Kousuke Tanegashima

Expression cloning of Xantivin, a Xenopus lefty/antivin-related gene, involved in the regulation of activin signaling during mesoderm induction.

In a screening for activin-responsive genes, we isolated a Xenopus lefty/antivin-related gene, called Xantivin (Xatv). In the animal cap assay, the expression of Xatv was induced by activin signaling, and in the embryo, by nodal-related genes. Overexpression of Xatv in the marginal zone caused suppression of mesoderm formation and gastrulation defects, and inhibited the secondary axis formation induced by Xnr1 and Xactivin, suggesting that Xatv acted as a feedback inhibitor of activin signaling. However, in the animal cap, Xatv failed to antagonize Xnr1 and Xactivin. This result suggested that Xatv has different responses in the marginal zone and in the animal region, and antagonizes to a higher degree activin signaling in the marginal zone.

FRL-1, a member of the EGF-CFC family, is essential for neural differentiation in Xenopus early development.

Recent studies indicate an essential role for the EGF-CFC family in vertebrate development, particularly in the regulation of nodal signaling. Biochemical evidence suggests that EGF-CFC genes can also activate certain cellular responses independently of nodal signaling. Here, we show that FRL-1, a Xenopus EGF-CFC gene, suppresses BMP signaling to regulate an early step in neural induction. Overexpression of FRL-1 in animal caps induced the early neural markers zic3, soxD and Xngnr-1, but not the pan-mesodermal marker Xbra or the dorsal mesodermal marker chordin. Furthermore, overexpression of FRL-1 suppressed the expression of the BMP-responsive genes, Xvent-1 and Xmsx-1, which are expressed in animal caps and induced by overexpressed BMP-4. Conversely, loss of function analysis using morpholino-antisense oligonucleotides against FRL-1 (FRL-1MO) showed that FRL-1 is required for neural development. FRL-1MO-injected embryos lacked neural structures but contained mesodermal tissue. It was suggested previously that expression of early neural genes that mark the start of neuralization is activated in the presumptive neuroectoderm of gastrulae. FRL-1MO also inhibited the expression of these genes in dorsal ectoderm, but did not affect the expression of chordin, which acts as a neural inducer from dorsal mesoderm. FRL-1MO also inhibited the expression of neural markers that were induced by chordin in animal caps, suggesting that FRL-1 enables the response to neural inducing signals in ectoderm. Furthermore, we showed that the activation of mitogen-activated protein kinase by FRL-1 is required for neural induction and BMP inhibition. Together, these results suggest that FRL-1 is essential in the establishment of the neural induction response.

ENDODERM DIFFERENTIATION AND INDUCTIVE EFFECT OF ACTIVIN-TREATED ECTODERM IN XENOPUS

When presumptive ectoderm is treated with high concentrations of activin A, it mainly differentiates into axial mesoderm (notochord, muscle) in Xenopus and into yolk‐rich endodermal cells in newt (Cynops pyrrhogaster). Xenopus ectoderm consists of multiple layers, different from the single layer of Cynops ectoderm. This multilayer structure of Xenopus ectoderm may prevent complete treatment of activin A and subsequent whole differentiation into endoderm. In the present study, therefore, Xenopus ectoderm was separated into an outer layer and an inner layer, which were individually treated with a high concentration of activin A (100 ng/mL). Then the differentiation and inductive activity of these ectodermal cells were examined in explantation and transplantation experiments. In isolation culture, ectoderm treated with activin A formed endoderm. Ectodermal and mesodermal tissues were seldom found in these explants. The activin‐treated ectoderm induced axial mesoderm and neural tissues, and differentiated into endoderm when it was sandwiched between two sheets of ectoderm or was transplanted into the ventral marginal zone of other blastulae. These findings suggest that Xenopus ectoderm treated with a high concentration of activin A forms endoderm and mimics the properties of the organizer as in Cynops.

Xnr2 and Xnr5 Unprocessed Proteins Inhibit Wnt Signaling Upstream of Dishevelled

Nodal and Nodal‐related proteins activate the Activin‐like signal pathway and play a key role in the formation of mesoderm and endoderm in vertebrate development. Recent studies have shown additional activities of Nodal‐related proteins apart from the canonical Activin‐like signal pathway. Here we report a novel function of Nodal‐related proteins using cleavage mutants of Xenopus nodal‐related genes (cmXnr2 and cmXnr5), which are known to be dominant‐negative inhibitors of nodal family signaling. cmXnr2 and cmXnr5 inhibited both BMP signaling and Wnt signaling without activating the Activin‐like signal in animal cap assays. Pro region construct of Xnr2 and Xnr5 did not inhibit Xwnt8, and pro/mature region chimera mutant cmActivin‐Xnr2 and cmActivin‐Xnr5 also did not inhibit Xwnt8 activity. These results indicate that the pro domains of Xnr2 and Xnr5 are necessary, but not sufficient, for Wnt inhibition, by Xnr family proteins. In addition, Western blot analysis and immunohistochemistry analysis revealed that the unprocessed Xnr5 protein is stably produced and secreted as effectively as mature Xnr5 protein, and that the unprocessed Xnr5 protein diffused in the extracellular space. These results suggest that unprocessed Xnr2 and Xnr5 proteins may be involved in inhibiting both BMP and Wnt signaling and are able to be secreted to act on somewhat distant target cells, if these are highly produced. Developmental Dynamics 234:900–910, 2005.

Tracing of Xenopus tropicalis germ plasm and presumptive primordial germ cells with the Xenopus tropicalis DAZ‐like gene

A gamete is derived initially from a presumptive primordial germ cell (pPGC) and transmits genetic potential to the next generation. Xenopus tropicalis, which is a close relative of Xenopus laevis, has a diploid genome and advantages for genetic and genomic research; however, little is known about the developmental mechanism of its germinal lineage. Here, we identified the Xenopus tropicalis DAZ‐like gene (Xtdazl), which encodes RNA‐binding proteins homologous to Xdazl in Xenopus laevis and examined the expression patterns of Xtdazl transcripts during embryogenesis. In this work, we showed that Xtdazl mRNA was localized in the germ plasm and was expressed from the previtellogenic oocyte to early tadpole, in testis and ovary. The same localization patterns have been reported in Xenopus laevis germ plasm and pPGCs. These results indicate that Xtdazl mRNA is the first specific marker of germ plasm and pPGCs in Xenopus tropicalis and is very useful to trace Xenopus tropicalis pPGCs, including germ plasm until the early tadpole stage. Developmental Dynamics 229:367–372, 2004.

Expression of the Xenopus GTP-binding protein gene Ran during embryogenesis

Abstract The Ran gene family encodes small GTP binding proteins that are associated with a variety of nuclear processes. We isolated a Xenopus Ran cDNA and analyzed the pattern of expression of this gene during embryogenesis. Ran is expressed maternally and later in the CNS, neural crest, mesenchyme, eyes, and otic vesicles. However, expression is not detected in the somites or the notochord.

XIer2 is required for convergent extension movements during Xenopus development.

Immediate early response 2 (Ier2) is a downstream target of fibroblast growth factor (FGF) signaling. In zebrafish, Ier2 is involved in left-right asymmetry establishment and in convergent extension movements. We isolated the Xenopus ier2 gene based on sequence similarity searches using multiple vertebrate species. Xenopus Ier2 has high homology in the N-terminal region to other vertebrate Ier2 proteins, and Xier2 transcripts were observed from oocytes through larval stages. Except for the maternal expression of xier2, the expression of this gene in the marginal region at gastrulation and in somites and the notochord at later stages is similar to the expression pattern of zebrafish ier2. XIer2 knockdown using antisense morpholinos resulted in defects of convergent extension leading to severe neural tube defects; overexpression of Ier2 showed similar, albeit milder phenotypes. Assays in animal cap explants likewise showed inhibition of elongation after blocking XIer2 expression. These results indicate that Xenopus Ier2 is essential for the execution of convergent extension movements during early Xenopus development.