tBid mediated activation of the mitochondrial death pathway leads to genetic ablation of the lens in Xenopus laevis.

Xenopus is a well proven model for a wide variety of developmental studies, including cell lineage. Cell lineage in Xenopus has largely been addressed by injection of tracer molecules or by micro-dissection elimination of blastomeres. Here we describe a genetic method for cell ablation based on the use of tBid, a direct activator of the mitochondrial apoptotic pathway. In mammalian cells, cross-talk between the main apoptotic pathways (the mitochondrial and the death domain protein pathways) involve the pro-death protein BID, the active form of which, tBID, results from protease truncation and translocation to mitochondria. In transgenic Xenopus, restricting tBID expression to the lens-forming cells enables the specific ablation of the lens without affecting the development of other eye structures. Thus, overexpression of tBid can be used in vivo as a tool to eliminate a defined cell population by apoptosis in a developing organism and to evaluate the degree of autonomy or the inductive effects of a specific tissue during embryonic development.

Generation of trangenic Xenopus laevis using the Sleeping Beauty transposon system.

Using the Sleeping Beauty (SB) transposon system, we have developed a simple method for the generation of Xenopus laevis transgenic lines. The transgenesis protocol is based on the co-injection of the SB transposase mRNA and a GFP-reporter transposon into one-cell stage embryos. Transposase-dependent reporter gene expression was observed in cell clones and in hemi-transgenic animals. We determined an optimal ratio of transposase mRNA versus transposon-carrying plasmid DNA that enhanced the proportion of hemi-transgenic tadpoles. The transgene is integrated into the genome and may be transmitted to the F1 offspring depending on the germline mosaicism. Although the transposase is necessary for efficient generation of transgenic Xenopus, the integration of the transgene occurred by an non-canonical transposition process. This was observed for two transgenic lines analysed. The transposon-based technique leads to a high transgenesis rate and is simple to handle. For these reasons, it could present an attractive alternative to the classical Restriction Enzyme Mediated Integration (REMI) procedure.

Developmental cell death during Xenopus metamorphosis involves BID cleavage and caspase 2 and 8 activation.

Elimination of tadpole organs during Xenopus metamorphosis is largely achieved through apoptosis, and recent evidence suggest involvement of the mitochondrial death route and bax-initiated caspase-3 and -9 deployment. However, events upstream of the activation of Bax are unknown. In other models, proteins of the BH3-only group such as BID are known to assure this function. We show that Xenopus bid transcript levels increase at metamorphosis in larval cells destined to disappear. This increase correlates with an abrupt rise in Caspase-2 and -8 mRNA levels and an enhanced activity of Caspase-2 and -8. In BIDGFP transgenic animal's tail regression is accelerated. The cleavage of BIDGFP fusion protein during natural or T(3)-induced metamorphosis was specifically inhibited by caspase-8 inhibitors. Our results show that tail regression at metamorphosis implicates an apoptotic pathway inducible by T(3) hormone in an organ autonomous manner and involving the cell death executioners BID and Caspases-2 and -8.

Xenopus Bcl-X(L) selectively protects Rohon-Beard neurons from metamorphic degeneration.

Amphibian metamorphosis involves extensive, but selective, neuronal death and turnover, thus sharing many features with mammalian postnatal development. The antiapoptotic protein Bcl-X(L) plays an important role in postnatal mammalian neuronal survival. It is therefore of interest that accumulation of the mRNA encoding the Xenopus Bcl-X(L) homologue, termed xR11, increases abruptly in the nervous system, but not in other tissues, during metamorphosis in Xenopus tadpoles. This observation raises the intriguing possibility that xR11 selectively regulates neuronal survival during postembryonic development. To investigate this hypothesis, we overexpressed xR11 in vivo as a green fluorescent protein (GFP)-xR11 fusion protein by using somatic and germinal transgenesis. Somatic gene transfer showed that the fusion protein was effective in counteracting, in a dose-dependent manner, the proapoptotic effects of coexpressed Bax. When GFP-xR11 was expressed from the neuronal beta-tubulin promoter by germinal transgenesis we observed neuronal specific expression that was maintained throughout metamorphosis and beyond, into juvenile and adult stages. Confocal microscopy showed GFP-xR11 to be exclusively localized in the mitochondria. Our findings show that GFP-xR11 significantly prolonged Rohon-Beard neuron survival up to the climax of metamorphosis, even in the regressing tadpole tail, whereas in controls these neurons disappeared in early metamorphosis. However, GFP-xR11 expression did not modify the fate of spinal cord motoneurons. The selective protection of Rohon-Beard neurons reveals cell-specific apoptotic pathways and offers approaches to further analyze programmed neuronal turnover during postembryonic development.

An autoradiographical study of binding sites for vasopressin located on corticotrophs in rat and sheep pituitary glands.

Arginine vasopressin (AVP) carried by hypophysial portal blood acts in the pituitary gland, in synergy with corticotrophin-releasing factor (CRF), to induce ACTH secretion. The relative importance of AVP and CRF in this secretory response depends upon the nature and intensity of stressful stimuli, and perhaps also on the species. The aim of the present work was to study, in isolated rat and sheep pituitary glands, the topography of binding sites for AVP and to establish whether they are indeed associated with corticotrophs. To this end, we performed contact autoradiography on tritium-sensitive film using 1.5-2.0 nmol [3H]AVP/l as ligand. ACTH immunoreactivity was detected on sections immediately adjacent to those used for autoradiography. In both species, specific binding sites for AVP were only present in the anterior lobe; the intermediate lobe was not labelled and the neural lobe showed non-specific labelling. Autoradiograms from experiments using [3H]AVP in competition with different synthetic structural analogues showed that, in rat and sheep anterior pituitary glands, receptors differ from the V1 and V2 subtypes. Specific [3H]AVP-binding sites formed an irregular, patchy pattern throughout the anterior lobe. In both species, this pattern was strikingly similar to that formed by cell clusters showing ACTH immunoreactivity, indicating that the AVP-binding sites are associated with the corticotrophs. Immunoreactive cells in the intermediate lobe had no [3H]AVP-binding sites. [3H]AVP binding was more intense in the sheep than in the rat anterior lobe, suggesting that AVP may be particularly important for ACTH secretion in the sheep.