A Maternal Product of the Punch Locus of Drosophila Melanogaster Is Required for Precellular Blastoderm Nuclear Divisions

Journal of Cell Science 107, 3501-3513 (1994) 3501 Printed in Great Britain © The Company of Biologists Limited 1994

A maternal product of the Punch locus of Drosophila melanogaster is required for precellular blastoderm nuclear divisions

Xiongying Chen1, Elaine R. Reynolds2,*, Gogineni Ranganayakulu1,† and Janis M. O’Donnell1,‡ 1Department of Biological Sciences, University of Alabama, Tuscaloosa, AL 35487, USA 2Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA *Present address: 201 Wellman Hall, ESPM, Division of Entomology, University of California, Berkeley, CA 94720, USA †Present address: Department of Biochemistry and Molecular Biology-117, MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA ‡Author for correspondence

SUMMARY

The Punch locus of Drosophila melanogaster encodes the dine, at a very early cleavage stage. Furthermore, an pteridine biosynthesis enzyme guanosine triphosphate inhibitor of a terminal step in pteridine biosynthesis cyclohydrolase. One class of Punch mutants is defective for produced an identical phenotype. Immunolocalization a maternal function that results in embryonic death. We experiments deﬁne expression of Punch protein in nurse demonstrate here that the embryos exhibit nuclear division cells during oogenesis. The protein is packaged into defects during the precellular blastoderm stage of develop- granules as it is transported into the oocyte cytoplasm. As ment. These defects include abnormal nuclear distribution, syncytial blastoderm nuclear divisions proceed, Punch mitotic asynchrony, and persisting chromatin bridges. protein levels decrease and disappear by cellularization. Daughter nuclei that do not complete chromosome separa- Defects in the expression of the protein in Punch maternal tion nevertheless initiate new interphase and mitotic cycles. effect mutants correlate well with the early phenotypes. As a result, interconnected mitotic ﬁgures are observed. These results show that a Punch product is directly Mitotic spindles and nuclear envelopes appear essentially involved in early nuclear divisions and suggest a possible normal. A mutant phenocopy was induced in wild-type role in chromosome separation. embryos by treatment with the guanosine triphosphate cyclohydrolase inhibitor, 2,4-diamino-6-hydroxypyrimi- Key words: Punch, GTP cyclohydrolase, pteridine, nuclear division

INTRODUCTION More than 100 loci, as identified by mutations, have been found to be involved in the cell cycle in Drosophila (for review Early embryogenesis in Drosophila is unusual with respect to see Glover, 1989; Foe et al., 1993). Some zygotic mitotic the rapidity of nuclear divisions and the absence of cytokine- mutants show no effects until the larval or pupal stage of devel- sis. The embryo is initially a syncytium in which there are 13 opment. One example is the mus-101 mutant, which has the rounds of synchronous nuclear divisions without cytokinesis mitotic phenotype of defects in condensation of heterochro- over a period of about two hours. Fertilization is followed by matin (Gatti and Baker, 1989). Other mutations, for example nine nuclear divisions in the center of the embryos at approx- in cyclin A (Lehner and O’Farrell, 1989) or string (Edgar and imately 10 minute intervals. The majority of the nuclei migrate O’Farrell, 1989), show a mitotic phenotype in the embryo to the periphery of the embryo during cycles 8 and 9, where when the cell cycle lengthens after cellularization, a time at they form an evenly spaced monolayer just below the plasma which their zygotic expression is normally initiated. Mutations membrane. The nuclei in this monolayer undergo four rounds disrupting these general mitotic genes have both maternal and of division and then become synchronously cellularized by zygotic lethal effects. Another group of mutants involved invaginations of the plasma membrane to form the cellular specifically in embryonic mitosis have strictly maternal effects. blastoderm, at which stage there are about 5,000 nuclei These include abnormal chromatin (Vessey et al., 1991), (Zalokar and Erk, 1976; Foe and Alberts, 1983; Karr and which is required only for nuclear cycles in the syncytial Alberts, 1986). Following cellularization, there are three addi- embryo, and gnu (Freeman et al., 1986; Freeman and Glover, tional mitotic cycles during gastrulation and germ band 1987), which leads to giant nuclei due to failure to suppress elongation; these are asynchronous and occur in spatially and DNA replication in the unfertilized egg. temporally regulated domains (Foe, 1989). After these three One class of maternal effect alleles of the Punch (Pu) locus, mitotic cycles, most cells in the embryo cease division five to a genetically and molecularly complex gene (Mackay and six hours after fertilization and shift to an endoreplication O’Donnell, 1983; Mackay et al., 1985), is defective for a pattern (Smith and Orr-Weaver, 1991). maternal function, resulting in death, much of which occurs

3502 X. Chen and others during the precellular blastoderm stage (Reynolds and envelope labeling, mouse monoclonal antibody T47 against lamin O’Donnell, 1987). Pu encodes GTP cyclohydrolase (GTPCH), (generated by H. Saumweber and given to us by E. Stephenson) was which catalyzes the first step in pteridine biosynthesis (Mackay used at a 1:50 dilution. Microtubules were labeled with a mouse mon- and O’Donnell, 1983). One of the major products of the oclonal antibody against β-tubulin (N 357, Amersham Corp.), used at pteridine pathway is the regulatory cofactor tetrahydro- a 1:250 dilution. Both mouse monoclonal antibodies were visualized biopterin (BH ). BH has well-characterized roles in neuro- with a 1:300 dilution of fluorescein-conjugated goat anti-mouse 4 4 antibody (Jackson Immunoresearch Labs). The level of background transmitter expression, where it is essential to, and in many was determined by incubating embryos in secondary antibody alone. cases rate-limiting for, the activities of the aromatic amino acid Labeling of nuclei was carried out using 0.5 µg/ml of 4,6-diamidino- hydroxylases and nitric oxide synthases (Nagatsu et al., 1964; 2-phenylindole (DAPI). Lovenberg et al., 1967; Tayeh and Marletta, 1989; Kwon et al., 1990; Mayer et al., 1991). Less well-understood are pteridine Application of enzyme inhibitors functions in proliferative diseases in humans where pteridine Wild-type female flies were allowed to lay eggs on agar plates for 20 expression is massively stimulated (Brew et al., 1990; Shaskan minutes at 25¡C, after a 90 minute pre-lay collection was discarded. et al., 1992) and in cytokine-stimulated proliferation of Appropriately staged embryos were manually dechorionated or the chorion was removed with 2.6% sodium hypochlorite. Dechorionated erythroid, astrocyte and macrophage cell lines in which BH4 production is required (Tanaka et al., 1989; Milstein et al., embryos were transferred to a siliconized depression slide, permeabilized with octane for 20 seconds, washed and incubated in a drop of 1990; Werner-Felmayer et al., 1993). The maternal effect an incubation medium (Limbourg and Zalokar, 1973) containing alleles of Pu affect GTPCH activity, suggesting that the various concentrations of the GTPCH inhibitor, 2,4-diamino-6- resulting lethality is due to a deficit in pteridine production; hydroxypyrimidine (DHAP, Sigma) or the sepiapterin reductase however, the physiological basis for the effects is not clear. In inhibitor, N-acetyl serotonin (NAS, Sigma) for 5 minutes (Katoh et order to explore the source of these defects, we have charac- al., 1982; Bräutigam et al., 1984). DHAP was dissolved in 0.01 mM terized the embryonic abnormalities associated with the Pu ascorbic acid to a concentration of 0.2 M, and NAS was dissolved in maternal effect. We also correlate these phenotypes with the water to a concentration of 10 mM. Both stock solutions were diluted temporal and spatial expression of Pu products in wild-type in incubation medium to make working solutions of the required con- and mutant ovaries and early embryos. We further demon- centrations. Embryos developed normally in control experiments, in strate, by using inhibitors of pteridine biosynthesis enzymes, which they were incubated only with incubation medium or incubation medium containing ascorbic acid without inhibitor. After removal that the mutant phenotypes are the direct result of disruption of the medium, the embryos were washed several times with incuba- of pteridine expression. Our results show that Punch has a role tion medium minus inhibitors and then covered with a drop of halo- in early embryonic nuclear division, affecting a function carbon oil. Slides were kept in moist chambers at 25¡C to allow the required for appropriate chromosome separation. embryos to develop to the required stage. After incubation, the embryos were handled as described above. Production of anti-Pu antibodies MATERIALS AND METHODS A cDNA fragment containing exon 2 to the EcoRI site in exon 6 (Fig. 1A) and representing most of the coding region of a 1.75 kb Pu tran- Strains and culture conditions script (O’Donnell et al., 1993; McLean et al., 1993) was cloned in All strains were maintained on standard medium at 25¡C unless frame, into the pET 3c vector and expressed in bacteria (Studier et otherwise noted. All mutations are described by Lindsley and Zimm al., 1990). Exon 2 is incorporated into a subset of Pu transcripts, while (1992). Further information on the Pu mutant strains is given by exons 3-6 are shared by all Pu transcripts characterized to date Mackay and O’Donnell (1983); Mackay et al. (1985); Reynolds and (O’Donnell et al., 1993). Rabbit antiserum was raised against gel- O’Donnell (1988); O’Donnell et al. (1989a). All homozygous lethal purified, expressed Pu product. The antiserum was affinity-purified mutations were maintained balanced against the SM1 or SM5 chro- by passage through a cyanogen bromide-activated Sepharose column mosome. by standard procedures. Western blotting was conducted to ascertain the specificity of the antibodies. The bacterial expression product from Immunofluorescence the cDNA described above and protein extracts from 0 to 2-hour Eggs were collected and aged to the desired stage at 25¡C. Embryos Canton-S embryos were separated by 12% SDS-PAGE and trans- were dechorionated and fixed as described by Patel et al. (1987). For ferred to nitrocellulose by standard methods. The filter was incubated labeling with anti-tubulin antibodies, fixation was performed in the with a 1:300 dilution of the affinity-purified antiserum, followed by presence of taxol (Sigma) as described by Karr and Alberts (1986) 1:500 dilution of peroxidase-conjugated goat anti-rabbit antibodies. with slight modifications, as follows. All procedures were carried out Bands were detected by chemiluminescence (DuPont). Fig. 1B shows at room temperature. Following dechorionation, embryos were trans- that the antibody detects a single fusion protein of the expected size ferred to a glass vial containing 5 ml of PEM buffer (0.1 M PIPES, (43 kDa) in the bacterial extract and a 52 kDa protein in 0-2 hour 1 mM EGTA, 2 mM MgSO4, pH 7.0). A 10 µl sample of 0.5 mM wild-type embryos. That this protein is in fact a product of the Pu taxol was added and the vial was shaken gently for 2 minutes. Then, locus was verified by detecting reduced proteins in western blots of 5 ml heptane was added, and the vial was shaken vigorously for 30 extracts from several Pu mutants. Details of this characterization will seconds. Fixation was done by adding 1 ml of 20% formaldehyde, be reported elsewhere; however, the reduction of cross-reacting followed by vigorous agitation for 20 minutes. The aqueous layer was materials in PuWE75 embryos described in this paper verifies the then removed, an equal volume of 90% methanol, 10% water, and specifity of the antibodies used in the present studies. 0.05 M EGTA was added, again followed by vigorous shaking for 1 minute. Embryos that had lost their vitelline membranes sank to the Localization of protein bottom of the vial and were rinsed twice in methanol. These embryos Fixation of embryos for the whole-mount immunolocalization of the were then rehydrated and washed in PBT (1× PBS, 0.2% BSA, 0.1% Pu product was performed as described above. Preparation of ovaries Triton X-100). Incubation with primary and secondary antibodies was was carried out as described by Patel et al. (1987). For localization of carried out according to the methods of Patel et al. (1987). For nuclear the protein in ovaries, ovarioles were teased apart in PBS and fixed

Punch maternal function 3503

A 0 +4 +8 structure, embryos were ﬁxed with glutaraldehyde and osmium S R R S B H R H R R tetroxide, and embedded in Spurr’s low-viscosity epoxy resin (Spurr, 1969). The sections were stained with uranyl acetate and lead citrate.

1 2 3 4 5 6

a b c d e RESULTS

The genetics of Punch maternal effect mutants kDa Fig. 1. Molecular map of a portion of the This report concerns a class of Pu mutations affecting early Punch region and specificity of the anti-Pu embryonic function, represented by three ethylmethanesul- antibody. (A) Two alternate forms of fonate-induced alleles, PuWE75, PuWE67 and PuK8-2 (Reynolds Punch adult transcripts are shown below the line designating genomic restriction and O’Donnell, 1987). We previously demonstrated that the enzyme recognition sites. Protein used to alleles primarily affect a maternal function and act in a semi- generate anti-Pu antibodies was obtained dominant fashion. They do not affect the later zygotic function by expression of a cDNA containing exons of catecholamine production or the adult function of eye 2-6, cloned into the pET 3c vector. pigment production. Two of the alleles, PuWE67 and PuK8-2, are (B) Western blot testing the specificity of homozygous lethal. Approximately one-half of all inviable antibody generated against the bacterially embryos die prior to the cellular blastoderm stage, while the expressed Pu product. Lane 1 contains 20 remaining half die later in embryogenesis. PuWE75, on the other µ g of extracts of bacterially expressed hand, is homozygous viable and the mutant effects appear to protein. The single band at 43 kDa is the be restricted to precellular blastoderm development. The size predicted for the translation product K8-2 WE75 of the expressed cDNA. Lane 2 contains Pu /Pu heteroallelic combination is viable at about 30 µg of crude protein extract from 0-2 10% of expected levels. The survivors also exhibit a maternal WE75 hour Canton S embryos. The antibody effect, similar to, but stronger than Pu . The following phe- detects a single protein of 52 kDa. The position of molecular mass notypic analyses concern embryos derived from PuWE67/Pu+, markers are shown to the left of the lanes. PuK8-2/Pu+, PuK8-2/PuWE75 and homozygous PuWE75 mothers. Nuclear phenotypes of precellular blastoderm Pu prior to reaction with the antisera. The primary antiserum was diluted embryos to 1:50 in PBT and incubated overnight at 4¡C. A peroxidase-coupled As an initial step in the determination of the basis for the early goat anti-rabbit secondary antibody (Jackson Immunoresearch) was developmental arrest of the Pu maternal effect mutants, the used at a dilution of 1:500. For double staining embryo with anti-Pu progeny of PuWE75 homozygous and PuWE75/PuK8-2 and protein antibody and DAPI, the antibody was used at 1:50 dilution PuWE67/Pu+ heterozygous mothers were examined by DAPI and secondary antibody conjugated with fluorescein was used at staining; all have similar phenotypes. Most embryos of all 1:300, followed by DAPI staining. In all cases, pre-immune serum mutant genotypes exhibit some degree of nuclear division and secondary antibody alone were used as controls for the specificity of antibody reactions. abnormality. The most severely affected embryos in each case die prior to formation of the cellular blastoderm. The range is Immunoelectron microscopy from approximately 70% of PuWE75/PuK8-2 embryos to about WE67 + Dechorionated 0-1 hour embryos were shaken in a mixture of 8 ml 15% of Pu /Pu embryos. In normal precellular blastoderm heptane and 2 ml 25% glutaraldehyde (Electron Microscopy Science) development, the nuclei divide synchronously and are distrib- in 0.1 M sodium cacodylate buffer, pH 7.2, for 10 minutes. Embryos uted evenly throughout the embryo (Fig. 2A,C). In the mutant were transferred to double stick tape and were covered with 2% glu- embryos, the distribution of nuclei within syncytial blastoderm taraldehyde in 0.1 M cacodylate buffer (fix A). The vitelline embryos is irregular (Fig. 2B,D). The uneven distribution of membrane was ruptured with a glass microelectrode and embryos nuclei results in a much lower nuclear density in some areas were fixed for another 20 minutes in fix A. After several washes in than is appropriate for a given stage. In addition, the mitotic 0.1 M cacodylate buffer, the embryos were dehydrated sequentially cycles of cleavage stage nuclei, from the earliest divisions, are in 50%, 75% and 90% dimethylformamide (DMF). The embryos were infiltrated with Lowicryl K4M (Polyscience): DMF and embedded in asynchronous, and all stages of the cell cycle are often repre- Lowicryl K4M (Altman et al., 1984). Sections were cut and immuno- sented simultaneously (Fig. 2D). Chromosomes initiate con- stained as follows: sections were incubated in blocking buffer (2% densation as they enter prophase, and they align on the normal goat serum, 1% BSA, 0.3% Triton X-100 in PBS) for 30 metaphase plate appropriately. However, throughout mitosis, minutes and with affinity-purified primary antibody diluted 1:100 in the chromosomes are rarely as discrete as in wild type, often blocking buffer overnight at 4¡C. For negative controls, sections appearing abnormally diffuse (Figs 2D, 3, 4). Chromatin received similar treatment except that the primary antibody was bridges between daughter nuclei are often observed, initially omitted. Sections were rinsed twice in blocking buffer for 2 minutes during anaphase and then persisting to later stages (Figs 2D, each, once in PBS, and incubated in the blocking buffer with goat 3C, 4G,K). Fragmented nuclei are occasionally observed (data anti-rabbit IgG conjugated with 10 nm colloidal gold (Sigma) for 1.5 not shown). hours. Grids were rinsed as above, then three times for 1 minute each with water. Sections were then post-stained with 2% aqueous uranyl Because some mutant embryos survive beyond the cellular- acetate and lead citrate for 10 minutes each, and examined and pho- ization stage during blastoderm formation, we examined the tographed with a Zeiss 10-A transmission electron microscope (Carl nuclear phenotypes of Pu embryos throughout embryogenesis. Zeiss) operated at 60 kV. When embryos were collected for 30 minute intervals and For transmission electron microscopic examination of granule allowed to develop at 25¡C for a total of 12 or 24 hours, nuclear 3504 X. Chen and others

Fig. 2. Embryonic DNA stained with DAPI during precellular blastoderm nuclear division in wild-type and Pu mutant embryos. (A) A wild- type precellular blastoderm embryo at nuclear cycle 10. Synchronous nuclei are evenly distributed throughout the embryo. (B) A PuWE67 embryo at approximately the same stage of development as the embryo in A. Nuclear divisions are asynchronous and the spatial organization of the nuclei is abnormal. (C) A high magnification view from a wild-type embryo at interphase of nuclear division cycle 11. (D) A PuWE67 embryo at approximately the same stage as the embryo in C. Nuclei are irregularly spaced, and all stages of mitosis can be observed in a single field. Chromatin bridges at anaphase and at telophase are indicated by arrows. i, interphase; p, prophase; m, metaphase; a, anaphase; t, telophase. Bar, 10 µm. division arrests were observed only in the precellular blasto- observe the mitotic spindle. The metaphase chromosomes and derm stage. Embryos surviving beyond this point show no spindles seen in Fig. 4A and B and the anaphase chromosomes further division arrests during the remaining three post-cellu- and spindle seen in Fig. 4E and F illustrate the appearance of larization nuclear cycles, but rather die after post-germ-band dividing nuclei in wild-type embryos. In Pu mutant embryos, shortening stages. We have not yet determined whether death the morphology of the spindle appears reasonably normal at during the later stages of embryogenesis is the result of early metaphase (Fig. 4D), at anaphase (Fig. 4H) and at telophase defects arising from the maternal effects or of a defect in a (Fig. 4L). These observations suggest that there are no major zygotic function. abnormalities in spindle component structure or function. However, we do observe extensive chromatin bridging and Nuclear envelope and microtubule structure in Pu chromatin remaining at the metaphase plate, initially at embryos anaphase and persisting into telophase (Fig. 4G,K). We also The cellular basis of the nuclear phenotypes observed after note that the chromatin at all stages of mitosis is less fully DAPI staining of Pu embryos is not clear. As the first step in condensed than it is in wild-type mitotic figures. Compare, for the analysis of the affected function, we examined other instance, late interphase chromatin in Fig. 3A and C, cellular components important in nuclear division. Pu and metaphase chromatin in Fig. 4A and C, and the anaphase wild-type embryos were double-stained with DAPI and anti- chromatin in Fig. 4E and G. Surprisingly, daughter nuclei that lamin antibody to detect nuclear envelopes. Apparently normal do not complete chromatin separation at the previous telophase nuclear envelopes were detected around all interphase nuclei still go on to initiate a new nuclear division cycle. The mitotic in Pu mutants (Fig. 3). However, the nuclei are variable in size spindles of adjacent sister nuclei at metaphase remain and often irregular in shape; the formation of the nuclear connected by chromatin bridges (Fig. 4C,D). Although we envelope mirrors these defects. We conclude that the nuclear observe high proportions of interconnected metaphase figures envelope formation per se is normal, with shape and size alter- in mutant embryos, we never observe chromatin-bridged ations depending upon the chromatin structure of each nucleus. figures that have moved into anaphase. It appears that division We next compared wild-type embryos with Pu mutant arrest generally occurs at this point. In some instances, we embryos for the distribution of tubulin during the cleavage observe multiple spindles arising from a common pole and stage. Fig. 4 shows the results of double staining embryos with some half-spindles (data not shown). However, the majority of DAPI to visualize DNA and an anti-β-tubulin antibody to spindles in the mutant embryos appear to be appropriately Punch maternal function 3505

Fig. 3. Nuclear envelope structure in wild-type and Pu embryos. Wild-type and Pu embryos were double-stained for DNA and lamin as described in Materials and Methods. Shown are high magnification views of the nuclei during late interphase of nuclear cycle 11. (A) Wild- type embryo, DAPI stain. (B) Wild-type embryo, lamin stain, same field as in A. (C) Pu embryo, DAPI stain. Nuclei are unevenly distributed and chromatin bridges can be observed (arrows). (D) Pu embryo, same field as in C, lamin stain. The nuclear envelope shape and size are correlated with the nucleus. The arrows indicate the frequently observed interconnections between nuclei. Bar, 10 µm. bipolar. Therefore, we infer that the multipolar phenotypes tal abnormalities, all experiments included mock-treated arise secondarily as a consequence of the incomplete separa- control embryos that were subjected to an identical regimen tion and abnormal spacing of nuclei. except that inhibitor was omitted. In early experiments, all embryos were hand-dechorionated prior to treatment. Induction of Pu phenocopies with inhibitors of However, we have since ascertained that chemically dechori- pteridine biosynthesis onated embryos respond identically to those dechorionated by The nuclear division phenotype of Pu mutant embryos could hand, and all later experiments were then performed using arise as a direct result of loss of GTPCH activity and pteridine chemical dechorionation. production, as a direct effect of a protein product of this Embryonic development was found to be affected in a con- complex locus with unknown functions, or as a secondary centration-dependent manner, with DHAP concentrations effect of metabolic disturbance in the oocyte. In order to between 10−3 M and 10−1 M (data not shown). Lower concen- address this issue, we examined the effects of a GTPCH trations had a minimal effect, and normal larvae hatched after inhibitor, 2,4-diamino-6-hydroxypyrimidine (DHAP), on exposure of the embryos to 10−4 M DHAP. These concentra- nuclear division in wild-type embryos. Briefly, dechorionated tions, although high due to the poor permeability of this early embryos were lightly permeabilized with a 20 second compound in Drosophila embryos, are consistent with the con- exposure to octane, followed by a 5 minute exposure to the centration of this inhibitor that disrupts proliferation of enzyme inhibitor. After a period of time ranging from 30 mammalian tissue culture lines (Tanaka et al., 1989). The minutes to several hours, the embryos were fixed and stained remaining experiments were conducted using 10−1 M DHAP with DAPI and anti-β-tubulin antibody. Because these manip- in the incubation medium. ulations have the potential to cause non-specific developmen- Treated embryos exhibited a single set of phenotypes. These 3506 X. Chen and others

Fig. 4. Tubulin and DNA double-stained nuclei. Wild- type control, Pu mutant, and DHAP-treated embryos were stained with DAPI (A,C,E,G,I and K) and with the anti-β-tubulin antibody (B,D,F,H,J and L). (A) Wild- type nuclei at metaphase. (B) Same field as in A, showing the spindle structure corresponding with the mitotic nuclei. (C) PuWE67 sister nuclei at metaphase connected by chromatin bridges. (D) Same field as in C. Spindles at metaphase are physically connected. (E) Wild-type nucleus at anaphase. (F) Same field as in C. (G) PuWE67 nucleus at anaphase, connected by chromatin bridges. (H) The spindle (s) corresponding to the anaphase nucleus seen in G is indicated. (I) Anaphase nucleus from wild-type embryo treated with the GTP cyclohydrolase inhibitor, DHAP (0.1 M). Chromatin bridges are similar to those of Pu mutant embryos. (J) Spindles associated with the nucleus shown in I. (K) PuWE67 nucleus at telophase, showing a persisting chromatin bridge. (L) Same field as in K, showing the corresponding spindle with mid-body. Bar, 5 µm. consisted of asynchronous nuclear division cycles, uneven embryos exhibiting the nuclear defects to mock-treated control nuclear distribution and chromatin bridges between daughter embryos. For this comparison, we set a minimum criterion for nuclei. In all respects, the appearance of these nuclei was indis- abnormality as defective nuclear spacing over at least 10-15% tinguishable from those of the Pu maternal effect mutant of the surface of the embryo. Table 1 shows the results of one embryos (Fig. 5C,D,G,H) and distinct from the nuclear mor- such series of experiments. Only 12 of the 139 mock-treated phology of the mock-treated embryos (Fig. 5A,B). Further- embryos exhibited any nuclear defects compared to 114 of 212 more, chromatin bridge morphology of mutant and inhibitor- DHAP-treated embryos. Previous experiments indicated that treated mitotic figures was similar at anaphase and telophase embryos at cellular blastoderm stage or beyond are refractory (Figs 4G,I and 5D). Although spindle morphology varies with to inhibitor treatment. When we corrected the results to the degree of aster preservation among preparations, we account for the proportion of embryos at the sensitive stages observed no obvious spindle abnormalities (Fig. 4J). While when treatment was initiated, we observed nuclear defects in inhibitor-treated embryos show defects that are apparently 77% of the inhibitor-treated embryos. Defects were seen in more severe than those of the mutant embryos, this difference only 12% of the mock-treated embryos. It is clear that these is likely to be related to variation in residual wild-type results are significantly different (P<0.001, two-tailed Fisher’s function. As in the case of the Pu maternal mutant embryos, exact test). However, the difference between inhibitor-treated those embryos that survived past the cellularization stage after and control embryos is even greater than is apparent from the DHAP treatment exhibited no obvious defects in nuclear mor- data in Table 1. Most of the control embryos that were scored phology. as abnormal had only minimal defects in nuclear spacing We also compared the proportion of inhibitor-treated and no other apparent defects. Asynchronous division and Punch maternal function 3507

Fig. 5. Effects of GTP cyclohydrolase and sepiapterin reductase inhibitors on nuclear division. (A and B) Two magnifications of a mock-treated wild-type embryo at division cycle 12. Synchronous nuclei are evenly distributed. (C and D) Two magnifications of a division cycle 11 wild- type embryo treated with 0.1 M DHAP within 30 minutes of egg deposition. Asynchronous nuclei are unevenly distributed; chromatin bridges are often present at anaphase and telophase. These are indicated by arrows in D. (E and F) Two magnifications of a division cycle 11 wild-type embryo treated with 1 mM NAS within 30 minutes of egg deposition. Arrows in F show chromatin bridges between daughter nuclei at telophase. (G and H) Two magnifications of PuWE67 embryo at division cycle 11, with arrows in H indicating chromatin bridges. Cycles of mutant and inhibitor-treated embryos were estimated on the basis of nuclear density within regions in which spacing of the nuclei was approximately normal. Bars: 50 µm (A,C,E,G); 10 µm (B,D,F,H). 3508 X. Chen and others

Table 1. Effect of inhibitors of pteridine biosynthesis on embryonic development Control DHAP NAS No. emb. Corrected %* No. emb. Corrected %* No. emb. Corrected %* Total 139 212 208 Nuclear defects 12 12 114 77 98 67

*Late blastoderm and post-blastoderm embryos are refractory to inhibitor treatment. The percentage of embryos affected by the treatments has been corrected for the proportion of embryos in precellular blastoderm stages at the initiation of the experiments. chromatin bridging were very rare, occurring in only two and transcripts, with certain exceptions that will be noted embryos. In contrast, virtually all embryos scored as defective below. Since the patterns are virtually identical, only the after DHAP treatment exhibited the full spectrum of nuclear protein data will be presented here. division defects. In sum, these results suggest that GTPCH is Pu protein appears initially in the germarium and persists at indeed involved in a nuclear division function during early low levels to stage 6-7 of oogenesis (data not shown). In stage Drosophila development. 10a, a second phase of expression occurs and by stage 10b the Because GTPCH catalyzes the first step in tetrahydro- protein signal is intense in the nurse cell cytoplasm (Fig. 6A). biopterin (BH4) synthesis, we next asked whether GTPCH per se, perhaps in its capacity as a GTP-binding protein, or its final product, BH4, is involved in early nuclear division. We reasoned that if BH4 synthesis were the critical function, then A B inhibition of any other enzymatic step in its biosynthesis would also produce the nuclear division phenotype. If the defect were dependent on the GTPCH protein, but not its ability to direct BH4 synthesis, then inhibition of BH4 production at a later step in the pathway should have no effect on the ability of the embryos to execute nuclear divisions. We consequently examined the effect of N-acetylserotonin (NAS), a potent inhibitor of sepiapterin reductase (Katoh et al., 1982), the terminal enzyme in BH4 biosynthesis. These experiments were performed identically to the previous inhibitor studies, except that the optimal concentration for NAS was 1 mM. The result of NAS treatment was a phenotype that was virtually indistinguishable from those of Pu mutants or DHAP-treated wild-type embryos, with respect to both nuclear (Fig. 5E,F) and spindle morphology (data not shown). Ninety-eight out of 208 NAS- CD treated embryos exhibited these defects. After correction of the data as above, 67% of susceptible (i.e. precellular blastoderm) embryos were affected. This effect is significantly different from that in the control (P<0.001). By contrast, the effects of DHAP and NAS were not significantly different from each other (P>0.025). We therefore conclude that BH4 itself is involved in these early nuclear divisions and that it is this function of the Pu locus that is disrupted in Pu maternal effect mutants. Localization of Pu protein in developing egg chambers Previous molecular characterization of the Punch locus has shown that Pu transcripts and protein are expressed during oogenesis and early embryogenesis (McLean et al., 1990, 1993; O’Donnell et al., 1993; and X. Chen, S. Krishnakumar Fig. 6. Spatial patterns of Punch protein expression during and J. O’Donnell, unpublished data). However, it is not known oogenesis. (A) Stage 10b egg chamber from wild-type female showing accumulation of Pu protein in the nurse cell cytoplasm. whether any of these products were expressed in a manner cor- WE75 responding to the functions suggested by phenotypic analysis. (B) Stage 10b egg chamber from homozygous Pu female. By stage 10b, some Punch protein has begun to move into the oocyte Experiments to localize Pu protein in egg chambers were cytoplasm and much of it is abnormally localized to the posterior conducted using polyclonal antibodies that recognize specific pole of the oocyte (note arrow). (C) Wild-type egg chamber at stage isoforms of the Pu product containing domains encoded by 14. The protein has been transferred to the oocyte cytoplasm and exons 2-6 (Fig. 1). Parallel in situ hybridization experiments packaged into granules. (D) PuWE75 egg chamber at stage 14. Protein were conducted using probes from the corresponding regions. is seen in the mutant oocyte, but fewer granules are formed. Bar The results show similar expression patterns for Pu proteins 100 µm. Punch maternal function 3509

The protein, but not Pu transcripts, moves into the oocyte into granules as it does so, but the protein granules are fewer in during the bulk dumping of nurse cell cytoplasm that begins number than in wild type and are not distributed uniformly. in stage 11. After the Pu product moves into the oocyte, it is Rather, they and unpackaged protein pass on to the posterior packaged into granules, which are uniformly distributed pole where some of the mutant protein usually resides through- throughout the egg cytoplasm (Fig. 6C). These granules persist out the remainder of oogenesis (Fig. 6B,D). It appears that some into the precellular blastoderm stage (see description of of this protein is also degraded prior to fertilization. We do not embryonic expression below). yet know the structural basis for the mislocalization of the We examined the patterns of Pu expression in the ovaries of protein, but we can infer that its aberrant behavior is not the females homozygous for PuWE75 and for females tran- result of general disruptions in cytoplasmic organization, since sheterozygous for the WE75 and K8-2 alleles. These genotypes both bcd transcripts (unpublished observations; Berleth et al., result in identical phenotypes. In situ hybridization to the 1988) and the Pu transcript itself are appropriately localized. mutant ovaries showed that a signiﬁcant amount of the Pu tran- Analysis of the mutant protein should provide information script was present in both the early and later expression periods relevant to the understanding of the localization mechanisms. (data not shown). In the early stages of oogenesis, no difference in the expression of Pu protein between mutant and wild-type Localization of Pu protein in precellular blastoderm ovaries could be distinguished. In stage 10a, however, marked embryos differences in the protein expression pattern are seen in many In early embryos the protein is retained in the granules that egg chambers, and these differences persist through the form in the oocyte; they are dispersed uniformly throughout remainder of oogenesis. The severity of the defects varied, but the cytoplasm (Fig. 7A). Ultrastructural immunolocalization the most extreme phenotypes were observed in approximately shows that the protein lies within the granules (Fig. 7D). Exam- 20-25% of all egg chambers. While the wild-type product ination of these granules by regular ﬁxation methods (Fig. 7C) exhibits no movement into the oocyte until the mass dumping shows that they are membrane-bound and have a morphology beginning in stage 11, the mutant product begins to appear in like that of α-yolk granules (Mahowald and Kambysellis, the anterior end of the oocyte cytoplasm as soon as the protein 1980). The unfertilized eggs and early embryos from PuWE75 can be detected in the nurse cells. It continues to pass into the homozygotes have far fewer protein-positive granules than oocyte throughout stage 10. Some of the protein is packaged wild-type eggs or embryos at the same stage (Fig. 7B).

A B

C D

Fig. 7. Wild-type and Punch mutant precellular blastoderm embryos stained with anti-Pu antibody. The anterior poles of the embryos in A and B are oriented to the left. (A) Wild-type embryo at stage 2. Granule staining can be observed throughout the embryo. (B) PuWE75 mutant embryo at stage 2. Granule staining can be observed but is less than that of wild type. (C) Electron micrograph of a region from 0- to 1-hour-old wild-type embryo. Part of the embryo shows membrane-bounded yolk granules (Y). (D) Thin section stained with anti-Pu antiserum and subsequently treated with secondary antibody conjugated with colloidal gold, 10 nm. Pu protein is located inside the yolk granules. Note that gold particles predominate in the yolk granules; there is little if any binding to the surrounding cytoplasm. Bars: 50 µm (A and B); 2 µm (C and D). 3510 X. Chen and others

Fig. 8. Wild-type embryos doubly stained with DAPI (A,C,E and G) and anti-Punch antibody (B,D,F and H), detected with ﬂuorescein- conjugated secondary antibody. (A,B) An unfertilized egg, showing large numbers of Punch protein-containing granules. (C,D) An embryo at the ﬁrst nuclear cycle, also containing very large numbers of granules. (E,F) An embryo at nuclear division 10. Fewer granules are observed. (G,H) Cellular blastoderm embryo. As nuclear divisions have proceeded, the numbers of granules detected by the antibody have decreased; they disappear altogether just after cellularization. Bar, 50 µm.

Correlation of Pu protein-containing granules and of the Pu protein has disappeared from the embryo. Therefore, progression of precellular blastoderm nuclear the number of cross-reacting granules and their staining division cycles intensity are inversely correlated with the number of precellu- We next examined the fate of the granules during embryoge- lar blastoderm nuclear division cycles. nesis. For these experiments, wild-type embryos were double- stained, with DAPI to detect nuclei and with fluorescein- labeled anti-Pu antibody to detect the granules. As the nuclear DISCUSSION divisions during precellular blastoderm proceed, the intensity of the signal in each granule and the number of cross-reacting Our results provide a foundation for the analysis of the role of granules diminish (Fig. 8). After the nuclei have migrated to the Pu locus in early development of Drosophila. The Pu the cell periphery at the onset of cellularization, a small amount product, GTP cyclohydrolase, is rate-limiting for the synthesis of the protein remains in cytoplasmic granules. By the time that of all pteridines and for many, if not all, of the subsequent cellularization of the embryo has been completed, almost all pteridine-requiring activities. A number of the phenotypes Punch maternal function 3511 arising at various developmental stages in Pu mutants can be direct perturbation in GTP pools or utilization. A direct link to interpreted as resulting from the direct effects on these target BH4 cofactor expression is also suggested by the observation activities (O’Donnell et al., 1989b). The connection is less that the maternal effect mutants have reduced cofactor levels obvious in regard to other Pu phenotypes, the maternal effect both in oogenesis and in very early (0-2 hour) embryogenesis nuclear division defect among them. These may be indirect (O’Donnell et al., 1993). The role of BH4 in the progression consequences of disruption of diverse cellular functions or they of the nuclear cycles remains unclear. However, it is likely that may reflect additional biochemical roles for this enzyme or for this function is shared by mammalian cells. There are now a pteridines in general. In an earlier effort to determine whether number of studies indicating BH4 participation in signal- the maternal effect mutant alleles define a normal function of induced mammalian cell proliferation (e.g., see Tanaka et al., Pu during oogenesis, we generated germ line clones, homozy- 1989; Milstein et al., 1990). In the studies cited, exposure of gous for null and maternal effect Pu alleles (Weischaus et al., erythroid and astrocyte cells to cytokines results in the 1981; Perrimon and Gans, 1983). Females in which clones expected proliferation of the cells, preceded by a sharp rise in were induced were capable of producing eggs, but the embryos BH4 levels. Blocking the pteridine biosynthetic pathway with do not develop past the first nuclear cycle (E. Reynolds, unpub- the inhibitors used here in the Drosophila study blocks cell lished observations). While these results indicate that a Pu proliferation. The block can be overcome by the addition of product is used early in development, the phenotype is not exogenous cofactor to the culture medium. Given the many informative with respect to the exact nature of the maternal conserved mechanisms that play roles in DNA replication and function. However, the further observations of the early devel- in mitosis, it seems likely that the disrupted mechanisms in opment of Pu maternal effect embryos reported here, coupled Drosophila are analogous to those operating in cell prolifera- with enzymatic studies of function have provided additional tion in these mammalian cell lines. No morphological studies insights. have been conducted on inhibited mammalian cells, so it is In the studies described here, we observe the nuclear difficult to assess the link further at this time, especially given division abnormalities in Pu mutant embryos from the earliest the complexity of the phenotype in Drosophila. However, nuclear cycles. The very early nature of these effects itelf Schott et al. (1992) observed that both GTP cyclohydrolase and argues for a more direct role for Pu. Not all nuclei are affected BH4 levels increase in a cyclic manner as interleukin-2 stimu- at every cycle, however, suggesting a reduction, rather than a lated rat thymocytes enter S phase. These experiments do not complete lack of functional product. The presence of Pu address directly the nature of the BH4 function, but they are protein in reduced amounts in PuWE75 embryos supports this consistent with a role for the pteridines in the cell cycle. idea. The timing and location of Pu expression are also con- While the Pu phenotype is complex, it is possible to make sistent with a specific precellular blastoderm function. The dis- several inferences concerning the defects. It seems likely that appearance of Pu protein-containing granules as nuclear cycles the abnormal nuclear distribution and asynchronous nuclear proceed in precellular blastoderm embryos is particularly divisions are consequences of the failure of chromosomes to striking. It should be noted, however, that these results do not separate properly. Abnormal nuclear placement might result demonstrate the possibility of a comparable role for Pu in later from lack of stabilizing cytoskeletal structures, but we find no embryogenesis or in imaginal cells. This particular product has strong evidence that this is the case. Examination of phalloidin- disappeared by cellularization, and we observe no further stained embryos reveals no consistent or extensive alteration nuclear defects in mutant- or inhibitor-treated embryos that in actin distribution (G. Ranganayakulu, unpublished observa- survive to this stage. This suggests that the product is specific tions). Moreover, mitotic spindles form in mutant embryos, to early nuclear division cycles. Nevertheless, Pu expresses and they appear capable of guiding the chromosomes through several protein isoforms in a precisely regulated fashion, and all of the mitotic stages, although it is not clear that do so with these are apparently unaffected in the maternal effect Pu normal efficiency. The spindles generally appear normally mutants (McLean et al., 1993). Any one of these could well bipolar, even when two daughter nuclei are interconnected by have a comparable role in later development. unseparated chromatin strands. Occasionally, however, tripolar These phenotypes and the pattern of Pu expression suggest spindles are observed in the mutants and in inhibitor-treated a specific role for a product of this locus in early nuclear embryos. The latter contain a somewhat higher incidence of divisions. The phenocopying of Pu defects by inhibitors of clustered spindles, but the number is variable. We interpret this GTP cyclohydrolase, the first enzyme in the pteridine pathway, variation to result from varying concentrations of functional and sepiapterin reductase, the terminal enzyme in BH4 pro- products in these embryos. It seems likely that these clusters duction, present a compelling case for the direct involvement arise because the nuclei are kept very close together by the of the locus. DHAP and NAS are specific competetive chromosome bridges, resulting in the physical proximity of inhibitors of GTP cyclohydrolase and sepiapterin reductase, centrosome nucleating centers. Similarly, it seems reasonable respectively. They have been used extensively in both in vivo that the physical impediment of unseparated, improperly and in vitro inhibition studies (Gal and Sherman, 1976; condensed, chromosomes is the source of division asynchrony. Bräutigam et al., 1984; Smith et al., 1990; Sung et al., 1994). While the biochemical basis for pteridine effects on the Only a five minute exposure to these inhibitors, or less, even nuclear division cycle are at present unknown, there are several when followed by extensive washing, is sufficient to produce features of GTP cyclohydrolase expression and function that widespread and immediate disruption of nuclear cycles in pre- allow us to construct a working hypothesis. Because GTP cellular blastoderm embryos. The resulting phenotypes are cyclohydrolase catalyzes the first of several steps required for indistinguishable from those of the maternal effect Pu alleles. the production of BH4 and that cofactor is used still further Moreover, these results argue strongly for the direct involve- downstream in target functions, it may seem that GTPCH is ment of the cofactor, tetrahydrobiopterin, as opposed to a less too far removed from the critical reactions to have anything 3512 X. Chen and others other than indirect effects on the phenotypes. Nevertheless, in Foe, V. E. and Alberts, B. M. (1983). Studies of the nuclear and cytoplasmic virtually all cases examined to date, the expression of GTPCH behavior during the five cycles that precede gastrulation in Drosophila is linked extremely tightly to the level of expression of down- embryogenesis. J. Cell Sci. 61, 31-70. Foe, V. E., O’Dell, G. M. and Black, B. A. (1993). Mitosis and morphogenesis stream activities. For instance, the production of L-DOPA, as in the Drosophila embryos: Point and counterpoint. In The Development of a consequence of the activity of the biopterin-requiring Drosophila melanogaster, vol. 1 (ed. M. Bate and A. Martinas Arias), pp. enzyme, tyrosine hydroxylase, in neuroblastoma and PC-12 149-300. Cold Spring Harbor Laboratory Press, New York. cell lines is tightly correlated with the level of BH4 synthesized Freeman, M. and Glover, D. M. (1986). The dissociation of nuclear and centrosomal division in gnu, a mutation causing giant nuclei in Drosophila. de novo in those cells (Bräutigam et al., 1984). The synthesis Cell 46, 457-468. of BH4 itself is tightly regulated by GTP CH activity (Fan and Freeman, M., Nüsslein-Volhard, C. and Glover, D. M. (1987). The gnu Brown, 1976; Fukushima et al., 1976). Werner-Felmayer et al. mutation of Drosophila causes inappropriate DNA synthesis in unfertilized (1993) demonstrated that the NO-stimulated increase in cGMP and fertilized eggs. Genes Dev. 1, 924-930. levels occurs as a consequence of increased nitric oxide Fukushima, K., Richter, W. E. Jr and Shiota, T. (1977). Partial purification of 6-(D-erthro-1′,2′,3′-trihydroxypropyl)-7,8-dihydropterin triphosphate synthase activity in the human cervix carcinoma cell line, ME- synthetase from chicken liver. J. Biol. Chem. 252, 5750-5755. 180. This increase is strongly dependent upon the levels of the Gal, E. M. and Sherman, A. D. (1976). Biopterin. II. Evidence for cerebral required BH4 cofactor. The nitric oxide synthase activity was synthesis of 7,8-dihydrobiopterin in vivo and in vitro. Neurochem. Res. 1, stimulated by addition of exogenous biopterin, and cGMP pro- 627-639. duction is rapidly reduced by the GTPCH inhibitor, DHAP. Gatti, M. and Baker, B. S. (1989). Genes controlling essential cell-cycle functions in Drosophila melanogaster. Genes Dev. 3, 438-453. This suggests a strong regulation of the pathway by pteridine Glover, D. M. (1989). Mitosis in Drosophila. J. Cell Sci. 92, 137-146. production. Furthermore, our preliminary results show that Karr, T. L. and Alberts, B. M. (1986). Organization of the cytoskeleton in both tyrosine hydroxylase and nitric oxide synthase activities Drosophila embryos. J. Cell Biol. 102, 1494-1509. in Drosophila are strongly dependent upon wild-type Pu Katoh, S., Sueoka, T. and Yamada, S. (1982). Direct inhibition of brain sepiapterin reductase by a catecholamine and an indoleamine. Biochem. dosage (Jane Rasco, unpublished observations). Given that Biophys. Res. Commun. 105, 75-81. cGMP-dependent kinases have been found associated with the Kwon, N. S., Nathan, C. F. and Stuehr, D. J. (1990). Reduced biopterin as a mitotic apparatus in cultured mammalian cells (Browne et al., cofactor in the generation of nitrogen oxides by murine macrophages. J. Biol. 1980), one reasonable possibility for the Pu phenotypes might Chem. 264, 20496-20510. be related to a pteridine-dependent regulation of cGMP- Lehner, C. F. and O’Farrell, P. H. (1989). Expression of function of Drosophila cyclin A during embryonic cell cycle progression. Cell 56, 957- dependent kinases in the nuclear division cycles. We are now 968. testing this idea directly. Limbourg, B. and Zalokar, M. (1973). Permeabilization of Drosophila eggs. Dev. Biol. 35, 382-387. This work was supported by NSF grant DCB-8608696 and NIH Lindsley, D. L. and Zimm, D. L. (1992). The Genome of Drosophila grant GM-26757 to J.M.O. We thank Drs Harriett Smith-Somerville melanogaster. Academic Press, Inc., San Diego. and Ed Stephenson for their technical advice and for their careful Lovenberg, W., Jequier, E. and Sjoerdsma, A. (1967). Tryptophan readings of the manuscript. We also acknowledge the expertise of hydroxylation: Measurement in pineal gland, brainstem and caranoid tumor. Jolanta Nunley, who prepared sections for the electron microscopic Science 153, 217-219. Mackay, W. J. and O’Donnell, J. M. (1983). A genetic analysis of the studies and advised us on other aspects of these experiments. We also pteridine biosynthetic enzyme, guanosine triphosphate cyclohydrolase in thank another member of our laboratory, Xin Hu, for stimulating dis- Drosophila melanogaster. Genetics 105, 35-53. cussions. Mackay, W. J., Reynolds, E. R. and O’Donnell, J. M. (1985). Tissue-specific and complex complementation patterns in the Punch locus of Drosophila melanogaster. Genetics 111, 885-904. REFERENCES Mahowald, A. P. and Kambysellis, M. P. (1980). Oogenesis. In The Genetics and Biology of Drosophila, vol. 2D (ed. M. Ashburner and T.R.F. Wright), Altman, L. G., Schneider, B. G. and Papermaster, D. S. (1984). Rapid pp. 141-225. Academic Press, Inc., New York. embedding of tissues in Lowicryl K4M for immunoelectron microscopy. J. Mayer, B., John, M., Heinze, B., Werner, E. R., Wachter, H., Schultz, G. Histochem. Cytochem. 32, 1217-1223. and Bohme, E. (1991). Brain nitric oxide synthase is a biopterin- and flavin- Berleth, T., Burri, M., Thoma, G., Pichstein, S., Frigerio, G., Noll, M. and containing multi-functional oxido-reductase. FEBS Lett. 288, 187-191. Nusslein-Volhard, C. (1988). The role of localization of bicoid RNA in McLean, J. R., Boswell, R. and O’Donnell, J. M. (1990). Cloning and organizing the anterior pattern of the Drosophila embryo. EMBO J. 7, 1749- molecular characterization of a metabolic gene with developmental functions 1756. in Drosophila. I. Analysis of the head function of Punch. Genetics 126, 007- Bräutigam, M., Dreesen, R. and Herken, H. (1984). Tetrahydrobiopterin and 1019. total biopterin content of neuroblastroma (NIE-115, N2A) and McLean, J. R., Krishnakuma, S. and O’Donnell, J. M. (1993). Multiple pheochromocytoma (PC12) clones and the dependence of catecholamine mRNAs from the Punch locus of Drosophila melangaster encode isoform of synthesis on tetrahydrobiopterin concentration in PC-12 cells. J. Neurochem. GTP cyclohydrolase I with distinct N-terminal domaions. J. Biol. Chem. 268, 42, 390-396. 27191-27197. Brew, B. J., Bhalla, R. B., Paul, M., Gallardo, H., McArthur, J. C., Milstein, S., Kaufman, S. and Tanaka, K. (1990). Regulation of cellular Schwartz, M. K. and Price, R. W. (1990). Cerebrospinal fluid neopterin in proliferation by tetrahydrobiopterin. In The Chemistry and Biology of human immunodeficiency virus type I infection. Ann. Neurol. 28, 556-560. Pteridines (ed. H-Ch. Curtius, S. Ghisla and N. Blau), pp. 505-510. Walter de Browne, C. L., Lockwood, A. H., Su, J.-L., Beavo, J. A. and Steiner, A. L. Gruyter, Berlin. (1980). Immunofluorescent localization of cyclic nucleotide-dependent Nagatsu, T., Levitt, M. and Udenfried, S. (1964). Tyrosine hydroxylase: The protein kinases on the mitotic apparatus of cultured cells. J. Cell Biol. 87, initial step in norepinephrine biosynthesis. J. Biol. Chem. 239, 2910-2917. 336-345. O’Donnell, J. M., Boswell, R., Reynolds, T. and Mackay, W. (1989a). A Edgar, B. A. and O’Farrell, P. H. (1989). Genetic control of cell division cytogenetic analysis of the Punch-tudor region of chromosome 2R in patterns in the Drosophila embryo. Cell 57, 177-187. Drosophila melanogaster. Genetics 121, 273-280. Fan, C. L. and Brown, G. M. (1976). Partial purification and properties of O’Donnell, J. M., McLean, R. J. and Reynold, E. R. (1989b). Molecular and guanosine triphosphate cyclohydrolase from Drosophila melanogaster. developmental genetics of the Punch locus, a pterin biosynthesis gene in Biochem. Genet. 14, 259-270. Drosophila melanogaster. Dev. Genet. 10, 273-286. Foe, V. (1989). Mitotic domains reveal early commitment of cells in O’Donnell, J. M., Ranganayakulu, G., Chen, X., Krishnakumar, S. and Drosophila embryo. Development 107, 1-22. Neckameyer, W. S. (1993). Drosophila GTP cyclohydrolase: Multiple Punch maternal function 3513

isoform products of a single gene derive from alternate transcripts that are Studier, F. W., Rosenberg, A. H., Dunn, J. J. and Dubendorff, J. W. (1990). developmentally regulated and functionally specific. In The Chemistry and Use of T7 RNA polymerase to direct expression of cloned genes. Meth. Biology of Pteridines and Folic Acid (ed. J. E. Ayling, M. G. Nair and C. M. Enzymol. 185, 60-89. Baugh), pp. 147-155. Plenum Press, New York. Sung, Y.-J., Hotchkiss, J. H. and Dietert, R. R. (1994). 2,4-diamino-6- Patel, N. H., Schafer, B., Goodman, C. S. and Holmgren, R. (1987). The role hydroxypyrimidine, an inhibitor of GTP cyclohydrolase I, suppresses nitric of segment polarity genes during Drosophila neurogenesis. Genes Dev. 3, oxide production by chicken macrophages. Int. J. Immunopharmacol. 16, 890-904. 101-108. Perrimon, N. and Gans, M. (1983). Clonal analysis of the tissue specificity of Tanaka, K., Kaufman, S. and Milstein, S. (1989). Tetrahydrobiopterin, the recessive female-sterile mutations of Drosophila melanogaster using cofactor for aromatic amino acid hydroxylases is synthesized by and dominant female-sterile mutation, Fs(1)K1237. Dev. Biol. 100, 365-373. regulates proliferation of erythroid cells. Proc. Nat. Acad. Sci. USA 86, 5864- Reynolds, E. R. and O’Donnell, J. M. (1987). An analysis of the embryonic 5867. defects in Punch mutants of Drosophila melanogaster. Dev. Biol. 123, 430- Tayeh, M. A. and Marletta, M. A. (1989). Macrophage oxidation of L- 441. arginine to nitric oxide, nitrate, and nitrite. Tetrahydrobiopterin is required as Reynolds, E. R. and O’Donnell, J. M. (1988). Characterization of new Punch a cofactor. J. Biol. Chem. 264, 19654-19658. mutations: Identification of two additional mutant classes. Genetics 199, Vessey, K. B., Ludwiczak, R. L., Briot, A. S. and Underwood, E. M. (1991). 609-617. abnormal chromatin (abc), a maternal-effect locus in Drosophila Schott, K., Brand, K., Hatakeyama, K., Kagamiyama, H., Maier, J., melanogaster. J. Cell Sci. 98, 233-243. Werner, T. and Ziegler, I. (1992). Control of cell-cycle-associated Werner-Felmayer, G., Werner, E. R., Fuchs, D., Hausen, A., Mayer, B., tetrahydrobiopterin synthesis in rat thymocytes. Exp. Cell Res. 200, 105-109. Reibnegger, G., Weiss, B. and Wachter, H. (1993). Ca2+/calmodulin- Shaskan, E. G., Brew, B. J., Rosenblum, M., Thompson, R. M. and Price, dependent nitric oxide synthase activity in the human cervix carcinoma cell R. W. (1992). Increased neopterin levels in brain of patients with human line ME-180. Biochem. J. 289, 357-361. immunodeficiency virus type I infection. J. Neurochem. 59, 1541-1546. Wieschaus, E., Audit, C. and Masson, M. (1981). A clonal analysis of the Smith, A. V. and Orr-Weaver, T. L. (1991). The regulation of the cell cycle roles of somatic cells and germline during oogenesis in Drosophila. Dev. during Drosophila embryogenesis: the transition to polyteny. Development Biol. 88, 92-103. 112, 997-1008. Zalokar, J. E. and Erk, I. (1976). Division and migration of nuclei during Smith, G. K., Duch, D. S., Edelstein, M. P. and Bigham, E. C. (1990). early embryogenesis of Drosophila melanogaster. J. Microsc. Biol. Cell. 25, Inhibitors of bovine adrenal medullary sepiapterin reductase. In The 97-106. Chemistry and Biology of Pteridines (ed. H.-Ch. Curtius, S. Ghisla and N. Blau), pp. 320-323. Plenum Press, New York. Spurr, A. R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-43. (Received 25 May 1994 - Accepted 15 August 1994)