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The aim of the paper focuses on generating insights into the competitiveness of public transport in Moscow City in relation to the use of personal cars, considering individual basic consumer values and environmental factors, including the impact of the COVID-19 pandemic. The research question in this study is: how can we assess the main indicators of public transport’s competitiveness, which can motivate personal car owners with specific basic values to change the transportation mode for personal mobility? This conceptual research model combines the content analysis of publications and large-scale online survey of Moscow City’s car owners—potential consumers of public transport services—on a representative quota sample of 1263 respondents. The methodology includes the axiological approach for the identification of the main factors of consumer behavior of personal car owners in the city with combinations of the modified Schwartz’s methodology of the basic individual values evaluations. Secondary data manifested the changes in the behavior of car owners due to the fact that they value metropolitan environmental degradation and changed their transportation preferences to public transport even during the COVID-19 pandemic. Based on the factor and cluster analysis of the value profile of the individuals, seven clusters of car owners were identified, and two large clusters were selected as target segments for public transport services. The conclusion was made that it is necessary to differentiate social and marketing programs to promote the competitiveness of public transport in the minds of consumers.

Background: Kommerell's diverticulum (KD) is associated with a high incidence of right-sided aortic arch (RAA). Hybrid thoracic endovascular aortic repair (TEVAR) is an effective and less invasive alternative to open repair. However, the long-term results regarding KD diameter regression or symptom improvement remain inadequately described., Methods: Nine patients underwent TEVAR for KD associated with RAA between January 2016 and September 2023 at our university hospital and affiliated institutions. A hybrid procedure was performed to exclude KD by blocking the proximal blood flow with TEVAR and distal blood flow with embolization of the aberrant subclavian artery. Simultaneously, extra-anatomical bypass surgery was performed to revascularize the covered supraarch vessels., Results: The patients' mean age was 65.2 years, and 6 patients were men. Two patients presented with dysphagia, whereas the rest were asymptomatic. The mean diameter and distance to the opposite aortic wall (OAW) of KD were 32.1 mm and 56.2 mm, respectively. For revascularization of the covered supraarch vessels, 6 and 2 patients underwent total debranching with sternotomy and extra-thoracic bypass (bilateral common carotid artery-axial artery bypass), respectively. The 30-day and in-hospital mortality rates were 0%, with no instances of cerebral infarction or spinal cord ischemia. The mean follow-up period was 3.2 years. The survival and avoidance rates of aortic events were 100% at 1 and 3 years. Follow-up computed tomography scans showed no endoleaks; however, 1 (11.1%) type 2 endoleak from the aberrant left subclavian artery occurred 1 week postoperatively, necessitating additional coiling. Seven patients were followed up for more than 1 year, with 5 experiencing reductions of more than 3 mm in KD diameter, distance to the OAW, or both., Conclusions: Although further follow-up and investigations are needed, TEVAR may be a safe and effective surgical treatment for KD associated with RAA., (Copyright © 2024 Elsevier Inc. All rights reserved.)

During anuran metamorphosis from herbivorous tadpoles to carnivorous frogs, the gastrointestinal (GI) tract undergoes drastic remodeling, such as the formation of the stomach-intestine boundary and the development of the pyloric sphincter at the posterior end of the stomach. However, the morphogenetic process and molecular mechanisms of how the pyloric sphincter is formed during metamorphosis, instead of during embryogenesis as in amniotes, are largely uninvestigated. Using the African clawed frog Xenopus laevis, we histologically examined the development of the pylorus region from embryonic to froglet stages and performed spatiotemporal gene expression analyses. We found that the pyloric sphincter is formed at a flexure within the pyloric region during metamorphic climax, and that the pyloric and duodenal epithelia, which are morphologically indistinguishable before sphincter formation, become clearly demarcated by the sphincter at the end of metamorphosis. Consistent with these morphological changes, expression domains of a stomach marker barx1 and an intestine marker cdx2 overlapped until late metamorphic climax, but became separated after metamorphosis. Despite the absence of the sphincter before metamorphosis, various genes crucial for sphincter formation in amniotes were already expressed in the pylorus region of Xenopus embryos. RNA-sequencing analysis at pre-metamorphic and metamorphic-climax stages suggest unappreciated roles of genes, such as those for retinoic acid signaling and various transcription factors, in suppressing or promoting sphincter formation. These data provide histological and molecular insights into the heterochrony of the pyloric sphincter formation in amniotes and anurans., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Dialysis patients undergoing transcatheter aortic valve replacement (TAVR) generally have poor prognosis compared with non-dialysis patients. Furthermore, there are few reliable risk models in this clinical setting. Therefore, we aimed to establish a risk model in dialysis patients undergoing TAVR that would be informative for their prognosis and the decision-making process of TAVR., Methods: A total 118 dialysis patients (full cohort) with severe aortic stenosis underwent TAVR in our institute between 2012 and 2022. The patients of the full cohort were randomly assigned to two groups in a 2:1 ratio to form derivation and validation cohorts. Risk factors contributing to deaths were analyzed from the preoperative variables and a risk model was established from Cox proportional hazard model., Results: There were 69 deaths following TAVR derived from infectious disease (43.5 %), cardiovascular-related disease (11.6 %), cerebral stroke or hemorrhage (2.9 %), cancer (1.4 %), unknown origin (18.8 %), and others (21.7 %) during the observational period (811 ± 719 days). The cumulative overall survival rates using the Kaplan-Meier method at 1 year, 3 years, and 5 years in the full cohort were 82.8 %, 41.9 %, and 24.2 %, respectively. An optimal risk model composed of five contributors: peripheral vascular disease, serum albumin, left ventricular ejection fraction < 40 %, operative age, and hemoglobin level, was established. The estimated C index for the developed models were 0.748 (95 % CI: 0.672-0.824) in derivation cohort and 0.705 (95 % CI: 0.578-0.832) in validation cohort. The prediction model showed good calibration [intraclass correlation coefficient = 0.937 (95%CI: 0.806-0.981)] between actual and predicted survival., Conclusions: The risk model was a good indicator to estimate the prognosis in dialysis patients undergoing TAVR., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The field of molecular embryology started around 1990 by identifying new genes and analyzing their functions in early vertebrate embryogenesis. Those genes encode transcription factors, signaling molecules, their regulators, etc. Most of those genes are relatively highly expressed in specific regions or exhibit dramatic phenotypes when ectopically expressed or mutated. This review focuses on one of those genes, Lim1/Lhx1, which encodes a transcription factor. Lim1/Lhx1 is a member of the LIM homeodomain (LIM-HD) protein family, and its intimate partner, Ldb1/NLI, binds to two tandem LIM domains of LIM-HDs. The most ancient LIM-HD protein and its partnership with Ldb1 were innovated in the metazoan ancestor by gene fusion combining LIM domains and a homeodomain and by creating the LIM domain-interacting domain (LID) in ancestral Ldb, respectively. The LIM domain has multiple interacting interphases, and Ldb1 has a dimerization domain (DD), the LID, and other interacting domains that bind to Ssbp2/3/4 and the boundary factor, CTCF. By means of these domains, LIM-HD-Ldb1 functions as a hub protein complex, enabling more intricate and elaborate gene regulation. The common, ancestral role of LIM-HD proteins is neuron cell-type specification. Additionally, Lim1/Lhx1 serves crucial roles in the gastrula organizer and in kidney development. Recent studies using Xenopus embryos have revealed Lim1/Lhx1 functions and regulatory mechanisms during development and regeneration, providing insight into evolutionary developmental biology, functional genomics, gene regulatory networks, and regenerative medicine. In this review, we also discuss recent progress at unraveling participation of Ldb1, Ssbp, and CTCF in enhanceosomes, long-distance enhancer-promoter interactions, and trans-interactions between chromosomes., (Copyright © 2021 Elsevier Inc. All rights reserved.)

On December 11, 2018, a single unengorged adult tick was found on the body surface of the trunk of an imported wild-caught Linnaeus's two-toed sloth (Choloepus didactylus) during a routine health check in an animal clinic in Tokyo, Japan. The tick was identified as Amblyomma geayi based on the morphological and molecular characteristics. This is the first case of the introduction of an Amblyomma species to Japan via an imported pet sloth. The present study highlights the current loopholes in Japan's regulatory system for animal imports., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Sox13, a group D member of the Sry-related high-mobility group box (Sox) transcription factor family, is expressed in various tissues including the hair follicle. However, its spatiotemporal expression patterns in the hair follicle and its role in hair development remain to be elucidated. To address these questions, we generated Sox13-LacZ-knock-in mice (Sox13 LacZ/+ ), in which the LacZ reporter gene was inserted in-frame into exon 2, which contains the translation initiation codon. X-gal staining in Sox13 LacZ/+ embryos revealed that Sox13 is initially expressed in the epithelial portion of the placode, and subsequently in the hair germ and the hair peg during early hair follicle development. In postnatal catagen and anagen, Sox13 was detected in the epithelial sheath, whereas in telogen, Sox13 was localized in the bulge region, where hair follicle stem cells reside. Immunohistochemistry with an anti-β-galactosidase antibody and anti-hair keratin antibodies that specifically mark the different layers of the hair follicle revealed that Sox13 was predominantly expressed in the outer root sheath in anagen. However, the integumentary structures of Sox13 LacZ/LacZ mice were grossly and histologically indistinguishable from those of wild type mice. These results suggest that although Sox13 is dispensable for epidermal and adnexal development, Sox13 is a useful marker for early hair follicle development., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Ehrlichiosis is a tick-borne bacterial disease caused by pathogens of the Ehrlichia genus. Although human ehrlichiosis has not been reported in Japan, Ehrlichia spp., which are closely related to Ehrlichia chaffeensis, were detected in several species of ixodid ticks. In this study, the presence of Ehrlichia spp. in ticks in Japan was studied by using isolation and molecular detection methods. In total, 1237 ticks were collected from vegetation in western, central, and eastern parts of Japan. The ticks were tested for detection of ehrlichial DNA with a nested polymerase chain reaction and/or isolation by inoculation of mice with the homogenate. Ehrlichial DNA was detected in 29 of these ticks. The ehrlichial DNAs, groEL and 16S rRNA genes, detected in Ixodes turdus showed a high similarity to those of E. chaffeensis with 94.7% and 99.2% identity, respectively. Ehrlichia sp. HF and Candidatus Neoehrlichia mikurensis were also detected in I. ovatus. Furthermore, Ehrlichia sp. HF was isolated from laboratory mice that were intraperitoneal inoculated with I. ovatus tick homogenate. Some ehrlichial agents detected in Ixodes ticks might be a previously unknown Ehrlichia species. In this study, Candidatus N. mikurensis was detected in I. ovatus ticks. Because I. ovatus is distributed widely and cases of its tick bite in humans are ubiquitously reported in Japan, there is a potential for ehrlichiosis to be endemic to Japan, necessitating intensive surveillance of this infectious disease., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Physical therapists (PTs) are required to obtain an accurate understanding of the physical and mental states of their patients through observational assessment. To perform comprehensive observational assessments of patients' movements, PTs likely need to engage their own neural systems involved in action understanding and theory of mind, such as the action observation network (AON) and the right temporoparietal junction (rTPJ). Both systems are modulated by the observer's actual experience with the observed movements. Although, most PTs do not have physical experience with neurological disabilities, they routinely examine hemiplegic movements in stroke patients, and are thus considered to have acquired pseudoexperience with hemiplegia. We hypothesized that the PTs' pseudoexperience with hemiplegia would modulate the neural system associated with the understanding of others to elaborately comprehend the physical and mental states associated with hemiplegia. To investigate our hypothesis, we recruited 19 PTs and 19 naïve participants (NPs) to undergo functional MRI (fMRI) for cortical activity measurement while viewing videos of hemiplegic (HHM) and non-hemiplegic (non-HHM) hand movements. The participants subsequently viewed the same videos again outside the MRI scanner, and evaluated the observed hand movements via a questionnaire. Compared to the NPs, the PTs showed greater activation in the AON and rTPJ while observing HHMs. Psychophysiological interaction analyses revealed increased connectivity between the rTPJ and AON when the PTs viewed the HHMs. Behavioral analyses further indicated that the PTs more accurately assessed feeling states associated with HHMs than did NPs. These findings suggest that the PTs' pseudoexperience modulates the AON and rTPJ, enabling them to better understand hemiplegia-associated feeling states., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Intimal sarcoma of the pulmonary artery is a rare and highly malignant neoplasm. This report describes a case of a 30-year-old woman with an extensive right pulmonary artery tumor who underwent an emergency operation. The tumor was aggressively resected with right pneumonectomy and reconstruction of the right ventricle outflow tract and left pulmonary artery. Although the resected margin at the left pulmonary artery was positive for disease, as confirmed by mouse double minute type 2 homologue staining, she is doing well and remains free of relapse at 16 months after the operation., (Copyright © 2018 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: This study aimed to identify the prognostic value of the preoperative stroke volume ratio (right ventricular stroke volume/left ventricular stroke volume) for redilatation of the right ventricle after pulmonary valve replacement in patients with repaired tetralogy of Fallot., Methods: From April 2004 to November 2013, 20 patients with repaired tetralogy of Fallot underwent pulmonary valve replacement for pulmonary valve regurgitation and right ventricular dilatation. Serial changes in ventricular volume were examined by cardiac magnetic resonance or computed tomography imaging. The redilatation ratio was calculated for right ventricular end-diastolic and end-systolic volume indices by dividing the increment in right ventricular volume from the first (median, 1.1 years) to the second (median, 3.2 years) evaluations after pulmonary valve replacement by the first evaluation. The relationships between the stroke volume ratio and redilatation ratio were assessed. The degree of right ventricular myocardial fibrosis was examined in 13 patients and compared with the stroke volume ratio., Results: Right ventricular volume (redilatation) significantly increased from a median of 1.1 to 3.2 years after pulmonary valve replacement. Significant positive correlations were detected between the stroke volume ratio and redilatation ratio of the right ventricular end-diastolic (r = 0.50. p = 0.02) and end-systolic volume indices (r = 0.49, p = 0.03). The stroke volume ratio also showed a significant positive correlation with the degree of right ventricular myocardial fibrosis (r = 0.73, p = 0.005)., Conclusions: The preoperative stroke volume ratio can predict redilatation of the right ventricle after pulmonary valve replacement and the degree of right ventricular myocardial fibrosis., (Copyright © 2017 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Inductive interactions mediated by the TGF-β and FGF-MAPK pathways are essential for specification of the germ layers and embryonic body axes during early vertebrate embryogenesis. TGF-β and FGF ligands signal through receptor Ser/Thr and Tyr kinases, respectively, and these signaling pathways cross-talk to regulate transcription and cell behavior. The allotetraploid Xenopus laevis and its ancestral diploid Xenopus tropicalis are versatile model organisms with which to study the inductive interactions and mechanisms of these signal transduction pathways. Here we have analyzed the draft genome of X. laevis with respect to the genomic organization and differential expression of genes in the TGF-β and FGF pathways. Genomic structure and gene expression analyses of pathway components in X. laevis revealed that genetic modulations, including deletions resulting in singletons and differential expression of homeologs, have occurred frequently among extracellular regulatory factors of the TGF-β pathway after allotetraploidization. Moreover, differential gene expression was found for factors regulating various cellular responses including co-receptors, decoy receptors, and intracellular negative regulators in both the TGF-β and FGF-MAPK pathways. We summarize the patterns of genetic alterations in the allotetraploid frog X. laevis and discuss the importance of these changes with regard to developmental processes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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&#95;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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cell signaling pathways, such as Wnt, Hedgehog (Hh), Notch, and Hippo, are essential for embryogenesis, organogenesis, and tissue homeostasis. In this study, we analyzed 415 genes involved in these pathways in the allotetraploid frog, Xenopus laevis. Most genes are retained in two subgenomes called L and S (193 homeologous gene pairs and 29 singletons). This conservation rate of homeologs is much higher than that of all genes in the X. laevis genome (86.9% vs 60.2%). Among singletons, 24 genes are retained in the L subgenome, a rate similar to the average for all genes (82.8% vs 74.6%). In addition, as general components of signal transduction, we also analyzed 32 heparan sulfate proteoglycan (HSPG)-related genes and eight TLE/Groucho transcriptional corepressors-related genes. In these gene sets, all homeologous pairs have been retained. Transcriptome analysis using RNA-seq data from developmental stages and adult tissues demonstrated that most homeologous pairs of signaling components have variable expression patterns, in contrast to the conservative expression profiles of homeologs for transcription factors. Our results indicate that homeologous gene pairs for cell signaling regulation have tended to become subfunctionalized after allotetraploidization. Diversification of signaling pathways by subfunctionalization of homeologs may enhance environmental adaptability. These results provide insights into the evolution of signaling pathways after polyploidization., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Extracellular factors belonging to the TGF-β family play pivotal roles in the formation and patterning of germ layers during early Xenopus embryogenesis. Here, we show that the vg1 and nodal3 genes of Xenopus laevis are present in gene clusters on chromosomes XLA1L and XLA3L, respectively, and that both gene clusters have been completely lost from the syntenic S chromosome regions. The presence of gene clusters and chromosome-specific gene loss were confirmed by cDNA FISH analyses. Sequence and expression analyses revealed that paralogous genes in the vg1 and nodal3 clusters on the L chromosomes were also altered compared to their Xenopus tropicalis orthologs. X. laevis vg1 and nodal3 paralogs have potentially become pseudogenes or sub-functionalized genes and are expressed at different levels. As X. tropicalis has a single vg1 gene on chromosome XTR1, the ancestral vg1 gene in X. laevis appears to have been expanded on XLA1L. Of note, two reported vg1 genes, vg1(S20) and vg1(P20), reside in the cluster on XLA1L. The nodal3 gene cluster is also present on X. tropicalis chromosome XTR3, but phylogenetic analysis indicates that nodal3 genes in X. laevis and X. tropicalis were independently expanded and/or evolved in concert within each cluster by gene conversion. These findings provide insights into the function and molecular evolution of TGF-β family genes in response to allotetraploidization., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Two siamois-related homeobox genes siamois (sia1) and twin (sia2), have been reported in Xenopus laevis. These genes are expressed in the blastula chordin- and noggin-expressing (BCNE) center and the Nieuwkoop center, and have complete secondary axis-inducing activity when over-expressed on the ventral side of the embryo. Using whole genome sequences of X. tropicalis and X. laevis, we identified two additional siamois-related genes, which are tandemly duplicated near sia1 and sia2 to form the siamois gene cluster. Four siamois genes in X. tropicalis are transcribed at blastula to gastrula stages. In X. laevis, the siamois gene cluster is present on both homeologous chromosomes, XLA3L and XLA3S. Transcripts from seven siamois genes (three on XLA3L and four on XLA3S) in X. laevis were detected at blastula to gastrula stages. A transcribed gene, sia1p. S, encodes an inactive protein without a homeodomain. When over-expressed ventrally, all siamois-related genes tested in this study except for sia1p. S induced a complete secondary axis, indicating that X. tropicalis and X. laevis have four and six active siamois-related genes, respectively. Of note, each gene required different amounts of mRNA for full activity. These results suggest the possibility that siamois cluster genes have functional redundancy to endow robustness and quickness to organizer formation in Xenopus species., (Copyright © 2017. Published by Elsevier Inc.)

Cell proliferation is strictly regulated by the dosage balance among cell-cycle regulators such as CDK/cyclin complexes and CDK-Inhibitors. Even in the allotetraploid genome of Xenopus laevis, the dosage balance must be maintained for animals to stay alive, and the duplicated homeologous genes seem to have gradually changed, through evolution, resulting in the best genes for them to thrive. In the Xenopus laevis genome, while homeologous gene pairs of CDKs are fundamentally maintained and a few cyclin genes are amplified, homeologous gene pairs of the important CDK-Inhibitors, CDKn1c and CDKn2a, are deleted from chromosomes L and S. Although losses of CDKn1c and CDKn2a can lead to diseases in humans, their loss in X. laevis does not affect the animals' health. Also, another gene coding CDKn1b is lost besides CDKn1c and CDKn2a in the genome of Xenopus tropicalis. These findings suggest a high resistance of Xenopus to diseases. We also found that CDKn2c.S expression is higher than that of CDKn2c.L, and a conserved noncoding sequence (CNS) of CDKn2c genomic loci on X. laevis chromosome S and X. tropicalis has an enhancement activity in regulating the different expression. These findings together indicate a surprising fragility of CDK inhibitor gene loci in the Xenopus genome in spite of their importance, and may suggest that factors other than CDK-inhibitors decelerate cell-cycling in Xenopus., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Studies using force-matching tasks have suggested that when we feel a "sense of effort," cortical regions may act to increase motor commands, and thus recruit additional motor units, in order to compensate for the exerted force. We hypothesized that suppressing activity in the primary motor cortex (M1), which is the source of the motor commands, would initiate the same process, and induce the same sense of effort. In a force-matching task, grip force was applied to 'right' hand and 10 healthy participants were asked to try to exert the same amount by using 'left' hand, with no visual feedback. On some trials, low-frequency, repetitive transcranial magnetic stimulation (lf-rTMS) was used to suppress the M1 and the primary somatosensory cortex (SI) in the left hemisphere, separately. Results showed that participants tended to overestimate the level of exerted force by up to 24%. In contrast, sham stimulation of the M1 and lf-rTMS over the SI did not significantly affect participants' estimations. Further, the M1 suppression resulted in a 42% reduction in motor-evoked potentials. Thus, the M1 suppression can affect our sense of effort, suggesting that compensatory neural mechanisms that increase the MI activity may play an important role in producing senses of effort., (Copyright © 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

We assessed the effect of teeth clenching on handgrip force behaviorally, and investigated cortical activity during the occurrence of facilitatory effects using functional magnetic resonance imaging (fMRI). Twenty-three participants were assessed as to whether they had habitual teeth clenching during maximal voluntary contraction (MVC) exertion, and 21 of them were identified to have such a habit. For those participants, behavioral testing showed that MVC with clenching was greater compared with without clenching (approximately 108% greater on average). Next, cortical activity was measured under gripping with clenching (GwC), gripping without clenching (GwoC), and teeth clenching (C) conditions. We found that the activity of the hand region in primary motor cortex (M1), cingulate motor area/supplementary motor area (CMA/SMA) and anterior cerebellum (AC) was greater in contrast of GwC vs. (GwoC+C). Furthermore, significant correlation was observed between the increasing ratio of the handgrip force and the % signal change in the hand region of M1 and AC, but not in CMA/SMA. These results suggest that the activation in the hand region of M1 and AC may facilitate the spinal motoneurons, and the activation in the hand region in M1 by clenching may be due to a signal from CMA/SMA., (Copyright © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

We previously observed that transient vascular occlusion in volunteers increased the estimation of force exertion with no change in peripheral nerves or muscles. We hypothesized that the primary factor responsible for the overestimation of force exertion during occlusion was the centrally generated motor command, as hypothesized by McCloskey et al. (1974) and McCloskey (1978, 1981). In the present study, we tested the hypothesis that transient vascular occlusion increases the excitability of the primary motor cortex (M1) during force exertion. Healthy human volunteers lay on a bed and squeezed a dynamometer in their right hand. Repetitive gripping forces were exerted at 20%, 40%, or 60% of maximum force, with or without transient (20s) vascular occlusion of the proximal portion of the right upper arm. During the task, single-pulse transcranial magnetic stimulation was applied to the contralateral M1 to induce motor evoked potentials (MEPs) in the flexor carpi ulnaris (FCU) muscle. The MEP amplitudes were enhanced with occlusion under all conditions, with the exception of 60% contraction. In contrast, no significant difference was observed between the MEP amplitudes obtained from the occluded or non-occluded, relaxed FCU muscle. These results suggest that transient vascular occlusion increases the excitability of M1 only during force exertion., (Copyright © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

In this study the effects of molecular structure of proteins on their adsorption behaviour and viscoelasticity are investigated using a QCM-D method. The adsorption measurements show that spherical lysozyme is rapidly adsorbed on the gold surface as a stiff monolayer, as indicated by a sharp drop in the oscillation frequency (Deltaf) of the sensor, and by a very small increase in the energy dissipation of the adlayer (DeltaD). Fibrous calfskin collagen (CSC) is, however, adsorbed on the same surface rather slowly in two steps to form a thick and soft multilayer (large Deltaf and DeltaD) at pH 3 in salt-free conditions. The two-step adsorption is not observed for stiffly aggregated CSC. This study clearly demonstrates that the polymer structure strongly affects not only how adsorption develops but also the viscoelastic properties of the adlayer., (2010 Elsevier B.V. All rights reserved.)

In the nervous system, transcription factor expression in progenitor and/or nascent neurons regulates cell type specification. Although the functions of these transcription factors at early stages are well established, whether or not they are required during late developmental periods remains an open question. To address this issue, we conditionally manipulated gene expression using a recently developed transposon-mediated gene transfer system combined with in ovo electroporation. In chicken retinas, horizontal cells are classified into three subtypes according to their characteristic neuronal morphology. Two LIM family transcription factors, Lim1 and Isl1, begin to be expressed in a distinct subset of nascent retinal neurons, which results in complementary expression of these genes in mature retinas in type I and type II/III horizontal cells, respectively. Overexpression of Isl1 in post-migratory horizontal cells represses endogenous Lim1 expression and increases the number of neurons with a dendritic morphology characteristic of type II horizontal cells, which normally express Isl1. Inhibition of Lim1 function by expression of a dominant negative form Lim1 perturbs axonal morphogenesis of type I horizontal cells. Therefore, we propose that LIM family transcription factors are required for subtype-specific morphogenesis of horizontal cells at later stages of retinal development.

To clarify the molecular mechanisms of neural development in vertebrates, we analyzed a novel gene, termed nemp1 (nuclear envelope integral membrane protein 1), which is expressed in the Xenopus anterior neuroectoderm at the neurula stage. Nemp1 has a putative signal peptide and five transmembrane domains, but does not have any other known domains. We show that Nemp1 is localized to the inner nuclear membrane (INM) with its evolutionarily conserved C-terminal region facing the nucleoplasm. Both overexpression and knockdown of Nemp1 in Xenopus embryos reduced the expression of early eye marker genes, rax, tbx3, and pax6, and later resulted mainly in severe eye defects at the tailbud stage. In contrast, the expression of a forebrain/midbrain marker, otx2, and a pan-neural marker, sox2, was largely unaffected. Deletion analysis of Nemp1 showed that nuclear envelope-localization of the C-terminal region is necessary for its eye-reducing activity. Furthermore, nemp1 is coexpressed with baf (barrier-to-autointegration factor) in the eye anlagen, and that Nemp1 interacts with BAF through the BAF-binding site in the C-terminal region and this site is required for Nemp1 activity. These data suggest that Nemp1 is involved in the expression of eye marker genes by functioning at the INM at least partly through BAF.

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.

A vascular occlusion by a tourniquet inflated at the proximal end of the upper arm is suggested to affect the estimation of exertion force level. In the first part of this study, subjects were asked to estimate the isometric force exerted by the occluded hand with that of the other hand (matching experiment). We found that the perceived force with arterial occlusion was always overestimated. To examine the underlying neural mechanism for this phenomenon, in the second part, the somatosensory evoked potentials (SEPs) and nerve action potential (NAP) were recorded following electrical median nerve stimulation with or without arterial occlusion. Moreover, the maximum motor response (M response) to median nerve stimuli at the axilla was recorded from the skin surface of the thenar eminence muscle of the hand during with arterial occlusion. The N20 of SEP and NAP at Erb's point were unaffected by the arterial occlusion, and the M response was also unchanged. These results suggest that the tourniquet-induced transient occlusion of the brachial artery does not seriously affect median nerve function. Thus, it is likely that the primary responsible factor for the overestimation of perceived force exertion during arterial occlusion is the centrally generated motor command as previously hypothesized by McCloskey [McCloskey, D.I., Ebeling, P., Goodwin, G.M., 1974. Estimation of weights and tensions and apparent involvement of a "sense of effort". Exp Neurol. 42, 220-232; McCloskey, D.I., 1978. Kinesthetic sensibility. Physiol. Rev. 58, 763-820; McCloskey, D.I., 1981. Corollary discharge and motor commands and perception. In: Brookhart, J.M., Mountcastle, V.B. (Eds.), Handbook of Physiology. American Physiological Society, Bethesda, pp. 1415-1447].

The Xenopus gene crescent encodes a member of the secreted Frizzled-related protein (sFRP) family and is expressed in the head organizer region. However, the target and function of Crescent in early development are not well understood. Here, we describe a role of Crescent in the regulation of convergent extension movements (CEMs) during gastrulation and neurulation. We show that overexpression of Crescent in whole embryos or animal caps inhibits CEMs without affecting tissue specification. Consistent with this, Crescent efficiently forms complexes with Xwnt11 and Xwnt5a, in contrast to another sFRP, Frzb1. As expected, the inhibitory effect of Crescent or Xwnt11 on CEMs is cancelled when both proteins are coexpressed in the neuroectoderm. Interestingly, when coexpressed in the dorsal mesoderm, the activity of Xwnt11 is rather enhanced by Crescent. Supporting this finding, the inhibition of CEMs by Crescent in mesodermalized but not neuralized animal caps is reversed by the dominant-negative form of Cdc42, a putative mediator of Wnt/Ca2+ pathway. Antisense morpholino oligos for Crescent impair neural plate closure and elicit microcephalic embryos with a shortened trunk without affecting early tissue specification. These data suggest a potential role for Crescent in head formation by regulating a non-canonical Wnt pathway positively in the adjacent posterior mesoderm and negatively in the overlying anterior neuroectoderm.

The midbrain-hindbrain boundary (MHB) acts as a local organizer in the development of the CNS in vertebrates. Previously, we identified an MHB-specific bHLH-WRPW transcriptional repressor gene, Xenopus Hes-related 1 (XHR1), which is initially expressed in the presumptive MHB (pre-MHB) region at the early gastrula stage. To better understand the gene cascades involved in MHB formation, we investigated the genes downstream from XHR1 by differential screening using a Xenopus cDNA macroarray and a dexamethasone (DEX)-inducible, dominant-negative transcriptional activator construct of XHR1 (XHR1-VP16-GR). Among the newly identified candidate target genes of XHR1 were Enhancer of split-related genes (ESR1, ESR3/7, and ESR9) and Xenopus laevis cleavage 2 (XLCL2). XHR1-VP16-GR induced the expression of the ESR genes and XLCL2 as well as Xdelta1, Xngnr1, and XHR1 itself in the presence of DEX even after pretreatment with the protein synthesis inhibitor, cycloheximide. This suggests that these genes are direct targets of XHR1. XHR1-knockdown experiments with antisense morpholino oligos and ectopic expression of wild-type XHR1 revealed that XHR1 is necessary and sufficient to repress ESR genes in the pre-MHB region. Misexpression of the ESR genes in the pre-MHB region repressed the MHB marker gene, Pax2, suggesting that the repression of the ESR genes by XHR1 is at least partly required for the early development of the pre-MHB. Our data also show that XHR1 is not activated by Notch signaling, differing from ESR genes. Taken together, we propose a model in which XHR1 defines the pre-MHB region as a prepattern gene by repressing those possible direct target genes.

Objective: We studied whether attention regulates information processing in the human primary somatosensory cortex (SI) by selective enhancement of relevant- and suppression of irrelevant information., Methods: Under successive and simultaneous electric stimuli to both the right index and middle fingers, tactile stimuli were randomly (20%) presented on one of the two fingers in separate two runs exchanging the finger. Subjects were requested to discriminate the tactile stimuli in an attention task to induce attention to one finger and to ignore the stimuli in a control task to avoid such an attention focus. Somatosensory evoked magnetic fields were measured only for the two-finger electric stimulation and an early component (M50) was analyzed., Results: In spite of the two-finger simultaneous stimulation, attention to either the index or middle finger lowered or heightened the M50-sourse location, respectively. The attention task did not increase the M50 amplitude., Conclusions: Attention to a finger enhanced selectively the representation of the finger in the SI cortex. However, this SI activity did not increase the M50 amplitude, suggesting that the attention suppressed another finger region receiving the unattended inputs., Significance: Attention regulates the SI activity by selectively enhancing the task-relevant information and by filtering out other noise inputs.

We can see things in three dimensions because the visual system re-constructs the three-dimensional (3D) configurations of objects from their two-dimensional (2D) images projected onto the retinas. The purpose of this paper is to give an overview of the psychological background and recent physiological findings concerning three-dimensional vision. Psychophysical and computational studies have suggested that in the visual system the 3D surface orientation is first estimated independently from individual depth cues--such as binocular disparity, as well as various monocular cues including texture gradients--and then the information from these different depth cues is integrated to construct a generalized representation of the 3D surface geometry. Neurons involved in low-level disparity processing, or the detection of local absolute disparity, were found mainly in the occipital cortex, whereas neurons involved in high-level disparity processing, or the reconstruction of 3D surface orientation through the computation of disparity gradients, were found mainly in the parietal area caudal intraparietal sulcus (CIP). Neurons sensitive to texture gradients, which is one of the major monocular cues, were also found in CIP. The majority of these neurons were sensitive to disparity gradients as well, suggesting their involvement in the computation of 3D surface orientation. In CIP, neurons sensitive to multiple depth cues were widely distributed together with those sensitive to a specific depth cue, suggesting CIP's involvement in the integration of depth information from different sources. In addition, human and monkey imaging studies have indicated convergence of multiple depth cues in CIP. These neurophysiological findings suggest that CIP plays a critical role in 3D vision by constructing a generalized representation of the 3D surface geometry of objects.

Cortical areas related to the information processing of binocular disparity-defined geometrical features of a surface, such as depth, orientation and shape are examined by functional magnetic resonance imaging while subjects discriminated these three types of geometrical feature in random dot stereograms. Results indicate that disparity-defined information of depth and that of orientation are processed in the parietal area. Furthermore, the visual system for 3D vision in the parietal area may be organized in a hierarchical manner and the posterior part of the right intraparietal sulcus may be involved in cognitive process of 3D vision. On the other hand, disparity-defined shape information seems to be processed in the occipital visual areas and the crucial involvement of human LOS for this process is suggested.

In this report, we provide evidence using a serial bone marrow transplantation (BMT) protocol that intra-bone marrow (IBM)-BMT (IBM-BMT) can efficiently reconstitute the hemopoietic system with cells of donor origin, in contrast to conventional intravenous (IV)-BMT (IV-BMT). Furthermore, the hematolymphoid system of secondary recipients that had received bone marrow cells (BMCs) from primary recipients treated with IBM-BMT recovered earlier than that of the secondary recipients of BMCs from primary recipients treated with IV-BMT. This was the case when the Lin-/c-kit+ progenitor cells of the secondary and tertiary recipients were examined. These findings indicate that IBM-BMT can facilitate the development of not only cells of various lineages but also the effective generation and, more importantly, the maintenance of the progenitor cells. Furthermore, we show that IBM-BMT can reconstitute the dendritic cell (DC) subsets (myeloid and lymphoid DCs), which are critical for the initiation of both adaptive and innate immune responses. The frequency of both myeloid and lymphoid DC subsets was approximately equal to that of normal age-matched untreated controls and, after second and third BMT, this ratio was close to that observed in the normal controls. However, the lymphoid DCs were clearly reduced in the secondary and tertiary recipients of BMCs from mice that had received IV-BMT. Therefore, the development of DC subsets is also normally maintained in the IBM-BMT group.

Objective: The digit representations in area 3b were studied to examine whether there is training-dependent reorganization in string players., Methods: Somatosensory evoked magnetic fields were recorded following electrical stimulation of digits 1 (D1), 2 (D2) and 5 (D5) of both hands in 8 string players and of the left hand in 12 control subjects. The N20m and P30m responses, and high-frequency oscillations (HFOs) were separated by 3-300 Hz and 300-900 Hz bandpass filtering., Results: The dipole locations on the coronal plane and strengths of D1, D2 and D5, and D1-D5 cortical distance estimated at the peak of N20m or P30m did not differ between left and right hand in string players or between left hand in string players and controls. On the other hand, the dipole locations of D2 estimated from N20m and P30m and of D1 from N20m were significantly anterior, the D2-D5 distance from P30m longer, and the number of HFO peaks larger for D5 in string players than controls., Conclusions/significance: With strong mutual competition among the fingering digits, the scale of reorganization should be much smaller as compared with the competition-free denervation-induced reorganizations. Taken together, the training-dependent reorganization of somatosensory cortex in string players is manifest not only in the enlarged cortical representation but also in the enhanced HFOs presumably representing activity of the fast-spiking interneurons.

To elucidate the molecular basis of organizer functions in Xenopus, we sought the target genes of the LIM homeodomain protein Xlim-1, which is one of the organizer-specific transcriptional activators. We found that an activated form of Xlim-1, Xlim-1/3m, initiates ectopic expression of the head-inducing organizer factor gene cerberus in animal caps. Thus, we analyzed the cerberus promoter using reporter assays. We show that three consecutive TAAT motifs of the homeodomain-binding sites between positions -141 and -118, collectively designated the "3xTAAT element," are crucial for the response of the cerberus promoter to Xlim-1/3m, and for its activation in the dorsal region of the embryo. Because cooperative activation of the cerberus promoter by Xnr1 and Xwnt8 also requires the 3xTAAT element, we focused on homeodomain transcriptional activators downstream from either Nodal or Wnt signaling. We found that wild-type Xlim-1 synergistically activates the cerberus promoter with Mix.1 and Siamois through the 3xTAAT element, and this synergy requires the LIM domains of Xlim-1. In contrast, Xotx2 acts synergistically with Mix.1 and Siamois through the TAATCT sequence at -95. Electrophoretic mobility shift assays revealed that Xlim-1, Siamois, and Mix.1 are likely to bind as a complex, in a LIM domain-dependent manner, to the region containing the 3xTAAT element. These data suggest that cerberus is a direct target for Xlim-1, Mix.1, Siamois, and Xotx2. Therefore, we propose a model for the molecular link in the inductive sequence from the formation of the organizer to anterior neural induction.

To study how epithelial layers are formed during early development in Xenopus embryos, we have focused on Claudin, the major component of the tight junction. So far, 19 claudin genes have been found in the mouse, expressed in different epithelial tissues. However, though a number of cytological studies have been done for the roles of Claudins, their expression patterns and functions during early embryogenesis are largely unknown. We found three novel Xenopus claudin genes, which are referred to as claudin-4L1, -4L2, and -7L1. At the early gastrula stage, claudin-4L1, -4L2, and -7L1 mRNAs were detected in the ectoderm and in the mesoderm. At the late gastrula stage, claudin mRNAs were detected in the ectoderm through the involuting archenteron roof. At the neurula stage, claudin-4L1/4L2 and -7L1 mRNAs were differentially expressed in the neural groove and the epidermal ectoderm. At the tailbud stage, the claudin mRNAs were found in the branchial arches, the otic vesicles, the sensorial layer of the epidermis, and along the dorsal midline of the neural tube. In addition, claudin-4L1/4L2 mRNAs were detected in the pronephros and the endoderm, whereas claudin-7L1 mRNA was observed in the epithelial layer of the epidermis.

The molecular mechanisms that govern early patterning of anterior neuroectoderm (ANE) for the prospective brain region in vertebrates are largely unknown. Screening a cDNA library of Xenopus ANE led to the isolation of a Hairy and Enhancer of split- (HES)-related transcriptional repressor gene, Xenopus HES-related 1 (XHR1). XHR1 is specifically expressed in the midbrain-hindbrain boundary (MHB) region at the tailbud stage. The localized expression of XHR1 was detected as early as the early gastrula stage in the presumptive MHB region, an area just anterior to the involuting dorsal mesoderm that is demarcated by the expression of the gene Xbra. Expression of XHR1 was detected much earlier than that of other known MHB genes, XPax-2 and En-2, and also before the formation of the expression boundary between Xotx2 and Xgbx-2, suggesting that the early patterning of the presumptive MHB is independent of Xotx2 and Xgbx-2. Instead, the location of XHR1 expression appears to be determined in relation to the Xbra expression domain, since reduced or ectopic expression of Xbra altered the XHR1 expression domain according to the location of Xbra expression. In functional assays using mRNA injection, overexpression of dominant-negative forms of XHR1 in the MHB region led to marked reduction of XPax-2 and En-2 expression, and this phenotype was rescued by coexpression of wild-type XHR1. Furthermore, ectopically expressed wild-type XHR1 near the MHB region enhanced En-2 expression only in the MHB region but not in the region outside the MHB. These data suggest that XHR1 is required, but not sufficient by itself, to initiate MHB marker gene expression. Based on these data, we propose that XHR1 demarcates the prospective MHB region in the neuroectoderm in Xenopus early gastrulae.

We describe here a systematic screen of an anterior endomesoderm (AEM) cDNA library to isolate novel genes which are expressed in the head organizer region. After removing clones which hybridized to labeled cDNA probes synthesized with total RNA from a trunk region of tailbud embryos, the 5' ends of 1039 randomly picked cDNA clones were sequenced to make expressed sequence tags (ESTs), which formed 754 tentative unique clusters. Those clusters were compared against public databases and classified according to similarities found to other genes and gene products. Of them, 151 clusters were identified as known Xenopus genes, including eight organizer-specific ones (5.3%). Gene expression pattern screening was performed for 198 unique clones, which were selected because they either have no known function or are predicted to be developmental regulators in other species. The screen revealed nine possible organizer-specific clones (4.5%), four of which appeared to be expressed in the head organizer region. Detailed expression analysis from gastrula to neurula stages showed that these four genes named crescent, P7E4 (homologous to human hypothetical genes), P8F7 (an unclassified gene), and P17F11 (homologous to human and Arabidopsis hypothetical genes) demarcate spatiotemporally distinct subregions of the AEM corresponding to the head organizer region. These results indicate that our screening strategy is effective in isolating novel region-specific genes., (Copyright 2001 Academic Press.)

The LIM class homeodomain protein Xlim-1 is specifically expressed in the organizer and later in the notochord in Xenopus. By seeking genes which are activated by Xlim-1 in animal caps, we isolated a gene which encodes a protein highly homologous to human PA26-T2, a p53-inducible nuclear factor, which we refer to as XPA26. Northern blot analysis showed that maternal transcripts of XPA26 are detected at cleavage stages and reduced during gastrulation. Whole-mount in situ hybridization showed that zygotic transcripts of XPA26 were initially detected in the anterior portion of developing notochord at neurula stages, and later in the entire notochord except its posterior region at tailbud stages. Thus the zygotic expression of XPA26 was detected exclusively in the notochord.

The Xenopus LIM homeodomain protein Xlim-1 is specifically expressed in the Spemann organizer region and assumed to play a role in the establishment of the body axis as a transcriptional activator. To further elucidate the mechanism underlying the regulation of its transcriptional activity, we focused on the region C-terminal to the homeodomain of Xlim-1 (CT239-403) and divided it into five regions, CCR1-5 (C-terminal conserved regions), based on similarity between Xlim-1 and its paralog, Xlim-5. The role of Xlim-1 CT239-403 in the Spemann organizer was analyzed by assaying the axis-forming ability of a series of CCR-mutated constructs in Xenopus embryos. We show that high doses of Xlim-1 constructs deleted of CCR1 or CCR2 initiate secondary axis formation in the absence of its coactivator Ldb1 (LIM-domain-binding protein 1), suggesting that CCR1 and CCR2 are involved in negative regulation of Xlim-1. In contrast, while Xlim-1 is capable of initiating secondary axis formation at low doses in the presence of Ldb1, deletion of CCR2 (aa 275-295) or substitution of five conserved tyrosines in CCR2 with alanines (CCR2-5YA) abolished the activity. In addition, UAS-GAL4 one-hybrid reporter assays in Xenopus showed that CCR2, but not CCR2-5YA, with its flanking regions (aa 261-315) functions as a transactivation domain when fused to the GAL4 DNA-binding domain. Finally, we show that none of the known transcriptional coactivators tested (CBP, SRC-1, and TIF2) interacts with the Xlim-1 transactivation domain (aa 261-315). Thus, Xlim-1 not only contains a unique tyrosine-rich activation domain but also contains a negative regulatory domain in CT239-403, suggesting a complex regulatory mechanism underlying the transcriptional activity of Xlim-1 in the organizer.

The Spemann organizer can be subdivided into head- and trunk-inducing tissues along the anteroposterior axis (Mangold, 1933. Naturwiisenschaften 43, 761-766; Spemann, 1931. Wilhelm Roux Arch. Entwicklungsmech. Org. 123, 389-517). Recent studies have suggested that head formation is brought about by repression of both Wnt and BMP signalling (Glinka et al., 1998. Nature 391, 357-362; Glinka et al., 1997. Nature 389, 517-519). Several Wnt inhibitors secreted from the head organizer region have been identified in Xenopus, such as Cerberus (Bouwmeester et al., 1996. Nature 382, 595-601), Frzb-1 (Leyns et al., 1997. Cell 88, 747-756; Lin et al., 1997. Proc. Natl. Acad. Sci. USA 94, 11196-11200), and Dkk-1 (Glinka et al., 1998. Nature 391, 357-362), supporting this two-inhibitor model. To isolate genes expressed in the head organizer, we screened a prechordal plate cDNA library by sequencing and expression pattern, and isolated the Xenopus ortholog of chick crescent encoding a Frizzled-like domain that is related to Wnt-binding regions of the Frizzled-family proteins. Expression of Xenopus crescent was first detected in the Spemann organizer region at the early gastrula stage and later in prechordal plate cells lining the boundary of mesoderm and ectoderm layers and in the anterior endoderm. At tailbud stages, the expression in the endomesoderm region was diminished, while expression in the pronephros became detectable. In animal cap assays, crescent gene was synergistically upregulated by coexpression of Xlim1, Ldb1, and Siamois, but not by Activin treatment.

The homeobox genes Xlim-1 and goosecoid (gsc) are coexpressed in the Spemann organizer and later in the prechordal plate that acts as head organizer. Based on our previous finding that gsc is a possible target gene for Xlim-1, we studied the regulation of gsc transcription by Xlim-1 and other regulatory genes expressed at gastrula stages, by using gsc-luciferase reporter constructs injected into animal explants. A 492-bp upstream region of the gsc promoter responds to Xlim-1/3m, an activated form of Xlim-1, and to a combination of wild-type Xlim-1 and Ldb1, a LIM domain binding protein, supporting the view that gsc is a direct target of Xlim-1. Footprint and electrophoretic mobility shift assays with GST-homeodomain fusion proteins and embryo extracts overexpressing FLAG-tagged full-length proteins showed that the Xlim-1 homeodomain or Xlim-1/Ldb1 complex recognize several TAATXY core elements in the 492-bp upstream region, where XY is TA, TG, CA, or GG. Some of these elements are also bound by the ventral factor PV.1, whereas a TAATCT element did not bind Xlim-1 or PV.1 but did bind the anterior factors Otx2 and Gsc. These proteins modulate the activity of the gsc reporter in animal caps: Otx2 activates the reporter synergistically with Xlim-1 plus Ldb1, whereas Gsc and PV.1 strongly repress reporter activity. We show further, using animal cap assays, that the endogenous gsc gene was synergistically activated by Xlim-1, Ldb1, and Otx2 and that the endogenous otx2 gene was activated by Xlim-1/3m, and this activation was suppressed by the posterior factor Xbra. Based on these data, we propose a model for gene interactions in the specification of dorsoventral and anteroposterior differences in the mesoderm during gastrulation.

Double-stranded RNA (dsRNA) interferes with gene expression in various species, a phenomenon known as RNA interference (RNAi). We show here that RNAi is also effective in modifying gene expression in Xenopus embryos. First, expression of an exogenous luciferase gene as a reporter in embryos was reduced by coinjection with dsRNA corresponding to the luciferase gene. Next, injection of dsRNA for Xlim-1, a homeobox gene suggested to be involved in Spemann organizer functions, reduced the endogenous level of Xlim-1 mRNA and produced embryos with reduced eyes or anterior truncation at high efficiency. In addition, injection of an antisense expression construct of Xlim-1 elicited phenotypes very similar to those of Xlim-1 dsRNA-injected embryos. These results indicate the effectiveness of RNAi for loss of function studies in Xenopus embryos, and the importance of Xlim-1 in head formation., (Copyright 2000 Academic Press.)

Members of the transforming growth factor-beta (TGF-beta) superfamily play various roles during development in both vertebrates and invertebrates. Two isoforms, TGF-beta2 and -beta5, have been isolated from Xenopus laevis. We describe here the localization of TGF-beta5 mRNA in early embryos of X. laevis, assessed by whole-mount in situ hybridization. The first detectable expression of TGF-beta5 was seen in the stage 14 embryo at the posterior tip of notochord, which continued to later stages, accompanied by the expression in bilateral regions of posterior wall in the tail region next to the notochord. At later stages, transient expression was seen in the cement gland (around stage 21) and in the somites (stages 24-27). In addition, expression was present in the branchial arches (stage 29-36) and olfactory placodes (stage 36).

Class II non-muscle myosins are implicated in diverse biological processes such as cytokinesis, cellularization, cell shape changes and gastrulation. Two distinct non-muscle myosin heavy chain genes have been reported in all vertebrates: non-muscle myosin heavy chain-A (NMHC-A) and -B (NMHC-B). We report here the isolation of the Xenopus homolog of NMHC-A and present a comparative analysis of the developmental and spatial expression patterns of NMHC-A and the previously isolated NMHC-B to address the role of NMHCs in Xenopus development. A 7.5 kb NMHC-A mRNA is present, maternally in unfertilized eggs and throughout embryogenesis, as well as in all adult tissues examined. An additional 8.3 kb zygotic transcript for NMHC-A is also detected, but only during embryonic stages. Whole mount in situ hybridization with tailbud stage embryos shows that NMHC-A mRNA is predominantly expressed in the epidermis, whereas NMHC-B mRNA is expressed in the somites, brain, eyes and branchial arches. Interestingly, the expression of NMHC-B in developing somites is gradually restricted to the center of each somite as differentiation proceeds. DAPI nuclear staining demonstrated that NMHC-B mRNA is colocalized with the nuclei or perinuclear area. In animal cap experiments, treatment with activin A or ectopic expression of Xbra and an activated form of Xlim1 markedly up-regulates NMHC-B as well as muscle actin mRNAs and slightly down-regulates NMHC-A mRNA, consistent with NMHC-B expression in the somitic muscle and NMHC-A expression in the epidermis., (Copyright 1998 Elsevier Science Ireland Ltd. All Rights Reserved)

The purpose of this study was to assess the computed tomography (CT) and plain radiographic findings of acute mesenteric ischemia, in an effort to elucidate its poor prognostic signs. The study group consisted of 26 cases with pathologically or angiographically proven mesenteric ischemia. The pathologically proven longitudinal extent of the bowel ischemia was graded using six degrees, and correlated with the radiographic findings. The mortality of the patients depended primarily on the extent of infarctions and the age of the patient. Patients with bowel dilation or abnormal gas in the bowel wall or portal system were prone to have wider extents of ischemia.