Multiple maternal influences on dorsal-ventral fate of Xenopus animal blastomeres.

Molecular asymmetries in the animal-vegetal axis of the Xenopus oocyte are well known to regulate the formation of gametes and germ layers. Likewise, many transplantation and explant studies demonstrate that maternal dorsalizing activities are localized to the future dorsal side of the embryo after fertilization, but to date only a few of the molecules involved in this process have been shown to be asymmetrically distributed. In this report, we identify two new aspects of the maternal regulation of dorsal-ventral fate asymmetry in Xenopus blastomeres: cytoplasmic polyadenylation of dorsal maternal mRNAs and localized Wnt8b signaling. Previous studies demonstrated that there are maternal, dorsal axis-inducing RNAs localized to dorsal animal blastomeres that become activated between the 8- and 16-cell stage (Hainski and Moody [1992] Development 116:347-355; Hainski and Moody [1996] Dev. Genet. 19:210-221). We report herein that the activation of these axis-inducing dorsal mRNAs is regulated by cytoplasmic polyadenylation. We also show that maternal wnt8b mRNA is concentrated in ventral animal blastomeres. These ventral cells and exogenous Wnt8b both inhibit the dorsal fate of neighboring blastomeres in culture, indicating that a maternal Wnt signal also contributes to segregating dorsal and ventral fates.     

From oocyte to 16-cell stage: cytoplasmic and cortical reorganizations that pattern the ascidian embryo.

The dorsoventral and anteroposterior axes of the ascidian embryo are defined before first cleavage by means of a series of reorganizations that reposition cytoplasmic and cortical domains established during oogenesis. These domains situated in the periphery of the oocyte contain developmental determinants and a population of maternal postplasmic/PEM RNAs. One of these RNAs (macho-1) is a determinant for the muscle cells of the tadpole embryo. Oocytes acquire a primary animal-vegetal (a-v) axis during meiotic maturation, when a subcortical mitochondria-rich domain (myoplasm) and a domain rich in cortical endoplasmic reticulum (cER) and maternal postplasmic/PEM RNAs (cER-mRNA domain) become polarized and asymmetrically enriched in the vegetal hemisphere. Fertilization at metaphase of meiosis I initiates a series of dramatic cytoplasmic and cortical reorganizations of the zygote, which occur in two major phases. The first major phase depends on sperm entry which triggers a calcium wave leading in turn to an actomyosin-driven contraction wave. The contraction concentrates the cER-mRNA domain and myoplasm in and around a vegetal/contraction pole. The precise localization of the vegetal/contraction pole depends on both the a-v axis and the location of sperm entry and prefigures the future site of gastrulation and dorsal side of the embryo. The second major phase of reorganization occurs between meiosis completion and first cleavage. Sperm aster microtubules and then cortical microfilaments cause the cER-mRNA domain and myoplasm to reposition toward the posterior of the zygote. The location of the posterior pole depends on the localization of the sperm centrosome/aster attained during the first major phase of reorganization. Both cER-mRNA and myoplasm domains localized in the posterior region are partitioned equally between the first two blastomeres and then asymmetrically over the next two cleavages. At the eight-cell stage the cER-mRNA domain compacts and gives rise to a macroscopic cortical structure called the Centrosome Attracting Body (CAB). The CAB is responsible for a series of unequal divisions in posterior-vegetal blastomeres, and the postplasmic/PEM RNAs it contains are involved in patterning the posterior region of the embryo. In this review, we discuss these multiple events and phases of reorganizations in detail and their relationship to physiological, cell cycle, and cytoskeletal events. We also examine the role of the reorganizations in localizing determinants, postplasmic/PEM RNAs, and PAR polarity proteins in the cortex. Finally, we summarize some of the remaining questions concerning polarization of the ascidian embryo and provide comparisons to a few other species. A large collection of films illustrating the reorganizations can be consulted by clicking on "Film archive: ascidian eggs and embryos" at http://biodev.obs-vlfr.fr/recherche/biomarcell/.     

Revisions to the Xenopus gastrula fate map: implications for mesoderm induction and patterning.

A revised fate map of the gastrula Xenopus embryo predicts the existence of patterning mechanisms that operate within the animal/vegetal axis of the mesoderm-forming marginal zone. We review here molecular and embryologic data that demonstrate that such mechanisms are present and that they operate independently of the Spemann organizer. Evidence suggests that polarized fibroblast growth factor activity in the animal/vegetal axis patterns this axis. We present a model of mesoderm induction and patterning that integrates the new data on Spemann organizer-independent animal/vegetal patterning with data on other inductive pathways known to act on the gastrula marginal zone.     

The carboxyl 35 amino acids of SV40 Vp3 are essential for its nuclear accumulation

To identify the moiety responsible for nuclear localization of the SV40 structural protein Vp3 in its natural environment, a transfection vector containing the entire coding regions of Vp2, Vp3, and agnoprotein, and one-third of the coding region of Vp1, was constructed. Several mutations were introduced into the plasmid and the subcellular distribution of Vp3 or mutant Vp3 was examined following DEAE-dextran-mediated DNA transfection into TC7 cells. Our study shows that Vp3 is synthesized and is transported into the nucleus in the absence of Vp2, agnoprotein, and intact Vp1. However, in the absence of its carboxyl-terminal 35 amino acids, the truncated Vp3 is limited to a cytoplasmic and perinuclear accumulation. Thus, the carboxyl 35 amino acids of Vp3 are required for its nuclear localization and may contain a nuclear accumulation signal.     

Aggregation of maternal pigment granules is induced by the cytosolic discoidin domain of the Xenopus Del1 protein.

Xenopus oocytes generate pigment granules (melanosomes) that predominantly localize to the animal hemisphere cortex. During embryonic development, these granules are located near the membranes of outer layer ectoderm cells. We report a novel phenotype found during an expression cloning screen in Xenopus laevis embryos. The phenotype is characterized by dissociation of pigment granules from the cell membrane to form large central aggregates. This phenomenon was induced by a truncated form of the Xenopus Del1 (XDel1) protein that contains only the C-terminal discoidin (D2) domain. This truncated form of XDel1 localized to membranes as shown by a chimeric enhanced green fluorescent protein construct. Although a similar localization occurred in immature oocytes, dissociation of pigment granules was limited to the oocyte vegetal hemisphere. The full-length XDel1 cDNA was cloned, and XDel1 mRNA expression was found to be ubiquitous and continuous from early oocyte to tail bud stages, with a transient enrichment in the cement gland. Ectopic expression of various deletion or full-length constructs or antisense morpholino oligonucleotides did not induce any significant developmental phenotypes.     

Dynamic changes in intranuclear and subcellular localizations of mouse Prrp/DAZAP1 during spermatogenesis: the necessity of the C-terminal proline-rich region for nuclear import and localization

Mouse Prrp (mPrrp)/DAZAP1 is a mouse ortholog of Xenopus Prrp, which is involved in vegetal pole localization of Vg1 mRNA in oocytes and is highly expressed in the testis. The mouse protein has been reported to be a shuttling protein which localizes in the nucleus of pre-meiotic spermatogenic cells and round spermatids, and shifts its location into the cytoplasm in elongating spermatids, suggesting that mPrrp may be involved in mRNA transport as well as that of the Xenopus ortholog. We reexamined immunohistochemical analyses of mPrrp/DAZAP1 during spermatogenesis utilizing a newly established monoclonal antibody and reconfirmed it to be a shuttling protein. We also carried out new observations that included remarkable intranuclear movement during spermatogenesis. In addition, we found that a long amino acid stretch which spanned over the C-terminal half of the protein was required for the nuclear import. These observations demonstrated dynamic changes in subnuclear and subcellular localization which might reflect specific functions during spermatogenesis.     

Beta-catenin signaling marks the prospective site of primitive streak formation in the mouse embryo.

Beta-catenin signaling has been shown to be involved in triggering axis formation in several organisms, including Xenopus and zebrafish. Genetic analysis has demonstrated that the Wnt/beta-catenin signaling pathway is also involved in axis formation in the mouse, since a targeted deletion of beta-catenin results in embryos that have a block in anterior-posterior axis formation, fail to initiate gastrulation, and do not form mesoderm. However, because beta-catenin is ubiquitously expressed, the precise time and cell types in which this signaling pathway is active during early embryonic development remain unknown. Thus, to better understand the role of the Wnt/beta-catenin signaling pathway in axis formation and mesoderm specification, we have examined both the distribution and signaling activity of beta-catenin during early embryonic development in the mouse. We show that the N-terminally nonphosphorylated form of beta-catenin as well as beta-catenin signaling is first detectable in the extraembryonic visceral endoderm in day 5.5 embryos. Before the initiation of gastrulation at day 6.0, beta-catenin signaling is asymmetrically distributed within the epiblast and is localized to a small group of cells adjacent to the embryonic--extraembryonic junction. At day 6.5 and onward, beta-catenin signaling was detected in the primitive streak and mature node. Thus, beta-catenin signaling precedes primitive streak formation and is present in epiblast cells that will go on to form the primitive streak. These results support a critical role for the Wnt/beta-catenin pathway in specifying cells to form the primitive streak and node in the mammalian embryo as well as identify a novel domain of Wnt/beta-catenin signaling activity during early embryogenesis.     

Simian virus 40 Vp2/3 small structural proteins harbor their own nuclear transport signal.

We have used a microinjection approach to identify a domain of the simian virus 40 (SV40) structural proteins Vp2 and Vp3(Vp2/3) responsible for their nuclear transport. By using both synthetic peptides, containing small regions of Vp2/3 conjugated to bovine serum albumin (BSA), and beta-galactosidase-Vp3 fusion proteins, we have narrowed this nuclear transport signal (NTS) to 9 amino acids (198 to 206 of Vp3 or 316 to 324 of Vp2), Gly-Pro-Asn-Lys-Lys-Lys-Arg-Lys-Leu. The porter proteins carrying the NTS or mutant NTS were microinjected into the cytoplasm of TC7 cells and their subcellular localization following the subsequent incubation period was determined immunologically using anti-BSA IgG or anti-beta-galactosidase. The 9-residue NTS peptide localized BSA into the nucleus of injected cells, changing lysine-202 to threonine or valine abolished this accumulation while changing arginine-204 to lysine did not grossly affect transport. A peptide containing the carboxyl-terminal 13 residues of Vp3 failed to localize BSA to the nucleus. Several single or double point mutations at Vp3 residues 202 and 204 have been introduced by site-directed mutagenesis. Vp3 residues 194-234, containing either a wild-type or mutated sequence at 202 and/or 204, were expressed in Escherichia coli as Vp3-beta-galactosidase fusion proteins. Addition of the carboxyl-terminal 40 residues, but not an internal 150 residues, to otherwise cytoplasmic beta-galactosidase promoted entry of the fusion protein into the nucleus. Changing lysine-202 into threonine, valine, or methionine abolished this nuclear accumulation as did changing arginine-204 into lysine. A double mutant at both positions was also blocked. We have also observed that the lectin wheat germ agglutinin inhibits the nuclear accumulation of BSA carrying the Vp2/3 NTS while the lectin concanavalin A had no effect. These data indicate that even small nuclear proteins can contain NTS's which most likely utilize a mechanism for nuclear import similar to that described for other larger proteins.     

Cytoarchitecture of Caudiverbera caudiverbera stage VI oocytes: a light and electron microscope study

The general characteristics and salient features of the full-grown stage VI Caudiverbera caudiverbera oocyte at the light and electron microscopy level are described. The oocyte is a huge cell with radial symmetry and distinct polarity. A black animal hemisphere, rich in pigment granules and containing the nucleus, is clearly distinguished from the unpigmented white-yellowish vegetal hemisphere. The cell is surrounded by a highly invaginated plasma membrane, with numerous microvilli. The cortex underlying the plasma membrane contains cortical and pigment granules, mitochondria, rough endoplasmic reticulum and coated vesicles. Cytoskeletal components, such as actin filaments and microtubules, are also found in this region. The predominant structures, distributed throughout the cell, are the yolk platelets, which show a gradient in size with small platelets in the animal half and very large ones in the vegetal zone. Mitochondria are also very abundant in both hemispheres and clouds of these organelles are found in the perinuclear region, frequently associated with microtubules. Developed Golgi complexes are present in the cytoplasm and occasionally, annulate lamellae appear towards the inner zones. The nucleus is a large structure containing numerous nucleoli. The nuclear envelope is highly invaginated, especially at the side facing the vegetal pole. It is regularly perforated by large nuclear pores. Our results show that the structural organization of Caudiverbera oocytes, although similar to that of other amphibian oocytes, differs from them especially concerning the spatial distribution of several structural components.     

Localization and loss-of-function implicates ciliary proteins in early, cytoplasmic roles in left-right asymmetry.

Left-right asymmetry is a crucial feature of the vertebrate body plan. While much molecular detail of this patterning pathway has been uncovered, the embryonic mechanisms of the initiation of asymmetry, and their evolutionary conservation among species, are still not understood. A popular recent model based on data from mouse embryos suggests extracellular movement of determinants by ciliary motion at the gastrulating node as the initial step. An alternative model, driven by findings in the frog and chick embryo, focuses instead on cytoplasmic roles of motor proteins. To begin to test the latter hypothesis, we analyzed the very early embryonic localization of ciliary targets implicated in mouse LR asymmetry. Immunohistochemistry was performed on frog and chick embryos using antibodies that have (KIF3B, Polaris, Polycystin-2, acetylated alpha-tubulin) or have not (LRD, INV, detyrosinated alpha-tubulin) been shown to detect in frog embryos only the target that they detect in mammalian tissue. Immunohistochemistry revealed localization signals for all targets in the cytoplasm of cleavage-stage Xenopus embryos, and in the base of the primitive streak in chick embryos at streak initiation. Importantly, several left-right asymmetries were detected in both species, and the localization signals were dependent on microtubule and actin cytoskeletal organization. Moreover, loss-of-function experiments implicated very early intracellular microtubule-dependent motor protein function as an obligate aspect of oriented LR asymmetry in Xenopus embryos. These data are consistent with cytoplasmic roles for motor proteins in patterning the left-right axis that do not involve ciliary motion.     

ELP3 localises to mitochondria and actin-rich domains at edges of HeLa cells

Motor neuron disease is associated with mutations in the ELP3 protein. Familial dysautonomia is a hereditary disease of the autonomous nervous system that occurs due to a mutation in the IκB kinase complex-associated protein (IKAP). Both ELP3 and IKAP are components of the ELONGATOR histone acetylase complex. This complex has six subunits ELP1 (IKAP)–ELP6. ELP3 is the acetylase component of the complex and is known to play a key role in histone acetylation. However, ELONGATOR components including IKAP also localise to cytoplasmic compartments, including actin-rich membrane ruffles. Therefore it is likely that the ELP3 acetylase may also acetylate cytoplasmic proteins. Here, we show using immunofluorescence with two different antibodies against ELP3 that it localises to mitochondria in HeLa cells as well as actin-like filaments and actin-rich sites at the edges of spreading cells. This suggests that ELP3, and possibly the ELONGATOR complex may play a role in mitochondrial function, as well as in actin organisation and cell motility.     

Morphogenetic domains in the yolk syncytial layer of axiating zebrafish embryos.

The yolk syncytial layer (YSL) of the teleostean yolk cell is known to play important roles in the induction of cellular mesendoderm, as well as the patterning of dorsal tissues. To determine how this extraembryonic endodermal compartment is subdivided and morphologically transformed during early development, we have examined collective movements of vitally stained YSL nuclei in axiating zebrafish embryos by using four-dimensional confocal microscopy. During blastulation, gastrulation, and early segmentation, zebrafish YSL nuclei display several highly patterned movements, which are organized into spatially distinct morphogenetic domains along the anterior-posterior and dorsal-ventral axes. During the late blastula period, with the onset of epiboly, nuclei throughout the YSL initiate longitudinal movements that are directed along the animal-vegetal axis. As epiboly progresses, nuclei progressively recede from the advancing margin of the epibolic YSL. However, a small group of nuclei is retained at the YSL margin to form a constricting blastoporal ring. During mid-gastrulation, YSL nuclei undergo convergent-extension behavior toward the dorsal midline, with a subset of nuclei forming an axial domain that underlies the notochord. These highly patterned movements of YSL nuclei share remarkable similarities to the morphogenetic movements of deep cells in the overlying zebrafish blastoderm. The macroscopic shape changes of the zebrafish yolk cell, as well as the morphogenetic movements of its YSL nuclei, are homologous to several morphogenetic behaviors that are regionally expressed within the vegetal endodermal cell mass of gastrulating Xenopus embryos. In contrast to the cellular endoderm of Xenopus, the dynamics of zebrafish YSL show that a syncytial endodermal germ layer can express a temporal sequence of morphogenetic domains without undergoing progressive steps of cell fate restriction.     

Cytoarchitecture of Caudiverbera caudiverbera stage VI oocytes: a light and electron microscope study

 The general characteristics and salient features of the full-grown stage VI Caudiverbera caudiverbera oocyte at the light and electron microscopy level are described. The oocyte is a huge cell with radial symmetry and distinct polarity. A black animal hemisphere, rich in pigment granules and containing the nucleus, is clearly distinguished from the unpigmented white-yellowish vegetal hemisphere. The cell is surrounded by a highly invaginated plasma membrane, with numerous microvilli. The cortex underlying the plasma membrane contains cortical and pigment granules, mitochondria, rough endoplasmic reticulum and coated vesicles. Cytoskeletal components, such as actin filaments and microtubules, are also found in this region. The predominant structures, distributed throughout the cell, are the yolk platelets, which show a gradient in size with small platelets in the animal half and very large ones in the vegetal zone. Mitochondria are also very abundant in both hemispheres and clouds of these organelles are found in the perinuclear region, frequently associated with microtubules. Developed Golgi complexes are present in the cytoplasm and occasionally, annulate lamellae appear towards the inner zones. The nucleus is a large structure containing numerous nucleoli. The nuclear envelope is highly invaginated, especially at the side facing the vegetal pole. It is regularly perforated by large nuclear pores. Our results show that the structural organization of Caudiverbera oocytes, although similar to that of other amphibian oocytes, differs from them especially concerning the spatial distribution of several structural components. Accepted: 27 October 1998