Differential tissue expression of a calpastatin isoform in Xenopus embryos.

This study is aimed at demonstrating the role played by a calpastatin isoform (Xcalp3) in Xenopus embryos. A specific monoclonal antibody (mAb) was raised against a glutathione S-transferase (GST)-Xcalp3 fusion protein and characterized by immunoblotting and confocal fluorescence microscopy on stage 20-36 embryos. Under these conditions, calpastatin reactivity is associated with a major 110kDa protein fraction and preferentially expressed by notochord and somitic cells. In notochord cells, anti-calpastatin reactive sites were initially restricted to the luminal space of the vacuoles and later became diffused throughout the cytoplasm. In contrast, anti-calpastatin reactive sites in somitic cells were initially diffused throughout the cytoplasm and became restricted to a few intracellular granules in the later developmental stages. At the ultrastructural level, notochord cells appeared as flattened discs containing several vacuoles and numerous electron-dense granules. During transition from stages 26 to 32, electron-dense granules were gradually reduced in number as vacuoles enlarged in size and losed their calpastatin reactivity. Electron-dense granules were also present in myoblast cells and their number gradually reduced during development. To determine whether these observations bear any causal relationship to the calpain/calpastatin system, a number of Xenopus embryos were examined both ultrastructurally and histochemically following exposure to a specific calpain inhibitor (CI3). Under these conditions, Xenopus embryos exhibited an altered right-left symmetry and an abnormal axial shortening. In CI3-treated stage 32 embryos, notochord cells had a reduced vacuolar extension and exhibited at the same time an increase in granular content. The overall morphology of the somites was also distorted and myoblasts were altered both in shape and granular content. Based on these findings, it is concluded that the calpain/calpastatin may play an important role in the control of notochord elongation and somite differentiation during Xenopus embryogenesis.

The serotonin 5-HT2B receptor from the puffer fish Tetraodon fluviatilis: cDNA cloning, genomic organization and alternatively spliced variants.

We cloned the 5-HT2B serotonin receptor from the puffer fish Tetraodon fluviatilis. Two cDNAs differing in length because of the use of alternative polyadenylation sites were isolated. We partly characterized the genomic organization of the 5-HT2B gene and we found two introns conserved in position between the puffer fish and mammals. In addition, four splice variants which would generate truncated forms of the receptor were detected.

Identification of different forms of calpastatin mRNA co-expressed in the notochord of Xenopus laevis embryos.

We isolated three Xenopus cDNA clones, Xcalp1, Xcalp2 and Xcalp3, which encode different forms of calpastatin mRNA. Compared to the canonical form of mammalian calpastatin, the predicted Xcalp3 protein contained a very long N-terminal domain L and an additional inhibitory domain. The other two deduced calpastatin proteins were truncated forms, both lacking domain L and containing four (Xcalp2) and two (Xcalp1) inhibitory domains, respectively. The presence of Xcalp1, Xcalp2 and Xcalp3 transcripts was detected by in situ hybridization in the notochord from the embryonic stage 20 to stage 36, afterwards the expression was only present in the growing tailbud. As shown by RT-PCR, the three calpastatin mRNAs were also expressed in the adult brain.

Characterization of a cloned xenopus laevis serotonin 5-HT1A receptor expressed in the NIH-3T3 cell line.

In a previous work we isolated a Xenopus 5-HT1A receptor gene and now report the characterization of this receptor. The HindIII-XbaI fragment of this gene was cloned into the pcDNA I NEO vector and stably transfected into eukaryotic cells (NIH-3T3). To determine the specific 5-HT1A receptor binding, [3H]8-OH-DPAT was used as radioligand. The selective 5-HT1A receptor agonist bound only a single class of saturable high-affinity binding sites with pharmacological characteristics similar to those of the mammalian 5-HT1A receptor. The effects of X5-HT1A receptor activation on cell growth were also investigated in stably transfected NIH-3T3 cells. The 5-HT1A agonist 8-OH-DPAT was found to increase DNA synthesis and accelerated cell growth.

Cloning and developmental expression of 5-HT1A receptor gene in Xenopus laevis.

The aim of our work is to investigate the potential involvement of serotonin and its G-protein-coupled receptors in neural differentiation or other developmental processes in Xenopus laevis. By using a RT-PCR strategy, we isolated a cDNA fragment from X. laevis brain showing high amino-acid similarity with the mammalian 5-HT1A receptor. We used this fragment to isolate a cDNA clone containing a single ORF of 408 amino-acids with an overall amino-acid identity of 73% with the human and rat 5-HT1A receptor. This structural similarity suggests that this clone encodes the Xenopus homolog of the mammalian 5-HT1A receptor (X5-HT1A). In order to establish a possible role for this receptor in development, we analyzed the pattern of its gene expression during embryogenesis, larval stages and in adult brain by in situ hybridization. The first signal of mRNA expression appears in the rostral part of brain stem at stage 22, when the first neurons start differentiation [38,21]. In later stages of development, the cells expressing X5-HT1A transcripts appear to correspond to serotonergic neurons. By stage 41, X5-HT1A mRNA is also detected in the inner nuclear layer (INL) of the developing retina. This pattern of expression is maintained until stage 46, i.e. at the beginning of metamorphosis. In adult, additional brain areas express X5-HT1A mRNA, particularly in telencephalon, diencephalon and mesencephalon. On the whole, our data show that the X5-HT1A receptor mRNA is developmentally regulated, with expression first appearing in differentiating serotonergic neurons, where this receptor may mediate, through an autocrine regulatory pathway, the trophic action of serotonin on developing serotonergic system.

Gypsy/Ty3-like elements in the genome of the terrestrial Salamander hydromantes (Amphibia, Urodela).

We have studied a family of long repetitive DNA sequences (Hsr1) interspersed in the large genome of the European plethodontid salamander Hydromantes. The sequence analysis of a 5-kb fragment (Hsr1A) of one member has revealed significant similarities with amino acidic domains of retroviruses and retrotransposons. The similarity of the reverse transcriptase domain and the gene organization identifies Hsr1A as a member of the gypsy/Ty3 class of retrotransposons. We hypothesize that Hsr1 sequences are vestiges of an invasion of the Hydromantes genome that occurred early in the evolutionary history of these European plethodontids. About 10(6) Hsr1 sequences are present in the large Hydromantes genome. This is the highest number of copies so far discovered for retrotransposon-like elements in eukaryote organisms.

A tandemly repeated DNA family originated from SINE-related elements in the European plethodontid salamanders (Amphibia, Urodela).

We have characterized a highly repetitive family, named Hy/Pol III, in the genome of the European salamanders Hydromantes (Plethodontidae). This family consists of short, tandemly repeated sequences organized in clusters, scattered through the genome as shown both by in situ hybridization to chromosomes and by Southern blot hybridization. The repeat unit is about 200 bp in length and it is a composite element since it contains a SINE-like retroposon with a tRNA structure, flanked by two short direct repeats. The whole element itself is bordered by two other direct repeats. The sequence data suggest that two elements, presumably derived from polymerase III transcripts, have been inserted one into the other, giving rise to the observed composite structure. During evolution the Hy/Pol III family was then amplified by tandem duplication at the DNA level. The inferred relationships between Hy/Pol III members from three representative species of the European Hydromantes suggests that a subfamily structure characterizes the evolutionary history of this family.

Localization and gene expression of serotonin 1A (5HT1A) receptors in human brain postmortem.

We investigated the binding parameters, i.e. the maximum binding capacity (Bmax) and the dissociation constant (Kd), of [3H]8-hydroxy-2-(di-N-propylamino)tetralin ([3H]8-OH-DPAT) labeling the serotonin receptor of the 1A type (5HT1A), and the distribution of the mRNA encoding it in some human brain areas obtained from autoptic samples. The results showed that the Bmax was significantly higher in the hippocampus than in the prefrontal cortex and the striatum, while the Kd had the inverse, although not significant, pattern. The expression study revealed that 5HT1A mRNA distribution in the hippocampus and prefrontal cortex was consistent with the data of the [3H]8-OH-DPAT binding. A different result was obtained in the striatum where no 5HT1A mRNA expression was detected, despite the measurement of specific [3H]8-OH-DPAT binding. These findings underline the different nature of [3H]8-OH-DPAT binding sites in different brain areas and the need for further studies on 5HT receptor gene expression in human brain.