Development of the Central Nervous System in the Larvacean Oikopleura Dioica and the Evolution of the Chordate Brain

Developmental Biology 285 (2005) 298 – 315 www.elsevier.com/locate/ydbio

Development of the central nervous system in the larvacean Oikopleura dioica and the evolution of the chordate brain

Cristian Can˜estro, Susan Bassham, John Postlethwait*

Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA

Received for publication 18 March 2005, revised 11 June 2005, accepted 17 June 2005 Available online 18 August 2005

Abstract

In non-vertebrate chordates, central nervous system (CNS) development has been studied in only two taxa, the Cephalochordata and a single Class (Ascidiacea) of the morphologically diverse Urochordata. To understand development and molecular regionalization of the brain in a different deeply diverging chordate clade, we isolated and determined the expression patterns of orthologs of vertebrate CNS markers (otxa, otxb, otxc, pax6, pax2/5/8a, pax2/5/8b, engrailed, and hox1)inOikopleura dioica (Subphylum Urochordata, Class Larvacea). The three Oikopleura otx genes are expressed similarly to vertebrate Otx paralogs, demonstrating that trans-homologs converged on similar evolutionary outcomes by independent neo- or subfunctionalization processes during the evolution of the two taxa. This work revealed that the Oikopleura CNS possesses homologs of the vertebrate forebrain, hindbrain, and spinal cord, but not the midbrain. Comparing larvacean gene expression patterns to published results in ascidians disclosed important developmental differences and similarities that suggest mechanisms of development likely present in their last common ancestor. In contrast to ascidians, the lack of a radical reorganization of the CNS as larvaceans become adults allows us to relate embryonic gene expression patterns to three subdivisions of the adult anterior brain. Our study of the Oikopleura brain provides new insights into chordate CNS evolution: first, the absence of midbrain is a urochordate synapomorphy and not a peculiarity of ascidians, perhaps resulting from their drastic CNS metamorphosis; second, there is no convincing evidence for a homolog of a midbrain–hindbrain boundary (MHB) organizer in urochordates; and third, the expression pattern of ‘‘MHB- genes’’ in the urochordate hindbrain suggests that they function in the development of specific neurons rather than in an MHB organizer. D 2005 Elsevier Inc. All rights reserved.

Keywords: Appendicularia; Forebrain; Midbrain; Hindbrain; Larvacea; Organizer; Chordate evolution of development; Tunicate; Subfunctionalization; Vertebrate; Urochordate; Rhombomere 4

Introduction surrounding hindbrain, at least in zebrafish (Maves et al., 2002; Walshe et al., 2002). In vertebrate embryos, the expression of Otx2 and Hoxb1 The isthmic (or midbrain–hindbrain boundary) organizer in the dorsal epiblast reveals the nascent forebrain + (MHB) develops in three phases: positioning, establishment, midbrain and hindbrain before these regions become and maintenance (Rhinn and Brand, 2001). In the position- morphologically distinct (Lumsden and Krumlauf, 1996). ing phase, the MHB arises during gastrulation just anterior Organizing centers then emerge along the anterior–posterior to the tip of the notochord between the posterior limit of the (AP) axis and play a crucial role in patterning the central Otx2 expression domain and the anterior limit of the nervous system (CNS). The isthmic organizer patterns the Gbx2 expression domain (Rubenstein et al., 1998). In the midbrain and hindbrain primordia (reviewed by Raible and establishment phase, Pax2, Fgf8, and Wnt1 expression Brand, 2004; Rhinn and Brand, 2001; Wurst and Bally-Cuif, initiates at the Otx2 –Gbx2 interface. During the mainte- 2001), and the rhombomere-4 (r4) organizer patterns the nance phase, genes already used in the establishment phase and their downstream targets, including En1, En2, Pax5, * Corresponding author. and Pax8, become mutually dependent for their continued E-mail address: [email protected] (J. Postlethwait). expression and maintain the boundary (Matsunaga et al.,

2000; Scholpp et al., 2003; Schwarz et al., 1999). Caudal to et al., 1999). This apparent conflict was not resolved by the MHB, the presence of a second organizing activity in recent studies of additional markers in ascidians, which zebrafish (Maves et al., 2002), and possibly in other revealed substantial variability in gene expression patterns vertebrates (Graham et al., 1993, Graham and Lumsden, among different ascidian species (Imai et al., 2002; Jiang 1996, Marin and Charnay, 2000a,b) has been suggested by and Smith, 2002; Mazet et al., 2003; Wada et al., 1998). The transplantation and ectopic expression studies; this activity absence of functional data leaves unclear the roles of these is located in rhombomere 4 (r4) of the hindbrain, and genes during ascidian and cephalochordate CNS develop- patterns the surrounding rhombomeres by FGF signaling in ment, or whether any portion of the ascidian CNS possesses the context of the Hox code. organizing activity. Many genes with roles in the MHB subsequently play Urochordate larvae possess the defining chordate fea- new roles in the progressive refinement of AP subdivisions tures of notochord, dorsal hollow nerve cord, gill slits, and a and in the differentiation of specific cell populations in the muscular, post-anal tail. In ascidians, an elaborate meta- CNS (Lumsden and Krumlauf, 1996). Most genes involved morphosis erases most of these chordate features as the in CNS patterning belong to multigene families, many of notochord is reabsorbed and the CNS is dramatically which arose during large-scale gene duplication events that restructured. It is possible that selective pressures leading accompanied, and likely facilitated, early vertebrate evolu- to rapid metamorphosis in ascidians have obscured or tion (Garcia-Ferna`ndez and Holland, 1994; Holland et al., compromised some developmental mechanisms present in 1994; Ohno, 1970). Understanding vertebrate CNS develop- stem chordates. In contrast, larvacean urochordates (Class ment is complicated because duplicated genes generally Appendicularia) retain a chordate body plan as adults. retain some redundancy but also acquire spatial and Phylogenetic relationships of urochordate classes are not temporal diversification of expression patterns (Force et yet known for certain, but the most accepted phylogeny al., 1999; McClintock et al., 2002; Postlethwait et al., 2004). places larvaceans as the basal sister group of ascidians + Nervous systems of the non-vertebrate chordates— thaliaceans (Christen and Braconnot, 1998; Holland et al., cephalochordates and urochordates—though simpler than 1988; Swalla et al., 2000; Wada, 1998; Wada and Satoh, the vertebrate CNS, share with vertebrates basic deve- 1994; but see Stach and Turbeville, 2002). Genomic lopmental genetic mechanisms (reviewed in Holland and analysis has uncovered substantial differences in genome Chen, 2001; Shimeld and Holland, 2000; Wada and Satoh, size and syntenic relationships between Oikopleura and 2001). Understanding developmental mechanisms in non- ascidians (Edvardsen et al., 2005; Seo et al., 2001; Seo et vertebrate chordates, which possess low gene redundancy al., 2004). For these reasons, the inclusion of larvaceans in due to their divergence before the large-scale duplication developmental genetic investigations is essential for obtain- events, can facilitate inferences for the roles of the founding ing a balanced view of development in the urochordate members of CNS gene families in the last common ancestor Subphylum. of chordates. The Subphylum Urochordata, or Tunicata, is In the work reported here, we present the first molecular the sister taxon of the Cephalochordata + Vertebrata clade genetic analysis of larvacean brain regionalization. Signifi- (but see Graham, 2004), and includes three Classes: cant homologies and differences between larvacean and ascidians, thaliaceans, and larvaceans. Among these, only ascidian CNS developmental mechanisms can be inferred ascidians have been intensively studied at the embryological from the comparison of gene expression patterns. Knowl- and molecular level. edge of Oikopleura CNS molecular regionalization provides The application of molecular and genomic tools has led new insights into chordate CNS evolution, helps illuminate to impressive progress in understanding ascidian develop- previous conflicting interpretations of ascidian CNS expres- ment (reviewed in Can˜estro et al., 2003; Corbo et al., 2001; sion data, extends within urochordates prior suggestions Holland and Gibson-Brown, 2003; Jeffery, 2002; Lemaire et about CNS organization that previously applied only to al., 2002; Meinertzhagen et al., 2004; Satoh, 2003; Sordino ascidians, and provides a basis for functional genetic et al., 2001). Ascidians and amphioxus share with verte- analysis. brates the early expression of otx anteriorly and hox1 posteriorly, with an intervening gap. In vertebrates, the MHB forms in this gap. The role and fate of this gap in non- Materials and methods vertebrate chordates, however, are problematic (reviewed in Holland and Holland, 1999; Wada and Satoh, 2001). The Animal culture, embryo staging, and imaging discovery that pax2/5/8a is expressed in the otx –hox1 gap in the ascidian Halocynthia roretzi led to the proposal that Oikopleura dioica individuals were collected with the ‘‘neck’’ of the ascidian CNS is homologous to the MHB plankton nets in the Pacific Ocean off Charleston, Oregon (Wada et al., 1998). In amphioxus, however, homologs of (Oregon Institute of Marine Biology) and Vancouver Island, vertebrate MHB markers Pax2/5/8, Engrailed, and Wnt1 are B.C. (Bamfield Marine Sciences Centre). Animals were not expressed in the otx –hox1 gap suggesting that cultured for several generations in 10-Am filtered seawater amphioxus lacks an MHB (Holland et al., 1997; Kozmik in 4-l plastic jars and fed with algal concentrates (‘‘Coral 300 C. Can˜estro et al. / Developmental Biology 285 (2005) 298–315 and Clam diet’’, Reed Mariculture). Embryos were grown at ature [e.g., fertilization to hatching requires less than 3 h at 13 T 3-C as described (Bassham and Postlethwait, 2000). 22-C, but about 12 h at 7-C(Fenaux, 1998)], we stage Because developmental rate varies strongly with temper- Oikopleura according to morphological landmarks. The

Fig. 1. CNS development in Oikopleura dioica. DIC images of live specimens with schematic insets depicting the CNS (yellow) and notochord (red). Anterior is to the left in lateral views of tailbud embryos (A–C) and hatchlings (D–F), and to the right in the adult (G–J). Different focal planes of incipient-tailbud (A) and mid-tailbud stages (B,C) show cell morphology and organization of developing structures. A dorsal optical section (boxed in C) shows the 2- to 4-cell width of the anterior brain at the level indicated in the schematic between panels B and C (dashed green line). An early hatchling (D), mid-hatchling (E), late hatchling (F), and adult (G–J) are shown with focal planes digitally integrated. Optical cross section (boxed in panel E) at the level of the sensory vesicle (green dashed line in insert sketch) shows the boundaries of the anterior brain, pharynx, and endostyle. Details of regions corresponding to the anterior brain (lateral in panel H, dorsal in panel I, green dashed lines) and caudal ganglion (J, blue dashed lines) are boxed in the schematic (G). In adults, the anterior brain (AB) contains about 70 cells (Martini, 1909), and includes a putative pituitary homolog (CF)(Bassham, 2002), which connects with the pharyngeal space, and a prominent sensory vesicle (SV), which includes a statolith (St) but, unlike ascidians, no pigmented cells (Georges et al., 1988; Holmberg, 1984). Paired afferent and efferent nerves (n1–n3) with sensory and motor functions emanate from the anterior brain (Bollner et al., 1991; Olsson et al., 1990). The trunk nerve cord (TNC), which lacks a central canal, bends ventrally between the stomach lobes and meets the caudal ganglion (CG) in the proximal tail. The approximately 25 neurons and other cells of the caudal ganglion coordinate activity of the caudal musculature, and receive inputs from the anterior brain and paired trunk mechanoreceptors (Bone and Mackie, 1975). A hollow spinal cord (SC) extends from the caudal ganglion down the tail (Bone, 1998). Schematic representations of panels F and I are shown in Figs. 5A,B. AB, anterior brain; CF, ciliary funnel; CG, caudal ganglion; CNS, central nervous system; E, esophagus; Ec, ectoderm; En, endoderm; Es, endostyle; Go, gonad; LL, lower lip; Mo, mouth; Mu, muscle; n1–3, paired nerves; No, notochord; Ph, pharynx; R, rectum; S, stomach; SC, spinal cord; SV, sensory vesicle; St, statocyte, Sl, statolith; TNC, trunk nerve cord; UL, upper lip; VO, ventral organ. C. Can˜estro et al. / Developmental Biology 285 (2005) 298–315 301 lack of a detailed blastomere fate map precludes an exact O. dioica genes by a name (usually based on its human description of expression patterns at cleavage stages ortholog) in italics and small-case letters (i.e., pax6), and (Nishino and Satoh, 2001). Stages prior to hatching include: proteins by a name with the first letter in upper-case (i.e., (i) incipient-tailbud stage (beginning about 3.5 h post- Pax6). When multiple paralogs are found, we add Latin fertilization (pf) at 13-C), characterized by an indentation at letters in alphabetical order (i.e., otxa, otxb, otxc) with no the initial demarcation of trunk and tail (Fig. 1A) and by an additional punctuation. Whole-mount in situ hybridization irregular, rod-shaped notochord. (ii) Mid-tailbud stage was performed as described (Bassham and Postlethwait, (beginning about 4 h pf at 13-C), in which a deep 2000). Riboprobe information is provided in Table S2. indentation divides trunk and tail (Figs. 1B,C), and a rod- shaped row of 20 notochord cells is easily distinguishable by DIC microscopy (Fig. 1C). A twisting of the tail with Results respect to the trunk (Delsman, 1910) is already pronounced at this stage. This flexure makes it impossible to position Isolation of Oikopleura CNS markers embryos for a true orthogonal image capture, but the notochord and large muscle cells provide helpful landmarks To study the development and regionalization of the to orient samples. Nuclear staining (i.e., DAPI, Hoechst) larvacean CNS, we characterized CNS markers that are was routinely included in expression analysis at tailbud highly conserved across bilaterians and play a central stages to confirm notochord cell positions. role in AP organization of the tripartite vertebrate brain Stages after hatching conform to Fenaux’s (1976), (Hirth et al., 2003; Reichert and Simeone, 2001). We including: (i) early hatchling (Fig. 1D) (Fenaux stage I), isolated eight O. dioica genes homologous to the Otx, in which the animals are cylindrical, without substantial Pax6, Pax2/5/8, Engrailed, and Hox1 vertebrate gene demarcation between trunk and tail, and, although the trunk families. Sequence comparisons and phylogenetic analyses still lacks obvious organs, the tail begins short bursts of unequivocally assigned each isolated Oikopleura gene movement; (ii) mid-hatchling (Fenaux II) (Fig. 1E), in to its gene family. We isolated single copies for Oiko- which the beginning of organogenesis is obvious, and pleura pax6 (AY870650), engrailed (AY870647), and animals can swim; (iii) late hatchling (Fenaux IV) (Fig. 1F), hox1 (AY871214) genes, three duplicated copies of otx in which movements of the heart and the cilia of the (AY886542, AY897556, AY897557) and two copies of digestive system and spiracles become visible, the trunk-tail pax2/5/8 (DQ020279, AY870648, AY870649). boundary is well defined, and coordinated swimming During the preparation of this manuscript, a list of O. improves; (iv) tailshift, characterized by the shift of the tail dioica homeobox genes inferred from genome sequences to an acute angle relative to the trunk, signaling the end of was reported (Edvardsen et al., 2005), and in the present embryonic development, and competence to secrete and work, we have adopted the same names for the three inflate a filter-feeding house (Figs. 1G–J). Oikopleura otx duplicate genes we independently isolated: The transparency of Oikopleura embryos and adults otxa, otxb, and otxc. Analysis of the predicted proteins for allows non-invasive study of internal morphology at the the three Oikopleura genes revealed the presence of a level of individual cells. For some images, we merged DIC homeodomain, a single C-terminal hexapeptide motif (in optical sections using Adobe-Photoshop software to contrast with two hexapeptide motifs in vertebrate OTX integrate images of structures that spanned focal planes proteins), and a moderately conserved WSP motif (Fig. (Figs. 1D–J). S1A). The small amount of evolutionary information provided by the conserved domains and variation in the Cloning and whole-mount in situ hybridization rest of the molecule hampered the construction of confident protein alignments and phylogenetic inferences. Among the Genomic DNA from about 50 Oikopleura individuals three Oikopleura Otx duplicates, Otxa showed the highest from Bamfield Marine Station was used to construct an overall similarity to ascidian Otx. Analysis of exon–intron arrayed fosmid library (Epicentre, CCFOS110). Complete organization of Oikopleura otx genes revealed multiple sequencing of targeted fosmid clones was performed by the introns in addition to the two introns conserved in all known DOE Joint Genome Institute (Walnut Creek, CA). mRNA otx genes (Fig. S1A). Remarkably, all ascidian and from about 1500 embryos was used to synthesize cDNA as Oikopleura otx genes are characterized by the presence of described (Bassham and Postlethwait, 2000). Genes of additional exons (yellow in Fig. S1A) between the interest were amplified by PCR with degenerate primers conserved exon containing the transcription origin and the (see Table S1 in supplementary materials) using cDNA or exon containing the N-terminal part of the homeobox. The genomic DNA as template. Gene-specific primers were still detectable sequence similarities among these additional designed for RACE PCR, and to screen the genomic library exons in urochordates suggest that these exons were using a pooling strategy. Coding sequence and gene originally gained before the separation of the ascidian and structures were inferred by sequence comparisons between larvacean lineages; thus, they are likely a synapomorphy of RACE products and fosmid genomic clones. We designate urochordate otx genes. Analysis of fosmid sequences from 302 C. Can˜estro et al. / Developmental Biology 285 (2005) 298–315 our Oikopleura genomic library (AY873983–AY873986) probably during gastrulation. Expression of otxb and hox1 shows that Oikopleura otxb and otxc are adjacent, is already detectable during cleavage stages (data not transcribed in opposite directions, and separated by a 17 shown), and probably provides the initial AP information kb stretch that contains no intervening putative genes. upon which subsequent AP regionalization is based. This result suggests that Oikopleura otxb and otxc At the incipient-tailbud stage, an anterior domain probably arose by tandem gene duplication during the expresses otxb and a posterior domain in the presumptive evolution of larvaceans after they separated from the CNS expresses hox1 (Figs. 2A,G,P). In addition to ascidian lineage. Evidence from protein sequence similar- epidermal expression, a broad otxb expression domain ities, gene structures, and phylogenetic analysis (data not may span the entire presumptive anterior brain (Fig. 2A). shown and Edvardsen et al., 2005), shows that the At this stage, we did not detect expression of otxa or duplication event that produced the third Oikopleura otx otxc. gene also probably occurred in the larvacean lineage after At the incipient-tailbud stage, the presumptive anterior it separated from the ascidian lineage, although an otx brain marked by otxb is subdivided by pax6 expression, gene lost during the evolution of the ascidian lineage which appeared in two separate domains (Figs. 2D,F,P). cannot be ruled out. At least two bilateral pairs of cells located at the midline We isolated from Oikopleura two pax2/5/8 genes, only constitute the most rostral pax6 domain (Fig. 2D.d1). one of which (pax2/5/8b) was reported by Edvardsen et al. The caudal pax6 domain, anterior to the tip of the (2005). Sequence similarities and phylogenetic analysis notochord, also consists of at least two bilateral pairs of showed that one Oikopleura pax2/5/8 gene (AY870648, cells (Fig. 2D.d1,d2). DQ020279) is orthologous to ascidian pax2/5/8a and the While otxb and pax6 mark the anterior CNS, bilateral other (AY870649) is orthologous to ascidian pax2/5/8b rows of cells express hox1 dorsolaterally and posteriorly (Fig. S1B). These results show that the duplication event to the anterior tip of the notochord, at the level of the that led to these two urochordate pax2/5/8 clades occurred prospective caudal ganglion and anterior spinal cord in the urochordate lineage after it diverged from the (Figs. 2G,P). cephalochordate + vertebrate lineage, but before the split Because cells expressing otxb, pax6, and hox1 in the of the ascidian and larvacean lineages; therefore, the pax2/5/ presumptive CNS were already internal and close to the 8a+pax2/5/8b duplicates appear to be a urochordate midline at incipient-tailbud stage, we conclude that by this synapomorphy. stage, neurulation is at or nearing completion. Contrary to expectations, neither of the two Oikopleura Development and AP regionalization of the Oikopleura pax2/5/8 genes is specifically expressed in the CNS. Pax2/ CNS 5/8a is expressed mainly in the trunk ectoderm (Fig. 2J). Complementary to pax2/5/8a expression, pax2/5/8b was Whole-mount in situ hybridization experiments revealed detected in most of the internal portion of the trunk, that the eight Oikopleura genes we isolated are transcribed probably including some presumptive CNS cells (Fig. 2M). in the embryonic CNS, endoderm, and epidermis. Here, we These diffuse pax2/5/8 expression patterns are likely not focus on expression patterns necessary to understand the artifactual since different non-overlapping probes render mechanisms of CNS development (Figs. 2–4). The complex identical patterns, and the same probes reveal distinctive patterns of other expression domains will be described in tissue-specificity at later stages (Fig. 2K and data not detail elsewhere. shown).

Incipient-tailbud stage Mid-tailbud stage Analysis of the expression of Oikopleura CNS markers At mid-tailbud stage, early broad gene expression revealed that AP regionalization of the CNS begins early, domains become refined and the expression of new CNS

Fig. 2. Expression of CNS genes before hatching. Whole-mount in situ hybridization in Oikopleura dioica incipient-tailbud (A,D,F,G,J,M,P) and mid-tailbud embryos (B,C,E,H,I,K,L,N,O,Q,R). otxb (yellow, A,B), otxa (yellow, C), pax6 (red, D–F), hox1 (light blue, G,H), hox1 + brachyury (I), pax2/5/8a (green, J,K), engrailed (dark blue, L,O), pax2/5/8b (M,N), and hox1 + engrailed (R). The central large image in each panel is a left lateral view, and the inset corner images (labeled with numbered lower case letters) are optical cross sections at the levels of the dashed lines, viewed from the aspect of the label relative to the line. In each panel, left insets are oriented with dorsal towards the top, and in right insets, anterior is towards the left. Double detection of hox1 and brachyury expression shows the relative position between the Oikopleura hindbrain and the notochord (I). Inspection of different specimens reveals variability of the cell position of the anterior hox1 expression domain (lateral and dorsal views, squared insets in panel I). The degree of twisting of the trunk relative to the tail is revealed by posterior CNS gene expression (i.e., H.h1,L.l1). Double in situ detection of the expression of hox1 and engrailed (R) demonstrates the hox1 –en – hox1 nested expression pattern (dorsal views oriented anterior towards the bottom; comparable to panels H.h2 and L.l2 optical sections). Arrowheads label expression in the presumptive CNS (yellow), epidermis (black), and endoderm (pink). The position of notochord cells, sometimes in a different focal plane, is indicated by white dots. The cell number and size, and notochord position relative to expression domains were routinely visualized by nuclear staining, as exemplified in panels F and O, which are the same individuals shown in panels D and L, respectively. Schematized summaries of gene expression patterns are projected onto incipient-tailbud (P) and mid-tailbud (Q) embryos in which Brachyury expression (Bra) labels notochord (white) (Bassham and Postlethwait, 2000). Scale bar = 20 Am. TB, tailbud stage. C. Can˜estro et al. / Developmental Biology 285 (2005) 298–315 303 markers appears. At this time, while otxb continues to domain (Figs. 2B,E,Q), apparently by down-regulation of label a row of cells in the midline of the presumptive the more posterior of the two domains seen in incipient- anterior brain, pax6 expression is reduced to a single tailbud embryos. At mid-tailbud stage, otxa expression was 304 C. Can˜estro et al. / Developmental Biology 285 (2005) 298–315 detected for the first time in the CNS, labeling asymmetri- tissues as early as incipient-tailbud stage (Figs. 1A–C), the cally one cell in the right side and at least four cells in the four main AP regions of the CNS—anterior brain (AB), left side of the presumptive anterior brain (Fig. 2C). At compact trunk nerve cord (TNC), caudal ganglion (CG), this stage, otxc expression was not yet detected within the and hollow tail nerve cord or spinal cord (SC)—do not CNS. become obviously demarcated from each other until early The posterior CNS becomes subdivided in mid-tailbud hatchling stages (Fig. 1D). This suggests that the primary embryos, with the appearance of a hox1 –engrailed –hox1 molecular definition of these regions is probably complete nested pattern. Analysis of this nested pattern by single and before hatching, and therefore, the later expression of otx, double in situ hybridization experiments, together with pax6, engrailed,andhox1 likely reflects secondary notochord cell positioning by detection of brachyury functions related to cell fate maintenance or to specifica- expression (Bassham and Postlethwait, 2000) and nuclear tion of neuron subpopulations, especially in the caudal staining (Figs. 2H,I,L,O,Q,R), revealed an anterior hox1 ganglion where motor neurons probably start to control tail domain spanning approximately from the AP level of the movements. Accordingly, while expression patterns of anterior tip of the notochord to the third notochord cell, a these genes are largely continuous from mid-tailbud stage, central engrailed domain at the level of the fourth notochord expression becomes restricted to specific cell subpopula- cell, and a posterior hox1 domain extending from the fifth to tions (Fig. 3). seventh notochord cells (Figs. 2H,I). Analysis of several In early and mid-hatchlings, pax6 is expressed in the dozen mid-tailbud embryos revealed slight variations in the anterior brain, outside the future sensory vesicle (Figs. positions of cells forming the anterior hox1 domain with 3A,B). A thin extension of expression reaches forward from respect to the tip of the notochord (Figs. 2H,I). At this stage, pax6-positive cells (rostral arrowhead in Fig. 3A), suggest- frontal views of embryos probed for CNS markers revealed ing the formation of neuronal processes. The internal pax2/ an approximately 60- counterclockwise twist of the dorso- 5/8a expression domain observed anterior to pax6 expres- ventral axis in the tail relative to the trunk, which causes the sion at mid-tailbud stage disappears by mid-hatchling stages posterior CNS to come to lie left of and somewhat dorsal to (data not shown). the notochord (Fig. 2e1,h1,k1,l1). Double detection of Otxb expression narrows in hatchlings, from initially hox1 and brachyury expression showed that some of the spanning the entire presumptive anterior brain (Fig. 2A) to cells of the anterior hox1 domain come to lie in the midline comprising only a few cells (Fig. 3D). Interestingly, as otxb of the trunk, dorsal and sometimes anterior to the expression declines in early hatchlings (Fig. 3D), the otxa notochord. We did not detect hox1 expression within the expression signal becomes intense (Fig. 3G), and for the notochord (Figs. 2H,I). first time, otxc transcripts begin to accumulate in CNS cells Although there is a gap between the posterior border of (Fig. 3J). This process, in which otxa + otxc expression the otxa + otxb expression domain and the hox1 expression seems to replace otxb expression over time, is finished by domain (see Figs. 2P,Q), we detected no pax2/5/8 late hatchling stages when otxb transcription is almost expression specific for the CNS in that gap. This contrasts undetectable in the anterior brain (Fig. 3F). with ascidians, in which pax2/5/8a expression fills the During hatchling stages, the expression of the three otx otx–hox1 gap (Wada et al., 1998). In contrast, two other duplicates is restricted to the central portion of the tissues express Oikopleura pax2/5/8a in the trunk: a small anterior brain, although there are differences in their patch of epidermal cells in the prospective mouth region, expression patterns; while some cells apparently co- and a group of internal cells at the boundary between the express multiple otx duplicates, other cells express only anterior brain and pharynx, rostral to the pax6 expression one, and in some cases the expression patterns are asym- domain (Fig. 2K, black, yellow arrowheads, respectively). metric (Figs. 3D,E,G,H,J,K). At this mid-tailbud stage, we could not discern the exact In early hatchling stages, in addition to strong epidermal anterior-most border of the anterior brain by morphology, hox1 expression at the trunk-tail transition (Fig. 4A, black and fate mapping is needed to confirm whether individual arrowheads), a row of hox1-expressing cells extends left of cells and their descendents in that region become part of the midline from anterior positions near the notochord tip the anterior brain or part of the pharynx roof. Broad (within the trunk) caudally to at least the level of the fourth expression of pax2/5/8b continues, restricted mainly to the notochord cell (Figs. 4A,B), probably spanning the trunk (Fig. 2N). prospective posterior TNC and anterior caudal ganglion (Fig. 1D). Additional hox1 signal with irregular shape and Early and mid-hatchling stages thin diameter appears at the level of the fifth and sixth During early hatchling stages, larvacean embryos notochord cells (Figs. 4A,B), and could mark caudal rapidly elongate and organs related to movement, such cellular extensions from anterior hox1-expressing cells. as the notochord, muscle cells, and CNS, are more mature These observations broadly confirm and extend those for than the digestive system (Fig. 1D). Although the trans- hox1 in Seo et al. (2004). In early hatchlings, in addition to parency of living Oikopleura embryos allowed us to the expression of hox1 in the anterior segment of the distinguish much of the developing CNS from surrounding caudal ganglion, we observed expression of engrailed (Fig. C. Can˜estro et al. / Developmental Biology 285 (2005) 298–315 305

Fig. 3. Expression of anterior CNS genes after hatching. Whole-mount in situ hybridization in Oikopleura dioica early hatchlings (left panels: A,D,G,J), mid- hatchlings (central panels: B,E,H,K), and late hatchlings (right panels: C,F,I,L). pax6 (red A–C), otxb (D–F), otxa (G–I), otxc (J–L). All panels have a left lateral view (lat) and additional features of the gene expression patterns are shown in dorsal (dor), ventral (vent), posterior (post), and frontal (front) perspectives, corresponding to planes labeled by dashed lines or numbered arrows on the lateral views. Anterior is towards the left and dorsal towards the top. Arrowheads label expression in the CNS (yellow), endoderm (pink), and epidermis (black). Scale bar = 20 Am. Abbreviations as in Fig. 1.

4D), and, transiently, pax6 (Fig. 3A) posterior to the caudal ganglion down-regulates hox1 expression (Fig. 4B). notochord tip. We found no expression of hox1 in the spinal cord, although In mid-hatchling stages, while precursor cells of the punctate, non-epidermal hox1 and engrailed signal appears posterior TNC continue to express hox1, the prospective broadly distributed along mid- and late-hatchling tails (Figs. 306 C. Can˜estro et al. / Developmental Biology 285 (2005) 298–315

Fig. 4. Expression of posterior CNS genes after hatching. Whole-mount in situ hybridization in Oikopleura dioica early hatchlings (left panels: A,D), mid-hatchlings (central panels: B,E), and late hatchlings (right panels: C,F). hox1 (light blue, A–C), engrailed (dark blue, D–F). Details of hox1 expression at the base of the Langerhans receptors (LR in F) and engrailed in the caudal ganglion (CG in O) are boxed. Arrowheads label expression in the CNS (yellow), endoderm (pink), epidermis (black), and hox1 and engrailed tail-specific expression (white). Scale bar = 20 Am. Abbreviations as in Figs. 1 and 3.

4B,E,F, white arrowheads). Although this punctate pattern is brain, based on fine structure and nerve positions (Olsson specific for these two genes, the pattern varies in different et al., 1990). Calling these regions AB1–3 avoids animals. potentially misleading homologies with the vertebrate brain (see Discussion). Late-hatchling stage In the posterior trunk of late hatchlings, the TNC bends In late hatchlings, the expression pattern of CNS 90- towards the caudal ganglion (Fig. 1F). Only the markers mapped to the four morphologically distinct posterior half of the TNC is labeled by hox1 expression regions of the CNS, the anterior brain (AB), trunk nerve (Figs. 4C and 5A). Hox1 signal was also detected in the cord (TNC), caudal ganglion, and spinal cord (Figs. 3–5), sensory field that includes the epidermal Langerhans and they divide the anterior brain into three subdivisions mechanoreceptors (Fig. 4C), which are innervated by (AB1–3)(Figs. 5A,B). AB1, the anterior subdivision, is axons from the caudal ganglion (Bone and Mackie, labeled by pax6 (Fig. 3C). The ciliary funnel and the 1975). At this late stage, the caudal ganglion itself does anterior extensions from AB1 that form the rostral paired not express hox1, and among the genes we studied, only nerve n1 appear to be free of pax6 expression. AB2, the engrailed expression appeared in the anterior part of the central subdivision, is broadly marked by the expression of caudal ganglion (Figs. 4C,F). pax6, otxa, and otxc (Figs. 3C,I,L). While some cells appear to co-express pax6, otxa, and otxc genes, the most dorsal AB2 cells appear not to express otx, and the most Discussion ventral cells appear not to express pax6. In addition, the statocyte expresses otxa, but not pax6 or otxc (Figs. Homologies and differences between larvacean and 3C,I,L). AB3, the posterior subdivision, was free of any ascidian CNS development pax6, otx, engrailed, pax2/5/8, and hox1 signal (Figs. 3C,F,I,L, 4C,F, and 5A). These three subdivisions of the This study of gene expression in the developing anterior brain broadly coincide with regions of the Oikopleura CNS reveals the molecular genetic region- Oikopleura brain previously called fore-, mid-, and hind- alization of the larvacean CNS, and allows comparison C. Can˜estro et al. / Developmental Biology 285 (2005) 298–315 307

Fig. 5. (A) Schematized summary of gene expression patterns projected onto the CNS of a late hatchling (lateral view). pax6 (red), otxa + otxc (yellow), hox1

(light blue), and engrailed (dark blue). Notochord in pink. (B) Schematic representation of the anterior brain AP subdivisions (AB1–3) in relation to neural circuits (colored cells) in the adult brain described by Olsson et al. (1990). Afferent and efferent nerve pathways (paired nerves, n1–n3, and dorsal nerves, dn) connecting the anterior brain (AB) with target cells (white: epidermal cells, EC; ciliary rings of spiracles, CR; ventral organ, VO; and upper and lower lips, UL,LL) are shown. Abbreviations are as in Fig. 1. (C) Schematic comparison of CNS anteroposterior regionalization to gene expression patterns among chordate Subphyla. Tree (bottom) indicates phylogenetic relationships. For larvaceans and ascidians, gene expression patterns are detailed during different developmental stages (grey boxes). Gene expression domains labeled with an asterisk appear relatively late in development. Grey spheres represent proximal notochord cells. Cephalochordate CNS regions are depicted relative to somite positions (s1–8). Vertebrate and urochordate data support homologies for forebrain (black) and hindbrain (blue), but not for midbrain or MHB.

with ascidians. The ascidian CNS has been divided into The anterior CNS four regions: the ‘‘brain’’ (which includes the sensory vesicle housing the statocyte and ocellus), neck, The Oikopleura anterior brain and ascidian ‘‘brain’’ are visceral ganglion, and spinal cord (Satoh, 2003). The homologous. Expression of Oikopleura otxb and ascidian following section highlights evidence that defines otx demarcates the anterior CNS in cleavage stages regional homologies in the CNS of two urochordate (Hinman and Degnan, 2000; Hudson and Lemaire, 2001; classes, and discusses the differences observed along Wada et al., 1996; Wada et al., 2004; and this study). Both the AP axis of the CNS, starting rostrally and moving urochordate classes have two pax6 expression domains, the caudally. posterior of which overlaps the expression of otx near the 308 C. Can˜estro et al. / Developmental Biology 285 (2005) 298–315 anterior tip of the notochord (Figs. 2P and 5C.c,f)(Glardon common ancestral pro-ortholog (Sharman, 1999)] converg- et al., 1997; Mazet et al., 2003). By the mid-tailbud stage ing on similar evolutionary outcomes. Functional analysis in Oikopleura, although the otxb posterior boundary does of otx regulation in Oikopleura duplicates will test whether not change relative to the notochord, pax6 is expressed this evolutionary convergence is due to parallel subfunc- only in the former anterior domain, suggesting down- tionalization events in conserved regulatory modules (Force regulation of pax6 expression in the posterior domain et al., 1999) present in the last common ancestor of (Figs. 2Q and 5C.d). Similarly, pax6 expression is absent vertebrates and urochordates, or whether phased otx from the posterior part of the ascidian brain at mid-tailbud regulation has been gained independently in the two stage (Glardon et al., 1997; Mazet et al., 2003). This chordate lineages. congruence of otx and pax6 expression patterns in the early tailbud stages of Oikopleura and ascidians supports Pax2/5/8 genes reveal differences between Oikopleura and homology of the larvacean anterior brain and the ascidian ascidian anterior CNS regions. In ascidians, the expres- ‘‘brain’’. sion of pax2/5/8a in the gap between otx and hox1 domains suggested homology with the vertebrate MHB (Wada et al., Expression of otx and pax6 reveals differences between the 1998). Despite the expression similarities between Oiko- Oikopleura anterior brain and the ascidian ‘‘brain’’. Al- pleura and ascidian pax2/5/8a and pax2/5/8b orthologs in though early expression of otx and pax6 is similar in the two several domains (Mazet et al., 2005; Wada et al., 1998), urochordate classes, at later stages, there are three major neither Oikopleura pax2/5/8 gene is expressed in the gap differences. First, pax6, which is typically expressed in between otx and hox1 domains. Therefore, pax2/5/8 photoreceptors (Callaerts et al., 1997), is expressed in the expression data argue against the presence of an MHB photosensitive ocellus in the ascidian sensory vesicle homolog in Oikopleura. (Glardon et al., 1997), but not in that of Oikopleura, which In mid-tailbud stages, ascidians lack pax2/5/8a expres- lacks pigmented photoreceptor cells. The second major sion similar to the Oikopleura pax2/5/8a internal domain at difference is that while pax6 is no longer expressed in the the brain–pharynx border anterior to the pax6 domain ascidian ‘‘brain’’ by late larval stages (Mazet et al., 2003), in (Mazet et al., 2003; Wada et al., 1998). In amphioxus, pax2/ Oikopleura, it continues to be expressed in the anterior brain 5/8 expression in the anterior cerebral vesicle has been until at least the late hatchling stage (Figs. 3C and 5A,C.e). compared to vertebrate Pax2 expression in development of This expression difference perhaps reflects the continuity of the optic stalk and optic nerve (Fig. 5C.a) (Kozmik et al., embryonic and adult Oikopleura CNS patterning (see 1999; Krauss et al., 1991; Macdonald et al., 1997). discussion below), while the ascidian CNS is drastically Additionally, it was proposed that pax2.1, in cooperation restructured during metamorphosis. with Fgf signaling, influences axon guidance and early The third major difference is that the three Oikopleura rostral midline development in the zebrafish forebrain otx genes, in contrast to the single ascidian otx gene, show (Shanmugalingam et al., 2000). Because AB1, the rostral ‘‘phased’’ expression dynamics, differing in their temporal subdivision of the anterior brain of Oikopleura, projects and spatial expression patterns during CNS development. axons rostrally via the paired nerves n1 and n2 to the ventral These results suggest the hypothesis that the three otx organ and sensory receptors in the lips (Figs. 1I and 5B), genes in Oikopleura have different developmental func- Oikopleura pax2/5/8a expression is consistent with a role in tions. The broad and uniform expression of the single otx axon guidance like its vertebrate homolog Pax2. Study of gene described in the ascidian brain (Wada et al., 1996) more markers and gain- and loss-of-function experiments would mask potential multiple functions. Like Oikopleura, are necessary to test these hypotheses for the role of the tetrapods have three OTX paralogs (OTX1, OTX2, and Oikopleura pax2/5/8a. OTX5/CRX) due to independent duplications within the vertebrate lineage. The vertebrate paralogs, like their Links between developmental expression patterns and the Oikopleura homologs, have also assumed separate early adult larvacean brain. In contrast to ascidians, the and late developmental roles. First, the earliest expression continuous transition of the Oikopleura CNS from hatch- of Oikopleura otxb in the anterior neuroectoderm and ling to adult provides direct links between embryonic gene endoderm is comparable to the expression of OTX2 during expression domains and the fine structure and function of vertebrate gastrulation (Reichert and Simeone, 2001). And the adult Oikopleura brain (Olsson et al., 1990). Figs. 5A,B second, the later appearance of Oikopleura otxa and otxb show the major anatomical features of the anterior CNS in expression in a reduced number of cells in the anterior the juvenile and adult Oikopleura. The rostral subdivision brain can be compared to the action of OTX1 and OTX2 in of the anterior brain (AB1) labeled by pax6 expression specifying identity and fate of specific cell populations in receives afferent pathways from anterior sensory cells, the vertebrate brain (Puelles et al., 2003; Puelles et al., including the ventral organ (via n1), and ciliated receptor 2004). This parallelism between vertebrates and Oiko- cells in the lips and pharynx (via n2) (Olsson et al., 1990; pleura provides an example of trans-homologs [genes and Fig. 5B). The efferent pathways described by Olsson et duplicated independently in different lineages from a al. (1990) may originate in the central and posterior C. Can˜estro et al. / Developmental Biology 285 (2005) 298–315 309

subdivisions AB2 and AB3.AB2, which is labeled by pax6, the CNS of the two urochordate classes, similar morpho- otxa, and otxc, probably includes three neurons that project genetic processes seem to be conserved underlying the axons via n3, a postulated motor nerve carrying fibers to development of the neural tube at the level of the anterior the spiracle’s ciliary rings and to ventral epidermal cells tip of the notochord. that might have a role in house secretion (Fig. 5B). The Engrailed expression in the posterior CNS is also similar posterior subdivision AB3, which does not express any of among urochordates. In Oikopleura, engrailed expression the genes in this study during late development, sends one begins at mid-tailbud stage between the two initial hox1 process via the left n3 to the ciliary ring and another into domains creating a hox1–engrailed–hox1 nested pattern the TNC, probably towards the caudal ganglion. Therefore, (Fig. 2R). Throughout development, engrailed expression the AB1–3 subdivisions revealed by molecular markers continues posterior to the anterior notochord tip, labeling the during development seem to correspond to different func- anterior part of the caudal ganglion (Figs. 2L,O,R and 4D– tional areas of the brain in charge of integrating afferent F). Expression of engrailed differs between the two and efferent pathways. congeneric ascidian species in which it has been described. In Ciona savignyi, a single bilateral pair of cells co- The posterior CNS expresses engrailed and pax2/5/8a (Jiang and Smith, 2002). The posterior urochordate CNS features a prominent In the congener C. intestinalis, pax2/5/8a is also expressed ganglion near the anterior tip of the notochord called the in a single pair of cells, but engrailed flanks the pax2/5/8 caudal ganglion in Oikopleura and the visceral ganglion expression domain in an engrailed-pax2/5/8a-engrailed in ascidians (Fig. 1)(Meinertzhagen et al., 2000). Variation nested pattern (Fig. 5C.g), in which the posterior engrailed in gene expression and morphology among ascidian species, domain labels the anterior part of the visceral ganglion (Imai however, hinders a straightforward assignment of homology et al., 2002). Although no direct comparison of hox1 and between larvacean and ascidian posterior CNS regions. engrailed expression has been published for any ascidian, Larvaceans and ascidians share a two-domain expression comparing the positions of hox1 and engrailed expression profile for hox1 (Fig. 5C.d,g): an anterior domain near the domains with the notochord (Ikuta et al., 2004; Imai et al., anterior tip of the notochord [compare dorsal views of 2002; Nagatomo and Fujiwara, 2003) makes it likely that Oikopleura in Fig. 4A to views of the ascidian in Fig. 5 of the hox1 –engrailed –hox1 nested pattern is shared by Katsuyama et al. (1995)] and a posterior, transient domain in Oikopleura and at least some ascidians (Fig. 5C.g). The the presumptive caudal/visceral ganglion and spinal cord fact that pax6 is expressed transiently at early hatchling (Figs. 2P–Q and 5A,C)(Ikuta et al., 2004; Katsuyama et al., stages in the presumptive caudal/visceral ganglion approx- 1995; Nagatomo and Fujiwara, 2003). In Oikopleura,we imately at the same level as engrailed (Glardon et al., can trace the anterior hox1 domain throughout development 1997; Mazet et al., 2003; and our Fig. 3A) bolsters the to the posterior TNC. In ascidians, although the hox1 correspondence of larvacean and ascidian posterior expression pattern is the same in different species, various engrailed domains. authors have differed in their interpretation of the fate of the The similar expression of engrailed, pax6, and hox1 anterior domain, assigning it either to the neck or to the during development of the caudal ganglion in Oikopleura visceral ganglion (Ikuta et al., 2004; Katsuyama et al., 1995; and the visceral ganglion in ascidians suggests that these Nagatomo and Fujiwara, 2003; Wada et al., 1998). This two structures are homologous. The present expression data controversy is probably due to the lack of a morphologically are also consistent with the homology of the ascidian neck distinguished neck in H. roretzi rather than a true fate and the posterior Oikopleura TNC. In agreement with the difference. interpretation that the ascidian neck is part of the visceral These expression data also indicate shared morphoge- ganglion (Meinertzhagen et al., 2000), we conclude that the netic behaviors of larvacean and ascidian axial tissues. The posterior TNC and the caudal ganglion derive from the same anterior hox1 expression domain shifts forward with embryonic CNS region labeled by the single hox1 domain at respect to the notochord during Oikopleura development, incipient-tailbud stage (Fig. 2G). Because Oikopleura as it does in ascidians (Katsuyama et al., 1995). This engrailed expression remains in a constant position poste- domain shift probably explains the variability of the cell rior to the tip of the notochord, while the hox1 domain shifts positions of the anterior hox1 domain observed in anteriorly, there may also be elongation or migration of Oikopleura mid-tailbud stages as different individuals are specific cells, or stationary cells turning on and off fixed at slightly different stages (Figs. 2H–I,R). In Ciona expression, rather than simply a general anterior shift of intestinalis, neck precursor cells apparently move from this entire part of the posterior CNS. behind the tip of the notochord in tailbud embryos to an anterior location in hatchlings, suggesting that the neck is Homologies and differences between urochordate and part of the visceral ganglion (Cole and Meinertzhagen, vertebrate CNS development 2004; Meinertzhagen et al., 2000). This shift applies also to H. roretzi pax2/5/8a-positive cells (Wada et al., 1998). Our comparative analysis of Oikopleura and ascidian Therefore, despite the morphological differences between urochordates has revealed homologies and differences in 310 C. Can˜estro et al. / Developmental Biology 285 (2005) 298–315 developmental genetic pathways, which now can be Which part of the vertebrate CNS is homologous to the compared to other chordate Subphyla. urochordate ‘‘anterior brain’’? In addition to the ‘‘urochordate hindbrain’’, we designate Which part of the vertebrate CNS is homologous to the the anterior part of the urochordate CNS as ‘‘anterior brain’’, urochordate spinal cord? rather than just ‘‘brain’’, because in vertebrates the term In contrast to ascidians and vertebrates, Oikopleura and ‘‘brain’’ includes the forebrain, midbrain, and hindbrain. cephalochordates (Glardon et al., 1998) lack pax6 expres- Data from ascidians have led to conflicting interpreta- sion along the length of their spinal cords. Because the tions concerning homologies between the ascidian ‘‘brain’’ posterior Drosophila CNS expresses ey, which is the fly and the vertebrate brain, sometimes because of differences ortholog of pax6 (Quiring et al., 1994), the most parsimo- in morphology and gene expression among ascidian species nious explanation for species-specific pax6 expression (Lemaire et al., 2002; Locascio et al., 1999; Meinertzhagen patterns is that the last common ancestor of extant chordates and Okamura, 2001; Meinertzhagen et al., 2004; Satoh, had pax6 expression in the spinal cord and it was 2003; Takahashi and Holland, 2004; Wada and Satoh, independently lost in the Oikopleura and cephalochordate 2001). For example, the posterior part of the ascidian lineages. ‘‘brain’’ has inconsistently been proposed to be homologous The similar expression pattern between ascidian hox5 to (i) the vertebrate metencephalon [based on the co- (Gionti et al., 1998) and the only Oikopleura central Hox- expression of Ci-fgf9/16/20 and Ci-engrailed (Meinertzha- subclass gene (called hox4, although equally related to gen et al., 2004)]; (ii) the vertebrate midbrain [based on the hox4/5/6/7)(Seo et al., 2004) suggests that these genes co-expression of Ci-otx and Ci-engrailed (Imai et al., may function to define the anterior boundary of the 2002)]; or (iii) the vertebrate forebrain [based on the urochordate spinal cord. Therefore, despite the variation presence of Ci-otx expression (Hudson and Lemaire, in pax6 expression patterns among chordates, we conclude 2001) and the absence of Ci-dmbx expression (Takahashi that the spinal cord of urochordates and vertebrates is and Holland, 2004)]. To address these conflicting conclu- homologous. sions, we first integrated larvacean and ascidian CNS gene expression patterns (see above), and now we compare that Which part of the vertebrate CNS is homologous to the result to vertebrate expression patterns. urochordate TNC/neck and caudal ganglion/visceral In vertebrates, forebrain and midbrain are labeled by Otx ganglion? expression (Fig. 5C.b). The expression of Pax6 in two Since the caudal ganglion of Oikopleura and the domains, one in the posterior forebrain and one in the ascidian visceral ganglion are probably homologous, as anterior hindbrain, has been used to define the midbrain, are the posterior TNC in Oikopleura and the ascidian which develops in the intervening gap (‘‘pax6-gap’’) and is neck, and since these structures derive from a posterior regulated by Pax2 and Engrailed expression (Fig. 5C.b) CNS region expressing hox1, we conclude that these (Matsunaga et al., 2000; Scholpp et al., 2003; Schwarz et structures are homologous to at least part of the al., 1999). vertebrate hindbrain (Fig. 5C.b,c). The presence of motor As in vertebrates, the prospective anterior brain of function in the vertebrate hindbrain (Lumsden and urochordates is labeled by otx expression (Fig. 5C.c,f), Krumlauf, 1996), the existence of motor neurons in the suggesting at first glance that the urochordate anterior brain caudal ganglion that coordinate muscular tail movements is homologous to the vertebrate forebrain + midbrain (Fig. in larvaceans (Bone, 1998), and the presence of motor 5C.b). In urochordates, however, the fact that the otx neurons in the neck and visceral ganglion of ascidians domain is subdivided along the AP axis by two expression (Katsuyama et al., 2005; Meinertzhagen et al., 2000; domains of pax6 at early tailbud stage (Fig. 5C.c,f), leads to Okada et al., 2002) are consistent with this proposed an alternative interpretation. In this alternative, the anterior homology; we will therefore refer to the posterior-TNC/ pax6 domain in urochordates labels the homolog of the neck plus caudal/visceral ganglion as the ‘‘urochordate vertebrate forebrain, while the posterior pax6 domain labels hindbrain’’. the homolog of the anterior hindbrain, and the pax6-gap In ascidians, hox3 expression suggests that the anterior could be the urochordate homolog of the vertebrate limit of the visceral ganglion corresponds to the anterior midbrain. This interpretation, however, conflicts with two limit of r4 of the vertebrate hindbrain (Locascio et al., facts. First, the pax6-gap of the urochordate anterior brain 1999). Unexpectedly, despite other gene expression fails to express the vertebrate midbrain markers pax2/5/8 similarities between the larvacean caudal ganglion and and engrailed (Fig. 5C.b,c,f). And second, the posterior the ascidian visceral ganglion (Fig. 5C.d,g), there does not expression domain of pax6 in the urochordate anterior brain appear to be a hox3 ortholog in the Oikopleura genome overlaps the otx expression domain, while Otx expression is (Seo et al., 2004). Analysis of additional hindbrain excluded from the vertebrate hindbrain (Fig. 5C.b,c,f). markers such as Kreisler and Krox20 could help us Therefore, these data lead to the conclusion that the understand the consequences of the loss of Oikopleura urochordate anterior brain is homologous to the vertebrate hox3. forebrain (Fig. 5C.b,c). C. Can˜estro et al. / Developmental Biology 285 (2005) 298–315 311

New perspectives on chordate CNS evolution from an Oikopleura CNS are also consistent with a different Oikopleura point of view hypothesis. The posterior overlap of otx and pax6 expression, the absence of gbx in urochordates, and the Gbx gene loss, an ancestral event in urochordate evolution variability or absence of the expression of orthologs of In vertebrates, the action of Gbx excludes the posterior vertebrate midbrain markers (such as engrailed and pax2/ boundary of Otx expression from the anterior hindbrain 5/8) in the urochordate anterior brain, suggest that the labeled by Pax6 (Fig. 5C.b) (reviewed in Rhinn and Brand, midbrain was present in the last common ancestor of 2001). We know that the boundary between Otx and Gbx extant chordates, but was modified or lost independently in expression domains is crucial to define the limit between the the urochordate and in the cephalochordate lineages. Study midbrain and the hindbrain in vertebrates because vertebrate of the developmental mechanisms underlying the posterior Gbx mutants show caudal expansion of Otx expression back subdivision of the anterior brain of Oikopleura (AB3) to the level of rhombomere 4. The finding that Gbx also could provide new data to better understand the evolution antagonizes Otx in Drosophila suggests that this gene of the urochordate brain, because AB3 is located just interaction is ancient in Bilateria (Hirth et al., 2003; Reichert anterior to the urochordate hindbrain, and its homology is and Simeone, 2001). Interestingly, the apparent absence as yet uncertain due to the absence of expression of all of any Gbx homolog from the genome projects of C. CNS markers so far analyzed. intestinalis (Wada et al., 2003) and C. savignyi (http:// www.broad.mit.edu/annotation/ciona/index.html), and the Do urochordates have a midbrain–hindbrain organizer failure to isolate a Gbx homolog from Oikopleura [despite (MHB)? our unpublished efforts and the genome sequencing traces In vertebrates, the MHB forms at the border of Otx and (Edvardsen et al., 2005)], strongly suggest that the loss of Gbx expression domains, and requires expression of Pax2, gbx is a urochordate synapomorphy. This gene loss might Engrailed, Wnt11, and Fgf8 genes (reviewed in Rhinn and have affected the evolution and regionalization of the Brand, 2001). The absence of a Gbx ortholog in urochor- urochordate CNS, perhaps by permitting a caudal expansion dates makes it difficult to recognize a putative MHB of the otx domain as observed in vertebrate Gbx mutants. A organizer in this Subphylum. In Oikopleura, the absence caudal expansion of otx expression in an ancestral urochor- of engrailed and pax2/5/8 expression immediately posterior date would be consistent with our comparative analysis of to the otx expression domain argues against the presence of Oikopleura and ascidian data, which shows that the an MHB organizer homolog in the larvacean CNS. posterior boundary of otx expression overlaps with the In ascidians, it was argued that because pax2/5/8 posterior pax6 expression domain in the anterior brain (see expression in the neck coincides with the gap between otx discussion above and Fig. 5C.b,c,f). The study of additional and hox1 expression domains (Wada et al., 1998), and urochordate orthologs of genes upstream and downstream of because engrailed and fgf8/17/18 expression appears in the Gbx function in the vertebrate anterior hindbrain (e.g., anterior visceral ganglion (Imai et al., 2002), the neck region Wnt1, Fgf8) will help assess the impact of the loss of gbx of the ascidian CNS is homologous to the vertebrate from the urochordate genome. MHB (Imai et al., 2002; Jiang and Smith, 2002; Meinertz- hagen et al., 2004; Satoh, 2003; Wada et al., 1998; Do urochordates have a homolog of the vertebrate Wada and Satoh, 2001). Close inspection of Ci-pax2/5/8, midbrain? Ci-engrailed, and Ci-fgf8/17/18, however, reveals that they In the Oikopleura CNS, absence of engrailed and pax2/ are not co-expressed in C. intestinalis (Imai et al., 2002). If 5/8 expression anterior to the hox1 expression domain that these genes are not co-expressed, then they cannot have the labels the hindbrain argues against the presence of midbrain same genetic interactions their orthologs have in the in the larvacean lineage. Thus, we conclude that the absence development of the vertebrate MHB. Even if Ci-fgf8/17/ of a midbrain in ascidians, recently proposed by T. 18 signaling might provide organizer function in the Takahashi and P.W.H. Holland (2004) based on the absence anterior part of the visceral ganglion in ascidians, that of dmbx expression in the anterior brain, is not just a function might not necessarily be homologous to the MHB peculiarity of the ascidian Class, resulting perhaps from its organizer specifically, because Fgf8 is also active in the r4 drastic CNS metamorphosis, but is a synapomorphy of the organizer in the hindbrain of the vertebrate Danio rerio urochordate Subphylum. (Maves et al., 2002; Walshe et al., 2002). The interpretation Absence of evidence supporting the existence of a that Fgf expression in the ascidian CNS could be midbrain in amphioxus (Holland et al., 1997) and ascidians homologous to the r4 organizer is compatible with our (Takahashi and Holland, 2004) led to the most parsimonious definition of the ‘‘urochordate hindbrain’’, and with the conclusion that the absence of a distinct midbrain was the correspondence of the anterior part of the visceral ganglion ancestral condition for chordates, and that the midbrain is a in ascidians with rhombomere 4 in vertebrates based on developmental innovation in the vertebrate lineage (Takaha- expression of Ci-hox3 (Locascio et al., 1999). shi and Holland, 2004). Although this conclusion continues Finally, the obvious differences in the expression patterns to be the most parsimonious explanation, our data on of engrailed and pax2/5/8a between Oikopleura and 312 C. Can˜estro et al. / Developmental Biology 285 (2005) 298–315 ascidians, and indeed among different ascidian species [i.e., MHB organizer. Functional experiments will be necessary even between the congeners C. savignyi and C. intestinalis to disprove this conclusion. (Imai et al., 2002; Jiang and Smith, 2002)], are not expected if the presence of an MHB organizer is fundamental for the regionalization of at least the ascidian CNS. Therefore, in Acknowledgments the light of our data from Oikopleura and published results in ascidians, and in the absence of any functional data about We are grateful to Skipper B. Young of the ‘‘Charming the roles of engrailed, pax2/5/8, and fgf8/17/18 in ascidians, Polly’’ for help in larvacean collecting. We thank F. Mazet we conclude that there is no convincing evidence for an for sharing with us her unpublished results on the expression MHB homolog in urochordates. Characterization and func- of Ci-pax2/5/8b in Ciona intestinalis. We thank L. Maves tional analysis of Fgf family members in Oikopleura and and W. Cresko for helpful suggestions and discussion, and ascidians would help to test whether organizer activity exists A. Amores for providing Hox and Engrailed primers. We in urochordates. thank undergraduate researchers T. Siriphatnaboon and T. Keopuhiwa for help with animal care. We appreciate the Alternative hypothesis for the function of ‘‘MHB genes’’ in work of two anonymous reviewers for helping to improve urochordates the manuscript. Complete fosmid sequencing was per- Urochordate tailbud stage embryos may correspond to formed under the auspices of the U.S. Department of much later developmental stages than the vertebrate gastrula Energy, Office of Biological and Environmental Research, and neurula stages in which the MHB forms. In vertebrates, in the University of California, Lawrence Berkeley National many of the genes that are involved in the development of Laboratory (contract DE-AC03-76SF00098). This material the MHB are also expressed later in the hindbrain, where is based on work supported by the National Science they perform different functions than they do in the MHB Foundation under Grant No. IBN-0345203 and an IGERT (Lumsden and Krumlauf, 1996). In the vertebrate hindbrain, grant in Evolution of Development DGE-9972830. Any Pax2, Pax5, Pax8, En1, En2, Pax6, Hox1, Dmbx1, and Lim opinions, findings, and conclusions or recommendations transcription factors, in conjunction with graded FGF expressed in this material are those of the author(s) and do signals, specify motor neurons and interneurons, and are not necessarily reflect the views of the National Science important for axon guidance (Burrill et al., 1997; Dasen et Foundation. We thank the Spanish Ministry of Education, al., 2003; Gavalas et al., 2003; Irving et al., 2002; Kawahara Culture and Sports for support for CC (EX2002-0059). et al., 2002; Pfeffer et al., 1998; Sapir et al., 2004; Segawa et al., 2001). In cephalochordates, the expression of pax2/5/8 and en that appears during late developmental stages in the Appendix A. Supplementary data amphioxus hindbrain, has been postulated to be related to functions in neuron specification (Holland and Holland, Supplementary data associated with this article can be 1999; Holland et al., 1997; Kozmik et al., 1999). The late found, in the online version, at doi:10.1016/j.ydbio.2005. expression of ‘‘MHB genes’’ in the vertebrate and cepha- 06.039. lochordate hindbrains raises the hypothesis that tailbud stage expression of the orthologs of these transcription factors in the ‘‘urochordate hindbrain’’ is related to specification of References neuron fate and axon guidance rather than an organizer function. Bassham, S., 2002. Molecular biology of a larvacean urochordate, This alternative hypothesis is consistent with several Oikopleura dioica, and the origin of chordate innovations. Ph.D. facts. 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