Microinjection of Xenopus

Microinjection of 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

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 oocytes at various developmental stages, and guidelines for maintaining oocytes in culture

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

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

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

Abstract 1541: The tumor suppressor BAP1 promotes a developmental switch from pluripotency to differentiation

Abstract Introduction: Our lab discovered that mutations in the tumor suppressor BAP1 are strongly associated with metastasis and death in patients with uveal melanoma. Subsequently, other cancers have been found to harbor BAP1 mutations, including skin melanoma, kidney cancer, mesothelioma and others. Germline BAP1 mutations are responsible for a newly described genetic cancer syndrome. Therapeutic molecules that reverse the effects of BAP1 mutations could represent a potent new treatment strategy for BAP1-mutant cancers. Unfortunately, there are several obstacles to developing such therapies. First, BAP1 is a tumor suppressor that is inactivated by mutations, such that targeted therapy would need to be directed against downstream effectors that are deregulated by BAP1 loss. Second, the effectors of BAP1 that are relevant to cancer are not known. Interestingly, most known proteins that interact with Bap1 are developmental epigenetic regulators such as Asxl1/2, Cbx1/3 and Kdm1b. Third, BAP1 is difficult to study in cultured cells because BAP1 loss results in cell cycle exit and stem cell-like behavior. These obstacles led us to shift to Xenopus laevis as an in vivo developmental model to study the functions of BAP1. Results and conclusions: Loss of BAP1 during embryo development results in a failure to turn off pluripotency genes such as ventx 1, ventx2 (Xenopus orthologues of the mammal gene nanog), oct 25, oct 91 (Xenopus orthologues of the mammal gene oct3/4) and pax3, and a failure to induce lineage specification genes such as the prospective epidermis marker keratin1 and the melanocyte precursor marker sox10. This block in the shift from pluripotency to differentiation programs results in a delay in gastrulation, neural crest specification and migration, mesodermal differentiation and other phenotypes. The BAP1-deficient phenotype can be rescued by Xenopus or human wildtype BAP1 or by the histone deacetylase inhibitor SAHA (vorinostat). We conclude that BAP1 is a fundamental regulator of multiple developmental lineages, including the neural crest from which melanomas arises, and that this in vivo model can be used to screen for novel therapeutic compounds that reverse the phenotypic effects of BAP1 loss. Citation Format: Jeffim N. Kuznetsov, Tristan Aguero, Stefan Kurtenbach, Matthew G. Field, Mary Lou King, J William Harbour. The tumor suppressor BAP1 promotes a developmental switch from pluripotency to differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1541. doi:10.1158/1538-7445.AM2017-1541</jats:p

Methods for Isolating the Balbiani Body/Germplasm from Xenopus laevis Oocytes Embryological, Cellular, and Genetic Methods

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

Time- vs. Frequency-domain Identification of Parametric Radiation Force Models for Marine Structures at Zero Speed

The dynamics describing the motion response of a marine structure in waves can be represented within a linear framework by the Cummins Equation. This equation contains a convolution term that represents the component of the radiation forces associated with fluid memory effects. Several methods have been proposed in the literature for the identification of parametric models to approximate and replace this convolution term. This replacement can facilitate the model implementation in simulators and the analysis of motion control designs. Some of the reported identification methods consider the problem in the time domain while other methods consider the problem in the frequency domain. This paper compares the application of these identification methods. The comparison is based not only on the quality of the estimated models, but also on the ease of implementation, ease of use, and the flexibility of the identification method to incorporate prior information related to the model being identified. To illustrate the main points arising from the comparison, a particular example based on the coupled vertical motion of a modern containership vessel is presented

Two Oppositely Localised Frizzled RNAs as Axis Determinants in a Cnidarian Embryo

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