Malgorzata Kloc

Mechanisms of Subcellular mRNA Localization

Localization of RNA is a widespread and efficient way to target gene products to a specific region of a cell or embryo. This strategy of posttranscriptional gene regulation utilizes a variety of distinct mechanisms to regulate the movement and anchoring of different transcripts.

Joint action of two RNA degradation pathways controls the timing of maternal transcript elimination at the midblastula transition in Drosophila melanogaster

Maternally synthesized RNAs program early embryonic development in many animals. These RNAs are degraded rapidly by the midblastula transition (MBT), allowing genetic control of development to pass to zygotically synthesized transcripts. Here we show that in the early embryo of Drosophila melanogaster, there are two independent RNA degradation pathways, either of which is sufficient for transcript elimination. However, only the concerted action of both pathways leads to elimination of transcripts with the correct timing, at the MBT. The first pathway is maternally encoded, is targeted to specific classes of mRNAs through cis‐acting elements in the 3′‐untranslated region and is conserved in Xenopus laevis. The second pathway is activated 2 h after fertilization and functions together with the maternal pathway to ensure that transcripts are degraded by the MBT.

The Balbiani body and germ cell determinants: 150 years later.

Publisher Summary This chapter describes the Balbiani body (Bb) in various animal species. The most comprehensive ultrastructural and molecular studies on the origin, composition, and function of Bb and its relationship to the germplasm have been done for the oocytes and embryos of Xenopus. Various RNAs and proteins have been discovered localized in the mitochondrial cloud (MC) and in the germplasm in Xenopus oocytes and embryos. The chapter discusses two major pathways of RNA localization in Xenopus: (1) Message Transport Organizer (METRO) or early pathway-localizing RNAs, (2) Late or Vg1 pathway-localizing RNAs. Emphasis is given on the localization of METRO pathway RNAs within the MC and the germplasm islands in the embryo. The chapter discusses the ultrastructural, molecular, and functional studies that have been carried out on polar granules and germ cells of Drosophila melanogaster . It reveals that the molecular composition of pole plasm, polar granules, nuage, and sponge bodies in Drosophila has been deduced from mutational and functional analyses and indirect genetic approaches.

RNA localization mechanisms in oocytes

In many animals, normal development depends on the asymmetric distribution of maternal determinants, including various coding and noncoding RNAs, within the oocyte. The temporal and spatial distribution of localized RNAs is determined by intricate mechanisms that regulate their movement and anchoring. These mechanisms involve cis-acting sequences within the RNA molecules and a multitude of trans-acting factors, as well as a polarized cytoskeleton, molecular motors and specific transporting organelles. The latest studies show that the fates of localized RNAs within the oocyte cytoplasm are predetermined in the nucleus and that nuclear proteins, some of them deposited on RNAs during splicing, together with the components of the RNA-silencing pathway, dictate the proper movement, targeting, anchoring and translatability of localized RNAs.

Acquisition of the heat-shock response and thermotolerance during early development of Xenopus laevis☆

The ability to synthesize a 68,000- to 70,000-Da protein (hsp) in heat-shocked early Xenopus laevis embryos is dependent on the stage of development. Whereas late blastula and later stage embryos synthesize hsp68-70 after heat shock, cleavage stages are incompetent with respect to hsp synthesis. In vitro translation experiments and RNA blot analyses demonstrate that enhanced synthesis of hsp68-70 is associated with an accumulation of hsp68-70 mRNA. Examination of the effect of heat shock on preexisting actin mRNA reveals that heat shock promotes a reduction in the levels of actin mRNA in cleavage embryos but has no discernible effect on actin mRNA levels in neurula embryos. Finally, the acquisition of the heat-shock response (i.e., synthesis of hsp68-70 and accumulation of hsp70 mRNA) during early Xenopus development is correlated with the acquisition of thermotolerance.

The cloning and characterization of a maternally expressed novel zinc finger nuclear phosphoprotein (xnf7) in Xenopus laevis

We report the cloning of a cDNA (xnf7) coding for a maternally expressed Xenopus protein that becomes highly enriched in nuclei of the central nervous system during later development and in nuclei of adult brain. The protein also shows stage-specific nuclear/cytoplasmic partitioning and phosphorylation that may be related to its function. In addition, it binds to double-stranded DNA in vitro. The conceptual protein produced by the xnf7 clone contains several acidic domains, a novel zinc finger domain, three putative p34cdc2 protein kinase phosphorylation sites, and a bipartite basic nuclear localization signal. The xnf7 mRNA was detected as a maternal transcript that decreased in abundance during development through the gastrula stage. It was reexpressed at the neural stage in mesoderm and neural tissues, and its reexpression was not dependent upon the normal juxtaposition of the mesoderm and ectoderm that occurs during neural induction as demonstrated by high titer in exogastrulae. In situ hybridization showed enrichment of the mRNA in the neural tube and a small amount in the mesoderm at the late neurula stage. Xnf7 is normally phosphorylated during oocyte maturation. The bacterially expressed xnf7 protein was phosphorylated in vitro by purified maturation-promoting factor at a threonine in a small N-terminal domain containing one of the p34cdc2 protein kinase phosphorylation sites, but not by several other protein kinases. The structural domains present in the protein and its localization in nuclei suggest that the xnf7 gene product performs an important nuclear function during early development, perhaps as a transcription factor or a structural component of chromatin.

Apparent continuity between the messenger transport organizer and late RNA localization pathways during oogenesis in Xenopus

The localization of RNAs at the vegetal cortex in Xenopus oocytes is a complex process, involving at least two different pathways. The early, or messenger transport organizer (METRO), pathway, localizes RNAs such as Xlsirts, Xcat2 and Xwnt11 during stages 1 and 2 of oogenesis, while the late pathway localizes RNAs such as Vg1 during stages 2-4. We demonstrate that the onset of Vg1 localization is characterized by its microtubule-independent binding to a subdomain of the endoplasmic reticulum (ER). The formation of this unique ER structure is intimately associated with the movement of the mitochondrial cloud toward the vegetal cortex. In addition, we demonstrate that the mitochondrial cloud contains a gamma-tubulin-positive structure that may function as a microtubule organizing center for establishing microtubule tracks for Vg1 localization. These data, support, although they do not prove, a model in which the development of the late pathway machinery relies upon the prior functioning of the early pathway.

Contribution of METRO pathway localized molecules to the organization of the germ cell lineage.

To elucidate the potential role of localized components in the specification of the germ cell lineage we analyzed the composition of the germ plasm in Xenopus laevis oocytes and early embryos with respect to the vegetally-localized RNAs. We focused on Xlsirts, Xcat2, and Xwnt11 transcripts that are localized to the vegetal cortex through a region of the mitochondrial cloud called the messenger transport organizer (METRO) that also contains the nuage or germ plasm. At the ultrastructural level Xcat2 mRNA was detected on germinal granules while Xlsirts and Xwnt11 were associated with a fibrillar network of the germ plasm in stage-1 and stage-4 oocytes. In embryos, we found that all three RNAs remained associated with the germ plasm. Vg1 mRNA, a transcript localized through the late pathway, was excluded from the germ plasm in oocytes and embryos. Addtionally, we detected the protein spectrin within 16 cell nests of germ cells, in a structure reminiscent of the Drosophila spectrosome. Spectrin was detected in the mitochondrial cloud and was found in the germ plasm during embryogenesis. These data indicate that the various RNAs found within METRO and the protein spectrin are integral components of the Xenopus germ plasm with the RNAs being associated with different subcellular structures. They also suggest that the pathway through which RNAs are localized during oogenesis may be an important factor in biasing their distribution into specific cell lineages. The presence of Xwnt11 in the germ cell lineage suggests that a wnt-directed signaling pathway may be involved in germ cell specification. differentiation or migration.

Hormad1 mutation disrupts synaptonemal complex formation, recombination, and chromosome segregation in mammalian meiosis.

Meiosis is unique to germ cells and essential for reproduction. During the first meiotic division, homologous chromosomes pair, recombine, and form chiasmata. The homologues connect via axial elements and numerous transverse filaments to form the synaptonemal complex. The synaptonemal complex is a critical component for chromosome pairing, segregation, and recombination. We previously identified a novel germ cell–specific HORMA domain encoding gene, Hormad1, a member of the synaptonemal complex and a mammalian counterpart to the yeast meiotic HORMA domain protein Hop1. Hormad1 is essential for mammalian gametogenesis as knockout male and female mice are infertile. Hormad1 deficient (Hormad1−/ −) testes exhibit meiotic arrest in the early pachytene stage, and synaptonemal complexes cannot be visualized by electron microscopy. Hormad1 deficiency does not affect localization of other synaptonemal complex proteins, SYCP2 and SYCP3, but disrupts homologous chromosome pairing. Double stranded break formation and early recombination events are disrupted in Hormad1−/ − testes and ovaries as shown by the drastic decrease in the γH2AX, DMC1, RAD51, and RPA foci. HORMAD1 co-localizes with γH2AX to the sex body during pachytene. BRCA1, ATR, and γH2AX co-localize to the sex body and participate in meiotic sex chromosome inactivation and transcriptional silencing. Hormad1 deficiency abolishes γH2AX, ATR, and BRCA1 localization to the sex chromosomes and causes transcriptional de-repression on the X chromosome. Unlike testes, Hormad1−/ − ovaries have seemingly normal ovarian folliculogenesis after puberty. However, embryos generated from Hormad1−/ − oocytes are hyper- and hypodiploid at the 2 cell and 8 cell stage, and they arrest at the blastocyst stage. HORMAD1 is therefore a critical component of the synaptonemal complex that affects synapsis, recombination, and meiotic sex chromosome inactivation and transcriptional silencing.

Premature ovarian failure in nobox-deficient mice is caused by defects in somatic cell invasion and germ cell cyst breakdown

PurposeTo understand the mechanism of premature ovarian failure (POF).MethodsThe ultrastructural (electron microscopy) analysis of primordial ovarian follicles in Nobox deficient mice.ResultsWe studied, for the first time, the fate of oogonia in embryonic (prenatal) mouse ovaries and showed that the abolishment of the transition from germ cell cysts to primordial follicles in the ovaries of Nobox deficient mice is caused by defects in germ cell cyst breakdown, leading to the formation of syncytial follicles instead of primordial follicles.ConclusionsThese results indicate that POF syndrome in Nobox deficient mice results from the faulty signaling between somatic and germ line components during embryonic development. In addition, the extremely unusual and abnormal presence of adherens junctions between unseparated oocytes within syncytial follicles indicates that faulty communication between somatic and germ cells is involved in, or leads to, abnormalities in the cell adhesion program.