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Germ granules in Drosophila

Abstract Germ granules are hallmarks of all germ cells. Early ultrastructural studies in Drosophila first described these membraneless granules in the oocyte and early embryo as filled with amorphous to fibrillar material mixed with RNA. Genetic studies identified key protein components and specific mRNAs that regulate germ cell‐specific functions. More recently these ultrastructural studies have been complemented by biophysical analysis describing germ granules as phase‐transitioned condensates. In this review, we provide an overview that connects the composition of germ granules with their function in controlling germ cell specification, formation and migration, and illuminate these mysterious condensates as the gatekeepers of the next generation.


| GERM PLASM AND GERM GRANULES OF DROSOPHILA-FIRST DESCRIPTIONS
In his search for a "heritable substance" that is transmitted from generation to generation, German biologist August Weismann proposed in 1893 that the offspring owes its origin to a "peculiar substance of extremely complicated structure" called the germ plasm. 1 It is this structure, he explained, that distinguishes cells that give rise to the next generation from those that produce the "perishable body." Experimental support for such a germ cell determinant was later provided by Robert Hegner's pricking experiments where he removed the germ plasm from the beetle Calligrapha punctate and found that the resulting embryos lacked morphologically discernable germ cells. 2 With microscopy techniques of the time, Hegner described the germ plasm as containing "special bodies," which were dense and stained like "chromatin." 3 However, it was not until the early 1960s, when the detailed electron microscopic (EM) studies of Drosophila melanogaster and other Drosophilid embryos by Tony Mahowald revealed the existence of morphologically unique structures found within the germ plasm, called the germ granules ( Figure 1A). Mahowald described these granules as round, membraneless bodies about 0.2 to 0.5 μm in diameter, which contained fibrous material and ribosomes and stained with nucleic acid markers. 8 These studies also revealed that granules could have a defined structure, with the periphery more electron dense than the core, and could often closely associate with mitochondria 9 ( Figure 1A 10,11 Nuclei located in the transplanted region formed cells with the morphology of "pole cells," as the primordial germ cells (PGCs) in Drosophila are called. Moreover, these ectopic pole cells gave rise to functional germ cells after being transferred into a host embryo. 10,11 Some of the key proteins and mRNAs contained in germ plasm were later genetically identified as the products of so-called maternal effect genes required maternally to regulate the assembly of germ plasm during oogenesis and the function of germ cells in the resulting embryo. Mutations in genes required for germ plasm assembly lead to a "grand-childless" phenotype due to the loss of germ cells in the progeny of mutant mothers. [12][13][14] Among these genes,  14,15 Thus, the anterior localization of osk's ORF and the resulting local production of Osk protein at the anterior pole was sufficient to attract other germ plasm components, leading to assembly of germ granules and specification of functional germ cells at the ectopic location. 15 Interestingly, Oskar is not conserved beyond dipterans 16  Early EM studies revealed that germ granules first appear as small and dense bodies at the posterior pole of the developing oocyte, are later inherited by the fertilized embryo and finally become engulfed by the newly formed pole cells. Modern molecular biology, microscopy and genetic tools revealed that these granules accumulate maternally provided messenger ribonucleic acids (mRNAs) and proteins critical for the establishment of PGCs and the germline; in their absence, germ cells do not form and the resultant embryo is sterile (reviewed in Reference 4). Thus, while Weismann was looking for the heritable substance, the DNA, he instead identified germ granules as the hallmark substance that provides continuity of the species, the subject discussed in this review. To ensure the continuity of the species, Drosophila evolved an intricate mechanism by which osk reaches the posterior pole (described in detail 4 ). Briefly, osk mRNA is synthesized by the nurse cells, which are  Barentsz. [20][21][22][23][24] Transport and mRNA localization to the posterior pole rely on the ability of osk mRNA to dimerize and are coordinated by cis-acting sequences positioned within the osk 3 0 UTR and the first exon junction in osk. 25 Additional sequences in the osk 3 0 UTR are specifically recognized by translational regulatory proteins such as the RNA-binding protein Bruno, which represses oskar translation during transport. [26][27][28] The switch from minus-to plus-end-directed microtubule transport depends on the germline-specific isoform of tropomyosin. [29][30][31] In addition to this early, motor-dependent enrichment, osk accumulation also relies on a motor-independent enrichment process during later stages of oogenesis and is mediated by the RNA-binding proteins Rump and Lost. 32 This accumulation of osk together with the continuous production of Oskar protein amplifies the amount of germ plasm available to form germ granules. 5,18,32,33 Upon localization, the repressive activity of Bruno is inhibited, allowing osk translation. Together these regulatory mechanisms ensure

| Organization of proteins in germ granules
Germ plasm is a specialized cytoplasm that forms at the embryo's posterior pole and is populated by core granule proteins Oskar, Vasa, Tud and Aub, a variety of RNA-binding proteins involved in various aspects of RNA biology, maternally deposited mRNAs, piRNAs and mitochondria ( Figure 1A-D) (reviewed in Reference 37). While most of the RNA-binding proteins enriched in germ plasm are also found elsewhere in the embryo, the core granule proteins are almost exclusively found only at the posterior pole. 38,39 The exact composition of germ plasm is not known, however, recent quantitative imaging data demonstrated that the vast majority of core germ plasm proteins (>94%) are condensed into granules, with very little of these proteins diffusing in the intergranular germ plasm space. 38 Indeed, their concentration in the intergranular space is similar to their concentration in somatic regions. 38 For germ plasm mRNAs, the majority of transcripts that enrich at the posterior remain dispersed in somatic regions of the embryo with only up to 4% of a particular mRNA enriched in germ plasm. 7,40 Despite this small fraction, however, these mRNAs become 8-to 10-fold more concentrated upon localization. 7 Some mRNAs that enrich in germ plasm also tend to preferentially accumulate in granules. For instance, whole-mount in situ labeling of 59 mRNAs revealed that these transcripts interact with germ granules; they arrange as crescents surrounding the dividing PGC nuclei in older embryos, a spatial organization driven entirely by germ granule proteins ( known granule-enriched mRNAs. 38 Nuclear germ granules are bigger and often appear hollow in EM and fluorescence images ( Figure 2B), 38,50 and some cytoplasmic germ granules seem to have similar protein lucid cores. In both types of granules, this core appears less electron dense in EM images than the outside granule rim ( Figure 2B). 8,38,50,51 A short nuclear localization sequence controls nuclear import of Osk and cotransport of Vasa into PGC nuclei where the two proteins condense into the same granules. As with cytoplasmic granules, the majority of Osk and Vasa is found in granules rather than freely diffusing in the nucleoplasm. 38 How these nuclear granules regulate of PCG number is currently unknown.
Short Osk contains two, structurally discernable protein domains.
The N-terminus forms the LOTUS domain named after the Limkain (a human autoantigen whose function and binding partners are unknown), Oskar, and Tudor-containing proteins 5 and 7 (TDRD5 and TDRD7 proteins, respectively), two mammalian members of the germline Tudor group. 52 The C-terminus is a novel RNA-binding domain that shares similarity to SGNH hydrolases but lacks its enzymatic activity. 53  In addition to mRNAs, germ granules recruit Tudor 58 and Aubergine 59 proteins ( Figure 1D). Aub is a member of the Argonaute/Piwi family of proteins and binds to small piRNAs that regulate transposable elements but has also been implicated in mRNA binding (see below 60 ).
Tud consists of 11 Tudor domains, each able to bind symmetrically methylated arginines found in Aub. Tud tethers Aub via its Tudor domain to the germ plasm, as mutations that affect Tudor's ability to bind dimethylated arginines strongly reduce the localization of Aub to the posterior pole ( Figure 1D). 61 51 indicating that Tud plays a central role in maintaining the integrity and the morphology of germ granules in Drosophila ( Figure 1D).
Oskar/Vasa and Tudor/Aubergine form specific complexes of high affinity interactions. However, these proteins also associate into large membraneless droplets that by other criteria such as high concentration, mobility of components and variable stochiometry resemble RNA droplets such as stress granules, processing bodies and germ granules called P-granules of the round worm Caenorhabditis elegans (C. elegans). These RNA granules, including germ granules in Drosophila, are composed of diverse RNA-binding proteins and mRNAs and form by phase separation, a process best described as oil-and-water demixing. 37 Biophysical studies further demonstrated that Drosophila germ granules display both liquid-like and hydrogel-like properties and are thus best described as phase-transitioned condensates. Specifically, a fraction of core granule proteins readily exchanges with the granule environment while a fraction appears highly immobile and is retained within granules. 38 Figure 1D). 34 Most enriched mRNAs reach the posterior and anchor into germ granules during mid-oogenesis by passive diffusion-entrapment mechanisms ( Figure 3). 36 Here, transcriptionally active nurse cells dump their content into a quiescent and growing oocyte. The oocyte forms microtubule bundles, which cause cytoplasmic streaming in the oocyte that swirls the cytoplasm through the oocyte enabling mRNAs to entrap as they brush along the germ granules formed at the posterior pole ( Figure 3). 36 Deep-sequencing studies have revealed that the initial entrapment of mRNAs into granules could be mediated by RNA:RNA interactions via short RNAs called piRNAs loaded into the Aubergine protein. In this model, partial complementarity is established between piRNAs and target mRNAs, which is sufficient to initially recruit and anchor a variety of transcripts in germ granules. 60 For several mRNAs, the sequences proposed to mediate complementarity between piRNAs and target transcripts reside within the mRNAs' 3 0 untranslated regions (UTRs). 60 This result agrees well with previous findings using transgenically expressed reporter mRNAs, which demonstrated that sequences necessary and sufficient for posterior localization of nos, gcl and pgc reside within their 3 0 UTR. 42,43,71 Additionally, recent highresolution microscopy studies have shed new light on the mechanism by which mRNAs such as nos, CycB, gcl and pgc may become highly enriched and organized within germ granules. While individual mRNAs localize as single transcripts, upon enrichment, however, between 2 to more than 40 mRNA molecules derived from the same gene organize into homotypic mRNA clusters within germ granules ( Figure 1C). 5-7 Importantly, these homotypic clusters occupy defined positions from the center to the periphery of granules, while the core granule proteins Osk, Vasa, Tud and Aub, that make the granules and recruit other granule components including mRNAs are homogeneously distributed within the same granule. 7 These results suggest that the assembly of homotypic clusters is driven at least in part, by the granule mRNAs themselves, possibly through direct RNA-RNA interactions. In support of a process mediated by RNA-RNA interactions, mRNAs seem more likely to associate with each other than equally distributing across all granules, leaving some granules devoid of specific mRNA species ( Figure 1C). 5-7 Additionally, granule mRNAs display an ability to self-recruit to granules, 5 46,72,73 (c) Translational regulation. A common feature of posteriorly localized mRNAs is that they are translated while the 96% of unlocalized counterparts distributed throughout the embryo that will give rise to future soma, are translationally repressed. 42,71 (d) mRNAs stored in the germ granules are translated temporally in the order of need ( Figure 2E). 42 For many mRNAs it has been shown that translational repression outside of germ granules depends on the RNA-binding protein Smaug, which recruits the CCR4/NOT deadenylases to the respective mRNAs. 74,75 For nanos mRNA, translational activation has been linked to germ plasm localization via Oskar, which displaces Smaug from nos thereby preventing the deadenylation of nos CCR4/NOT complex and increasing the stability of the mRNA while simultaneously also relieving translational repression imposed by Smaug. 76 It is unlikely that this is a general regulatory mechanism for germ plasm-localized mRNAs as not all posteriorly localized mRNAs are Smaug targets. 46 (Figure 2A,E). 42 Curiously, the spatial organization of homotypic mRNA clusters within granules does not predict when an mRNA becomes translated nor how effectively it will be protected from somatic mRNA decay. 7 Whether this mRNA self-organization may influence other aspects of posttranscriptional control of localized transcripts is presently unclear.

| Functions of germ plasm and germ granules
Components enriched in the germ plasm and germ granules control critical germ cell functions. Several of these functions can be directly linked to specific mRNAs, including those required for germ cell fate specification (nos), germ cell formation ( gcl), transcriptional silencing (pgc), germ cell migration (tre1) and germ cell survival (wun2). Other germ cell functions, such as mitochondrial inheritance and germ cell genome integrity also rely on maternally synthesized factors. The latter, however, are not linked to a single gene but to the deposition of small RNAs, called piRNAs, which protect germ cells against transposable elements.

| Nanos suppresses somatic fate in germ cells
Germ cell specification requires the function of the nanos gene. nanos translation is initiated specifically at the posterior pole as soon as the mRNA becomes localized (Figure 2A,D,E) and the resulting Nanos protein forms a posterior-to-anterior gradient. Germ granule localization of nos followed by local translation and formation of the gradient plays a critical role in establishing the anterior-posterior segmental patterning of the embryo. 43 Nanos together with its cofactor Pumilio inhibits the translation of maternal hunchback (hb) mRNA 77 and the absence of Hb protein in the posterior region allows the expression of gap genes required for abdomen formation. 78,79 Thus, in the absence of maternally provided Nos, embryos fail to form an abdomen. 13 Additionally, failure to localize nos and defects in posterior Nos protein translation account for the abdominal phenotype of the maternal effect genes required for germ plasm assembly (so called posterior group phenotype). 13,79 Nanos is not required for germ cell formation per se, but it is required to preserve a germ cell-specific cell cycle program. As a consequence, in nanos mutant embryos, pole cells express somatic genes, undergo apoptosis, migrate aberrantly and lose their ability to give rise to functional germ cells. 80,81 How Nanos promotes germ cell specification remains unclear, however, recent experiments have provided some insight into this question. The tumor suppressor L(3)mbt was shown to secure somatic cellular identity in Drosophila ovaries and larval brains by repressing germline-specific genes, including nanos. 82 Given its well-documented role in translational regulation, it is likely that the primary role of Nanos is to repress factors that promote somatic development rather than to actively promote germ cell development, akin to its recently reported role in C. elegans. 83 Whether Nanos also has an independent role in actively promoting germ cell fate is less clear.  45 GCL is not directly involved in transcriptional silencing or centrosome segregation during germ cell formation as previously suggested. 44,86,87 Instead, GCL functions as a substrate-specific adaptor for the Cullin 3-Ubiquitin-ligase complex. 45 GCL targets the receptor tyrosine kinase (RTK) Torso for degradation via the conserved GCL substrate-binding motif. During pole cell formation, GCL translocates from the inner nuclear membrane to the cell membrane where it leads to the degradation of Torso. Amazingly, pole cell formation is fully restored in gcl, torso double mutants, indicating that Torso is the single critical target of GCL in the early embryo and involved in the formation of basal constriction promoted by GCL. 44,45 How Torso activation inhibits this constriction is not fully understood. Preliminary studies suggest that this process is independent of the conventional downstream components of the Torso RTK including MAP kinase-mediated transcriptional activation of target genes. 45 These studies have also suggested that additional, yet unknown germ plasm-localized factors can promote basal furrow constrictions in the absence of gcl and torso.

| Pgc represses transcription in germ cells
Major transcriptional activity in germ cells is not observed until they reach the gonad, although PGCs become transcriptionally competent shortly after they form. 88  The ability to survive and reach the gonad is directly correlated with the amount of germ plasm inherited. 94 Indeed, the levels of a single germ plasm-localized mRNA, Wunen 2, which encodes a protein that is a homolog of mammalian lipid-phosphate phosphatase 3, was shown to be a quantitative regulator of germ cell survival. 94 Another regulator of germ cell migration, the G-protein-coupled receptor Tre1 is also synthesized maternally and deposited as a localized mRNA to the germ plasm and translated there. Tre1 receptor activation orients germ cells as they exit the gut and directs their migration toward the somatic gonad. 95 Thus, heterogeneity of germ plasm, inherited at the time primordial germ cells form, predetermines the success of these cells for future generations.

| Mitochondria and germ plasm
The transmission of mitochondria through the germ line is an essential component of maternal inheritance. Mitochondria are passed from the mother to the progeny in most organisms and specific mechanisms exist to eliminate paternal mitochondria. 96 Consequently, the mitochondria of PGCs will constitute the pool from which all mitochondria of the next generation originate. The original EM images suggested that germ granules and mitochondria could closely associate in the germ plasm. 68 Subsequently, it was proposed that mitochondrial large and small ribosomal RNAs (mtrRNA), which are transcribed by the mitochondrial genome, were extra-mitochondrial in the germ plasm and associated with germ granules. 97 It was hypothesized that these extra-mitochondrial mtrRNAs induced the assembly of "mitochondrial-like" ribosomes within the germ plasm and directed translation of germ plasm mRNAs with mitochondrial codon usage. 98,99 While intriguing, recent high-resolution imaging showed that mtrRNA localizes separate from germ granule markers such as nanos mRNA and is strictly confined to the mitochondria at the posterior pole as well as elsewhere in the embryo. 48 This study shows that mitochondria are enriched at the posterior pole, consistent with the previous EM observations. Closer examination of the mechanisms by which mitochondria become enriched at the posterior pole showed that this localization requires the Long Osk ( Figure 1D) and is independent of the germ plasm inducing function of Short Osk. 48 Both Long Osk as well as just the 138 aa N-terminal extension of Oskar, which distinguishes Long Osk from Short Osk, are sufficient to localize mitochondria to an ectopic location. 48 Previous studies have suggested that Long Osk enhances the cortical recruitment and maintenance of osk mRNA at the posterior pole by mediating the formation of actin filaments and recruitment of endosomes. 19,100 The new studies further show that Long Osk also directly associates with actin cytoskeleton and that this function is critical for mitochondrial enrichment at the posterior pole. 48 The functional significance of this mitochondrial enrichment in PGCs is unclear. Germ cells form in the absence of Long Osk on condition that Short Osk is provided separately, however, their number is reduced and they contain fewer mitochondria. This reduction in PGC number could be an indirect consequence of loss of Long Osk diminishing the ability of osk mRNA to anchor at the cortex, thereby translating less Short Osk and forming fewer germ granules. Alternatively, this finding may suggest a more direct requirement for increased mitochondrial activity during PGC formation. The later idea is supported by experiments that interfered with prokaryotic (and mitochondrial) translation specifically at the posterior pole and resulted in a reduction in germ cell number. 99 Beyond a potential functional role during the formation of germ cells, enrichment of mitochondria could impact germ cell development in other ways. Germ granules are closely associated with mitochondria ( Figure 1B) 105 Recent studies using immunoprecipitation followed by RNA sequencing suggested that in addition to targeting TEs, maternally deposited piRNAs associated with Aubergine and Tudor proteins also enriched for germ plasm localized mRNA. 60 In this scenario, piRNA-mRNA recognition would trap mRNAs in the germ plasm. In these experiments, piRNA-mRNA pairs did not show any sequence specificity for germ plasm localized RNAs and it was therefore proposed that enrichment of localized mRNAs is achieved by special mRNA features such as longer 3 0 UTRs. 60 While intriguing, other studies have suggested that Aubergine may bind directly and preferentially to germ plasm-enriched mRNAs to stabilize them. In contrast, in the soma Aubergine/mRNA interactions lead to mRNA degradation. 106 What are the conserved and unique principles of composition, structure and function of germ granules compared to other droplets formed by phase separation? New quantitative, super-resolution imaging studies combined with biochemistry and genetics are providing a framework to begin addressing some of these questions. These methods allow us to dissect the function of germ granules and explore how concentration of protein and mRNA components in granules establishes a highly specialized program that controls posttranscriptional regulation of mRNAs required for many aspects of early germ cell biology. As for the past century, with modern technology and approaches these mysterious condensates will continue to fascinate us.

ACKNOWLEDGMENTS
We would like to acknowledge NYULH Microscopy Lab for providing electron microscopy services, whose resources are partially supported by Cancer Center Support Grant P30 CA016087-38 (Laura and Isaac Perlmutter Cancer Center). This work was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development K99HD088675 grant awarded to T.T. RL is an HHMI investigator.