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Plasticity of Drosophila germ granules during germ cell development [1]

['Anna C. Hakes', 'Department Of Molecular Biology', 'Princeton University', 'Princeton', 'New Jersey', 'United States Of America', 'Elizabeth R. Gavis']

Date: 2023-04

Compartmentalization of RNAs and proteins into membraneless structures called granules is a ubiquitous mechanism for organizing and regulating cohorts of RNAs. Germ granules are ribonucleoprotein (RNP) assemblies required for germline development across the animal kingdom, but their regulatory roles in germ cells are not fully understood. We show that after germ cell specification, Drosophila germ granules enlarge through fusion and this growth is accompanied by a shift in function. Whereas germ granules initially protect their constituent mRNAs from degradation, they subsequently target a subset of these mRNAs for degradation while maintaining protection of others. This functional shift occurs through the recruitment of decapping and degradation factors to the germ granules, which is promoted by decapping activators and renders these structures P body-like. Disrupting either the mRNA protection or degradation function results in germ cell migration defects. Our findings reveal plasticity in germ granule function that allows them to be repurposed at different stages of development to ensure population of the gonad by germ cells. Additionally, these results reveal an unexpected level of functional complexity whereby constituent RNAs within the same granule type can be differentially regulated.

Here, we have investigated a role for germ granules in regulating mRNA stability in pole cells. We found that during a period when germ granules gain the ability to fuse with each other, they sequentially recruit mRNA decay factors typically found in P bodies, and nos and pgc are destabilized. In contrast, CycB is maintained throughout embryogenesis, despite residing within the same granules. These findings suggest that in contrast to their protective role prior to pole cell formation, germ granules play a more complex role in pole cells, selectively protecting some mRNAs while promoting the degradation of others. Overexpression of an activating subunit of the decapping complex, DCP1, disrupts the protection of CycB, suggesting DCP1 levels are limiting for CycB degradation. We show that the decapping activators Edc3 and Patr-1 are necessary to localize the decapping complex to germ granules and disruption of decapping complex recruitment leads to aberrant stabilization of nos and pgc. Furthermore, disrupting the selective mRNA protection or degradation by germ granules leads to defects in pole cell migration, suggesting both of these germ granule functions are necessary for proper gonad formation. Overall, these findings reveal a shift in germ granule function after pole cell formation that is required for proper mRNA regulation and pole cell development. Such plasticity allows the same RNP granules to be repurposed for distinct functions at different stages of development.

After the pole cells have formed and their contents are physically separated from the soma, the need for protection from the somatic MZT is eliminated. Furthermore, once the pole cells enter mitotic quiescence in advance of gastrulation [ 26 ], the role of germ granules in distributing mRNAs to daughter cells is no longer required. However, the germ granules persist into later stages of embryogenesis [ 27 ]. Interestingly, germ granules increase in size in the pole cells [ 28 – 30 ]. The significance of this morphological change and what roles the germ granules play throughout the remainder of germ cell development have yet to be determined. Since germ granules are a conserved feature of differentiated germ cells, deciphering their regulatory functions at these stages is of particular interest.

During embryogenesis, the germ plasm induces the formation of membrane buds around nuclei at the posterior of the syncytial embryo, which then pinch off to form the germ cell progenitors—called pole cells [ 20 ]. The germ granules accumulate around these nuclei and their associated centrosomes by dynein-dependent transport. Co-packing of many different mRNAs into each granule ensures efficient trafficking and segregation of mRNAs to the pole cells as they bud and divide [ 21 ]. Germ granules also play a role in stabilizing constituent RNAs during the maternal to zygotic transition (MZT), when a majority of maternal mRNAs are degraded in the somatic region of the embryo. This degradation allows zygotic transcription to be activated, which is necessary for cellularization and differentiation of the soma and for body patterning [ 22 , 23 ]. In contrast, numerous transcripts, including important germline determinants, are stabilized in the germ plasm [ 23 – 25 ]. Although not all stabilized mRNAs are localized to the germ granules, sequestration within germ granules may be a mechanism to stabilize a subset of these RNAs by making them less accessible to mRNA decay factors.

In Drosophila, the germ granules form at the posterior of the oocyte within a specialized cytoplasm called the germ plasm. There, the germ granule proteins Oskar (Osk), Vasa (Vas), Tud, and Aubergine (Aub) form a scaffold [ 11 – 13 ] to recruit mRNAs that are necessary for germ cell development, including nanos (nos), polar granule component (pgc), and Cyclin B (CycB) [ 14 – 16 ]. Within the germ granules, these RNAs self-associate to form spatially distinct homotypic clusters [ 17 – 19 ]. The germ granules become anchored to the posterior cortex by the end of oogenesis and persist there into embryogenesis.

One class of RNP granules, called germ granules, is a characteristic feature of germ cells across animal species [ 5 ]. Germ granules contain mRNAs and proteins that are necessary for germ cell development and are therefore thought to play important roles in germline development and function [ 6 , 7 ]. In some animals, like Drosophila, Xenopus, and zebrafish, germ granules form during oogenesis from maternally expressed proteins and RNAs. During early embryogenesis, these maternally supplied granules are segregated to a subset of cells that will give rise to the germline. In other animals, including mammals, germ granules are not acquired maternally, but similar granules containing necessary germline determinants form de novo after the germ cells have been specified [ 5 , 8 ]. Despite differences in how germ granules arise, shared components like the RNA-binding protein Nanos (Nos), Tudor (Tud) domain proteins, and RNA helicases [ 5 , 9 , 10 ] suggest a conserved role in RNA metabolism.

Ribonucleoprotein (RNP) granules are biomolecular condensates containing RNAs and RNA-binding proteins that create cytoplasmic compartments without the use of membranes. Compartmentalization in granules concentrates RNAs together with regulatory proteins, such as those involved in RNA localization, translational control, RNA processing, and control of mRNA stability [ 1 – 3 ]. Thus, RNP granules are hypothesized to be hubs of posttranscriptional regulation. Several types of RNP granules—such as processing bodies (P bodies), stress granules, and neuronal transport granules—are found in many different cell types and species, suggesting a common and highly conserved regulatory strategy [ 1 , 4 ].

Results

Germ granules enlarge and persist in pole cells through embryogenesis Within the pole cells, germ granules become larger and undergo shape changes [28,29,31]. To determine precisely when germ granules grow in size and how long they persist, we visualized germ granules throughout embryogenesis using Osk as a marker. To ensure that changes we observed in the germ granules reflect their normal physiology, we used CRISPR-Cas9 genome editing to endogenously tag Osk with sfGFP at its C terminus. Individuals homozygous for the endogenously tagged Osk-sfGFP are fertile and show no phenotypic abnormalities, indicating that the protein is fully functional. We visualized Osk-sfGFP by anti-GFP immunofluorescence, using somatic nuclear cycles (nc) [32] or Bownes stages [33] to measure developmental time, starting with pole cell budding at nc9 and ending when the pole cells coalesce in the gonad (Bownes stage 14). In addition to marking the germ granules, endogenously tagged Osk is also present in nuclear puncta beginning at nc10 (Fig 1B) as previously reported for transgenically expressed Osk-GFP [30]. We focused on the cytoplasmic granules, as only they contain mRNAs [30]. During nc9, these granules appear as diffraction limited spots that cluster around the budding nuclei (Fig 1A). A few larger germ granules, with diameters between 500 and 700 nm, first begin to appear in the pole cells starting at nc12, approximately 10 min after pole cell formation (Fig 1A–1D). Over the next 90 min, there is a trend toward larger granules such that by the end of nc14, most granules appear much larger than those first segregated to the pole cells. The majority of these granules are at least 1 μm in diameter, with some reaching over 1.5 μm (Fig 1E and 1F). PPT PowerPoint slide

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TIFF original image Download: Fig 1. Germ granules increase in size and persist through gonad formation. Single confocal sections, showing endogenously tagged Osk-sfGFP (green) in the pole cells after pole cell budding (A–F), throughout gastrulation (G–H) and during pole cell migration (I–L). Embryos were staged by nuclear cycle (nc, A–F) or Bownes stage (st, G–L) according to nuclear density or morphological features, respectively. Osk-sfGFP was detected by anti-GFP immunofluorescence and nuclei were stained with DAPI (blue). The brightness and contrast were adjusted individually for each image to best show the features of the germ granules at that stage. Arrows indicate examples of the larger germ granules that first appear at nc12. Scale bar: 5 μm. https://doi.org/10.1371/journal.pbio.3002069.g001 At the end of nc14, gastrulation begins. Cellular movements carry the pole cells into the posterior midgut primordium, where they respond to chemotactic cues directing them to migrate throughout the midgut epithelium and attach to adjacent mesoderm cells. Once aligned, the germline and mesodermal cells migrate and coalesce to form the gonad [34]. In the gonad, the pole cells resume cell division and ultimately generate the germline stem cells capable of producing eggs or sperm. We visualized Osk-sfGFP throughout these migratory movements to determine how long germ granules persist. Both large (≥1 μm diameter) and small granules are visible as the pole cells begin to migrate through the midgut and toward the presumptive mesoderm (Bownes stage 9). These granules persist throughout gastrulation and at least until the end of pole cell migration at stage 14 (Fig 1G–1L). Similar results were obtained using another endogenously tagged germ granule marker, Vas-EGFP [35] (S1 Fig). Their persistence suggests that the larger granules are stable and that the germ granules could play a role in germ cell development throughout embryogenesis.

Germ granules grow through fusion in the pole cells During this period of germ granule growth, we observed a decrease in the number of granules that coincided with their increase in size (Fig 1). Therefore, we hypothesized that germ granules enlarge via fusion of smaller granules. To test this hypothesis, we performed time lapse confocal imaging of Osk-sfGFP during nc14, when the majority of germ granule growth occurs. Throughout nc14, we observed examples of apparent fusion between 2 granules, occurring over the course of 2 to 3 min (Fig 2A–2E, S1 Video). These events are slower than fusion of the more liquid-like germ granules in Caenorhabditis elegans, which occurs in seconds [36]. Furthermore, this analysis cannot distinguish true fusion from granules docking together without exchanging their materials. To confirm that fusion does occur, we endogenously tagged Osk with the photoconvertible fluorescent protein Dendra2 at its C terminus. Osk-Dendra2 was then photoconverted from green to red (shown here as green to magenta) within a small region of a pole cell to generate differentially labeled germ granules (Fig 2F) that were tracked using time lapse imaging. During nc14, we observed almost entirely photoconverted (red) and unconverted green granules that appear to “stick” together for a period of about 1 to 3 min without mixing (Fig 2F–2H, S2 and S3 Videos). After this initial period, the 2 colors begin to mix, resulting in a granule with green and red signals distributed uniformly throughout (Fig 2I–2K, S2 and S3 Videos). Together, these data suggest that germ granule growth at nc14 occurs at least in part by the slow fusion of smaller granules. Since fusion has not been observed at earlier stages [30,37], these results suggest that the germ granules are more dynamic at nc14. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Germ granules grow by fusion in the pole cells. Maximum intensity confocal z-projections of a 1 μm region of a representative pole cell at nc14 with endogenously tagged Osk-sfGFP (A–E) or Osk-Dendra2 (F–K). Osk-sfGFP and Osk-Dendra2 images were taken from a 5-min period of S1 Video and a 4-min period of S2 Video, respectively. Yellow arrows and boxes indicate germ granules that undergo fusion. Enlargements of the boxed regions in (F), (H), and (J), show the mixing of green and red (shown here in magenta) fluorescent Osk-Dendra2 signal over time. White arrows indicate a region of a granule where the magenta labeled and green labeled contents have yet not mixed after fusion. Time stamps indicate minutes:seconds. Scale bar: 1 μm. https://doi.org/10.1371/journal.pbio.3002069.g002

The mRNA decay machinery is sequentially recruited to germ granules The loss of nos and pgc could result from their selective degradation within the germ granules, or from their selective release and subsequent degradation in the cytoplasm. To distinguish between these 2 possibilities, we visualized proteins involved in each major step of the 5′ to 3′ mRNA decay pathway by immunofluorescence during the period when nos and pgc degradation begins. CCR4, a component of the CCR4-NOT deadenylation complex, forms puncta that do not colocalize with the germ granules at any point during nc9 to nc14 (S3 Fig), suggesting that deadenylation is not occurring in the germ granules at these stages and may have preceded pole cell formation. At nc10, decapping protein 1 (DCP1), an activating subunit of the mRNA decapping complex, and Pacman (Pcm), the Drosophila homolog of the 5′ to 3′ XrnI exonuclease, form puncta in the pole cells that do not overlap with germ granules (Fig 4A and 4B). However, colocalization of germ granules with DCP1 can be detected beginning at nc12. At this time, 1 to 2 germ granules per pole cell appear to colocalize with DCP1 (Fig 4A). Interestingly, this initial colocalization occurs at the same nuclear cycle when larger germ granules first appear (Fig 1D). The overlap between DCP1 and germ granules further increases in nc13 and nc14 such that 30% of granules associate with DCP1 (Fig 4A and 4C). Pcm follows a similar pattern, but its recruitment to germ granules is delayed by 1 nuclear cycle relative to DCP1. Pcm is first detected in a few germ granules per pole cell at nc13 (Fig 4B). Colocalization increases at nc14, when approximately 30% of granules are associated with Pcm (Fig 4B and 4D). The finding that a decapping co-factor and the Pcm exonuclease associate with germ granules just before nos and pgc levels decrease suggests that germ granules play a role in promoting mRNA degradation in pole cells, which contrasts with their stabilizing role in early embryos. PPT PowerPoint slide

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TIFF original image Download: Fig 4. mRNA decapping proteins and degradation factors localize to germ granules prior to mRNA degradation. (A, B) Single confocal sections of the posterior region of syncytial blastoderm stage embryos expressing a vas-efgp transgene to mark the germ granules. Vas-EGFP (green) was detected by direct fluorescence together with anti-DCP1 immunofluorescence or anti-Pcm immunofluorescence (magenta). Enlargements of the boxed regions show examples of the earliest germ granule colocalization detected at nc12 or 13 (blue) and the strong colocalization at nc14 (yellow) for DCP1 (A) or Pcm (B). The percent of cytoplasmic Vas puncta that colocalize with DCP1 (C) and Pcm (D) puncta was quantified at each nc, n = 5–7 embryos per nc. Nuclear Vas puncta were masked using Imaris software. Values for individual embryos and means are shown. *p < 0.01, **p < 0.01, ***p < 0.001 as determined by Welch ANOVA with Dunnett’s T3 post hoc test. Source data for the graphs in Fig 4C and 4D are provided in S1 Data. Scale bar: 5 μm. DCP1, decapping protein 1; Pcm, Pacman. https://doi.org/10.1371/journal.pbio.3002069.g004

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002069

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