The expression pattern of shu during oogenesis, spermatogenesis, and embryogenesis was examined using whole mount in situ hybridization with RNA probes made from the cDNA clone corresponding to its transcript. The mRNA can first be detected at the apical tip of the germarium in the germline stem cells and cystoblasts. The level of expression decreases in the remainder of region 1, where the cystocytes are dividing to produce 2-, 4-, 8-, and 16-cell cysts. However, in region 2b, where 16-cell cysts are present, strong levels of expression are again seen. In region 3, equivalent levels of expression can be seen in all 16 germ cells of the stage 1 egg chamber. This pattern of expression corresponds well with the earliest phenotypes of shu and confirms the results of the clonal analysis that indicate that shu function is required in the germline. Subsequent to stage 4 of oogenesis, there appears to be an abrupt downregulation of shu expression until stage 10, where the transcript can be detected at high levels in the nurse cells. In early cleavage-stage embryos uniform levels of SHU mRNA are detected, suggesting a possible maternal function for shu. Interestingly, by the cellular blastoderm-stage shu mRNA is exclusively found in the pole cells. The mRNA can be detected in the germ cells throughout their migration through the midgut and into the gonadal mesoderm and is present in the embryonic gonad of stage 15 embryos. No staining above background level can be detected in any tissues other than the gonads. The shu mRNA can also be detected at the apical tip of the testes where the stem cells are dividing to produce cysts of primary spermatocytes (Munn, 2000).

Rat antisera were raised against a 6x His-tagged protein containing amino acids 31-392 of Shu. Western analysis reveals that the antisera specifically recognize a 52-kD band in wild-type ovary extracts. This protein is not detected in ovary extracts from hemizygous shuWQ41 females, confirming the specificity of the antisera. To determine the subcellular localization of the Shu protein, affinity-purified antibodies were used for immunofluorescent staining of ovaries. The Shu protein is expressed in the germline stem cells and cystoblasts. Low levels of staining were observed in dividing cystocytes in the posterior of region 1. In region 2b, staining is evident in all cells of the newly formed 16-cell cysts. Like the shu mRNA, this persists until stage 4-5 egg chambers. The staining is present in the cytoplasm of the germ cells. Only background levels are detected in the follicle cells. Germline staining is eliminated in shuWQ41 ovaries. Cytoplasmic staining is also seen later in the nurse cells of stage 10 and older egg chambers. However, since the strong loss-of-function alleles of shu do not develop normal eggs at this stage it is difficult to ascertain if this later staining is specific. Staining in the germ cells of embryos could not be detected with this antiserum. This could be due to technical reasons, such as the antibody sensitivity or specificity; only very low levels of the protein can be detected on immunoblots of embryonic extracts (Munn, 2000).

Effects of Mutation or Deletion

All three shu alleles cause recessive female sterility with no effects on zygotic viability (Schupbach, 1991). The two strong loss-of-function alleles, shuWQ41 and shuWM40, also result in male sterility while the weaker allele, PB70, does not affect male fertility. Comparison of the phenotypes of shuWQ41 and shuWM40 as homozygotes and hemizygotes [over Df(2R)b23] reveals that shuWQ41 is likely to be a null allele while shuWM40 is a very strong loss-of-function allele. Due to the presence of a closely linked recessive lethal mutation on the shuWQ41 and shuPB70 chromosomes, few homozygous adults could be isolated. The phenotype of shuWQ41 was therefore studied in hemizygous flies [over Df(2R)b23], is referred to here as shuWQ41, while that of shuPB70 was analyzed in hemizygotes or in the heteroallelic combination with shuWM40 (Munn, 2000).

To analyze the shuWQ41 ovarian phenotype, ovaries were dissected from 0- to 1- and 2- to 4-day-old females and stained with the nuclear stain Hoechst and with antibodies recognizing several different markers of germ cell differentiation. The severity of the phenotypes observed in shuWQ41 can vary between females, and even between ovarioles, suggesting that there may be some functional redundancy for shu. In contrast to wild-type ovaries, 40%-60% of the ovarioles from newly eclosed shuWQ41 females do not contain any developing egg chambers. Usually, the other ovarioles contain only one to three egg chambers and their numbers fail to increase as the females age. Although some egg chambers with 15 nurse cells and an apparently normal oocyte nucleus are observed, subsequent egg chambers have fewer than 16 germ cells, e.g., 2, 4, 8, 10, or 12. None of the egg chambers develop into normal eggs but instead degenerate by mid- to late oogenesis. Those egg chambers with the correct number of germ cells may have arisen through the differentiation of prestem cells, i.e., germ cells that developed as cystoblasts without first being established as stem cells. The remainder of the shuWQ41 ovarioles contain no developing egg chambers, only germaria-containing clusters of germline cells that can sometimes appear pycnotic, indicating that they may be dying (Munn, 2000).

The strong cytoplasmic staining of these germ cells with antibodies recognizing the Sex Lethal protein indicates that shu function is dispensable for the establishment of the female mode of sexual differentiation of the germ cells. Staining of these germ cell clusters with antibodies that recognize spectrosomes and fusomes reveals that they do not develop branched fusomes characteristic of older cysts. To determine the developmental stage of these shu germ cells, they were further analyzed using an antibody specific for the cytoplasmic form of the Bam protein. In wild-type germaria, the cytoplasmic form of Bam accumulates in mitotic cystoblasts and cystocytes. However, in newly eclosed shuWQ41 females no staining was observed in any of the germaria analyzed. This suggests that, after the first few egg chambers are formed, no further mitotic cystoblasts develop. Instead, these germ cells appear to redivide to form clusters of abnormal germ cells that degenerate or occasionally produce tumorous cysts. In older females (>4 days), ovarioles in which the cells at the tip do not express Vasa, apparently failing to maintain germline cells at the tip, are frequently observed consistent with defects in stem cell renewal or maintenance. In addition, some mispackaged egg chambers have also been observed (Munn, 2000).

Females heteroallelic for the weak loss-of-function allele PB70 and the strong loss-of-function allele WM40 lay only ~30% as many eggs as wild-type females, none of which develop. Typically, these eggs are abnormally shaped and dorsal appendages, if present, are reduced or fused. Inspection of the ovaries of these females reveals at least five developing egg chambers in the majority of ovarioles. About 80% of these egg chambers contain the correct 15:1 ratio of nurse cells to oocyte but never develop into wild-type eggs. Although an oocyte is established, as seen by the localization of Bic-D and Orb proteins to the posterior-most cell in the cyst, its identity is not maintained and the oocyte appears to fail in its further development. Subsequent to stage 2 or 3, the distribution of the two proteins becomes diffuse and accumulation of the proteins is observed in nurse cells. This pattern is particularly striking for the Bic-D protein and suggests that the transport system from the nurse cells to the oocyte has collapsed in these egg chambers. Subsequently, oocyte development fails and the egg chambers degenerate. These observations corroborate the results of Tirronen (1993). At low frequency (5%-10%) egg chambers were observed in which the oocyte is not correctly positioned and egg chambers in which the oocyte nucleus appears polyploid rather than diploid, although not to the same extent as the nurse cells. This is similar to phenotypes observed in Bic-D partial loss-of-function mutations. These observations suggest that there may be a later requirement for shu function during egg chamber development (Munn, 2000).

The phenotype of shu indicates that its function is required for the normal activity of the germline stem cell. To address whether shu functions autonomously in the germline, use was made of the FRT-mediated, dominant female sterile (DFS) technique and mutant clones were generated in the germline by mitotic recombination. Females heterozygous for autosomal insertions of the DFS mutation ovoD1 fail to lay eggs and their ovaries contain no egg chambers that have developed beyond stage 4-5 of oogenesis. In contrast, females of the genotype y w P[FLP]/w; FRT ovoD1/FRT shuPB70, which were subjected to heat shock, lay abnormal eggs. These eggs are similar to those laid by females hemizygous or heteroallelic for the PB70 allele. Inspection of ovaries from females in which germline clones of the strong loss-of-function alleles WQ41 and WM40 were induced reveals the presence of abnormal egg chambers that appear, by Hoechst staining, to be degenerating by mid- to late-oogenesis. These results indicate that shu functions in the germline. They do not, however, exclude the possibility of an additional somatic function (Munn, 2000).

To investigate the nature of the shu mutations, the genomic DNA from flies hemizygous for each allele was sequenced. The WQ41 allele has a C to T transversion at position 88 that creates a premature stop codon (Q11 to STOP). This confirms the prediction, based on genetic analysis, that WQ41 is a complete loss-of-function allele. The WM40 allele has a G to A transversion at position 1142 that creates a premature termination of translation 31 amino acids before the end of the protein (W342 to STOP). The weakest loss-of-function allele of shu, PB70, is due to a G to A transversion at position 1051 that results in an alanine (332) to threonine substitution within the TPR motif. This result suggests that the TPR motif is likely to be important for shu function. Interestingly, this mutation does not affect male fertility (Munn, 2000).


Search PubMed for articles about Drosophila Shutdown

Brown, E. J. and Schrieber, S. L. (1996). A signaling pathway to translational control. Cell 86: 517-520. PubMed ID: 8752206

Ding, et al. (1993). Dynamic Hsp83 RNA localization during Drosophila oogenesis and embryogenesis. Mol. Cell. Biol. 13: 3773-3781. PubMed ID: 7684502

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Handler, D., Olivieri, D., Novatchkova, M., Gruber, F. S., Meixner, K., Mechtler, K., Stark, A., Sachidanandam, R. and Brennecke, J. (2011). A systematic analysis of Drosophila TUDOR domain-containing proteins identifies Vreteno and the Tdrd12 family as essential primary piRNA pathway factors. EMBO J 30: 3977-3993. PubMed ID: 21863019

Iki, T., Yoshikawa, M., Nishikiori, M., Jaudal, M. C., Matsumoto-Yokoyama, E., Mitsuhara, I., Meshi, T. and Ishikawa, M. (2010). In vitro assembly of plant RNA-induced silencing complexes facilitated by molecular chaperone HSP90. Mol Cell 39: 282-291. PubMed ID: 20605502

Iki, T., Yoshikawa, M., Meshi, T. and Ishikawa, M. (2012). Cyclophilin 40 facilitates HSP90-mediated RISC assembly in plants. EMBO J 31: 267-278. PubMed ID: 22045333

Iwasaki, S., Kobayashi, M., Yoda, M., Sakaguchi, Y., Katsuma, S., Suzuki, T. and Tomari, Y. (2010). Hsc70/Hsp90 chaperone machinery mediates ATP-dependent RISC loading of small RNA duplexes. Mol Cell 39: 292-299. PubMed ID: 20605501

Kay, J. E. (1996). Structure-function relationships in the FK506-binding protein (FKBP) family of peptidylprolyl cis-trans isomerases. Biochem. J. 314: 361-385. PubMed ID: 8670043

Lamb, J. R., Tugendreich, S. and Hieter, P. (1995) Tetratricopeptide repeat interactions: to TPR or not to TPR? Trends Biochem. Sci. 20: 257-259. PubMed ID: 7667876

Mamane, Y., et al. (2000). Posttranslational regulation of IRF-4 activity by the immunophilin FKBP52. Immunity 12(2): 129-40. PubMed ID: 10714679

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Meng, X., et al. (1998). A novel human gene FKBP6 is deleted in Williams syndrome. Genomics 52: 130-137. PubMed ID: 9782077

Miyoshi, T., Takeuchi, A., Siomi, H. and Siomi, M. C. (2010). A direct role for Hsp90 in pre-RISC formation in Drosophila. Nat Struct Mol Biol 17: 1024-1026. PubMed ID: 20639883

Munn, K. and Steward, R. (2000). The shut-down gene of Drosophila melanogaster encodes a novel FK506-binding protein essential for the formation of germline cysts during oogenesis. Genetics 156: 245-256. PubMed ID: 10978289

Olivieri, D., Senti, K. A., Subramanian, S., Sachidanandam, R. and Brennecke, J. (2012). The cochaperone shutdown defines a group of biogenesis factors essential for all piRNA populations in Drosophila. Mol Cell 47: 954-969. PubMed ID: 22902557

Peattie, D. A., et al. (1992). Expression and characterization of human FKBP52, an immunophilin that associates with the 90-kDa heat shock protein and is a component of steroid receptor complexes. Proc. Natl. Acad. Sci. 89: 10974-10978. PubMed ID: 1279700

Preall, J. B., Czech, B., Guzzardo, P. M., Muerdter, F. and Hannon, G. J. (2012). shutdown is a component of the Drosophila piRNA biogenesis machinery. RNA 18: 1446-1457. PubMed ID: 22753781

Ratajczak, T. and Carrello, A. (1996). Cyclophilin 40 (CyP-40), Mapping of its hsp90 binding domain and evidence that FKBP52 competes with CyP-40 for hsp90 binding. J. Biol. Chem. 271: 2961-2965. PubMed ID: 8621687

Sanchez, E. R., et al. (1990). The 56-59 kilodalton protein identified in steroid hormone receptor complexes is a unique protein that exists in cytosol in a complex with both the 70- and 90-kilodalton heat shock proteins. Biochem. J. 29: 5145-5152. PubMed ID: 2378870

Schupbach, T. and Wieschaus, E. (1991). Female sterile mutations on the second chromosome of Drosophila melanogaster. II. Mutations blocking oogenesis or altering egg morphology. Genetics 129: 1119-1136. PubMed ID: 1783295

Silverstein, A. M., et al. (1999). Different regions of the immunophilin FKBP52 determine its association with the glucocorticoid receptor, hsp90, and cytoplasmic dynein. J. Biol. Chem. 274: 36980-36986. PubMed ID: 10601253

Tirronen, M., et al. (1993). Analyses of the Drosophila quit, ovarian tumor and shut down mutants in oocyte differentiation using in situ hybridisation. Mech. Dev. 40: 113-126. PubMed ID: 8443104

Zamparini, A. L., Davis, M. Y., Malone, C. D., Vieira, E., Zavadil, J., Sachidanandam, R., Hannon, G. J. and Lehmann, R. (2011). Vreteno, a gonad-specific protein, is essential for germline development and primary piRNA biogenesis in Drosophila. Development 138: 4039-4050. PubMed ID: 21831924

shutdown: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation

date revised: 25 May 2013

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