Three amphioxus Wnt genes (AmphiWnt3, AmphiWnt5, and AmphiWnt6) associated with the tail bud: the evolution of somitogenesis in chordates.

The amphioxus tail bud is similar to the amphibian tail bud in having an epithelial organization without a mesenchymal component. We characterize three amphioxus Wnt genes (AmphiWnt3, AmphiWnt5, and AmphiWnt6) and show that their early expression around the blastopore can subsequently be traced into the tail bud; in vertebrate embryos, there is a similar progression of expression domains for Wnt3, Wnt5, and Wnt6 genes from the blastopore lip (or its equivalent) to the tail bud. In amphioxus, AmphiWnt3, AmphiWnt5, and AmphiWnt6 are each expressed in a specific subregion of the tail bud, tentatively suggesting that a combinatorial code of developmental gene expression may help generate specific tissues during posterior elongation and somitogenesis. In spite of similarities within their tail buds, vertebrate and amphioxus embryos differ markedly in the relation between the tail bud and the nascent somites: vertebrates have a relatively extensive zone of unsegmented mesenchyme (i.e., presomitic mesoderm) intervening between the tail bud and the forming somites, whereas the amphioxus tail bud gives rise to new somites directly. It is likely that presomitic mesoderm is a vertebrate innovation made possible by developmental interconversions between epithelium and mesenchyme that first became prominent at the dawn of vertebrate evolution.     

Characterization of amphioxus AmphiWnt8: insights into the evolution of patterning of the embryonic dorsoventral axis

SUMMARY The full-length sequence and developmental expression of an amphioxus Wnt gene ( AmphiWnt8 ) are described. In amphioxus embryos, the expression patterns of AmphiWnt8 suggest patterning roles in the forebrain, in the hindgut, and in the paraxial mesoderm that gives rise to the muscular somites. Phylogenetic analysis indicates that a single Wnt8 subfamily gene in an ancestral chordate duplicated early in vertebrate evolution into a Wnt8 clade and a Wnt8b clade. Coincident with this gene duplication, the functions of the ancestral AmphiWnt8 -like gene appear to have been divided between vertebrate Wnt8b (exclusively neurogenic, especially in the forebrain) and vertebrate Wnt8 (miscellaneous, especially in early somitogenesis). Amphioxus AmphiWnt8 and its vertebrate Wnt8 homologs probably play comparable roles in the early dorsoventral patterning of the embryonic body axis.     

Axial patterning interactions in the sea urchin embryo: suppression of nodal by Wnt1 signaling

Wnt and Nodal signaling pathways are required for initial patterning of cell fates along anterior-posterior (AP) and dorsal-ventral (DV) axes, respectively, of sea urchin embryos during cleavage and early blastula stages. These mechanisms are connected because expression of nodal depends on early Wnt/β-catenin signaling. Here, we show that an important subsequent function of Wnt signaling is to control the shape of the nodal expression domain and maintain correct specification of different cell types along the axes of the embryo. In the absence of Wnt1, the posterior-ventral region of the embryo is severely altered during early gastrulation. Strikingly, at this time, nodal and its downstream target genes gsc and bra are expressed ectopically, extending posteriorly to the blastopore. They override the initial specification of posterior-ventral ectoderm and endoderm fates, eliminating the ventral contribution to the gut and displacing the ciliary band dorsally towards, and occasionally beyond, the blastopore. Consequently, in Wnt1 morphants, the blastopore is located at the border of the re-specified posterior-ventral oral ectoderm and by larval stages it is in the same plane near the stomodeum on the ventral side. In normal embryos, a Nodal-dependent process downregulates wnt1 expression in dorsal posterior cells during early gastrulation, focusing Wnt1 signaling to the posterior-ventral region where it suppresses nodal expression. These subsequent interactions between Wnt and Nodal signaling are thus mutually antagonistic, each limiting the range of the other's activity, in order to maintain and stabilize the body plan initially established by those same signaling pathways in the early embryo.     

Topographic changes in nascent and early mesoderm in amphioxus embryos studied by DiI labeling and by in situ hybridization for a Brachyury gene

 In amphioxus embryos, the nascent and early mesoderm (including chorda-mesoderm) was visualized by expression of a Brachyury gene (AmBra-2). A band of mesoderm is first detected encircling the earliest (vegetal plate stage) gastrula sub-equatorially. Soon thereafter, the vegetal plate invaginates, resulting in a cap-shaped gastrula with the mesoderm localized at the blastoporal lip and completely encircling the blastopore. As the gastrula stage progresses, DiI (a vital dye) labeling demonstrates that the entire mesoderm is internalized by a slight involution of the epiblast into the hypoblast all around the perimeter of the blastopore. Subsequently, during the early neurula stage, the internalized mesoderm undergoes anterior extension mid-dorsally (as notochord) and dorsolaterally (in paraxial regions where segments will later form). By the late neurula stage, AmBra-2 is no longer transcribed throughout the mesoderm as a whole; instead, expression is detectable only in the posterior mesoderm and in the notochord, but not in paraxial mesoderm where definitive somites have formed. Received: 28 November 1996 / Accepted: 2 January 1997     

Profilin is an effector for Daam1 in non-canonical Wnt signaling and is required for vertebrate gastrulation

Non-canonical Wnt signaling plays important roles during vertebrate embryogenesis and is required for cell motility during gastrulation. However, the molecular mechanisms of how Wnt signaling regulates modification of the actin cytoskeleton remain incompletely understood. We had previously identified the Formin homology protein Daam1 as an important link between Dishevelled and the Rho GTPase for cytoskeletal modulation. Here, we report that Profilin1 is an effector downstream of Daam1 required for cytoskeletal changes. Profilin1 interacted with the FH1 domain of Daam1 and was localized with Daam1 to actin stress fibers in response to Wnt signaling in mammalian cells. In addition, depletion of Profilin1 inhibited stress fiber formation induced by non-canonical Wnt signaling. Inhibition or depletion of Profilin1 in vivo specifically inhibited blastopore closure in Xenopus but did not affect convergent extension movements, tissue separation or neural fold closure. Our studies define a molecular pathway downstream of Daam1 that controls Wnt-mediated cytoskeletal reorganization for a specific morphogenetic process during vertebrate gastrulation.     

Xenopus glucose transporter 1 (xGLUT1) is required for gastrulation movement in Xenopus laevis

Glucose transporters (GLUTs) are transmembrane proteins that play an essential role in sugar uptake and energy supply. Thirteen GLUT genes have been described and GLUT1 is the most abundantly expressed member of the family in animal tissues. Deficiencies in human GLUT1 are associated with many diseases, such as metabolic abnormalities, congenital brain defects and oncogenesis. It was suggested recently that Xenopus GLUT1 (xGLUT1) is upregulated by Activin/Nodal signaling, although the developmental role of xGLUT1 remains unclear. Here, we investigated the expression pattern and function of xGLUT1 during Xenopus development. Whole-mount in situ hybridization analysis showed expression of xGLUT1 in the mesodermal region of Xenopus embryos, especially in the dorsal blastopore lip at the gastrula stage. From the neurula stage, it was expressed in the neural plate, eye field, cement gland and somites. Loss-of-function analyses using morpholino antisense oligonucleotides against xGLUT1 (xGLUT1MO) caused microcephaly and axis elongation error. This elongation defect of activin-treated animal caps occurred without downregulation of early mesodermal markers. Moreover, dorsal-marginal explant analysis revealed that cell movement was suppressed in dorsal marginal zones injected with xGLUT1MO. These findings implicate xGLUT1 as an important player during gastrulation cell movement in Xenopus.     

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.     

Ndst1, a heparan sulfate modification enzyme, regulates neuroectodermal patterning by enhancing Wnt signaling in Xenopus

Neural tissue is derived from three precursor regions: neural plate, neural crest, and preplacodal ectoderm. These regions are determined by morphogen-mediated signaling. Morphogen distribution is generally regulated by binding to an extracellular matrix component, heparan sulfate (HS) proteoglycan. HS is modified by many enzymes, such as N-deacetyl sulfotransferase 1 (Ndst1), which is highly expressed in early development. However, functions of HS modifications in ectodermal patterning are largely unknown. In this study, we analyzed the role of Ndst1 using Xenopus embryos. We found that ndst1 was expressed in anterior neural plate and the trigeminal region at the neurula stage. ndst1 overexpression expanded the neural crest (NC) region, whereas translational inhibition reduced not only the trigeminal region, but also the adjacent NC region, especially the anterior part. At a later stage, ndst1 knocked-down embryos showed defects in cranial ganglion formation. We also found that Ndst1 activates Wnt signaling pathway at the neurula stage. Taken together, our results suggest that N-sulfonated HS accumulates Wnt ligand and activates Wnt signaling in ndst1-expressing cells, but that it inhibits signaling in non-ndst1-expressing cells, leading to proper neuroectodermal patterning.     

Xenopus laevis POU91 protein, an Oct3/4 homologue, regulates competence transitions from mesoderm to neural cell fates

Cellular competence is defined as a cell's ability to respond to signaling cues as a function of time. In Xenopus laevis, cellular responsiveness to fibroblast growth factor (FGF) changes during development. At blastula stages, FGF induces mesoderm, but at gastrula stages FGF regulates neuroectoderm formation. A Xenopus Oct3/4 homologue gene, XLPOU91, regulates mesoderm to neuroectoderm transitions. Ectopic XLPOU91 expression in Xenopus embryos inhibits FGF induction of Brachyury (Xbra), eliminating mesoderm, whereas neural induction is unaffected. XLPOU91 knockdown induces high levels of Xbra expression, with blastopore closure being delayed to later neurula stages. In morphant ectoderm explants, mesoderm responsiveness to FGF is extended from blastula to gastrula stages. The initial expression of mesoderm and endoderm markers is normal, but neural induction is abolished. Churchill (chch) and Sip1, two genes regulating neural competence, are not expressed in XLPOU91 morphant embryos. Ectopic Sip1 or chch expression rescues the morphant phenotype. Thus, XLPOU91 epistatically lies upstream of chch/Sip1 gene expression, regulating the competence transition that is critical for neural induction. In the absence of XLPOU91 activity, the cues driving proper embryonic cell fates are lost.     

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.     

Retinoic acid metabolizing factor xCyp26c is specifically expressed in neuroectoderm and regulates anterior neural patterning in Xenopus laevis

Anterior-posterior neural patterning is determined during gastrulation when head structure is induced. Induction of anterior neural structures requires inhibition of Wnt signaling by several Wnt antagonists. We performed microarray analysis to isolate genes regulated by canonical Wnt signaling and abundantly expressed in the anterior neuroectoderm at the early neurula stage. We identified xCyp26c, a Cyp26 (RA-metabolizing protein)-family gene. In situ hybridization showed xCyp26c expression restricted to the anterior region of neurula, while xCyp26a was expressed in both anterior and posterior regions. At the tadpole stage, xCyp26c was also expressed in restricted sets of cranial nerves. Microarray, RT-PCR and in situ hybridization analyses revealed decreased xCyp26c expression with overexpression of beta-catenin, suggesting regulation by Wnt/beta-catenin signaling. We also assessed the effects of retinoic acid (RA) on xCyp26c expression. Embryos treated with 10(-7) M RA showed an anterior shift in the spatial expression of xCyp26c, reflecting a posteriorization effect. Conversely, expression patterns in embryos treated with more than 10(-6) M RA were less affected and remained restricted to the most anterior region. Moreover, injection of xCyp26c mRNA into animal poles caused head defects, and exogenous expression of xCyp26c rescued the posteriorizing effect of RA treatment. Taken together, these results implicated a role for xCyp26c in anterior patterning via RA signaling.     

A phylogenetic tree of the Wnt genes based on all available full-length sequences, including five from the cephalochordate amphioxus

The WNT: gene family is large, and new members are still being discovered. We constructed a parsimony tree for the WNT: family based on all 82 of the full-length sequences currently available. The inclusion of sequences from the cephalochordate amphioxus is especially useful in comprehensive gene trees, because the amphioxus genes in each subfamily often mark the base of the vertebrate diversification. We thus isolated full-length cDNAs of five amphioxus WNT: genes (AmphiWnt1, AmphiWnt4, AmphiWnt7, AmphiWnt8, and AmphiWnt11) for addition to the overall WNT: family tree. The analysis combined amino acid and nucleotide sequences (excluding third codon positions), taking into account 97% of the available data for each sequence. This combinatorial method had the advantage of generating a single most-parsimonious tree that was trichotomy-free. The reliability of the nodes was assessed by both jackknifing and Bremer support (decay index). A regression analysis revealed that branch length was strongly correlated with branch support, and possible reasons for this pattern are discussed. The tree topology suggested that in amphioxus, at least an AmphiWnt5 and an AmphiWnt10 have yet to be discovered.     

Methylation of Wnt7a is modulated by DNMT1 and cigarette smoke condensate in non-small cell lung cancer

Wnt7a is known to be a tumor suppressor that is lost in NSCLC, but no mechanism of loss has been established. Methylation of promoter regions has been established as a common mechanism of loss of tumor suppressor expression in NSCLC. We previously demonstrated that loss of Wnt7a in non-transformed lung epithelial cell lines led to increased cell growth, altered 3-D culture growth, and increased migration. The Wnt7a promoter has a higher percentage of methylation in NSCLC tumor tissue compared to matched normal lung tissue and methylation of the promoter region leads to decreased activity. We treated H157 and H1299 NSCLC cell lines with 5-Aza-2'-deoxycytidine and detected loss of Wnt7a promoter methylation, increased Wnt7a expression, and increased activity of the Wnt7a lung signaling pathway. When DNMT1 expression was knocked down by shRNA, expression of Wnt7a increased and methylation decreased. Together these data suggest that in NSCLC, Wnt7a is lost by methylation in a subset of tumors and that this methylation is maintained by DNMT1. Restoration of Wnt7a expression through demethylation could be an important therapeutic approach in the treatment of NSCLC.     

Movements of cellular material of the dorsal wall in clawed-toad embryos during gastrulation and neurulation

The pieces of dorsal ectoderm of the Rana temporaria embryos at the early and midgastrula stages were transplated onto the dorsal surface of the X. laevis embryos of the same age and the movements and changes in the form and area of the transplants were followed from early gastrula to neurula. During the first period (early--midgastrula) all movements of the transplants were directed towards the blastopore and related ma- In the beginning of the second period the transplants moved toward the blastopore only in the most caudal region, whereas in all other regions the material was markedly displaced craniad. Until the early neurula stage these movements were related to the longitudinal expansion of the material in the dorsal area and later, during neurulation, to its transverse compression. The head region material was first markedly expanded in the transverse direction and then also contracted. Alternation of active contractions and expansion of the suprablastopore material has been revealed and mediocaudal (gastrulation) vs. craniopetal (neurulation) cell movements were distinctly shown.