Differential expression of synapsin genes during early zebrafish development.

Synapsins are a family of synaptic vesicle (SV)-associated phosphoproteins that have been identified in several vertebrates and invertebrates. We report here the cloning and expression of synapsin family genes in the zebrafish Danio rerio. We identified the complete coding sequence of synapsin 3, which is not present in the currently available genome, and characterized and annotated the synapsin gene family in the zebrafish D. rerio. By means of whole-mount in situ hybridization, we showed the spatiotemporal expression of synapsin genes at three different time points during early embryonic development: 20-24 h postfertilization (hpf), 30-33 hpf, and 3 days postfertilization (dpf). As very few data are available describing the expression of synapsin family genes during CNS development in vertebrate models, our results may help to achieve a better understanding of the complex functions of these molecules. Finally, new interesting evidence from our temporal gene expression studies suggests that synapsins have also maternal functions.

Efficiency of two different transfection reagents for use with human NTERA2 cells.

The teratocarcinoma cell line NTERA2 is recently used in a wide range of researches (from developmental biology to toxicology, for their ability to be induced to neural differentiation. In order to study the genetic potential of these cells, it is needed to use methods for gene silencing and/or mRNA interference, allowing cell viability and further differentiation. To check these features, we simultaneously tested the transfection efficiency of NTERA2, A549 and HeLa cells with Metafectene PRO (Biontex, Germany) and another optimal transfection reagent currently used in our Laboratory, using as a reporter gene the DsRed2 vector (Clontech, Mountain View, CA). Under our culture conditions for NTERA2 and HeLa cells, Metafectene PRO transfection method was found to possess high throughput performance, that allows low concentration rate and low exposure time to excitation light source, thus reducing both toxicity and phototoxicity.

Hormonal regulation of vasa-like messenger RNA expression in the ovary of the marine teleost Sparus aurata.

The vasa gene is an important maternal regulator of primordial germ cell (PGC) development in both vertebrate and invertebrate models. It is also expressed in the mature gonads, but its role in these tissues is still unclear. In oviparous species, oogenesis is a complex process under hormonal control: estrogens, gonadotropins, and other hormones operate at different stages of oogenesis, regulating meiosis, vitellogenesis, follicle maturation, and egg release. The aim of this work is the determination of a regulative role of hormones controlling oocyte maturation on vasa mRNA expression in the sea bream ovary through a molecular biology approach. By in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR), reaction (the vasa mRNA in the sea bream ovary was found to be expressed at higher levels in the advanced stages of oocyte maturation. After in vivo hormonal treatment, the effect on ovarian vasa mRNA expression was studied through semiquantitative RT-PCR. The quantification of vasa-like mRNA expression in sea bream ovary demonstrates that estradiol (E2), growth hormone (GH), and the combination of gonadotropin-releasing hormone (GnRH) with GH are able to induce an increase in vasa mRNA expression. In contrast, the treatments with GnRH alone or E2 plus GH significantly decreased vasa mRNA expression. These data suggest a regulative interplay between the vasa gene expression and the endocrine system that controls the oogenesis in the ovary of the sea bream.

Serotonin localization in Phallusia mammillata larvae and effects of 5-HT antagonists during larval development.

The neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) plays an important role in a wide range of non-neural processes. Using immunofluorescence with an antiserotonin antibody, 5-HT was localized in the brain and in some neurons of the larval tail of Phallusia mammillata. To test the effect of 5-HT on development, we treated embryos with two different 5-HT receptor subtype antagonists. Treatment at the gastrula stage with 10 microM ondansetron, an antagonist of the 5-HT(3) receptor, induced anterior truncation and a short tail. At 10 microM, ritanserin, a 5-HT(2B) receptor antagonist, induced larval phenotypes characterized by a roundish trunk region with flat papillae. The juveniles developed from these larvae had an abnormal cardiocirculatory system: their heart contractions were ineffective and their blood cells accumulated in the heart cavity. We conclude that an appropriate level of 5-HT is necessary for correct development and morphogenesis. Moreover, a different key role for multiple receptors in modulating the morphogenetic effects of 5-HT is suggested.

Immunocytochemical localization of serotonin in embryos, larvae and adults of the lancelet, Branchiostoma floridae.

Serotonin (5-hydroxytryptamine) is a biogenic amine distributed throughout the metazoans and has an old evolutionary history. It is involved as a developmental signal in the early morphogenesis of both invertebrates and vertebrates, whereas in adults it acts mainly as a neurotransmitter and gastrointestinal hormone. In vertebrates, serotonin regulates the morphogenesis of the central nervous system and the specification of serotonergic as well as dopaminergic neurons. The present study uses, as an experimental model, an invertebrate chordate, the lancelet Branchiostomafloridae, characterized by its remarkable homologies with vertebrates that allows the 'bauplan' of the probable ancestor of vertebrates to be outlined. In particular, the involvement of serotonin as a developmental signal in embryos and larvae, as well as a neurotransmitter and gastrointestinal hormone in adult specimens of Branchiostoma floridae, gives further support to a common origin of cephalocordates and vertebrates.

Immunoreactive endozepine-like peptides in the brain and pituitary of the Atlantic hagfish, Myxine glutinosa.

Endozepines are a family of peptides capable of displacing benzodiazepines from their specific binding sites, to which belong the diazepam-binding inhibitor and the octadecaneuropeptide (ODN). This paper reports the distribution of ODN-related peptides, investigated for the first time by immunocytochemistry, in different brain and pituitary regions of the Atlantic hagfish, Myxine glutinosa. Immunoreactive ODN-like material was found in the telencephalon at the level of bundles of different olfactory nerve fibres. Moreover, at the level of the pallium, immunoreactive multipolar neurons were observed in the pars parvocellularis of the stratum griseum superficialis. Similar immunopositive nerve cell bodies were found in the nucleus medialis of the central prosencephalic complex. In the mesencephalon, few immunoreactive neurons lining and contacting the mesencephalic ventricle were detected; such nerve cells could be involved in the regulation of cerebrospinal fluid homeostasis. Dorsally in the mesencephalon, numerous ODN-containing cell bodies were present in the area praetectalis. The rhomboencephalon was immunostained only in the octavolateral area and in the nucleus motorius magnocellularis of the trigeminal nerve. Furthermore, ODN immunoreactivity was also present in the nerve cells of ganglia of the ophthalmic division of the trigeminal nerve complex. The immunocytochemical patterns described here in the brain of M. glutinosa suggest an involvement of ODN-like peptides as neuromodulators in sensory pathways, such as olfactory and visual. Finally, ODN-like substances were localized in discrete populations of adenohypophysial cells and in tanycytes lining the neurohypophyseal walls, suggesting for endozepines a paracrine and/or endocrine control of pituitary hormones release and a neurohormone role respectively. These results could give new insights into the chemioarchitecture of the brain of myxinoids.

Distribution and localization of immunoreactive FMRFamide-like peptides in the lancelet.

Immunofluorescence was used to study the distribution of FMRFamide (Phe-Met-Arg-Phe-NH2) in premetamorphic larvae and adults of the lancelet, Branchiostoma lanceolatum. In the larvae, FMR-Famide-containing presumably neuronal perikarya and fibers were limited to the anterior third of the dorsal nerve cord. Throughout this region, most of the immunoreactive perikarya and fibers were located ventrolaterally and ventrally within the nerve cord; in addition, in the caudal part of the cerebral vesicle, some of the immunofluorescent cells projected cytoplasmic extensions across the slot-like neural canal. In adult lancelets, immunofluorescence was detected in cells of the Hatschek's pit (a probable homologue of the anterior hypophysis of vertebrates); however, no immunofluorescence was detected in the larval preoral pit, which is the ontogenetic precursor of Hatschek's pit. Moreover, the FMR-Famide-containing elements do not show immunoreactivity to other peptides of the FaRPs family such as pancreatic polypeptide (PP). The results suggest that FMRF-amide may be involved in neuroendocrine functions of lancelets.

Proopiomelanocortin (POMC) mRNA and POMC-derived peptides immunolocalization in the skin of Protopterus annectens, an African lungfish.

Antisera against adrenocorticotropic hormone (ACTH), alpha-melanocyte stimulating hormone (alpha-MSH) and beta-endorphin were used to localize, by immunohistochemistry, proopiomelanocortin (POMC)-derived peptides in the skin excised from different regions of the African lungfish Protopterus annectens. Immunoreactivity was observed in the epidermis mainly in the germinal layer. Using human POMC cDNA as hybridization probe, POMC-like mRNA was identified in situ in epidermal cells. The demonstration in the same cells of POMC mRNA and POMC-related peptides immunoreactivity indicates a local production of opiate hormones.

The tissue-specific transcription factor Pit-1 is expressed in the spinal cord of the lancelet, Branchiostoma lanceolatum.

The spinal cord of the lancelet Branchiostoma lanceolatum was studied by using a monoclonal antibody to the rat tissue-specific transcription factor, Pit-1. Our previous studies have demonstrated Pit-1 immunoreactivity in different nervous and endocrine structures of the head region of adults and in the rostral central nervous system (CNS) of larval lancelet. Our present results show the presence of Pit-1-like protein in dorso-lateral nerve cells and ependymocytes of the adult spinal cord. Using double immunofluorescence techniques, we have revealed the coexistence of the glial fibrillary acidic protein (GFAP) with Pit-1 in groups of laterally located ependymocytes. The occurrence of GFAP, a specific marker of mammalian astrocytes and radial glia, in some lancelet ependymocytes confirms that glial elements are also present in protochordates. Furthermore, other ependymocytes, located in the roof of the central canal and containing Pit-1-like protein exclusively, could be considered as ependymal tanycytes.

Expression of pro-opiomelanocortin (POMC) in the cerebral ganglion and ovary of a protochordate.

The distribution of neurones expressing POMC mRNA in the cerebral ganglion of the protochordate ascidian, Styela plicata, was investigated using a non-radioactive in situ hybridization technique. Nerve cell bodies of mono and bipolar types expressing POMC mRNA, were observed mainly in the outer layer of the ganglion. Discrete groups of neurones containing POMC mRNA were also localized in the inner portion of the ganglion, and few small monopolar perykaria expressing POMC mRNA were visible at the emergence of the main nerve trunks. POMC mRNA labeling was also found at level of the cytoplasm of previtellogenic and vitellogenic oocytes, and of follicular cells. Our results demonstrate the expression of one or more genes in the cerebral ganglion and ovary, that may be similar to one or more regions of the mammalian POMC gene. Therefore POMC-related molecules seem to be involved in neuromodulatory pathways and regulatory mechanisms of the oogenesis of ascidians.

Evidence for the presence of the tissue-specific transcription factor Pit-1 in lancelet larvae.

Recent molecular studies have noted the affinity among cephalochordates and vertebrates. In particular, a cluster of vertebrate-like homeobox genes regulates the development of the lancelet Branchiostoma lanceolatum. A previous study has outlined the expression pattern of the pituitary-specific transcription factor Pit-1 in adult lancelets. Pit-1 belongs to the POU family of transcription factors, which, like homeotic proteins, are members of the helix-turn-helix superfamily of proteins. POU is an acronym for Pit-1, Oct-1 and Oct-2, and Unc-86. In the present work, we investigated the head region of premetamorphic larvae of B. lanceolatum, by means of scanning electron microscopy, wholemount and tissue sections immunocytochemistry, and Western blotting assay, to verify the presence and distribution of Pit-1. Immunoreactive Pit-1 protein was detected in the rostral nerves and in a cluster of photoreceptor cells of the frontal eye. At the same time, an electrophoretic band of 33 kDa was shown from extracts of premetamorphic larvae and recognized by a monoclonal antibody to rat Pit-1. On the basis of the immunocytochemical and electrophoretic results, we can assume that Pit-1 may play a neuromodulatory role in the larval central nervous system. Moreover, the spatial and temporal distribution of Pit-1 protein in larva and adult lancelets agrees only in part with that described in embryonic and adult mice, suggesting different molecular controls of regional identity in the nervous system of cephalochordates and vertebrates.

Expression of the tissue-specific transcription factor Pit-1 in the lancelet, Branchiostoma lanceolatum.

Lancelets, known also as amphioxus, are protochordates that share common archetypal features with vertebrates. Recently, several developmental and molecular biology studies have pointed out homologies between anatomical structures of lancelets and vertebrates. We have studied the head region of the lancelet, Branchiostoma lanceolatum, by means of scanning electron microscopy, immunocytochemistry, and Western blotting techniques, to localize the pituitary-specific transcription factor, Pit-1. Immunoreactive Pit-1 protein has been found in cells of two typical structures of the lancelets, the Kölliker's and Hatschek's pits. Moreover, the frontal eye complex, neurons, and the rostral nerves show Pit-1 immunoreactivity. A band of 33 kilodaltons has been resolved in lancelet extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and after Western blotting, the bands have been probed by a monoclonal antibody to rat Pit-1. Our results demonstrate that Pit-1 is expressed in both neurones and receptosecretory epithelial cells of adult lancelets, and that the cells lining the two pits display ultrastructural and immunocytochemical features typical of chemoreceptosecretory/olfactory- and adenohypophyseal-like structures.

Localization of interleukin-1 beta mRNA in the cerebral ganglion of the protochordate, Styela plicata.

There is evidence that interleukin 1 beta (IL-1 beta) plays an important role in several biological functions in mammals where it is synthesized by cells of haematological, dermal and neural origin. Moreover, production of cytokine-like molecules has been demonstrated in some blood cells of non-mammalian vertebrates and invertebrates in which also nerve cells are demonstrated to be IL-1 beta immunoreactive. The purpose of the present study is to demonstrate the IL-1 beta mRNA expression in nerve cells of the ascidian Styela plicata by use of non-isotopic in situ hybridization technique. The expression of IL-1 beta messenger was demonstrated in monopolar neurons in the cortical layer of the cerebral ganglion. The neuronal expression of cytokine-like molecules in tunicates suggests that IL-1 beta is an ancestral and functionally conserved molecule, and that a neuroimmune axis appeared early during the metazoan phylogeny.

Pro-opiomelanocortin (POMC) expression and immunolocalization of POMC-related peptides in the ovary of Protopterus annectens, an African lungfish.

Antisera against adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha MSH) and beta-endorphin were used to localize pro-opiomelanocortin (POMC)-derived peptides in the ovary of the African lungfish Protopterus annectens by immunohistochemistry. Immunoreactivity was observed in the granulosa and the internal theca of the vitellogenic follicles. No immunoreactivity was observed in immature follicles. Using human POMC cDNA as the hybridization probe POMC-like mRNA was identified in situ in cells of the granulosa and internal theca of the vitellogenic follicles. No labeling was observed in primordial follicles. The demonstration in the same cells of POMC mRNA and POMC-related peptides immunoreactivity indicates a local production of the opiate hormones.

Acetyl salmon endorphin-like immunoreactivity in the ovary of two teleostean species: changes with environmental conditions.

The presence of salmon acetylated endorphin (acetyl sEP) in the ovary of seabream and sea bass was investigated through immunocytochemical and biochemical techniques in order to compare aquatic species with terrestrial ones. Endorphin-like immunoreactivity was found in the cytoplasm of oogonia and similar immunostaining was present in the granulosa layer of mature follicles. In both pituitary and ovarian extracts of the two teleostean species, acetyl sEP-like immunoreactivity was distributed over three main peaks, the second one corresponding to the elution time of the reference synthetic peptide. Serial dilutions of HPLC fraction II of the ovaries of both fishes ran parallel with the standard curve obtained with reference peptide. The ovarian content of acetyl sEP, obtained by calculating the integrated area of the fraction II peak, indicates large and highly significant (p < 0.01) differences in the amount of peptide found in ovarian tissues of wild seabream in comparison with that of farmed fish. Increased peptide values in wild animals with respect to farmed fish were also found in the sea bass. These data indicate that not only the pituitary, but also the ovary is sensitive to environmental cues, and strongly suggest the role of opioid peptides in adaptation.

Ectocellular in vitro and in vivo metabolism of cADP-ribose in cerebellum.

CD38, a type II transmembrane glycoprotein predominantly expressed in blood cells, is a bifunctional ectoenzyme directly involved in the metabolism of cADP-ribose (cADPR). This is a potent Ca2+ mobilizer in several types of cells. The relationship between the ectocellular site of cADPR production and its intracellular calcium-related functions is poorly understood. Cultured rat cerebellar granule cells showed both enzymic activities of CD38, ADP-ribosyl cyclase and cADPR hydrolase, at a ratio of 16 to 1 respectively, and were immunostained by the anti-(human CD38) monoclonal antibody IB4. In these cells externally added cADPR and beta-NAD+ (the precursor of cADPR), but not alpha-NAD+ or ADP-ribose, enhanced the peak of the depolarization-induced rise in intracellular Ca2+ concentration. This effect was inhibited by 1 microM ryanodine, suggesting a potentiation of calcium-induced calcium release by cADPR. CD38 ectoenzyme activities, ADP-ribosyl cyclase and cADPR hydrolase, were also demonstrated in vivo by microdialysis of adult rat cerebellum, where IB4 bound to granule neurons selectively. Trace amounts (11.5 +/- 3.8 nM) of NAD+ were detected by microdialysis sampling and sensitive assays in the basal interstitial fluid of the cerebellum. These results provide a link between ectocellular cADPR turnover and intracellular calcium mobilization in cerebellum.

Immunoreactive Phe-Met-Arg-Phe-NH2-like peptides in the brain of the antarctic icefish, Chionodraco hamatus.

The distribution of Phe-Met-Arg-Phe-NH2 (FMRFamide)-like immunoreactive peptides was investigated in the brain of the antarctic icefish, Chionodraco hamatus, using the indirect immunofluorescence technique. Three main groups of immunoreactive perikarya were respectively localized in the nucleus entopeduncularis of the telencephalon, the nucleus preopticus periventricularis of the hypothalamus, and within the nucleus oculomotorius of the mesencephalon. Delicate FMRFamide positive nerve fibers were distributed in several brain regions of the forebrain and brainstem. In particular, these fibers densely innervated the caudal part of the dorsomedial pallium, the hypothalamus, the thalamus, the mesencephalic tegmentum and the optic tectum. The distribution pattern of the FMRFamide-like immunoreactivity was compared with that reported in previous studies in other teleost species. The anatomical organization of the FMRFamide-like immunoreactive peptidergic system in the brain of Chionodraco hamatus suggests that a FMRFamide-like peptide may play a role as a neuromodulator in fish adapted to the extreme Antarctic environment.

Immunocytochemical localization and biochemical characterization of melanotropin-like peptides in the gonads of a protochordate.

The compound gonads of the protochordate ascidian Styela plicata were investigated by immunocytochemistry, HPLC, and radioimmunoassay to verify the presence of melanotropin-like peptides, alpha-MSH-like immunoreactivity is localized in the follicular cells and in the perinuclear cytoplasm of different types of ovaric follicles, as well as in the spermatogonia and spermatocytes of testicular lobules. The ascidian immunoreactive peptides occurring in the gonads consist of alpha-MSH and ACTH(1-13)-NH2 and their amounts are higher in summer than in winter.

Opioid peptides and testicular activity in the lizard Podarcis s. sicula Raf.

In mammals endorphinergic systems have been shown to modulate reproductive processes and beta-endorphin (beta-EP) has been found to influence sexual functions, acting at the hypothalamus-pituitary-gonadal axis level. Using immunocytochemical and in vitro studies, evidence for a diffuse pro-opiomelanocortin-related opioid system in the lizard Podarcis s. sicula was produced. In the testis, beta-EP immunoreactivity showed seasonal variation, being most pronounced in the interstitial cells of sexually quiescent lizards (December). Reverse-phase high-performance liquid chromatography, coupled with radioimmunoassay and immunocytochemistry, showed that beta-EP and acetyl beta-EP increased during December, while their concentrations were low during April, when the highest testicular activity occurred. Using in vivo studies, it was found that naltrexone treatment, blocking pituitary opioid receptor, increased androgen levels in the plasma and in the testis. It was also found with in vitro studies that the endogenous opioid system inhibits gonadotrophin release and therefore androgen production by the testis. The data reported here provide evidence for the physiological role played by opioid peptides at the pituitary level to regulate the seasonal reproductive activity of the lizard Podarcis s. sicula.

Presence and steroidogenetic activity of beta-endorphin in the ovary of the lizard, Podarcis s. sicula raf.

In mammals, proopiomelanocortin (POMC)-related peptides are involved in reproductive processes at both the hypothalamopituitary and ovarian levels. Through immunocytochemical and physiological in vitro studies, evidence for a diffuse POMC-related opioid system in the lizard Podarcis s. sicula is provided. In the lizard ovary, beta-endorphin (beta-EP)-like immunoreactive cells were observed within the granulosa layer; the immunoresponse showed seasonal variation, being most pronounced in the winter ovary. HPLC followed by immunoassay showed that acetyl beta-EP is the main form of POMC-related peptide in both pituitary and ovary. In vitro studies showed that picomolar amounts of beta-EP stimulate follicular estrogen production during both the reproductive and winter phases; induction was found to be higher in the reproductive phase. The data reported here provide evidence for the physiological role played by beta-EP in the reproductive function of Podarcis s. sicula via induction of ovarian production of estradiol-17 beta, which is the main factor responsible for the vitellogenic process.