Developmental expression and transcriptional regulation of Ci-Pans, a novel neural marker gene of the ascidian, Ciona intestinalis Christian Alfano 1,2 , Monia Teresa Russo 1 , Antonietta Spagnuolo ⁎ Laboratory of Biochemistry and Molecular Biology, Stazione Zoologica “A. Dohrn”, Villa Comunale, 80121, Naples, Italy Received 30 January 2007; received in revised form 11 May 2007; accepted 18 May 2007 Available online 16 June 2007 Abstract A novel gene, named Ci-Pans, was isolated and characterized from the ascidian Ciona intestinalis. It is an 885-bp cDNA, is thought to encode a protein with no sequence similarities to known proteins and shows a spatial and temporal specific expression pattern. In fact, besides a transient early localization in the muscle precursors, it is expressed in a dynamic fashion in the nervous system, during C. intestinalis embryogenesis, reaching very high level of expression as the development proceeds. To study Ci-Pans transcriptional control, we isolated the predicted promoter region of C. intestinalis Ci-Pans using databases for this species. Analysis of transgenic embryos, with a green fluorescence protein (GFP) reporter, showed that approximately 1 kb of the 5′-flanking sequence of the Ci-Pans gene was implicated in its specific expression in the CNS. The data on the expression pattern of Ci-Pans together with the strong activity exhibited by the 1 kb promoter region we have identified, indicate that a more deeply investigation on Ci-Pans could provide clues for exploring the complex network of nervous system-specific genes. © 2007 Elsevier B.V. All rights reserved. Keywords: Ciona; Ci-Pans; Nervous system; In situ hybridization; Promoter; Transgenesis; Electroporation 1. Introduction Ascidians are popular organisms for developmental and evolutionary studies because of their small genomes, invariant cell lineages, simple tadpole larvae and phylogenetic affinity to vertebrates (Satoh and Jeffery, 1995; Jeffery, 2000). The tadpole has a primitive central nervous system (CNS) that provides us a chordate nervous system in miniature. The ascidian larval CNS consists, indeed, of a sensory vesicle, neck, visceral ganglion and tail nerve cord distributed along the anterior posterior axis (for reviews, see Meinertzhagen and Okamura, 2001; Lemaire et al., 2002; Meinertzhagen et al., 2004). Despite the complex organization, however, it is composed of only around 330 cells including 215 in the brain, 50 in the tail ganglion, and 65 ependymal cells of the caudal neural tube (Nicol and Meinertzha- gen, 1991). This relative simplicity of organization compared with that of vertebrates is very fascinating, considering also that the expression of some genes along the dorsal–ventral and anterior–posterior axes of the CNS is conserved between ascidian and vertebrate embryos. Orthologues of Otx, Pax2/5/8 and Hox genes are differentially expressed along the anterior–posterior axis, genes encoding Hedgehog and HNF3 are expressed in the ventral neural tube while Snail, Pax3/7, BMP2/4 and Msx are expressed in the lateral or dorsal part of the neural tube (Corbo et al., 1997; Miya et al., 1997; Wada et al., 1997, 1998; Aniello et al., 1999; Wada and Saiga, 1999a,b; Hudson and Lemaire, 2001; Imai et al., 2002; Takatori et al., 2002). Therefore, the two peculiar characteristics of ascidian, that is a small number of cells and far fewer genes than vertebrates, coupled with the nervous system architecture very close to that of vertebrates, make them an ideal model for exploring molecular mechanisms underlying the development of a complex nervous system, applicable to those of higher animals. Available online at www.sciencedirect.com Gene 406 (2007) 36 – 41 www.elsevier.com/locate/gene Abbreviations: CNS, Central Nervous System; cDNA, DNA complementary to RNA; PCR, polymerase chain reaction; GFP, green fluorescent protein; bp, base pair(s); kb, kilobases; nt, nucleotide(s); ORF, open reading frame. ⁎ Corresponding author. Tel.: +39 081 5833252; fax: +39 081 7641355. E-mail address: nietta@szn.it (A. Spagnuolo). 1 These two authors contributed equally to the work. 2 Present address. TIGEM-Telethon Institute of Genetics and Medicine, Via P. Castellino 111, 80131 - Naples, Italy.3 0378-1119/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2007.05.026