Using transgenic animal models in neuroendocrine research: lessons from Xenopus laevis.

Transgenic animals are commonly employed to explore the function of individual proteins. Transgenic animal models include the mouse, the zebrafish, and the South African clawed toad Xenopus laevis. In contrast to mice and zebrafish, with Xenopus transgenesis DNA integration is mostly achieved in the one-cell stage. Moreover, Xenopus (as well as zebrafish) eggs are relatively large, the embryos are transparent, a large offspring is generated, and maintenance of the offspring is easy. In our transgenic studies in Xenopus, we focus on the well-characterized neuroendocrine melanotrope cells of the pituitary pars intermedia that are regulated during the process of adaptation of Xenopus to a changing environment. When the animal is placed on a black background, the melanotrope cells produce and process large amounts of the prohormone proopiomelanocortin (POMC). We apply stable melanotrope-specific transgenesis that is achieved by mixing a Xenopus POMC-promoter/transgene construct with sperm nuclei and injecting this mixture into unfertilized eggs. Since in the melanotrope cells the POMC promoter is much more active in black-adapted animals, the level of transgene expression can be manipulated by placing the animal on either a black or a white background. In this paper we review the possibilities of the Xenopus melanotrope-specific transgenic approach. Following a brief overview of the functioning of Xenopus melanotrope cells, stable melanotrope-specific transgenesis is discussed and our transgenic studies on brain-derived neurotrophic factor and secretory pathway components are described as examples of the transgenic approach in a physiological context and close to the in vivo situation.

Identification of proSAAS homologs in lower vertebrates: conservation of hydrophobic helices and convertase-inhibiting sequences.

The prohormone convertases (PCs) 1/3 and 2 accomplish the major proteolytic cleavage events in neuroendocrine tissues; each of these convertases has a small associated binding protein that inhibits convertase action in the secretory pathway. The proSAAS protein binds to PC1/3, whereas the 7B2 protein binds to PC2. However, both convertase-binding proteins are more widely expressed than their cognate enzymes, suggesting that they may perform other functions as well. All known mammalian proSAASs are over 85% conserved; thus, identifying functionally important segments has been impossible. Here, we report the first identification of nonmammalian proSAAS molecules, from Xenopus and zebrafish (Danio rerio). Although these two proteins show an overall amino acid sequence identity of only 29 and 30% with mouse proSAAS, two 14-16 residue hydrophobic segments (predicted to form alpha-helices) and two, nine through 11 residue sequences containing basic convertase cleavage sites are highly conserved; therefore, these sequences may be of functional importance. Confidence that these nonmammalian molecules represent authentic proSAAS is supported by the finding that both inhibit mouse PC1/3 with nanomolar inhibition constants; human furin was not inhibited. In vitro, the two proteins were cleaved by PC2 and furin to three or more peptide products. Both zebrafish and Xenopus proSAAS exhibited neural and endocrine distributions, as assessed by in situ and PCR experiments, respectively. In summary, the identification of proSAAS molecules in lower vertebrates provides clues as to functional regions within this widely expressed neuroendocrine protein.