Expression of Heart-Specific Constructs in \u3cem\u3eCiona intestinalis\u3c/em\u3e Embryos

Ciona intestinalis is a useful animal model system for studying developmental processes. It is particularly helpful in studies of heart development since many of the developmental steps and genes are evolutionarily-conserved in C. intestinalis. This system replicates early heart development in other chordates, such as vertebrates. In addition to evolutionary conservation of genes and developmental features, there are many advantages to using this model system including rapid development and simple maintenance. Our main focus is the process of myocardial growth in the heart of C. intestinalis. In order to monitor the growth of the heart during development, we have constructed an expression vector using a fluorescently-labeled, heart-specific gene (BC030863/Micalcl, transcript model ci0100139114 from the ANISEED database). Previous studies have shown that development of C. intestinalis embryos is altered if the PI3K/AKT signaling pathway is disrupted. C. intestinalis embryos treated with PI3K- or AKT-specific inhibitory drugs at the larval stage just prior to metamorphosis and heart formation have a reduced heart size and delayed development. We will quantitatively assess heart growth using the reporter plasmid we constructed that contains a heart-specific promoter to generate fluorescently labeled hearts in juveniles. In addition, we also have obtained similar reporter constructs from the C. intestinalis transgenic line resource (CITRES, Japan). The requested plasmids, pMiCiTnIG and pMiCiTnIGCiprmG, are specifically expressed in muscle cells, including the heart. Electroporation of these plasmids has been successful and we have generated transgenic juveniles. Currently, we are optimizing the inhibitory drug treatments and will monitor heart growth by fluorescence microscopy