Figure 1.
(A) Diagrams of longitudinal and cross sections through one arm of the adult hermaphrodite gonad with germ nuclei indicated in dark blue; somatic sheath cells that surround the gonad are not shown. Optical sectioning planes referred to in this paper are indicated in the cross-section (dashed lines). During development, germ cells move from the distal (mitotic) end of the gonad toward the proximal end, where they differentiate as oocytes. (B) Enlarged diagram of two germ cells with ring channels opening to the gonad core. Basket and core microtubules (MTs) are drawn in red. (C) Linear representation of germ cell development. Red shading indicates region-specific conditions in germ cells that might impede viral replication. These include the state of the nuclear envelope [67], P granules [21], transcription [68], chromatin [69], and cytoplasmic flow [27]; ooc = oocytes, emb = embryos. (D–E) Electron micrographs of C. japonica VLPs. Low magnification in panel E shows a small cluster of VLPs (arrow) on top of a P granule; arrowheads indicate examples of nuclear pores. See also Supplemental Figure S1A, S1B, S1C). (F) C. elegans VLPs showing variation in internal electron density; note curved, rod-like bodies within the VLPs in the middle panel. Scale bars: D (0.2 µm), E (0.5 µm), F (0.1 µm).
Table 1.
Electron microscopy of VLPs in Caenorhabditis.
Table 2.
Electron microscopy of VLPs in C. elegans strains.
Figure 2.
Cer1 GAG expression is temperature dependent.
(A) Diagram of the Cer1 retrotransposon, mRNA products confirmed by sequencing in this study, and the locations of probes used for Northern and in situ experiments. Colored boxes within POL correspond to homology regions for Protease (PR), Reverse Transcriptase (RT), Ribonuclease H (RNH), and Integrase (IN) as described [30]. mRNAs beginning 5′ to the splice acceptor used for SL1 trans-splicing were demonstrated by RT-PCR using primer pairs KE409F, KE416R and KE409F, KE401BR (Materials and Methods). (B–D) Gonads immunostained with αGAG; gonads were dissected from day 1 adults raised at 25°C, then shifted to 15°C for the times indicated. (E) Western blot of total proteins from adults grown at either 15°C, 20°C, or 25°C; blots were stained with αGAG. (F) Northern blots of poly(A)+-selected RNA from adults grown at either 15°C or 25°C; blots were hybridized with probes (I–III) as diagrammed in panel A. Normalization relative to a control actin probe showed that the difference in signal between 15°C and 25°C is at least tenfold for the 8 kb band. (G–I) RNA in situ hybridization (alkaline phosphatase detection) of gonads and intestines (int) from adults cultured at the indicated temperatures, using probes for sense or antisense (control) gag-containing mRNA; each asterisk indicates the distal, mitotic region of the gonad. Bottom panels are higher magnifications of the same gonads showing gag-containing RNA concentrated in paired, nuclear dots (double arrows). (J) Fluorescence in situ hybridization (FISH) of a 15°C gonad using the gag-specific probe (green); the image shows a longitudinal, optical section through the gonad core. Note that RNA is concentrated in the superapical region of the core (brackets), and in paired dots within nuclei (blue, DAPI). (K–L) High magnification of FISH with the rt-specific probe; images show two nuclear dots in wild-type germ cells (panel K, two copies of Cer1) and one dot in nuclei from N2/CB4856 heterozygotes (panel L, one copy of Cer1); control CB4856 homozygotes which lack Cer1 show no staining (data not shown). Scale bars: B–D (20 µm); G–I (20 µm), inserts (5 µm); J–L(5 µm).
Table 3.
Cer1 GAG particles detected by immunostaining.
Table 4.
Cer1 GAG particles in C. elegans wild strains.
Figure 3.
Cer1 capsids show stage-specific changes in localization.
(A) Low magnification, wide-field image of a gonad from a 15°C adult, immunostained with αGAG to detect Cer1 capsids (green). Note that capsids in the mid-pachytene region are enriched along the superapical zone of the core (compare with Figure 2J), rather than within germ cells at the periphery (arrow). (A′) High magnification showing the crescent-shaped aggregates of capsids by oogonia nuclei. (B,C) Newly fertilized eggs showing the maternal chromosomes at diakinesis (B), and at metaphase of the meiosis I division (C). Arrows point to concentrations of capsids associated with the meiosis I spindle, and arrowheads indicate small foci of capsids at the periphery. The examples shown for these images contain unusually high numbers of capsids; most eggs show the same localization pattern but with fewer capsids, and many fertilized eggs contain no detectable capsids. (D) 28-cell stage embryo; the arrow points to capsids within the first polar body. The asterisk is below the germ cell precursor, showing that capsids do not concentrate in this cell. The eggshell is outlined in panels B–C (dashed line), and the cellular boundary of the 28-cell embryo is indicated in panel D (white line). (E) Capsid localization in a dhc-1/dynein mutant; this gonad was scored as “+” in Table 3. Scale bars: A–E (20 µm).
Figure 4.
Cer1 capsids localize on microtubules.
(A–F) Electron microscopy of gonads from 15°C adults; regions of the gonad are as indicated at the top of the panels. Capsids localize predominantly to the core in the mid-pachytene region, but concentrate near nuclei in late-pachytene germ cells (C–E) and in oogonia (F). Note that many capsids localize with microtubules (arrowheads) both in the core (panel A) and in germ cells (panel C). P granules are visible in panels C (arrow) and in panel D; arrowheads in panel D indicate examples of nuclear pores. (E) Cluster of capsids near the nucleus of an early oogonium. (F) A “crescent” of capsids by an oogonium nucleus; there are at least 64 capsids and numerous microtubules visible at higher magnifications of this single thin section (data not shown). Scale bars: A–F (0.2 µm).
Figure 5.
Capsids accumulate in the mid-pachytene gonad core as adults age at 15°C.
(A–D) Longitudinal, optical sections through gonads showing increased accumulation of capsids (green) with adult age, as indicated. The gonad in panel B is immunostained for HIM-4/hemicentin (red) to visualize the apical membrane (see Figure 1B). Note that capsids localize predominately in the core, outside the ring channel and away from nuclei (blue, DAPI). (C) Capsids grouped in wavy lines and tangles of lines in the mid-pachytene region of a day 3 adult. (D) Low magnification of the mid-pachytene region. Note variation in capsid abundance between the two sides of the gonad core (double-headed arrow), and that the wavy lines of capsids disappear as germ cells move proximally into late pachytene (bracketed region). (E–G) The late-pachytene/diplotene region, stained and imaged as for panel B; two of the somatic sheath cells that surround the gonad are visible in this image. Germ cells from regions F and G are shown at high magnification, oriented as in Figure 1B. An optical rotation of a similar region of the gonad is shown in Supplemental Video S1. (H) Optical section through germ nuclei in the late-pachytene/diplotene region showing capsids (green) and P granules (red, αPGL-1). Note that capsids localize close to the nuclear envelope, but most are not directly on, or within, P granules. Electron micrographs of capsids within P granules are shown in Supplemental Figure S1H. Scale bars: A–C, F–H (5 µm), D–E (10 µm).
Figure 6.
A subset of microtubules in the mid-pachytene region are inhibitor-resistant, or stable.
(A) Optical section through the superapical plane of an untreated day 1 adult gonad showing the dense network of long microtubules (see Figure 1A for section orientation). The inset shows a high magnification of the dashed box, with an arrowhead marking the end of a single microtubule traced from the indicated germ cell (asterisk). (B,C) Optical sections through the central planes of untreated, day 1 adult gonads; the gonads are immunostained for both microtubules and Cer1 capsids. Note that capsids are concentrated in linear or irregular shapes, while the microtubules are distributed uniformly. (D) Optical section through the superapical plane of a day 1 adult gonad treated with nocodazole; the bracketed region is the same late-pachytene/diplotene region, and the same optical plane, as for the untreated gonad in panel A. Note that most microtubules have disappeared from this region after nocodazole treatment, in contrast to the numerous “stable” microtubules that remain in the mid-pachytene region. (E,F) Microtubules in the distal, mitotic regions of gonads before (E) and after (F) nocodazole treatment. The arrow in panel E indicates an example of a mitotic spindle. Most of the brightest spots of tubulin visible in panel F co-localize with centrosomes (data not shown). (G) Optical section through the central plane of a nocodazole-treated gonad. This sectional view combined with that in panel D illustrates that most of the stable microtubules in the mid-pachytene region are in the superapical zone of the core (arrowheads). Note that most of the microtubules in the late-pachytene/diplotene germ cells have depolymerized; stable microtubules that appear at the periphery of the gonad (arrow) are outside of germ cells and within the thin cell bodies of somatic sheath cells. (H) Stable microtubules in nocodazole-treated oocytes; the oocytes advance in age right to left (see Figure 1A). Note the focus of stable microtubules (arrow) near the oocyte nucleus, and the abrupt disappearance of all stable microtubules in the more mature oocyte to the left; compare with oocyte progression panel D, where numbers indicate oocyte position relative to ovulation. Scale bars: A–C (5 µm), D–H (10 µm).
Figure 7.
Cer1 capsids accumulate on stable microtubules in the mid-pachytene region.
(A–F) All images are of the mid-pachytene region of day 3 adult gonads. Panels A–C and F show central optical planes, and panels D,E show superapical optical planes. (A) Untreated wild-type gonad, showing the dense population of total microtubules in the core; panel A′ indicates a basket microtubule (arrowhead) traced back to the germ cell of origin (asterisk). (B) Stable microtubules in a nocodazole-treated, Cer1(-) gonad. Most of the core microtubules have depolymerized, and the stable microtubules that remain are enriched in the superapical zone (arrow); the arrowhead points to stable microtubules in sheath cells. Stable microtubules in a nocodazole-treated, wild-type gonad. Note the multiple aggregates of stable microtubules (arrows) and the neighboring regions devoid of microtubules (arrowhead). Note also that capsids are strongly concentrated in the microtubule aggregates (see also panel D below). (D–E) Images of stable microtubules as in panels B and C, respectively, but viewed from a superapical plane. Note the thick and irregular staining of the wild-type stable microtubules, and their association with Cer1 capsids. Panels at right are high magnifications of the dashed, boxed regions at left. (F) High magnification image of an aggregate from the gonad shown in panel C. Note the abrupt loss of stable microtubules, and most capsids, to the right of the panel as germ cells exit pachytene (see also Figure 8B). (G) Quantitation showing mean and standard deviation in the number of dead eggs laid by wild-type and Cer1(-) adults grown for the indicated number of days at 15°C. This analysis was performed on 20–25 separate pools of 5 adults each that were cultured in the constant presence of males to maintain ovulation. Brood sizes remained comparable up to day 5 (WT = 437 +/− 57 total progeny, Cer1(-) = 442 +/− 83), after which some adults in both sets stopped laying eggs. (H) Percentage of gonads without aggregates of stable microtubules in wild-type (green) and Cer1(-) (orange) animals as a function of adult age. Note that nearly all wild-type gonads contain 1 or more aggregates by day 5. (I, J) Analysis of dead eggs and total progeny produced within 24 hours after shifting 3 day adults from 15°C to 25°C. Dead eggs were scored in panel I only if they were approximately normal size. The wild-type adults also produced significantly more small, “partial” eggs than Cer1(-) [WT = 2.9%+/− 0.4 partial eggs, Cer1(-) = 1.6% +/− 0.2; P<.0001]. Scale bars: A–F (5 µm).
Figure 8.
Capsid localization near late-pachytene nuclei requires inhibitor-sensitive microtubules.
(A) Late-pachytene/diplotene region of a nocodazole-treated, day 3 wild-type gonad showing the near absence of stable microtubules and capsids; the arrowhead indicates a germ cell shown a higher magnification in the lower panels. Compare these images with the abundant microtubules and capsids in the same region of untreated gonads (Figure 6A, boxed region, and Figure 5G, respectively). (B) Low magnification image of a nocodazole-treated, day 4 wild-type gonad, oriented as in Figure 6D. Note the rare example of a large aggregate of stable microtubules and capsids in an early oogonium. Because oogonia in this region begin to receive cytoplasmic flow from the gonad core, it is possible that this focus broke off from the mid-pachytene region, rather than surviving the normal developmental progression through late pachytene. Note also that many of the oogonia and early oocytes lack crescents of capsids (arrowhead; compare with Figure 3A), and the few crescents of capsids co-localize with stable microtubules. (C) Untreated, wild-type oocyte showing enrichment of microtubules near the nuclear envelope. (D) Nocodazole-treated, wild-type oocyte showing a focus of stable microtubules and a crescent of capsids. Oocytes shown in both panels occupied the -5 position in the ovulation sequence. Scale bars: A (5 µm), B (10 µm), C (8 µm).
Figure 9.
Load-Release-Transfer model for capsid traffic toward late-pachytene/diplotene nuclei.
Cartoon of capsids and microtubules as germ cells progress into late pachytene/diplotene. Capsids that had accumulated on stable microtubules (black) in the core (top) are released as these microtubules depolymerize. The released capsids transfer to surrounding labile, or dynamic, microtubules (red) and traffic into germ cells. In an alternative model, capsids could remain on the original microtubule as it becomes destabilized. Traffic could involve microtubule dynamics, or microtubule motor proteins.