Developmental expression of ascidian neurotransmitter synthesis genes

To identify cholinergic neurons, we isolated a choline acetyltransferase (Ci-ChAT) gene from Ciona intestinalis by PCR methods. In the cloning process, we also obtained the gene encoding the vesicular acetylcholine transporter (Ci-vAChTP). These two genes shared the same 5'-UTR sequence as well as similar expression patterns. In both cases, the gene expression was first detected by whole-mount in situ hybridization in the anterior-dorsal region of the caudal nerve cord at the early tailbud stage. In the larva, the expression was seen in several cells of the visceral ganglion. These results suggest that ascidian larval motor neurons exist in the visceral ganglion.     

Ascidian arrestin (Ci-arr), the origin of the visual and nonvisual arrestins of vertebrate.

Arrestin is one of the key proteins for the termination of G protein signaling. Activated G protein-coupled receptors (GPCRs) are specifically phosphorylated by G protein-coupled receptor kinases (GRKs) and then bind to arrestins to preclude the receptor/G protein interaction, resulting in quenching of the following signal transduction. Vertebrates possess two types of arrestin; visual arrestin expressed exclusively in photoreceptor cells in retinae and pineal organs, and beta-arrestin, which is expressed ubiquitously. Unlike visual arrestin, beta-arrestin contains the clathrin-binding domain at the C-terminus, responsible for the agonist-induced internalization of GPCRs. Here, we isolated a novel arrestin gene (Ci-arr) from the primitive chordate, the ascidian Ciona intestinalis larvae. The deduced amino acid sequence suggests that Ci-Arr be closely related to vertebrate arrestins. Interestingly, this arrestin has the feature of both visual and beta-arrestin. Whereas the expression of Ci-arr was restricted to the photoreceptors in the larvae similarly to visual arrestin, the gene product, containing the clathrin-binding domain, promoted the GPCR internalization in HEK293tsA201 cells similarly to beta-arrestin. The phylogenetic tree shows that Ci-Arr is branched from a common root of visual and beta-arrestins. Southern analysis suggests that the Ciona genome contains only one gene for the arrestin family. These results suggest that the visual and beta-arrestin genes were generated by the duplication of the prototypical arrestin gene like Ci-arr in the early evolution of vertebrates.     

The ascidian homolog of the vertebrate homeobox gene Rx is essential for ocellus development and function

The tadpole larvae prosencephalon of the ascidian Ciona intestinalis contains a single large ventricle, along the inner walls of which lie two sensory organs: the otolith (a gravity-sensing organ) and the ocellus (a photo-sensing organ composed of a single cup-shaped pigment cell, about 20 photoreceptor cells, and three lens cells). Comparison has been drawn between the morphology and physiology of photoreceptor cells in the ascidian ocellus and the vertebrate eye. The development of vertebrate and invertebrate eyes requires the activity of several conserved genes and it is regulated by precise expression patterns and cell fate decisions common to several species. We have isolated a Ciona homeobox gene (Ci-Rx) that belongs to the paired-like class of homeobox genes. Rx genes have been identified from a variety of organisms and have been demonstrated to have a role in vertebrate eye formation. Ci-Rx is expressed in the anterior neural plate in the middle tailbud stage and subsequently in the larval stage in the sensory vesicle around the ocellus. Loss of Ci-Rx function leads to an ocellus-less phenotype that shows a loss of photosensitive swimming behavior, suggesting the important role played by Ci-Rx in basal chordate photoreceptor cell differentiation and ocellus formation. Furthermore, studies on Ci-Rx regulatory elements electroporated into Ciona embryos using LacZ or GFP as reporter genes indicate the presence of Ci-Rx in pigment cells, photoreceptors, and neurons surrounding the sensory vesicle. In Ci-Rx knocked-down larvae, neither basal swimming activity nor shadow responses develop. Thus, Rx has a role not only in pigment cells and photoreceptor formation but also in the correct development of the neuronal circuit that controls larval photosensitivity and swimming behavior. The results suggest that a Ci-Rx "retinal" territory exists, which consists of pigment cells, photoreceptors, and neurons involved in transducing the photoreceptor signals.     

Introduction and Expression of Recombinant Genes in Ascidian Embryos

In order to examine the expression of exogenous genes introduced into ascidian eggs, two recombinant plasmids pmiwZ and pHrMA4aCAT were microinjected into the cytoplasm of fertilized eggs of Ciona savignyi and Halocynthia roretzi , respectively. The plasmid pmiwZ contains the coding sequence of bacterial β-galactosidase gene ( lac-Z ) fused with animal gene promoters, while pHrMA4aCAT was constructed by fusing about 1.4-kb long 5' flanking region of H. roretzi muscle actin gene HrMA4a with bacterial chloramphenicol acetyltransferase gene ( CAT ). Injection of approximately 160 pl of 10 μg/ml pmiwZ DNA into Ciona eggs did not affect the embryogenesis, although introduction of the same volume of 30 μg/ml pmiwZ DNA resulted in abnormal development of injected eggs. When the expression of lac-Z was examined by histochemical detection of the enzyme activity, the expression was evident in the early tailbud embryos and later stage embryos, and larvae, irrespective of linear or circular form of the plasmid. The enzyme activity appeared in various cell-types including epidermis, nervous system, endoderm, mesenchyme, notochord, and muscle. In contrast, when pHrMA4aCAT was introduced into Halocynthia eggs and the appearance of CAT protein was examined later by the anti-CAT antibody, the CAT expression was restricted to muscle cells. These results indicate that the recombinant genes introduced into ascidian eggs could express during embryogenesis and that the 1.4-kb long 5' flanking region of HrMA4a contains regulatory sequences enough for the appropriate spatial and temporal expression of the gene.     

High-throughput enhancer trap by remobilization of transposon Minos in Ciona intestinalis

The enhancer trap approach utilizing transposons yields us information about gene functions and gene expression patterns. In the ascidian Ciona intestinalis, transposon-based transgenesis and insertional mutagenesis were achieved with a Tc1/mariner transposon Minos. We report development of a novel technique for enhancer trap in C. intestinalis. This technique uses remobilization of Minos in the Ciona genome. A Minos vector for enhancer trap was constructed and a tandem array insertion of the vector was introduced into the Ciona genome to create a mutator line. Minos was remobilized in Ciona chromosomes to create new insertions by providing transposases. These transposase-introduced animals were crossed with wild-type animals. Nearly 80% of F1 families showed novel GFP expression patterns. This high-throughput enhancer trap screen will be useful to create new marker transgenic lines showing reporter gene expression in specific tissues and to identify novel patterns of gene expression.     

Genomic organization and the 5' upstream sequences associated with the specific spatio-temporal expression of HrEpiC, an epidermis-specific gene of the ascidian Halocynthia roretzi.

An epidermis-specific gene HrEpiC of the ascidian Halocynthia roretzi is activated in all presumptive blastomeres by the 64-cell stage. To explore the molecular mechanisms underlying the regulation of the timing of activation of HrEpiC, we studied the genomic organization and the 5' upstream sequences of HrEpiC associated with specific spatio-temporal expression of the gene. The restriction site mapping and sequencing of genomic clones showed that the H. roretzi genome contained two copies of HrEpiC gene, HrEpiC1 and HrEpiC2, aligned tandemly in about 8 kb of the genome. Analysis of various deletion constructs with the 5' flanking sequences of HrEpiC1 revealed that 103 bp of the 5' flanking region was sufficient for the minimal epidermis-specific expression of HrEpiC1 and that the region between -281 bp and -198 bp of the 5' flanking region was associated with the amplification of the minimal expression of the reporter gene in the epidermis. This module between -281 bp and -198 bp was also shown to be associated with the timing of the activation of HrEpiC1 by the 64-cell stage. We discussed how the spatio-temporal expression pattern of HrEpiC1 is regulated by the two modules.     

羊BLG基因5′和3′调控区克隆及其调控GFP基因在乳腺细胞的表达

乳球蛋白 (ＢＬＧ)是反刍家畜的一种主要乳清蛋白质。将外源基因置于ＢＬＧ 5′调控区下游 ,能够控制外源基因在动物乳腺的组织特异性表达。现已清楚在ＢＬＧ 5′端有多个乳核因子 1(ＮＦ1)和转录因子Ｓｔａｔ5的识别位点 ,此外还存在多个激素作用位点[1 ] 。具有这些调控成分的ＢＬＧ 5′翼端 (5′ｆｌａｎｋｉｎｇ)能够控制外源基因在乳腺的表达 ,但受整合位点、外源基因结构以及转基因拷贝数及其排列方式的影响[2 ] 。研究结果表明 ,仅含有 5′调控区的乳腺表达构件 ,还不能使外源基因的表达达到内源性ＢＬＧ的表达水平 ,因为在 5′远端(…     

An enhancer trap in the ascidian Ciona intestinalis identifies enhancers of its Musashi orthologous gene

The enhancer trap technique, established in Drosophila melanogaster, is a very sophisticated tool. Despite its usefulness, however, there have been very few reports on enhancer traps in other animals. The ascidian Ciona intestinalis, a splendid experimental system for developmental biology, provides good material for developmental genetics. Recently, germline transgenesis of C. intestinalis has been achieved using the Tc1/mariner superfamily transposon Minos. During the course of that study, one Minos insertion line that showed a different GFP expression pattern from other lines was isolated. One fascinating possibility is that an enhancer trap event occurred in this line. Here we show that a Minos insertion in the Ci-Musashi gene was responsible for the altered GFP expression. Ci-Musashi showed a similar expression pattern to GFP. In addition, introns of Ci-Musashi have enhancer activity that can alter the expression pattern of nearby genes to resemble that of GFP in this line. These results clearly demonstrate that an enhancer trap event that entrapped enhancers of Ci-Musashi occurred in C. intestinalis.     

Ascidian larva reveals ancient origin of vertebrate-skeletal-muscle troponin I characteristics in chordate locomotory muscle

Ascidians are protochordates related to vertebrate ancestors. The ascidian larval tail, with its notochord, dorsal nerve cord, and flanking rows of sarcomeric muscle cells, exhibits the basic chordate body plan. Molecular characterization of ascidian larval tail muscle may provide insight into molecular aspects of vertebrate skeletal muscle evolution. We report studies of the Ci-TnI gene of the ascidian Ciona intestinalis, which encodes the muscle contractile regulatory protein troponin I (TnI). Previous studies of a distantly related ascidian, Halocynthia roretzi, showed that different TnI genes were expressed in larval and adult muscles, the larval TnI isoforms having an unusual C-terminal truncation not seen in any vertebrate TnI. Here we show that, in contrast with Halocynthia, Ciona does not have a specialized larval TnI; the same TnI gene that is expressed in the heart and body-wall muscle of the sessile adult is also expressed in embryonic/larval tail muscle cells. Moreover the TnI isoform produced in embryonic/larval muscle is identical to that produced in adult body-wall muscle, i.e., a 182-residue protein with the characteristic chain length and overall structure of vertebrate skeletal muscle TnI isoforms. Phylogenetic analyses indicate that the unique features of Halocynthia larval TnI likely represent derived features, and hence that the vertebrate-skeletal-muscle -like TnI of Ciona is a closer reflection of the ancestral ascidian larval TnI. Our results indicate that characteristics of vertebrate skeletal muscle TnI emerged early in the evolution of chordate locomotory muscle, before the ascidian/vertebrate divergence. These features could be related to a basal chordate locomotory innovation-e.g., swimming by oscillation of an internal notochord skeleton-or they may be of even greater antiquity within the deuterostomes.