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.

PLAUF binding to the 3'UTR of the H3.3 histone transcript affects mRNA stability.

In P. lividus sea urchin the H3.3 histone variant is coded by an mRNA characterized by a long 3'UTR containing ARE (AU-Rich element) motifs. RNA stability assays performed in rabbit reticulocyte lysate showed that such 3'UTR affects the degradation rate of the transcripts. In fact, chimeric molecules containing the 3'UTR of H3.3 transcript, ligated to the coding region of the rabbit beta-globin transcript, were unstable whereas chimeric molecules containing mainly the coding region of the H3.3 transcript were stable as the wild-type globin mRNA. Three proteins (45kDa, 32kDa and 25kDa) that bind specifically the 3'UTR have been revealed in the whole protein extracts of embryos at different stages of development. PLAUF, a P. lividus RNA-binding protein similar to human and rodent AUF1 proteins, was identified as the 32kDa factor using anti-PLAUF antibody in Western blot and supershift mobility assays. Moreover the recombinant GST-PLAUF protein specifically binds part of the H3.3 3'UTR and in vitro affects the half-life of the transcript. In addition in situ hybridization experiments demonstrated that PLAUF and H3.3 histone mRNAs co-localize in embryos at different stages of development. In conclusion all the reported results suggest that PLAUF can bind in vivo the 3'UTR of the H3.3 histone mRNA and plays some role in the stability of the mRNA.

PLAUF is a novel P. lividus sea urchin RNA-binding protein.

Preliminary results have shown that various proteins bind long 3'UTR of the transcript for Paracentrotus lividus sea urchin H3.3 histone variant and are probably implicated in mRNA instability. In order to identify these RNA-binding proteins, we screened a lambda-ZAPII cDNA expression library prepared from poly(A) mRNA extracted from sea urchin embryos at blastula stage. We isolated a cDNA that codes for a novel RNA-binding protein homologous to rat and human AUF1 family proteins and we refer to it as PLAUF. Proteins present in the whole lysate of the phages expressing PLAUF bound specifically in vitro the 3'UTR of the H3.3 histone transcript. Northern blot analysis revealed three PLAUF transcripts that are already present in unfertilized eggs; during development their amount increased starting from 4-blastomere embryos and reached the plateau at blastula stage. While the transcription start point was unique, longer 3'UTRs were revealed by 3'RACE approach and further cDNA library screening. Moreover RT-PCR showed the presence of at least one alternative spliced mRNA that codes for a protein with different COOH terminus. The structure of the PLAUF gene was determined by screening a P. lividus sea urchin genomic library with the PLAUF cDNA as probe. Analysis of the positive clones showed that the PLAUF gene is split in 10 exons and 9 introns spanning a distance of about 10 kb. Moreover we demonstrated that the exon 9 was alternative spliced during mRNA processing.

A new family of "H3L-like" histone genes.

The H3L histone variant gene in Paracentrotus lividus (sea urchin) shows almost all typical features of the replication-dependent histone genes, but it codes for the H3.3 histone protein with the S.//. A.IG amino acid motif, which is typical of the variants synthesized in a replication-independent manner. "H3L-like" histone genes have been found in several unrelated organisms. These genes are intronless and encode for the typical H3.3 histone proteins. The newly described family of H3L-like variants, nearly ubiquitous within the animal kingdom, could represent the common ancestor of H3 and H3.3 histone genes.

DNA (cytosine-5) methyltransferase turnover and cellular localization in developing Paracentrotus lividus sea urchin embryo.

The turnover and localization of the enzyme DNA (cytosine-5) methyltransferase (Dnmt1) were studied during Paracentrotus lividus sea urchin embryo development using antibody preparations against the NH(2) and COOH-terminal regions of the molecule. The antibodies reveal, by Western blots and whole-mount analyses, that the enzyme is differently required during embryonic development. The changeover point is at blastula stage, where a proteolytic mechanism hydrolyses the enzyme present in all embryonic cells by removing a peptide of about 45 kDa from the amino terminal region of the 190 kDa enzyme initially synthesized on maternal transcripts. The resulting 145 kDa enzyme shows modified catalytic properties, different antibody reactivity and is rapidly destroyed in the few hours before gastrulation. At more advanced stages of development the enzyme is newly synthesized but only in particular cell types, among which neurons. The data show that Dnmt1 is removed from embryonic cells before gastrulation to be synthesized again at different levels in different cell types, indicating that the concentration of Dnmt1 is critical for the various differentiated cells of the developing sea urchin embryo.

Isolation and characterization of a novel member of the relaxin/insulin family from the testis of the frog Rana esculenta.

A complementary DNA (cDNA) encoding a frog relaxin/insulin member family (fRLX) from testis cDNA library was isolated and characterized. The fRLX cDNA predicted a 155-amino acid protein with a low homology to mammalian RLF and relaxin. Northern blot analysis revealed a single transcript expressed in the interstitial compartment, RT-PCR, evidenced that fRLX is expressed at low levels in the oviduct and ovary too. The predicted mature fRLX protein, composed of the signal peptide, B, C, and A domains, has conserved amino acid sequences in the characteristic functional domains. A different expression of the transcript was found during the frog reproductive cycle, with a peak in Spring. After administration of ethane dimethane sulfonate, by in situ hybridization, fRLX messenger RNA disappeared from the interstitial compartment and reappeared again at the time of generating of a new population of Leydig cells (LC), strongly indicating that LC are the interstitial cell type expressing fRLX. Preliminary results obtained by in situ hybridization, performed on testis of hypophysectomized frogs evidenced a pituitary control of fRLX expression. This study is the first cloning of a relaxin/insulin family member in a nonmammalian vertebrate. In addition, because fRLX expression changes during the annual cycle suggesting its involvement in spermatogenesis, fRLX may be considered a new marker for the study of spermatogenesis in the Rana esculenta.

The replacement H3.3 histone gene in Paracentrotus lividus sea urchin: structure and regulatory elements.

We have isolated the Paracentrotus lividus sea urchin H3.3 histone gene and characterized the nucleotide sequences of the gene and its proximal promoter. Band shift experiments showed that two cAMP/PMA responsive elements (CRE/TRE), present in the proximal promoter, bind nuclear factors present in embryos at the blastula and gastrula stages (CRE1) and at the blastula stage (CRE2). The putative H3.3 coding region activating sequences (CRAS) failed to bind nuclear factors while the corresponding elements of the two replication-dependent genes (H3L and late H3) clearly recognized nuclear proteins. These results suggest some role of the CRE/TRE elements but not CRAS elements in the transcriptional regulation of the replication-independent histone genes in invertebrates.

Ci-IPF1, the pancreatic homeodomain transcription factor, is expressed in neural cells of Ciona intestinalis larva.

We describe the cloning and the expression pattern of insulin promoter factor 1 in the ascidian Ciona intestinalis (Ci-IPF1). Northern blot analysis showed that transcripts appeared at the late tailbud stage and increased at the larval stage. We have raised a specific antibody against the Ci-IPF1-GST fusion protein to determine the spatial expression of this gene. The protein is immunodetected at the larval stage in the sensory vesicle, in the visceral ganglion and in the mesenchymal cells. Our results support the hypothesis that IPF1/IDX1 might have extrapancreatic functions during animal development, particularly in neural cells.

Identification and developmental expression of three Distal-less homeobox containing genes in the ascidian Ciona intestinalis.

Several homeobox-containing genes related to Drosophila Distal-less (Dll) have been isolated from a wide variety of organisms and have been shown to function as developmental regulators. While in Drosophila only one Dll gene has been described so far, in Vertebrates many components of the Dlx multigenic family have been characterized. This suggests that, during the evolution of the Chordate phylum, the Dlx genes arose from an ancestral Dll/Dlx gene via gene duplication. We have previously reported the isolation of two Dll-related homeoboxes from the protochordate Ciona intestinalis, and described their clustered arrangement (Gene 156 (1995) 253). Here we present the detailed genomic organization and spatial-temporal expression of these two genes, Ci-Dll-A and Ci-Dll-B, and describe the isolation and characterization of another member of the ascidian family of Dll-related genes, which we tentatively named Ci-Dll-C.

Isolation and characterization of the cDNA for a Ciona intestinalis RNA binding protein: spatial and temporal expression during development.

A cDNA encoding an RNA binding protein has been isolated from a cDNA library prepared from larvae of the ascidian Ciona intestinalis. The putative protein of 162 amino acids contained in the N-terminal region one copy of the consensus sequence RNA binding domain and in the C-terminal region a glycine-rich domain. The in vitro translated protein bound various RNA homopolymers, preferentially polyU, polyA, and polyG, and the binding was affected by increasing ionic strength. Northern blot analysis revealed a single transcript of about 0.7 kb in length that was present during embryonic development with two major peaks of accumulation at gastrula and larval stages. Whole-mount in situ hybridization experiments on embryos at different stages of development showed gene expression mainly in mesenchymal cells and in neural tissue.

Characterization of a new variant DNA (cytosine-5)-methyltransferase unable to methylate double stranded DNA isolated from the marine annelid worm Chaetopterus variopedatus.

The enzyme S-adenosylmethionine-DNA (cytosine-5)-methyltransferase has been identified, first time for invertebrates, in embryos of the marine polychaete annelid worm Chaetopterus variopedatus. The molecule has been isolated from embryos at 15 h of development. It is a single peptide of about 200 kDa molecular weight, cross-reacting with antibodies against sea urchin DNA methyltransferase. The enzymatic properties of the molecule are similar to those of Dnmt1 methyltransferases isolated from other organisms, but with the peculiarity to be unable to make 'de novo' methylation on double stranded DNA.

Identification and developmental expression of Ci-msxb: a novel homologue of Drosophila msh gene in Ciona intestinalis.

We report the cloning and expression pattern of Ci-msxb the second Ciona intestinalis homeobox gene homologue to the Drosophila muscle segment homeobox (msh) gene. Northern blot analysis showed that transcripts appeared at gastrula stage, peaked in the early tailbud and decreased during the tailed stages. Whole mount in situ hybridization showed that the Ci-msxb expression first is detected at 110 cell-stage in the blastomeres that are precursors of different tissue (muscle, spinal cord, endodermal strand, brain, mesenchyme, pigmented cells and primordial pharynx). Transcript level declined in mesoderm cells after the completion of gastrulation, but mRNAs were still present in the folding neural plate during neurulation and in the pigmented cells. Later, at larval stage, transcripts were present around the otolith and ocellus, in a restricted part of the nervous system and in the primordial pharynx; the gene expression was conserved after metamorphosis in the juvenile.

Patterning the ascidian nervous system: structure, expression and transgenic analysis of the CiHox3 gene.

Hox genes play a fundamental role in the establishment of chordate body plan, especially in the anteroposterior patterning of the nervous system. Particularly interesting are the anterior groups of Hox genes (Hox1-Hox4) since their expression is coupled to the control of regional identity in the anterior regions of the nervous system, where the highest structural diversity is observed. Ascidians, among chordates, are considered a good model to investigate evolution of Hox gene, organisation, regulation and function. We report here the cloning and the expression pattern of CiHox3, a Ciona intestinalis anterior Hox gene homologous to the paralogy group 3 genes. In situ hybridization at the larva stage revealed that CiHox3 expression was restricted to the visceral ganglion of the central nervous system. The presence of a sharp posterior boundary and the absence of transcript in mesodermal tissues are distinctive features of CiHox3 expression when compared to the paralogy group 3 in other chordates. We have investigated the regulatory elements underlying CiHox3 neural-specific expression and, using transgenic analysis, we were able to isolate an 80 bp enhancer responsible of CiHox3 activation in the central nervous system (CNS). A comparative study between mouse and Ciona Hox3 promoters demonstrated that divergent mechanisms are involved in the regulation of these genes in vertebrates and ascidians.

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.

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.

Molecular cloning of a peroxidase mRNA specifically expressed in the ink gland of Sepia officinalis.

A full-length cDNA encoding a peroxidase has been assembled from two different overlapping cDNA clones isolated from the cDNA library of Sepia officinalis ink gland and 5' RACE procedure. The cDNA of 2898 bp contains an open reading frame of 2778 bp corresponding to a protein of 926 amino acids. The protein shows 76% identity with a peroxidase isolated from the light organ of the squid Euprymna scolopes and approximately 30% identity with mammalian peroxidases. Northern blot analysis of mRNA from various tissues of Sepia officinalis reveals that peroxidase is exclusively expressed in the ink gland. These results represent the first characterization of a melanogenic peroxidase whose possible involvement in the biosynthesis of melanin is discussed.

Specific cellular localization of tyrosinase mRNA during Ciona intestinalis larval development.

A Ciona intestinalis cDNA clone that encodes a protein highly homologous to other tyrosinases was isolated. Northern blot analysis showed that expression of Ciona tyrosinase starts at the early neurula stage and continues throughout the tail-bud and tadpole larval stages. The earliest tyrosinase expression was detected, by in situ hybridization, at the neural plate stage, in pigment precursor cells located along the two neural folds, in the animal region of the embryo. In the course of embryonic development the strong hybridization signal was always localized, within the rostral part of the developing brain, in the pigment precursor cells and was later detected in the otolith and ocellus. These results are discussed in relation to tyrosinase as an early marker of neural induction.

The S.//.A.IG amino acid motif is present in a replication dependent late H3 histone variant of P. lividus sea urchin.

A novel gene encoding a new H3 histone varian (H3L) has been identified in P. lividus sea urchin embryo. It encodes a H3 histone protein showing the S.//.A.IG amino acid motif typical of the replication independent H3.3 variants but in a mRNA showing the 3' terminal stem-loop nucleotide sequence that is typical of the replication dependent variants. The gene is intronless, the corresponding short transcript is non-polyadenyl ated and its expression is replication dependent with a timing of late variant. The new H3 variant is expressed as a minor component with respect to a major replication dependent late H3 histone here identified by partial cDNA sequence. These results show that classification of histones in replication dependent and independent variants only on the basis of their amino acid sequences should be reconsidered.

Isolation of cDNA clones encoding DNA methyltransferase of sea urchin P. lividus: expression during embryonic development.

A full-length cDNA, encoding a DNA (cytosine-5)-methyltransferase (DNA MTase), has been assembled from a series of overlapping cDNA clones isolated from P. lividus sea urchin embryo cDNA libraries. The cDNA contains 103 bp 5'-UTR, 4839 bp open reading frame corresponding to a 1612 amino acids (aa) protein and 2240 bp 3'-UTR including a terminal 18-bp poly(A) tail. Both the cDNA and the encoded protein are the longest so far reported for DNA MTases. The protein shows five distinct and sequential regions of identity with the other animal DNA MTases, with values of identity from zero to 80%. Northern blot analyses reveal a single RNA band of about 7.5 kb in length showing a highly regulated concentration pattern during development with peak value at the four blastomere stage.