Existence of tenascin-C isoforms in rat that contain the alternatively spliced AD1 domain are developmentally regulated during hippocampal development.

Tenascin-C (TN-C) is a multimodular glycoprotein of the extracellular matrix which is important for the development of the nervous system and has a range of different functions which are mediated by the different protein domains present. TN-C contains eight constitutive fibronectin type III (FNIII) domains and a region of alternatively spliced FNIII domains. In the mouse and chick, six of these domains have been described and characterized, whereas in human there are nine of them. In this report, we show that seven alternatively spliced FNIII domains exist in rat and describe the differential expression pattern of the additional domain AD1 during embryonic and postnatal rat brain development. The AD1 domain of rat is homologous to the ones described in human and chick proteins but does not exist in mouse. Its expression can be located to the developing rat hippocampus and the lining of the lateral ventricle, regions where the TN-C protein may affect the behavior of stem and progenitor cells. During hippocampal development AD1 and the other alternatively spliced domains are differentially expressed as shown by RT-PCRs, immunocytochemistry and in situ hybridizations.

The Rho guanine nucleotide exchange factors Intersectin 1L and ß-Pix control calcium-regulated exocytosis in neuroendocrine PC12 cells.

GTPases of the Rho family are molecular switches that play an important role in a wide range of membrane-trafficking processes including neurotransmission and hormone release. We have previously demonstrated that RhoA and Cdc42 regulate calcium-dependent exocytosis in chromaffin cells by controlling actin dynamics, whereas Rac1 regulates lipid organisation. These findings raised the question of the upstream mechanism activating these GTPases during exocytosis. The guanine nucleotide exchange factors (GEFs) that catalyse the exchange of GDP for GTP are crucial elements regulating Rho signalling. Using an RNA interference approach, we have recently demonstrated that the GEFs Intersectin-1L and ß-Pix, play essential roles in neuroendocrine exocytosis by controlling the activity of Cdc42 and Rac1, respectively. This review summarizes these results and discusses the functional importance of Rho GEFs in the exocytotic machinery in neuroendocrine cells.

Expression of the Idefix retrotransposon in early follicle cells in the germarium of Drosophila melanogaster is determined by its LTR sequences and a specific genomic context.

Retrotransposons are transcriptionally activated in different tissues and cell types by a variety of genomic and environmental factors. Transcription of LTR retrotransposons is controlled by cis-acting regulatory sequences in the 5' LTR. Mobilization of two LTR retroelements, Idefix and ZAM, occurs in the unstable RevI line of Drosophila melanogaster, in which their copy numbers are high, while they are low in all other stocks tested. Here we show that both a full-length and a subgenomic Idefix transcript that are necessary for its mobilization are present in the Rev1 line, but not in the other lines. Studies on transgenic strains demonstrate that the 5' LTR of Idefix contains sequences that direct the tissue-specific expression of the retroelement in testes and ovaries of adult flies. In ovaries, expression occurs in the early follicle and in other somatic cells of the germarium, and is strictly associated with the unstable genetic context conferred by the RevI line. Control of tissue-specific Idefix expression by interactions between cis-acting sequences of its LTR and trans-acting genomic factors provides an opportunity to use this retroelement as a tool for the study of the early follicle cell lineage in the germarium.

Impact of multiple insertions of two retroelements, ZAM and Idefix at an euchromatic locus.

Transposable elements represent a large fraction of eukaryotic genomes and they are thought to play an important role in chromatin structure. ZAM and Idefix are two LTR-retrotransposons from Drosophila melanogaster very similar in structure to vertebrate retroviruses. In all the strains where their distribution has been studied, ZAM appears to be present exclusively in the intercalary heterochromatin while Idefix copies are mainly found in the centromeric heterochromatin with very few copies in euchromatin. Their distribution varies in a specific strain called RevI in which the mobilization of ZAM and Idefix is highly induced. In this strain, 15 copies of ZAM and 30 copies of Idefix are found on the chromosomal arms in addition to their usual distribution. Amongst the loci where new copies are detected, a hotspot for their insertion has been detected at the white locus where up to four elements occurred within a 3-kb fragment at the 5' end of this gene. This property of ZAM and Idefix to accumulate at a defined site provides an interesting paradigm to bring insight into the effect exerted by multiple insertions of transposable elements at an euchromatic locus.

Mobilization of two retroelements, ZAM and Idefix, in a novel unstable line of Drosophila melanogaster.

We describe a novel transposition system in a line of Drosophila melanogaster called RevI in which two retroelements are mobilized. These elements are the retroelement ZAM, recently described in the literature, and a novel element designated Idefix. Like ZAM, Idefix displays the structural features of a vertebrate retrovirus. Its three open reading frames encode predicted products resembling the products of the gag, pol, and env genes of retroviruses. In situ hybridization and Southern analyses performed on the RevI genome revealed the presence of some 20 copies of ZAM and Idefix, whereas ZAM is absent and Idefix is present in only four copies on the chromosomal arms of the original parental line. From RevI, a series of mutations affecting eye coloration has been recovered. The genetic and molecular analyses of these mutations have shown that most of them affected the white locus through three rounds of mutational events. The first mutational event was previously shown to be caused by a ZAM insertion 3 kb upstream of the transcription start site of white. It confers a red-brick phenotype to the orange eye coloration of the parental line. The second event results from the insertion of an Idefix copy 1.7 kb upstream of the transcription start site of the white gene, which modifies the red-brick phenotype to orange. This second mutational event was recovered as a recurrent specific mutation in 11 independent individuals. The third event results from an additional Idefix located 1.7 kb upstream of white that is responsible for the full reversion of the orange phenotype to red-brick. The fact that such mutations due to recurrent appearances of both ZAM and Idefix at the white locus result in such a variety of phenotypes brings to light a new molecular system in which the interference of mobile elements with the correct expression of the host gene can be tested.

GEBF-I activates the Drosophila Sgs3 gene enhancer by altering a positioned nucleosomal core particle.

The enhancer region of the Drosophila melanogaster ecdysone-regulated glue gene, Sgs3, shows dramatic modifications of chromatin structure in strict correlation with changes in gene expression during development. We show that there is a positioned nucleosomal core particle over the enhancer which is displaced or disrupted during gene activation. This transition is prevented in Drosophila larvae mutated in the ecdysone-dependent 2B5 locus, in which Sgs3 is inactive and GEBF-I, a Glue Enhancer Binding Factor, is missing. We have defined the GEBF-I binding sites in vitro and shown that mutation of these sequences abolishes the enhancer activity in vivo. This combined in vitro and in vivo approach reveals new aspects of the dynamic organization of a regulatory element during development and highlights the potential of this model for studies of the relation between chromatin structure and gene activity.