject to sumoylation (Desterro, 1998), which suggests that the Cact protein may interact with the Lwr protein. Since both dl and Dif are expressed in the hematopoietic tissues, the entrance of Dl proteins were used as an indicator for Tl signaling activity. At this moment, it is not yet clear whether other Tl-like receptors are involved in hematopoiesis. Hereby, the term Tl signaling in this report does not exclude possible signaling inputs from other Tl-like receptors (Huang, 2005).
Nuclear localization of Dl protein was observed in many lwr mutant hemocytes as well as in Tl10B hemocytes. In lwr mutants, nearly half of hemocytes showed accumulation of Dl proteins in the nuclei, which was comparable to that of Tl10B mutants . In contrast, nuclear localization of Dl protein was observed in only a few control heterozygous and wild type hemocytes, e.g., 3.6% in a total of 1291 cells from 10 Canton-S larvae. These observations indicate that the activation of the Tl pathway is a consequence of the loss of lwr function, which is manifested as abnormal production of larval hemocytes. Although some small lamellocytes were positive for Dl nuclear staining, little nuclear localization of Dl protein was observed in large matured lamellocytes of lwr and Tl10B mutants. Thus, there is not a mechanism to allow entrance of Dl proteins to the nucleus in mature lamellocytes (Huang, 2005).
To further investigate the relationship between the lwr mutation and Tl signaling, genetic interactions were examined of lwr with dl and Dif single mutants as well as with dl Dif double mutants. The rationale here is that genetic interactions between lwr and dl as well as Dif would be clearly detected if lwr function modulates signaling from Tl receptor and possibly from other Tl-like receptors. The results of these three combinations were examined separately because the dl and the Dif mutations showed different effects on the hematopoietic defects of the lwr mutation. Nonetheless, the results below indicate that the majority of lwr's mutant effects on larval hematopoiesis are manifested through the Tl signal transduction pathway, which agrees with the results of the immunohistochemical study (Huang, 2005).
A double mutant combination of lwr with dl [lwr4-3 dl1/lwr5 Df(2L)J4] was created. The total hemocyte counts and lamellocyte percentages were measured. The total hemocyte counts were significantly reduced in the lwr dl double mutant background. The numbers of plasmatocytes and lamellocytes were then calculated based on the total hemocyte counts and lamellocyte percentages. It was noticed that plasmatocyte counts were reduced by approximately 50% in the double mutant combination. Interestingly, the production of lamellocytes was not overly affected in the same mutant background. which indicates that dl plays a minor role in lamellocyte production (Huang, 2005).
Although the production of plasmatocytes was suppressed by the complete loss of dl function, this suppression was not absolute, suggesting that Dif may contribute equally to larval plasmatocyte production. If Dif only played a minor role in this process, the lwr mutation would affect plasmatocyte production via another pathway besides the Tl pathway. Note that the latter hypothesis was proven to be very unlikely (Huang, 2005).
To examine the effect of Dif in the lwr mutant background, a double mutant combination of lwr and Dif [lwr4-3 Dif2/lwr5 Df(2L)J4] were created and the total hemocyte counts and lamellocyte percentages was measured. The loss of Dif function in the lwr background significantly reduces total hemocyte counts, and the effects were observed on both plasmatocyte and lamellocyte levels. Plasmatocyte population was reduced to half the level caused by the lwr mutation, while the number of lamellocytes was similar to that found in wild type larvae (Huang, 2005).
Similar to what was observed in the lwr dl double mutants, the reduction of the plasmatocyte population due to the loss of Dif function in the lwr background was approximately 50%. This result strongly suggests, in conjunction with the results of the lwr dl double mutants, that the increase of hemocytes observed in the lwr mutation is mediated by the functions of both dl and Dif (Huang, 2005).
In contrast to the lwr dl double mutant, the lamellocyte population was considerably affected by the introduction of the Dif mutation into the lwr mutant background, and the number of lamellocytes observed in the lwr Dif double mutants was almost identical to those in wild type larvae. Therefore, lamellocyte production is primarily controlled by Dif function when the Tl pathway is activated. This in turn suggests that dl and Dif play different roles in hematopoiesis when it is stimulated by the lwr mutation (Huang, 2005).
To obtain a more conclusive answer as to whether the effects of the lwr mutation are observed mostly through Rel-related proteins, Dl and Dif, lwr dl Dif triple mutants [lwr4-3 Df(2L)J4/lwr5 Df(2L)TW119] were examined. The loss of both gene functions almost completely cancels the effects of the lwr mutation on hematopoiesis (Huang, 2005).
The functions of dl and Dif were eliminated by combining the deficiencies Df(2L)J4 and Df(2L)TW119. Total hemocyte counts of lwr5 Df(2L)J4/lwr4-3 Df(2L)TW119 heterozygotes were significantly reduced from those of the lwr single mutants and were indistinguishable from those in wild type larvae as well as Df(2L)J4/ Df(2L)TW119 heterozygotes. The number of lamellocytes was almost identical to those of wild type larvae. These results indicate that the hematopoietic defects of the lwr mutation are manifested through dl and Dif function (Huang, 2005).
The loss of both dl and Dif functions with and without the lwr mutation did not lead to complete loss of hemocytes. This observation agrees with the fact that loss-of-function mutations of the Tl gene do not eliminate all hemocytes completely. Low levels of hemocytes in these mutant backgrounds can be explained by hemocyte production using other pathways such as JAK/STAT and Ras, which are known to be involved in hematopoiesis. Therefore, these observations do not rule out the importance of dl and Dif functions in hematopoiesis (Huang, 2005).
In order to obtain additional evidence that supports different but overlapping roles of dl and Dif in hematopoiesis, UAS-dl and UAS-Dif transgenes were overexpressed in the lymph glands and hemocytes using the CgGAL4 driver. Dif promote lamellocyte production more effectively than do dl, which shows good agreement with genetic analysis (Huang, 2005).
Since the CgGAL4 driver expresses the GAL4 transcription factor in the fat body, the Lsp2GAL4 driver, which is fat body specific, was used to express the UAS constructs used in this study. The combinations of all UAS transgenes with the Lsp2GAL4 serve as a control to assess any possible additional effect of the CgGAL4 driver. In all cases examined, while the differences between controls and experimental sets were statistically significant in some cases, total hemocyte counts fell in the range of wild type, Oregon-R and Canton-S. Therefore, the results with the CgGAL4 driver, which are described below, are most likely to represent the effects of these genes in the hematopoietic tissues, the lymph gland and hemocytes (Huang, 2005).
Both dl and Dif exhibited significant increases in total hemocyte counts when they were overexpressed by the CgGAL4 driver. Interestingly, overexpression of Dif produced more hemocytes than dl. After dividing the total hemocyte population into plasmatocytes and lamellocytes, it was observed that plasmatocyte counts increased to levels similar to those of lwr and Tl10B mutants in both UAS-dl/CgGAL4 and UAS-Dif/CgGAL4 combinations. Unlike the plasmatocyte population, lamellocytes responded differently. Overexpression of dl showed no effect on lamellocyte production. In contrast, overexpression of Dif promoted lamellocyte production and its effect was similar to that of a Tl10Btransgene. These results indicate that dl and Dif share a similar function in plasmatocyte production, and that Dif is likely to be a sole factor for lamellocyte production in the Tl pathway (Huang, 2005).
To investigate how coordinately dl and Dif function in hematopoiesis, effects on hemocyte production were examined by overexpressing Tl10B and both dl and Dif simultaneously. It was found that dl may be dispensable in the production of both plasmatocytes and lamellocytes when there were enough Dif proteins around. Furthermore, the activation of Tl signaling by Tl10B, a constitutively active form of the Tl receptor, showed an additional effect on plasmatocyte production compared to those by the simultaneous expression of dl and Dif (Huang, 2005).
dl and Dif were overexpressed simultaneously with the CgGAL4 driver and the total hemocyte number and the proportion of lamellocytes in the total hemocyte population was estimated. Even though dl and Dif showed significant effects on hemocyte production when they were individually overexpressed, they did not show any synergistic effect when they were induced at the same time. When both genes were expressed together, the total hemocyte counts did not differ from those when Dif was overexpressed by the CgGAL4 driver, but were statistically higher than those when dl was overexpressed by the same driver. Thus, Dif is sufficient to represent the effect of the dl and Dif double expression combination, and dl did not suppress the effect of Dif (Huang, 2005).
As far as lamellocyte production is concerned, the combination of dl and Dif showed the highest lamellocyte estimate among all constructs tested including UAS-Tl10B. Although a statistical test could not be applied, the differences among the constructs seemed to be marginal, indicating that the effect of dl on lamellocyte production may be small, if there is any. Taken together, dl only plays a minimal role in hematopoiesis when both dl and Dif are highly induced (Huang, 2005).
In order to verify whether the dl-Dif double combination represents the activation of the Tl pathway, a UAS-Tl10B transgene was overexpressed with the CgGAL4 driver. While lamellocyte production appeared to be very similar to that of a dl–Dif double combination, the overexpression of Tl10B exhibited the most pronounced effect on plasmatocyte production among all the combinations used in this study. The results indicate that there are abundant Dl and Dif proteins in the CgGAL4 expressing cells and that these transcription factors can fully respond to the activation of the Tl pathway, i.e., the overexpression of Tl10B. It is also possible that the activation of the Tl receptor may stimulate plasmatocyte production, in part bypassing the Dl and Dif transcription factors. This possible bypass indicates that the Tl receptor might use a different set of transducers and transcription factors to control plasmatocyte production. Alternatively, the differences might be due to the different levels of UAS transgene expression (Huang, 2005).
Highly conserved during evolution, the enzyme Ubc9 activates the small ubiquitin-like modifier (SUMO) prior to its covalent ligation to target proteins. Mutations in the Drosophila Ubc9 (dUbc9) gene have been used to understand Ubc9 functions in vivo. Loss-of-function mutations in dUbc9 cause strong mitotic defects in larval hematopoietic tissues, an increase in the number of hematopoietic precursors in the lymph gland and of mature blood cells in circulation, and an increase in the proportion of cyclin-B-positive cells. Some blood cells are polyploid and multinucleate, exhibiting signs of genomic instability. Also, an overabundance of highly differentiated blood cells (lamellocytes), normally not found in healthy larvae, are observed. Lamellocytes in mutants are either free in circulation or recruited to form tumorous masses. Hematopoietic defects of dUbc9 mutants are strongly suppressed in the absence of the Rel/NF-κB-family transcription factors Dorsal and Dif or in the presence of a non-signaling allele of Cactus, the IκB protein in Drosophila. In the larval fat body, dUbc9 negatively regulates the expression of the antifungal peptide gene Drosomycin, which is constitutively expressed in dUbc9 mutants in the absence of immune challenge. dUbc9-mediated drosomycin expression requires Dorsal and Dif. Together, these results support a role for dUbc9 in the negative regulation of the Drosophila NF-κB signaling pathways in larval hematopoiesis and humoral immunity (Chiu, 2005).
Ubc9 was discovered in Saccharomyces cerevisiae based on its sequence similarity to other known ubiquitin-conjugating enzymes (Seufert, 1995). A loss of function of Ubc9 causes an increase in the S- and M (B type)-phase cyclins, resulting in an arrest of the cell cycle at the G2 or early M phase. This cessation of the cell cycle causes an accumulation of large budded cells with a single nucleus and replicated DNA. Ubc9 was also identified in a screen for DNA damage checkpoint control in S. pombe (Al-Khodairy, 1995; Tanaka, 1999). Although not directly involved in the checkpoint control, Ubc9 (encoded by hus5) is required for the efficient recovery from DNA damage or S-phase arrest, and for chromosome segregation. Yeast mutants display severe impairment in growth and exhibit a high frequency of failed mitosis. Further studies in yeast revealed that unlike other E2 ubiquitin-conjugating enzymes, Ubc9 is unable to form a thioester bond with ubiquitin; instead it conjugates the ubiquitin-like protein SUMO/Smt3 to specific targets in a yeast extract (Johnson, 1997; Chiu, 2005 and references therein).
Vertebrate homologs of Ubc9 were subsequently identified in many laboratories, showing that Ubc9 may interact with a wide variety of cellular proteins, regulating cellular processes such as cell division, protein trafficking, signal transduction, and transcriptional regulation (Pichler, 2002; Muller, 2000). Studies designed to identify biochemical targets of Ubc9 highlighted a role for sumoylation in the regulation of chromatin organization, gene expression, and genome surveillance (Melchoir, 2002; Muller, 2004; Zhao, 2004; Chiu, 2005 and references therein).
While SUMO-1 is structurally similar to ubiquitin and sumoylation and ubiquitination are enzymatically similar processes, the conjugation of SUMO-1 or ubiquitin to the same protein can have opposite effects. For example, ubiquitin or SUMO-1 can be directly conjugated to lysine residues 21 and 22 of mammalian IκBα in reactions catalyzed by activating enzymes Ubch5 and Ubc9, respectively (Scherer, 1995; Desterro, 1998). IκBα is a cytoplasmic inhibitor of the transcription factor NF-κB. In unstimulated cells, cytoplasmic NF-κB, complexed with IκBα, remains inactive. Activation of NF-κB is achieved by ubiquitination and proteasome-mediated degradation of IκBα, allowing NF-κB translocation to the nucleus. However, when the same lysine residues in IκBα are conjugated to SUMO-1/Smt3, ubiquitination is blocked, thereby stabilizing the cytoplasmic pool of this protein. This increase in stabilization of IκB sequesters NF-κB in the cytoplasm, leading to a downregulation of the NF-κB pathway (Tashiro, 1997; Desterro, 1998; Hay, 1999). Thus, while ubiquitination targets proteins for degradation, SUMO-1 modification acts antagonistically to render proteins resistant to degradation. Given that SUMO modification alters the ability of proteins to interact with their partners, alters their subcellular localization, and controls their stability, understanding the role of sumoylation in different cellular processes is of fundamental importance in normal and diseased cells (Chiu, 2005).
Drosophila has been used as a model system to understand the functions of Ubc9 in vivo. An alignment of the Drosophila Ubc9 (dUbc9) with its counterparts in yeast, C. elegans, and humans shows that dUbc9 shares a higher level of structural similarity with the human Ubc9 (84% identical) than with either the C. elegans (76% identical) or yeast (35% identity) proteins. Strikingly, expression of either human or Drosophila Ubc9 can rescue an S. cerevisiae Ubc9ts mutant (Yasugi, 1996; Joanisse, 1998). dUbc9, also called semushi and lesswright, (lwr), has many biological functions. For example, mutation in dUbc9 disrupts anterior segmentation in embryogenesis by interfering with the nuclear uptake of the homeodomain transcription factor Bicoid (Epps, 1998). Mutants in dUbc9 also suppress the nodDTW (dominant antimorphic allele of no distributive disjunction) phenotype, implying a role in chromosome segregation during meiosis in females (Apionishev, 2001). Biochemically, the SUMO-1/Ubc9 pathways are conserved between flies and humans: (1) dSmt3 and dUbc9 are coexpressed during development, (2) dSmt3 can be processed and conjugated in human cells, and (3) human transcription factor PML can be modified by dSmt3 in Drosophila SL2 or human HeLa cells. Like their human counterparts, dSmt3 and dUbc9 colocalize in nuclear foci (Lehembre, 2000; Chiu, 2005 and references therein).
Drosophila Ubc9 was also identified in a yeast two-hybrid screen for Dorsal-interacting proteins (Bhaskar, 2000). Dorsal is one of three Drosophila Rel/NF-κB-family proteins. The nuclear localization of Dorsal is controlled by the Toll receptor. Toll activation leads to signal transduction via Tube and Pelle, as well as the phosphorylation and degradation of Cactus, the Drosophila IκB protein. Components of the Toll-Dorsal pathway were first identified as maternal-effect genes controlling the development of the embryonic dorsal-ventral axis. Although the precise mechanism underlying Cactus degradation in the embryo is still unclear, in vivo studies suggest that, like mammalian IκBα, Cactus degradation is regulated by Toll signal-dependent phosphorylation. dUbc9 conjugates a Drosophila SUMO/Smt3 to lysine 382 of Dorsal (Bhaskar, 2002). Whether Cactus also serves as a sumoylation target is not known (Chiu, 2005 and references therein).
In Drosophila, the NF-κB pathway regulates many biological processes at different developmental stages. The Toll-Dorsal/Dif pathway activates transcription of antifungal and antibacterial (Gram-positive) peptide genes in the larval and adult fat body. Dif (Dorsal-related immunity factor) belongs to the NF-κB family . The regulation of genes encoding antibacterial peptides that kill Gram-negative bacteria (e.g., diptericin) is under the control of Relish, the third Drosophila Rel/NF-κB protein similar to the mammalian p100/p105 proteins. Relish activation is Toll-independent. Instead, the intracellular Relish phosphorylation and activation are regulated by activities of Immune deficiency (Imd) pathway including proteins of the IKK complex and the Dredd caspase. This pathway is negatively regulated by the ubiquitin proteasome system (Chiu, 2005 and refenreces therein).
The Toll-IκB pathway also contributes to proliferation of blood cells (hemocytes) during normal larval hematopoiesis and during the hematopoietic proliferation that accompanies immune challenge. Unchallenged Drosophila larvae have two hemocyte types in circulation: the plasmatocyte and the crystal cell, both of which are specified and formed during embryonic stages. More than 90% of all hemocytes in circulation are plasmatocytes. Plasmatocytes are phagocytic cells, ridding the larvae of microbial infections. The remaining hemocytes in circulation (5% or less), the crystal cells, carry prominent crystalline inclusions and, when activated, lyse and release their contents, melanizing target cells. Mutations that upregulate the Toll pathway (loss-of-function mutations in cactus, gain-of-function mutations in Toll, or overexpression of Dorsal in larval hemocytes) result in overproliferation of hemocytes, whereas mutations that downregulate the pathway (loss-of-function in Toll, tube, or pelle) lead to a reduction in the number of circulating hemocytes. Changes in hemocyte counts in mutations affecting either dorsal or Dif are unremarkable (Chiu, 2005 and references therein).
A third kind of hemocyte, the lamellocyte, appears in the larval hemolymph only in response to infections by naturally occurring endoparasitoid wasps and other foreign bodies. The parasitoids constitute a major threat to the Drosophila population in nature, as they hijack the larval body for their own development. Lamellocytes are adhesive, and rapidly aggregate around a parasite egg to form a cellular capsule. Parasite-induced lamellocyte differentiation in the lymph gland is accompanied by a modest increase in the number of plasmatocytes and crystal cells. The encapsulated wasp egg is melanized. Lamellocyte precursors are normally quiescent. However, mutations that lead to the overproliferation of hemocytes (loss-of-function cactus alleles, gain-of-function Toll alleles) also result in constitutive differentiation of lamellocytes, resulting in the encapsulation of self-tissue in the absence of wasp infection (innate autoimmune response). Because of their dark appearance, these capsules are called melanotic tumors (Chiu, 2005 and references therein).
This study reports that larvae carrying loss-of-function mutations in dUbc9 show strong hematopoietic proliferation and differentiation defects. Furthermore, the antifungal gene drosomycin is constitutively active in developmentally delayed dUbc9 mutants. Both constitutive humoral and cellular immune defects are rescued by mutations in dorsal and Dif. These results suggest that dUbc9 contributes to the regulation of both humoral immunity and hemocyte proliferation by acting as a negative regulator of the Toll pathway (Chiu, 2005).
Genetic experiments in Drosophila emphasize the central regulatory role for Ubc9 function and sumoylation in different cells and during different life cycle stages. lwr adults exhibit defects in eye, wing, and leg morphogenesis: mutant males die and the few surviving females are sterile, revealing roles for dUbc9 in cell division and differentiation, in tissue patterning, and in oogenesis. This paper describes additional defects in dUbc9 mutants, based on studies of mutant larvae. Phenotypes are described that point to the involvement of dUbc9 in larval metamorphosis, proliferation, and differentiation of hematopoietic precursors, in antimicrobial gene expression, and in NF-κB signal transduction. These observations show that diverse biological processes share a common regulatory mechanism involving sumoylation and the variety of the phenotypes observed suggest the possibility of multiple biochemical targets in vivo. Drosophila is an excellent model for the identification of cell- and tissue-specific sumoylation targets of Ubc9, and it is very likely that their conserved mammalian counterparts will be similarly modified and regulated (Chiu, 2005).
Like many larval lethals (e.g., cact mutants), mutations in dUbc9 result in a prolonged third instar period followed by larval death. Hematopoietic defects are observed as early as 4 days after egg lay, whereas the immune defects are evident 6 days after egg lay. Defects of both types become severe as mutant animals persist in larval stages even 10 days after their birth. Indeed, constitutive expression of antimicrobial genes during larval stages continues through lwr4-3/lwr5 pupae and adults. How dUbc9 affects the rate of development is currently not known. Epistasis experiments between lwr and mutants of the ecdysone genetic hierarchy may reveal a role for dUbc9 sumoylation during development and metamorphosis. Also not known is whether hemocyte survival or their apoptosis is affected in lwr mutants. One study of salivary gland apoptosis in Drosophila during pupariation provides evidence that the Rel family members are not required for salivary gland cell death during metamorphosis. The current results provide a link between lwr negative regulation of dl and Dif in the larval fat body and hematopoietic tissues. In any case, the hematopoietic and immune defects in lwr mutants may be tied to abnormalities in larval development as these defects are most pronounced in older animals. The rescue of the immune defects by expression of the dUbc9 protein in the fat body suggests that the misregulation of drom expression in lwr mutants is due to a specific reduction or an absence of the Ubc9 function. Analysis of mutant clones in the fat body or lymph gland will reveal if the requirement of this enzyme is cell autonomous or not (Chiu, 2005).
Four distinct hematopoietic defects affecting hemocyte abundance, differentiation, and morphology are observed in both the lymph gland and circulating hemocyte populations in 4-day-old lwr larvae. The mean CHC values in mutants are significantly higher than those of the pooled control class of larvae; in two of the three lwr genetic combinations studied, this increase in abundance correlates with an increase in cyclin-B-positive hemocytes. Such increase in cyclin B-positive hemocyte population is reminiscent of increase in the B type cyclins in mutant yeast lacking Ubc9 (Seufert, 1995). The current observations suggest that dUbc9 negatively regulates the rate at which hematopoietic cells divide. The prehemocyte classes influenced by the dUbc9 mutation are not known, however, it is interesting that while there is an increase in circulating plasmatocyte and lamellocyte percentages, there is a clear reduction in the number of crystal cells. The opposite effect of lwr on crystal cell numbers indicates a distinct role for dUbc9 function in this hemocyte lineage. Prohemocytes following the crystal cell fate require a combination of signals from the transcription factors Serpent (Srp; human GATA-2) and Lozenge (lz; human AML-1), along with permissive signals from Ser/Notch. Perhaps dUbc9 asserts a role in crystal cell development by propagating the above required signals or alleviating suppression of this cellular fate exerted by the combinatorial interaction of SrpNC (Srp isoform containing both N- and C-terminal Zinc finger domains) and the U-shaped protein. These observations suggest multiple requirements for dUbc9 in the hematopoietic tissue. Furthermore, as mitotic defects are observed in both lymph gland hemocytes and circulating hemocytes, and since these groups of hemocytes originate independently, it is likely that dUbc9 function is independently required in each hemocyte population (Chiu, 2005).
The presence of large numbers of lamellocytes accounts for a significant fraction (10%-20%) of the increase of CHC in lwr mutants. The coincident expansion of the plasmatocyte population and constitutive differentiation of lamellocytes are hallmarks of melanotic tumor mutants in which affected genes are not necessarily related by either structure or function. Yet, hemocyte proliferation and differentiation have distinct genetic requirements. For example, while the Drosophila kinase Hopscotch and transcription factor STAT92E are required for lamellocyte differentiation, Toll and Tube proteins are not, even though upregulation of either the JAK/STAT or the Toll/NF-κB pathways results in the production of melanotic tumors. One explanation for the simultaneous expansion of the plasmatocyte and lamellocyte populations in melanotic tumor mutants such as lwr, cact, and Toll10b is that these mutations affect proliferation of precursors in the lymph gland (or in circulation) that differentiate as plasmatocytes or lamellocytes (Chiu, 2005).
The aberrant nuclear morphologies observed in lwr4-3/lwr14 and lwr4-3/lwr5 hemocytes are variable in appearance and fall into four categories: aneuploidy, abnormal nuclear shapes, presence of multiple nuclei, and presence of fragmented nuclear material (additional smaller Hoechst-positive structures). Genetic evidence suggests that dUbc9 is required for the proper disjunction of homologous chromosomes in meiosis I (Apionishev, 2001). It is thus possible that dUbc9 is also involved in chromosome segregation in mitotic divisions. Indeed, defects in lwr hemocytes are strikingly similar to proliferation defects in cultured chicken cells conditionally depleted of Ubc9 protein, in which cells with multiple or fragmented nuclei are also observed (Hayashi, 2002). The defects in chicken cells arise due to chromosomal loss during chromosome segregation. In both Drosophila and chicken, the frequency of cells with multiple or fragmented nuclei increases with age (Hayashi, 2002). Thus, it is possible that the biochemical targets of Ubc9 in chromosome segregation in Drosophila and chicken cells (and possibly other vertebrates) are conserved and that chromosomal damage accumulates in Ubc9-depleted cells because of similar molecular processes. The proportion of multinucleate hemocytes (in lwr4-3 larvae) is reduced in lwr4-3 dl− as well as lwr4-3 Dif− dl− mutant larvae. The identity of the extra chromosomes and extrachromosomal DNA in dUbc9 mutant hemocytes is not known nor the specificity of this phenotype to lwr allele 4-3. The presence of multinucleate hemocytes in circulation within hemocyte overproliferation mutants is unique to lwr and, to date, has not been reported in other mutants where similar overproliferation and lamellocyte differentiation defects have been documented (e.g., hopTum-l; Toll10b/+; cactus−), reflecting the multiplicity of effects of dUbc9 on the cell cycle (Chiu, 2005).
The genetic interaction and immunohistochemical studies presented in this study constitute the first clear evidence of a role for Dorsal and Dif during hemocyte proliferation in Drosophila larvae, even though these functions were predicted from previous experiments. Like Cactus, the cellular function of dUbc9 is to regulate the nuclear localization of NF-κB proteins. Interestingly, Dorsal and Dif appear to have somewhat redundant functions as suppression of lwr phenotypes is stronger in triple mutants than in double mutants and suppression appears earlier in development in triple mutants than in double mutants. Functional redundancy may also explain why clear hematopoietic phenotypes (e.g., lowered hemocyte concentration or reduced encapsulation of wasp egg) have not been observed in dl or Dif single mutants. Similar redundancy in Dorsal and Dif function has been reported for antimicrobial peptide gene activation in the larval fat body (Chiu, 2005).
Genetic experiments here support a model for negative regulation by dUbc9 of antifungal peptide-encoding genes drosomycin and Cecropin, making it the second negative regulator of the Toll pathway to be identified so far. In general, the effects of lwr mutants on drosomycin expression are stronger than on that of Cecropin. These effects are evident by characterization using promoter-driven GFP reporters representative of the Toll and Imd downstream antimicrobial genes and subsequent confirmation by Northern analysis in whole animals. Perhaps the expression pattern of Cecropin A observed in lwr mutants is partially independent of the Toll pathway. cecropin A has been shown to be regulated by both Toll and Imd immunity pathways. Indeed, dynamic expression patterns of the two variant Cec A1 and A2 transcripts are detected in adult flies, after specific microbial infection regiments, further delineating their expression into the two immune pathways. A detailed analysis of Cecropin A regulation in lwr mutant larvae in combination with loss of function alleles of the Imd pathway may elucidate control of this gene further (Chiu, 2005).
The constitutive activation of drosomycin and Cecropin in lwr mutants is also dependent on Dorsal and Dif, whose roles and functional redundancy in the larval fat body are already recognized. Furthermore, genetic epistasis experiments described here place dUbc9 function upstream of Dorsal/Dif and Cactus. These observations are largely consistent with previous biochemical experiments on these proteins (Bhaskar, 2000; Bhaskar, 2002) and provide additional support for a model in which Dorsal, Dif, Cactus and dUbc9 exist in a complex that is activated by a Toll-dependent signal. Significantly, however, the results suggest that dUbc9 blocks the nuclear localization of Dorsal and Dif and differ from observations made in Drosophila S2 cell cultures, in which dUbc9 facilitates the nuclear localization of Dorsal-GFP (Bhaskar, 2000; Bhaskar, 2002). This divergence in experimental results is likely to be due to different experimental models used in the two studies. The genetic results are consistent with Ubc9's role in IκB sumoylation and downregulation of the mammalian NF-κB pathway (Hay, 1999). Consistent with this model of mammalian Ubc9 function, it is likely that sumoylation of Cactus by dUbc9 protects it from phosphorylation, assisting the retention of Dorsal/Dif in the cytoplasm. This model requires biochemical support as Cactus sumoylation has not been demonstrated (Chiu, 2005).
The intracellular Toll-Dorsal/Dif pathways in both the fat body and in hemocytes include Toll, Tube, Pelle, Ubc9, Dorsal, and Dif, and in both cases, dUbc9 appears to function upstream of Cactus and Dorsal/Dif. These observations with GFP reporter constructs suggest that the effect of dUbc9 is restricted to the Toll pathway and it is possible that the Imd signaling cascade is not regulated by sumoylation. Indeed, a ubiquitin proteasome pathway involving function of SkpA and Slimb has been identified and shown to repress the Imd pathway. Thus, sumoylation and ubiquitination of specific targets appear to have parallel but specific effects in downregulating these pathways (Chiu, 2005).
In conclusion, strong evidence is presented that dUbc9 is a negative regulator of the Toll-NF-κB pathways that control both the humoral and cellular aspects of immune responses in Drosophila. Spatzle activates the Toll pathway in the fat body; however, a role for Spatzle function in hemocyte proliferation or differentiation has not been demonstrated, and therefore the mechanism of Toll activation in the hemocytes is not known. Similarly, target genes of the NF-κB pathway in hemocytes (besides the antimicrobial peptide genes) have not yet been identified. The differences in phenotypes observed in lwr and cact mutants are likely to arise from differences in gene expression programs in the two mutants. These differences provide a unique opportunity to resolve issues of biological specificity in the regulation of NF-κB activation and gene expression in vivo (Chiu, 2005).
In Saccharomyces cerevisiae and other organisms, the UBC9 (ubiquitin-conjugating 9) protein modifies the function of many different target proteins through covalent attachment of the ubiquitin-like protein SMT-3/SUMO. In normal female meiosis, a protein encoded by no distributive disjunction (nod) is responsible for preventing the nondisjunction of chromosome pairs that do not have an exchange. Using a second-site suppression screen of a mutation in the locus with a variable meiotic phenotype, mutations were identified in the Drosophila melanogaster UBC9 homologue, encoded by the gene lesswright (lwr). lwr mutations dominantly suppress the nondisjunction and cytological defects of female meiotic mutations that affect spindle formation. The lwr lethal phenotype is rescued by a Drosophila UBC9/lwr transgene. It is suggested that LWR mediates the dissociation of heterochromatic regions of homologues at the end of meiotic prophase I. A model proposes that when there is less LWR protein, homologues remain together longer, allowing for more normal spindle formation in mutant backgrounds and therefore more accurate meiotic chromosome segregation (Apionishev, 2001).
Genetic and cytological data show that lwr partially suppresses several mutations that affect chromosome segregation in Drosophila female meiosis. For the meiotic mutations that affect spindle formation, nod and ncd, lwr suppresses the nondisjunction of both of the chromosomes measured, the X and the 4th. In the case of two other meiotic mutations, AxsD and mei-218, lwr only suppresses 4th chromosome nondisjunction. It is not clear what the reason for this differential effect is, although it is clear that lwr suppresses nondisjunction to some extent in each meiotic mutant tested. This is a striking observation, because gene products affected by the four meiotic mutations tested act at different times and on different targets during meiosis (Apionishev, 2001).
The model for how the lwr mutation suppresses nondisjunction in several different classes of meiotic mutation is based on the hypothesis that UBC9 plays a role in freeing the 'glue' that holds chromosomes together as the spindle forms. After the homologues are aligned, that 'glue' dissolves, so that the homologues may dissociate from each other and disjoin. Thus, once the nuclear envelope breaks down and the spindle begins to form, the homologues begin to move apart. When oocytes in metaphase are observed cytologically in the wild-type background, exchange homologues are held together by chiasmata, while the nonexchange bivalents have already come apart and have moved poleward, held on the spindle by the NOD protein (Theurkauf, 1992). Thus, nonexchange chromosomes are particularly prone to nondisjunction. For example, if the spindle formation is impaired (ncd) or if there is no nod, the nonexchange chromosomes can move apart from the mass, and not end up on the major spindle (Theurkauf, 1992; Apionishev, 2001).
It is hypothesized that nonexchange homologues are more tightly associated in a lwr mutant background (the 'glue' remains longer than normal), thus delaying the separation of the nonexchange homologues from the chromosome mass. According to this model, spindle formation has a chance to 'catch up' to keep nonexchange homologues in the chromosome mass. This hypothesis would explain how lwr mutations suppress several different kinds of mutations, including those that impair spindle formation (Apionishev, 2001).
The following model is for the sequence of meiotic events. Chromosomes pair and form a synaptonemal complex, which then breaks down. All that remains are heterochromatic associations, primarily in the centromeric region, mediated by vestiges of the synaptonemal complex and/or by a distinct protein ('glue') complex specific for this function (see Dernburg, 1996). The homologues remain associated with each other, either because of chiasmata or because of the heterochromatic associations (Apionishev, 2001).
However, it is very likely that there is not enough of the material mediating the heterochromatic associations ('glue') to hold more than two pairs of nonexchange homologues together. Consistent with this model, nondisjunction rises as more chromosomes enter the nonexchange pool. Females heterozygous for a single balancer have relatively little nondisjunction (0.6%); heterozygosity for two balancers raises the rate (4%-5%), and heterozygosity for the X and both major autosomes leads to enormous rates (21%). This observation suggests that there is enough material to hold only one or two nonexchange pairs together (Apionishev, 2001).
Therefore, it is hypothesized that the proper redistribution of the 'glue' materials must occur to ensure strong associations between nonexchange homologues. The cytological manifestation of this process is the diplotene-diakinesis repulsion that is seen in many organisms as chiasmata become apparent. Although repulsion is not seen in Drosophila females, cytological evidence suggests that there is, in fact, a modification of the pairing, because euchromatic regions dissociate as prophase progresses (Dernburg, 1996), leaving the heterochromatic regions attached. Thus, such redistribution of the 'glue' materials may occur in Drosophila. In this model, LWR facilitates the redistribution of the 'glue' materials (Apionishev, 2001).
Thus it is hypothesized that reduced exchange, as in multiple balancer stocks, or recombination-defective mutants, leads to nondisjunction because the limited amount of material on each bivalent is not enough to hold them together. They separate and then behave as univalents. The genetic consequence of this premature breakdown of homologue association would be nondisjunction with an apparently normal spindle. This is exactly the phenotype that is seen for mei-218 and AxsD (Dernburg, 1996; McKim, 1993). This model resolves the long-standing issue of why decreased exchange leads to increased nondisjunction (Apionishev, 2001).
The vertebrate Cor1 synaptonemal complex protein behaves strikingly like the postulated 'glue' target of LWR. It associates with homologues as they begin to synapse and form a mature synaptonemal complex. However, as the prophase ends, Cor1 does not dissociate from the chromosomes. Instead, its distribution becomes discontinuous as it moves to heterochromatic centromeric regions. The fact that Cor1 also interacts with UBC9 in two-hybrid studies strengthens this assertion (Tarsounas, 1997). Furthermore, UBC9 localizes to the midregion of mouse bivalents in spermatocytes during the meiotic prophase (Kovalenko, 1996). Therefore, it is speculated that Cor1, which is apparently regulated by phosphorylation, is also modified by SMT-3 conjugation mediated by UBC9. This modification might promote the prompt redistribution of Cor1 to the centromere. According to this model, in lwr mutant backgrounds, the redistribution process may be delayed, allowing the homologues to stay together a little longer at the end of prophase I. Currently, experiments are being carried out to test this model of meiotic chromosome segregation and lwr function (Apionishev, 2001).
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date revised: 22 June 2006
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