EVOLUTION & DEVELOPMENT 12:4, 404–415 (2010) DOI: 10.1111/j.1525-142X.2010.00427.x Evolutionary origin of rhopalia: insights from cellular-level analyses of Otx and POU expression patterns in the developing rhopalial nervous system Nagayasu Nakanishi,a,Ã David Yuan,a Volker Hartenstein,b and David K. Jacobsa aDepartment of Ecology and Evolutionary Biology, UCLA, 621 Young Drive South, Los Angeles CA 90095-1606, USA bDepartment of Molecular, Cellular and Developmental Biology, UCLA, 621 Young Drive South, Los Angeles CA 90095- 1606, USA ÃAuthor for correspondence (email: [email protected]) SUMMARY In Cnidaria, the medusae of Scyphozoa and its rhopalium, within which distinct populations of AurBrn3- and sister-group Cubozoa uniquely possess rhopalia at their bell AurPit1-expressing sensory cells develop. Thus, despite the margin. These sensory centers coordinate behavior and unique attributes of rhopalial evolution, we suggest that the development. We used fluorescent in situ hybridization and rhopalial nervous system of scyphozoan medusae involves confocal microscopy to examine mRNA expression patterns in similar patterns of differential expression of genes that Aurelia sp.1 (Cnidaria, Scyphozoa) during early medusa function in bilaterian cephalic structure and neuroendocrine formation, while simultaneously visualizing the develop- system development. We propose that rhopalia evolved ing nervous system by immunofluorescence. The genes from preexisting sensory structures that developed distinct investigated include AurOtx1, and the POU genes, AurPit1, populations of sensory cells differentially expressing POU and AurBrn3, homologs of genes known to function in ceph- genes within Otx oral-neuroectodermal domains. This implies alar neural organization and sensory cell differentiation across some commonality of developmental genetic functions Bilateria. Our results show that AurOtx1 expression defines involving these genes in the still poorly constrained common the major part of the oral neuroectodermal domain of the ancestor of bilaterians and cnidarians. INTRODUCTION growth. Here we report the first detailed study of develop- mental gene expression in scyphozoan rhopalia. In Cnidaria, the medusae of Scyphozoa and its sister-group The scyphozoan and cubozoan jellyfish belong to the clade Cubozoa uniquely possess rhopalia (Fig. 1) at their bell Cnidaria that also contains Anthozoa (sea anemones and margin. Rhopalia receive sensory input from the sensory corals), Staurozoa (stalked jellyfishes), and Hydrozoa (e.g., nerve net of the umbrella as well as from specialized sensory Hydra and Portuguese Man o’ War). Cnidarians are generally domains within rhopalia, and generate sensory-input-depen- characterized as having two germ layers, ectoderm, and en- dent, as well as spontaneous and regular, electrical impulses to doderm, separated by extracellular matrix, the mesoglea. The coordinate bell contractions (reviewed in Lesh-Laurie and jellyfish life cycle typically consists of the swimming planula Suchy 1991). The rhopalium often has a statolith-containing stage, the asexually reproducing polyp stage, and the free- sac here referred to as the lithocyst (also called the statocyst), swimming, sexual medusa stage. The likely phylogenetic re- sensory epithelia, and ocelli (the presumptive photoreceptors). lationship within Cnidaria is (Anthozoa, (Staurozoa, (Hydro- Cubozoan ocelli can be morphologically complex, with cor- zoa, (Scyphozoa, Cubozoa)))) (Collins et al. 2006). This nea, lens, and multilayered retina with pigmented ciliary pho- suggests that rhopalia likely evolved after the divergence of toreceptor cells (Yamasu and Yoshida 1976), and may be Hydrozoa and the Scyphozoa/Cubozoa clade and before the capable of image-formation (Nilsson et al. 2005). Less well divergence of Scyphozoa and Cubozoa (Collins et al. 2006), known are the roles in development; removal of rhopalia although other histories of evolutionary loss cannot be ruled leads to overall weight loss and a reduction in the rate of out (Jacobs and Gates 2003; Jacobs et al. 2007). tissue regeneration in scyphozoan medusae (Cary 1916a, b), We sought to gain a better understanding of rhopalial suggesting that rhopalia may also regulate regeneration and evolution via the comparative analysis of nervous system 404 & 2010 Wiley Periodicals, Inc. Nakanishi et al. Otx and POU expression patterns in Aurelia 405 development and gene expression across Cnidaria and primarily due to the lack of a neuro-anatomical framework Bilateria, the most likely sister-group to Cnidaria (Medina and/or spatial and temporal resolution for interpretation of et al. 2001; Wallberg et al. 2004; Putnam et al. 2007). We here the expression patterns in the context of nervous system present evidence indicating that the rhopalial nervous system development. POU gene expression patterns have not been of scyphozoan medusae develops via differential expression of reported previously in Cnidaria. genes related to bilaterian genes Otx, class I POU (POU-I/Pit-1) We have recently described the pattern of rhopalial ner- and class IV POU (POU-IV/Brn-3). Members of Otx and vous system development in the scyphozoan Aurelia sp.1 POU gene groups encode homeodomain-containing tran- (Nakanishi et al. 2009; summarized in Fig. 1). With this scription factors that regulate bilaterian head nervous/endo- anatomical framework, and using in situ hybridization com- crine system development. The paired-type homeobox gene, bined with immunohistochemistry and confocal microscopy, Otx (orthodenticle,orotd,inDrosophila) is expressed in an- we analyzed mRNA expression patterns of Otx, POU-I/Pit-1, terior parts of the developing nervous system (neuroectoderm) and POU-IV/Brn-3 in A. sp.1 during early medusa formation. in the majority of bilaterians examined to date, including We used PCR to isolate A. sp.1 sequences closely related to ecdysozoans (Finkelstein et al. 1990; Tomsa and Langeland Otx (named AurOtx1 and 2; Fig. S1); the POU-class genes, 1999; Lanjuin et al. 2003; Schroder 2003), lophotrochozoans POU-I/Pit-1 (AurPit1), and POU-IV/Brn-3 (AurBrn3) (Fig. (Bruce and Shankland 1998; Umesono et al. 1999; Arendt S2) were recovered previously (Jacobs and Gates 2001). Our et al. 2001; Nederbragt et al. 2002), and deuterostomes (Sim- results show that AurOtx1 expression defines the major part eone et al. 1993; Williams and Holland 1996; Wada et al. of the oral neuroectodermal domain of the rhopalium, within 1998; Tomsa and Langeland 1999; Hudson and Lemaire which distinct populations of AurBrn3- and AurPit1-express- 2001; Lowe et al. 2003). Within the anterior Otx domain ing sensory cells develop. emerge cephalic organ primordia such as cranial ectodermal placodes of vertebrates (reviewed in Schlosser 2006) and eye- antennal imaginal discs of Drosophila. POU class gene prod- RESULTS ucts control differentiation of specific cell types in developing cephalic organs. In particular, class IV POU (POU-IV or Structure and development of the rhopalial Brn-3) regulates the development of sensory neuron subtypes, nervous system including hair cells in the ear (an otic placode derivative) and The neuro-sensory system of scyphozoan medusae is primar- retinal ganglion cells in the eye (a lens-placode and optic- ily ectodermal, consisting of rhopalia, the motor nerve net vesicle derivative) in vertebrates (reviewed in Ryan and Rose- (MNN) and the diffuse nerve net (DNN) (Fig. 1A). As the nfeld 1997), and chemosensory neurons in the olfactory organ swimming pacemaker, the rhopalium generates regular elec- (an antennal disc derivative) of Drosophila (Clyne et al. 1999). trical impulses conducted via the ectodermal MNN to elicit In addition, POU-IV/Brn-3 is expressed in neurons in devel- periodic swimming contractions (Romanes 1885; Horridge oping cephalic structures (heads) in a diverse array of bila- 1954; Horridge 1956). The DNN is distributed in the terians including the acoel Neochildia fusca (Ramachandra et ectoderm (in Aurelia) outside of the rhopalium and acts as al. 2002), the cephalochordate Brachiostoma floridae (Cand- a sensory nerve net to elicit feeding as well as escape response iani et al. 2006), the urochordate Ciona intestinalis (Candiani behaviors upon sensory stimuli (Horridge 1956). et al. 2005), the lophotrochozoan Haliotis asinina (O’Brien The Aurelia rhopalium is divided into a terminal, inter- and Degnan 2002) and the ecdysozoan Caenorhabditis elegans mediate, and basal segment along the distal–proximal axis (Finney and Ruvkun 1990). In vertebrates, class I POU (Schafer 1878; Nakanishi et al. 2009) (Fig. 1B). The statolith- (POU-I or Pit-1) regulates the development of subsets of containing lithocyst (also called the statocyst) occupies the peptide-hormone-producing cells in the anterior pituitary (an terminal segment (Fig. 1, B and C). The intermediate segment adenohypophyseal placode derivative) (reviewed in Andersen contains the pigment-cup ocellus, the pigment-spot ocellus, and Rosenfeld 1994). In the cephalochordate B. floridae, the mechanosensory touch plate, and several populations of POU-I/Pit-1 is expressed in developing sensory-neurosecreto- ciliated sensory cells (Fig. 1C). Morphological and physio- ry cells in the anterior end of the developing pharynx (Cand- logical evidence supports the photosensory function of the iani et al. 2008; see ‘‘Discussion’’), where early anterior Otx pigment-cup ocellus but not of the pigment-spot ocellus expression persists (Williams and Holland 1996). To authors’ (Horstmann 1934;