The ascidian pigmented sensory organs: structures and developmental programs.

The recent advances on ascidian pigment sensory organ development and function represent a fascinating platform to get insight on the basic programs of chordate eye formation. This review aims to summarize current knowledge, at the structural and molecular levels, on the two main building blocks of ascidian light sensory organ, i.e. pigment cells and photoreceptor cells. The unique features of these structures (e.g., simplicity and well characterized cell lineage) are indeed making it possible to dissect the developmental programs at single cell resolution and will soon provide a panel of molecular tools to be exploited for a deep developmental and comparative-evolutionary analysis.

Expression and functional analysis of Cititf1, an ascidian NK-2 class gene, suggest its role in endoderm development.

In solitary ascidians the fate of endoderm is determined at a very early stage of development and depends on cytoplasmic factors whose nature has not been determined. We have isolated a member of the NK-2 gene family, Cititf1, from the ascidian Ciona intestinalis, showing high sequence homology to mammalian TITF1. The Cititf1 gene was expressed in all endodermal precursors at the pregastrula and gastrula stages, and is thus the first specific regulatory endodermal marker to be isolated from an ascidian. Cititf1 expression was downregulated at the end of gastrulation to reappear at middle tailbud and larval stages in the most anterior and ventral parts of head endoderm, regions which give rise, after metamorphosis, to the adult endostyle, where Cititf1 mRNA was still present. Microinjection of Cititf1 mRNA into fertilized eggs resulted in tadpole larvae with abnormalities in head-trunk development consequent to the formation of excess endoderm, perhaps due to recruitment of notochord precursors to an endodermal fate. These data suggest that Cititf1 plays an important role in normal endoderm differentiation during ascidian embryogenesis.

The midbrain-hindbrain boundary genetic cascade is activated ectopically in the diencephalon in response to the widespread expression of one of its components, the medaka gene Ol-eng2.

In vertebrates, the engrailed genes are expressed at early neurula stage in a narrow stripe encompassing the midbrain-hindbrain boundary (MHB), a region from which a peculiar structure, the isthmus, is formed. Knock-out experiments in mice demonstrated that these genes are essential for the development of this structure and of its derivatives. In contrast, little is known about the effect of an overexpression of engrailed genes in vertebrate development. Here we report the isolation of Ol-eng2, a medaka fish (Oryzias latipes) engrailed gene. We have monitored the effects of its widespread expression following mRNA injections in 1- and 2-cell medaka and Xenopus embryos. We found that the ectopic expression of Ol-eng2 predominantly results in an altered development of the anterior brain, including an inhibition of optic vesicle formation. No change in the patterns of mesencephalic and telencephalic markers were observed. In contrast, expressions of markers of the diencephalon were strongly repressed in injected embryos. Furthermore, the endogenous Ol-eng2, Pax2, Wnt1 and Fgf8, which are essential components of the MHB genetic cascade, were ectopically expressed in this region. Therefore, we propose that Ol-eng2 induces de novo formation of an isthmus-like structure, which correlates with the development of ectopic midbrain structures, including optic tectum. A competence of the diencephalon to change to a midbrain fate has been demonstrated in isthmic graft experiments. Our data demonstrate that this change can be mimicked by ectopic engrailed expression alone.

Expression of the medaka (Oryzias latipes) Ol-Rx3 paired-like gene in two diencephalic derivatives, the eye and the hypothalamus.

Here we report the expression pattern of the homeobox Ol-Rx3 gene, a medaka gene homologous to the mouse, Xenopus, zebrafish and Drosophila Rx genes. Ol-Rx3 starts to be expressed, at late gastrula stages, in the presumptive territories of the anterior brain. Subsequently, transcripts are localised in an antero-ventral region of the prosencephalon and in the primordia of the optic vesicles. During organogenesis, distribution of Ol-Rx3 transcripts are gradually restricted to the floor of the diencephalon, the prospective territory of the hypothalamus and the neurohypophysis. During late development and in adult, Ol-Rx3 expression is maintained in hypothalamic nuclei bordering the third ventricle. In the optic vesicles, Ol-Rx3 expression is temporarily switched off when the eye cup morphogenesis is complete, but it is turned on again in the inner nuclear layer of the retina. Thus, the early expression pattern of Ol-Rx3 is in agreement with a conserved role in the specification of the ventral forebrain and eye field. Putative functions linked to late expression domains are discussed in light of the different hypothesis concerning the involvement of vertebrate Rx genes in the maintenance of particular cell fate.

Developmental regulation and tissue-specific localization of calmodulin mRNA in the protochordate Ciona intestinalis.

A full-length cDNA encoding a highly conserved calmodulin was isolated from a cDNA library prepared from hatched larvae of the ascidian Ciona intestinalis. Sequence analysis has identified a 447 b.p. open reading frame, encoding a putative protein of 149 amino acid residues, with a predicted molecular weight of 16.8 kDa, showing 85-98% identity to known calmodulins. Northern blot analysis revealed a single transcript of about 0.8 kb in length, which was maternally expressed and progressively increased during development, until late tail-bud stage. Whole-mount in situ hybridizations, carried out on embryos at different stages of development, showed that starting from the neurula stage, the C. intestinalis calmodulin (Ci-CaM) expression became restricted to the neuroectoderm and that in larvae it was specifically detected in the nervous system.

Cihox5, a new Ciona intestinalis Hox-related gene, is involved in regionalization of the spinal cord.

In this paper we report the cloning, sequence and expression analysis of a new Ciona intestinalis hox gene. On the basis of sequence comparison with mammalian and Amphioxus homologues, we called this gene Cihox5. Northern blot analysis reveals a single transcript of about 1.3 kb in length, that is present from neurula until larva stage. Whole-mount in situ hybridization shows restricted expression of this gene in putative blood cells precursors and in a regional domain of the spinal chord. Expression in the spinal cord is attributed to ependymal cells. This result implies a role for this gene in primitive regionalization of spinal cord along the anteroposterior axis in the absence of neuronal bodies.

A new transglutaminase-like from the ascidian Ciona intestinalis.

A cDNA clone encoding a transglutaminase (TGase) was isolated from a cDNA library prepared from the larval stage of Ciona intestinalis. The cDNA sequence has an open reading frame encoding a protein of 696 amino acids and is about 36% identical to 11 other TGase sequences. In addition, the critical residues thought to form the catalytic center are conserved. The Ciona TGase (CiTGase) has an extension of 39 amino acids in the NH2-terminal region similar to that reported for keratinocyte TGases. A phylogenetic analysis among other types of TGases demonstrated that CiTGase represents a new type of the enzyme.

Cloning of ascidian homeobox genes provides evidence for a primordial chordate cluster.

In order to isolate genes important in controlling embryonic development in Tunicates, a genomic library from the ascidian Ciona intestinalis was screened with a degenerate oligodeoxyribonucleotide encoding the third helix of Antennapedia-type homeoboxes. Fourteen C. intestinalis homeobox genes, corresponding to several classes of homeodomains, have been identified. Five of the isolated homeoboxes show their highest homology to members of the Vertebrate HOX clusters. mRNAs for two of the isolated homeoboxes are present in unfertilized C. intestinalis eggs.

Ultrastructural immuno-localization of CUT-1 and CUT-2 antigenic sites in the cuticles of the nematode Caenorhabditis elegans.

CUT-1 and CUT-2 are two distinct proteins found in cuticlin, the insoluble residue of the cuticles of the nematode Caenorhabditis elegans. They are the products of genes which have been previously characterized molecularly. These proteins have been expressed as recombinant in Escherichia coli and specific antisera have been raised against them. The experiments reported here regard their ultrastructural immuno-gold localization either on purified cuticles or on whole worms of various stages of development of Caenorhabditis elegans. A location in the cortical layer of the isolated cuticles is common to all stages, whereas there is a dauer specific location in the fibrous ribbon underneath the alae. These localizations are compared with immuno-labelling obtained using a serum raised against the whole cuticlin residue. CUT-1 and CUT-2 epitopes are easily and specifically lost during conventional chemical fixation.

Amphid defective mutant of Caenorhabditis elegans.

Studies are reported on a chemoreception mutant which arose in a mutator strain. The mutant sensory neurons do not stain with fluoresceine isothiocyanate (Dyf phenotype), hence the name, dyf-1, given to the gene it identifies. The gene maps on LGI, 0.4 map units from dpy-5 on the unc-11 side. The response of mutant worms to various repellents has been studied and shown to be partially altered. Other chemoreception based behaviors are less affected. The cilia of the sensory neurons of the amphid are shorter than normal and the primary defect may be in the capacity of the sheath cells to secrete the matrix material that fills the space between cilia in the amphid channel. Progress toward the molecular cloning of the gene is also reported. Relevant results from other laboratories are briefly reviewed.