-
[
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.
-
[
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.
-
[
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.
-
[
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.
-
[
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.
-
[
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.
-
[
Mol Reprod Dev,
2017]
Insulin signaling regulates various aspects of physiology, such as glucose homeostasis and aging, and is a key determinant of female reproduction in metazoans. That insulin signaling is crucial for female reproductive health is clear from clinical data linking hyperinsulinemic and hypoinsulinemic condition with certain types of ovarian dysfunction, such as altered steroidogenesis, polycystic ovary syndrome, and infertility. Thus, understanding the signaling mechanisms that underlie the control of insulin-mediated ovarian development is important for the accurate diagnosis of and intervention for female infertility. Studies of invertebrate and vertebrate model systems have revealed the molecular determinants that transduce insulin signaling as well as which biological processes are regulated by the insulin-signaling pathway. The molecular determinants of the insulin-signaling pathway, from the insulin receptor to its downstream signaling components, are structurally and functionally conserved across evolution, from worms to mammals - yet, physiological differences in signaling still exist. Insulin signaling acts cooperatively with gonadotropins in mammals and lower vertebrates to mediate various aspects of ovarian development, mainly owing to evolution of the endocrine system in vertebrates. In contrast, insulin signaling in Drosophila and Caenorhabditis elegans directly regulates oocyte growth and maturation. In this review, we compare and contrast insulin-mediated regulation of ovarian functions in mammals, lower vertebrates, C. elegans, and Drosophila, and highlight conserved signaling pathways and regulatory mechanisms in general while illustrating insulin's unique role in specific reproductive processes. This article is protected by copyright. All rights reserved.
-
[
J Vis Exp,
2016]
The evolutionarily conservedextracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.
-
[
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.
-
[
International Worm Meeting,
2015]
Understanding the organization of a tissue provides a solid foundation for genetic and cell biological analyses. A comprehensive map of the somatic lineage of Caenorhabditis elegans has laid a foundational groundwork that aided in determining of the organization of its tissues through understanding how they are derived. Unfortunately, due to technical limitations, a germline lineage has yet to be investigated. In the mitotic zone, this leaves many questions unanswered such as how many times mitotic cells divide before entering meiosis, how long are they exposed to differentiation signals before entering meiosis, and whether alteration of these parameters will alter the viability or maturation of a germ cell. To answer these questions and further resolve the organization of the germline, I am developing methods to lineage trace in the germline through conditional recombination and live imaging. Here I present my progress to date.