Gene Structure Analysis of Chemokines and Their Receptors in Allotetraploid Frog, Xenopus laevis.

Chemokines, relatively small secreted proteins, are involved in cell migration and function in various biological events, including immunity, morphogenesis, and disease. Due to their nature, chemokines tend to be a target of hijacking of immunity by virus and therefore show an exceptionally high mutation rate. Xenopus laevis is considered an excellent model to investigate the effect of whole-genome duplication for gene family evolution. Because its allotetraploidization occurred around 17-18 million years ago, ancestral subgenomes L and S were well conserved. Based on the gene model of human and diploid frog Xenopus tropicalis, we identified 52 chemokine genes and 26 chemokine receptors in X. laevis. The retention rate of the gene in the X. laevis L and S subgenomes was 96% (45/47) and 68% (32/47), respectively. We conducted molecular phylogenetic analysis and found clear orthologies in all receptor genes but not in the ligand genes, suggesting rapid divergences of the ligand. dN/dS calculation demonstrated that dN/dS ratio greater than one was observed in the four ligand genes, cxcl8b.1.S, cxcl18.S, ccl21.S, and xcl1.L, but nothing in receptor genes. These results revealed that the whole-genome duplication promotes diversification of chemokine ligands in X. laevis while conserving the genes necessary for homeostasis, suggesting that selective pressure also supports a rapid divergence of the chemokines in amphibians.

A simple culture method for liver and intestinal tissue-resident macrophages from neonatal mice.

The liver and intestine contain a remarkably large portion of tissue-resident macrophage cells representing a phenotype that downregulates inflammation and initiates tissue repair. Here, liver and intestinal tissues obtained from neonatal mice were minced, enzymatically digested, and incubated in RPMI1640-based media. In a 2-wk culture, spherical floating cells emerged on a fibroblastic sheet. These cells showed phagocytic activity and F4/80+-CD11b+-CD206+-Arg1+-iNOS--CD209a- phenotype, suggesting that these cells are tissue-resident macrophages. These macrophages proliferated in the co-culture system in the presence of fibroblastic feeder cell layer and absence of supplemental cytokines; the co-culture system did not cause a significant change in the phenotype of cells grown in a 4-wk culture. On the feeder cells, macrophage density was approximately 1.5 × 104/cm2 and the doubling time was approximately 70 h. Based on these observations, we present a simple method for the isolation and propagation of tissue-resident macrophages resembling M2 macrophage from neonatal mice, and this method provides a useful platform for in vitro studies of tissue-resident macrophages.

A comprehensive reference transcriptome resource for the Iberian ribbed newt Pleurodeles waltl, an emerging model for developmental and regeneration biology.

Urodele newts have unique biological properties, notably including prominent regeneration ability. The Iberian ribbed newt, Pleurodeles waltl, is a promising model amphibian distinguished by ease of breeding and efficient transgenic and genome editing methods. However, limited genetic information is available for P. waltl. We conducted an intensive transcriptome analysis of P. waltl using RNA-sequencing to build and annotate gene models. We generated 1.2 billion Illumina reads from a wide variety of samples across 12 different tissues/organs, unfertilized egg, and embryos at eight different developmental stages. These reads were assembled into 1,395,387 contigs, from which 202,788 non-redundant ORF models were constructed. The set is expected to cover a large fraction of P. waltl protein-coding genes, as confirmed by BUSCO analysis, where 98% of universal single-copy orthologs were identified. Ortholog analyses revealed the gene repertoire evolution of urodele amphibians. Using the gene set as a reference, gene network analysis identified regeneration-, developmental-stage-, and tissue-specific co-expressed gene modules. Our transcriptome resource is expected to enhance future research employing this emerging model animal for regeneration research as well as for investigations in other areas including developmental biology, stem cell biology, and cancer research. These data are available via our portal website, iNewt (http://www.nibb.ac.jp/imori/main/).

Enhancement of humoral and cell mediated immune response to HPV16 L1-derived peptides subsequent to vaccination with prophylactic bivalent HPV L1 virus-like particle vaccine in healthy females.

Currently prophylactic HPV16/18 L1 virus-like particle (VLP) vaccines are employed with great success for the prevention of HPV infection. However, limited information is available regarding the immune responses against human papillomavirus (HPV) 16/18 L1 subsequent to HPV16/18 L1 VLP vaccination, primarily due to the lack of widely used assays for immune monitoring. The aim of the present study was to identify HPV16 L1-derived B and T cell epitopes for monitoring the immune responses after HPV16/18 L1 VLP vaccination in healthy females. The levels of immunoglobulin G (IgG), IgE, IgA and IgM reactive to HPV16 L1-derived peptides were measured by multiplex bead suspension assay. Following detailed B cell epitope mapping, T cell responses specific to HPV16 L1-derived peptides were evaluated by an IFN-γ ELISPOT assay. The levels of IgG, IgM and IgA reactive to 20-mer peptides (PTPSGSMVTSDAQIFNKPYW) at positions 293-312 and 300-319 of HPV16 L1 were significantly increased in the plasma after 2, 7, and 12 months after first vaccination. Detailed epitope mapping identified the amino acid sequence (TSDAQIFNKP) at position 301-310 of HPV16 L1 as an immunogenic B cell epitope. In addition, T cell responses to an HLA-A2- and HLA-A24-restricted epitope (QIFNKPYWL) at position 305-313 of HPV16 L1 were increased following immunization, suggesting that the HPV16/18 L1-VLP vaccination as able to induce specific immune responses in T and B cells simultaneously. The identified B and T cell epitopes may be useful as a biomarker for monitoring the immune responses subsequent to HPV16/18 L1 VLP vaccination. Thus, the present study may provide novel information to improve the understanding of the immune responses to HPV16 L1.

Clustered Xenopus keratin genes: A genomic, transcriptomic, and proteomic analysis.

Keratin genes belong to the intermediate filament superfamily and their expression is altered following morphological and physiological changes in vertebrate epithelial cells. Keratin genes are divided into two groups, type I and II, and are clustered on vertebrate genomes, including those of Xenopus species. Various keratin genes have been identified and characterized by their unique expression patterns throughout ontogeny in Xenopus laevis; however, compilation of previously reported and newly identified keratin genes in two Xenopus species is required for our further understanding of keratin gene evolution, not only in amphibians but also in all terrestrial vertebrates. In this study, 120 putative type I and II keratin genes in total were identified based on the genome data from two Xenopus species. We revealed that most of these genes are highly clustered on two homeologous chromosomes, XLA9_10 and XLA2 in X. laevis, and XTR10 and XTR2 in X. tropicalis, which are orthologous to those of human, showing conserved synteny among tetrapods. RNA-Seq data from various embryonic stages and adult tissues highlighted the unique expression profiles of orthologous and homeologous keratin genes in developmental stage- and tissue-specific manners. Moreover, we identified dozens of epidermal keratin proteins from the whole embryo, larval skin, tail, and adult skin using shotgun proteomics. In light of our results, we discuss the radiation, diversification, and unique expression of the clustered keratin genes, which are closely related to epidermal development and terrestrial adaptation during amphibian evolution, including Xenopus speciation.

Sex chromosome differentiation and the W- and Z-specific loci in Xenopus laevis.

Genetic sex-determining systems in vertebrates include two basic types of heterogamety; XX (female)/XY (male) and ZZ (male)/ZW (female) types. The African clawed frog Xenopus laevis has a ZZ/ZW-type sex-determining system. In this species, we previously identified a W-specific sex (female)-determining gene dmw, and specified W and Z chromosomes, which could be morphologically indistinguishable (homomorphic). In addition to dmw, we most recently discovered two genes, named scanw and ccdc69w, and one gene, named capn5z in the W- and Z-specific regions, respectively. In this study, we revealed the detail structures of the W/Z-specific loci and genes. Sequence analysis indicated that there is almost no sequence similarity between 278kb W-specific and 83kb Z-specific sequences on chromosome 2Lq32-33, where both the transposable elements are abundant. Synteny and phylogenic analyses indicated that all the W/Z-specific genes might have emerged independently. Expression analysis demonstrated that scanw and ccdc69w or capn5z are expressed in early differentiating ZW gonads or testes, thereby suggesting possible roles in female or male development, respectively. Importantly, the sex-determining gene (SDG) dmw might have been generated after allotetraploidization, thereby indicating the construction of the new sex-determining system by dmw after species hybridization. Furthermore, by direct genotyping, we confirmed that diploid WW embryos developed into normal female frogs, which indicate that the Z-specific region is not essential for female development. Overall, these findings indicate that sex chromosome differentiation has started, although no heteromorphic sex chromosomes are evident yet, in X. laevis. Homologous recombination suppression might have promoted the accumulation of mutations and transposable elements, and enlarged the W/Z-specific regions, thereby resulting in differentiation of the W/Z chromosomes.

Conservatism and variability of gene expression profiles among homeologous transcription factors in Xenopus laevis.

Xenopus laevis has an allotetraploid genome of 3.1Gb, in contrast to the diploid genome of a closely related species, Xenopus tropicalis. Here, we identified 412 genes (189 homeolog pairs, one homeologous gene cluster pair, and 28 singletons) encoding transcription factors (TFs) in the X. laevis genome by comparing them with their orthologs from X. tropicalis. Those genes include the homeobox gene family (Mix/Bix, Lhx, Nkx, Paired, POU, and Vent), Sox, Fox, Pax, Dmrt, Hes, GATA, T-box, and some clock genes. Most homeolog pairs for TFs are retained in two X. laevis subgenomes, named L and S, at higher than average rates (87.1% vs 60.2%). Among the 28 singletons, 82.1% were deleted from chromosomes of the S subgenome, a rate similar to the genome-wide average (82.1% vs 74.6%). Interestingly, nkx2-1, nkx2-8, and pax9, which reside consecutively in a postulated functional gene cluster, were deleted from the S chromosome, suggesting cluster-level gene regulation. Transcriptome correlation analysis demonstrated that TF homeolog pairs tend to have more conservative developmental expression profiles than most other types of genes. In some cases, however, either of the homeologs may show strongly different spatio-temporal expression patterns, suggesting neofunctionalization, subfunctionalization, or nonfunctionalization after allotetraploidization. Analyses of otx1 suggests that homeologs with much lower expression levels have undergone greater amino acid sequence diversification. Our comprehensive study implies that TF homeologs are highly conservative after allotetraploidization, possibly because the DNA sequences that they bind were also duplicated, but in some cases, they differed in expression levels or became singletons due to dosage-sensitive regulation of their target genes.

Genome evolution in the allotetraploid frog Xenopus laevis.

To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of 'fossil' transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17-18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

Real-time imaging of actin filaments in the zebrafish oocyte and embryo.

Dynamic changes of cytoplasmic and cortical actin filaments drive various cellular and developmental processes. Although real-time imaging of actin filaments in living cells has been developed, imaging of actin filaments in specific cells of living organisms remains limited, particularly for the analysis of gamete formation and early embryonic development. Here, we report the production of transgenic zebrafish expressing the C-terminus of Moesin, an actin filament-binding protein, fused with green fluorescent protein or red fluorescent protein (GFP/RFP-MoeC), under the control of a cyclin B1 promoter. GFP/RFP-MoeC was expressed maternally, which labels the cortical actin cytoskeleton of blastula-stage cells. High levels of GFP/RFP fluorescence were detected in the adult ovary and testis. In the ovaries, GFP/RFP-MoeC was expressed in oocytes but not in follicle cells, which allows us to clearly visualize the organization of actin filaments in different stages of the oocyte. Using full-grown oocytes, we revealed the dynamic changes of actin columns assembled in the cortical cytoplasm during oocyte maturation. The number of columns slightly decreased in the early period before germinal vesicle breakdown (GVBD) and then significantly decreased at GVBD, followed by recovery after GVBD. Our transgenic fish are useful for analyzing the dynamics of actin filaments in oogenesis and early embryogenesis.

Molecular Role of RNF43 in Canonical and Noncanonical Wnt Signaling.

Wnt signaling pathways are tightly regulated by ubiquitination, and dysregulation of these pathways promotes tumorigenesis. It has been reported that the ubiquitin ligase RNF43 plays an important role in frizzled-dependent regulation of the Wnt/ß-catenin pathway. Here, we show that RNF43 suppresses both Wnt/ß-catenin signaling and noncanonical Wnt signaling by distinct mechanisms. The suppression of Wnt/ß-catenin signaling requires interaction between the extracellular protease-associated (PA) domain and the cysteine-rich domain (CRD) of frizzled and the intracellular RING finger domain of RNF43. In contrast, these N-terminal domains of RNF43 are not required for inhibition of noncanonical Wnt signaling, but interaction between the C-terminal cytoplasmic region of RNF43 and the PDZ domain of dishevelled is essential for this suppression. We further show the mechanism by which missense mutations in the extracellular portion of RNF43 identified in patients with tumors activate Wnt/ß-catenin signaling. Missense mutations of RNF43 change their localization from the endosome to the endoplasmic reticulum (ER), resulting in the failure of frizzled-dependent suppression of Wnt/ß-catenin signaling. However, these mutants retain the ability to suppress noncanonical Wnt signaling, probably due to interaction with dishevelled. RNF43 is also one of the potential target genes of Wnt/ß-catenin signaling. Our results reveal the molecular role of RNF43 and provide an insight into tumorigenesis.

Application of Multichannel Collagen Gels in Construction of Epithelial Lumen-like Engineered Tissues.

Introduction of epithelial lumen-like structures such as blood and lymphatic vessels, as well as renal tubules, is a prerequisite for successful construction and function of artificially engineered giant tissues. Here, we demonstrate a methodology for construction of various epithelial lumen-like structures by using multichannel collagen gels (MCCGs). MCCGs were prepared and used as template scaffolds for constructing epithelial lumen structures in a controlled fashion. The effect of NaCl concentration on the multichannel structure of MCCGs was investigated by using confocal laser scanning microscopy along with fluorescent staining. The channel diameter increased with increasing NaCl concentrations in the collagen solution and the phosphate buffer solution. In contrast, the channel number decreased with increasing NaCl concentrations. Engineered tissues with various lumen-like structures were constructed by seeding and culturing Madin-Darby canine kidney cells on MCCGs. The diameter of the lumen and the number of lumens per unit area were controllable by regulating the multichannel structure of cylindrical MCCG. We believe that our methodology for the construction of engineered tissues possessing epithelial lumen-like structures will prove helpful in regeneration of giant tissues with various hierarchical structures.

Validity of intraoperative diagnosis at laparoscopic surgery for ovarian tumors.

STUDY OBJECTIVE: To evaluate the accuracy and usefulness of intraoperative diagnosis of ovarian tumor during laparoscopic surgery. DESIGN: Retrospective cohort study (Canadian Task Force classification II-3). SETTING: Tertiary care university hospital. PATIENTS: We reviewed the cases of 262 patients who underwent laparoscopic surgery at our institution between January 2005 and December 2011 in whom a benign ovarian tumor was diagnosed intraoperatively. INTERVENTIONS: Intraoperative pathologic assessment of frozen sections. MEASUREMENTS AND MAIN RESULTS: Intraoperative diagnosis of ovarian tumors demonstrated sensitivity of 80%, specificity of 99.6%, positive predictive value of 80%, and diagnostic accuracy of 99.2%. Mucinous tumors diagnosed intraoperatively showed differing intraoperative and final pathologic diagnoses significantly more frequently than did other types of tumors. CONCLUSION: Intraoperative pathologic assessment of benign ovarian tumors during laparoscopic surgery is reliable. However, clinicians should recognize that it is possible to make an incorrect diagnosis in some situations and should exercise caution accordingly.

Multiscale analysis of changes in an anisotropic collagen gel structure by culturing osteoblasts.

Mimicking the complicated anisotropic structures of a native tissue is extremely important in tissue engineering. In a previous study, we developed an anisotropic collagen gel scaffold (ACGS) having a hierarchical structure and a properties gradient. In this study, our objective was to see how cells remodel the scaffolds through the cells-ACGS interaction. For this purpose, we cultured osteoblastic cells on ACGS, which we regarded as a model system for the cells-extracellular matrix (cell-ECM) interaction. Changes in the ACGS-cell composites structure by cell-ECM interactions was investigated from a macroscopic level to a microscopic level. Osteoblastic cells were also cultured on an isotropic collagen gel (ICGS) as a control. During the cultivation, mechanical stimuli were applied to collagen-cell composites for adequate matrix remodeling. Confocal laser scanning microscope (CLSM) was used to observe macroscopic changes in the ACGS-cell composite structure by osteoblastic cells. Small-angle X-ray scattering (SAXS) measurements were performed to characterize microscopic structural changes in the composites. Macroscopic observations using CLSM revealed that osteoblastic cells remained only in the diluted phase in ACGS and they collected collagen fibrils or formed a toroidal structure, depending on the depth from the ACGS surface in the tubular diluted phase. The cells were uniformly distributed in ICGS. SAXS analysis suggests that collagen fibrils were remodeled by osteoblastic cells, and this remodeling process would be affected by the structure difference between ACGS and ICGS. These results suggest that we directly regulate cell-ECM interaction by the unique anisotropic and hierarchical structure of ACGS. The cell-gel composite presented in this study would promise an efficient scaffold material in tissue engineering.

Expression of xSDF-1α, xCXCR4, and xCXCR7 during gastrulation in Xenopus laevis.

Chemokines play a crucial role in developmental processes and recent studies have revealed that they also control gastrulation movements. In this paper, we report the expression patterns of xSDF-1α, xCXCR4 and xCXCR7 and regulation of the expression of xSDF-1α and xCXCR4 during gastrulation. We performed whole mount in situ hybridization (WISH) and quantitative real-time RT-PCR (qRT-PCR) analyses to examine the distribution of transcripts. The effect of activin/nodal signaling on the expression of xSDF-1α and its receptors was examined by animal cap assay and microinjection of cer-s mRNA. We have demonstrated that the xSDF-1αtranscript is increased in the blastocoel roof during gastrulation, but not in the involuted mesoderm. xCXCR4 was expressed in the mesendoderm at late blastula and was retained throughout gastrulation. xCXCR7 was found in the dorsal lip around the blastopore in the early gastrula stage and became localized in the presumptive notochord later. We also show that the expression of xCXCR4 and xSDF-1transcript is increased in the blastocoel roof during gastrulation, but not in the involuted mesoderm. xCXCR4 was expressed in the mesendoderm at late blastula and was retained throughout gastrulation. xCXCR7 was found in the dorsal lip around the blastopore in the early gastrula stage and became localized in the presumptive notochord later. We also show that the expression of xCXCR4 and xSDF-1α were reciprocally regulated by activin/nodal signaling. These results suggest that xSDF-1α and its receptors contribute to the cell arrangement of mesoderm cells and their expression patterns are partially regulated by activin/nodal signaling.

Changes in the viscoelastic properties of cortical bone by selective degradation of matrix protein.

We have studied stress relaxation of bovine femoral cortical bone specimens treated with KOH aqueous solution which had been known to degrade selectively protein molecules in bone. With the KOH treatment, we found an increase in specimens' volume. This increase was regarded as swelling of the bone specimen, presumably due to matrix protein network degradation including that of collagen. In an analogy of bone to gel structure, an increasing ratio of specimen volume was used as an indicating parameter for the matrix protein network degradation by the treatment. Although an empirical equation with a linearly combined form of two Kohlrausch-Williams-Watts (KWW) functions has been shown to describe the stress relaxation of bone specimens, a single KWW function was suitable for the bone specimens treated with KOH solution for as little as 3h. In KOH treated specimens, both the initial modulus and the relaxation time decreased with the volume-increasing ratio, while the relaxation time distribution did not change. A chemo-rheological consideration attributed the reduction of modulus values to the network degradation in the organic matrix phase. The relaxation time of KOH treated specimens was thought to be related to the longer relaxation time of untreated bones, although there was a discontinuity between the extrapolated relaxation time values for KOH treated specimens and untreated specimens. This discontinuity may have originated from the release of residual stress existing in the bone by the matrix protein degradation. The results of the present study suggest that the state of matrix protein is crucial for integrating the mechanical properties of bone.

Identification of B cell epitopes reactive to human papillomavirus type-16L1- derived peptides.

BACKGROUND: Persistent infection of human papillomavirus (HPV) types 16 and 18 causes cervical cancer. To better understand immune responses to the prophylactic vaccine, HPV 16/18 L1 virus-like particles (HPV-VLPs), we investigated B cell epitopes of HPV16 L1-derived peptides. METHODS: Sera from mice immunized with HPV-16/18 L1 VLPs were analyzed for their IgG titers against 10 different HPV16 L1-derived peptides (20-mer) that contain human leukocyte antigen (HLA)-class I A-2, A-24 and class II DR. RESULTS: One 20-mer peptide at positions 300 to 319 was identified as a common B cell epitope in both Balb/c (H-2d) and C57BL/6 (H-2b) mice. Mapping analysis showed that the 10-amino-acid sequence at positions 304 to 313 was an immunogenic portion. It is of note that the binding capability of this 10-mer peptide to the HLA-A2 and HLA-A24 molecules was confirmed by the HLA class I stabilization assay. In addition, one unique 20-mer was determined as a B cell epitope in each strain. CONCLUSIONS: These results might provide new information for better understanding of immune responses to HPV 16 L1.

Studies on the formation mechanism and the structure of the anisotropic collagen gel prepared by dialysis-induced anisotropic gelation.

We have found that dialysis of 5 mg/mL collagen solution into the phosphate solution with a pH of 7.1 and an ionic strength of 151 mM [corrected] at 25 °C results in a collagen gel with a birefringence and tubular pores aligned parallel to the growth direction of the gel. The time course of averaged diameter of tubular pores during the anisotropic gelation was expressed by a power law with an exponent of 1/3, suggesting that the formation of tubular pores is attributed to a spinodal decomposition-like phase separation. Small angle light scattering patterns and high resolution confocal laser scanning microscope images of the anisotropic collagen gel suggested that the collagen fibrils are aligned perpendicular to the growth direction of the gel. The positional dependence of the order parameter of the collagen fibrils showed that the anisotropic collagen gel has an orientation gradient.

A case of minimal uterine serous carcinoma with distant lymph node metastasis without peritoneal dissemination.

A 61-year old woman underwent total abdominal hysterectomy and pelvic lymph node dissection under the diagnosis of endometrial cancer. Although pelvic lymph nodes were positive for adenocarcinoma with psamomma bodies, no other lesion that was a primary lesion was verified. A postoperative study revealed the existence of para-aortic lymph node and supraclavicular lymph node metastases. Therefore, the endometrial biopsy specimen was reviewed. With the findings of p53 positivity by immunohistochemistry in the papillary part, the final histopathological diagnosis was changed to endometrial serous adenocarcinoma. Postoperative chemotherapy followed by radiotherapy for supraclavicular lymph node metastasis achieved complete response. This type of tumor must be considered in a differential diagnosis when metastatic papillary serous carcinoma is detected, but the primary site remains unknown.

Xenopus furry contributes to release of microRNA gene silencing.

A transcriptional corepressor, Xenopus furry (Xfurry), is expressed in the chordamesodermal region and induces secondary dorsal axes when overexpressed on the ventral side of the embryo. The N-terminal furry domain functions as a repressor, and the C-terminal leucine zipper (LZ) motifs /coiled-coil structure, found only in vertebrate homologs, contributes to the nuclear localization. The engrailed repressor (enR)+LZ repressor construct, which has properties similar to Xfurry, induced several chordamesodermal genes. In contrast, an antisense morpholino oligonucleotide, Xfurry-MO, and the activating construct, herpes simplex virus protein (VP16)+LZ, had effects opposite those of Xfurry overexpression. Because blocking protein synthesis with cycloheximide superinduced several Xfurry transcriptional targets, and because expression of enR+LZ induced such genes under cycloheximide treatment, we analyzed the role of an Xfurry transcriptional target, microRNA miR-15. Cycloheximide reduced the expression of primary miR-15 (pri-miR-15), whereas miR-15 reduced the expression of genes superinduced by cycloheximide treatment. These results show that Xfurry regulates chordamesodermal genes by contributing to repression of pretranscriptional gene silencing by miR-15.

Highly conserved functions of the Brachyury gene on morphogenetic movements: insight from the early-diverging phylum Ctenophora.

Brachyury, a member of the T-box transcription family identified in a diverse array of metazoans, was initially recognized for its function in mesoderm formation and notochord differentiation in vertebrates; however, its ancestral role has been suggested to be in control of morphogenetic movements. Here, we show that morpholino oligonucleotide knockdown of Brachyury (MlBra) in embryos of a ctenophore, one of the most ancient groups of animals, prevents the invagination of MlBra expressing stomodeal cells and is rescued with corresponding RNA injections. Injection of RNA encoding a dominant-interfering construct of MlBra causes identical phenotypes to that of RNA encoding a dominant-interfering form of Xenopus Brachyury (Xbra) in Xenopus embryos. Both injected embryos down-regulate Xbra downstream genes, Xbra itself and Xwnt11 but not axial mesodermal markers, resulting in failure to complete gastrulation due to loss of convergent extension movements. Moreover, animal cap assay reveals that MlBra induces Xwnt11 like Xbra. Overall results using Xenopus embryos show that these two genes are functionally interchangeable. These functional experiments demonstrate for the first time in a basal metazoan that the primitive role of Brachyury is to regulate morphogenetic movements, rather than to specify endomesodermal fates, and the role is conserved between non-bilaterian metazoans and vertebrates.