Interplay of RNA-binding proteins controls germ cell development in zebrafish.

The specification of germ cells in zebrafish mostly relies on an inherited mechanism by which localized maternal determinants, called germ plasm, confer germline fate in the early embryo. Extensive studies have partially allowed the identification of key regulators governing germ plasm formation and subsequent germ cell development. RNA-binding proteins, acting in concert with other germ plasm components, play essential roles in the organization of the germ plasm and the specification, migration, maintenance, and differentiation of primordial germ cells. The loss of their functions impairs germ cell formation and causes sterility or sexual conversion. Evidence is emerging that they instruct germline development through differential regulation of mRNA fates in somatic and germ cells. However, the challenge remains to decipher the complex interplay of maternal germ plasm components in germ plasm compartmentalization and germ cell specification. Because failure to control the developmental outcome of germ cells disrupts the formation of gametes, it is important to gain a complete picture of regulatory mechanisms operating in the germ cell lineage. This review sheds light on the contributions of RNA-binding proteins to germ cell development in zebrafish and highlights intriguing questions that remain open for future investigation.

Caenorhabditis elegans germ granules accumulate hundreds of low translation mRNAs with no systematic preference for germ cell fate regulators.

In animals with germ plasm, embryonic germline precursors inherit germ granules, condensates proposed to regulate mRNAs coding for germ cell fate determinants. In Caenorhabditis elegans, mRNAs are recruited to germ granules by MEG-3, a sequence non-specific RNA-binding protein that forms stabilizing interfacial clusters on germ granules. Using fluorescence in situ hybridization, we confirmed that 441 MEG-3-bound transcripts are distributed in a pattern consistent with enrichment in germ granules. Thirteen are related to transcripts reported in germ granules in Drosophila or Nasonia. The majority, however, are low-translation maternal transcripts required for embryogenesis that are not maintained preferentially in the nascent germline. Granule enrichment raises the concentration of certain transcripts in germ plasm but is not essential to regulate mRNA translation or stability. Our findings suggest that only a minority of germ granule-associated transcripts contribute to germ cell fate in C. elegans and that the vast majority function as non-specific scaffolds for MEG-3.

Dzip1 is dynamically expressed in the vertebrate germline and regulates the development of Xenopus primordial germ cells.

Primordial germ cells (PGCs) are the precursors of sperms and oocytes. Proper development of PGCs is crucial for the survival of the species. In many organisms, factors responsible for PGC development are synthesized during early oogenesis and assembled into the germ plasm. During early embryonic development, germ plasm is inherited by a few cells, leading to the formation of PGCs. While germline development has been extensively studied, how components of the germ plasm regulate PGC development is not fully understood. Here, we report that Dzip1 is dynamically expressed in vertebrate germline and is a novel component of the germ plasm in Xenopus and zebrafish. Knockdown of Dzip1 impairs PGC development in Xenopus embryos. At the molecular level, Dzip1 physically interacts with Dazl, an evolutionarily conserved RNA-binding protein that plays a multifaced role during germline development. We further showed that the sequence between amino acid residues 282 and 550 of Dzip1 is responsible for binding to Dazl. Disruption of the binding between Dzip1 and Dazl leads to defective PGC development. Taken together, our results presented here demonstrate that Dzip1 is dynamically expressed in the vertebrate germline and plays a novel function during Xenopus PGC development.

Germ plasm dynamics during oogenesis and early embryonic development in Xenopus and zebrafish.

Specification of the germline and its segregation from the soma mark one of the most crucial events in the lifetime of an organism. In different organisms, this specification can occur through either inheritance or inductive mechanisms. In species such as Xenopus and zebrafish, the specification of primordial germ cells relies on the inheritance of maternal germline determinants that are synthesized and sequestered in the germ plasm during oogenesis. In this review, we discuss the formation of the germ plasm, how germline determinants are recruited into the germ plasm during oogenesis, and the dynamics of the germ plasm during oogenesis and early embryonic development.

Solubility phase transition of maternal RNAs during vertebrate oocyte-to-embryo transition.

The oocyte-to-embryo transition (OET) is regulated by maternal products stored in the oocyte cytoplasm, independent of transcription. How maternal products are precisely remodeled to dictate the OET remains largely unclear. In this work, we discover the dynamic solubility phase transition of maternal RNAs during Xenopus OET. We have identified 863 maternal transcripts that transition from a soluble state to a detergent-insoluble one after oocyte maturation. These RNAs are enriched in the animal hemisphere, and many of them encode key cell cycle regulators. In contrast, 165 transcripts, including nearly all Xenopus germline RNAs and some vegetally localized somatic RNAs, undergo an insoluble-to-soluble phase transition. This phenomenon is conserved in zebrafish. Our results demonstrate that the phase transition of germline RNAs influences their susceptibility to RNA degradation machinery and is mediated by the remodeling of germ plasm. This work thus identifies important remodeling mechanisms that act on RNAs to control vertebrate OET.

Phase transition of maternal RNAs during vertebrate oocyte-to-embryo transition.

The oocyte-to-embryo transition (OET) is regulated by maternal products stored in the oocyte cytoplasm, independent of transcription. How maternal products are precisely remodeled to dictate the OET remains an open question. In this work, we discover the dynamic phase transition of maternal RNAs during Xenopus OET. We have identified 863 maternal transcripts that transition from a soluble state to a detergent-insoluble one after oocyte maturation. These RNAs are enriched in the animal hemisphere and many of them encode key cell cycle regulators. In contrast, 165 transcripts, including nearly all Xenopus germline RNAs and some vegetally localized somatic RNAs, undergo an insoluble-to-soluble phase transition. This phenomenon is conserved in zebrafish. Our results demonstrate that the phase transition of germline RNAs influences their susceptibility to RNA degradation machinery and is mediated by the remodeling of germ plasm. This work thus uncovers novel remodeling mechanisms that act on RNAs to regulate vertebrate OET.

Specialized germline P-bodies are required to specify germ cell fate in Caenorhabditis elegans embryos.

In animals with germ plasm, specification of the germline involves 'germ granules', cytoplasmic condensates that enrich maternal transcripts in the germline founder cells. In Caenorhabditis elegans embryos, P granules enrich maternal transcripts, but surprisingly P granules are not essential for germ cell fate specification. Here, we describe a second condensate in the C. elegans germ plasm. Like canonical P-bodies found in somatic cells, 'germline P-bodies' contain regulators of mRNA decapping and deadenylation and, in addition, the intrinsically-disordered proteins MEG-1 and MEG-2 and the TIS11-family RNA-binding protein POS-1. Embryos lacking meg-1 and meg-2 do not stabilize P-body components, misregulate POS-1 targets, mis-specify the germline founder cell and do not develop a germline. Our findings suggest that specification of the germ line involves at least two distinct condensates that independently enrich and regulate maternal mRNAs in the germline founder cells. This article has an associated 'The people behind the papers' interview.

The germ plasm is anchored at the cleavage furrows through interaction with tight junctions in the early zebrafish embryo.

The zebrafish germline is specified during early embryogenesis by inherited maternal RNAs and proteins collectively called germ plasm. Only the cells containing germ plasm will become part of the germline, whereas the other cells will commit to somatic cell fates. Therefore, proper localization of germ plasm is key for germ cell specification and its removal is crucial for the development of the soma. The molecular mechanism underlying this process in vertebrates is largely unknown. Here, we show that germ plasm localization in zebrafish is similar to that in Xenopus but distinct from Drosophila. We identified non muscle myosin II (NMII) and tight junction (TJ) components, such as ZO2 and claudin-d (Cldn-d) as interaction candidates of Bucky ball (Buc), which is the germ plasm organizer in zebrafish. Remarkably, we also found that TJ protein ZO1 colocalizes with germ plasm, and electron microscopy of zebrafish embryos uncovered TJ-like structures at the cleavage furrows where the germ plasm is anchored. In addition, injection of the TJ receptor Cldn-d produced extra germ plasm aggregates, whereas expression of a dominant-negative version inhibited germ plasm aggregate formation. Our findings support for the first time a role for TJs in germ plasm localization.

Germline specification and axis determination in viviparous and oviparous pea aphids: conserved and divergent features.

Aphids are hemimetabolous insects that undergo incomplete metamorphosis without pupation. The annual life cycle of most aphids includes both an asexual (viviparous) and a sexual (oviparous) phase. Sexual reproduction only occurs once per year and is followed by many generations of asexual reproduction, during which aphids propagate exponentially with telescopic development. Here, we discuss the potential links between viviparous embryogenesis and derived developmental features in the pea aphid Acyrthosiphon pisum, particularly focusing on germline specification and axis determination, both of which are key events of early development in insects. We also discuss potential evolutionary paths through which both viviparous and oviparous females might have come to utilize maternal germ plasm to drive germline specification. This developmental strategy, as defined by germline markers, has not been reported in other hemimetabolous insects. In viviparous females, furthermore, we discuss whether molecules that in other insects characterize germ plasm, like Vasa, also participate in posterior determination and how the anterior localization of the hunchback orthologue Ap-hb establishes the anterior-posterior axis. We propose that the linked chain of developing oocytes and embryos within each ovariole and the special morphology of early embryos might have driven the formation of evolutionary novelties in germline specification and axis determination in the viviparous aphids. Moreover, based upon the finding that the endosymbiont Buchnera aphidicola is closely associated with germ cells throughout embryogenesis, we propose presumptive roles for B. aphidicola in aphid development, discussing how it might regulate germline migration in both reproductive modes of pea aphids. In summary, we expect that this review will shed light on viviparous as well as oviparous development in aphids.

Sinhcaf-dependent histone deacetylation is essential for primordial germ cell specification.

Primordial germ cells (PGCs) are the progenitor cells that give rise to sperm and eggs. Sinhcaf is a recently identified subunit of the Sin3 histone deacetylase complex (SIN3A-HDAC). Here, we provide evidence that Sinhcaf-dependent histone deacetylation is essential for germ plasm aggregation and primordial germ cell specification. Specifically, maternal-zygotic sinhcaf zebrafish mutants exhibit germ plasm aggregation defects, decreased PGC abundance and male-biased sex ratio, which can be rescued by re-expressing sinhcaf. Overexpression of sinhcaf results in excess PGCs and a female-biased sex ratio. Sinhcaf binds to the promoter region of kif26ab. Loss of sinhcaf epigenetically switches off kif26ab expression by increasing histone 3 acetylation in the promoter region. Injection of kif26ab mRNA could partially rescue the germ plasm aggregation defects in sinhcaf mutant embryos. Taken together, we demonstrate a role of Sinhcaf in germ plasm aggregation and PGC specialization that is mediated by regulating the histone acetylation status of the kif26ab promoter to activate its transcription. Our findings provide novel insights into the function and regulatory mechanisms of Sinhcaf-mediated histone deacetylation in PGC specification.

Primordial Germ Cell Development in the Poeciliid, Gambusia holbrooki, Reveals Shared Features Between Lecithotrophs and Matrotrophs.

Metazoans exhibit two modes of primordial germ cell (PGC) specification that are interspersed across taxa. However, the evolutionary link between the two modes and the reproductive strategies of lecithotrophy and matrotrophy is poorly understood. As a first step to understand this, the spatio-temporal expression of teleostean germ plasm markers was investigated in Gambusia holbrooki, a poecilid with shared lecitho- and matrotrophy. A group of germ plasm components was detected in the ovum suggesting maternal inheritance mode of PGC specification. However, the strictly zygotic activation of dnd-ß and nanos1 occurred relatively early, reminiscent of models with induction mode (e.g., mice). The PGC clustering, migration and colonisation patterns of G. holbrooki resembled those of zebrafish, medaka and mice at blastula, gastrula and somitogenesis, respectively-recapitulating features of advancing evolutionary nodes with progressive developmental stages. Moreover, the expression domains of PGC markers in G. holbrooki were either specific to teleost (vasa expression in developing PGCs), murine models (dnd spliced variants) or shared between the two taxa (germline and somatic expression of piwi and nanos1). Collectively, the results suggest that the reproductive developmental adaptations may reflect a transition from lecithotrophy to matrotrophy.

Germ plasm and the origin of the primordial germ cells in the oriental river prawn Macrobrachium nipponense.

Germ plasm is a special cytoplasmic component containing special RNAs and proteins, and is located in specific regions of oocytes and embryos. Only the blastomeres inheriting the germ plasm can develop into primordial germ cells (PGCs). By using Vasa mRNA as a germline marker, we previously demonstrated that germline specification followed the preformation mode in the prawn Macrobrachium nipponense. In this study, we raised the Vasa antibody to identify germ plasm in the oocyte and trace the origin and migration of PGCs. In previtellogenic oocytes, Vasa protein was detected in the perinuclear region, in which electron-dense granules associated with numerous mitochondria were mostly visualized under a transmission electron microscope. In mature oocytes, immunosignal was localized to a large granule under the plasma membrane. During early embryogenesis, the granule was inherited by a single blastomere from 1-cell to 16-cell stages, and thereafter was segregated into two daughter blastomeres at the 32-cell stage. In gastrula, the Vasa-positive cells were large with typical PGC characteristics, containing a big round nucleus and a prominent nucleolus. The immunosignal was localized to the perinuclear region again. In the zoea stage, the Vasa-positive cells migrated toward the genital ridge and clustered in the dorsomedial region close to the yolk portion. Accordingly, we concluded that the prawn PGCs could be specified from the 16-cell stage by inheriting the germplasm. To our knowledge, this is the first report on the identification of the prawn germ plasm and PGCs. The continuous expression of Vasa protein throughout oogenesis and embryogenesis suggests that Vasa protein could be an important factor in germ plasm that functions in early germ cell specification.

Tools to Image Germplasm Dynamics During Early Zebrafish Development.

During the first day of zebrafish development, ribonucleoprotein (RNP) complexes called germplasm form large aggregates that initially segregate asymmetrically during cleavage stages. After zygotic genome activation, the granules break into smaller fragments that associate with the nuclear membrane as perinuclear (germ) granules toward the end of gastrulation. The mechanisms underlying the highly dynamic behavior of germ granules are not well studied but thought to be facilitated by the cytoskeleton. Here, we present efficient mounting strategies using 3d-printed tools that generate wells on agarose-coated sample holders to allow high-resolution imaging of multiplexed embryos that are less than one day post-fertilization (dpf) on inverted (spinning disk confocal) as well as upright (lattice light-sheet and diSPIM) microscopes. In particular, our tools and methodology allow water dipping lenses to have direct access to mounted embryos, with no obstructions to the light path (e.g., through low melting agarose or methyl cellulose). Moreover, the multiplexed tight arrays of wells generated by our tools facilitate efficient mounting of early embryos (including cleavage stages) for live imaging. These methods and tools, together with new transgenic reporter lines, can facilitate the study of germ granule dynamics throughout their lifetime in detail, at high resolution and throughput, using live imaging technologies.

Evolution of a Cytoplasmic Determinant: Evidence for the Biochemical Basis of Functional Evolution of the Novel Germ Line Regulator Oskar.

Germ line specification is essential in sexually reproducing organisms. Despite their critical role, the evolutionary history of the genes that specify animal germ cells is heterogeneous and dynamic. In many insects, the gene oskar is required for the specification of the germ line. However, the germ line role of oskar is thought to be a derived role resulting from co-option from an ancestral somatic role. To address how evolutionary changes in protein sequence could have led to changes in the function of Oskar protein that enabled it to regulate germ line specification, we searched for oskar orthologs in 1,565 publicly available insect genomic and transcriptomic data sets. The earliest-diverging lineage in which we identified an oskar ortholog was the order Zygentoma (silverfish and firebrats), suggesting that oskar originated before the origin of winged insects. We noted some order-specific trends in oskar sequence evolution, including whole gene duplications, clade-specific losses, and rapid divergence. An alignment of all known 379 Oskar sequences revealed new highly conserved residues as candidates that promote dimerization of the LOTUS domain. Moreover, we identified regions of the OSK domain with conserved predicted RNA binding potential. Furthermore, we show that despite a low overall amino acid conservation, the LOTUS domain shows higher conservation of predicted secondary structure than the OSK domain. Finally, we suggest new key amino acids in the LOTUS domain that may be involved in the previously reported Oskar-Vasa physical interaction that is required for its germ line role.

Diversity of Balbiani body formation in internally and externally fertilizing representatives of Osteoglossiformes (Teleostei: Osteoglossomorpha).

During the early stages of oogenesis, the Balbiani body is formed in the primary oocytes. It consists of the Golgi apparatus, endoplasmic reticulum (ER), and numerous mitochondria aggregated with germ plasm, but its form may differ among animals. Hypothetically, during oogenesis oocytes become adapted to future development in two different environments depending on internal or external fertilization. We aimed to investigate, using light and transmission electron microscopy, the development of the Balbiani body during oogenesis in representatives of Osteoglossiformes, one of the most basal Teleostei groups. We analyzed the structure of oogonia and primary oocytes in the internally fertilizing butterflyfish Pantodon buchholzi and the externally fertilizing Osteoglossum bicirrhosum and Arapaima gigas to compare formation of the Balbiani body in relation to modes of fertilization. We demonstrated that the presence of the germ plasm as well as the fusion and fission of mitochondria are the conserved features of the Bb. However, each species exhibited also some peculiar features, including the presence of three types of ooplasm with different electron density and mitochondria-associated membranes in P. buchholzi; annulate lamellae, complexes of the Golgi apparatus, ER network, and lysosome-like bodies in O. bicirrhosum; as well as karmellae and whorls formed by the lamellae of the ER in A. gigas. Moreover, the form of the germ plasm observed in close contact with mitochondria differed between osteoglossiforms, with a "net-like" structure in P. buchholzi, the presence of numerous strings in O. bicirrhosum, and irregular accumulations in A. gigas. These unique features indicate that the extreme diversity of gamete structure observed so far only in the spermatozoa of osteoglossiforms is also characteristic for oocyte development in these basal teleosts. Possible reason of this variability is a period of about 150 million years of independent evolution of the lineages.

Protein-based condensation mechanisms drive the assembly of RNA-rich P granules.

Germ granules are protein-RNA condensates that segregate with the embryonic germline. In Caenorhabditis elegans embryos, germ (P) granule assembly requires MEG-3, an intrinsically disordered protein that forms RNA-rich condensates on the surface of PGL condensates at the core of P granules. MEG-3 is related to the GCNA family and contains an N-terminal disordered region (IDR) and a predicted ordered C-terminus featuring an HMG-like motif (HMGL). We find that MEG-3 is a modular protein that uses its IDR to bind RNA and its C-terminus to drive condensation. The HMGL motif mediates binding to PGL-3 and is required for co-assembly of MEG-3 and PGL-3 condensates in vivo. Mutations in HMGL cause MEG-3 and PGL-3 to form separate condensates that no longer co-segregate to the germline or recruit RNA. Our findings highlight the importance of protein-based condensation mechanisms and condensate-condensate interactions in the assembly of RNA-rich germ granules.

Early germline differentiation in bivalves: TDRD7 as a candidate investigational unit for Ruditapes philippinarum germ granule assembly.

The germline is a key feature of sexual animals and the ways in which it separates from the soma differ widely across Metazoa. However, at least at some point during germline differentiation, some cytoplasmic supramolecular structures (collectively called germ plasm-related structures) are present and involved in its specification and/or differentiation. The factors involved in the assembly of these granular structures are various and non-ubiquitous among animals, even if some functional patterns and the presence of certain domains appear to be shared among some. For instance, the LOTUS domain is shared by Oskar, the Holometabola germ plasm master regulator, and some Tudor-family proteins assessed as being involved in the proper assembly of germ granules of different animals. Here, we looked for the presence of LOTUS-containing proteins in the transcriptome of Ruditapes philippinarum (Bivalvia). Such species is of particular interest because it displays annual renewal of gonads, sided by the renewal of germline differentiation pathways. Moreover, previous works have identified in its early germ cells cytoplasmic granules containing germline determinants. We selected the orthologue of TDRD7 as a candidate involved in the early steps of germline differentiation through bioinformatic predictions and immunohistological patterning (immunohistochemistry and immunofluorescence). We observed the expression of the protein in putative precursors of germline cells, upstream to the germline marker Vasa. This, added to the fact that orthologues of this protein are involved in the assembly of germ granules in mouse, zebrafish, and fly, makes it a worthy study unit for investigations on the formation of such structures in bivalves.

Visualization of Transcriptional Activity in Early Zebrafish Primordial Germ Cells.

Here, we describe a fast and straightforward methodology to in vivo detect transcriptional activity in the early zebrafish germ line. We report how fluorescently labeled morpholinos, targeted to nascent early transcripts, can be used to track the onset of transcriptional events during early embryogenesis. This method could be applied to any tagged cell line in a developing early zebrafish embryo as long as the gene of interest is expressed at high enough level for morpholino detection and is expressed at the first and main wave of genome activation, for which the protocol has been verified. The protocol, in combination with genetic manipulation, allows studies of mechanisms driving zygotic genome activation (ZGA) in individual cells. The reported procedures apply to a broad range of purposes for zebrafish embryo manipulation in view of imaging nuclear molecules in specific cell types.

Glyoxal Fixation as an Alternative for Zebrafish Embryo Immunostaining.

Immunohistochemistry has been widely used as a robust technique to determine the cellular and subcellular localization of proteins. This information ultimately helps to understand the function of these proteins and how biological processes are regulated. Antibodies applicable for labeling in zebrafish are limited, making immuno-staining challenging. Recently glyoxal fixation was rediscovered in tissue culture, mouse, rat, and Drosophila, expanding the list of effective antibodies for these species. Here, we compare a protocol for zebrafish staining using glyoxal as a fixative agent with PFA. We demonstrate that glyoxal fixation improves the antigenicity of some epitopes thereby increasing the number of useful antibodies in zebrafish.

m6A reader Igf2bp3 enables germ plasm assembly by m6A-dependent regulation of gene expression in zebrafish.

Bucky ball (Buc) is involved in germ plasm (GP) assembly during early zebrafish development by regulating GP mRNA expression via an unknown mechanism. The present study demonstrates that an m6A reader Igf2bp3 interacts and colocalizes with Buc in the GP. Similar to the loss of Buc, the genetic deletion of maternal igf2bp3 in zebrafish leads to abnormal GP assembly and insufficient germ cell specification, which can be partially restored by the injection of igf2bp3 mRNA. Igf2bp3 binds to m6A-modified GP-organizer and GP mRNAs in an m6A-dependent manner and prevents their degradation. These findings indicate that the functions of Igf2bp3, a direct effector protein of Buc, in GP mRNA expression and GP assembly involve m6A-dependent regulation; these results emphasize a critical role of m6A modification in the process of GP assembly.