Localized maternal proteins in Xenopus revealed by subtractive immunization

It has long been appreciated that the localization of cytoplasmic determinants in the egg can provide the foundation for patterning in the embryo. Differences in cell fate among the early blastomeres are thus a consequence of asymmetric distributions of informational molecules prior to fertilization. The frog egg has a single axis of asymmetry present prior to fertilization, the animal/vegetal axis, and the localization of developmental information appears to be polarized along this axis. Such developmental information can be localized as either RNA or protein; localized RNAs are well documented in the Xenopus oocyte, and some are thought to play roles in axial patterning. While it is apparent that not all of the localized maternal components are RNAs, much less is known about maternal proteins that might be localized in the egg. In the present study, we have taken a novel approach to identify localized maternal proteins within the Xenopus egg. Using a subtractive immunization strategy, we have generated monoclonal antibodies which recognize antigens that are restricted to the vegetal cortex of fertilized eggs. Analysis of biogenesis during oogenesis reveals two distinct patterns of localization to the cortex. At least three of these localized antigens are proteins, and these localized proteins could represent maternal determinants with roles in patterning.     

Localization of Xenopus Vg1 mRNA by Vera protein and the endoplasmic reticulum

In many organisms, pattern formation in the embryo develops from the polarized distributions of messenger RNAs (mRNAs) in the egg. In Xenopus, the mRNA encoding Vg1, a growth factor involved in mesoderm induction, is localized to the vegetal cortex of oocytes. A protein named Vera was shown to be involved in Vg1 mRNA localization. Vera cofractionates with endoplasmic reticulum (ER) membranes, and endogenous Vg1 mRNA is associated with a subcompartment of the ER. Vera may promote mRNA localization in Xenopus oocytes by mediating an interaction between the Vg1 3' untranslated region and the ER subcompartment.     

Localization of mitochondrial large ribosomal RNA in germ plasm of Xenopus embryos

In Xenopus, factors with the ability to establish the germ line are localized in the vegetal pole cytoplasm, or germ plasm, of the early embryo [1-3]. The germ plasm of Xenopus, and of many other animal species including Drosophila, contains electron-dense germinal granules which may be essential for germ-line formation [4-5]. Several components of the germinal granules have so far been identified in Drosophila [6-10]. One of these is mitochondrial large ribosomal RNA (mtlrRNA), which is present in the germinal granules (polar granules) during the cleavage stage until the formation of the germ-line progenitors or pole cells [8-9]. MtlrRNA has been identified as a factor that induces pole cells in embryos that have been sterilized by ultraviolet radiation [11]. The reduction of mtlrRNA in germ plasm by injecting anti-mtlrRNA ribozymes into embryos leads to the inability of these embryos to form pole cells [12]. These observations clearly show that mtlrRNA is essential for pole cell formation in Drosophila. Here, we report that mtlrRNA is enriched in germ plasm of Xenopus embryos from the four-cell stage to the blastula. Furthermore, our electron microscopic studies show that this mtlrRNA is present in the germinal granules during these stages. Thus, mtlrRNA is a common component of germinal granules in Drosophila and Xenopus, suggesting that the mtlrRNA has a role in germ-line development across phylogenetic boundaries.     

Low-cost serum-free medium for the BTI-Tn5B1-4 insect cell line

Ascidians show a highly determinate mode of development. In particular, components of the posterior-vegetal cytoplasm of fertilized eggs are responsible for the establishment of the embryonic axis. Recent studies have, however, also revealed significant roles of cell-cell interactions during embryogenesis. Proteins encoded by the Wnt family of genes act as signals and have been shown to play important roles in a wide range of developmental processes. Here we have isolated and characterized an ascidian Wnt gene, HrWnt-5, from Halocynthia roretzi. HrWnt-5 mRNA is present in the vegetal cortex in unfertilized eggs. After fertilization, HrWnt-5 mRNA moves to the equatorial region to form a crescent-like structure, after which the mRNA is concentrated in the posteriormost region of the embryo. This early pattern of HrWnt-5 mRNA localization coincides with another posterior-vegetally localized mRNA, pem, isolated from Ciona savignyi. In the gastrula, the zygotic HrWnt-5 mRNA is found in a variety of blastomeres, suggesting multiple roles of the gene.     

Identification and localization of a sea urchin Notch homologue: insights into vegetal plate regionalization and Notch receptor regulation

The specifications of cell types and germ-layers that arise from the vegetal plate of the sea urchin embryo are thought to be regulated by cell-cell interactions, the molecular basis of which are unknown. The Notch intercellular signaling pathway mediates the specification of numerous cell fates in both invertebrate and vertebrate development. To gain insights into mechanisms underlying the diversification of vegetal plate cell types, we have identified and made antibodies to a sea urchin homolog of Notch (LvNotch). We show that in the early blastula embryo, LvNotch is absent from the vegetal pole and concentrated in basolateral membranes of cells in the animal half of the embryo. However, in the mesenchyme blastula embryo LvNotch shifts strikingly in subcellular localization into a ring of cells which surround the central vegetal plate. This ring of LvNotch delineates a boundary between the presumptive secondary mesoderm and presumptive endoderm, and has an asymmetric bias towards the dorsal side of the vegetal plate. Experimental perturbations and quantitative analysis of LvNotch expression demonstrate that the mesenchyme blastula vegetal plate contains both animal/vegetal and dorsoventral molecular organization even before this territory invaginates to form the archenteron. Furthermore, these experiments suggest roles for the Notch pathway in secondary mesoderm and endoderm lineage segregation, and in the establishment of dorsoventral polarity in the endoderm. Finally, the specific and differential subcellular expression of LvNotch in apical and basolateral membrane domains provides compelling evidence that changes in membrane domain localization of LvNotch are an important aspect of Notch receptor function.     

Identification of Miranda protein domains regulating asymmetric cortical localization, cargo binding, and cortical release

An important question in cellular and developmental biology is how a cell divides to produce daughter cells with different fates. Drosophila neuroblasts are a model system for studying asymmetric cell division: at each division, neuroblasts retain stem cell-like features, whereas their sibling ganglion mother cell (GMC) has a more restricted fate. Establishing neuroblast/GMC differences involves the asymmetric localization of proteins (Inscuteable, Miranda, Prospero, and Staufen) and RNA (prospero). All of these factors are apically localized during interphase, and all except Inscuteable move to the basal cortex at mitosis prior to being partitioned solely into the GMC. In this study, we show that Miranda is colocalized with Staufen and Prospero in neuroblasts, and is required for the asymmetric cortical localization of both proteins. Analysis of miranda mutants reveals three functional domains within the Miranda protein: (1) an N-terminal domain (1-290 aa) sufficient for association of Miranda with the cell cortex and basal localization in mitotic neuroblasts; (2) a central domain (446-727 aa) necessary for apical localization in interphase neuroblasts as well as for "cargo binding" of Prospero, Staufen, and prospero mRNA; and (3) a C-terminal domain (727-830 aa) necessary for the timely degradation of Miranda and release of its cargo from the cortex of the newborn GMC. In addition, Miranda is asymmetrically localized in epithelial cells that lack Inscuteable and divide symmetrically; thus the mechanism regulating Miranda localization is common to epithelial cells and neuroblasts, and Inscuteable is not an obligate component. Finally, we define a C-terminal domain of Staufen sufficient for Miranda-dependent cortical localization in neuroblasts.     

The vegetal determinants required for the Spemann organizer move equatorially during the first cell cycle

Embryos with no dorsal axis were obtained when more than 15% of the egg surface was deleted from the vegetal pole of the early 1-cell embryo of Xenopus laevis. The timing of the deletion in the first cell cycle was critical: dorsal-deficient embryos were obtained when the deletion began before time 0.5 (50% of the first cell cycle) whereas normal dorsal axis usually formed when the deletion was done later than time 0.8. The axis deficiency could be restored by lithium treatment and the injection of vegetal but not animal cytoplasm. Bisection of the embryo at the 2-cell stage, which is known to restore the dorsal structures in the UV-ventralized embryos, had no effect on the vegetal-deleted embryos. These results show clearly that, in Xenopus, (1) the dorsal determinants (DDs) localized in the vegetal pole region at the onset of development are necessary for dorsal axis development and (2) the DDs move from the vegetal pole to a subequatorial region where they are incorporated into gastrulating cells to form the future organizing center. A model for the early axis formation process in Xenopus is proposed.     

The organization and animal-vegetal asymmetry of cytokeratin filaments in stage VI Xenopus oocytes is dependent upon F-actin and microtubules

Confocal immunofluorescence microscopy with anti-cytokeratin antibodies revealed a continuous and polarized network of cytokeratin (CK) filaments in the cortex of stage VI Xenopus oocytes. In the animal cortex, CK filaments formed a dense meshwork that both was thicker and exhibited a finer mesh than the network of CK filaments previously observed in the vegetal cortex (Klymkowsky et al., 1987). CK filaments first appeared in association with germinal vesicle (GV) and mitochondrial mass (MM) of oocytes in early mid stage I, indicating that CK filaments are the last of the three cytoskeletal networks to be assembled. By late stage I, CK filaments formed complex networks surrounding the GV, surrounding and penetrating the MM, and linking these networks to a meshwork of CK filaments in the oocyte cortex. During stage III-early IV, CK filaments formed a highly interconnected, apparently unpolarized, radial array linking the perinuclear and cortical CK filament networks. Polarization of the CK filament network was observed during mid stage IV-stage V, as first the animal, then the vegetal CK filament networks adopted the organization characteristic of stage VI oocytes. Treatment of stage VI oocytes with cytochalasin B disrupted the organization of both cortical and cytoplasmic CK filaments, releasing CK filaments from the oocyte cortex and inducing formation of numerous cytoplasmic CK filament aggregates. CB also disrupted the organization of cytoplasmic microtubules (MTs) in stage VI oocytes. Disassembly of oocyte MTs with nocodazole resulted in loss of the characteristic A-V polarity of the cortical CK filament network. In contrast, disruption of cytoplasmic CK filaments by microinjection of anti-CK antibodies had no apparent effect on cytoplasmic or MT organization. We propose a model in which the organization and polarization of the cortical network of CK filaments in stage VI Xenopus oocytes are dependent upon a hierarchy of interactions with actin filaments and microtubules.     

Detection of endothelin-1 and its receptors in rat liver endothelial cells by in situ reverse transcriptase-polymerase chain reaction

Bep mRNAs are localized at the animal pole of P. lividus eggs. In the present communication the secondary structures of the 3'UTRs of the bep1, bep3 and bep4 mRNAs are reported. The minimal lengths of these regions required to bind the 54-kDa protein, previously shown to be involved in localization and anchoring of these RNAs, is estimated. Microinjection of the bep3 3'UTR into egg shows that this RNA fragment is also able to become localized to one of the egg poles, as happens for the entire bep3 RNA.     

Polynucleotide phosphorylase is a component of a novel plant poly(A) polymerase

We have isolated cDNA clones encoding a novel RNA-binding protein that is a component of a multisubunit poly(A) polymerase from pea seedlings. The encoded protein bears a significant resemblance to polynucleotide phosphorylases (PNPases) from bacteria and chloroplasts. More significantly, this RNA-binding protein is able to degrade RNAs with the resultant production of nucleotide diphosphates, and it can add extended polyadenylate tracts to RNAs using ADP as a donor for adenylate moieties. These activities are characteristic of PNPase. Antibodies raised against the cloned protein simultaneously immunoprecipitate both poly(A) polymerase and PNPase activity. We conclude from these studies that PNPase is the RNA-binding cofactor for this poly(A) polymerase and is an integral player in the reaction catalyzed by this enzyme. The identification of this RNA-binding protein as PNPase, which is a chloroplast-localized enzyme known to be involved in mRNA 3'-end determination and turnover (Hayes, R., Kudla, J., Schuster, G., Gabay, L., Maliga, P., and Gruissem, W. (1996) EMBO J. 15, 1132-1141), raises interesting questions regarding the subcellular location of the poly(A) polymerase under study. We have reexamined this issue, and we find that this enzyme can be detected in chloroplast extracts. The involvement of PNPase in polyadenylation in vitro provides a biochemical rationale for the link between chloroplast RNA polyadenylation and RNA turnover which has been noted by others (Lisitsky, I., Klaff, P., and Schuster, G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13398-13403).     

Localization of HIV-1 co-receptors CCR5 and CXCR4 in the brain of children with AIDS

The neuropeptide Y Y2 receptor is one of six receptor subtypes mediating the multiform physiological actions of neuropeptide Y. The Y2 receptor has been demonstrated to be the most predominant receptor subtype in the human brain and appears to be involved in many neuropeptide Y actions, such as the regulation of locomotor activity, cardiovascular functions, memory processing, circadian rhythms and release of other neurotransmitters. We have recently demonstrated the widespread and abundant distribution of neuropeptide Y Y1 receptor messenger RNA in the human cerebral cortex (different laminar patterns within distinct cortical regions), hippocampal dentate gyrus and striatum. To assess a possible differential distribution of Y1 and Y2 receptor messenger RNAs, the regional expression of neuropeptide Y Y2 messenger RNA-containing cells in the human brain was analysed, in particular within the cerebral cortex and striatum. In situ hybridization experiments revealed the localization of the Y2 messenger RNA signal throughout all cortical regions, with the highest intensity per cell apparent in lamina IV, with the exception of the striate cortex, which showed an intense labelling primarily in layer VI. The striatum expressed low to undetectable levels of the Y2 receptor messenger RNA. The dentate gyrus and the CA2 region presented the highest hybridization signals, while a very weak Y2 messenger RNA expression was found in the CA1 region and subiculum. Positive Y2 messenger RNA hybridization signals were also detected in the lateral geniculate nucleus, amygdala, substantia nigra, hypothalamus, cerebellum and choroid plexus. These results demonstrate the widespread distribution of neuropeptide Y Y2 receptor messenger RNA in the human brain, with a pattern of expression distinct from the Y1 subtype, suggesting that these two receptor subtypes may mediate different neuropeptide Y functions in the human brain, mainly through actions on different neuronal systems.     

beta-Catenin is essential for patterning the maternally specified animal-vegetal axis in the sea urchin embryo

In sea urchin embryos, the animal-vegetal axis is specified during oogenesis. After fertilization, this axis is patterned to produce five distinct territories by the 60-cell stage. Territorial specification is thought to occur by a signal transduction cascade that is initiated by the large micromeres located at the vegetal pole. The molecular mechanisms that mediate the specification events along the animal-vegetal axis in sea urchin embryos are largely unknown. Nuclear beta-catenin is seen in vegetal cells of the early embryo, suggesting that this protein plays a role in specifying vegetal cell fates. Here, we test this hypothesis and show that beta-catenin is necessary for vegetal plate specification and is also sufficient for endoderm formation. In addition, we show that beta-catenin has pronounced effects on animal blastomeres and is critical for specification of aboral ectoderm and for ectoderm patterning, presumably via a noncell-autonomous mechanism. These results support a model in which a Wnt-like signal released by vegetal cells patterns the early embryo along the animal-vegetal axis. Our results also reveal similarities between the sea urchin animal-vegetal axis and the vertebrate dorsal-ventral axis, suggesting that these axes share a common evolutionary origin.     

A conserved 90 nucleotide element mediates translational repression of nanos RNA

Correct formation of the Drosophila body plan requires restriction of nanos activity to the posterior of the embryo. Spatial regulation of nanos is achieved by a combination of RNA localization and localization-dependent translation such that only posteriorly localized nanos RNA is translated. Cis-acting sequences that mediate both RNA localization and translational regulation lie within the nanos 3' untranslated region. We have identified a discrete translational control element within the nanos 3' untranslated region that acts independently of the localization signal to mediate translational repression of unlocalized nanos RNA. Both the translational regulatory function of the nanos 3'UTR and the sequence of the translational control element are conserved between D. melanogaster and D. virilis. Furthermore, we show that the RNA helicase Vasa, which is required for nanos RNA localization, also plays a critical role in promoting nanos translation. Our results specifically exclude models for translational regulation of nanos that rely on changes in polyadenylation.     

Expression of the CB1 and CB2 receptor messenger RNAs during embryonic development in the rat

We mapped the distribution of CB1 and CB2 receptor messenger RNAs in the developing rat to gain insight into how cannabinoids may affect embryogenesis. In situ hybridization histochemistry studies were done using riboprobes specific for CB1 or CB2 receptor messenger RNAs. We found that CB1 and CB2 receptor messenger RNAs are expressed in the placental cone and in the smooth muscle of the maternal uterus at the earliest gestational periods studied [from eight days of gestation (E8) through E12]. In the embryo, as early as E11, CB1 receptor messenger RNA is expressed in some cells of the neural tube and, at later embryological stages (from E15 to E21), in several distinct structures within the central nervous system. In addition, high levels of CB1 receptor messenger RNA were also found in areas of the peripheral nervous system such as the sympathetic and parasympathetic ganglia, in the retina and in the enteric ganglia of the gastrointestinal tract. In addition to neural structures, high levels of the CB1 receptor messenger RNA were also present in two endocrine organs, the thyroid gland and the adrenal gland. On the other hand, CB2 receptor messenger RNA is expressed exclusively in the liver of the embryo as early as E13. The region-specific expression of CB1 and CB2 receptor messenger RNAs suggests that these receptors have a functional role during embryogenesis.