Identification and cloning of localized maternal RNAs from Xenopus eggs

A central question in developmental biology is to explain how cells in different regions of an embryo acquire different developmental fates. We have begun to address this question by investigating whether specific RNAs are localized within a frog egg. Differential screening of a cDNA library shows that most maternal RNAs are uniformly distributed along the animal-vegetal axis. However, we find that a rare class of maternal RNAs is localized. cDNA clones of four localized RNAs have been characterized. Three of these cDNAs are derived from maternal RNAs that are concentrated in the animal hemisphere of unfertilized eggs and remain localized through the early blastula stage. One cDNA is derived from a maternal RNA found almost exclusively in the vegetal hemisphere at both stages. These studies show that some informational molecules, specifically RNAs, are localized in eggs and are inherited by particular blastomeres.     

Maternal information and localized maternal mRNAs in eggs and early embryos of the ascidian Halocynthia roretzi

Summary

The mosaic behavior of blastomeres isolated from ascidian embryos has been taken as evidence that localized ooplasmic factors (cytoplasmic determinants) specify tissue precursor cells during embryogenesis. Experiments involving the transfer of egg cytoplasm have revealed the presence and localization of various kinds of cytoplasmic determinants in eggs of Halocynthia roretzi. Three cell fates, epidermis, muscle and endoderm, are fixed by cytoplasmic determinants. The three kinds of tissue determinants move in different directions during ooplasmic segregation. Prior to the onset of the first cleavage the three kinds of determinants reside in egg regions that correspond to the future fate map of the embryo and then they are differentially partitioned into specific blastomeres. In addition to tissue-specific determinants, there is evidence suggesting that ascidian eggs contain localized cytoplasmic factors that are responsible for controlling the cleavage pattern and morphogenetic movements. Transplantation of posterior-vegetal egg cytoplasm to an anterior-vegetal position causes a reversal of the anterior-posterior polarity of the cleavage pattern. Localized cytoplasmic factors in the posterior-vegetal region are involved in the generation of a unique cleavage pattern. When vegetal pole cytoplasm is transplanted to the animal pole or equatorial position of the egg, ectopic gastrulation occurs at the site of transplantation. This finding supports the idea that vegetal pole cytoplasm specifies the site of gastrulation. Recently, we started a cDNA project to analyze maternal mRNAs. An arrayed cDNA library of fertilized eggs of H. roretzi was constructed, and more than 2000 clones have been partially sequenced so far. To estimate the proportion of the maternal mRNAs that are localized in the egg and embryo, 150 randomly selected clones were examined by in situ hybridization. We found eight mRNAs that are localized in the eight-cell embryo, of which three were localized to the myoplasm (a specific region of the egg cytoplasm that is partitioned into muscle-lineage blastomeres) of the egg, and then to the postplasm of cleavage-stage embryos. These results indicate that the proportion of localized messages is much higher than we expected. These localized maternal messages may be involved in the regulation of various developmental processes.     

Synergistic induction of mesoderm by FGF and TGF-beta and the identification of an mRNA coding for FGF in the early Xenopus embryo

The primary patterning event in early vertebrate development is the formation of the mesoderm and its subsequent induction of the neural tube. Classic experiments suggest that the vegetal region signals the animal hemisphere to diverge from the pathway of forming ectoderm to form mesoderm such as muscle. Here we show that bovine basic FGF has a limited capacity to induce muscle actin expression in animal hemisphere cells. This level of expression can be raised to levels normally induced in the embryo by another mammalian growth factor, TGF-beta, which by itself will not induce actin expression. We show that the Xenopus embryo contains an mRNA encoding a protein highly homologous to basic FGF. These results together with the identification of a maternal mRNA with strong homology to TGF-beta, suggest that molecules closely related to FGF and TGF-beta are the natural inducers of mesoderm in vertebrate development.     

Animal-vegetal polarity in the plasma membrane of a molluscan egg: a quantitative freeze-fracture study

Using freeze-fracture electron microscopy, the numerical particle distribution in the fertilized Nassarius egg plasma membrane has been analyzed in four areas at different positions along the animal-vegetal axis of the egg. These areas can be distinguished by distinct microvilli patterns and differences in microvilli densities. In all areas, more IMPs (intramembrane particles) are present on the P face than on the corresponding E face. The ratio of the number of IMPs present on E and P face is similar in all areas (0.48-0.55) except for the most animal part of the vegetal hemisphere, where relatively more IMPs remain attached to the exterior half of the fractured membrane (E/P ratio = 0.88). The IMP density at the vegetal pole of the egg is considerably higher than in the animal hemisphere and in the animal part of the vegetal hemisphere. This difference is due to an increased number of IMPs in all size classes (4-18 nm). In the area adjacent to the vegetal pole the density of particles is also higher than in the two more animal areas, but here the difference is exclusively due to the smaller IMP size classes (4-8 nm). Statistical analysis of our data reveals that the area adjacent to the vegetal pole patch is significantly different from the other areas with respect to the distribution of the IMPs over the different IMP size classes. These results demonstrate the polar organization of the Nassarius egg plasma membrane. The possible role of this surface heterogeneity in the spatial organization of the egg cell and the later embryo is discussed.     

Patterning of an ascidian embryo along the anterior-posterior axis through spatial regulation of competence and induction ability by maternally localized PEM

In ascidian embryos, anterior-posterior (A-P) patterning of the vegetal cells is regulated by posteriorizing activities of a localized egg region known as posterior-vegetal cortex/cytoplasm (PVC). PEM is an essential component of the PVC and is involved in the posterior-specific cell cleavage pattern. Here we report a novel function of PEM independently of its function in cleavage regulation; it controls cell fate by excluding competence to respond to the FGF signal for notochord induction from posterior-vegetal cells. PEM was found to regulate the nuclear accumulation of β-catenin, an upstream activator of the competence factor. PEM also influences A-P patterning in the animal hemisphere. It was found to regulate FGF signal expression and restrict the occurrence of brain induction only in the anterior region. Our results suggest a model in which PEM patterns the embryo along the A-P axis through regulation of the spatial distribution of competence and induction ability.     

Wnt6 activates endoderm in the sea urchin gene regulatory network

In the sea urchin, entry of β-catenin into the nuclei of the vegetal cells at 4th and 5th cleavages is necessary for activation of the endomesoderm gene regulatory network. Beyond that, little is known about how the embryo uses maternal information to initiate specification. Here, experiments establish that of the three maternal Wnts in the egg, Wnt6 is necessary for activation of endodermal genes in the endomesoderm GRN. A small region of the vegetal cortex is shown to be necessary for activation of the endomesoderm GRN. If that cortical region of the egg is removed, addition of Wnt6 rescues endoderm. At a molecular level, the vegetal cortex region contains a localized concentration of Dishevelled (Dsh) protein, a transducer of the canonical Wnt pathway; however, Wnt6 mRNA is not similarly localized. Ectopic activation of the Wnt pathway, through the expression of an activated form of β-catenin, of a dominant-negative variant of GSK-3β or of Dsh itself, rescues endomesoderm specification in eggs depleted of the vegetal cortex. Knockdown experiments in whole embryos show that absence of Wnt6 produces embryos that lack endoderm, but those embryos continue to express a number of mesoderm markers. Thus, maternal Wnt6 plus a localized vegetal cortical molecule, possibly Dsh, is necessary for endoderm specification; this has been verified in two species of sea urchin. The data also show that Wnt6 is only one of what are likely to be multiple components that are necessary for activation of the entire endomesoderm gene regulatory network.     

A maternal mRNA localized to the vegetal hemisphere in Xenopus eggs codes for a growth factor related to TGF-beta

We report that Vg1, a maternal mRNA localized to the vegetal hemisphere of frog eggs, encodes a member of the transforming growth factor-beta (TGF-beta) family of proteins. Furthermore, we show that Vg1 mRNA is distributed to presumptive endodermal cells after fertilization. Previous studies had shown that the vegetal end of a frog egg produces a signal that induces the overlying animal pole cells to form mesodermal tissue. More recently it has been shown that fibroblast growth factor (FGF) and TGF-beta can participate in the induction of muscle. Together, these results lead us to propose that the formation of mesoderm during frog development is specified by the products of localized maternal mRNAs, including Vg1.     

Early development and axis specification in the sea anemone Nematostella vectensis

We investigated the early development of the sea anemone Nematostella vectensis, an emerging model system of the Cnidaria. Early cleavage stages are characterized by substantial variability from embryo to embryo, yet invariably lead to the formation of a coeloblastula. The coeloblastula undergoes a series of unusual broad invaginations-evaginations which can be blocked by cell cycle inhibitors suggesting a causal link of the invagination cycles to the synchronized cell divisions. Blastula invagination cycles stop as cell divisions become asynchronous. Marking experiments show a clear correspondence of the animal-vegetal axis of the egg to the oral-aboral axis of the embryo. The animal pole gives rise to the concave side of the blastula and later to the blastopore of the gastrula, and hence the oral pole of the future polyp. Asymmetric distribution of granules in the unfertilized egg suggest an animal-vegetal asymmetry in the egg in addition to the localized position of the pronucleus. To determine whether this asymmetry reflects asymmetrically distributed determinants along the animal-vegetal axis, we carried out blastomere isolations and embryonic divisions at various stages. Our data strongly indicate that normal primary polyps develop only if cellular material from the animal hemisphere is included, whereas the vegetal hemisphere alone is incapable to differentiate an oral pole. Molecular marker analysis suggests that also the correct patterning of the aboral pole depends on signals from the oral half. This suggests that in Nematostella embryos the animal hemisphere contains organizing activity to form a normal polyp.     

A highly localized activation current yet widespread intracellular calcium increase in the egg of the frog, Discoglossus pictus

Sperm entry in the egg of the painted frog, Discoglossus pictus, occurs only at a specialized region of the animal hemisphere called the animal dimple, a structure not found in other species of frog. An extracellular vibrating electrode was used to measure the activation current to determine if the ion channels that open to generate the fertilization potential are localized in this region. Eggs that were activated by microinjecting inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) exhibited activation potentials very similar to those of fertilized eggs. There was a delay between the time of Ins(1,4,5)P3 injection and the initiation of the activation potential that was proportional to the distance between the site of the activating stimulus and the animal dimple, similar to the delay previously observed in prick-activated eggs (R. Talevi, B. Dale, and C. Campanella (1985). Dev. Biol. 111, 316-323). The delay lasted 30 sec when the stimulus site was 20 degrees (300 micron) from the animal dimple and 14 min when it was 150 degrees C from the dimple. Once the activation potential was initiated, there was an excellent temporal correlation between the time of depolarization and the time of the first detectable current entering the dimple region. This inward current was typically 60 microA/cm2 in amplitude and was found only in the central 200 micron of the dimple region. The outward current was distributed over the remainder of the egg surface and was much smaller in amplitude. The activation current was carried by Cl- efflux in the animal dimple region, and was reduced by DIDS and reversed by high external Cl- or I-. The occurrence of inward current only at the dimple region indicates that Cl- channels which open to produce the activation potential are localized there. Using Ca2+-specific microelectrodes, we found that [Ca2+]i increased from 0.25 to 2 microM following both fertilization and activation and returned to the unactivated level after about 37 min. Immature oocytes of D. pictus were also studied with the vibrating probe and the inward current in these cells was much less localized than that in the activating egg. A steady transcellular current of up to 4 microA/cm2 entered the entire animal hemisphere of the oocyte and exited the vegetal hemisphere.     

Specification process of animal plate in the sea urchin embryo

The most animal part of the ciliated band of sea urchin larvae, the animal plate, is a specialized region in which elongated cells form long and non-beating cilia. To learn how this region is specified, animal halves were isolated from the early cleavage to pregastrulation stages. As is well known, the animal half that is isolated at the eight-cell stage develops into a 'dauerblastula', which forms long and non-beating cilia all around the surface. The region with long cilia, however, became restricted toward the animal pole when separation was delayed. If separated before primary mesenchyme ingression, even a small animal-pole-side fragment formed a normal-sized animal plate. Thus, the prospective animal plate region is gradually restricted by some signal from the vegetal hemisphere, and the specification process terminates before the mesenchyme blastula stage. It was also known that a normal-sized animal plate was formed in micromere-less embryos, indicating that the signal does not depend on micromeres or their descendants. Further, the animal-pole-side fragments were isolated from embryos in which the third cleavage plane was shifted toward the vegetal pole. They formed a normal-sized animal plate, containing more than 75% of the egg volume from the animal pole. This indicates that the egg cytoplasm delivered to veg₁ -lineage blastomeres plays an important role in the animal plate specification. Interestingly, the an1-less embryo formed long and non-beating cilia at its top region, but thickening did not occur. The cytoplasm near the animal pole might contain some factors necessary for the animal plate to become thick.     

Intracellular expression profiles measured by real-time PCR tomography in the Xenopus laevis oocyte

Real-time PCR tomography is a novel, quantitative method for measuring localized RNA expression profiles within single cells. We demonstrate its usefulness by dissecting an oocyte from Xenopus laevis into slices along its animal-vegetal axis, extracting its RNA and measuring the levels of 18 selected mRNAs by real-time RT-PCR. This identified two classes of mRNA, one preferentially located towards the animal, the other towards the vegetal pole. mRNAs within each group show comparable intracellular gradients, suggesting they are produced by similar mechanisms. The polarization is substantial, though not extreme, with around 5% of vegetal gene mRNA molecules detected at the animal pole, and around 50% of the molecules in the far most vegetal section. Most animal pole mRNAs were found in the second section from the animal pole and in the central section, which is where the nucleus is located. mRNA expression profiles did not change following in vitro fertilization and we conclude that the cortical rotation that follows fertilization has no detectable effect on intracellular mRNA gradients.     

Differentiation of the animal-vegetal axis in Xenopus laevis oocytes. I. Polarized intracellular translocation of platelets establishes the yolk gradient

The animal-vegetal axis of the oocyte of Xenopus laevis is recognizable not only by the pattern of surface pigmentation, but also by the distribution of yolk platelets, with the largest platelets (congruent to 14 microns in diameter) and 70% of the total yolk protein localized in the vegetal hemisphere. We have used fluorescent and radioactive vitellogenins (yolk protein precursors) to study the spatial and temporal patterns of yolk deposition along this axis. We find that the rate of uptake of vitellogenin is nearly uniform over the surface of vitellogenic oocytes of all sizes. Once formed, yolk platelets in the animal hemisphere move inward, around the germinal vesicle, and into the central region of the vegetal hemisphere. Yolk platelets of the vegetal hemisphere do not actively move but are slowly displaced from the surface by successive layers of younger platelets arising and enlarging near the surface. The oldest yolk platelets, which arise circumcortically at the beginning of vitellogenesis in stage II and III oocytes, eventually come to reside in the vegetal hemisphere of stage VI oocytes, in the upper portion of the cup-shaped region of largest platelets. The vegetal hemisphere thus gains the majority of yolk protein by directed intracellular transport from the animal hemisphere adding to the amount directly sequestered by the vegetal hemisphere.