Muscle development in Ciona intestinalis requires the b-HLH myogenic regulatory factor gene Ci-MRF

The activity of myogenic regulatory factor (MRF) genes is essential for vertebrate muscle development, whereas invertebrate muscle development is largely independent of MRF function. This difference indicates that myogenesis is controlled by distinct regulatory mechanisms in these two groups of animals. Here we used overexpression and gene knockdown to investigate the role in embryonic myogenesis of the single MRF gene of the invertebrate chordate Ciona intestinalis (Ci-MRF). Injection of Ci-MRF mRNA into eggs resulted in increased embryonic muscle-specific gene activity and revealed the myogenic activity of Ci-MRF by inducing the expression of four muscle marker genes, Acetylcholinesterase, Actin, Troponin I, and Myosin Light Chain in non-muscle lineages. Conversely, inhibiting Ci-MRF activity with antisense morpholinos down-regulated the expression of these genes. Consistent with the effects of morpholinos on muscle gene activity, larvae resulting from morpholino injection were paralyzed and their "muscle" cells lacked myofibrils. We conclude that Ci-MRF is required for larval tail muscle development and thus that an MRF-dependent myogenic regulatory network probably existed in the ancestor of tunicates and vertebrates. This possibility raises the question of whether the earliest myogenic regulatory networks were MRF-dependent or MRF-independent.     

Effects of U0126 and fibroblast growth factor on gene expression profile in Ciona intestinalis embryos as revealed by microarray analysis

Fibroblast growth factor (FGF) induces the notochord and mesenchyme in ascidian embryos, via extracellular signal-regulated kinase (ERK) that belongs to the mitogen-activated protein kinase (MAPK) family. A cDNA microarray analysis was carried out to identify genes affected by an inhibitor of MAPK/ERK kinase (MEK), U0126, in embryos of the ascidian Ciona intestinalis. Data obtained from the microarray and in situ hybridization suggest that the majority of genes are downregulated by U0126 treatment. Genes that were downregulated in U0126-treated embryos included Ci-Bra and Ci-Twist-like1 that are master regulatory genes of notochord and mesenchyme differentiation, respectively. The plasminogen mRNA was downregulated by U0126 in presumptive endoderm cells. This suggests that a MEK-mediated extracellular signal is necessary for gene expression in tissues whose specification does not depend on cell-to-cell interaction. Among 85 cDNA clusters that were not affected by U0126, 30 showed mitochondria-like mRNA localization in the nerve cord/muscle lineage blastomeres in the equatorial region. The expression level and asymmetric distribution of these mRNA were independent of MEK signaling.     

Identification and expression analysis of EST-based genes in the bud of Lycoris longituba

To obtain a primary overview of gene diversity and expression pattern in Lycoris longituba, 4,992 ESTs (Expressed Sequence Tags) from L. longituba bud were sequenced and 4,687 cleaned ESTs were used for gene expression analysis. Clustered by the PHRAP program, 967 contigs and 1,343 singlets were obtained. Blast search showed that 179 contigs and 227 singlets (totally 1,066 ESTs) had homologues in GenBank and 3,621 ESTs were novel.     

EST analysis of gene expression in testis of the ascidian Ciona intestinalis

To explore the gene expression underlying spermatogenesis, a large-scale analysis has been done on the cDNAs from testis of the ascidian, Ciona intestinalis. A set of 5,461 expressed sequence tags was analyzed and grouped into 2,806 independent clusters. Approximately 30% of the clusters showed significant sequence matches to the proteins reported in DDBJ/GenBank/EMBL database including a set of proteins closely related to the gene regulation during spermatogenesis, functional and morphological changes of spermatogenic cells during spermiogenesis, and physiological functions of sperm, as well as those with housekeeping functions commonly expressed in other cells. Some clones show similarities to the proteins present in vertebrate lymphocytes, suggesting a primitive immune system in ascidians. We have also found some genes that are known to participate in hormonal regulation of spermatogenesis in vertebrates. The large majority of the genes expressed in Ciona testis show no significant matches to known proteins and the further analysis of these genes may shed new light on the molecular mechanism of spermatogenesis and sperm functions.     

Localization of hatching enzyme in embryos and larvae of the sea-squirt Ciona intestinalis

Summary

A polyclonal antibody raised against the hatching enzyme of Ciona intestinalis (D'Aniello et al., 1997) was used on larvae of different ages in whole mount immunofluorescence experiments in order to localize the cells secreting the enzyme. After staining with FITC-conjugated second antibody, the larvae were observed by confocal microscopy. Larvae just before hatching (9–10 hours after fertilization) showed the presence of the enzyme in the peripheral cells of the adhesive papillae. The newly hatched larvae showed fluorescence also in the epidermal cells of the tip of the tail. Higher magnification confocal images of the papillae revealed bright fluorescence both in peripheral cells of the papillae and in the cavity between the tunic and the apex of the papillae (hyaline cap).

The swimming larvae maintain the fluorescence in the peripheral cells and in the hyaline cap for some hours until the beginning of metamorphosis, whereas the fluorescence of the tip of the tail disappears.

Following application of the antibody to Phallusia mamillata, the peripheral cells of the papillae of the newly hatched larvae were fluorescent and a bright fluorescence was also present between the two layers of the tunic above the papillae and the anterior part of the cephalenteron. We never observed fluorescence in the cells of the epidermis of the tail.

Retinoic acid (RA) treatment has been used to confirm the localization on the papillae of the cells secreting the hatching enzyme. The larvae of Ciona intestinalis were able to hatch because the cells of the tip of the tail positively reacted to immunofluorescence stain with anti-hatching enzyme antibody. On the contrary Phallusia tnamillata larvae failed to hatch and did not show anti-hatching enzyme reaction in the tail.     

Pax gene expression in the developing central nervous system of Ciona intestinalis

We compare the expression patterns in Ciona intestinalis of three members of the Pax gene family, CiPax3/7, CiPax6 and Cipax2/5/8. All three genes are expressed in restricted patterns in the developing central nervous system. At the tailbud stage, CiPax3/7 is present in three patches in the brain and along the posterior neural tube, CiPax6 throughout the anterior brain and along the posterior neural tube and CiPax2/5/8 in a restricted region of the posterior brain. Double in situ hybridisations were used to identify areas of overlap between the expression of different genes. This showed that CiPax3/7 overlaps with the boundaries of CiPax6 expression in the anterior brain, and with CiPax2/5/8 in the posterior brain. The overlap between CiPax3/7 and CiPax2/5/8 is unlike that described in the ascidian Halocynthia rorezti.     

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.     

EST-based analysis of gene expression in the porcine brain

Since pig is an important livestock species worldwide, its gene expression has been investigated intensively, but rarely in brain. In order to study gene expression profiles in the pig central nervous system, we sequenced and analyzed 43,122 highquality 5’ end expressed sequence tags (ESTs) from porcine cerebellum, cortex cerebrum, and brain stem cDNA libraries, involving several different prenatal and postnatal developmental stages. The initial ESTs were assembled into 16,101 clusters and compared to protein and nucleic acid databases in GenBank. Of these sequences, 30.6% clusters matched protein databases and represented function known sequences; 75.1% had significant hits to nucleic acid databases and partial represented known function; 73.3% matched known porcine ESTs; and 21.5% had no matches to any known sequences in GenBank. We used the categories defined by the Gene Ontology to survey gene expression in the porcine brain.