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Results Probl Cell Differ,
2017]
Generation of healthy oocytes requires coordinated regulation of multiple cellular events and signaling pathways. Oocytes undergo a unique developmental growth and differentiation pattern interspersed with long periods of arrest. Oocytes from almost all species arrest in prophase I of oogenesis that allows for long period of growth and differentiation essential for normal oocyte development. Depending on species, oocytes that transit from prophase I to meiosis I also arrest at meiosis I for fairly long periods of time and then undergo a second arrest at meiosis II that is completed upon fertilization. While there are species-specific differences in C. elegans, D. melanogaster, and mammalian oocytes in stages of prophase I, meiosis I, or meiosis II arrest, in all cases cell signaling pathways coordinate the developmental events controlling oocyte growth and differentiation to regulate these crucial phases of transition. In particular, the ERK MAP kinase signaling pathway, cyclic AMP second messengers, and the cell cycle regulators CDK1/cyclin B are key signaling pathways that seem evolutionarily conserved in their control of oocyte growth and meiotic maturation across species. Here, I identify the common themes and differences in the regulation of key meiotic events during oocyte growth and maturation.
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Dev Cell,
2003]
Engulfment of apoptotic cells requires presentation of new cell surface ligands by the dying cells. Using a differential proteomics technology, we identify that annexin I is a caspase-dependent engulfment ligand; it is recruited from the cytosol and exported to the outer plasma membrane leaflet, colocalizes with phosphatidylserine, and is required for efficient clearance of apoptotic cells. Furthermore, phosphatidylserine receptor (PSR) clustering around apoptotic cells indicates a requirement for annexin I. In the nematode Caenorhabditis elegans, downregulation of the annexin homolog prevents efficient engulfment of pharyngeal cell corpses. These results provide novel mechanistic insights into how apoptotic cells are removed and may explain a pathogenic mechanism of chronic inflammatory diseases where annexin I autoantibodies have been described.
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Dev Cell,
2011]
Tissues that generate specialized cell types in a production line must coordinate developmental mechanisms with physiological demand, although how this occurs is largely unknown. In the Caenorhabditis elegans hermaphrodite, the developmental sex-determination cascade specifies gamete sex in the distal germline, while physiological sperm signaling activates MPK-1/ERK in the proximal germline to control plasma membrane biogenesis and organization during oogenesis. We discovered repeated utilization of a self-contained negative regulatory module, consisting of NOS-3 translational repressor, FEM-CUL-2 (E3 ubiquitin ligase), and TRA-1 (Gli transcriptional repressor), which acts both in sex determination and in physiological demand control of oogenesis, coordinating these processes. In the distal germline, where MPK-1 is not activated, TRA-1 represses the male fate as NOS-3 functions in translational repression leading to inactivation of the FEM-CUL-2 ubiquitin ligase. In the proximal germline, sperm-dependent physiological MPK-1 activation results in phosphorylation-based inactivation of NOS-3, FEM-CUL-2-mediated degradation of TRA-1 and the promotion of membrane organization during oogenesis.
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Proc Natl Acad Sci U S A,
2009]
RAS-extracellular signal regulated kinase (ERK) signaling governs multiple aspects of cell fate specification, cellular transitions, and growth by regulating downstream substrates through phosphorylation. Understanding how perturbations to the ERK signaling pathway lead to developmental disorders and cancer hinges critically on identification of the substrates. Yet, only a limited number of substrates have been identified that function in vivo to execute ERK-regulated processes. The Caenorhabditis elegans germ line utilizes the well-conserved RAS-ERK signaling pathway in multiple different contexts. Here, we present an integrated functional genomic approach that identified 30 ERK substrates, each of which functions to regulate one or more of seven distinct biological processes during C. elegans germ-line development. Our results provide evidence for three themes that underlie the robustness and specificity of biological outcomes controlled by ERK signaling in C. elegans that are likely relevant to ERK signaling in other organisms: (i) multiple diverse ERK substrates function to control each individual biological process; (ii) different combinations of substrates function to control distinct biological processes; and (iii) regulatory feedback loops between ERK and its substrates help reinforce or attenuate ERK activation. Substrates identified here have conserved orthologs in humans, suggesting that insights from these studies will contribute to our understanding of human diseases involving deregulated ERK activity.
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MicroPubl Biol,
2018]
PCNA (proliferating cell nuclear antigen) is the DNA polymerase processivity factor that loads onto the chromatin during S phase of the cell-cycle (Brauchle et al. 2003). Thus, nuclear localization of PCNA (PCN-1 in C. elegans) is used as a marker for the S phase of the cell cycle (Brauchle et al. 2003). GFP::PCN-1 has been shown to label S phase in C. elegans embryo when driven through the germline and embryonic promoter
pie-1 (Brauchle et al. 2003). We assayed GFP::PCN-1 (allele isIs17, GZ264 (Brauchle et al. 2003)) as a marker for S phase in adult germline progenitor zone cells. If this reagent were a faithful marker of S phase in germline progenitor zone cells, we would expect nuclear localization during S phase, and nuclear exclusion in the other phases of the cell-cycle, as is the case in the C. elegans embryo. We would also expect a perfect overlap with EdU which marks S phase of the cell cycle. EdU is incorporated in ~55-60% of the adult hermaphroditic wild-type progenitor zone cells (Fox et al. 2011; Furuta et al. 2018). We found that GFP::PCN-1 was nuclear in almost all of the progenitor zone cells, irrespective of whether they were EdU positive or EdU negative (arrowhead, Figure 1). The only cells that excluded GFP::PCN-1 from the nucleus were the metaphase cells (arrow) during M phase, when the nuclear envelope breaks down. Thus, GFP::PCN-1 does not overlap with EdU labeling in adult progenitor zone cells. These data suggest that nuclear localization of GFP::PCN-1 is not a good marker of S phase dynamics in the C. elegans adult germline progenitor cells. This could either be because GFP signal perdures in the nucleus in this context, or that GFP::PCN-1 is nuclear localized throughout the cell cycle in germline progenitor zone cells, unlike in the C. elegans embryo.
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MicroPubl Biol,
2023]
<i>Caenorhabditis elegans</i> gene <i>
sart-3</i> was first identified as the homolog of human SART3 ( S quamous cell carcinoma A ntigen R ecognized by T -cells 3). In humans, expression of SART3 is associated with squamous cell carcinoma, thus most of the studies focus on its potential role as a target of cancer immunotherapy (Shichijo et al. 1998; Yang et al. 1999). Furthermore, SART3 is also known as Tip110 (Liu et al. 2002; Whitmill et al. 2016) in the context of HIV virus host activation pathway. Despite these disease related studies, the molecular function of this protein was not revealed until the yeast homolog was identified as spliceosome U4/U6 snRNP recycling factor (Bell et al. 2002). The function of SART3 in development, however, remains unknown. Here we report that the <i>C. elegans</i> <i>
sart-3</i> mutant hermaphrodites exhibit a Mog ( M asculinization O f the G ermline) phenotype in adulthood suggesting that <i>
sart-3</i> normally functions to regulate the switch from spermatogenic to oogenic gametic sex.
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International Worm Meeting,
2017]
MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression via post-transcriptional repression of target mRNAs. Since their discovery in C. elegans in 1993, miRNAs have been found to play a critical role in varied biological processes across many systems. Germ cells in particular rely on post-transcriptional and translational control, in part because oocyte development is largely transcriptionally silent. Despite a key role for post-transcriptional and translational mechanisms in controlling oogenesis, a role for miRNAs is highly debated in this process. To investigate whether miRNAs regulate oocyte development, and female germ cell specification, I systematically profiled all 399 mature C. elegans miRNAs during oocyte development using two different genomic assays. A microarray analysis on whole adult C. elegans (wild type and germline-deficient) revealed that 28 of the 399 miRNAs are germline-expressed. Strikingly, a Fireplex TM analysis on dissected germlines revealed that ~95% of the miRNAs are not expressed in the oogenic germline, suggesting a global repression of miRNA production. Surprisingly, of the 28 germline-expressed miRNAs, 19 are Drosha-independent. Thus, a majority of miRNAs generated in the germline seem to be generated through a non-canonical miRNA biogenesis pathway. To determine the spatial and temporal expression pattern of each of the 28-germline miRNAs, a locked nucleic acid based in situ hybridization was performed. In situ analysis thus far reveals that a large proportion of the germline miRNAs are expressed in arrested oocytes, and maybe maternally contributed to the developing embryo. This suggests that miRNAs may not mediate meiotic progression, but rather regulate oocyte meiotic maturation and early embryo/larval development. Together these analyses reveal a novel class of Drosha-independent germline-expressed miRNAs, which reveals that those expressed during oogenesis largely regulate oocyte maturation and/or early embryogenesis.
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Mol Reprod Dev,
2022]
During oogenesis, oocytes arrest at meiotic prophase I to acquire competencies for resuming meiosis, fertilization, and early embryonic development. Following this arrested period, oocytes resume meiosis in response to species-specific hormones, a process known as oocyte maturation, that precedes ovulation and fertilization. Involvement of endocrine and autocrine/paracrine factors and signaling events during maintenance of prophase I arrest, and resumption of meiosis is an area of active research. Studies in vertebrate and invertebrate model organisms have delineated the molecular determinants and signaling pathways that regulate oocyte maturation. Cell cycle regulators, such as cyclin-dependent kinase (CDK1), polo-like kinase (PLK1), Wee1/Myt1 kinase, and the phosphatase CDC25 play conserved roles during meiotic resumption. Extracellular signal-regulated kinase (ERK), on the other hand, while activated during oocyte maturation in all species, regulates both species-specific, as well as conserved events among different organisms. In this review, we synthesize the general signaling mechanisms and focus on conserved and distinct functions of ERK signaling pathway during oocyte maturation in mammals, non-mammalian vertebrates, and invertebrates such as Drosophila and Caenorhabditis elegans.
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Curr Protoc Mol Biol,
2019]
MicroRNAs (miRNAs) are key regulators of cell and tissue development. However, spatial resolution of miRNA heterogeneity and accumulation patterns in vivo remains uncharted. Next-generation sequencing methods assay miRNA abundance in tissues, yet these analyses do not provide spatial resolution. A method to assay miRNA expression at single-cell resolution in vivo should clarify the cell-autonomous functions of miRNAs, their roles in influencing the cellular microenvironment, and their perdurance and turnover rate. We present an in situ hybridization protocol to map miRNA subcellular expression in single cells in vivo in four days. Using this protocol, we mapped distinct miRNAs that accumulate in the cytoplasm of one sibling oocyte but not another, dependent on the oocyte developmental stage. Thus, this method provides spatial and temporal resolution of the heterogeneity in expression of miRNAs during Caenorhabditis elegans oogenesis. This protocol can generally be adapted to any tissue amenable to dissection and fixation. 2019 by John Wiley & Sons, Inc.
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[
Methods Mol Biol,
2017]
The Rat Sarcoma (RAS) GTPAse-mediated extracellular signal-regulated kinase (ERK) pathway regulates multiple biological processes across metazoans. In particular during Caenorhabditis elegans oogenesis, ERK signaling has been shown to regulate over seven distinct biological processes in a temporal and sequential manner. To fully elucidate how ERK signaling cascade orchestrates these different biological processes in vivo, identification of the direct functional substrates of the pathway is critical. This chapter describes the methods that were used to identify ERK substrates in a global manner and study their functions in the germline. These approaches can also be generally applied to study ERK-dependent biological processes in other systems.