Dicyema Pax6 and Zic: tool-kit genes in a highly simplified bilaterian

BMC Evolutionary Biology BioMed Central Research article Open Access Dicyema Pax6 and Zic: tool-kit genes in a highly simplified bilaterian Jun Aruga*1, Yuri S Odaka1, Akiko Kamiya1 and Hidetaka Furuya2 Address: 1Laboratory for Comparative Neurogenesis, RIKEN Brain Science Institute, Wako 351-0198, Japan and 2Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan Email: Jun Aruga* - ; Yuri S Odaka - ; Akiko Kamiya - ; Hidetaka Furuya - * Corresponding author Published: 25 October 2007 BMC Evolutionary Biology 2007, 7:201 doi:10.1186/1471-2148-7-201 Received: 4 July 2007 Accepted: 25 October 2007 This article is available from: http://www.biomedcentral.com/1471-2148/7/201 © 2007 Aruga et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Dicyemid mesozoans (Phylum Dicyemida) are simple (8–40-cell) cephalopod endoparasites. They have neither body cavities nor differentiated organs, such as nervous and gastrointestinal systems. Whether dicyemids are intermediate between Protozoa and Metazoa (as represented by their "Mesozoa" classification) or degenerate species of more complex metazoans is controversial. Recent molecular phylogenetic studies suggested that they are simplified bilaterians belonging to the Lophotrochozoa. We cloned two genes developmentally critical in bilaterian animals (Pax6 and Zic), together with housekeeping genes (actin, fructose-bisphosphate aldolase, and ATP synthase beta subunit) from a dicyemid to reveal whether their molecular phylogeny supported the "simplification" hypothesis, and to clarify evolutionary changes in dicyemid gene structure and expression profiles. Results: Genomic/cDNA sequence analysis showed that 1) the Pax6 molecular phylogeny and Zic intron positions supported the idea of dicyemids as reduced bilaterians; 2) the aa sequences deduced from the five genes were highly divergent; and 3) Dicyema genes contained very short introns of uniform length. In situ hybridization analyses revealed that Zic genes were expressed in hermaphroditic gonads, and Pax6 was expressed weakly throughout the developmental stages of the 2 types of embryo and in the hermaphroditic gonads. Conclusion: The accelerated evolutionary rates and very short and uniform intron may represent a part of Dicyema genomic features. The presence and expression of the two tool-kit genes (Pax6 and Zic) in Dicyema suggests that they can be very versatile genes even required for the highly reduced bilaterian like Dicyema. Dicyemids may be useful models of evolutionary body plan simplification. Background Dicyemid mesozoans (Phylum Dicyemida) are typically found in the kidneys of cephalopod mollusks [For general reviews on Dicyemid, [1]]. They have neither body cavities nor differentiated organs, such as nervous and gastrointestinal systems (Fig. 1A). Their life cycle consists of two phases (Fig. 1B). One is the vermiform stage, in which the dicyemid exists as a vermiform embryo formed asexually from an agamete to form worms in the renal sac of the host. The other is the infusoriform larva, which develops from a fertilized egg produced around hermaphroditic gonads called infusorigens and can escape from the host Page 1 of 16 (page number not for citation purposes) BMC Evolutionary Biology 2007, 7:201 http://www.biomedcentral.com/1471-2148/7/201 Figure 1acuticephalum Dicyema Dicyema acuticephalum. (A) Light micrograph of a rhombogen. Note that embryos develop in the axial cell. Scale bar, 10 μm. (B) Life cycle of the dicyemid (after Furuya and Tsuneki [1]). The dotted line indicates an unknown process. The vermiform stage includes the nematogen, rhombogen, and vermiform embryo. The development of infusorigens, gametogenesis around the infusorigen, and development of the 2 types of embryo all proceed within the axial cell.AG, agamete; AX, axial cell; CL, calotte; D, developing infusoriform embryo; DV, developing vermiform embryo;H, hermaphroditic gonad (infusorigen); IN, infusoriform embryo; P, peripheral cell. into seawater. How the infusoriform larvae enter the vermiform stage in the new host is unknown. However, high population density in the cephalopod phase may cause the shift from an asexual mode to a sexual mode of reproduction. Notably, both fertilization and embryonic development occur within the worm body. The viviparous mode of reproduction makes this organism a good subject for developmental analysis [2]. The original classification of dicyemids as Mesozoa reflects their intermediate position between the Protozoa (unicellular eukaryotic organisms) and Metazoa (multicellular animals) in body organization. The phylogeny of the dicyemids is controversial, and some researchers consider that dicyemids represent truly primitive multicellular organisms. However, several zoologists regard the simple body plan of dicyemids as the result of specialization of parasitism [references in [1]]. Recent molecular studies suggest that dicyemids are not truly primitive ani- Page 2 of 16 (page number not for citation purposes) BMC Evolutionary Biology 2007, 7:201 http://www.biomedcentral.com/1471-2148/7/201 mals. 18s rDNA phylogenetic analyses by Katayama et al. [3] showed that the dicyemids belong among the bilateria, and from the structure of the amino acid (aa) sequence in the carboxy-terminal flanking region of the Antennapedia protein in Dicyema orientale, Kobayashi et al. [4] suggested their affinity to the Lophotrochozoa. A limited number of genes are available for phylogenetic analysis, and the phylogenetic relationships of the dicyemids will need further evaluation when the sequences of more genes become available. cDNA and genomic DNA. Both genomic and cDNA fragments containing entire open reading frames were cloned and sequenced to reveal the entire aa sequences encoded and the exon-intron boundaries (Fig. 2). Two types of Zic and actin genes were identified; they were termed ZicA/ ZicB and actin1/actin2, respectively. In the course of the cloning procedure, we fortunately obtained sequences representing 2 more genes, the housekeeping genes ATPS and aldolase, both of which were fully cloned to supplement the analysis (Fig. 2). If reduction of body plan complexity secondary to parasitism truly happened in dicyemids, we expect to find genomic features in the dicyemid genome that are associated with the adaptive simplification of body organization. However, such features have not been described in dicyemids. In broader terms, the genomic basis of the simplification that occurs during the course of evolution is poorly understood. We first examined the conserved domains of the putative aa sequence of Dicyema Pax6 (408 aa), which conta (...truncated)