Xenopus tropicalis nodal-related gene 3 regulates BMP signaling: an essential role for the pro-region

In vertebrates, nodal-related genes are crucial for specifying mesendodermal cell fates. Six nodal-related genes have been identified in Xenopus, but only one, nodal, has been identified in the mouse. The Xenopus nodal-related gene 3 (Xnr3), however, lacks the mesoderm-inducing activity of the other five nodal-related genes in Xenopus, and can directly induce neural tissue in animal caps by antagonizing BMP signals. In this study, we isolated three clones of the Xenopus (Silurana) tropicalis nodal-related gene 3 (Xtnr3) and analyzed their function. The Xtnr3 genes show high homology to Xnr3 and have the same activity. Southern blot and genomic PCR analyses indicate that the X. tropicalis genome has duplications in the Xtnr3 gene sequences and our three clones represent separate gene loci. We also found a partial clone of Xtnr3 that coded for the N-terminal part of its pro-region. Surprisingly, this sequence also induced neural tissue by antagonizing BMP signals, and its coded protein physically associated with BMP4 mature protein. Furthermore, we showed that the pro-region of Xnr5 has the same activity. Together, these findings indicate that the pro-region of nodal-related genes acts antagonistically towards BMP signals, which identifies a novel mechanism for the inhibition of BMP signaling.     

Endocannabinoid system in Xenopus laevis development: CB1 receptor dynamics

This study investigates for the first time the dynamics of endocannabinoid system appearance during low vertebrate Xenopus laevis development. We observed that the CB1 gene started to be expressed during the organogenesis period (+/- 1 dpf, st. 28) and expression persisted throughout the three further stages analyzed. Attention was focused on the localization of the CB1 messenger that was found both at the central level (in romboencephalon and in olfactory placods) and at the peripheral level (in the gastrointestinal tract) at +/- 3 dpf (st. 41), +/- 4 dpf (st. 46) and +/- 12 dpf (st. 49). We also considered the synthesis of CB1 protein that occurred from st. 41 onwards and, from this stage, we tested the receptor functionality in response to anandamide using cytosensor microphysiometry. CB1 functionality increased with development at both central and peripheral level. These data provide sufficient evidence to encourage further analysis on endocannabinoid physiological roles during embryonic and larval X. laevis growth.     

A Xenopus tropicalis oligonucleotide microarray works across species using RNA from Xenopus laevis

Microarrays have great potential for the study of developmental biology. As a model system Xenopus is well suited for making the most of this potential. However, Xenopus laevis has undergone a genome wide duplication meaning that most genes are represented by two paralogues. This causes a number of problems. Most importantly the presence of duplicated genes mean that a X. laevis microarray will have less or even half the coverage of a similar sized microarray from the closely related but diploid frog Xenopus tropicalis. However, to date, X. laevis is the most commonly used amphibian system for experimental embryology. Therefore, we have tested if a microarray based on sequences from X. tropicalis will work across species using RNA from X. laevis. We produced a pilot oligonucleotide microarray based on sequences from X. tropicalis. The microarray was used to identify genes whose expression levels changed during early X. tropicalis development. The same assay was then carried out using RNA from X. laevis. The cross species experiments gave similar results to those using X. tropicalis RNA. This was true at the whole microarray level and for individual genes, with most genes giving similar results using RNA from X. laevis and X. tropicalis. Furthermore, the overlap in genes identified between a X. laevis and a X. tropicalis set of experiments was only 12% less than the overlap between two sets of X. tropicalis experiments. Therefore researchers can work with X. laevis and still make use of the advantages offered by X. tropicalis microarrays.     

Wnt/beta-catenin signaling controls Mespo expression to regulate segmentation during Xenopus somitogenesis

The vertebral column is derived from somites, which are transient segments of the paraxial mesoderm that are present in developing vertebrates. The strict spatial and temporal regulation of somitogenesis is of crucial developmental importance. Signals such as Wnt and FGF play roles in somitogenesis, but details regarding how Wnt signaling functions in this process remain unclear. In this study, we report that Wnt/beta-catenin signaling regulates the expression of Mespo, a basic-helix-loop-helix (bHLH) gene critical for segmental patterning in Xenopus somitogenesis. Transgenic analysis of the Mespo promoter identifies Mespo as a direct downstream target of Wnt/beta-catenin signaling pathway. We also demonstrate that activity of Wnt/beta-catenin signaling in somitogenesis can be enhanced by the PI3-K/AKT pathway. Our results illustrate that Wnt/beta-catenin signaling in conjunction with PI3-K/AKT pathway plays a key role in controlling development of the paraxial mesoderm.     

Asymmetric Nodal expression in the mouse is governed by the combinatorial activities of two distinct regulatory elements

In all vertebrates, invariant left/right (L/R) positioning and organization of the internal viscera is controlled by a conserved pathway. Nodal, a member of the TGFbeta superfamily is a critical upstream component responsible for initiating L/R axis determination. Asymmetric Nodal expression in the node preceeds and foreshadows morphological L/R asymmetry. Here we address the mechanism of Nodal activation in the left LPM by studying the function of a novel enhancer element, the AIE. We show this element is exclusively active in cells of the left lateral plate mesoderm (LPM) and is not itself responding to Nodal asymmetry. To test the hypothesis that this element may initiate asymmetric Nodal expression in the LPM, we deleted it from the mouse germ line. Mice homozygous for the AIE deletion (Nodal(deltaaie/deltaaie)) show no defects. However, we find that the AIE contributes to regulating the level of asymmetric Nodal activity; analysis of transheterozygous embryos (Nodal(deltaaie/null)) shows reduced Nodal expression in the left LPM associated with a low penetrance of L/R defects. Our findings point to the existence of two independent pathways that control Nodal expression in the left LPM.     

Spatially and temporally regulated alpha6 integrin cleavage during Xenopus laevis development

The α6 integrin is essential for early nervous system development in Xenopus laevis. We have previously reported a uPA cleaved form of integrin α6 (α6p), in invasive human prostate cancer tissue, whose presence correlates with increased migration and invasive capacity. We now report that α6 is cleaved during the normal development of Xenopus in a spatially and temporally controlled manner. In addition, unlike normal mammalian tissues, which lack α6p, the major form of the α6 integrin present in adult Xenopus is α6p. The protease responsible for the cleavage in mammals, uPA, is not involved in the cleavage of Xenopus α6. Finally, overexpression of a mammalian α6 mutant which cannot be cleaved leads to developmental abnormalities suggesting a potential role for the cleavage in development.     

Highly efficient transgenesis in Xenopus tropicalis using I-SceI meganuclease

In this study, we report a highly efficient transgenesis technique for Xenopus tropicalis based on a method described first for Medaka. This simple procedure entails co-injection of meganuclease I-SceI and a transgene construct flanked by two I-SceI sites into fertilized eggs. Approximately 30% of injected embryos express transgenes in a promoter-dependent manner. About 1/3 of such embryos show incorporation of the transgene at the one-cell stage and the remainder are 'half-transgenics' suggesting incorporation at the two-cell stage. Transgenes from both classes of embryos are shown to be transmitted and expressed in offspring. The procedure also works efficiently in Xenopus laevis. Because the needle injection procedure does not significantly damage embryos, a high fraction develop normally and can, as well, be injected with a second reagent, for example an mRNA or antisense morpholino oligonucleotide, thus allowing one to perform several genetic manipulations on embryos at one time. This simple and efficient technique will be a powerful tool for high-throughput transgenesis assays in founder animals, and for facilitating genetic studies in the fast-breeding diploid frog, X. tropicalis.     

Expression of Sox1 during Xenopus early embryogenesis

Sox B1 group genes, Sox1, Sox2, and Sox3 (Sox1-3), are involved in neurogenesis in various species. Here, we identified the Xenopus homolog of Sox1, and investigated its expression patterns and neural inducing activity. Sox1 was initially expressed in the anterior neural plate of Xenopus embryos, with expression restricted to the brain and optic vesicle by the tailbud stage. Expression subsequently decreased in the eye region by the tadpole stage. Sox1 expression in animal cap explants was induced by inhibition of BMP signaling in the same manner as Sox2, Sox3, and SoxD. In addition, overexpression of Sox1 induced neural markers in ventral ectoderm and in animal caps. These results implicate Xenopus Sox1 in neurogenesis, especially brain and eye development.     

Foxj1 regulates asymmetric gene expression during left-right axis patterning in mice

Mice with a targeted mutation of the foxj1 gene demonstrate either D- or L-looping of the embryonic cardiac tube. Foxj1 is expressed in ventral cells of the embryonic node prior to asymmetric, left-right expression of other genes. Despite an absence of 9+2 cilia in foxj1(-/-) mice, 9+0 cilia are present in the node of foxj1(-/-) embryos. In foxj1(-/-) embryos, the patterns of expression of the TGF-beta family member nodal and the homeobox family member pitx2 are randomized. No expression of the TGF-beta family member lefty-2 is observed in any foxj1(-/-) early somite stage embryos. Foxj1 thus acts early in left-right axis patterning and regulates asymmetric gene expression. This regulation does not appear to be the result of a direct interaction between Foxj1 and the genes examined.     

Identification and preliminary function study of Xenopus laevis DRR1 gene

Xenopus laevis has recently been determined as a novel study platform of gene function. In this study, we cloned Xenopus DRR1 (xDRR1), which is homologous to human down-regulated in renal carcinoma (DRR1) gene. Bioinformatics analysis for DRR1 indicated that xDRR1 shared 74% identity with human DRR1 and 66% with mouse DRR1, and the phlogenetic tree of DRR1 protein was summarized. The xDRR1 gene locates in nuclei determined by transfecting A549 cells with the recombinant plasmid pEGFP-N1/xDRR1. RT-PCR analysis revealed that xDRR1 gene was expressed in all stages of early embryo development and all kinds of detected tissues, and whole-mount in situ hybridization showed xDRR1 was mainly present along ectoderm and mesoderm. Furthermore, xDRR1 expression could suppress A549 cell growth by transfecting with plasmid pcDNA3.1(+)/xDRR1. xDRR1 probably plays important roles involving in cell growth regulation and Xenopus embryo development.     

ALK4 functions as a receptor for multiple TGF beta-related ligands to regulate left-right axis determination and mesoderm induction in Xenopus

In Xenopus, several TGF betas, including nodal-related 1 (Xnr1), derriere, and chimeric forms of Vg1, elicit cardiac and visceral organ left-right (LR) defects when ectopically targeted to right mesendoderm cell lineages, suggesting that LR axis determination may require activity of one or more TGF betas. However, it is not known which, if any, of these ligands is required for LR axis determination, nor is it known which type I TGF beta receptor(s) are involved in mediating left-side TGF beta signaling. We report here that similar to effects of ectopic TGF betas, right-side expression of constitutively active activin-like kinase (ALK) 4 results in LR organ reversals as well as altered Pitx2 expression in the lateral plate mesoderm. Moreover, left-side expression of a kinase-deficient, dominant-negative ALK4 (DN-ALK4) or an ALK4 antisense morpholino also results in abnormal embryonic body situs, demonstrating a left-side requirement for ALK4 signaling. To determine which TGF beta(s) utilize the ALK4 pathway to mediate LR development, biochemical and functional assays were performed using an Activin-Vg1 chimera (AVg), Xnr1, and derriere. Whereas ALK4 can co-immunoprecipitate all of these TGF betas, including endogenous Vg1 protein from embryo homogenates, functional assays demonstrate that not all of these ligands require an intact ALK4 signaling pathway to modulate LR asymmetry. When AVg and DN-ALK4 are co-expressed, LR defects otherwise induced by AVg alone are attenuated by DN-ALK4; however, when functional assays are performed with Xnr1 or derriere, LR defects otherwise elicited by these ligands alone still occur in the presence of DN-ALK4. Intriguingly, when any of these TGF betas is expressed at a higher concentration to elicit primary axis defects, DN-ALK4 blocks gastrulation and dorsoanterior/ventroposterior defects that otherwise occur following ligand-only expression. Together, these results suggest not only that ALK4 interacts with multiple TGF betas to generate embryonic pattern, but also that ALK4 ligands differentially utilize the ALK4 pathway to regulate distinct aspects of axial pattern, with Vg1 as a modulator of ALK4 function in LR axis determination and Vg1, Xnr1, and derriere as modulators of ALK4 function in mesoderm induction during primary axis formation.     

Xenopus ADAMTS1 negatively modulates FGF signaling independent of its metalloprotease activity

We have isolated the Xenopus ortholog of ADAMTS1 (a disintegrin and metalloprotease with thrombospondin motifs), XADAMTS1, which is expressed in the presumptive ectoderm, then the Spemann organizer, and later in the trunk organizer region and posterior ectoderm in the Xenopus embryo. We show that, when overexpressed in the dorsal marginal zone or in the anterior ectoderm by mRNA injection, XADAMTS1 inhibits gastrulation or generates embryos with an enlarged cement gland, respectively. XADAMTS1 also reduces the expression of Xbra in both whole embryos and FGF-treated animal caps. These effects of XADAMTS1 are likely to be due to its inhibition of the Ras-MAPK cascade because XADAMTS1 inhibits the phosphorylation of ERK by FGF4 in animal caps. Deletion analysis of XADAMTS1 revealed that a combination of the signal peptide and the C-terminal region containing the thrombospondin type 1 repeats is necessary and sufficient for this function, whereas the metalloprotease domain is dispensable. In addition, loss-of-function analysis with antisense morpholino oligos showed that knockdown of XADAMTS1 sensitizes animal caps to Xbra induction by FGF2. These data suggest that secreted XADAMTS1 negatively modulates FGF signaling in the Xenopus embryo.     

Xvelo1 uses a novel 75-nucleotide signal sequence that drives vegetal localization along the late pathway in Xenopus oocytes

Vegetally localized RNAs in Xenopus laevis oocytes are involved in the patterning of the early embryo as well as in cell fate specification. Here we report on the isolation and characterization of a novel, vegetally localized RNA in Xenopus oocytes termed Xvelo1. It encodes a protein of unknown biological function and it represents an antisense RNA for XPc1 over a length of more than 1.8 kb. Xvelo1 exhibits a localization pattern reminiscent of the late pathway RNAs Vg1 and VegT; it contains RNA localization elements (LE) which do not match with the consensus structural features as deduced from Vg1 and VegT LEs. Nevertheless, the protein binding pattern as observed for Xvelo1-LE in UV cross-linking experiments and coimmunoprecipitation assays is largely overlapping with the one obtained for Vg1-LE. These observations suggest that the structural features recognized by the protein machinery that drives localization of maternal mRNAs along the late pathway in Xenopus oocytes must be redefined.