Nop60B RNA is found at high levels in nurse cells and in the oocyte, and is present throughout development. Nop60B protein is localized primarily to the nucleolus of interphase cells, and is absent from the chromosomes during mitosis (Phillips, 1998).
When the 2.0-kb mRNA was used on Northern blots, a probe derived from these two exons detects exclusively the 2.0-kb subform, specifically present in embryos and adult female RNAs. Hybridization of this probe to whole mount preparations of wild-type ovaries reveals that the female transcript accumulates in germ line cells from the early germarial until last oogenesis stages. The accumulation profile of both mfl mRNAs were followed during embryogenesis by developmental Northern blot analysis of carefully synchronized embryos. mRNAs are detected in very early, 0-2 h embryos. However, while the zygotic 1.8-kb mRNA persists at later stages, the level of female transcript drops subsequently, and becomes very low in 4-6 h embryos. This developmental pattern is very similar to that of other stable maternally supplied RNAs, which persist from early stages up to gastrulation (Giordano, 1999).
Nop60B mutants were generated and shown to be homozygous lethal. The Drosophila gene can rescue the lethal phenotype of yeast chf5 mutations, showing that the function of this protein has been conserved from yeast to Drosophila (Phillips, 1998).
The first minifly allele, mfl1, was isolated in the course of a PZ-element mutagenesis screen on the second chromosome as a viable, recessive mutation causing a variety of phenotypic abnormalities. The mfl1 pleiotropic phenotype included an extreme reduction of body size, developmental delay, essentially due to a 4-5-d prolongation of the larval life, defects in the abdominal cuticle, strong reduction in the length and thickness of abdominal bristles, and reduced female fertility. Most traits of the mfl1 phenotype largely overlapped those caused by the Drosophila Minute or bobbed mutations that affect, respectively, the synthesis of ribosomal proteins, 5S, or 18S and 28S rRNAs. This similarity suggested for mfl a possible role in ribosome biogenesis, encouraging an attempt at the the molecular cloning of the gene (Giordano, 1999).
The mfl1 mutation is caused by a single P-element insertion, which, by in situ hybridization of a P-specific probe to salivary gland polytene chromosomes of mfl1 heterozygous larvae, was mapped on the chromosome arm 2R, at the 60B-60C polytene subdivisions boundary. Given that wild-type revertants could be recovered from dysgenic crosses after precise excision of the element, mfl1 mutation appears to be directly caused by this single PZ insertion. Complementation analysis assigned the gene to the region covered by the Df(2R)Px4 deficiency. Among a number of P-induced lethal mutations deposited as part of the Berkeley Drosophila Genome Project, five mapped at the 60B-60C polytene subdivisions boundary. These mutations were all tested in a complementation analysis, by crossing each of them to mfl1 heterozygous flies. Two lines, l(2)k05318 and l(2)k06308, yielded transheterozygous flies with a strong mfl phenotype at the expected ratio, leading to the conclusion that they belong to the mfl complementation group and represent lethal mfl alleles. Accordingly, these lines were renamed, respectively, mfl05 and mfl06. Previous cytological mapping by the Berkeley Drosophila Genome Project assigned these two mfl alleles to the polytene interval 60B11-C2. By lethal phase analysis it was observed that mfl05 homozygotes die mainly as first instar larvae, while most of the mfl06 animals die later, either as second or mainly as young third-instar larvae. Both mfl05 and mfl06 animals fail to increase their size as compared with their wild-type heterozygous siblings and survive for an additional 4-5 d as first or third instar larvae, respectively (Giordano, 1999).
Since a feature of the mfl1 pleiotropic phenotype was represented by reduced female fertility, the structure of mutant ovaries was examined. Morphological abnormalities were often observed, with some of the egg chambers beginning to degenerate beyond approximately stage 7 of oogenesis. In the degenerating egg chambers, fragmented or condensed nurse cell nuclei with irregular shape are frequently found. These observations raise the possibility that apoptotic cell death may occur in mfl1 abnormal ovaries. This possibility was investigated by staining egg chambers with acridine orange (AO). AO is a vital dye that is known to selectively stain apoptotic cells in insects and has successfully been used to study the distribution of apoptosis in Drosophila ovaries. Wild-type ovaries exhibit a diffuse green fluorescence, whereas highly fluorescent yellow spots are detected in mfl1 degenerating egg chambers. These yellow spots are known to correspond to apoptotic, AO highly positive nuclei, thus confirming the occurrence of apoptosis in mfl1 ovaries (Giordano, 1999).
As a consequence of the gonadic abnormalities observed, mfl1 homozygous females lay a reduced number of mature eggs, and ~15% of the embryos produced fail to hatch. Such degenerating embryos show asynchronous and atypical development, invariantly accompanied by diffuse apoptotic cell death. Many mutations causing partial loss-of-function of vital genes interfere with the proper development of the egg, causing female sterility. Inadequate rate of protein synthesis is also known to affect Drosophila oogenesis, by slowing the level of yolk production and retarding egg chamber progression into vitellogenesis, beginning at stage 8. This effect is common to mutants unable to produce large amount of proteins, having reduced levels of either ribosomal proteins, 18S, 28S, or 5S rRNAs (Giordano, 1999).
When mfl mutants were checked for gene expression, they were found to have reduced levels of mfl mRNAs. While the viable, hypomorphic mfl1 allele shows only a modest reduction, mfl expression is strongly disrupted in mfl05 and mfl06, the two alleles causing larval lethality. MFL protein accumulation strictly parallels the level of mfl mRNAs, so that it is strongly reduced in mfl06 and nearly null in mfl05. Remarkably, the developmental time at which lethality is achieved in these two mutants correlates well with MFL level since mfl05 homozygotes die mainly as first instar larvae, while mfl06 animals die as second or early third-instar larvae. Note that, considering the timing of persistence of maternal rRNA, mortality at the first instar is that which may be expected for mutations causing severe loss of function of a gene essential for rRNA processing. Taken together, all these data indicated that MFL level appear to be critical for Drosophila viability (Giordano, 1999).
Attempts were made to rescue mfl lethal phenotype by ectopically expressing MFL from the heat-inducible hsp70 promoter. mfl05 and mfl06 transgenic animals were then obtained and daily treated at 37°C for 30 min. These heat-shock conditions usually produce amounts of the ectopically expressed protein that largely exceed the wild-type level. However, in these experiments they produce a MFL level just comparable to that present in wild-type flies, even though the induced protein remains quite stable from 6 h to as long as 24 h from the heat-shock pulse. Nevertheless, the level of induced MFL is sufficient to allow mfl05 and mfl06 transformed animals to developed synchronously with their wild-type siblings and to show a normal increase in their size. Moreover, 30% of the mfl05 and 80% of the mfl06 transgenic animals develop up to the pupal stage, although these transgenic pupae all failed to eclose adult flies. A possible explanation for this partial rescue of the mfl mortality is that the level of ectopically expressed MFL may be inadequate with respect to the rate of protein synthesis required in specific cell types during metamorphosis. An alternative possibility is suggested by the observation that, in yeast, Cbf5p is required for the stability of other components of the H/ACA class of RNPs, such as Gar1p and box H/ACA snoRNAs (Lafontaine, 1998). It is thus possible that the MFL level reached under heat-shock conditions may not be constant and this affects the stability of other essential RNP components. However, since no member of the H/ACA class of RNPs has yet been described in Drosophila, this hypothesis cannot be tested at present (Giordano, 1999).
The Nop60B gene is required in the maintenance of the germ line during spermatogenesis. Mutations impeding spermatogenesis often lead to reduced testis size in addition to male sterility. In general, defects that occur earlier in spermatogenesis cause a more severe size reduction. This appears to be because continued maintenance and division of germline stem cells is necessary to fill the testis with successive families of developing germ cells. Fly lines containing single P-element inserts were screened for male sterility and reduced testis size. This study focuses on sterile males with the profound testicular atrophy consistent with a defect in the maintenance of the stem cells. Two P{LacW} insertions, DL2011 and mM176, yielded such a phenotype and failed to complement each other. Precise excision of the P-element restored a wild type phenotype, demonstrating that the P insertion was responsible for the sterile phenotype. Flanking DNA was cloned and the insertion site was found to be in the 5' UTR of the Nop60B gene. Nop60B has been shown by sequence similarity to be a member of the TruB family of proteins, all of which show homology to Escherichia coli TruB, a tRNA pseudouridine synthase (Phillips, 1998). Several 'imprecise' excisions were analyzed; this analysis yielded lethal and semiviable alleles, as well as several viable but sterile alleles, such as nop60BDL4, which showed atrophic testes similar to DL2011 (Kauffman, 2003).
Testes from nop60BDL4 mutants are severely reduced in size compared with a wild-type testis, suggesting a significant reduction in the number of germline cells. This was verified by immunostaining with mAB-1B1, which marks the fusome, a germ-line-specific cytoskeletal organelle. In a wild type testis, germ-line stem cells adjacent to the hub exhibit dot fusomes, while later stage germ cells at a distance from the hub show progressively more branched fusomes, reflecting mitotic amplification of the gonial cells. Testes from nop60BDL4 mutants exhibited a near absence of fusome signal (Kauffman, 2003).
To verify that the testis phenotype was due to the insertion of the P element into the Nop60B gene, rescue experiments were performed using a HS-Nop60B transgenic line (Giordano, 1999). Daily heat shocks began 0-1 day after egg laying (AEL) and continued until adult flies eclosed. Testes were then dissected, fixed, and immunostained with mAB-1B1. nop60BDL4 homozygotes carrying the rescue construct showed dramatic heat shock-dependent rescue of testicular atrophy, as shown by the reappearance of fusomes. Ninety-one percent (91%) of testes from young males heat shocked until the day of dissection showed more than 10 branched fusomes per testis. Importantly, germ-line stem cells were also rescued, as judged by the presence of germ cells with dot fusomes adjacent to the hub. In contrast, only 17% of testes from age-matched nop60BDL4 flies raised without heat shock showed any evidence of germ cells, while 75% showed none. The rescue confirms that the testicular atrophy phenotype is due to a defect in the Nop60B gene. Thus, Nop60B is required in some manner for the maintenance of the germ line during spermatogenesis (Kauffman, 2003).
A panel of molecular markers was used to address the nature of the nop60BDL4 phenotype at the cellular level and ascertain the effect of this mutation on the various cell populations in the testis. Using such markers, it was found that nop60BDL4 testes showed a dramatic reduction in the number of germ cells relative to wild type, without exhibiting a similar loss of somatic cells. The fusome stain first suggested a strong germ cell phenotype. This was verified by testing for expression of the germ-line-specific protein Vasa. No germ cells are apparent in the mutant testis, as judged by the lack of signal. The lack of germ cells included an apparent lack of germ-line stem cells. This was confirmed by taking advantage of lacZ enhancer traps that mark specific cell types and stages during spermatogenesis. Of particular use in this analysis was the recovery of Nop60B alleles, such as nop60BDL4, which lost the ability to express lacZ, allowing analysis of the expression of lacZ markers that were introduced into nop60BDL4 flies. In wild-type testes, enhancer trap line M5-4 marks the hub, germ-line stem cells, and their immediate daughters, the gonialblast cells. In mutant testes, hub cells were present, as judged by the presence of groups of M5-4-expressing cells, although it was noted that these cells had an elongate appearance, which is similar to the disorganization in hub cells observed in agametic testes. Another similarity between agametic and nop60BDL4 mutant testes is that hub cells accumulate Fasciclin III protein to levels lower than those observed in wild type. Although hub cells were observed, usually no evidence for germ-line stem cells or their gonialblast progeny were found. Occasionally, a few lacZ-expressing germ cells were observed, but these were some distance from the hub. It was not clear whether these cells were wandering gonialblasts or germ-line stem cells. There are currently no markers with expression restricted solely to germ-line stem cells. It is concluded from these analyses that testes are atrophic due to severe loss of germ-line cells, including the germ-line stem cells (Kauffman, 2003).
Despite the loss of the germ cell population, the cyst cells, which derive from somatic stem cells, were observed in mutant testes, as judged by both an enhancer trap marker and the expression of Eyes Absent (Eya). In wild type, enhancer trap S2-11, which marks hub and early-stage cyst cells, shows an organized hub and an appropriately robust early-stage cyst cell population. In mutant testes, hub cells are apparent, and exhibit a similar loss of organization as that observed with enhancer trap M5-4 and in agametic testes. However, unlike the germ cell population, which is quite depleted, significant numbers of cyst cells are observed. That there is little effect on the cyst cell population was confirmed by examining the expression of Eya, a cyst cell-specific marker. Eya expression is first induced in cyst cells associated with midamplification stage gonia, approximately at the four- to eight-cell stage. Thus, Eya expression is not only specific to the cyst cell lineage, but its expression can be taken as a measure that the cyst cells have progressed to a certain point in their developmental program. Indeed, in mutant testes, large numbers of Eya-positive cyst cells accumulate. This confirms that there is not as large an effect on the somatic cells, as compared with the germ line. It was not possible to assay directly for cyst progenitor cells (the somatic stem cells), since the only marker for these cells is a P enhancer trap that resides on the CyO balancer chromosome, which cannot be used in nop60BDL4 homozygotes. Nevertheless, it is clear from these data that, in nop60BDL4 mutant testes, the behavior of the somatic stem cell lineage is not affected to the same degree as is the germ-line stem cell population and its descendants. Specific deletion of germ cells, achieved in agametic testes, leads to a loss of hub organization, expansion in the number of hub cells, and continued cyst cell proliferation. Thus, it appears that, in nop60BDL4 testes, the primary defect may be loss of the germ-line cells, leading to associated changes in somatic cell behavior (Kauffman, 2003).
Next addressed was the question of whether the Nop60B gene product is required for the maintenance of the germ-line stem cell population. Three different approaches to this question indicate that continued Nop60B gene function is necessary for the maintenance of the stem cell lineage (Kauffman, 2003).
It is concluded that the testicular atrophy observed in Nop60B gene mutation is due to an almost complete lack of germ cells in adult flies. The somatic cyst cell population, however, is not significantly reduced. By several criteria, the germ-line stem cells, at least some of which are initially specified, are prematurely lost, accounting for the virtual loss of the germ-line lineage in adult testes. In addition, the requirement for Nop60B is intrinsic to the germ-line; removing Nop60B activity leads to loss of germline stem cells and death of gonial cells. This implicates the Nop60B gene in the maintenance of the germ line during spermatogenesis (Kauffman, 2003).
In principle, it is possible that Nop60B plays a special role in germ cells, or in germ-line stem cells, which would explain the loss of this lineage in the hypomorphic mutants investigated here. An explanation such as this would justify screening for germ-line loss mutants on a large scale in order to identify genes governing the behavior of stem cell lineages. Alternatively, and more likely in this particular case, the hypomorphic nature of the Nop60B alleles may account for this apparent requirement solely in a renewing lineage. For instance, there may be enough gene product both from maternal stores and residual zygotic expression to support larval life and metamorphosis, but not enough to maintain the germ-line lineage in spermatogenesis. An explanation such as this makes one hesitate in trying to identify interesting genes governing the behavior of stem cell lineages from among mutants showing germ cell loss phenotypes (Kauffman, 2003).
Indeed, Nop60B encodes a protein likely involved centrally in cellular metabolism. It contains homology to a class of pseudouridine synthases, and, although this activity has yet to be directly demonstrated, the Nop60B protein is localized to the nucleolus, the site of rRNA processing, in all cell populations (Phillips, 1998), and a previous analysis of null mutant larvae strongly suggests defects in rRNA processing and pseudouridinylation (Giordano, 1999). It is not surprising then that strong mutations in this gene are not stem cell- or germ-line-specific, but are rather lethal to the organism at a very early stage. That hypomorphic alleles have a dramatic effect on the germline is likely due to the need to sustain proliferation in this adult tissue, or more particularly, sustain high rates of protein synthesis. This is a similar conclusion to that arrived at previously in order to explain the small body size phenotype of the 'mini-fly' class of Nop60B alleles (Giordano, 1999). The hypomorphic alleles used on this study did not exhibit a mini-fly phenotype, and, thus, do not appear to be generally compromising the fly. This has afforded the ability to investigate the requirement for Nop60B in tissue homeostasis and stem cell function, a role which might relate to that affected in a human disease (Kauffman, 2003).
Within the testis, the data suggest that the requirement for Nop60B gene product may be highest in the germ-line stem cells. The testes from most rescued hypomorphic Nop60B mutant flies initially have a functioning germline, and the germ cells are lost if the flies are aged without continued production of rescuing Nop60B activity. The fact that a few germ-line stem cells are apparent in the third larval instar gonad, but not in adults, also supports the proposition that stem cells are initially specified but cannot be maintained. Interestingly, it was also found that the somatic stem cells and their daughter cyst cells appear to be less affected than the germ line under these hypomorphic conditions. It is possible that the P insertion affects the expression of Nop60B in the germ line more than in the somatic lineage. However, the current understanding of the gene structure of Nop60B provides no easy explanation for this, such as a testis-specific promoter (Giordano, 1999; Phillips, 1998). Alternatively, the data suggest that there is a higher requirement for Nop60B gene product for the steady-state operation of the germ-line stem cells. Although it has been shown that removing Nop60B function completely from the germ-line stem cells leads to their loss, similar mosaic analysis could not be performd on the somatic stem cell population because the somatic markers are not as robust (Kauffman, 2003).
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date revised: 30 March 2003
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