In Drosophila melanogaster, two genes, Prat and Prat2, encode the enzyme, amidophosphoribosyltransferase, that performs the first and limiting step in purine de novo synthesis. Only Prat mRNA is present in the female germline and 0- to 2-hr embryos prior to the onset of zygotic transcription. We studied the maternal-effect phenotype caused by Prat loss-of-function mutations, allowing us to examine the effects of decreased purine de novo synthesis during oogenesis and the early stages of embryonic development. In addition to the purine syndrome previously characterized, we found that Prat mutant adult females have a significantly shorter life span and are conditionally semisterile. The semisterility is associated with a pleiotropic phenotype, including egg chamber defects and later effects on embryonic and larval viability. Embryos show mitotic synchrony and/or nuclear content defects at the syncytial blastoderm stages and segmentation defects at later stages. The semisterility is partially rescued by providing Prat mutant females with an RNA-enriched diet as a source of purines. Our results suggest that purine de novo synthesis is a limiting factor during the processes of cellular or nuclear proliferation that take place during egg chamber and embryonic development.

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PURINE nucleotides are involved in many cellular functions as components of DNA and RNA, as sources of energy, as enzyme cofactors in metabolic pathways, and as components of signal transduction. Purine nucleotides are produced by three interrelated pathways: the inter-conversion pathway, which converts hypoxanthine to guanine and adenine; the salvage pathway, which uses preexisting bases and sugars; and the de novo biosynthesis pathway, which produces IMP, the common precursor of AMP and GMP, in 10 enzymatic steps (ZALKIN and DIXON 1992).

Phosphoribosylamidotransferase (PRAT) performs the first and limiting step in the de novo purine biosynthesis pathway. PRAT converts 5-phosphoribosyl-1-pyrophosphate (PRPP) into 5-phosphoribosyl-1-amine (PRA) using ammonia or glutamine as the source of the amide group. PRAT enzyme activity is regulated by feedback inhibition by pathway end products and by the availability of the substrate PRPP, which is used competitively by the salvage pathway (BECKER and KIM 1987). The gene that encodes PRAT has been characterized in unicellular and multicellular organisms, and the enzyme has two conserved domains. One domain is required for glutamine amide transfer and the second domain is required for synthesis of PRA from PRPP and ammonia (ZALKIN and DIXON 1992).

In Drosophila melanogaster, mutant alleles of genes involved in the de novo purine synthesis of IMP were first recovered in screens for purine auxotrophy (NASH et al. 1981; JOHNSTONE et al. 1985). As well, recessive lethal alleles of ade3 and ade2 were isolated in EMS mutagenesis screens (TIONG et al. 1989; TIONG and NASH 1990) and ade5 alleles were isolated in P-element and gamma-radiation mutagenesis screens (O'DONNELL et al. 2000). Phenotypic analysis of the different mutant alleles led to the definition of the purine syndrome, an adult phenotype that includes (1) reduced red eye pigments, (2) tarsal leg defects, and (3) wing vein pattern alterations with decreased vein length and/or incomplete posterior cross-veins. Null alleles of genes in the pathway are associated with strong pupal lethality (TIONG et al. 1989), while semilethal alleles show pupal lethality with a proportion of adult escapers with the purine syndrome. While auxotrophic alleles of genes that operate in downstream steps of the pathway were isolated, no auxotrophic alleles of the Prat gene were isolated. Instead, using information from sequence analysis of Prat in several model organisms and the purine syndrome phenotype, the Prat gene was characterized at the molecular and genetic levels (CLARK 1994). The five recessive loss-of-function mutations in Prat are semilethal to various degrees with adult escapers showing the purine syndrome.

While it appears that the Prat gene is essential for D. melanogaster development, the genome sequence project revealed the presence of a second Prat gene, Prat2, and this gene duplication is conserved in D. virilis (MALMANCHE et al. 2003). In both species, only the Prat gene is expressed in the female germline and Prat mRNA and PRAT protein are present in 0- to 2-hr embryos, prior to the onset of zygotic gene expression. Expression of all other genes involved in the pathway, except for the Prat2 gene, has also been detected in 0- to 2-hr embryos (TOMANCAK et al. 2002). Therefore, Prat and the other purine de novo synthesis genes appear to have an important role in oogenesis and embryonic development.

To understand the specific function of Prat during Drosophila development and to begin to address the relationship between the purine pathway and the events of cell proliferation and development, we investigated Prat function in the Drosophila female germline using two hypomorphic Prat EMS alleles, Prat^12A19 and Prat^16A6, where the purine syndrome phenotype is stronger for Prat^12A19 (CLARK 1994). Each EMS-mutagenized chromosome is recessive lethal, while a few adult escapers are produced when the chromosome is hemizygous with Df(3R)dsx43, a deficiency uncovering the Prat locus. In addition to the purine syndrome previously characterized, we have found that hemizygous females for a Prat mutation and heteroallelic females for Prat alleles show a pleiotropic mutant phenotype associated with (1) reduced mean and maximum life span, (2) reduced fertility that is conditional on diet and associated with alteration of the egg chambers, and (3) a semilethal maternal effect that appears to be due to defects in nuclear proliferation during the early stages of embryonic development and results in segmental defects.

Drosophila stocks:

Flies were raised at 25°, 60% relative humidity with a light/dark cycle of 12 hr. Prat^12A19 and Prat^16A6 are described by CLARK (1994). The wild-type Canton-S stock was provided by Steven Henikoff and the Df(3R)dsx43 and Efh23 stocks were provided by Bruce Baker (BAKER et al. 1991).

Nutrition:

Standard fly food consists of a mix of 560 g cornmeal, 126 g yeast extract, 70 g soy flour, 140 g light malt extract, and 240 ml of molasses mixed with 7 liters of water containing 56 g agar. The solution is boiled, autoclaved for 20 min, and allowed to cool to 60°, and then 5.8 ml of propionic acid mix (500 ml propionic and 32 ml phosphoric acid) is added per liter to inhibit microbial growth. Each batch of food was split in two, with half kept as "normal food" and half kept as "RNA food," to which 4 g RNA (R6625; Sigma, St. Louis) was added per liter of solution.

Drosophila crosses:

Due to second-site lethal mutations associated with each EMS-induced Prat allele's third chromosome, experiments were done with hemizygous F₁ females carrying a deficiency of the Prat region, Df(3R)dsx43, with cytogenetic breakpoints 84D13-14; 84E6-8 (BAKER et al. 1991) or with females carrying both Prat alleles in a heteroallelic genetic combination. Prat maps cytogenetically within these breakpoints to 84E1-2 (CLARK 1994). A recombinant chromosome carrying Df(3R)dsx43 and the e¹¹ marker was constructed by crossing Df(3R)dsx43/TM3, Sb males with v;e¹¹ females, selecting non-Sb F₁ females, backcrossing them to Df(3R)dsx43/TM3, Sb males, selecting a single recombinant v;Df(3R)dsx43 e¹¹/TM3, Sb male, and then backcrossing to Df(3R)dsx43/TM3, Sb to generate female sibs carrying the recombinant chromosome. The e¹¹ chromosome is the parental chromosome of the Prat mutant alleles (CLARK 1994).

Life-span experiment:

We followed the life span of five replicates of 15 females crossed with 10 Canton-S males on normal and RNA food for the following genotypes: v;Prat^12A19 e¹¹/Df(3R)dsx43 e¹¹, v; Prat^16A6 e¹¹/Df(3R)dsx43 e¹¹, v; Prat^16A6 e¹¹/Prat^12A19 e¹¹, v/+; Prat^12A19 e¹¹/+, v/+; Prat^16A6 e¹¹/+, v/+; Df(3R)dsx43 e¹¹/+, v; e¹¹, and Canton-S. For each replicate experiment, flies were transferred every 2 days and the number of dead females was counted. Males were not replenished as they died since they were all of the same genotype. The data were analyzed using a log-rank test for which n = 75 [survival estimate, Systat 10 software (Systat Software, Point Richmond, CA)].

Survival experiment:

We analyzed the embryonic and adult viabilities of progeny resulting from a cross between v; Prat^12A19 e¹¹/Df(3R)dsx43 e¹¹, v; Prat^16A6 e¹¹/Df(3R)dsx43 e¹¹, v; Prat^16A6 e¹¹/Prat^12A19 e¹¹, v/+; Prat^12A19 e¹¹/+, v/+; Prat^16A6 e¹¹/+, v/+; Df(3R)dsx43 e¹¹/+, Canton-S, and v; e¹¹ females and Canton-S males on normal and RNA food. We let the flies lay eggs at 25° for a period of 8 hr. For embryonic viability, we counted the number of eggs and, after 26 hr at 25°, the number of hatched eggs. For adult viability, we counted the number of eggs, and the number of adults was counted after 14 days at 25°. The data were analyzed using a two-factor ANOVA and a post hoc Tukey HSD test using Systat 10, after normalization of the raw data by an arcsin square transformation.

In situ hybridization:

In situ hybridization to whole-mount ovaries was done using a digoxigenin-labeled DNA probe. The probe template was a Prat cDNA isolated from a lambda-ZAP D. melanogaster 0- to 24-hr embryo cDNA library (Stratagene, La Jolla, CA; D. CLARK, unpublished results) and subcloned into pVZ1 (HENIKOFF and EGHTEDARZADEH 1987). Cross-hybridization of this probe with Prat2 mRNA is not a concern since Prat2 is not expressed in ovaries (MALMANCHE et al. 2003). After digestion by EcoRI (New England Biolabs, Beverly, MA), a 1.7-kb fragment was gel purified (QIAGEN, Valencia, CA) and 100 ng of DNA were used to produce a Dig-UTP probe using random primers (Roche, Indianapolis). Ovaries were dissected in 1x PBS on ice and prepared as described by EPHRUSSI et al. (1991). A 20-min proteinase K treatment was done at room temperature. The remaining steps were performed as for embryos, as described by TAUTZ and PFEIFLE (1989).

Nuclear staining:

v; Prat^16A6 e/Df(3R)dsx43 e and v; Prat^16A6 e/Prat^12A19 e embryos (0–4 hr) were collected on normal food plates. For controls, 0- to 4-hr Canton-S embryos were collected on grape agar plates supplemented with yeast. The nuclear staining on embryos was performed as described in SULLIVAN et al. (2000). For ovaries, the dissection was done in cold 1x PBS. Fixation was performed in 4% paraformaldehyde-1x PBS solution for 20 min at room temperature. After several washes in PBT 0.1%, a 20-min RNAse H treatment at 37° was performed and propidium iodide (Sigma) was added to the ovaries at 1 µg/ml in 90% glycerol-10% 1x PBS mounting media. Alternatively, after several washes in PBT 0.1% (PBS with 0.1% Triton X-100), 1 µg/ml DAPI (Sigma) nuclear stain was added to the ovaries and incubated for 5 min.

Cuticle preparation:

Flies were allowed to lay eggs for 4 hr on normal food plates for v; Prat^12A19 e/Df(3R)dsx43 e and v; Prat^16A6 e/Df(3R)dsx43 e genotypes and on grape agar plates for Canton-S. The embryonic cuticle preparations were performed as described by SULLIVAN et al. (2000).

Antibody production and purification:

To produce an antiserum specific to the Prat gene product that does not cross-react with the Prat2 gene product, a 16-amino-acid peptide of sequence LKHRDRGDSKSKGTGH was synthesized and conjugated to the carrier protein keyhole limpet hemocyanin, and antiserum was raised in rabbits by Alpha Diagnostics International. To generate antiserum D4, crude preimmune and immune (R7) sera were affinity purified using the methods described by CLARK and MACAFEE (2000). Immunostaining of Western blots of purified recombinant proteins (PRAT and PRAT2) and Drosophila protein extracts show that this antiserum, R5127, binds specifically to PRAT and to a 55-kD protein, respectively (MACAFEE and CLARK, unpublished observations).

Immunostaining:

Ovaries were dissected in cold 1x PBS. Fixation was performed in 4% paraformaldehyde-1x PBS solution for 20 min at room temperature. Following 2-hr permeabilization in 1x PBS-Triton X-100 1%, ovaries were washed in PBT 0.1% for 15 min three times. PRAT antibody was used at a dilution of 1/250 in PBT 0.1% and detected using an anti-rabbit fluorescein conjugated antibody (Jackson) at a dilution of 1/500. Nuclei were stained with TOTO-3 (Molecular Probes, Eugene, OR).

Immunostaining of Schneider cells (SCHNEIDER 1972) was performed by fixing cells in 4% paraformaldehyde-1x PBS for 20 min at room temperature, blocking cells for 30 min (0.2 M glycine, 2.5% fetal bovine serum, 0.1% Triton X-100, 0.02% sodium azide in 1x PBS), incubating with primary antibody in blocking solution for 30 min, detecting using an anti-rabbit fluorescein conjugated antibody (Jackson) at a dilution of 1/500, and staining nuclei with DAPI (Sigma) at 1 µg/ml in 1x PBS for 5 min.

Microscopy:

PRAT immunostaining in ovaries was observed using a Leica confocal microscope and a 3-D projection was produced using the stack of images. Immunostaining in Schneider cells was observed using a DeltaVision microscope (Applied Precision, Issaquah, WA). Propidium iodide nuclear staining of egg chambers was observed using a Zeiss 410 inverted confocal microscope with a plan neofluar x20 or x40 objective. DAPI nuclear staining of embryos and egg chambers was observed using a Leica compound epifluorescent microscope equipped with DAPI filters. In situ hybridization to whole-mount ovaries was observed using a Zeiss microscope equipped with Nomarski optics. Cuticle preparations were observed using an Olympus microscope equipped with dark field optics.

Prat mutant females have a reduced life span:

Phenotypic characterization of females hemizygous for Prat mutations was performed using the deficiency Df(3R)dsx43, where the purine syndrome phenotype for Prat^12A19 is more severe than that for Prat^16A6 (CLARK 1994) and for the heteroallelic combination (data not shown). The genetic characterization of both Prat alleles was performed using the Df(3R)dsx43 chromosome, which was recombined with the e¹¹ allele (see MATERIALS AND METHODS). Each EMS-mutagenized Prat mutation-carrying chromosome is fully lethal when homozygous, whereas a few adult escapers are produced when the Prat mutations are hemizygous with Df(3R)dsx43 or in a heteroallelic combination. Thus, the associated recessive lethality of the Prat^16A6 and Prat^12A19 chromosomes is partly due to complementing second-site mutations external to the Df(3R)dsx43 region. During analysis of the various Prat mutant phenotypes, each Prat mutation or deficiency-carrying chromosome was used in a control experiment, to test for the presence of dominant effects, by outcrossing mutation-carrying stocks to the wild-type Canton-S stock.

In addition to the purine syndrome phenotype previously reported (CLARK 1994), Prat mutant adult females, as hemizygotes or as heteroallelic heterozygotes, have a significantly shorter life span (Figure 1C). While one-half of the population died within the first week after the start of the experiment (flies were collected over a period of 48 hr), the other half showed a wider distribution of life span (Figure 1C).

View larger version (19K): In this window In a new window Download PPT slide   FIGURE 1.— Life-span experiment. For each genotype, survival of five replicates of 15 adult females with 10 Canton-S males (n = 75) on standard cornmeal medium was followed at 25° with enumeration and transfer of survivors on fresh food every 2 days. The data set was analyzed using a log-rank test.

 
Previous work on Drosophila de novo purine synthesis genes showed that 4 mg/ml RNA supplementation can rescue the phenotype of auxotrophic mutants fed a defined medium (HENIKOFF et al. 1986). We explored the effect of RNA supplementation on the preadult lethality and the early death of adult escapers for both Prat alleles. We performed the cross to generate Prat mutant hemizygotes on food supplemented with 4 mg/ml RNA as a source of nucleotides (see MATERIALS AND METHODS). Under this condition, no difference was observed in the production of the Prat mutant adult females on the normal food compared to the RNA supplemented food (data not shown) indicating that the development of the pupal lethal phenotype is independent of the food provided during previous stages of development. As well, we measured the life span of each genotype on the RNA-supplemented food (data not shown). We used the same experimental procedures as for the normal food. All three Prat mutant female genotypes have the same life span on both food types, indicating that the aging process also is not affected by the food supply at the adult stages and that a nonvisible phenotype occurs during the pupal developmental stage.

We found a dominant effect on the aging process for both Prat e¹¹ chromosomes but not for the Df(3R)dsx43 e¹¹ chromosome. Prat^12A19 e¹¹/+ and Prat^16A6 e¹¹/+ females have a reduced mean life span (the effect being stronger for the Prat^12A19 e¹¹ than for the Prat^16A6 e¹¹ chromosome, Figure 1B), while maximum life span was almost unchanged for both alleles. To ensure that genetic background was not influencing life span, in a separate experiment we found no significant difference between the mean and maximum life spans of v;e¹¹ and Canton-S females (data not shown). Since the EMS-induced Prat mutations were independently isolated, we can conclude that the reduction in mean life span is associated with the Prat mutations rather than with a dominant effect of a linked second-site mutation or with the e¹¹ allele used in the genetic background.

Prat mutant females are conditional semisterile:

Previously, Prat mRNA expression was found to be specific for oogenesis and 0- to 2-hr embryos in comparison to Prat2 (MALMANCHE et al. 2003). To explore this phenomenon further, we examined the fecundity of Prat mutant females and the embryonic development of their progeny resulting from a cross with Canton-S males. Here, the embryonic phenotype reflects the purine de novo biosynthesis contribution during early embryonic development, prior to the onset of zygotic transcription. Both the embryonic and the adult survival of the progeny from the three Prat mutant female genotypes was measured (Tables 1 and 2).

View this table: In this window In a new window   TABLE 1 Embryonic survival

 

View this table: In this window In a new window   TABLE 2 Adult survival

 
After normalizing the raw data by an arcsin square transformation, we analyzed them using a two-factor ANOVA with a post hoc Tukey HSD test (Tables 1 and 2). Four observations can be made from this experiment. First, we observed no change in the embryonic and adult survival among the five control genetic backgrounds tested (Tables 1 and 2), showing, first, that no dominant mutations are involved in the phenotype observed and, second, that the RNA addition is not providing some general supplementation of nutrients lacking in the normal food used during our experiments. Second, we observed a strong reduction in embryonic and adult survival of the progeny from the three Prat mutant female genotypes when they were outcrossed with Canton-S males, indicating that they are semisterile (Tables 1 and 2). Third, we observed a partial rescue of the semisterility phenotype when the food was supplemented with RNA (Tables 1 and 2); however, the rate of egg production and total number of eggs produced by the three Prat mutant genotypes did not differ between the two types of food (data not shown). This finding suggests that the effect of the RNA supplementation on sterility is not associated with increased embryo production or with improved somatic maintenance. The rescue of the semisterility by an exogenous source of nucleotides demonstrates that the effect is indeed caused by the reduction in purine nucleotide synthesis due to Prat loss-of-function mutations and not by a second-site mutation on the EMS-mutagenized chromosome. Fourth, it is interesting to note that the partial rescue of the phenotype results in the same level of embryonic and adult survival for each of the three mutant genotypes, indicating that the RNA addition can only partially cancel the genotype effect. Note that the RNA food did not significantly rescue the adult survival of the progeny for two mutant genotypes (Prat^16A6 e¹¹/Df(3R)dsx e¹¹ and Prat^12A19 e¹¹/Prat^16A6 e¹¹), although the observed effect is in the expected direction. We suspect that this result is due to the low replication used for this experiment (two replicates for each genotype on each food type).

To demonstrate that the maternal-effect embryonic and larval lethality is due to the Prat loss-of-function mutation, rather than to a dominant zygotic effect associated with the Df(3R)dsx43 e¹¹ region, we performed a cross to determine whether Prat and Df(3R)dsx43 were equally represented in the adult progeny. To test this idea, we backcrossed every male from the aging experiment with the Efh23 e¹¹/TM3, Sb e stock (BAKER et al. 1991; see MATERIALS AND METHODS). The result showed the Mendelian segregation of Prat e¹¹ and Df(3R)dsx e¹¹ in the surviving male progeny of the Prat mutant hemizygous females (Table 3). Therefore, the embryonic and larval phenotypes we observed are due to the maternal genotype and a decrease in the purine de novo biosynthesis activity during early embryogenesis, rather than to the zygotic genotype.

View this table: In this window In a new window   TABLE 3 Segregation of the maternal Prat allele and the deficiency to surviving male progeny

 

Detection of Prat mRNA and protein in ovaries and Schneider cells:

Prat is expressed at two times during egg chamber development. Prat mRNA can be first detected from the tip to the end of region 3 in the germarium (Figure 2B). A lower signal can be detected in stage 1–2 egg chambers following their exit from the germarium. Then, Prat mRNA can be detected in the nurse cells by stage 8 until stage 10 (Figure 2C). However, Prat mRNA does not have specific localization in the oocyte by stage 10.

View larger version (69K): In this window In a new window Download PPT slide   FIGURE 2.— Prat gene expression and PRAT protein localization in female ovaries and in Schneider S2 cells. (A) Diagram of a germarium [reprinted from Figure 7 of MAHOWALD and KAMBYSELLIS (1980) with permission from Elsevier]. (B and C) Prat RNA in situ hybridization in ovaries. (B) Prat RNA is first detected from the tip to region 3 in the germarium. Some weak expression can be detected in stages 1–2. (C) Prat is then detected in the nurse cells starting at stage 8 until stage 10. The RNA does not localize specifically within the oocyte at stage 10 and at later stages. (D–L) PRAT immunodetection in ovaries with higher magnification of germarium (x63). (D) PRAT is detected in both the germline and the somatic cells in the germarium until stage 6. (E) Nuclei are stained with TOTO-3. (F) Merged D and E images. (G and J) PRAT is detected in the follicle cells surrounding the oocyte at stage 10. Most of the PRAT staining is coincident with the optical sections including the follicle cells rather than with the oocyte. (H and K) Nuclei are stained with TOTO-3. (I and L) Merged G and H images and J and K images, respectively. (M) R7 preimmune serum staining of egg chambers. (N) Nuclei are stained with TOTO-3. (O) Merged M and N images. (P) PRAT immunodetection in Schneider cells. (Q) Nuclei are stained with DAPI. (R) Merged P and O images. (S) R7 preimmune serum staining of Schneider cells. (T) Nuclei are stained with DAPI. (U) Merged S and T images.

 
Using a specific antiserum that was raised against a peptide unique to PRAT (R7; see MATERIALS AND METHODS), PRAT was detected in both the somatic and the germline cells in the early stages of oogenesis: in the germarium and during stages 1–6 (Figure 2D and F). The intracellular distribution of PRAT has a speckled appearance in egg chambers in both cell types, suggesting the presence of a protein complex. The speckled pattern of staining has also been observed in Drosophila Schneider tissue culture cells using the same antiserum (R7) and a second antiserum that recognizes both PRAT and PRAT2 (D4; CLARK and MACAFEE 2000; Figure 2, P and R). In stage 10 egg chambers, PRAT can be detected in the follicle cells surrounding the oocyte (Figure 2, G, I, J, and L). The speckled pattern is specific to the anti-PRAT antiserum, since such staining was not observed using pre-immune sera or the fluorescein-conjugated secondary antibody alone at the same gain settings on the confocal microscope (Figure 2, M and O) or with Schneider cells using a DeltaVision microscope (Figure 2, S and U). We suspect that Prat transcription takes place in follicle cells but the level of Prat RNA is certainly under the detection threshold to be visualized using the alkaline phosphatase anti-digoxigenin antibody.

Prat mutant females have a pleiotropic egg chamber phenotype:

The reduction in egg production combined with the observation of Prat expression in the germarium suggests a function for the purine de novo synthesis pathway in egg chamber development. We therefore asked whether Prat mutant females' oocytes had alterations in cell organization occurring during egg chamber development. Using propidium iodide or DAPI as a nuclear stain, we observed for stage 9–10 egg chambers four main phenotypes in different proportions for the three Prat mutant female genotypes (Figure 3; Table 4): (1) the follicular layer at stage 9–10 is disorganized and the border cell migration and localization are abnormal (Figure 3, C and D), (2) the nurse cell polyploid nuclei are pycnotic and the follicle cells migrate into the oocyte (Figure 3, E–G), (3) the cyst is disorganized such that the oocyte is not localized at the posterior pole of the egg chamber and it is not surrounded by a complete follicle cell layer (Figure 3H), and (4) the number of oocytes and nurse cells within an egg chamber is increased (Figure 3I). The proportion of the different phenotypes is presented in Table 4 for the three mutant genotypes.

View larger version (76K): In this window In a new window Download PPT slide   FIGURE 3.— Inappropriate egg chamber organization in v;Prat12A19 e/Df(3R)dsx43 e, in v;Prat16A6 e/Df(3R)dsx43 e, and in v; Prat12A19 e/Prat16A6 e. A and B show wild-type egg chambers. C, D, G, H, and I show the v;Prat16A6 e/Df(3R)dsx43 e phenotype. E and F show the v; Prat12A19 e/Prat16A6 e phenotype. Samples in A, H, and I were stained with propidium iodide and images were taken using a Zeiss 410 confocal microscope. The remaining samples were stained with DAPI and pictures were taken using a Leica epifluorescent microscope. (A) Propidium iodide staining of nuclei showing the cellular organization of a wild-type egg chamber (g, germarium; st, stage). (B) DAPI staining of a wild-type stage 10 egg chamber showing the nurse cells (thick arrow), the localization of the border cells (fine arrow), and the localization of the oocyte (short arrow). The abnormal egg chamber organization produced by Prat loss of function involves a sequential series of effects in both cell types with a variation in the distribution in function of the female genotypes (Table 4). (C and D) Mislocalization of the border cells and the movement of the follicle cells surrounding the oocyte (fine arrow). (E, F, and G) Pycnotic nurse cell nuclei associated with migration of the follicle cells surrounding the oocyte (thick arrow in E and G). Sample F is a bright field exposure of sample E. (H) Modification of the cyst organization. A nurse cell nucleus is mislocalized relative to the oocyte position (short arrow). (I) Modification of the encapsulation process, where two cysts are present within one egg chamber.

 

View this table: In this window In a new window   TABLE 4 Egg chamber phenotype distribution

 

The Prat maternal phenotype shows a decrease and desynchronization of nuclear proliferation in Drosophila embryos:

The cleavage stages in Drosophila embryogenesis take place in a syncytium in which 10 synchronous and 3 metasynchronous nuclear divisions lead to the formation of a cellular blastoderm (FOE and ALBERTS 1983). The process of nuclear division and nuclear migration is under the control of the maternal information until division 13 (EDGAR and O'FARRELL 1989) and is associated with a complex modification of the cytoskeleton (FOE et al. 2000; JI et al. 2002). The increased survival of embryos from Prat mutant females on RNA food (Table 1) suggests that the nucleotide pool size is reduced in these embryos and this reduction affects nuclear proliferation and/or cytoskeleton modification.

To explore the idea that a decrease in the purine de novo biosynthesis activity can affect the nuclear divisions, we investigated the nuclear proliferation pattern in fixed preparations of 0- to 4-hr Drosophila embryos from two Prat mutant genotypes crossed with Canton-S males on normal food, v; Prat^16A6 e/Df(3R)dsx43 e and v; Prat^16A6 e/Prat^12A19 e. We stained nuclei with DAPI and visualized them with an epifluorescence microscope.

Whereas the 10 first nuclear divisions are synchronous in wild-type embryos (FOE and ALBERTS 1983), several alterations of the pattern of division in embryos were produced by Prat^16A6 e/Df(3R)dsx43 e and Prat^16A6 e/Prat^12A19 e females that appeared after cycle 7 (Figure 4; Table 5). First, there was a high level of unfertilized embryos: 19% for Prat^16A6 e/Df(3R)dsx43 e females and 16% for Prat^16A6 e/Prat^12A19 e females. For the remaining embryos, a portion showed a normal pattern and quantity of nuclei reaching the blastoderm periphery (Table 5). However, many of the embryos showed an alteration in the nuclear division pattern. The phenotype distribution is presented in Table 5 for two mutant genotypes. When looking at the nuclei using DAPI, we first observed a delay in the loss of the polar body (Figure 4E). Whereas in our four control genotypes the polar body remained until cycle 5–6, in the mutant genotypes the polar body remained until cycle 7–8. Second, the resolution of the mitoses was abnormal and led to the presence of chromosome bridges during anaphase or telophase and some nuclei became desynchronized (compare Figure 4, F and B). Third, we observed the presence of nuclear gaps at the blastoderm periphery, and these gaps were associated with increased yolk DNA content (compare Figure 4, G and H with Figure 4, C and D). However, a large portion of the embryos (14% for both genotypes analyzed; Table 5) show an increased yolk DNA content with a normal distribution of the nuclei at the blastoderm periphery. Finally, we observed an alteration of mitotic synchrony (Figure 4, I and J). The modification was observed along either the anteroposterior or the dorsoventral axis. For the embryos showing such a defect, it is interesting to note that a group of nuclei are desynchronized relative to the remaining nuclei, which indicates the presence of a mitotic check point that can delay nuclei not yet ready to perform mitosis.

View larger version (126K): In this window In a new window Download PPT slide   FIGURE 4.— Nuclear morphology of Drosophila embryos during the different phases of the mitotic cycles. Wild-type embryos are shown at cycle 9 in prophase (A), at cycle 10 in anaphase (B), at the blastoderm stage showing the nuclear density at the periphery (C), and at blastoderm stage showing the yolk DNA density (D). Note that the nuclei are homogeneous in their morphology, indicating that they are cycling synchronously. All other embryos shown are from Prat16A6/Df(3R)dsx43 females outcrossed to Canton-S males. (E) The polar body nucleus is present at late cycle 7–8. (F) The nuclei have abnormal anaphase and telophase resolution. Some chromosome bridges and some nuclei are desynchronized. (G) The syncytial blastoderm shows a nonhomogeneous distribution of nuclei at the periphery, where a gap of nuclei is formed in the anterior part of the embryo. (H) The yolk DNA content is increased and the number of nuclei at the blastoderm periphery is decreased. (I) Nuclei are desynchronized along the anterior-posterior axis. (J) The nuclei are desynchronized along the dorsoventral axis with chromosome bridges.

 

View this table: In this window In a new window   TABLE 5 Embryo phenotype distribution

 

The Prat maternal phenotype includes segmentation pattern defects:

The establishment of the cuticle pattern occurs under the control of maternal gene products that establish the anterior-posterior axis and define the expression of three successive sets of segmentation genes (NUSSLEIN-VOLHARD and WIESCHAUS 1980). The function of these maternal gene products depends on a sequence of nuclear divisions in the early embryo. Thus, alterations in the cuticle patterns of late-stage embryos or first instar larvae reflect the modification of embryonic patterning events that take place in 0- to 4-hr embryos (NUSSLEIN-VOLHARD and WIESCHAUS 1980).

We analyzed the cuticles from late-stage embryo and first instar larval progeny of both hemizygous Prat mutant female genotypes crossed with Canton-S males. The cuticles show different alterations of segmentation with a stronger effect on late-stage embryos than on first instar larvae (Figure 5). The most common phenotype in embryos is segmental fusion covering, in the most extreme cases, all the abdominal segments or the segments from A3 to A6 (Figure 5, B–D). Other weaker segmental fusions are present in first instar larvae and they involve a modification of the segmentation around A4, with different degrees in the severity of the fusion (Figure 5G). Two additional phenotypes are less common; however, they also both involve the abdominal segment A4 (Figure 5, E and F). The first phenotype shows an absence or a decrease in the size of the segment A4 and it is misplaced along the anterior-posterior axis (Figure 5E). The second phenotype shows an alteration in the lateral part of the segment, where the cuticle is disrupted and the remaining lateral part is no longer aligned with the ventral part (Figure 5F).

View larger version (67K): In this window In a new window Download PPT slide   FIGURE 5.— Cuticle morphology of late-stage embryos (B, C, D, and E) or larval stage 1 (F and G) from Prat16A6/Df(3R)dsx43 females (anterior is at the top). (A) Late-stage embryo wild-type cuticle showing the thoracic (T1–T3) and the abdominal (A1–A8) segments. (B–D) Alteration of the segmentation varies from strong gap fusion to weaker abdominal fusion. (E) Alteration of right/left symmetry, where the abdominal segment A4 is not aligned along the anterior-posterior axis. (F) The segments are discontinuous in their lateral portion, with the most lateral portion being more anterior. (G) Weak abdominal fusion is observed in the first larval instar where the segments are connected in their most ventral region.

 

We have shown that Prat loss-of-function mutations, when hemizygous with the deficiency Df(3R)dsx43 or in a heteroallelic combination, cause a reduction in the mean and maximum life span of females. Since the life span is variable in length, Prat mutant females that die early may have, in addition to the visible purine syndrome phenotype, some nonvisible defects that arise during metamorphosis in the pupal stages. In addition, a dominant effect is associated with both Prat EMS alleles for the first half of the adult population. The mean life span is reduced 25% for Prat^16A6 e¹¹/+ and 65% for Prat^12A19 e¹¹/+, while there is no reduction in mean life span for Df(3R)dsx43 e¹¹/+ relative to Canton-S control (Figure 1, A and B). Our results suggest that the reduction in the mean life span is due to the Prat mutations rather than to second-site mutations, due to the fact that both Prat mutations were independently selected during an EMS screen for the purine syndrome (CLARK 1994). Consistent with our observations, shorter life span has been reported for mutant flies deficient in other purine de novo synthesis enzymes encoded by the ade2 (TIONG et al. 1989) and ade3 (TIONG and NASH 1990) genes.

PRAT forms an inactive tetramer and functions as a dimer in Bacillus subtilis and Escherichia coli, as determined by X-ray crystallography (SMITH et al. 1994; MUCHMORE et al. 1998) and biochemical studies (HOLMES 1981; YAMAOKA and YAMASHITA 1993). In Drosophila, we do not know if the Prat and Prat2 genes have redundant function in some cells or if they function as a heterodimer. However, we do know that both genes are expressed in the pupal stages. Both Prat^16A6 and Prat^12A19 are missense mutations that are associated with partial loss of enzyme activity in hemizygous adults (CLARK 1994). One explanation for the dominant effect we observe is that the Prat missense allele gene products are having an antimorphic effect on PRAT activity during the pupal and/or adult stages. The effect could be occurring through formation of homodimers of the Prat gene products and/or through formation of heterodimers between Prat mutant and Prat2 gene products, where they are expressed in the same cell. Interestingly, we do not see a dominant effect during oogenesis, where only Prat is expressed. There are precedents for homodimeric enzymes where dominant-negative missense mutations affect enzyme activity. For example, various purified mutant-wild-type heterodimers of E. coli biotin carboxylase have 28- to 285-fold reduced activity (JANIYANI et al. 2001). Other such examples are found with Cu/Zn superoxide dismutase (PRAMATAROVA et al. 1995) and the purine pathway enzyme inosine monophosphate dehydrogenase 1 in humans (BOWNE et al. 2002).

The aging process is a complex series of events that leads to cell apoptosis and death. Genetic screens and transgenic technology have allowed the characterization of some genes involved in the reduction and/or extension of the mean and maximum life span in Drosophila (AIGAKI et al. 2002). As well, caloric restriction (PLETCHER et al. 2002) and drug treatment, acting on histone modification (KANG et al. 2002), can increase the mean and maximum life span of Drosophila. At the cellular level, the first known phenotype produced by the aging process occurs in the early adult (ARKING et al. 2000a,b), at days 5–7, and it is associated with changes in Krebs cycle enzymes, in mitochondrial electron transport chain activity, and in the resistance to various stresses and a decrease in reactive oxygen species (ROS) production (ARKING et al. 2000a,b; BHASKAR et al. 2002; KIRBY et al. 2002; MANDAVILLI et al. 2002). Hypotheses of aging predict that an increased production of ROS will cause a decrease in energy production and an accumulation of mtDNA damage in postmitotic cells (ARKING et al. 2002; BHASKAR et al. 2002), leading to a general decrease in cell stability. It will be of interest to investigate whether the premature aging we observe for Prat mutant heterozygous and hemizygous adults is associated with a change in stress resistance and ROS production.

The Prat-specific mRNA and protein expression in the female germline (Figure 2) and Prat mRNA expression in 0- to 2-hr embryos (MALMANCHE et al. 2003) motivated us to study the developmental effects of a decrease in purine de novo synthesis in Drosophila oogenesis and embryogenesis, a well-known model system for studying cell proliferation and differentiation in animals. We found that Prat loss-of-function causes a semisterile phenotype in which (1) some egg chambers show abnormal patterns of cell organization in the somatic and germline tissues and (2) some of the progeny die at the embryonic and first larval stages.

In the ovaries, Prat is first expressed in the germarium. In these regions, the formation of the cyst by incomplete cell divisions produces the 15 nurse cells and oocytes localized at the posterior pole of the newly formed egg chamber (VAN EEDEN and ST JOHNSTON 1999). In region 3, an induction from the cyst to the somatic layer allows the formation of a stage 1 egg chamber, in which the somatic cells migrate to surround and isolate individual cysts. The encapsulation of the cyst leads to the definition of three somatic cell types by the activity of the Delta, Notch, and fringe genes: the polar cells, the stalk cells, and the epithelial follicle cells (GRAMMONT and IRVINE 2001; LOPEZ-SCHIER and ST JOHNSTON 2001). In general, Prat mutant ovarioles have phenotypes that involve proliferation and migration of both the germline and the somatic cells. The phenotypes produced indicate that purine de novo biosynthesis, controlled by PRAT activity, somehow influences cell replication, division, migration, and differentiation and they lead us to speculate that Prat is a potential target gene of the Notch and Delta pathway during oogenesis.

The Prat expression we observed in stage 10 egg chambers leads to the accumulation of Prat mRNA in the oocyte and 0- to 2-hr embryos. We analyzed the embryonic phenotype caused by a reduction in purine de novo biosynthesis by examining the progeny of three genotypes of Prat mutant females crossed to wild-type males. In this way, we could examine the maternal contribution to the phenotype independently from the zygotic contribution. For the three genotypes, the lethal phase is pleiotropic, from early embryo to adult. For the group of progeny with developmental arrest at the embryonic stage, we found most of the 0- to 4-hr embryos showed defects in the process of nuclear division and nuclear migration. We speculate that some early defects in nuclear division lead to the segmental defects we observed in late-stage embryos and first instar larvae. These defects ranged from a full segmental fusion to a weaker cuticle phenotype associated with abdominal segment A4.

The first stages of development occur in a syncytium in which 13 nuclear divisions take place before the induction of cellularization (FOE and ALBERTS 1983). These divisions have specific features: (1) a rapid succession of S and M phases, with a loss of some cell cycle check points, (2) rapid DNA replication, and (3) a dynamic interaction between the phases of the mitotic cycle and the cytoskeleton (FOE et al. 2000; KOLONIN and FINLEY 2000; JI et al. 2002). During these events, the purines available must be limited to the nucleotide pool and to the potential activity of pathway enzymes dumped into the oocyte by the nurse cells. In our model, the decrease in purine de novo synthesis causes an alteration in the timing and synchrony of the nuclear divisions and in the quantity of nuclei reaching the blastoderm membrane, through a reduction in efficiency of DNA replication and the energy-requiring processes associated with cytoskeleton organization, respectively. Despite numerous studies on mammalian cell lines that have linked purine pathway activity to cell proliferation, none has determined whether the pool of purine nucleotides produced by the pathway is limiting for DNA replication and/or the rapid modification in the cytoskeleton network. We suggest that the use of RNA interference technologies and Drosophila Schneider cells, combined with manipulation of defined growth medium components, will allow us to dissect the complex interactions between regulation of purine nucleotide pools and the cell cycle in a simpler system. The results from such work will provide us with a basis for further analysis of the cell cycle in Prat maternal mutant Drosophila embryos, where we can address the role of the nucleotide pool present during the rapid nuclear divisions of early embryogenesis.

We thank Steven Heard, Myriam Barbeau, Andre Breton, and Michelle Gray for advice on statistical analyses; Kendra Corey and Ryan MacDonald for assistance with the aging experiments; Nancy MacAfee for antibody purification; Ping Lee for the confocal; and Claudio Sunkel, Jean Francois Colas, and Jay Penney for their comments on the manuscript. This work was funded by a Canadian Institutes of Health Research/New Brunswick Institute of Health Research grant to D.V.C.

¹ Present address: Instituto de Biologia Molecular e Celular, Laboratorio de Genetica Molecular da Mitose, Universidade do Porto, 4150-080 Porto, Portugal.

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