Evolutionary Origin of Rhopalia: Insights from Cellular-Level Analyses of Otx and POU Expression Patterns in the Developing Rhopalial Nervous System

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 developmental 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; Yoshida and Yoshino 1980; Takasu and knowledge, there are currently no reports of POU-I/Pit-1 Yoshida 1984; Nakanishi et al. 2009). The basal segment expression patterns from protostome taxa. forms the attachment to the medusa body and contains Although Otx mRNA expression patterns have been in- sensory- and ganglion-cell groups in the ectoderm (Fig. 1B). vestigated in several cnidarian taxa (Muller et al. 1999; Smith Regular electrical-impulse-generating pacemaker neurons et al. 1999; de Jong et al. 2006; Mazza et al. 2007), roles of Otx likely reside here (Passano 1973; Nakanishi et al. 2009). in cnidarian nervous system development remained unclear, FMRFamide- and Tubulin/Taurine-IR neuronal processes 406 EVOLUTION&DEVELOPMENT Vol. 12, No. 4, July--August 2010

Fig. 1. Neuromuscular system and rhopalia of Aurelia sp.1 larval medusa. (A) Confocal sections of an Aurelia sp.1 ephyra labeled with the fluorescent dye phalloidin and antibodies against Tyros- inated Tubulin (tyrTub) and FMRF- amide (FMRF). The specimen is viewed from the oral side. Phalloidin shows radial (rm) and circular (cm) muscle fibers. The tyrTub antibody shows the motor nerve net (MNN) along the musculature, and the FMRFamide antibody shows the diffuse nerve net (DNN) throughout the ectoderm. Note that lappets (la) are folded toward the oral side and their tips are pointing toward the center (manubrium, mn). (B, C) Schematic represen- tation of the rhopalial nervous system. The rhopalium is divided into three segments terminal (te), intermediate (in), and basal (ba). (B) Oral surface view with the terminal/distal end up, the basal/proxi- malenddown.(C)Lateralviewthrough the center of the rhopalium with the oral side facing down; only the terminal and intermediate segments, and the distal part of the basal segment, are shown. The terminal segment contains the lithocyst (LT). At least seven clusters of ectodermal neurons can be distinguished in the intermediate and basal segments (Nak- anishi et al. 2009). The intermediate segment has FMRFamide- and Taurine-IR oral-distal sensory cells (O-D), pigment- cup ocellus (CO) containing FMRF- amide-IR subepidermal photoreceptor cells, GLWamide-IR oral-central sensory cells (O-C) on the oral side, and the pigment-spot ocellus (SO) containing FMRFamide-IR epidermal sensory cells, the touch plate (TP), and FMRFamide- IR aboral-lateral sensory cells (A-L) on the aboral side. The basal segment contains FMRFamide-IR oral-central sensory cells (O-C) at the distal portion and FMRFamide- and GLWamide-IR oral- proximal sensory and ganglion cells (O-P) preferentially toward the proximal end. The MNN is connected to Taurine-IR oral-distal sensory cells, whereas the DNN is connected to the FMRFamide-IR oral-proximal neurons. GLWamide-IR neuronal processes connect GLW- amide-IR oral-central cluster to GLWamide-IR oral-proximal cluster, but do not leave the rhopalium. Note bilaterally symmetrical arrangement of the rhopalial nervous system organization (B). mn, manubrium; gf, gastric filaments; la, lappet (feeding appendage); ra, rhopalar arm; LC, lithocyst; te, terminal segment; in, intermediate segment; bs, basal segment; EN, endoderm; O-D, oral-distal group; A-L, aboral-lateral group; O-C, oral-central group; O-P, oral-proximal group; SO, spot-ocellus; TP, touch plate; CO, cup-ocellus; MNN, mnn, motor nerve net; DNN, dnn diffuse nerve net. Scale bar: 50 mm. connect to the DNN and the MNN, respectively, at the base the pacemaker activity of the rhopalium (Prephyra III) of the rhopalium (Fig. 1B; Horridge 1956; Nakanishi et al. (Schwab 1977; Nakanishi et al. 2009). The pigment-cup 2009). ocelli develop in the free-swimming ephyra (Ephyra), and During the transformation of the polyp into larval me- the pigment-spot ocelli subsequently begin to form at the dusae (ephyrae), referred to as strobilation, the lithocysts, metephyra stage (Yoshida and Yoshino 1980; Nakanishi the touch plates, and the DNN begin differentiation early et al. 2009). For definition of developmental stages, see (Prephyra I, II), followed by development of the MNN and (Nakanishi et al. 2009). Nakanishi et al. Otx and POU expression patterns in Aurelia 407

Initial AurOtx1 expression defines broad domains anishi et al. 2009), the boundary of the AurOtx1 expression of the oral neurogenic ectoderm (neuroectoderm), domain over the region of the MNN sharpens and the in- which later becomes spatially restricted tensity of expression reaches its maximum level (arrows in Early AurOtx1 expression occurs broadly in parts of the oral Fig. 2C). High levels of ectodermal AurOtx1 expression neuroectoderm, including the subumbrellar ectoderm in are maintained in a narrow stripe in the middle region of which the MNN develops (Prephyra I), the proximal half of the basal segment of the rhopalium just proximal to the the rhopalium in which oral-proximal and oral-central neu- FMRFamide-IR oral-central sensory-cell group through rons develop (Prephyra I), and the region of the rhopalium themetephyrastage(Fig.2,D,E,andG).Theexpression where oral-distal sensory cells develop (Prephyra II) (Fig. 2, A in the oral-distal region is also maintained through the mete- and B). Toward the end of the Prephyra stage (III) when the phyra stage, occurring in individual FMRFamide-IR oral- MNN develops into a functional conducting system (Nak- distal sensory cells (arrows in Fig. 2, D, F, and H).

Within the AurOtx1 domain in the rhopalium, high levels of AurBrn3 and AurPit1 expression occur in distinct subdomains Within the initial AurOtx1 oral neuroectodermal domain (Prephyra I, II), AurBrn3 is strongly expressed in the region where oral-central sensory cells develop (Fig. 3A), whereas strong AurPit1 expression occurs proximal to this oral-central domain (Fig. 3B). At the Prephyra III stage, AurBrn3 expression begins to occur in the region of the oral-distal sensory-cell group (Fig. 3C), and strong AurPit1 expression becomes restricted to the middle region of the basal segment (Fig. 3D). High levels of AurBrn3 expression are maintained in FMRFamide-IR oral-distal and oral-central sensory cells through the metephyra stage (Fig. 3, C, E, and G). Also,

Fig. 2. mRNA expression patterns of AurOtx1 in the oral ectoderm during rhopalial nervous system development. Confocal sections of ephyrae from the Prephyra I stage through the metephyra stage, labeled with the anti-sense riboprobe AurOtx1 and antibodies against FMRFamide (FMRF) and/or Acetylated Tub- ulin (acTub). (A) Prephyra I–II. (B) Prephyra II. (C, D) Prephyra III. (E, F) Ephyra. (G, H) Metephyra. In all panels, specimens are viewed from the oral side and the terminal/distal end of the rhopalium is up, except that in H the distal half of the rhopalium is twisted so that the oral side of the distal region facing the right. (A– E, G) Sections through the entire specimen. (F, H) Sections at medial levels. Rhopalia (A, C, E), the presumed region of MNN development (A), contours of the larvae (C), and the endodermal pigment cell layer (pi) of the pigment-cup ocellus (H) are outlined in white. Arrows in (A) and (C) show AurOtx1 expression in the domain of MNN development. Boxes outlined in white in (D), (E), and (G) indicates the site of FMRFamide-IR oral-central sensory cell development. An arrow in (G) shows the late AurOtx1 domain in the basal segment proximal to the FMRFamide-IR oral-central sensory-cell group. Insets with arrows in (B), (F), and (H) show magnified views of labeled cells. AurOtx1 expression occurs in differentiating/differentiated FMRFamide-IR oral-distal sensory cells (B, D, F, H). AurOtx1 transcripts were not detectable in ectodermal sensory cells (sc) of the pigment-cup (pi) ocellus (H). en, endoderm; rh, rhopalium; la, lappet; lc, lithocyst; tr, terminal segment; in, intermediate segment; bs, basal segment; mn, manubrium; o-p, oral-proximal neuron; o-c, oral-central neuron; o-d, oral-distal neuron; sc, sensory cells in the pigment cup ocellus; pi, endodermal pigment cells in the pigment cup ocellus; ci, cilia of the sensory cells; so, pigment-spot ocellus; tp, touch plate. Scale bar: 50 mm. 408 EVOLUTION&DEVELOPMENT Vol. 12, No. 4, July--August 2010 strong AurPit1 expression in the middle region of the basal AurBrn3 expression (Prephyra II) occurs in regions of the segment continues into the metephyra stage, within which ectoderm where aboral-lateral groups of neurons develop (Fig. AurPit1-expressing sensory cells that are neither FMRF- S3C), whereas AurPit1 expression occurs in lithocyst ectoder- amide- nor GLWamide-IR develop (Fig. 3, F and H). mal cells (Fig. S3D). Strong levels of AurBrn3 expression are maintained in individual differentiating/differentiated sensory cells in aboral-lateral (Fig. S3E) and spot-ocellus/touch-plate AurBrn3 and AurPit1 expression outside the regions (Fig. S3F) through the metephyra stage. AurOtx1 domain in the rhopalium Outside the AurOtx1 domain, AurBrn3 and AurPit1 initially share their mRNA expression domain in the aboral ectoderm DISCUSSION of rhopalia where the touch plate and the pigment-spot ocellus develop (Prephyra I, II) (Fig. S3, A and B). In addition, early mRNA expression patterns (Fig. 4A) thus suggest that AurOtx1 defines the major parts of the oral neuroectodermal domain within the rhopalium, from which a high density of neurons including AurBrn3-expressing oral-distal and oral- central sensory cells and AurPit1-expressing sensory cells develop. In addition, persistent expression of AurOtx1 in some of the developing and differentiated ectodermal sensory cells (e.g., oral-distal group) implicates roles in conferring identities of certain neuronal cell types within the expression domain. Below we discuss evolutionary implications of our results and existing comparative data, assuming parsimony for evolutionary history reconstruction. The inferred roles of Otx in A. sp.1 parallel those in Bilateria. As mentioned, Otx homologs are expressed broadly

Fig. 3. mRNA expression patterns of AurBrn3 (A, C, E, G) and AurPit1 (B, D, F, H) in the oral ectoderm during rhopalial nervous system development. Confocal sections of ephyrae from the Prep- hyra I stage through the metephyra stage, labeled with the antisense riboprobe AurBrn3 or AurPit1 and antibodies against FMRFamide (FMRF) or GLWamide (GLW) and/or Acetylated Tubulin (acTub). (A, B) Prephyra I-II. (C, D) Prephyra III. (E, F) Ephyra. (G, H) Metephyra. In all panels, specimens are viewed from the oral side and the terminal/distal end of the rhopalium is up. (A) Sections through oral ectoderm. (B–D) Sections through the entire specimen. (E, F, H) Sections at medial levels. (G) A section at the level of FMRFamide-IR oral-central group of neurons. The rhopalium in D is outlined in white. Boxes outlined in white in (A), (B), (D), and (G) indicates the site of FMRFamide-IR oral-central sensory cell development. Insets with arrows in (C), (E–H) show magnified views of labeled cells. AurBrn3 expression occurs in differentiating/differentiated FMRFamide-IR oral-distal sensory cells (C, E). In addition, AurBrn3 expression occurs in FMRFamide-IR oral-central sensory cells (G). AurPit1 expression occurs in cells with sensory-cell-like morphology (F, H); Cnidarian sensory cells generally have a spindle-shaped cell body oriented perpendicular to the mesoglea with the apical cell surface exposed to the external environment (Thomas and Edwards 1991). Note the morphological resemblance of AurPit1-expressing cells to FMRF- amide-IR oral-central sensory cell (red arrow in F0) and GLW- amide-IR oral-proximal sensory cells (H0). AurPit1 transcripts and FMRFamide- or GLWamide-like neuropeptides rarely co-local- ized in individual cells. en, endoderm; rh, rhopalium; la, lappet; lc, lithocyst; tr, terminal segment; in, intermediate segment; bs, basal segment; ca, rhopalar canal; o-p, oral-proximal neuron; o-c, oral- central neuron; o-d, oral-distal neuron. Scale bar: 50 mm. Nakanishi et al. Otx and POU expression patterns in Aurelia 409

Fig. 4. Summary. (A) Schematic repre- sentation of AurOtx1, AurBrn3, and AurPit1 expression patterns within AurOtx1 oral neuroectodermal domains during rhopalial nervous system development. Only the domains of high-level expression are shown. Oral ectodermal neuronal groups are highlighted in gray. LC, lithocyst; O-D, oral-distal group; O-C, oral-central group; O-P, oral-proximal group (B) Oral view of a free-swimming ephyra showing a ring of AurOtx1 expression in the subumbrellar ectoderm surrounding the base of the manubrium (MN), as well as a pair of radial projections of AurOtx1 expressing domains in each rhopalar arm (RA) reaching lappets (LA). Expression in each rhopalium (RH) is not shown. Only six rhopalar arms are drawn for simplicity; norm- ally there are eight. (C) Our proposed evolutionary model. Ancestral circum- oral sensory structures, whose nervous system development was controlled by Otx and POU, evolved before Cnidaria/ Bilateria split. Following the divergence, rhopalia evolved from these preexisting sensory structures early in the Scyphozoa/Cubozoa lineage. The phylogeny is derived from Collins et al. (2006). in anterior neuroectoderm in the majority of bilaterians (see cephalochordate B. floridae (larval frontal eyes; Williams and ‘‘Introduction’’). In Drosophila melanogaster, orthodenticle Holland 1996; Candiani et al. 2006) and the ecdysozoan (otd), an Otx homolog, initially specifies the dorso-anterior Drosophila (chemosensory neurons in olfactory organs, Clyne neuroectoderm during embryogenesis (Hirth et al. 1995; You- et al. 1999). Also, class I POU (POU-I or Pit-1) is expressed in nossiHartenstein et al. 1997). Loss of otd function results in a subset of anterior-pituitary cells in vertebrates (reviewed in loss and/or defects of central and sensory neuronal elements Andersen and Rosenfeld 1994), and in presumptive sensory- from the entire otd expression domain. In addition, otd is neurosecretory cells of the developing pharynx in the amp- required at a later stage for normal development of photo- hioxus B. floridae (Candiani et al. 2008); both the anterior receptor cells (Vandendries et al. 1996). In vertebrates, Otx2 is pituitary of vertebrates and the pharynx of B. floridae develop required for the early specification of the anterior neural plate, within the anterior Otx-expressing domain. Although data for whereasOtx1isnecessaryfornormalinnerearandeye POU-I/Pit-1 from protostomes are wanting (the gene is absent development (reviewed in Acampora et al. 2001), and Crx, from genomes of two ecdysozoan models, Drosophila and another Otx homolog, is specifically involved in differentia- C. elegans; Jacobs and Gates 2003), we propose that distinct tion of rods and cones in retina (Chen et al. 1997). In populations of POU-IV/Brn-3- and POU-I/Pit-1-expressing addition, persistent expression of Otx in differentiating/differ- sensory cells developed from Otx-expressing neuroectoderm entiated sensory cells/organs within the anterior neuroecto- in the last common ancestor of Cnidaria and Bilateria. dermal expression domain occurs in the cephalochordate B. floridae (Williams and Holland 1996), the lophotrochozoan Dugesia japonica (Umesono et al. 1999), and the ecdysozoan Ancestral POU-IV/Brn-3 may have been involved C. elegans (Lanjuin et al. 2003). Thus, the role of Otx in in the development of sensory cells with microvilli defining parts of the neuroectodermal domain that generates a or stereocilia at the apical cell surface concentration of neurons, including Otx-expressing sensory surrounding the base of the cilium cells, may be ancestral in Cnidaria and Bilateria. In A. sp.1, AurBrn3-expressing cells occur in regions where Within the early Otx domain, distinct populations of the touch plate and FMRFamide-IR oral-distal sensory cells AurBrn3- and AurPit1-expressing sensory cells differentiate in differentiate. Cells in these regions are characterized by the developing rhopalia of A. sp.1. Similarly, class IV POU presence of the specialized apical sensory apparatus, with a (POU-IV or Brn-3) is expressed in developing sensory cells/ cilium surrounded at its base by a collar of microvilli/stereo- organs within the anterior Otx domain in vertebrates (hair cilia, and are believed to be mechanosensory (Spangenberg cells and retinal ganglion cells, Ryan and Rosenfeld 1997), the et al. 1996; Nakanishi et al. 2009). This type of sensory cells is 410 EVOLUTION & DEVELOPMENT Vol. 12, No. 4, July--August 2010 structurally similar to choanoflagellates. In Metazoa, these specialization associated with the apical cellular structure such cells are often compared with choanocytes of sponges as well as the ‘‘paraciliary fibrillar processes’’ and cilia with ‘‘dilated as the metanephridia and other cell types of bilaterian groups tips’’ is reminiscent of the apical sensory apparatus of meta- (Salvini-Plawen and Splechtna 1979; Jacobs et al. 2007). In zoan sensory cells (Schlosser 2005). Taken together, these ob- Bilateria, a subset of POU-IV/Brn-3-expressing sensory cells servations suggest that POU-I/Pit-1 is likely to be involved in has elaborate microvilli/stereocillia structures at the apical the development of sensory and/or neurosecretory cells with cell surface, often forming a ring around each cilium. Exam- exocrine and/or endocrine activities in chordates. ples include mechanosensory hair cells in molluscan stato- In A. sp.1, AurPit1 is expressed in differentiating/differ- cysts (Barber and Dilly 1969; Stahlsch and Wolff 1972; entiated sensory cells in the ectoderm of rhopalia, which are O’Brien and Degnan 2002), rhabdomeric photoreceptor cells largely distinct from those that express AurBrn3. This in polychaete larval eyes (with reduced cilia; Rhode 1992; suggests that sensory cell development may be an ancestral Arendt et al. 2004), type I primary sensory cells of the amp- function of POU-I/Pit-1 in Cnidaria and Bilateria, and that hioxus (Lacalli and Hou 1999; Candiani et al. 2006), and hair POU-I/Pit-1 and POU-IV/Brn-3 may have directed the cells of vertebrate ears (Ryan and Rosenfeld 1997). On the development of (at least partially) distinct populations of basis of these parallels between A. sp.1 and bilaterians, we sensory cells in the last common ancestor. It is as yet unclear propose that a subset of POU-IV/Brn-3-expressing cells whether AurPit1-expressing presumptive sensory cells have developed a collar of microvilli around each cilium in the last neurosecretory function. common ancestor of Cnidaria and Bilateria. It will be Ablation experiments suggest that scyphozoan rhopalia important to examine POU-IV/Brn-3-expressing cells in may regulate regeneration and growth (Cary 1916a b), and additional taxa, particularly from nonbilaterian groups such this could be mediated by production and distribution of as sponges and placozoans, in order to gain further insight hormone(s). In vertebrates, Otx1 and POU-I/Pit-1 control into evolution of sensory cells in Metazoa. growth hormone production in a specific cell type (somatotropes) in the anterior pituitary (Li et al. 1990; Acampora et al. 1998). In A. sp.1, AurPit1 expression domain overlaps Ancestral POU-I/Pit-1 may have functioned to with AurOtx1 expression domain in a narrow stripe in the develop sensory cells that are largely distinct middle region of the basal segment of the rhopalium (com- from POU-IV/Brn-3-expressing sensory cells pare Figs. 2, G and 3D; the same pattern is observed when the POU-I/Pit-1 is involved in the development of specific same developmental stages are compared). Do cells co- peptide-hormone-producing cells in the anterior pituitary expressing AurOtx1 and AurPit1 in rhopalia produce the (adenohypophysis) in vertebrates (reviewed in Andersen and growth-stimulating-hormone like vertebrate somatotropes? Rosenfeld 1994), and is expressed in cells that develop into the Identification of growth-related hormones and their spatial pit region of the preoral organ in the amphioxus B. floridae distribution, coupled with AurPit1 gene knockdown experi- (Candiani et al. 2008). The preoral organ is the larval form of ments to see if it would retard growth and regeneration, will the Hatschek’s pit, the slight depression located in the dorsal be needed in order to gain further insight into the mechanism region of the mouth cavity, which is believed to be a chemo- controlling growth and regeneration in scyphozoan medusae. sensory and neurosecretory organ of amphioxus possibly homologous to the adenohypophysis of vertebrates (Nozaki and Gorbman 1992; Gorbman 1995; Candiani et al. 2008). Consideration of Otx expression pattern with The POU-I/pit-1-expressing region of the preoral organ con- regard to its position tains the mucus cells, which are enriched with vesicles apically, It is noteworthy that spatial patterns of Otx expression appear and the cells with stereocilia-like, ‘‘paraciliary fibrillar pro- similar across Cnidaria and Bilateria. In Aurelia sp.1,Otx cesses’’ (FP cells) (Lacalli 2008). Following metamorphosis, (AurOtx1) expression forms a contiguous ring-like domain the preoral organ-derived cells in the Hatschek’s pit are seen to around the mouth (manubrium) during medusa development have cilia with ‘‘dilated tips,’’ and some have accumulation of (Fig. 4B). Similar patterns of Otx expression around the electron-dense granules at the basement membrane, under- mouth are observed in other cnidarians as well, including neath which lies loosened collagen layer, likely allowing the developing medusae of the hydrozoan Podocoryne carnea release of the secretory materials into the blood spaces located (Muller et al. 1999) and developing polyps of the anthozoans basally (Sahlin and Olsson 1986). Consistent with the potential Nematostella vectensis (Mazza et al. 2007) and Acropora mill- secretory function, cells in the Hatschek’s pit are immunore- epora (de Jong et al. 2006). In Bilateria, distinct Otx-expressing active to antibodies against chorionic gonadotropin (Nozaki domains surrounding the (future) mouth occur in the annelids and Gorbman 1992) and neuropeptides such as FMRFamide Helobdella triserialis (Bruce and Shankland 1998) and Platy- (Massari et al. 1999) and neuropeptide Y (Castro et al. 2003). nereis dumerilii (Arendt et al. 2001), the echinoderm Stichopus Although neuronal processes were not observed in these cells, japonicus (Shoguchi et al. 2000) and the hemichordate Nakanishi et al. Otx and POU expression patterns in Aurelia 411

Ptychordera flava (Harada et al. 2000). Developmental genetic patterns in developing tentacles of Nematostella and evidence based on brachyury and goosecoid expression pat- other anthozoans will be critical. Likewise, comparable terns supports homology of mouths across Cnidaria and gene expression studies must be conducted additionally for Bilateria (except for chordates) (Hejnol and Martindale 2008). rhopalioids of enigmatic staurozoans. If the similar pattern of Otx expression around the mouth in Cnidaria and Bilateria were due to common ancestry, the last common ancestor of cnidarians and bilaterians may have MATERIALS AND METHODS used Otx for pattern formation around the mouth as well as for specification of the nervous system that developed in this Animals and fixation region. Although less parsimonious, an alternative hypothesis Polyps, strobilae, and ephyrae of Aurelia sp.1 (sensu Dawson and Jacobs 2001) were obtained from the Cabrillo aquarium (San would be to assume independent evolution. The ancestral Pedro, CA, USA). Animals were anesthetized in 7.3% MgCl be- Otx may have merely functioned to specify neuroectoderm 2 fore fixation in 4% formaldehyde for 1 h at room temperature (RT). somewhere (other than the domain around the mouth), and the convergence in Otx expression domains around the mouth Nucleic acid extraction, species identification, and cDNA might have occurred in Cnidaria and Bilateria due to selective synthesis advantage of having a high density of neurons around Genomic DNA and total RNA were simultaneously extracted ac- the mouth, for instance, for more effective feeding and/or prey cording to the published protocol (Schroth et al. 2005). First-strand capture. cDNAs were synthesized using SuperScript III First-Strand Syn- thesis System for RT-PCR (Invitrogen, Carlsbad, CA, USA) or BD SMART RACE cDNA Amplification Kit (BD Biosciences, Ancestral neuro-sensory system development San Jose, CA, USA). and evolution of rhopalia Our data from A. sp.1 combined with the existing data from Degenerate PCR, RACE, cloning, and sequencing Bilateria thus suggest that the last common ancestor of Cnid- Homologous sequences to bilaterian Otx genes were recovered aria and Bilateria may have used Otx to define neuroectoderm from the A. sp.1 genome via PCR with degenerate primers, using around the mouth from which distinct sets of sensory cells polyp and ephyra cDNAs as the PCR templates. Sequences of the 0 0 differentially expressing POU-I/Pit-1 and POU-IV/Brn-3 5 and 3 regions of genes of interest were obtained via RACE developed. Some of the POU-IV/Brn-3-expressing cells may using ephyra cDNAs as templates. PCR products were cloned into the pCRII-TOPO vector using the TOPO TA cloning Dual have had a collar of microvilli at the apical surface. Ectoderm Promotor kit (Invitrogen) and sequenced at the UCLA Genotyp- of developing rhopalia expresses genes closely related to sine ing and Sequencing Core facility. The alignment of the sequences oculis/Six1/2 and eyes absent/Eya (Nakanishi et al. in prep.), and assembly of contigs were performed using the CodonCode which are known to specify cranial ectodermal placodes (sen- Aligner (v.1.5.2, CodonCode Corporation, Dedham, MA, USA). sory structure precursors) in vertebrates and eyes in Droso- phila. We suggest that the ancestor used a developmental Immunohistochemistry and confocal microscopy genetic program involving Otx, POU, SIX, and EYA genes Immunohistochemistry was performed as previously described to generate sensory-neuronal structures around the mouth (Yuan et al. 2008). Primary antibodies that were used for this study (Fig. 4C). We further propose that, following the divergence were reactive against FMRFamide (‘‘FMRF’’; US Biological), of Cnidaria and Bilateria, rhopalia evolved from these GLWamide (‘‘GLW’’; Schmich et al. 1998; Schmich et al. 1998), preexisting ancestral sensory structures early in the Scypho- Acetylated Tubulin (‘‘acTub’’; Sigma, St. Louis, MO, USA), and zoa/Cubozoa lineage (Fig. 4C). Thiel (Thiel 1966) argued that Tyrosinated Tubulin (‘‘tyrTub’’; Sigma). Filamentous actin was scyphozoan and cubozoan rhopalia were homologous with labeled using phalloidin conjugated to AlexaFluor 568 (‘‘Pha’’; staurozoan rhopalioids and polyp tentacles as ‘‘tentaculous Molecular Probe). Secondary antibodies that were used for this study were AlexaFluor 488 (mouse, Molecular Probes, Carlsbad, (tentacle-derived) organs.’’ In addition, developmental genetic CA, USA), AlexaFluor 568 (rabbit, Molecular Probes), Alex- as well as ontogenetic evidence suggests that rhopalia and aFluor 633 (mouse, Molecular probe), and Cy5 (rabbit, Jackson hydrozoan marginal sensory structures (e.g., eyes and tentacle Immunoresearch Laboratories, West Grove, PA, USA). bulbs) might be related (see Nakanishi et al. 2009). Compar- ative analyses of developmental gene expression patterns Fluorescent mRNA in situ hybridization with among these potentially homologous sensory structures will immunohistochemistry be important for clarifying early histories of cnidarian sensory The following protocol was developed by modifying published in structure evolution. Interestingly, Otx is expressed in devel- situ hybridization protocols for invertebrate species (Gates et al. oping tentacles of the anthozoan Nematostella vectensis 2002; Finnerty et al. 2003; Okamoto et al. 2005). Fixed specimens (Mazza et al. 2007). Investigation of POU, SIX, and were washed in PBSTr (PBS10.3% Triton-X100), then in 1% EYA, together with more detailed analyses of Otx expression triethanolamine in PBS, followed by addition and mixing of 0.6% 412 EVOLUTION&DEVELOPMENT Vol. 12, No. 4, July--August 2010 acetic anhydride. The specimens were washed in PBSTr and were Bruce, A. E. E., and Shankland, M. 1998. Expression of the head gene refixed in 4% formaldehyde, followed by washes in PBSTr. The Lox22-Otx in the leech Helobdella and the origin of the bilaterian body specimens were then incubated in the hybridization solution (HB: plan. Dev. Biol. 201: 101–112. Candiani, S., et al. 2005. Ci-POU-IV expression identifies PNS neurons in 50% formamide, 5 SSC, 1 Denhardt’s solution, 100 mg/ml embryos and larvae of the ascidian Ciona intestinalis. Dev. Genes Evol. Heparin, 200 mg/ml tRNA, 0.3%, 30 mg/ml salmon sperm DNA, 215: 41–45. and Triton-X100) for 10 min at RT. Then 1% SDS was added and Candiani, S., et al. 2006. Expression of AmphiPOU-IV in the developing the specimens were incubated at 551C for 1 h. The specimens were neural tube and epidermal sensory neural precursors in amphioxus sup- incubated with a digoxigenin-labeled riboprobe synthesized using ports a conserved role of class IVPOU genes in the sensory cells development. Dev. Genes Evol. 216: 623–633. RACE products as the template (Megascript kit; Ambion Inc., Candiani, S., et al. 2008. Expression of the amphioxus Pit-1 gene (Amp- Austin, TX, USA), at the final probe concentration of 1 ng/mlfor hiPOU1F1/Pit-1) exclusively in the developing preoral organ, a putative 40 h at 551C. The specimens underwent a series of washes in HB/ homolog of the vertebrate adenohypophysis. Brain Res. Bull. 75: 324–330. 2 SSC solutions (HB; 100%, 75%, 50%, 25%, and 0%) at 551C. Cary, L. R. 1916a. The influence of the marginal sense organs on metabolic The specimens were finally washed in 0.05 SSC at 551Cand activity in Cassiopea xamachana bigelow. Proc.Natl.Acad.Sci.USA2: 709–712. underwent a series of washes in 0.05 SSC/TNT wash buffer Cary, L. R. 1916b. The influence of the marginal sense organs on the rate of (25%, 50%, 75%, and 100% TNT wash buffer: 0.1 M Tris-HCl, regeneration in Cassiopea xamachana. J. Exp. Zool. 21: 1–32. pH7.5, 0.15 M NaCl, 0.3% Triton X-100) at RT. The specimens Castro, A., et al. 2003. Distribution of neuropeptide Y immunoreactivity in were blocked in TNB blocking buffer (TNT10.5% blocking the central and peripheral nervous systems of amphioxus (Branchios- reagent; ParkinElmer, Covina, CA, USA; cat# FP1020), and were toma lanceolatum Pallas). J. Comp. Neurol. 461: 350–361. Chen, S. M., et al. 1997. Crx, a novel Otx-like paired-homeodomain pro- incubated with anti-digoxygenin horseradish peroxidase (Boeh- tein, binds to and transactivates photoreceptor cell-specific genes. Neuron ringer Mannheim) together with other primary antibodies over- 19: 1017–1030. night at 41C. After antibodies were washed in TNT wash buffer, Clyne, P. J., et al. 1999. The odor specificities of a subset of olfactory specimens were incubated in fluorophore tyramide amplification receptor neurons are governed by Acj6, a POU-domain transcription reagent (TSA kit, PerkinElmer). The specimens were then washed factor. Neuron 22: 339–347. Collins, A. G., et al. 2006. Medusozoan phylogeny and character evolution in PBSTr, and were then blocked in 3% normal goat serum in clarified by new large and small subunit rDNA data and an assessment PBSTr. Following secondary antibody incubation overnight at of the utility of phylogenetic mixture models. Syst. 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SUPPORTING INFORMATION (white boxes in A, B). Aboral AurBrn3 expression is maintained in differentiating and differentiated FMRFamide-IR Additional Supporting Information may be found in the on- aboral-lateral sensory cells (a-l; C, arrow in E; arrowhead in line version of this article: inset in E shows basal neuronal process), pigment-spot cells (so; arrow in F) and the touch-plate cells (tp). Little AurBrn3 Fig. S1. Analysis of the Otx gene family protein sequences. expression is observed in the ectodermal sensory cells (sc) of A: partial protein sequence alignment with selected taxa. The the pigment-cup (pi) ocellus (Fig. 3F0). Signal in the gas- black line indicates the sites that correspond to the homeo- troderm (gd) seen in (B) occurred in the sense control, and domain (HD), and the green line indicates the sites that were thus is likely to be non-specific. Abbreviations: la lappet; ex used for phylogenetic analyses. Red arrowhead indicates the exumbrellar ectoderm; st lithocyst; rh rhopalium; ec ectoderm; ninth position of the recognition helix 3/4. B: Maximum like- en endoderm; in intermediate segment; bs basal segment; te lihood phylogenetic tree, using PRD-class homeodomain terminal segment; a-l aboral-lateral neuron; ci cilium; bt bul- sequences as the outgroup. Internal branches with low sup- bous tip of a cilium; gc opening into the gastrovascular cavity; port values have been collapsed. Support for relevant nodes so pigment-spot ocellus; tp touch plate; pi endodermal pig- are shown above internal branches with posterior probability/ ment cells in the pigment-cup ocellus; sc ectodermal sensory Neighbor-Joining (N-J) bootstrap support/Maximum likeli- cells in the pigment-cup ocellus; gd gastroderm (endodermal). hood (ML) bootstrap support (maximum support, 1). A. sp.1 Bar: 50 mm. sequences are highlighted in red. If the node was not present Fig. S4. AurBrn3 and AurPit1 mRNA expression patterns at in Bayesean, N-J and/or ML tree(s), ‘‘-’’ was given. The unit the ephyra stage. Confocal sections of ephyrae, labeled with of the branch length is the number of substitutions per site. antisense riboprobe (AurBrn3 in A; AurPit1 in B) and For abbreviations and accession numbers, see Materials and antibodies against FMRFamide (FMRF) and Acetylated Methods. Tubulin (acTub). Strong signals are observed in rhopalia (rh) in both (A) and (B). In (A), AurBrn3 signal is seen in the Fig. S2. Analysis of the POU gene family protein sequences. ectoderm outside of the rhopalia (arrows). In (B), staining A: partial protein sequence alignment with selected taxa. The occurred in the gastroderm (gd) in one of the arms, but this is black line indicates the sites that correspond to the homeo- likely to be artifactual, as the sense control showed a similar domain (HD), the blue line corresponds to the POU domain pattern (data not shown). Abbreviations: mn manubrium; la (POU), and the green line indicates the sites that were used for lappet; gd gastroderm; rh rhopalium. phylogenetic analyses. B: Unrooted Bayesean consensus phylogenetic tree. A. sp.1 sequences are highlighted in red. Fig. S5. NBT/BCIP staining of mRNA transcripts for Support values and the unit of the branch length are as AurOtx1 (A), AurBrn3 (B) and AurPit1 (C) in strobilae. described in Fig. 2. If the node was not present in Bayesean, Insets show specimens at the free-swimming ephyra stage. N-J and/or ML tree(s), ‘‘-’’ was given. The analysis supports Arrows point to individual rhopalia. the presence of five distinct clusters and the placement of AurPit1 and AurBrn3 within the POU-I/Pit-1 and POU-IV/ Table S1. List of primers used in this study. Brn-3 clusters, respectively. For abbreviations and accession numbers, see Materials and Methods. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied Fig. S3. mRNA expression patterns of AurBrn3 (A, C, E, F) by the authors. Any queries (other than missing material) and AurPit1 (B, D) outside the AurOtx1 oral neuroectoder- should be directed to the corresponding author for the article. mal domain during rhopalial nervous system development. Confocal sections of ephyrae from the Prephyra I stage Supplementary References through the Metephyra (late ephyra) stage, labeled with the Chevenet, F., et al. 2006. TreeDyn: towards dynamic antisense riboprobe AurBrn3 or AurPit1 and antibodies graphics and annotations for analyses of trees. BMC Bioin- against FMRFamide (FMRF) and Acetylated Tubulin formatics 7: Article No.: 439. (acTub). A: Prephyra I. B-D: Prephyra II. E: Prephyra III. Clamp, M., et al. 2004. The Jalview Java alignment editor. F: Metephyra. In all panels, the terminal/distal end of the Bioinformatics (Oxford) 20: 426–427. rhopalium is up, and specimens are viewed from the oral side Dereeper, A., et al. 2008. Phylogeny. fr: robust phyloge- except for A and F; A, aboral view; F, lateral view with the netic analysis for the non-specialist. Nucleic Acids Res. 36: oral side to the right. B: a section at the level of aboral W465–W469. ectoderm.C-E:sectionsatthemediallevels.ArrowsinD Edgar, R. C. 2004. MUSCLE: multiple sequence align- show ectodermal lithocyst cells. Note the occurrence of early ment with high accuracy and high throughput. Nucleic Acids AurBrn3 and AurPit1 expression in the aboral ectoderm Res. 32: 1792–1797. Nakanishi et al. Otx and POU expression patterns in Aurelia 415

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