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.

In vitro methylation of histones in sea urchin nuclei during early embryogenesis.

Nuclei isolated from sea urchin embryos incubated in vitro in the presence of S-adenosyl-[methyl-3H]methionine, methylate their own basic proteins. The protein methylase activity varies during the embryonic development with two peaks of activity at mesenchymal blastula and at young gastrula. Histones H3 and H4 are the main substrates of the reaction. The extent of methylation of the two histones depends on the S-adenosylmethionine concentration. At low S-adenosylmethionine concentrations, the in vitro methyl-accepting ability of H3 is 10-times that of H4, while at high concentrations it is 3-times that of H4. This finding is clearly evident in the equilibrium saturation experiments with blastula and gastrula nuclei, which both show two distinct Km values for S-adenosylmethionine. The major and perhaps only product of methylation is epsilon-N-methyl-lysine. Enzyme activity is clearly correlated with specific embryonic stages, while no correlation is apparent between enzyme activity and the amount of DNA in the embryos.

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.

Histone-lysine methyltransferase activity from sea-urchin embryo nuclei. Changes in substrate specificity upon purification.

The S-adenosylmethionine:histone-lysine methyltransferase (EC 2.1.1.43) enzyme activity, present in the chromatin of sea-urchin embryo nuclei, has been purified about 300-fold with 30% overall yield. The initial activity in the nucleus transfers methyl groups to the epsilon-amino group of lysines and acceptor proteins are chromatin-bound H3 and H4 histones. In contrast, the purified enzyme activity transfers methyl groups to the arginines and acceptor proteins are soluble H3 and H4 histones. The two changes in substrate specificity do not occur at the same time. The variation of acceptor protein from chromatin-bound to soluble histones occurs at the first step, upon nuclei sonication, when no protein fractionation has yet been performed. At that step, lysine is still the only methylated side-chain. The variation of the methylated amino acid from lysine to arginine occurs gradually with increasing enzyme purification. The enzyme activity has a molecular mass of about 200 kDa. Saturation curves for H3 and H4 histones, used as substrate either individually or in total histones, and for AdoMet show no substantial dependence on enzyme purification. Maximal activity for the enzyme, at all purification levels, occurs at about pH 8 for all substrate histones. An increase in the relative concentrations of di- and trimethyllysine derivatives is observed with the more purified enzyme preparations, while the ratio of mono- and dimethylarginine derivatives remains constant. The data are taken as evidence that the same protein molecule is responsible for the two activities.

Isolation of a new H3.3 histone variant cDNA of P. lividus sea urchin: sequence and embryonic expression.

A cDNA encoding a new H3 histone variant has been isolated from a Paracentrotus lividus sea urchin embryo cDNA library. The encoded protein is identical to the H3.3 histone subtype identified in other species, with the difference that E replaces D at position 81. The clone corresponds to a transcript of about 1.6 kb, not dependent on DNA replication, present in the unfertilized egg and at all stages of embryonic development. The coding part of the cDNA cross-reacts also with a 0.5 kb H3 late histone mRNA.

Proteolytic enzymes as structural probes for ribonuclease BS-1.

Trypsin, pepsin and subtilisin have been used as conformational probes for the structure of bovine seminal ribonuclease BS-1 by studying, under definite conditions, their effects on the seminal enzyme, a dimeric protein made up to two identical subunits; on bovine pancreatic monomeric ribonuclease A (EC 3.1.4.22) with a polypeptide chain homologous to that of the seminal ribonuclease subunit chain; and on a monomeric, active and stable derivative of seminal ribonuclease. The results show: (1) that the C-terminal regions of the pancreatic and the seminal proteins are very similar as they appear to fit in an identical way to the active site of pepsin; (2) that the resistance of the N-terminal region of ribonuclease BS-1 to subtilisin is not due to the dimeric structure of the protein, but to the conformation of this region, where an essential feature is the presence of a proline residue at position 19; (3) that the monomer of ribonuclease BS-1 is resistant to tryptic action only when bound to the partner monomer in the quaternary structure of the protein. This indicates that dissociation of the seminal ribonuclease makes some potentially susceptible susceptible bond or bonds available to trypsin either through a conformational change of the protein subunit, or by simply exposing the protein area hidden at the intersubunit interfaces.

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.

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.

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.

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.

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.

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.

Cloning and characterization of a developmentally regulated sea urchin cDNA encoding glutamine synthetase.

A 2935-bp cDNA clone encoding glutamine synthetase (GS) was isolated from a cDNA library prepared from four-blastomere Paracentrotus lividus sea urchin embryos. The sequence consists of a 75-bp 5' untranslated region (5'-UTR) followed by a 1095-bp coding region corresponding to a 365-amino-acid (aa) protein, a 1747-bp 3'-UTR and a terminal 18-bp poly(A) tail. The encoded protein shows about 66% identical residues, as compared with human and lobster class-II GS. The sequence contains the Mn(2+)-binding aa and the highly conserved aa regions observed in other GS. Northern blot analyses show that the GS mRNA is present in the sea urchin egg and is developmentally regulated in the embryo.

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.

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.

DNA methyltransferase activity in the early stages of a sea urchin embryo. Evidence of differential control.

The specific activity of DNA methyltransferase increases in the nuclei of Sphaerechinus granularis sea urchin embryos at increasing stages of development. The activity reaches maximal value at about 20 h of growth, when embryos are at the mesenchyme blastula stage, then abruptly decreases and is essentially zero at about 35 h of development, when embryos are at the early gastrula stage. Both the increase and the drop of the activity are faster than embryonic cell duplication indicating that the enzyme is under strict control during development and that, in the more advanced embryo, a mechanism is activated to specifically block its activity.

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.

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.