Binary specification of nerve cord and notochord cell fates in ascidian embryos

In the ascidian embryo, the nerve cord and notochord of the tail of tadpole larvae originate from the precursor blastomeres for both tissues in the 32-cell-stage embryo. Each fate is separated into two daughter blastomeres at the next cleavage. We have examined mechanisms that are responsible for nerve cord and notochord specification through experiments involving blastomere isolation, cell dissociation, and treatment with basic fibroblast growth factor (bFGF) and inhibitors for the mitogen-activated protein kinase (MAPK) cascade. It has been shown that inductive cell interaction at the 32-cell stage is required for notochord formation. Our results show that the nerve cord fate is determined autonomously without any cell interaction. Presumptive notochord blastomeres also assume a nerve cord fate when they are isolated before induction is completed. By contrast, not only presumptive notochord blastomeres but also presumptive nerve cord blastomeres forsake their default nerve cord fate and choose the notochord fate when they are treated with bFGF. When the FGF-Ras-MAPK signaling cascade is inhibited, both blastomeres choose the default nerve cord pathway, supporting the results of blastomere isolation. Thus, binary choice of alternative fates and asymmetric division are involved in this nerve cord/notochord fate determination system, mediated by FGF signaling

Cis-regulatory control of the SM50 gene, an early marker of skeletogenic lineage specification in the sea urchin embryo

The SM50 gene encodes a minor matrix protein of the sea urchin embryo spicule. We carried out a detailed functional analysis of a cis-regulatory region of this gene, extending 440 bp upstream and 120 bp downstream of the transcription start site, that had been shown earlier to confer accurate skeletogenic expression of an injected expression vector. The distal portion of this fragment contains elements controlling amplitude of expression, while the region from −200 to +105 contains spatial control elements that position expression accurately in the skeletogenic lineages of the embryo. A systematic mutagenesis analysis of this region revealed four adjacent regulatory elements, viz two copies of a positively acting sequence (element D) that are positioned just upstream of the transcription start site; an indispensable spatial control element (element C) that is positioned downstream of the start site; and further downstream, a second positively acting sequence (element A). We then constructed a series of synthetic expression constructs. These contained oligonucleotides representing normal and mutated versions of elements D, C, and A, in various combinations. We also changed the promoter of the SM50 gene from a TATA-less to a canonical TATA box form, without any effect on function. Perfect spatial regulation was also produced by a final series of constructs that consisted entirely of heterologous enhancers from the CyIIIa gene, the SV40 early promoter, and synthetic D, C, and A elements. We demonstrate that element C exercises the primary spatial control function of the region we analyzed. We term this a ‘locator’ element. This differs from conventional ‘tissue-specific enhancers’ in that while it is essential for expression, it has no transcriptional activity on its own, and it requires other, separable, positive regulatory elements for activity. In the normal configuration these ancillary positive functions are mediated by elements A and D. Only positively acting control elements were observed in the SM50 regulatory domain throughout this analysis

Preparation and Use of "Authentic English" Web Materials at The University of Tokushima (Tokushima, Japan)

This paper outlines a pilot project funded by The President’s Fund of the University of Tokushima that was designed to create English educational material to motivate students to acquire “Authentic English.” Recording was at sites in Honolulu (Hawaii, USA), Storrs (Connecticut, USA), Montreal (Canada) and Brisbane (Australia). Material was designed for use in teaching EFL students at our university who are taking general English courses. Section One of this paper refers to the aims and process of making English educational material for the Web. Sections Two, Three, and Four refer to how the materials were created. Section Five introduces technical aspects of the Web application. Sections Six and Seven discuss merits and problems related to the “Authentic English” material and its production

Neural-tube specific paralogous genes and their upstream regulatory sequences

Ascidians are primitive chordates, and their critical evolutionary position expected to offer an excellent experimental system to understand the origin and the evolution of vertebrates (Satoh, 1994). Especially, neural tube is one of the characteristic features of chordates. In this study, we focused on the mechanism of the neural tube formation, and described the detailed expression profiles of two neural tube specific genes, CiNutl and CiNut2. Although the amount of CiNut2 expression is about 1/1000 compared to that of CiNutl, both two genes are expressed in the entire neural plate and neural tube during the course of the development. Both two genes are situated in an adjacent position of the chromosome in the same direction. Comparative analysis of the upstream regulatory sequence of two genes revealed the conserved sequences, which suggested having a role for the neural tube specific expression

コトナル タシャ トノ カカワリカタ ニ カンレン スル ショヨウイン ノ ケンキュウ : トクシマ ダイガク ソウゴウ カガクブ 1ネンセイ オ タイショウ ニ シタ アンケート チョウサ カラ

This study aimed to determine factors related to the level of acceptance for people who are regarded as being different in terms of country of origin, age and sexual norms. Questionnaires were distributed to first year undergraduate students at The University of Tokushima in Japan and promax rotation factor analysis yielded five factors: 1) orientation toward foreign cultures, 2) openness toward multiculturalism, 3) openness toward discussing sex, 4) openness toward having friendship with members of the opposite sex and 5) orientation toward educational achievement. Results showed that gender and course of study were the most important variables related to openness toward foreign cultures and openness toward multiculturalism, with female students and liberal arts students having a relatively high level of acceptance for people regarded as being different. Results suggest that university curricula should focus more on multiculturalism training for male students and students majoring in mathematics and science

多毛類巨大ヘモグロビンの一次構造比較

Amino acid sequences of four types of globin chains a, A, b, and B of the giant Hb from the polychaete Perinereis aibuhitensis, which were collected at the delta of the Yangtze River in the People's Republic of China, were determined by the combination of Edman degradation and RT-PCR method. The primary structures of Perinereis globin chains are compared with those of globins isolated from the polychaete Tylorrhynchus heterochaetus which was collected at the delta of the River Yoshinogawa in Tokushima City. Perinereis globin chains appeared to have 140 amino acid residues for the chain a, 144 for A, 147 for b and B, respectively. These sequences were completely in agreement with the partial cDNA sequences by RT-PCR which covered 119～137 amino acid residues. Some heterogeneities were found for the chain A by protein sequencing. On the other hand, RT-PCR revealed a few polymorphisms in the sequences of chains a, A and b. According to the phylogenetic tree constructed by UPGMA method, Perinereis globin chains were appeared to be separated into two strains A and B at the time later than the separation of vertebrate Mb and Hb but earlier than the separation of the alpha and beta chains of vertebrate Hb. Perinereis globins b and B appeared to be separated from globin A after the time when the trimeric globin chains A, b, and B had been separated clearly from the derivative of globin a according to the phylogenetic tree constructed by NJ method. The homologies between the corresponding globin chains of Perinereis and Tylorrhynchus Hbs were estimated to be within the range of 48～84%

Neural Marker Genes Differently Expressed in Subsets of Embryonic Neural Cells of the Ascidian Halocynthia roretzi

Ascidians are lower chordates that possess a possible prototype of the vertebrate nervous system. The central and peripheral nervous systems of ascidian larvae are composed of only a few hundred cells (Nicol and Meinertzhagen, 1991). To investigate how these ascidian nervous systems develop, dissection at the molecular level using subset-specific markers is essential. Here we describe four new genes zygotically expressed in subsets of the ascidian neural cells. The spatial expression domains of these genes overlap in some parts but not in other parts of the nervous systems. Our results suggest that there are functionally different regions in the nervous systems owing to the gene expression differences. Further analyses of these genes will enable us to determine the molecular neuro-developmental characteristics of various clusters of neural cells

Expression Patterns of Smad Family Members during Embryogenesis of the Ascidian Halocynthia roretzi

The ascidian embryo has been long thought to show a mosaic mode of development. However, recent studies revealed significance of cell-cell communication during cleavage stages of embryogenesis. FGF and BMP signalings play critical roles in determination of cell types. Little is, however, known about regulation of competence of cells to the signals. Here we report the isolation of ascidian smad genes; Hrsmad4 which encodes a homolog of smad4 of vertebrates, Hrsmad6/7 which encodes a homologous gene of smad 6 and smad7 of vertebrates, and Hrsmad2/3 which encodes a homolog of smad2 and smad3 of vertebrates. The mRNAs of the isolated smad family genes were maternally inherited in egg and early embryos. While Hrsmad4 and Hrsmad6/7 RNAs distributed broadly in the early embryos, Hrsmad2/3 RNA was preferentially accumulated in the animal hemisphere

A Large-Scale Whole-Mount in situ Hybridization System: Rapid One-Tube Preparation of DIG-Labeled RNA Probes and High Throughput Hybridization using 96-Well Silent Screen Plates

Recent progress in multiple and automated-sequencing technology allows large-scale random cDNA sequencing, the so-called EST project, in various fields. In addition to the EST collection, the cDNA project requires analysis of spatiotemporal patterns of gene expression of a large number of clones by whole-mount in situ hybridization (WISH). To facilitate the multiple WISH procedures, we developed a protocol for rapid and uniform synthesis of multiple probes and multi-well based WISH processing. A DIG-labeled RNA probe for WISH was synthesized from a PCR-amplified template which contained an RNA promoter. All reactions of PCR and subsequent RNA synthesis were performed in a single tube by sequential addition of the reagents without phenol extraction or ethanol precipitation steps. An RNA probe was purified and condensed by a centrifugal ultrafilter to achieve high and stable purification efficiency. WISH of 96 samples were performed simultaneously in a 96-well plate attached to silent screen filters that were connected with a vacuum exhausting system. These processes eliminated the labor-intensive steps of WISH and provided opportunities to search for novel genes