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doi: 10.1242/10.1242/dev.00310

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Maelstrom, a Drosophila spindle-class gene, encodes a protein that colocalizes with Vasa and RDE1/AGO1 homolog, Aubergine, in nuage

Department of Biochemistry, Box 357350, University of Washington, Seattle, WA 98195-7350, USA

View larger version (81K): [in a new window]   Fig. 5. Dependencies of nuage particle assembly. Maelstrom (A1) and Vasa (A1') localization in wild-type, aubHN2/N11 (A4, A4') and spn-E616/hls125 (A5,A5') ovarioles. Maelstrom (A2) and Vasa (A2') localization in maelM391/Df (3L)79E-F ovarioles; and Maelstrom localization in vasa ovarioles (A3). (Vasa signal in vasa ovarioles was not examined in this experiment, but in similar experiments performed, no signal is detected.) (B,C) Enlargements of nurse cells (B) and stem cells (C) from the correspondingly numbered panels in A. (A1) Distribution of Maelstrom in a wild-type ovariole. The highest concentration of Maelstrom is in perinuclear granules, evident in the germline from stem cells and cystoblasts (enlarged in C1) to nurse cells (enlarged in B1). In the maelstrom ovariole, virtually no Maelstrom immunoreactive signal is present (A2); yet Vasa is largely maintained in nuage (A2'). Maelstrom protein level in the germline of vasa null ovaries (A3, B3, C3) shows little preferential distribution to perinuclear particles in any cell type. (A4) Maelstrom in an aubHN2/N11 ovariole. Maelstrom's preferential localization to perinuclear particles is abolished. Instead, Maelstrom often accumulates on membranes between germline cells [B4 (black arrow), C4]. (A5) Maelstrom in a spn-E616/hls125 ovariole. Maelstrom shows no preferential accumulation in perinuclear particles in any cell type (B5,C5). Differential localization of Vasa to nuage is largely maintained in mael (A2', B2',C2'), aubHN2/N11 (A4',B4',C4') and spn-E616/hls125 (A5',B5',C5') backgrounds. Anterior is towards the top in all panels.

View larger version (19K): [in a new window]   Fig. 1. maelM391 is a null allele. (A) Organization of the maelstrom gene. Exons are indicated by boxes in which coding regions are black and UTRs are white. The P [w+lacZ]11A4 insertion is indicated by a lavender arrow, and the region deleted () in maelM391 is ochre. (B) Wild-type genomic DNA sequence at the junctions of the deletion. The P element is inserted between basepairs corresponding to nucleotides 123/124 of the mRNA. The DNA encoding nucleotides encoding 129-1266 of the mRNA is indicated by (dots). (C) The imprecise P-element excision in maelM391 results in the deletion of 1319 basepairs of genomic DNA corresponding to nucleotides 124-1270 (including the initiation codon) of the mRNA. The residual P-element sequence is indicated by the purple background. (D) Maelstrom staining in a wild-type ovariole. Immunoreactive signal is predominant in cells throughout the germline. (E) Maelstrom or (F) Maelstrom (green) and Adducin (red) staining of a maelM391/Df(3L)79E-F ovariole. Virtually all anti-Maelstrom immunoreactive signal is lost in the mutant. In the Western blot (G) probed with anti-Maelstrom antiserum, neither the full-length wild-type (left, at 50 kDa) nor potential truncated forms (not shown) of Maelstrom is detected in maelM391/Df(3L)79E-F ovaries lane (right).

View larger version (72K): [in a new window]   Fig. 2. Maelstrom is a spindle-class gene. (A) Distribution of synaptonemal complex component C(3)G (green) in a wild-type ovariole labeled with DAPI (blue) and for Adducin (red), which marks fusomes and cell membranes. C(3)G is normally acquired by germline chromosomes in region 1 of the germarium (top left) and is restricted to the oocyte nucleus by stage 1 (lower right). (B) The localization of C(3)G on chromosomes is unperturbed in most maelstrom null ovaries. (C) Magnified views of oocyte nuclei from wild-type and maelstrom oocytes. Top row (WT), wild type; bottom row (M), maelstrom (left to right): stage 1 C(3)G (green channel) and DNA (blue channel); stage 1 DNA channel; and stage 5/6 DNA. Wild-type (D) and maelstrom (E) stage 5/6 oocytes labeled with DAPI (green) and for nuclear lamin (red). Chromosomes within the wild-type oocyte nucleus (D) have condensed to form the karyosome. A maelstrom stage 5/6 oocyte (E), in which the karyosome never forms. (F) Distribution of BicD (green) in a wild-type stage 5/6 oocyte. Follicle cells are labeled with -Spectrin (red). BicD appears in a distinct gradient emanating from the posterior oocyte cortex. In maelstrom oocytes at this stage, BicD appears in a randomly localized focus (G) or unlocalized (not shown). Several RNAs, such as gurken, which localize to the posterior cortex of the wild-type stage 5/6 oocyte (H), are present at normal levels, but fail to localize in the maelstrom mutant (I) (oocytes indicated by broken blue lines). (J) Distribution of Gurken protein (green) in a wild-type stage 5/6 oocyte with follicle cells labeled for -Spectrin (red). Gurken is present at either low (K) or undetectable (not shown) levels in both maelstrom (K) and mei-W681; maelM391/Df(3L)79E-F (L) oocytes. (M) Western blot of Vasa in wild-type (WT), maelM391/Df(3L)79E-F(M) and spn-B153/Df(3R)trxE12* (B) backgrounds. In wild-type ovaries, Vasa runs as a 72.4 kDa singlet. In maelstrom, Vasa runs as a doublet, the slower migrating band of which has a larger apparent mass (74.4 kDa) than that of spn-B mutant. (MW markers are indicated on the right of the panel, the upper dot is at 83.6 kDa, and the lower dot is at 72 kDa). Nurse cell to the oocyte transport of oskar mRNA in maelstrom (O) ovarioles is comparable with wild-type (N). Scale bars: in L, 10 µm for A-D,F-L; in O, 50 µm for N,O.

View larger version (98K): [in a new window]   Fig. 3. Distribution of Maelstrom protein during oogenesis. (A) Left panel: three-dimensional projection of a partial confocal z-series stack showing a wild-type ovariole double-labeled (left) for Vasa (middle, green channel) and Maelstrom (right, red channel). Maelstrom colocalizes with Vasa in highly abundant perinuclear particles of all germline cells. Arrows indicate individual stem cell nuclei (SCN), nurse cell nuclei (NC) and an oocyte (OO). (B) From left to right: double labeling of Maelstrom (red) and nuclear Lamin (green) in a stage 4 egg chamber; enlargement of a single nurse cell nucleus; single Maelstrom and single Lamin channels. Maelstrom particles are closely apposed to the cytoplasmic face of the nuclear envelope. Scale bar: 10 µm. (C) An Aub-GFP germarium stained for Maelstrom and Vasa. Upper left: merged image of Vasa (green, upper right), Maelstrom (red, lower right) and Aub-GFP (blue, lower left) channels. Each discrete perinuclear particle labels for the three nuage components. Scale bar: 10 µm. (D) Localization of Vasa (green) and Maelstrom in a stage 10 egg chamber. Within nurse cells both proteins are present in nuage and cytoplasm. Maelstrom, unlike Vasa, shows no accumulation in the oocyte posterior. (E) Localization of Vasa and Maelstrom within a stage 2 oocyte nucleus. Panels from left to right: diagram of confocal field, with nurse cell nucleus (NCN) and oocyte nucleus (ON) indicated. Vasa (green in merged image) frequently localizes to intranuclear punctae (white arrow) ringed by concentrated Maelstrom (red in merged image). Scale bar: 2 µm. Anterior is towards the left in all panels. (F) Dorsal anterior corner of a stage 10B egg chamber labeled for 1B1 (green) and Maelstrom (red), which is present as diffuse staining in the oocyte nucleus. Scale bar: 20 µm.

View larger version (62K): [in a new window]   Fig. 4. Leptomycin B inhibits the nuclear export of Maelstrom. Ovaries were incubated for 20 minutes with or without 50 nM LMB, then stained for Vasa and Maelstrom. Two representative double-labeled ovarioles (A-D) and enlarged fields (E-H) are shown. The distribution of Vasa between nucleoplasm, nuage and cytoplasm of nurse cells is unperturbed during the 20 minute incubation without LMB (A, enlarged in E). There is a slight nuclear elevation of Vasa in LMB-treated samples (B, enlarged in F). Maelstrom maintains a normal distribution in the control ovariole (C, enlarged in G); upon LMB treatment (D, enlarged in H), Maelstrom is simultaneously reduced within cytoplasm of nurse cells and the oocyte, and enhanced within nuclei of the same cells. Anterior is towards the top in all panels. Scale bar: 20 µm in A-D.

View larger version (66K): [in a new window]   Fig. 6. Maelstrom is implicated in miRNA/RNAi pathways. Dicer in a wild-type ovariole (A, enlarged in A') is ubiquitously expressed and primarily cytoplasmic in both germline and somatic cells. In maelstrom ovarioles (B, enlarged in B'), Dicer is ectopically localized to discrete punctae, which are frequently perinuclear in stages 3-9 nurse cells. A wild-type ovariole (C, enlarged in C') labeled for Vasa (red channel) and AGO2 (green channel). AGO2 appears as small, irregular particles distributed randomly in the cytoplasm of germ cells. (E, enlarged in E') Wild-type AGO2 channel alone. In maelstrom ovarioles (D, enlarged in D'), AGO2 (green channel) aggregates in perinuclear halos that overlap Vasa (red channel). (F, enlarged in F') maelstrom AGO2 channel alone. AGO1 shows no discernable subcellular localization in either wild-type (G, enlarged in G') or maelstrom ovarioles (H, enlarged in H').

View larger version (26K): [in a new window]   Fig. 7. Nuage is implicated in the miRNA pathway. Vasa (V, green circle), a pivotal organizer of nuage (large gray circle) is required to recruit (black arrows) both Aubergine (A, blue circle) and Maelstrom (M, red circle) into particles (large gray circle). In nurse cells, sponge bodies (orange circle) are ultrastructurally interfaced with nuage particles, and contain RNPs required for proper translation and localization of mRNAs. MicroRNA maturation or assembly is suggested (pink shading) in the nuage/sponge body interface by the requirement for both Spn-E (gray circle) and Aubergine for recruitment of Maelstrom to nuage, as well as the ectopic localization of Dicer (pink circle) and Argonaute2 (not shown) to the perinuclear zone. Either microRNA processing (bottom) or recruitment of miRNA-directed specificity factors (small darker orange circle in sponge body) into RNPs could occur in the vicinity of nuage.