Expression of Insl3 Protein in Adult Danio rerio.

Insulin-like peptide 3 (INSL3) is a biomarker for Leydig cells in the testes of vertebrates, and it is principally involved in spermatogenesis through specific binding with the RXFP2 receptor. This study reports the insl3 gene transcript and the Insl3 prepropeptide expression in both non-reproductive and reproductive tissues of Danio rerio. An immunohistochemistry analysis shows that the hormone is present at a low level in the Leydig cells and germ cells at all stages of Danio rerio testis differentiation. Considering that the insl3 gene is transcribed in Leydig cells, our results highlight an autocrine and paracrine function of this hormone in the Danio rerio testis, adding new information on the Insl3 mode of action in reproduction. We also show that Insl3 and Rxfp2 belonging to Danio rerio and other vertebrate species share most of the amino acid residues involved in the ligand-receptor interaction and activation, suggesting a conserved mechanism of action during vertebrate evolution.

Expression pattern of zebrafish rxfp2 homologue genes during embryonic development.

RXFP2 is one of the 4 receptors for relaxin insulin-like peptides, in particular it binds with high affinity the INSL3 peptide. INSL3/RXFP2 pair is essential for testicular descent during placental mammalian development. The evolutionary history of this ligand/receptor pair has received much attention, since its function in vertebrate species lacking testicular descent, such as the fishes, remains elusive. Herein, we analyzed the expression pattern of three rxfp2 homologue genes in zebrafish embryonic development. For all the three rxfp2 genes (rxfp2a, rxfp2b, and rxfp2-like) we showed the presence of maternally derived transcripts. Later in the development, rxfp2a is only expressed at larval stage, whereas rxfp2b is expressed in all the analyzed stage with highest level in the larvae. The rxfp2-like gene is expressed in all the analyzed stage with a transcript level that increased starting at early pharyngula stage. The spatial localization analysis of rxfp2-like gene showed that it is expressed in many cell clusters in the developing brain. In addition, other rxfp2-like-expressing cells were identified in the retina and oral epithelium. This analysis provides new insights to elucidate the evolution of rxfp2 genes in vertebrate lineage and lays the foundations to study their role in vertebrate embryonic development.

Expression analysis of five zebrafish RXFP3 homologues reveals evolutionary conservation of gene expression pattern.

Relaxin peptides exert different functions in reproduction and neuroendocrine processes via interaction with two evolutionarily unrelated groups of receptors: RXFP1 and RXFP2 on one hand, RXFP3 and RXFP4 on the other hand. Evolution of receptor genes after splitting of tetrapods and teleost lineage led to a different retention rate between mammals and fish, with the latter having more gene copies compared to the former. In order to improve our knowledge on the evolution of the relaxin ligands/receptors system and have insights on their function in early stages of life, in the present paper we analyzed the expression pattern of five zebrafish RXFP3 homologue genes during embryonic development. In our analysis, we show that only two of the five genes are expressed during embryogenesis and that their transcripts are present in all the developmental stages. Spatial localization analysis of these transcripts revealed that the gene expression is restricted in specific territories starting from early pharyngula stage. Both genes are expressed in the brain but in different cell clusters and in extra-neural territories, one gene in the interrenal gland and the other in the pancreas. These two genes share expression territories with the homologue mammalian counterpart, highlighting a general conservation of gene expression regulatory processes and their putative function during evolution that are established early in vertebrate embryogenesis.

Developmental expression pattern of two zebrafish rxfp3 paralogue genes.

In mammals, the RXFP3 is the cognate receptor of the relaxin-3 peptide (RLN3). In teleosts, many different orthologue genes for RXFP3 are present. In particular, two paralogue genes, rxfp3-2a and rxfp3-2b, likely encode the receptors for the Rln3a peptide. The transcription of these two rxfp3 genes is differentially regulated early during zebrafish embryogenesis. Indeed, reverse transcription-polymerase chain reaction analyses show that the rxfp3-2b transcript is always present during embryo development, while the rxfp3-2a transcript is detectable only at larval stage. By in situ hybridization experiments on embryos and larvae, the rxfp3-2b transcript was revealed in the brain and in the retinal ganglion cell layer and thymus. Particularly in the brain, many territories are involved in the rxfp3-2b expression, among them the optic tectum, thalamus, preoptic area, different nerve nuclei, habenula and pineal gland. The RXFP3 spatiotemporal expression pattern appears to be conserved between Danio rerio and mammals, as also previously showed for the corresponding ligand, the RLN3. Interestingly, the brain areas expressing the rxfp3-2b receptor gene are involved in the visual system, emotional behaviors and circadian rhythm and could be functionally related to the neurotransmitter Rln3a-expressing territories.

A network system for vitellogenin synthesis in the mussel Mytilus galloprovincialis (L.).

The aim of this study is to assess, by RT-PCR, in situ hybridization, electron microscopy, and immunohistochemistry, the site/s of vitellogenin (VTG) synthesis in the mussel Mytilus galloprovincialis. Our investigations demonstrate that, among the analyzed tissues, the synthesis of VTG occurs only in the female gonad, that is, within the oocyte and follicle and connective cells. Such a synthesis is just evident in early vitellogenic oocytes, whose cytoplasm is characterized by numerous RER cisternae and an extended Golgi complex surrounded by nascent yolk platelets. The synthesis of VTG goes on in vitellogenic oocytes assuming a pear form, and progressively reduces once the oocyte shows the pear or polygonal form, typical of those oocytes that have concluded the growth. The expression of VTG occurs also within follicle (auxiliary) and connective cells. In particular, it is noteworthy that follicle cells are characterized by numerous RER cisternae and an active Golgi complex surrounded by numerous vesicles and vacuoles containing electron dense material. The same material is also present along their plasma membrane, within the intercellular space between oocyte and follicle cells, and finally within invaginations of the oocyte surface, thus suggesting a VTG transfer to the oocyte via endocytosis. Differently, no VTG synthesis was observed within digestive gland. All together the findings here reported strongly suggest that in M. galloprovincialis, inside the gonad, the VTG synthesis occurs in the oocyte (autosynthesis) and in the follicle and adipogranular cells (heterosynthesis).

Characterization, cDNA cloning and expression pattern of relaxin gene during embryogenesis of Danio rerio.

We report the identification, the cDNA cloning, the temporal and spatial expression pattern analysis of the rln gene in the zebrafish Danio rerio. The deduced Rln B and A domains show different evolutionary conservation. Rln B domain shows higher similarity when compared to zebrafish and human RLN3 B domain than human RLN1 and RLN2 B domain. Differently, the zebrafish Rln A domain shows relatively low amino acid sequence similarity when compared with the same sequences. The rln gene is transcribed both during embryogenesis and in adult organism, where higher transcript level has been particularly evidenced in the brain. Moreover, we provide the first description of rln spatial expression pattern during embryonic development. In particular, we show restricted transcript localization starting at the pharyngula stage in olfactory placode, branchial arch region, and in a cell cluster near to otic vesicle. In larval stage, new transcription territories have been detected in both neural and non-neural regions. In particular, in the brain, rln expression has been revealed in telencephalic region around anterior commissure, in the preoptic area, and in restricted rombencephalic cell clusters. Expression of rln gene in extra-neural territories has been detected in the pancreatic and thyroid gland regions. Danio rerio rln expression pattern analysis reveals shared features with the mammalian RLN gene, particularly in the brain, where it might have a role in the neurophysiological processes. In addition, expression in the thyroid and pancreas region suggests a function as a paracrine and endocrine hormone.

Design and production of a chimeric resilin-, elastin-, and collagen-like engineered polypeptide.

Protein-inspired biomaterials have gained great interest as an alternative to synthetic polymers, in particular, for their potential use as biomedical devices. The potential inspiring models are mainly proteins able to confer mechanical properties to tissues and organs, such as elasticity (elastin, resilin, spider silk) and strength (collagen, silk). The proper combination of repetitive sequences, each of them derived from different proteins, represents a useful tool for obtaining biomaterials with tailored mechanical properties and biological functions. In this report we describe the design, the production, and the preliminary characterization of a chimeric polypeptide, based on sequences derived from the highly resilient proteins resilin and elastin and from collagen-like sequences. The results show that the obtained chimeric recombinant material exhibits promising self-assembling properties. Young's modulus of the fibers was determined by AFM image analysis and lies in the range of 0.1-3 MPa in agreement with the expectations for elastin-like and resilin-like materials.

Characterization and developmental expression pattern of the relaxin receptor rxfp1 gene in zebrafish.

We report the gene characterization, the cDNA cloning and the temporal and spatial expression pattern of the relaxin receptor rxfp1 gene in the zebrafish Danio rerio. The zebrafish rxfp1 gene has the same syntenic genomic organization, and a similar exon-intron structure to the homologue human gene. Furthermore, the deduced Rxfp1 protein sequence shows a high degree of amino acid similarity when compared with the human protein and the conservation of all amino acid identity necessary for the binding with relaxin. Our results show that rxfp1 gene is active either during embryogenesis or in the adult organism, showing a wide expression pattern. Moreover, we provide the first description of rxfp1 spatial expression pattern during embryo development, showing that the transcript is already present at the early developmental stage and is distributed in all of the embryonic cells until somitogenesis. Starting at the pharyngula stage the gene expression becomes mainly restricted in the brain territories. In fact, at the larval stage, the transcript is detectable in the epiphysis, postoptic region, posterior tuberculum, hypothalamus, optic tectum, tegmentum/pons, medulla and also in the structure of a peripheral nervous system, the terminal nerve. The rxfp1 expression pattern in Danio rerio embryos is very similar to that reported in the adult mammalian brain, suggesting a pivotal role of this receptor in the neurophysiology processes already at very early developmental stages.

Evidence of genetic instability in tumors and normal nearby tissues.

BACKGROUND: Comprehensive analyses have recently been performed on many human cancer tissues, leading to the identification of a number of mutated genes but providing no information on the variety of mutations present in each of them. This information is of interest to understand the possible origin of gene mutations that cause tumors. METHODOLOGY/PRINCIPAL FINDINGS: We have analyzed the sequence heterogeneity of the transcripts of the human HPRT and G6PD single copy genes that are not considered tumor markers. Analyses have been performed on different colon cancers and on the nearby histologically normal tissues of two male patients. Several copies of each cDNA, which were produced by cloning the RT-PCR-amplified fragments of the specific mRNA, have been sequenced. Similar analyses have been performed on blood samples of two ostensibly healthy males as reference controls. The sequence heterogeneity of the HPRT and G6PD genes was also determined on DNA from tumor tissues. The employed analytical approach revealed the presence of low-frequency mutations not detectable by other procedures. The results show that genetic heterogeneity is detectable in HPRT and G6PD transcripts in both tumors and nearby healthy tissues of the two studied colon tumors. Similar frequencies of mutations are observed in patient genomic DNA, indicating that mutations have a somatic origin. HPRT transcripts show genetic heterogeneity also in healthy individuals, in agreement with previous results on human T-cells, while G6PD transcript heterogeneity is a characteristic of the patient tissues. Interestingly, data on TP53 show little, if any, heterogeneity in the same tissues. CONCLUSIONS/SIGNIFICANCE: These findings show that genetic heterogeneity is a peculiarity not only of cancer cells but also of the normal tissue where a tumor arises.

Differential expression of duplicated genes for prothymosin alpha during zebrafish development.

We show that ptma, a single copy gene found in all organisms investigated so far, is duplicated in zebrafish. The two genes, ptmaa and ptmab, are individually controlled as indicated by their different expression patterns during embryonic development. Only the ptmab transcript is observed at 4 and 8 hpf of development in all embryonic cells, whereas both genes are expressed at later stages as revealed by in situ hybridization studies. In most cases, the two genes are expressed in the same territories, but only the ptmaa transcript was found in the trigeminal ganglion and in endodermal pouches. In the eye, at 72 hpf, the ptmaa and ptmab transcripts were found in amacrine cells, whereas only the ptmab transcript appeared in horizontal cells. The existence of two prothymosin genes indicates that their function in cell proliferation and differentiation is more complex in fishes than in mammals.

A possible flip-flop genetic mechanism for reciprocal gene expression.

Innexins are a family of transmembrane proteins involved in the formation of gap junctions, specific intercellular channels, in invertebrates. Analyses of the entire innexin family during Drosophila melanogaster embryonic development shows the occurrence of complex and specific patterns of expression of the different genes. Innexins inx-2 and inx-7, in general, do not appear to exhibit extensive co-expression in different D. melanogaster cellular compartments. We propose here a new and robust mechanism, based on our analysis of the genomic organization of inx-2 and inx-7, that structurally justifies the reciprocal expression of genes.

The expression of de novo DNA methylase DNMT3b, of the methyl-CpG binding protein MBD2b and of 5-MCDG glycosylase shows two waves of induction during CaCO-2 cell differentiation.

DNA methylation plays a central role in the control of gene expression during development and cell differentiation, thus DNA methylation and demethylation processes are expected to be strictly regulated during these events. We have explored the expression levels of the genes encoding DNA methylases, methyl-CpG binding proteins and demethylases during in vitro differentiation of human carcinoma colon cells (CaCO-2) used as a model system. The results show that the global DNA methylation pattern remains constant during CaCO-2 cells differentiation indicating that required genome methylation pattern in cell differentiation was already established in the seeded cells. On the contrary, the timing of expression of several of the explored genes is tightly regulated, suggesting they are involved in the regulation of the differentiation program. In particular, the timing of expression of DNMT3b and of MBD2b and 5-MCDG shows two peaks not observed in the time courses of the expression of other genes belonging to the same families. These events, not dependent on the cell cycle synchronization, have apparently no significant impact on the overall methylation status of the genome.

Specificity of cellular expression of C. variopedatus polychaete innexin in the developing embryo: evolutionary aspects of innexins' heterogeneous gene structures.

Innexins are a family of membrane proteins involved in the formation of gap junctions in invertebrates. They have been found to participate in several aspects of cell differentiation and in embryonic patterning through the formation of specific intercellular communication channels. We present here data showing that the recently identified innexin of the marine worm Chaetopterus variopedatus is expressed only in particular cells of the early stage, demonstrating cell specificity of innexin expression also in polychaete annelids. Phylogenetic analysis of all known innexins results in a phylogenetic tree clearly distinguishing insect, nematode, and other invertebrate innexins. Comparative analysis of proteins and known related genes shows that the apparent similarity of protein composition, overall structural organization, and specificity of cellular expression, typical of innexins of all studied organisms, correspond to highly heterogeneous gene structures even for genes that are in close contiguity on the same chromosome. A possible evolutionary motive producing this situation is discussed.

Cloning and molecular characterization of the first innexin of the phylum annelida-expression of the gene during development.

A novel member of the innexin family (cv-inx) has been isolated from the annelid polychaete worm Chaetopterus variopedatus using a PCR approach on genomic DNA and sequence analysis on genomic DNA clones. The gene is present in a HindIII-HindIII segment of 2250 bp containing an uninterrupted open reading frame of 1196 bp encoding a protein of 399 amino acids. The predicted protein shows the typical structural features of innexins and consensus sites for phosphorylation. Analyses on genomic DNA demonstrate that cv-inx is a single copy gene with no introns in the coding region, exactly corresponding to the cDNA sequence. The gene expression is regulated during development as shown by Northern blots analyses of the RNA and by immunoreaction with antibodies against the protein at several embryonic stages. The finding of an innexin in the phylum Annelida, outside of the Ecdysozoa clade, and its peculiar gene structure suggest the necessity to reconsider the current hypothesis on the origin and evolution of gap junctional proteins.