A gradient of poly(A)+ RNA sequences in Xenopus laevis eggs and embryos

Xenopus laevis eggs and gastrula stage embryos were fractionated into three equal sections normal to the animal-vegetal axis, and poly(A)⁺ RNA was isolated from each section. Hybridization of these poly(A)⁺ RNAs with [³²P]cDNA synthesized using animal or vegetal poly(A)⁺ RNAs showed no detectable differences in the extents or rates of reaction. Thus, the vast majority of poly(A)⁺ RNAs are not segregated along the animal-vegetal axis. To increase the sensitivity of these experiments, [³²P]cDNAs were prepared which had reduced levels of RNA sequences from the animal region of the gastrula stage embryo or spawned unfertilized egg. Hybridization reactions with these probes showed that 3 to 5% of the input cDNA represents poly(A)⁺ RNA sequences enriched 2- to 20-fold in the vegetal region of the egg or gastrula stage embryo.     

Ectopic expression of Xenopus noggin RNA induces complete secondary body axes in embryos of the direct developing frog Eleutherodactylus coqui

We tested the effects of noggin RNA from Xenopus laevis on axis induction in embryos of a direct developing frog, Eleutherodactylus coqui. We microinjected noggin RNA into one blastomere of 4-cell embryos at the site close to the animal pole, and found that overexpression of noggin RNA is not only sufficient to induce additional axes but also induces heads with eyes. We also injected noggin RNA into 8-cell or 16-cell embryos in various sites, including the marginal zone, above the marginal zone, and the vegetal pole, and found the formation of a complete secondary axis in all three types of injection. These effects of X. laevis noggin RNA on the E. coqui embryo are remarkably different from those found in the X. laevis embryo itself. It has been shown previously that overexpression of noggin RNA on the ventral side of the normal X. laevis embryo induces only a partial axis, with no head structures. We show here that the failure of noggin induction of a complete axis when overexpressed on the ventral side of the X. laevis embryos is not due to an insufficient amount of RNA injected. Also, the failure is unlikely due to inhibition from the primary axis since noggin RNA can induce duplicated head structures on opposite sides of UV-irradiated X. laevis embryos. There appear to be fundamental differences in the responses of E. coqui and X. laevis embryos to exogenous noggin RNA. We propose that these differences stem from an alteration in cytoplasmic arrangements that occurred during evolution of this large egg. Received: 26 July 1999 / Accepted: 1 September 1999     

Activin redux: specification of mesodermal pattern in Xenopus by graded concentrations of endogenous activin B

Mesoderm formation in the amphibian embryo occurs through an inductive interaction in which cells of the vegetal hemisphere of the embryo act on overlying equatorial cells. The first candidate mesoderm-inducing factor to be identified was activin, a member of the transforming growth factor type beta family, and it is now clear that members of this family are indeed involved in mesoderm and endoderm formation. In particular, Derrière and five nodal-related genes are all considered to be strong candidates for endogenous mesoderm-inducing agents. Here, we show that activin, the function of which in mesoderm induction has hitherto been unclear, also plays a role in mesoderm formation. Inhibition of activin function using antisense morpholino oligonucleotides interferes with mesoderm formation in a concentration-dependent manner and also changes the expression levels of other inducing agents such as Xnr2 and Derrière. This work reinstates activin as a key player in mesodermal patterning. It also emphasises the importance of checking for polymorphisms in the 5' untranslated region of the gene of interest when carrying out antisense morpholino experiments in Xenopus laevis.     

Two essential processes in the formation of a dorsal axis during gastrulation ofCynops embryo

The isolated upper marginal zone from the initial stage ofCynops gastrulation is not yet determined to form the dorsal axis mesoderm: notochord and muscle. In this experiment, we will indicate where the dorsal mesoderm-inducing activity is localized in the very early gastrula, and what is an important event for specification of the dorsal axis mesoderm during gastrulation. Recombination experiments showed that dorsal mesoderm-inducing activity was localized definitively in the endodermal epithelium (EE) of the lower marginal zone, with a dorso-ventral gradient; and the EE itself differentiated into endodermal tissues, mainly pharyngeal endoderm. Nevertheless, when dorsal EE alone was transplanted into the ventral region, a secondary axis with dorsal mesoderm was barely formed. However, when dorsal EE was transplanted with the bottle cells which by themselves were incapable of mesoderm induction, a second axis with well-developed dorsal mesoderm was observed. When the animal half with the lower marginal zone was rotated 180° and recombined with the vegetal half, most of the rotated embryos formed only one dorsal axis at the primary blastopore side. The present results suggest that there are at least two essential processes in dorsal axis formation: mesoderm induction of the upper marginal zone by endodermal epithelium of the lower marginal zone, and dorsalization of the upper dorsal marginal zone evoked during involution.     

Inhibition of mesoderm formation by follistatin

 Mesoderm induction requires interaction between cells of the animal and vegetal hemispheres of the embryo. Several molecules have been proposed as candidates for mesoderm-inducing signals, with activin a particularly strong candidate. However, it has not been possible to inhibit mesoderm formation in vivo by specifically blocking activin action. Follistatin is able to inhibit the action of activin but not that of the mature region of Vg1, a member of the transforming growth factor β family. Follistatin therefore provides a useful tool for distinguishing between signalling by these two factors. We have overexpressed Xenopus follistatin mRNA and analysed the expression of several mesodermal markers. Our results show an inhibition of mesodermal formation by follistatin in a concentration-dependent manner, showing the requirement of activin for mesodermal induction. Received: 22 August 1997 / Accepted: 16 January 1998     

Vegetal pole cells and commitment to form endoderm in Xenopus laevis

In order to compare their states of commitment with their normal developmental fate, single vegetal pole cells from early Xenopus embryos were labeled and transplanted into the blastocoels of host embryos. In a previous study we showed, using this single cell transplantation assay, that vegetal pole cells become committed to endoderm by the early gastrula stage. In this paper we examine some properties of the commitment process. First, we show that it is gradual. When vegetal blastomeres are taken from progressively older embryos an increasing number of them enter only the endoderm, until by the early gastrula stage they all do. Second, we show that commitment can continue in vitro when an appropriate tissue mass is present. We suggest that commitment to form endoderm may be, in the right conditions, a cell autonomous process.     

Lbx1 expression and frog limb development

In order to identify prospective limb muscle cells in a frog, we cloned Lbx1 from the direct developing frog Eleutherodactylus coqui. Like in embryos of the frog Xenopus laevis but unlike in other vertebrates, EcLbx1 is expressed in all trunk somites. Like in embryos of chick, mouse, and zebrafish, cells expressing EcLbx1 are then found in limb buds, consistent with migration of those cells from somites. EcLbx1 is also expressed in the dorsal spinal cord as in other vertebrates.     

Quantification of Cell Proliferation and Alpha-Toxin Gene Expression of Clostridium perfringens in the Development of Necrotic Enteritis in Broiler Chickens

Cell proliferation and alpha-toxin gene expression of Clostridium perfringens in relation to the development of necrotic enteritis (NE) were investigated. Unlike bacitracin-treated chickens, non-bacitracin-treated birds exhibited typical NE symptoms and reduced growth performance. They also demonstrated increased C. perfringens proliferation and alpha-toxin gene expression that were positively correlated and progressed according to the regression model y = b₀ + b₁X − b₂X². The average C. perfringens count of 5 log₁₀ CFU/g in the ileal digesta appears to be a threshold for developing NE with a lesion score of 2.     

Delayed transition to new cell fates during cellular reprogramming

In many embryos specification toward one cell fate can be diverted to a different cell fate through a reprogramming process. Understanding how that process works will reveal insights into the developmental regulatory logic that emerged from evolution. In the sea urchin embryo, cells at gastrulation were found to reprogram and replace missing cell types after surgical dissections of the embryo. Non-skeletogenic mesoderm (NSM) cells reprogrammed to replace missing skeletogenic mesoderm cells and animal caps reprogrammed to replace all endomesoderm. In both cases evidence of reprogramming onset was first observed at the early gastrula stage, even if the cells to be replaced were removed earlier in development. Once started however, the reprogramming occurred with compressed gene expression dynamics. The NSM did not require early contact with the skeletogenic cells to reprogram, but the animal cap cells gained the ability to reprogram early in gastrulation only after extended contact with the vegetal halves prior to that time. If the entire vegetal half was removed at early gastrula, the animal caps reprogrammed and replaced the vegetal half endomesoderm. If the animal caps carried morpholinos to either hox11/13b or foxA (endomesoderm specification genes), the isolated animal caps failed to reprogram. Together these data reveal that the emergence of a reprogramming capability occurs at early gastrulation in the sea urchin embryo and requires activation of early specification components of the target tissues.     

Biochemical research on oogenesis: Oocytes and liver cells of the teleost fish Tinca tinca contain different kinds of 5S RNA

Previtellogenic oocytes of Tinca tinca accumulate very large amounts of 5S RNA. We show here that 5S RNA stored in oocytes differs from liver 5S RNA in 3 out of 120 nucleotides. Liver and oocyte 5S RNAs, therefore, are produced by different genes. Both kinds of 5S genes are active in oocytes. However, only 5S RNA of the oocyte type accumulates in these cells. In Tinca tinca as in Xenopus laevis, oocyte-type and somatic-type 5S RNAs differ by three properties, ie., primary structure, conformation, and metabolic stability. Nucleotide substitutions occur in different positions in oocyte and somatic 5S RNAs of Tinca tinca and Xenopus laevis. We do not understand how different sets of nucleotide substitutions confer to 5S RNAs of both species similar properties in vivo, namely, increased metabolic stability.     

Injected Xwnt-8 RNA acts early in Xenopus embryos to promote formation of a vegetal dorsalizing center.

Expression cloning from a pool of gastrula cDNAs identified the Wnt family member Xwnt-8 as having dorsal axis-inducing activity in Xenopus embryos. Microinjected Xwnt-8 mRNA was able to rescue the development of a dorsally complete anterior-posterior axis in embryos ventralized by exposure to UV light. Axis induction was observed in embryos injected in either marginal or vegetal blastomeres at the 32-cell stage. Vegetal blastomeres receiving Xwnt-8 mRNA contributed progeny not to the induced dorsal axis, but to the endoderm, a result consistent with Xwnt-8 causing cells to act as a Nieuwkoop center (the vegetal-inducing component of normal dorsal axis formation), rather than as a Spemann organizer (the induced dorsal marginal zone component that directly forms the dorsal mesoderm). Xwnt-8, which is normally expressed ventrally in midgastrula and neurula embryos, appears to mimic, when injected, maternally encoded dorsal mesoderm-inducing factors that act early in development.     

L-bodies are RNA–protein condensates driving RNA localization in Xenopus oocytes

Ribonucleoprotein (RNP) granules are membraneless compartments within cells, formed by phase separation, that function as regulatory hubs for diverse biological processes. However, the mechanisms by which RNAs and proteins interact to promote RNP granule structure and function in vivo remain unclear. In Xenopus laevis oocytes, maternal mRNAs are localized as large RNPs to the vegetal hemisphere of the developing oocyte, where local translation is critical for proper embryonic patterning. Here we demonstrate that RNPs containing vegetally localized RNAs represent a new class of cytoplasmic RNP granule, termed localization-bodies (L-bodies). We show that L-bodies contain a dynamic protein-containing phase surrounding a nondynamic RNA-containing phase. Our results support a role for RNA as a critical component within these RNP granules and suggest that cis-elements within localized mRNAs may drive subcellular RNA localization through control over phase behavior.     

Effects of cell heterogeneity on production of polypeptide growth factors and mesoderm-inducing activity by Xenopus laevis XTC cells

The Xenopus laevis XTC cell line has been analyzed for the production of polypeptide growth factors and mesoderm-inducing activity. By the use of specific biological assays, it is shown that XTC cells produce a growth factor functionally related to the platelet-derived growth factor (PDGF) and two transforming growth factor (TGF)β-like activities. Mesoderm-inducing activity, as measured on X. laevis ectodermal explants from stage 10 embryos, was found to coelute on a Bio-Gel P-100 column with one of the TGFβ-like activities at an apparent molecular weight of 6–10 kDa. Analysis of the DNA content from XTC cells by flow cytometry demonstrated that the cell line is heterogeneous and consists of both tetraploid and diploid cells. Cloning of the XTC cells and selecting single-cell colonies on the basis of their ability to grow in soft agar resulted in the isolation of several homogeneous, morphologically different clonal derivatives. Analysis of conditioned medium from these clonal derivatives showed that only one of them, the only diploid line among six investigated, produced a strong heat- and acid-stable mesoderm-inducing activity that induced notochord and muscle formation in stage 10 X. laevis ectodermal expiants. The relation between this activity and a recently described TGFβ-like mesoderm-inducing factor obtained from XTC-conditioned medium will be discussed. In conclusion, a clonal cell line derived from X. laevis XTC cells which provides a good source for further characterization of mesoderm-inducing factors has been established.     

Fates and states of determination of single vegetal pole blastomeres of X. laevis

Vegetal pole cells of Xenopus morulae contribute progeny to all three germ layers, but from the midblastula stage onward they contribute only to the endoderm. We have investigated whether this restriction in fate reflects cell determination by implanting labeled vegetal pole cells into the blastocoels of host embryos and asking which structures later include labeled progeny. Single vegetal pole cells from the morula and also from the midblastula stage can contribute progeny to all germ layers. At the early gastrula stage the cells can contribute only to the endoderm. Thus the restriction of fate in the midblastula does not reflect cell determination. However, the cells do become determined by the beginning of gastrulation.