The fate of cells in the tailbud of Xenopus laevis

The vertebrate tailbud and trunk form very similar tissues. It has been a controversial question for decades whether cell determination in the developing tail proceeds as part of early axial development or whether it proceeds by a different mechanism. To examine this question more closely, we have used photoactivation of fluorescence to mark small neighborhoods of cells in the developing tailbud of Xenopus laevis. We show that, in one region of the tailbud, very small groups of adjacent cells can contribute progeny to the neural tube, notochord and somitic muscle, as well as other identified cell types within a single embryo. Groups averaging three adjacent cells at a later stage can contribute progeny with a similar distribution. Our data suggest that the tailbud contains multipotent cells that make very late germ-layer decisions.     

Transport of different RNA species from the nucleus to the cytoplasm in Xenopus laevis neurula cells

Isolated cells from Xenopus laevis neurulae were labeled, and the RNAs extracted from their nuclear and soluble cytoplasmic fractions were analyzed on polyacrylamide gels. In the soluble cytoplasm, 4S RNA emerged very rapidly, and this was immediately followed by the emergence of poly(A)-containing RNA and 18S ribosomal RNA. In contrast, the emergence of 28S ribosomal RNA was delayed by about 2 hr. The size distribution of cytoplasmic poly(A)-containing RNA was much smaller as compared to that of nuclear poly(A)-containing RNA. These results indicate that the newly synthesized RNAs in Xenopus neurula cells are transported from the nucleus to the cytoplasm in a characteristic sequence.     

Deep cytoplasmic rearrangements during early development in Xenopus laevis

The egg of the frog Xenopus is cylindrically symmetrical about its animal-vegetal axis before fertilization. Midway through the first cell cycle, the yolky subcortical cytoplasm rotates 30 degrees relative to the cortex and plasma membrane, usually toward the side of the sperm entry point. Dorsal embryonic structures always develop on the side away from which the cytoplasm moves. Details of the deep cytoplasmic movements associated with the cortical rotation were studied in eggs vitally stained during oogenesis with a yolk platelet-specific fluorescent dye. During the first cell cycle, eggs labelled in this way develop a complicated swirl of cytoplasm in the animal hemisphere. This pattern is most prominent on the side away from which the vegetal yolk moves, and thus correlates in position with the prospective dorsal side of the embryo. Although the pattern is initially most evident near the egg's equator or marginal zone, extensive rearrangements associated with cleavage furrowing (cytoplasmic ingression) relocate portions of the swirl to vegetal blastomeres on the prospective dorsal side.     

Early cellular interactions promote embryonic axis formation in Xenopus laevis

We have attempted to define the location and mode of action of axial determinants in the egg of Xenopus laevis. To this end, we transplanted small numbers of blastomeres from normal 64-cell stage embryos into synchronous recipient embryos which had been irradiated with ultraviolet light prior to first cleavage. Without transplantation, such embryos fail to develop dorsal structures of the embryonic body axis. We found that one to three blastomeres transplanted from the vegetal-most octet of cells can effect complete or partial rescue of of axis development in a recipient, provided that the donor cells derive from the quadrant just under the prospective dorsal marginal region. These same cells, when transplanted into the ventral vegetal quadrant of a normal 64-cell embryo, cause the formation of a complete second body axis. In contrast, other cells from the vegetal octet of normal donors fail to cause axis formation. When the rescuing donor cells are labeled with a lineage-restricted fluorescent marker, we find that their progeny do not contribute to the axial structures of the recipient. Progeny of the transplanted cells are found below the level of the blastopore in the early gastrula and eventually give rise to portions of the gut, as is their fate in normal development. These results, in agreement with those of Nieuwkoop (P.D. Nieuwkoop, 1977, Curr. Top. Dev. Biol. 11, 115-132), imply that the dorsal-most vegetal cells of the 64-cell embryo receive from the egg cytoplasm a set of determinants enabling them to induce neighboring cells to undertake axis formation. We discuss the relationship between axis induction in rescued irradiated embryos and axis determining processes in normal embryogenesis.     

The role of Xmsx-2 in the anterior-posterior patterning of the mesoderm in Xenopus laevis

Many molecules are involved in defining mesodermal patterning of the Xenopus embryo. In this paper, evidence is provided that a member of the msx family of genes, the Xmsx-2 gene, is involved in anterior-posterior patterning of the mesoderm. A comparison of its sequence to another previously cloned msx-2 Xenopus homolog, Xhox-7.1' [45] showed that they are closely related. The Xmsx-2 gene is first expressed at midgastrulation predominantly in the dorsal part of the embryo. It showed a complex pattern of spatial expression, consistent with a role in patterning of the anterior-posterior axis. This inference is confirmed by gain-of-function experiments in which overexpressed msx-2 mRNA in developing Xenopus embryos resulted in embryos lacking anterior structures. Analysis of markers in mutant embryos showed that genes involved in ventral-posterior patterning such as Xhox-3, Xwnt-8, and Xvent-1 were upregulated, confirming the posteriorized nature of the embryos. We believe that the Xmsx-2 gene is involved in refining the patterning of the anterior-posterior part of the dorsal mesoderm after the initial signals determining the dorsal or ventral nature of the mesoderm have been specified.     

Effects of various ammonium salts, amines, polyamines and alpha-methylornithine on rRNA synthesis in neurula cells of Xenopus laevis and Xenopus borealis

Xenopus neurula cells were cultured in a medium that contained ammonium salts, amines, polyamines or alpha-methylornithine, and their rRNA synthesis was examined. All the ammonium salts and amines, but not polyamines, were strong and selective inhibitors of rRNA synthesis at 1.25-5.0 mM. alpha-Methylornithine did not inhibit rRNA synthesis, although it inhibited ornithine decarboxylase, an enzyme claimed to be a direct stimulator of rRNA synthesis. During the treatment ammonium ions and monomethylamines were accumulated within the treated cells. However, monomethylamines did not induce the accumulation of ammonium ions, and vice versa. Ammonium salts and amines also selectively inhibited rRNA synthesis in Xenopus borealis neurula cells.     

Progressive determination during formation of the anteroposterior axis in Xenopus laevis

The cement gland is an ectodermal organ in the head of frog embryos, lying anterior to any neural tissue. As analyzed by specific RNA expression, cement gland, like neural tissue, was induced by the dorsal mesoderm. Interestingly, mesoderm with the highest cement gland-inducing potential lay posterior to the ectoderm fated to form this organ, indicating that its induction occurred at a distance from the inducer source. Cement gland induction first occurred during early gastrulation. However, most initially induced cells did not contribute to the mature cement gland, but instead formed part of the neural plate. This change in fate could be reconstituted in vitro. These results suggest that determination of part of the anteroposterior axis occurs progressively, where future neural ectoderm is first induced to a cement glandlike state. As gastrulation proceeds, further induction by mesoderm may override this state, which persists only in the extreme anterior of the embryo.     

Designation of the anterior/posterior axis in pregastrula Xenopus laevis

A new fate map for mesodermal tissues in Xenopus laevis predicted that the prime meridian, which runs from the animal pole to the vegetal pole through the center of Spemann's organizer, is the embryo's anterior midline, not its dorsal midline (M. C. Lane and W. C. Smith, 1999, Development 126, 423-434). In this report, we demonstrate by lineage labeling that the column 1 blastomeres at st. 6, which populate the prime meridian, give rise to the anterior end of the embryo. In addition, we surgically isolate and culture tissue centered on this meridian from early gastrulae. This tissue forms a patterned head with morphologically distinct ventral and dorsal structures. In situ hybridization and immunostaining reveal that the cultured heads contain the anterior tissues of all three germ layers, correctly patterned. Regardless of how we dissect early gastrulae along meridians running from the animal to the vegetal pole, both the formation of head structures and the expression of anterior marker genes always segregate with the prime meridian passing through Spemann's organizer. The prime meridian also gives rise to dorsal, axial mesoderm, but not uniquely, as specification tests show that dorsal mesoderm arises in fragments of the embryo which exclude the prime meridian. These results support the hypothesis that the midline that bisects Spemann's organizer is the embryo's anterior midline.     

Positive and Negative Regulation of the Differentiation of Ventral Mesoderm for Erythrocytes in Xenopus laevis

To elucidate the mechanism of determination and regulation of hemopoiesis in the early Xenopus embryo, explants of dorsal and ventral mesoderm from various stage embryos were cultured alone or combined with various tissues derived from the same stage embryo. Western blot analysis of larvae-specific globin expression using monoclonal antibody L5.41 revealed that extensive erythropoiesis occurred in the explants of ventral mesoderm from st. 22 tailbud embryo, but not in those of dorsal mesoderm. Experiments using combined explants at this stage demonstrated that the in vitro differentiation of erythrocytes in the ventral mesoderm could be completely inhibited by the dorsal tissue, including neural tube, notochord, and somite mesoderm, but not by other mesoderms, gut endoderm, or forebrain. Subsequent explant studies showed that the notochord alone is sufficient for this inhibition. Furthermore, the ventral mesoderm explant from the st. 10⁺ early gastrula embryo was not able to differentiate into erythroid cells. However, small amounts of globin were expressed if ventral mesoderm of this stage was combined with animal pole cells which were mainly differentiated to epidermis. This stimulation was enhanced when both tissues were excised together without separation, while none of the other parts of st. 10⁺ embryo had this stimulatory effect. These observations found in the combined explants suggest that in vivo interactions between the ventral mesoderm and adjacent tissues are important for normal development of erythroid precursor cells.     

Contribution of Ventral Blood Island (VBI)-Derived Cells to Postembryonic Liver Erythropoiesis in Xenopus laevis

The ventral blood islands (VBI) of Xenopus laevis embryos are known as the hemopoietic site where the initial erythropoiesis takes place at st. 28. To determine the site of postembryonic erythropoiesis, larvae were induced anemic by phenylhydrazine (PHZ) at st. 31 and 40, and the tissue distribution of regenerating erythrocytes was determined with an anti-larval hemoglobin (LHb) monoclonal antibody. Three days after total anemia induction, the LHb⁺cells were detected first in the liver and the digestive tract, followed by the appearance of a few LHb⁺cells in the blood vessels. The lavae which had been hepatectomized and cardiectomized before the PHZ treatment showed a remarkable reduction in recovery of the LHb⁺cells. Induction of anemia in the chimeric individuals containing cytogenetically labelled VBI tissues demonstrated that the VBI-derived cells contribute to the regenerating LHb⁺cells in all experimental individuals. These results suggest that the larval liver is the major site where the VBI-derived hemopoietic cells reside and differentiate into erythrocytes.     

Polyethylene glycol- and lysolecithin-induced cell fusion between follicle cell and very small oocyte in Xenopus laevis

Cell fusion between follicle cells and very small oocytes in Xenopus laevis was induced by means of polyethylene glycol 6000 (PEG) and lysolecithin (LL) treatments. Ovarian pieces from toadlets 1 month after metamorphosis were pretreated with trypsin and collagenase and then treated with fusogens. The survival rates of oocytes, degrees of cell fusion and morphological changes of the follicle cell nuclei in the oocyte cytoplasms were examined on the histological sections after 24 h culture in vitro at 23 °C. The fusogenic ability of PEG was found to be much higher than LL in follicle cell-oocyte fusion. The follicle cell nuclei which were surrounded by cytoplasm of the very small oocyte (<100 μm Ø) showed a morphology similar to the zygotene and pachytene chromosomes of the original oocytes.     

Coordination of cell proliferation and anterior-posterior axis establishment in the mouse embryo

During development, the growth of the embryo must be coupled to its patterning to ensure correct and timely morphogenesis. In the mouse embryo, migration of the anterior visceral endoderm (AVE) to the prospective anterior establishes the anterior-posterior (A-P) axis. By analysing the distribution of cells in S phase, M phase and G2 from the time just prior to the migration of the AVE until 18 hours after its movement, we show that there is no evidence for differential proliferation along the A-P axis of the mouse embryo. Rather, we have identified that as AVE movements are being initiated, the epiblast proliferates at a much higher rate than the visceral endoderm. We show that these high levels of proliferation in the epiblast are dependent on Nodal signalling and are required for A-P establishment, as blocking cell division in the epiblast inhibits AVE migration. Interestingly, inhibition of migration by blocking proliferation can be rescued by Dkk1. This suggests that the high levels of epiblast proliferation function to move the prospective AVE away from signals that are inhibitory to its migration. The finding that initiation of AVE movements requires a certain level of proliferation in the epiblast provides a mechanism whereby A-P axis development is coordinated with embryonic growth.     

Generation of the germ layers along the animal-vegetal axis in Xenopus laevis

After completion of gastrulation, typical vertebrate embryos consist of three cell sheets, called germ layers. The outer layer, the ectoderm, which produces the cells of the epidermis and the nervous system; the inner layer, the endoderm, producing the lining of the digestive tube and its associated organs (pancreas, liver, lungs etc.) and the middle layer, the mesoderm, which gives rise to several organs (heart, kidney, gonads), connective tissues (bone, muscles, tendons, blood vessels), and blood cells. The formation of the germ layers is one of the earliest embryonic events to subdivide multicellular embryos into a few compartments. In Xenopus laevis, the spatial domains of three germ layers are largely separated along the animal-vegetal axis even before gastrulation; ectoderm in the animal pole region; mesoderm in the equatorial region and endoderm in the vegetal pole region. In this review, we summarise the recent advances in our understanding of the formation of the germ layers in Xenopus laevis.     

Involvement of the Xenopus homeobox gene Xhox3 in pattern formation along the anterior-posterior axis

The Xenopus homeobox gene Xhox3 shows a graded expression in the axial mesoderm, with the highest concentration in the posterior end of frog gastrula and neurula embryos. To investigate the function of the Xhox3 gene, synthetic Xhox3 mRNA was injected into different regions of developing embryos. In particular, Xhox3 was supplied in excess to anterior cells, which normally have the lowest levels of Xhox3 RNA. The results show that injection of Xhox3, but not control, mRNA into prospective anterior regions of developing embryos produces a series of graded axial defects. The injected embryos gastrulate normally but fail to form anterior (head) structures. Our findings suggest that Xhox3 is involved in establishing anterior-posterior cell identities during pattern formation of the axial mesoderm in early embryonic development.     

Comparison of induction during development between Xenopus tropicalis and Xenopus laevis

Several in vitro systems exist for the induction of animal caps using growth factors such as activin. In this paper, we compared the competence of activin-treated animal cap cells dissected from the late blastulae of Xenopus tropicalis and Xenopus laevis. The resultant tissue explants from both species differentiated into mesodermal and endodermal tissues in a dose-dependent manner. In addition, RT-PCR analysis revealed that organizer and mesoderm markers were expressed in a similar temporal and dose-dependent manner in tissues from both organisms. These results indicate that animal cap cells from Xenopus tropicalis have the same competence in response to activin as those from Xenopus laevis.