Xerl is a secreted protein required for establishing the neural plate/neural crest boundary in Xenopus embryo

We have previously isolated a CNS-specific gene, Xerl. The prospective amino acid sequence and functional analysis had shown that Xerl might act as the secretory protein for determining the neural plate/neural crest boundary. However, we had not yet characterized the Xerl protein. In the present study we examined the distribution and function of Xerl protein using anti-Xerl polyclonal antibody. Western blot analysis revealed that Xerl exists as 150 kDa protein in soluble fraction from the neurula stage. In comparison with gene expression of Xerl, Xerl protein showed a diffusive distribution from the neural tissue to the neighboring notochord and somite. Immunostaining of endogenous Xerl protein and subcellular localization of GFP-tagged Xerl demonstrated the extracellular secretion of Xerl protein. With functional blocking by antibody injection, the injected anti-Xerl antibody caused an inhibitory effect on the neural plate formation, whereas neural crest formation was promoted in the antibody-injected embryo. These results suggest that Xerl is a secreted protein required for establishing the neural plate/neural crest boundary in Xenopus embryo.     

Expression of Brachyury-like T-box transcription factor, Xbra3 in Xenopus embryo

The Xenopus Brachyury-like Xbra3 gene is a novel T-box gene that is closely associated with Xenopus Brachyury. The expression pattern of Xbra3 during development is similar to that of Xbra. During gastrulation Xbra3 is expressed in the marginal zone, with a gradient of increasing expression from ventral to dorsal. In the early neurula stage Xbra3 is expressed in the notochord and posterior mesoderm, but by the tailbud stage its expression is restricted to the forming tailbud and the posterior portion of the notochord.     

Expression pattern of BXR suggests a role for benzoate ligand-mediated signalling in hatching gland function

The Xenopus laevis nuclear receptor BXR has recently been shown to be activated by a class of endogenous benzoate metabolites, indicating the presence of a novel and unsuspected benzoate ligand-dependent signalling pathway. The receptor is expressed ubiquitously in blastula and gastrula stage embryos, and its expression declines during neurula stages. In order to examine further this novel vertebrate signalling system, we have examined the expression of the BXR gene in tailbud stage embryos and adults. We show here that in Xenopus tailbud stage embryos expression is restricted to the hatching gland, suggesting a role in hatching gland function. Neither BXR nor a BXR-VP16 fusion is sufficient to specify hatching gland in neurally-induced tissue. In adults, BXR expression is abundant in the brain and gonads. This expression pattern in adults is distinct from any of the putative mammalian homologues. A nuclear receptor that mediates benzoate signalling has yet to be found in mammals.     

Developmental expression of Shisa-2 in Xenopus laevis

Shisa is an antagonist of Wnt and FGF signaling, that functions cell autonomously in the endoplasmic reticulum (ER) to inhibit the post-translational maturation of Wnt and FGF receptors. In this paper we report the isolation of a second Xenopus shisa gene (Xshisa-2). Xenopus Shisa-2 shows 30.7% identity to Xshisa. RT-PCR analysis indicated that Xshisa-2 mRNA is present throughout early development and shows an increased expression during neurula and tailbud stages. At neurula stages Xenopus shisa-2 is initially expressed in the presomitic paraxial mesoderm and later in the developing somites. The expression profiles and pattern of Xshisa and Xshisa-2 differ significantly. During gastrulation only Xshisa mRNA is present in the Spemann-Mangold organizer and later on becomes restricted to the neuroectoderm and the prechordal plate.     

Expression of Axwnt-8 and Axszl in the urodele, axolotl: comparison with Xenopus

In both the urodele axolotl and the anuran Xenopus, Wnt-8 is expressed in posterior lateral plate mesoderm (LPM) in neurula and tailbud stages. In contrast to Xenopus, expression in axolotl is more prominent in gastrula endoderm, is not initiated in mesoderm until late gastrulation, and is present in the tailbud and in the brain at tailbud stages. Sizzled is expressed in axolotl in the ventral region, similar to its pattern in Xenopus. In axolotl, the Wnt-8-expressing LPM remains relatively dorsal through tailbud stages, while ventral blood island (VBI) markers appear in a wide ventral arc.     

Xerl, a novel CNS-specific secretory protein, establishes the boundary between neural plate and neural crest.

A novel gene, Xerl, has been found as a CNS-specific gene encoding a secretory protein. In order to clarify a function of Xerl, we first examined Xerl-expressing areas during early development. Comparison with XlSox-2-positive neural plate and ADAM13-positive neural crest showed that Xerl expression was limited within the neural plate area. Microinjection of Xerl mRNA into 2- or 4-cell stage embryos indicated that Xerl overexpression caused the regional expansion of XlSox-2- and NCAM-positive neural plate, which was concomitant with the outer shift of ADAM13-positive region. The Xerl injection resulted in incomplete neural closure because of the local overproduction of the neuroepithelium. In contrast, loss of function analysis of Xerl indicated that Xerl inhibition caused the ectopic differentiation of neural crest cells. In the conjugation experiment using chordin-injected animal caps, Xerl promoted chordin-induced XlSox-2 expression, whereas Xerl inhibition caused ADAM13expression even in the injection with a high dose of chordin. Animal cap assays also showed that Xerl expression was induced by chordin. In the functional analysis using truncated forms of Xerl, Xerl deltaL (lacking LNS domain) worked as a dominant negative form that induced the overproduction of neural crest cells. These results suggest that Xerl is involved in the boundary formation of the neural plate through exclusion of neural crest cell differentiation.     

Expression of the Xenopus homologue of the receptor for activated C-kinase 1 (RACK1) in the Xenopus embryo

We have isolated the Xenopus homologue of the receptor for activated C-kinase 1 (RACK1), whose amino acid sequence shows significant similarity with other vertebrate RACK1s. XRACK1 is a maternally expressed gene and its zygotic expression is detected in the antero-dorsal region and dorsal midline in the late neurula. At tailbud stage, rather diffuse staining is seen in the somite and head. Later, XRACK1 mRNA is expressed highly in ventrally migrating abdominal muscle anlagen, where it remains expressed during subsequent stages.     

Morphogenesis and regulated gene activity are independent of DNA replication in Xenopus embryos.

Xenopus embryos were transferred into media containing aphidicolin at late blastula, mid-gastrula, and early neurula stages. In each case, embryos continued to differentiate in the absence of DNA replication. When the inhibitor was added at late blastula, embryos continued to develop for about 8 h. However, when aphidicolin was added at the early neurula stage, development could be seen for up to 40 h after addition. The influence of replication on embryonic gene activity was studied by RNA blot analysis. Of the genes we examined only histone gene expression was down regulated by the addition of aphidicolin. The expression of various embryo-specific genes was unaffected by the lack of DNA synthesis. Even after several hours of treatment with aphidicolin, replication-inhibited tailbud and tadpole stages showed the same levels of specific mRNAs as control embryos containing 4-5 times more DNA. We conclude that morphogenesis and embryo-specific gene activity are independent of both DNA replication and a precise amount of DNA per embryo.     

Developmental expression of FoxJ1.2, FoxJ2, and FoxQ1 in Xenopus tropicalis

Members of the Fox gene family exhibit remarkably restricted patterns of expression where they have interesting, required functions during development. We have analyzed the developmental expression patterns of three members of the Fox gene family, FoxJ1.2, FoxJ2, and FoxQ1, which have not been previously described in Xenopus. FoxJ1.2 is expressed in the otic vesicle during late neurula stages and is then also expressed in the presumptive nephrostomes of the pronephros during tailbud stages. FoxJ2 is expressed in the notochord and ventral portion of the neural tube. FoxQ1 is expressed specifically in the pharyngeal pouches as early as neurula stages and remains on in pharyngeal tissue throughout the tailbud stages. At later stages, FoxQ1 is also expressed in the anterior gut. FoxJ1.2, FoxJ2, and FoxQ1 may prove to be useful tissue-specific markers of these embryonic structures.     

Molecular cloning and characterization of a novel inhibitor of apoptosis protein from Xenopus laevis

A novel inhibitor of apoptosis protein family member termed SIX was identified in Xenopus containing a single baculoviral IAP repeat (BIR) domain and no COOH-terminal RING finger domain. It exhibited striking amino acid sequence similarity with human survivin, mouse TIAP, and recently found Xenopus survivin, especially a part of BIR domain was highly conserved. Interestingly, SIX interacted with RXRalpha through the AF2 domain in the absence of ligand, which was weakened when the ligand was present. Northern blot analysis demonstrated that SIX mRNA was not detectable in adult with exception of the ovary and testis, and whole-mount in situ hybridization and Northern blot analyses revealed strong and homogeneous expression of SIX in the developing oocytes. In the embryos, the expression of SIX was observed in the animal hemisphere from one-cell to yolk plug stages and high level of expression was detected in the future brain and dorsal region of the neural tube at the neurula stage and early tail-bud stage. These results strongly support the fact that survivin is evolutionarily conserved in structure and SIX is likely to be the Xenopus counterpart of human and mouse survivin.     

Human MUC4 mucin cDNA and its variants in pancreatic carcinoma

The human MUC4 gene is not expressed in normal pancreas; however, its dysregulation results in high levels of expression in pancreatic tumors. To investigate the tumor-associated expression, MUC4 cDNA was cloned from a human pancreatic tumor cell line cDNA expression library using a polyclonal antibody raised against human deglycosylated mucin and RT-PCR. Pancreatic MUC4 cDNA shows differences in 12 amino acid residues in the non-tandem repeat coding region with no structural rearrangement as compared with tracheal MUC4. The full-length MUC4 cDNA includes a leader sequence, a serine and threonine rich non-tandem repeat region, a central large tandem repeat domain containing 48 bp repetitive units, regions rich in potential N-glycosylation sites, two cysteine-rich domains, EGF-like domains, and a transmembrane domain. We also report the presence of a new EGF-like domain in MUC4 cDNA, located in the cysteine-rich region upstream from the first EGF-like domain. Four distinct splice events were identified in the region downstream of the central tandem repeat domain that generate three new MUC4 cDNA sequences (sv4, sv9, and sv10). The deduced amino acid sequences of two of these variants lack the transmembrane domain. Furthermore, two unique forms of MUC4 (MUC4/Y and MUC4/X) generated as a result of alternative splicing lack the salient feature of mucins, the tandem repeat domain. A high degree of polymorphism in the central tandem repeat region of MUC4 was observed in various pancreatic adenocarcinoma cell lines, with allele sizes ranging from 23.5 to 10.0 kb. MUC4 mRNA expression was higher in differentiated cell lines, with no detectable expression in poorly differentiated pancreatic tumor cell lines.     

Selective expression of angiopoietin 1 and 2 in mesenchymal cells surrounding veins and arteries of the avian embryo

We have isolated a new Wnt receptor frizzled family member from Xenopus laevis, Xenopus frizzled-5 (Xfz5), a likely ortholog of human frizzled-5. Based on Northern and whole-mount in situ hybridization data, Xfz5 is first detected at the late neurula stage in retinal primordia. Throughout the tailbud stage Xfz5 is expressed exclusively in the neural retina within the optic vesicles. During tadpole stage Xfz5 expression becomes restricted to the ciliary marginal zone. This highly restrictive expression pattern makes Xfz5 an excellent marker for neural retinal tissue.     

Xenopus neurula left-right asymmetry is respeficied by microinjecting TGF-beta5 protein

A variety of TGF-beta-related ligands regulate the left-right asymmetry of vertebrates but the involvement of TGF-betas in left-right specification has not been reported. We assessed whether TGF-beta signaling is involved in the left-right specification of Xenopus post-gastrula embryos by microinjecting Xenopus TGF-beta5 protein into the left or right flank of neurula-tailbud embryos. Injection on the right side of neurulae caused left-right reversal of the internal organs in 93% of the embryos, while injection on the left side caused less than 5% left-right reversal. Expression of Xenopus nodal related-1 (Xnr-1 ), Xenopus antivin and Xenopus Pitx2, which are normally expressed on the left, was unaltered by the left-side injection. In contrast, right-side injection into neurulae induced the expression of these genes predominantly on the right side. Right-side injection into tailbud embryos caused bilateral expression of these handed genes. Time course analysis of asymmetric gene expression revealed that Xnr-1 could be induced by TGF-beta5 at late neurula stage, while antivin and Pitx2 could be induced by TGF-beta5 at the latertail bud stage. Injection of the antisense morpholino oligonucleotide against Xenopus TGF-beta5 into the left dorsal blastomere inhibited the normal left-handed expression of Xnr-1 and Pitx2, and caused the organ reversal in the injected embryos. These results suggest that normal left-right balance of endogenous TGF-beta5 signaling in the neurula embryo may be needed to determine the laterality of the asymmetric genes and to generate the correct left-right axis.