BAP1 regulates epigenetic switch from pluripotency to differentiation in developmental lineages giving rise to BAP1-mutant cancers.

The BAP1 tumor suppressor is mutated in many human cancers such as uveal melanoma, leading to poor patient outcome. It remains unclear how BAP1 functions in normal biology or how its loss promotes cancer progression. Here, we show that Bap1 is critical for commitment to ectoderm, mesoderm, and neural crest lineages during Xenopus laevis development. Bap1 loss causes transcriptional silencing and failure of H3K27ac to accumulate at promoters of key genes regulating pluripotency-to-commitment transition, similar to findings in uveal melanoma. The Bap1-deficient phenotype can be rescued with human BAP1, by pharmacologic inhibition of histone deacetylase (HDAC) activity or by specific knockdown of Hdac4. Similarly, BAP1-deficient uveal melanoma cells are preferentially vulnerable to HDAC4 depletion. These findings show that Bap1 regulates lineage commitment through H3K27ac-mediated transcriptional activation, at least in part, by modulation of Hdac4, and they provide insights into how BAP1 loss promotes cancer progression.

Novel functions of the ubiquitin-independent proteasome system in regulating Xenopus germline development.

In most species, early germline development occurs in the absence of transcription with germline determinants subject to complex translational and post-translational regulations. Here, we report for the first time that early germline development is influenced by dynamic regulation of the proteasome system, previously thought to be ubiquitously expressed and to serve 'housekeeping' roles in controlling protein homeostasis. We show that proteasomes are present in a gradient with the highest levels in the animal hemisphere and extending into the vegetal hemisphere of Xenopus oocytes. This distribution changes dramatically during the oocyte-to-embryo transition, with proteasomes becoming enriched in and restricted to the animal hemisphere and therefore separated from vegetally localized germline determinants. We identify Dead-end1 (Dnd1), a master regulator of vertebrate germline development, as a novel substrate of the ubiquitin-independent proteasomes. In the oocyte, ubiquitin-independent proteasomal degradation acts together with translational repression to prevent premature accumulation of Dnd1 protein. In the embryo, artificially increasing ubiquitin-independent proteasomal degradation in the vegetal pole interferes with germline development. Our work thus reveals novel inhibitory functions and spatial regulation of the ubiquitin-independent proteasome during vertebrate germline development.

Methods for Isolating the Balbiani Body/Germplasm from Xenopus laevis Oocytes.

The Balbiani body (Bb) is a large membrane-less organelle, densely packed with mitochondria, endoplasmic reticulum, proteins, and RNA. The Bb is present in many vertebrate female gametes. In frogs, the Bb is established early during oogenesis and operates as a maternal inherited embryonic determinant that specifies germline identity through the formation of germplasm. We describe here two techniques to isolate the Bb/germplasm from Xenopus laevis primary oocytes.

Combined functions of two RRMs in Dead-end1 mimic helicase activity to promote nanos1 translation in the germline.

Dead-end1 (Dnd1) expression is restricted to the vertebrate germline where it is believed to activate translation of messenger RNAs (mRNAs) required to protect and promote that unique lineage. Nanos1 is one such germline mRNA whose translation is blocked by a secondary mRNA structure within the open reading frame (ORF). Dnd1 contains a canonical RNA recognition motif (RRM1) in its N-terminus but also contains a less conserved RRM2. Here we provide a mechanistic picture of the nanos1 mRNA-Dnd1 interaction in the Xenopus germline. We show that RRM1, but not RRM2, is required for binding nanos1. Similar to the zebrafish homolog, Xenopus Dnd1 possesses ATPase activity. Surprisingly, this activity appears to be within the RRM2, different from the C-terminal region where it is found in zebrafish. More importantly, we show that RRM2 is required for nanos1 translation and germline survival. Further, Dnd1 functions as a homodimer and binds nanos1 mRNA just downstream of the secondary structure required for nanos1 repression. We propose a model in which the RRM1 is required to bind nanos1 mRNA while the RRM2 is required to promote translation through the action of ATPase. Dnd1 appears to use RRMs to mimic the function of helicases.

Microinjection of Xenopus Oocytes.

Microinjection of Xenopus oocytes has proven to be a valuable tool in a broad array of studies that require expression of DNA or RNA into functional protein. These studies are diverse and range from expression cloning to receptor-ligand interaction to nuclear programming. Oocytes offer a number of advantages for such studies, including their large size (∼1.2 mm in diameter), capacity for translation, and enormous nucleus (0.3-0.4 mm). They are cost effective, easily manipulated, and can be injected in large numbers in a short time period. Oocytes have a large maternal stockpile of all the essential components for transcription and translation. Consequently, the investigator needs only to introduce by microinjection the specific DNA or RNA of interest for synthesis. Oocytes translate virtually any exogenous RNA regardless of source, and the translated proteins are folded, modified, and transported to the correct cellular locations. Here we present procedures for the efficient microinjection of oocytes and their subsequent care.

Isolation of Xenopus Oocytes.

Xenopus oocytes and oocyte extracts are the starting material for a variety of experimental approaches. Oocytes are obtained by surgical removal of the ovary from anesthetized females. Although oocytes may be used while they remain within their ovarian follicle, it is more practical to work with defolliculated oocytes. Defolliculation can be performed either manually or enzymatically. Here we present a protocol for the isolation and separation of Xenopus oocytes at various developmental stages, and guidelines for maintaining oocytes in culture.

A novel role for sox7 in Xenopus early primordial germ cell development: mining the PGC transcriptome.

Xenopus primordial germ cells (PGCs) are determined by the presence of maternally derived germ plasm. Germ plasm components both protect PGCs from somatic differentiation and begin a unique gene expression program. Segregation of the germline from the endodermal lineage occurs during gastrulation, and PGCs subsequently initiate zygotic transcription. However, the gene network(s) that operate to both preserve and promote germline differentiation are poorly understood. Here, we utilized RNA-sequencing analysis to comprehensively interrogate PGC and neighboring endoderm cell mRNAs after lineage segregation. We identified 1865 transcripts enriched in PGCs compared with endoderm cells. We next compared the PGC-enriched transcripts with previously identified maternal, vegetally enriched transcripts and found that ∼38% of maternal transcripts were enriched in PGCs, including sox7 PGC-directed sox7 knockdown and overexpression studies revealed an early requirement for sox7 in germ plasm localization, zygotic transcription and PGC number. We identified pou5f3.3 as the most highly expressed and enriched POU5F1 homolog in PGCs. We compared the Xenopus PGC transcriptome with human PGC transcripts and showed that 80% of genes are conserved, underscoring the potential usefulness of Xenopus for understanding human germline specification.

Maternal Dead-end 1 promotes translation of nanos1 by binding the eIF3 complex.

In the developing embryo, primordial germ cells (PGCs) represent the exclusive progenitors of the gametes, and their loss results in adult infertility. During early development, PGCs are exposed to numerous signals that specify somatic cell fates. To prevent somatic differentiation, PGCs must transiently silence their genome, an early developmental process that requires Nanos activity. However, it is unclear how Nanos translation is regulated in developing embryos. We report here that translation of nanos1 after fertilization requires Dead-end 1 (Dnd1), a vertebrate-specific germline RNA-binding protein. We provide evidence that Dnd1 protein, expression of which is low in oocytes, but increases dramatically after fertilization, directly interacts with, and relieves the inhibitory function of eukaryotic initiation factor 3f, a repressive component in the 43S preinitiation complex. This work uncovers a novel translational regulatory mechanism that is fundamentally important for germline development.

Maternal messages to live by: a personal historical perspective.

In the 1980s, the study of localized maternal mRNAs was just emerging as a new research area. Classic embryological studies had linked the inheritance of cytoplasmic domains with specific cell lineages, but the underlying molecular nature of these putative determinants remained a mystery. The model system Xenopus would play a pivotal role in the progress of this new field. In fact, the first localized maternal mRNA to be identified and cloned from any organism was Xenopus vg1, a TGF-beta family member. This seminal finding opened the door to many subsequent studies focused on how RNAs are localized and what functions they had in development. As the field moves into the future, Xenopus remains the system of choice for studies identifying RNA/protein transport particles and maternal RNAs through RNA-sequencing.

High-throughput analysis reveals novel maternal germline RNAs crucial for primordial germ cell preservation and proper migration.

During oogenesis, hundreds of maternal RNAs are selectively localized to the animal or vegetal pole, including determinants of somatic and germline fates. Although microarray analysis has identified localized determinants, it is not comprehensive and is limited to known transcripts. Here, we utilized high-throughput RNA-sequencing analysis to comprehensively interrogate animal and vegetal pole RNAs in the fully grown Xenopus laevis oocyte. We identified 411 (198 annotated) and 27 (15 annotated) enriched mRNAs at the vegetal and animal pole, respectively. Ninety were novel mRNAs over 4-fold enriched at the vegetal pole and six were over 10-fold enriched at the animal pole. Unlike mRNAs, microRNAs were not asymmetrically distributed. Whole-mount in situ hybridization confirmed that all 17 selected mRNAs were localized. Biological function and network analysis of vegetally enriched transcripts identified protein-modifying enzymes, receptors, ligands, RNA-binding proteins, transcription factors and co-factors with five defining hubs linking 47 genes in a network. Initial functional studies of maternal vegetally localized mRNAs show that sox7 plays a novel and important role in primordial germ cell (PGC) development and that ephrinB1 (efnb1) is required for proper PGC migration. We propose potential pathways operating at the vegetal pole that highlight where future investigations might be most fruitful.

Mechanisms of Vertebrate Germ Cell Determination.

Two unique characteristics of the germ line are the ability to persist from generation to generation and to retain full developmental potential while differentiating into gametes. How the germ line is specified that allows it to retain these characteristics within the context of a developing embryo remains unknown and is one focus of current research. Germ cell specification proceeds through one of two basic mechanisms: cell autonomous or inductive. Here, we discuss how germ plasm driven germ cell specification (cell autonomous) occurs in both zebrafish and the frog Xenopus. We describe the segregation of germ cells during embryonic development of solitary and colonial ascidians to provide an evolutionary context to both mechanisms. We conclude with a discussion of the inductive mechanism as exemplified by both the mouse and axolotl model systems. Regardless of mechanism, several general themes can be recognized including the essential role of repression and posttranscriptional regulation of gene expression.

Primordial Germ Cell Isolation from Xenopus laevis Embryos.

Primordial germ cells (PGCs) are the precursors to the gametes and have the unique ability to retain full developmental potential. However, the mechanism(s) and gene-network(s) necessary for their proper specification and development are poorly understood. This is due, in part, to the challenges that must be overcome in order to identify and isolate PGCs during critical stages of development. Two distinct mechanisms have been characterized to specify the germ cell lineage in vertebrates: induction and inheritance. Regardless of mechanism, there are common developmental features shared among all vertebrates in forming the germ cell lineage. Xenopus offers several advantages for understanding the molecular mechanisms necessary to establish the germ line. Here, we provide detailed methods for isolating live PGCs at different time points: 1) just after they have segregated from the endodermal lineage, and 2) while they are migrating towards the presumptive gonad. Isolation of PGCs at these critical developmental stages will allow for the investigation of the mechanism(s) and gene-network(s) necessary for their proper specification and development.

Hermes (Rbpms) is a Critical Component of RNP Complexes that Sequester Germline RNAs during Oogenesis.

The germ cell lineage in Xenopus is specified by the inheritance of germ plasm that assembles within the mitochondrial cloud or Balbiani body in stage I oocytes. Specific RNAs, such as nanos1, localize to the germ plasm. nanos1 has the essential germline function of blocking somatic gene expression and thus preventing Primordial Germ Cell (PGC) loss and sterility. Hermes/Rbpms protein and nanos RNA co-localize within germinal granules, diagnostic electron dense particles found within the germ plasm. Previous work indicates that nanos accumulates within the germ plasm through a diffusion/entrapment mechanism. Here we show that Hermes/Rbpms interacts with nanos through sequence specific RNA localization signals found in the nanos-3'UTR. Importantly, Hermes/Rbpms specifically binds nanos, but not Vg1 RNA in the nucleus of stage I oocytes. In vitro binding data show that Hermes/Rbpms requires additional factors that are present in stage I oocytes in order to bind nanos1. One such factor may be hnRNP I, identified in a yeast-2-hybrid screen as directly interacting with Hermes/Rbpms. We suggest that Hermes/Rbpms functions as part of a RNP complex in the nucleus that facilitates selection of germline RNAs for germ plasm localization. We propose that Hermes/Rbpms is required for nanos RNA to form within the germinal granules and in this way, participates in the germline specific translational repression and sequestration of nanos RNA.

The Xenopus Maternal-to-Zygotic Transition from the Perspective of the Germline.

In Xenopus, the germline is specified by the inheritance of germ-plasm components synthesized at the beginning of oogenesis. Only the cells in the early embryo that receive germ plasm, the primordial germ cells (PGCs), are competent to give rise to the gametes. Thus, germ-plasm components continue the totipotent potential exhibited by the oocyte into the developing embryo at a time when most cells are preprogrammed for somatic differentiation as dictated by localized maternal determinants. When zygotic transcription begins at the mid-blastula transition, the maternally set program for somatic differentiation is realized. At this time, genetic control is ceded to the zygotic genome, and developmental potential gradually becomes more restricted within the primary germ layers. PGCs are a notable exception to this paradigm and remain transcriptionally silent until the late gastrula. How the germ-cell lineage retains full potential while somatic cells become fate restricted is a tale of translational repression, selective degradation of somatic maternal determinants, and delayed activation of zygotic transcription.

Evidence-based stillbirth prevention strategies: combining empirical and theoretical paradigms to inform health planning and decision-making.

INTRODUCTION: A global health project undertaken in Qatar on the Arabian Peninsula immersed undergraduate nursing students in hands-on learning to address the question: What strategies are effective in preventing stillbirth? Worldwide stillbirth estimates of 2.6 million per year and the high rate in the Eastern Mediterranean Region of 27 per 1,000 total live births provided the stimulus for this inquiry. METHODS: We used a dual empirical and theoretical approach that combined the principles of evidence-based practice and population health planning. Students were assisted to translate pre-appraised literature based on the 6S hierarchical pyramid of evidence. The PRECEDE-PROCEED (P-P) model served as an organizing template to assemble data extracted from the appraisal of 21 systematic literature reviews ± meta-analyses, 2 synopses of synthesized reports, and 9 individual studies summarizing stillbirth prevention strategies in low, middle, and high income countries. Consistent with elements of the P-P model, stillbirth prevention strategies were classified as social, epidemiological, educational, ecological, administrative, or policy. RESULTS: Ten recommendations with clear evidence of effectiveness in preventing stillbirth in low, middle, or high income countries were identified. Several other promising interventions were identified with weak, uncertain, or inconclusive evidence. These require further rigorous testing. LINKING EVIDENCE TO ACTION: Two complementary paradigms--evidence-based practice and an ecological population health program planning model--helped baccalaureate nursing students transfer research evidence into useable knowledge for practice. They learned the importance of comprehensive assessments and evidence-informed interventions. The multidimensional elements of the P-P model sensitized students to the complex interrelated factors influencing stillbirth and its prevention.

Maternal Dead-End1 is required for vegetal cortical microtubule assembly during Xenopus axis specification.

Vertebrate axis specification is an evolutionarily conserved developmental process that relies on asymmetric activation of Wnt signaling and subsequent organizer formation on the future dorsal side of the embryo. Although roles of Wnt signaling during organizer formation have been studied extensively, it is unclear how the Wnt pathway is asymmetrically activated. In Xenopus and zebrafish, the Wnt pathway is triggered by dorsal determinants, which are translocated from the vegetal pole to the future dorsal side of the embryo shortly after fertilization. The transport of dorsal determinants requires a unique microtubule network formed in the vegetal cortex shortly after fertilization. However, molecular mechanisms governing the formation of vegetal cortical microtubule arrays are not fully understood. Here we report that Dead-End 1 (Dnd1), an RNA-binding protein required for primordial germ cell development during later stages of embryogenesis, is essential for Xenopus axis specification. We show that knockdown of maternal Dnd1 specifically interferes with the formation of vegetal cortical microtubules. This, in turn, impairs translocation of dorsal determinants, the initiation of Wnt signaling, organizer formation, and ultimately results in ventralized embryos. Furthermore, we found that Dnd1 binds to a uridine-rich sequence in the 3'-UTR of trim36, a vegetally localized maternal RNA essential for vegetal cortical microtubule assembly. Dnd1 anchors trim36 to the vegetal cortex in the egg, promoting high concentrations of Trim36 protein there. Our work thus demonstrates a novel and surprising function for Dnd1 during early development and provides an important link between Dnd1, mRNA localization, the microtubule cytoskeleton and axis specification.

Repressive translational control in germ cells.

The earliest stages of embryonic development in many animals proceed without zygotic transcription. Genetic control is executed by maternally inherited mRNAs that are under translational control. To set aside the future germ cell lineage, it is pivotal to both exert translational regulation of maternal germline mRNAs and to repress maternal signals in those same cells that drive somatic cell-fate determination. Here we review repressive translational regulation in the germline from the perspective of the conserved RNA binding proteins Pumilio and Nanos, and discuss common themes that have emerged.

Activity of long-chain acyl-CoA synthetase is required for maintaining meiotic arrest in Xenopus laevis.

In most vertebrates, fully grown oocytes are arrested in meiotic prophase I and only resume the cell cycle upon external stimuli, such as hormones. The proper arrest and resumption of the meiotic cycle is critical for reproduction. A Galpha(S) signaling pathway essential for the arrest is conserved in organisms from Xenopus to mouse and human. A previous gene association study implicated that mutations of human ACSL6 may be related to premature ovarian failure. However, functional roles of ACSL6 in human infertility have yet to be reported. In the present study, we found that triacsin C, a potent and specific inhibitor for ACSL, triggers maturation in Xenopus and mouse oocytes in the absence of hormone, suggesting ACSL activity is required for the oocyte arrest. In Xenopus, acsl1b may fulfill a major role in the process, because inhibition of acsl1b by knocking down its RNA results in abnormal acceleration of oocyte maturation. Such abnormally matured eggs cannot support early embryonic development. Moreover, direct inhibition of protein palmitoylation, which lies downstream of ACSLs, also causes oocyte maturation. Furthermore, palmitoylation of Galpha(s), which is essential for its function, is inhibited when the ACSL activity is blocked by triacsin C in Xenopus. Thus, disruption of ACSL activity causes inhibition of the Galpha(s) signaling pathway in the oocytes, which may result in premature ovarian failure in human.

Xenopus Nanos1 is required to prevent endoderm gene expression and apoptosis in primordial germ cells.

Nanos is expressed in multipotent cells, stem cells and primordial germ cells (PGCs) of organisms as diverse as jellyfish and humans. It functions together with Pumilio to translationally repress targeted mRNAs. Here we show by loss-of-function experiments that Xenopus Nanos1 is required to preserve PGC fate. Morpholino knockdown of maternal Nanos1 resulted in a striking decrease in PGCs and a loss of germ cells from the gonads. Lineage tracing and TUNEL staining reveal that Nanos1-deficient PGCs fail to migrate out of the endoderm. They appear to undergo apoptosis rather than convert to normal endoderm. Whereas normal PGCs do not become transcriptionally active until neurula, Nanos1-depleted PGCs prematurely exhibit a hyperphosphorylated RNA polymerase II C-terminal domain at the midblastula transition. Furthermore, they inappropriately express somatic genes characteristic of endoderm regulated by maternal VegT, including Xsox17α, Bix4, Mixer, GATA4 and Edd. We further demonstrate that Pumilio specifically binds VegT RNA in vitro and represses, along with Nanos1, VegT translation within PGCs. Repressed VegT RNA in wild-type PGCs is significantly less stable than VegT in Nanos1-depleted PGCs. Our data indicate that maternal VegT RNA is an authentic target of Nanos1/Pumilio translational repression. We propose that Nanos1 functions to translationally repress RNAs that normally specify endoderm and promote apoptosis, thus preserving the germline.

Nanos1 functions as a translational repressor in the Xenopus germline.

Nanos family members have been shown to act as translational repressors in the Drosophila and Caenorhabditis elegans germline, but direct evidence is missing for a similar function in vertebrates. Using a tethered function assay, we show that Xenopus Nanos1 is a translational repressor and that association with the RNA is required for this repression. We identified a 14 amino acid region within the N-terminal domain of Nanos1 that is conserved in organisms as diverse as sponge and Human. The region is found in all vertebrates but notably lacking in Drosophila and C. elegans. Deletion and substitution analysis revealed that this conserved region was required for Nanos1 repressive activity. Consistent with this observation, deletion of this region was sufficient to prevent abnormal development that results from ectopic expression of Nanos1 in oocytes. Although Nanos1 can repress capped and polyadenylated RNAs, Nanos1 mediated repression did not require the targeted RNA to have a cap or to be polyadenylated. These results suggest that Nanos1 is capable of repressing translation by several different mechanisms. We found that Nanos1, like Drosophila Nanos, associates with cyclin B1 RNA in vivo indicating that some Nanos targets may be evolutionarily conserved. Nanos1 protein was detected and thus available to repress mRNAs while PGCs were in the endoderm, but was not observed in PGCs after this stage.