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Developmental Biology 2

Development of the central nervous system in the larvacean

Oikopleura dioica and the evolution of the chordate brain

Cristian Canestro, 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) in Oikopleura 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

In vertebrate embryos, the expression of Otx2 and Hoxb1

in the dorsal epiblast reveals the nascent forebrain +

midbrain and hindbrain before these regions become

morphologically distinct (Lumsden and Krumlauf, 1996).

Organizing centers then emerge along the anterior–posterior

(AP) axis and play a crucial role in patterning the central

nervous system (CNS). The isthmic organizer patterns the

midbrain and hindbrain primordia (reviewed by Raible and

Brand, 2004; Rhinn and Brand, 2001; Wurst and Bally-Cuif,

2001), and the rhombomere-4 (r4) organizer patterns the

0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.ydbio.2005.06.039

* Corresponding author.

E-mail address: jpostle@uoneuro.uoregon.edu (J. Postlethwait).

surrounding hindbrain, at least in zebrafish (Maves et al.,

2002; Walshe et al., 2002).

The isthmic (or midbrain–hindbrain boundary) organizer

(MHB) develops in three phases: positioning, establishment,

and maintenance (Rhinn and Brand, 2001). In the position-

ing phase, the MHB arises during gastrulation just anterior

to the tip of the notochord between the posterior limit of the

Otx2 expression domain and the anterior limit of the

Gbx2 expression domain (Rubenstein et al., 1998). In the

establishment phase, Pax2, Fgf8, and Wnt1 expression

initiates at the Otx2 –Gbx2 interface. During the mainte-

nance phase, genes already used in the establishment phase

and their downstream targets, including En1, En2, Pax5,

and Pax8, become mutually dependent for their continued

expression and maintain the boundary (Matsunaga et al.,

85 (2005) 298 – 315

C. Canestro et al. / Developmental Biology 285 (2005) 298–315 299

2000; Scholpp et al., 2003; Schwarz et al., 1999). Caudal to

the MHB, the presence of a second organizing activity in

zebrafish (Maves et al., 2002), and possibly in other

vertebrates (Graham et al., 1993, Graham and Lumsden,

1996, Marin and Charnay, 2000a,b) has been suggested by

transplantation and ectopic expression studies; this activity

is located in rhombomere 4 (r4) of the hindbrain, and

patterns the surrounding rhombomeres by FGF signaling in

the context of the Hox code.

Many genes with roles in the MHB subsequently play

new roles in the progressive refinement of AP subdivisions

and in the differentiation of specific cell populations in the

CNS (Lumsden and Krumlauf, 1996). Most genes involved

in CNS patterning belong to multigene families, many of

which arose during large-scale gene duplication events that

accompanied, and likely facilitated, early vertebrate evolu-

tion (Garcia-Fernandez and Holland, 1994; Holland et al.,

1994; Ohno, 1970). Understanding vertebrate CNS develop-

ment is complicated because duplicated genes generally

retain some redundancy but also acquire spatial and

temporal diversification of expression patterns (Force et

al., 1999; McClintock et al., 2002; Postlethwait et al., 2004).

Nervous systems of the non-vertebrate chordates—

cephalochordates and urochordates—though simpler than

the vertebrate CNS, share with vertebrates basic deve-

lopmental genetic mechanisms (reviewed in Holland and

Chen, 2001; Shimeld and Holland, 2000; Wada and Satoh,

2001). Understanding developmental mechanisms in non-

vertebrate chordates, which possess low gene redundancy

due to their divergence before the large-scale duplication

events, can facilitate inferences for the roles of the founding

members of CNS gene families in the last common ancestor

of chordates. The Subphylum Urochordata, or Tunicata, is

the sister taxon of the Cephalochordata + Vertebrata clade

(but see Graham, 2004), and includes three Classes:

ascidians, thaliaceans, and larvaceans. Among these, only

ascidians have been intensively studied at the embryological

and molecular level.

The application of molecular and genomic tools has led

to impressive progress in understanding ascidian develop-

ment (reviewed in Canestro et al., 2003; Corbo et al., 2001;

Holland and Gibson-Brown, 2003; Jeffery, 2002; Lemaire et

al., 2002; Meinertzhagen et al., 2004; Satoh, 2003; Sordino

et al., 2001). Ascidians and amphioxus share with verte-

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-

vertebrate chordates, however, are problematic (reviewed in

Holland and Holland, 1999; Wada and Satoh, 2001). The

discovery that pax2/5/8a is expressed in the otx –hox1 gap

in the ascidian Halocynthia roretzi led to the proposal that

the ‘‘neck’’ of the ascidian CNS is homologous to the MHB

(Wada et al., 1998). In amphioxus, however, homologs of

vertebrate MHB markers Pax2/5/8, Engrailed, andWnt1 are

not expressed in the otx –hox1 gap suggesting that

amphioxus lacks an MHB (Holland et al., 1997; Kozmik

et al., 1999). This apparent conflict was not resolved by

recent studies of additional markers in ascidians, which

revealed substantial variability in gene expression patterns

among different ascidian species (Imai et al., 2002; Jiang

and Smith, 2002; Mazet et al., 2003; Wada et al., 1998). The

absence of functional data leaves unclear the roles of these

genes during ascidian and cephalochordate CNS develop-

ment, or whether any portion of the ascidian CNS possesses

organizing activity.

Urochordate larvae possess the defining chordate fea-

tures of notochord, dorsal hollow nerve cord, gill slits, and a

muscular, post-anal tail. In ascidians, an elaborate meta-

morphosis erases most of these chordate features as the

notochord is reabsorbed and the CNS is dramatically

restructured. It is possible that selective pressures leading

to rapid metamorphosis in ascidians have obscured or

compromised some developmental mechanisms present in

stem chordates. In contrast, larvacean urochordates (Class

Appendicularia) retain a chordate body plan as adults.

Phylogenetic relationships of urochordate classes are not

yet known for certain, but the most accepted phylogeny

places larvaceans as the basal sister group of ascidians +

thaliaceans (Christen and Braconnot, 1998; Holland et al.,

1988; Swalla et al., 2000; Wada, 1998; Wada and Satoh,

1994; but see Stach and Turbeville, 2002). Genomic

analysis has uncovered substantial differences in genome

size and syntenic relationships between Oikopleura and

ascidians (Edvardsen et al., 2005; Seo et al., 2001; Seo et

al., 2004). For these reasons, the inclusion of larvaceans in

developmental genetic investigations is essential for obtain-

ing a balanced view of development in the urochordate

Subphylum.

In the work reported here, we present the first molecular

genetic analysis of larvacean brain regionalization. Signifi-

cant homologies and differences between larvacean and

ascidian CNS developmental mechanisms can be inferred

from the comparison of gene expression patterns. Knowl-

edge of Oikopleura CNS molecular regionalization provides

new insights into chordate CNS evolution, helps illuminate

previous conflicting interpretations of ascidian CNS expres-

sion data, extends within urochordates prior suggestions

about CNS organization that previously applied only to

ascidians, and provides a basis for functional genetic

analysis.

Materials and methods

Animal culture, embryo staging, and imaging

Oikopleura dioica individuals were collected with

plankton nets in the Pacific Ocean off Charleston, Oregon

(Oregon Institute of Marine Biology) and Vancouver Island,

B.C. (Bamfield Marine Sciences Centre). Animals were

cultured for several generations in 10-Am filtered seawater

in 4-l plastic jars and fed with algal concentrates (‘‘Coral

C. Canestro et al. / Developmental Biology 285 (2005) 298–315300

and Clam diet’’, Reed Mariculture). Embryos were grown at

13 T 3-C as described (Bassham and Postlethwait, 2000).

Because developmental rate varies strongly with temper-

Fig. 1. CNS development in Oikopleura dioica. DIC images of live specimens wit

is to the left in lateral views of tailbud embryos (A–C) and hatchlings (D–F), and

and mid-tailbud stages (B,C) show cell morphology and organization of develop

width of the anterior brain at the level indicated in the schematic between panels B

hatchling (F), and adult (G–J) are shown with focal planes digitally integrated. O

(green dashed line in insert sketch) shows the boundaries of the anterior brain, ph

(lateral in panel H, dorsal in panel I, green dashed lines) and caudal ganglion (J, bl

(AB) contains about 70 cells (Martini, 1909), and includes a putative pituitary hom

a prominent sensory vesicle (SV), which includes a statolith (St) but, unlike asc

afferent and efferent nerves (n1–n3) with sensory and motor functions emanate f

nerve cord (TNC), which lacks a central canal, bends ventrally between the stom

approximately 25 neurons and other cells of the caudal ganglion coordinate activi

paired trunk mechanoreceptors (Bone and Mackie, 1975). A hollow spinal cord (S

representations of panels F and I are shown in Figs. 5A,B. AB, anterior brain; C

esophagus; Ec, ectoderm; En, endoderm; Es, endostyle; Go, gonad; LL, lower lip;

R, rectum; S, stomach; SC, spinal cord; SV, sensory vesicle; St, statocyte, Sl, sta

ature [e.g., fertilization to hatching requires less than 3 h at

22-C, but about 12 h at 7-C (Fenaux, 1998)], we stage

Oikopleura according to morphological landmarks. The

h schematic insets depicting the CNS (yellow) and notochord (red). Anterior

to the right in the adult (G–J). Different focal planes of incipient-tailbud (A)

ing structures. A dorsal optical section (boxed in C) shows the 2- to 4-cell

and C (dashed green line). An early hatchling (D), mid-hatchling (E), late

ptical cross section (boxed in panel E) at the level of the sensory vesicle

arynx, and endostyle. Details of regions corresponding to the anterior brain

ue dashed lines) are boxed in the schematic (G). In adults, the anterior brain

olog (CF) (Bassham, 2002), which connects with the pharyngeal space, and

idians, no pigmented cells (Georges et al., 1988; Holmberg, 1984). Paired

rom the anterior brain (Bollner et al., 1991; Olsson et al., 1990). The trunk

ach lobes and meets the caudal ganglion (CG) in the proximal tail. The

ty of the caudal musculature, and receive inputs from the anterior brain and

C) extends from the caudal ganglion down the tail (Bone, 1998). Schematic

F, ciliary funnel; CG, caudal ganglion; CNS, central nervous system; E,

Mo, mouth;Mu, muscle; n1–3, paired nerves; No, notochord; Ph, pharynx;

tolith; TNC, trunk nerve cord; UL, upper lip; VO, ventral organ.

C. Canestro et al. / Developmental Biology 285 (2005) 298–315 301

lack of a detailed blastomere fate map precludes an exact

description of expression patterns at cleavage stages

(Nishino and Satoh, 2001). Stages prior to hatching include:

(i) incipient-tailbud stage (beginning about 3.5 h post-

fertilization (pf) at 13-C), characterized by an indentation at

the initial demarcation of trunk and tail (Fig. 1A) and by an

irregular, rod-shaped notochord. (ii) Mid-tailbud stage

(beginning about 4 h pf at 13-C), in which a deep

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

respect to the trunk (Delsman, 1910) is already pronounced

at this stage. This flexure makes it impossible to position

embryos for a true orthogonal image capture, but the

notochord and large muscle cells provide helpful landmarks

to orient samples. Nuclear staining (i.e., DAPI, Hoechst)

was routinely included in expression analysis at tailbud

stages to confirm notochord cell positions.

Stages after hatching conform to Fenaux’s (1976),

including: (i) early hatchling (Fig. 1D) (Fenaux stage I),

in which the animals are cylindrical, without substantial

demarcation between trunk and tail, and, although the trunk

still lacks obvious organs, the tail begins short bursts of

movement; (ii) mid-hatchling (Fenaux II) (Fig. 1E), in

which the beginning of organogenesis is obvious, and

animals can swim; (iii) late hatchling (Fenaux IV) (Fig. 1F),

in which movements of the heart and the cilia of the

digestive system and spiracles become visible, the trunk-tail

boundary is well defined, and coordinated swimming

improves; (iv) tailshift, characterized by the shift of the tail

to an acute angle relative to the trunk, signaling the end of

embryonic development, and competence to secrete and

inflate a filter-feeding house (Figs. 1G–J).

The transparency of Oikopleura embryos and adults

allows non-invasive study of internal morphology at the

level of individual cells. For some images, we merged DIC

optical sections using Adobe-Photoshop software to

integrate images of structures that spanned focal planes

(Figs. 1D–J).

Cloning and whole-mount in situ hybridization

Genomic DNA from about 50 Oikopleura individuals

from Bamfield Marine Station was used to construct an

arrayed fosmid library (Epicentre, CCFOS110). Complete

sequencing of targeted fosmid clones was performed by the

DOE Joint Genome Institute (Walnut Creek, CA). mRNA

from about 1500 embryos was used to synthesize cDNA as

described (Bassham and Postlethwait, 2000). Genes of

interest were amplified by PCR with degenerate primers

(see Table S1 in supplementary materials) using cDNA or

genomic DNA as template. Gene-specific primers were

designed for RACE PCR, and to screen the genomic library

using a pooling strategy. Coding sequence and gene

structures were inferred by sequence comparisons between

RACE products and fosmid genomic clones. We designate

O. dioica genes by a name (usually based on its human

ortholog) in italics and small-case letters (i.e., pax6), and

proteins by a name with the first letter in upper-case (i.e.,

Pax6). When multiple paralogs are found, we add Latin

letters in alphabetical order (i.e., otxa, otxb, otxc) with no

additional punctuation. Whole-mount in situ hybridization

was performed as described (Bassham and Postlethwait,

2000). Riboprobe information is provided in Table S2.

Results

Isolation of Oikopleura CNS markers

To study the development and regionalization of the

larvacean CNS, we characterized CNS markers that are

highly conserved across bilaterians and play a central

role in AP organization of the tripartite vertebrate brain

(Hirth et al., 2003; Reichert and Simeone, 2001). We

isolated eight O. dioica genes homologous to the Otx,

Pax6, Pax2/5/8, Engrailed, and Hox1 vertebrate gene

families. Sequence comparisons and phylogenetic analyses

unequivocally assigned each isolated Oikopleura gene

to its gene family. We isolated single copies for Oiko-

pleura pax6 (AY870650), engrailed (AY870647), and

hox1 (AY871214) genes, three duplicated copies of otx

(AY886542, AY897556, AY897557) and two copies of

pax2/5/8 (DQ020279, AY870648, AY870649).

During the preparation of this manuscript, a list of O.

dioica homeobox genes inferred from genome sequences

was reported (Edvardsen et al., 2005), and in the present

work, we have adopted the same names for the three

Oikopleura otx duplicate genes we independently isolated:

otxa, otxb, and otxc. Analysis of the predicted proteins for

the three Oikopleura genes revealed the presence of a

homeodomain, a single C-terminal hexapeptide motif (in

contrast with two hexapeptide motifs in vertebrate OTX

proteins), and a moderately conserved WSP motif (Fig.

S1A). The small amount of evolutionary information

provided by the conserved domains and variation in the

rest of the molecule hampered the construction of confident

protein alignments and phylogenetic inferences. Among the

three Oikopleura Otx duplicates, Otxa showed the highest

overall similarity to ascidian Otx. Analysis of exon–intron

organization of Oikopleura otx genes revealed multiple

introns in addition to the two introns conserved in all known

otx genes (Fig. S1A). Remarkably, all ascidian and

Oikopleura otx genes are characterized by the presence of

additional exons (yellow in Fig. S1A) between the

conserved exon containing the transcription origin and the

exon containing the N-terminal part of the homeobox. The

still detectable sequence similarities among these additional

exons in urochordates suggest that these exons were

originally gained before the separation of the ascidian and

larvacean lineages; thus, they are likely a synapomorphy of

urochordate otx genes. Analysis of fosmid sequences from

C. Canestro et al. / Developmental Biology 285 (2005) 298–315302

our Oikopleura genomic library (AY873983–AY873986)

shows that Oikopleura otxb and otxc are adjacent,

transcribed in opposite directions, and separated by a 17

kb stretch that contains no intervening putative genes.

This result suggests that Oikopleura otxb and otxc

probably arose by tandem gene duplication during the

evolution of larvaceans after they separated from the

ascidian lineage. Evidence from protein sequence similar-

ities, gene structures, and phylogenetic analysis (data not

shown and Edvardsen et al., 2005), shows that the

duplication event that produced the third Oikopleura otx

gene also probably occurred in the larvacean lineage after

it separated from the ascidian lineage, although an otx

gene lost during the evolution of the ascidian lineage

cannot be ruled out.

We isolated from Oikopleura two pax2/5/8 genes, only

one of which (pax2/5/8b) was reported by Edvardsen et al.

(2005). Sequence similarities and phylogenetic analysis

showed that one Oikopleura pax2/5/8 gene (AY870648,

DQ020279) is orthologous to ascidian pax2/5/8a and the

other (AY870649) is orthologous to ascidian pax2/5/8b

(Fig. S1B). These results show that the duplication event

that led to these two urochordate pax2/5/8 clades occurred

in the urochordate lineage after it diverged from the

cephalochordate + vertebrate lineage, but before the split

of the ascidian and larvacean lineages; therefore, the pax2/5/

8a+pax2/5/8b duplicates appear to be a urochordate

synapomorphy.

Development and AP regionalization of the Oikopleura

CNS

Whole-mount in situ hybridization experiments revealed

that the eight Oikopleura genes we isolated are transcribed

in the embryonic CNS, endoderm, and epidermis. Here, we

focus on expression patterns necessary to understand the

mechanisms of CNS development (Figs. 2–4). The complex

patterns of other expression domains will be described in

detail elsewhere.

Incipient-tailbud stage

Analysis of the expression of Oikopleura CNS markers

revealed that AP regionalization of the CNS begins early,

Fig. 2. Expression of CNS genes before hatching. Whole-mount in situ hybridizat

embryos (B,C,E,H,I,K,L,N,O,Q,R). otxb (yellow, A,B), otxa (yellow, C), pax6 (r

J,K), engrailed (dark blue, L,O), pax2/5/8b (M,N), and hox1 + engrailed (R). The

images (labeled with numbered lower case letters) are optical cross sections at the

line. In each panel, left insets are oriented with dorsal towards the top, and in right

expression shows the relative position between the Oikopleura hindbrain and the n

position of the anterior hox1 expression domain (lateral and dorsal views, squared

revealed by posterior CNS gene expression (i.e., H.h1,L.l1). Double in situ detectio

hox1 nested expression pattern (dorsal views oriented anterior towards the bottom

expression in the presumptive CNS (yellow), epidermis (black), and endoderm (pi

indicated by white dots. The cell number and size, and notochord position relati

exemplified in panels F and O, which are the same individuals shown in panels D

projected onto incipient-tailbud (P) and mid-tailbud (Q) embryos in which Brach

2000). Scale bar = 20 Am. TB, tailbud stage.

probably during gastrulation. Expression of otxb and hox1

is already detectable during cleavage stages (data not

shown), and probably provides the initial AP information

upon which subsequent AP regionalization is based.

At the incipient-tailbud stage, an anterior domain

expresses otxb and a posterior domain in the presumptive

CNS expresses hox1 (Figs. 2A,G,P). In addition to

epidermal expression, a broad otxb expression domain

may span the entire presumptive anterior brain (Fig. 2A).

At this stage, we did not detect expression of otxa or

otxc.

At the incipient-tailbud stage, the presumptive anterior

brain marked by otxb is subdivided by pax6 expression,

which appeared in two separate domains (Figs. 2D,F,P).

At least two bilateral pairs of cells located at the midline

constitute the most rostral pax6 domain (Fig. 2D.d1).

The caudal pax6 domain, anterior to the tip of the

notochord, also consists of at least two bilateral pairs of

cells (Fig. 2D.d1,d2).

While otxb and pax6 mark the anterior CNS, bilateral

rows of cells express hox1 dorsolaterally and posteriorly

to the anterior tip of the notochord, at the level of the

prospective caudal ganglion and anterior spinal cord

(Figs. 2G,P).

Because cells expressing otxb, pax6, and hox1 in the

presumptive CNS were already internal and close to the

midline at incipient-tailbud stage, we conclude that by this

stage, neurulation is at or nearing completion.

Contrary to expectations, neither of the two Oikopleura

pax2/5/8 genes is specifically expressed in the CNS. Pax2/

5/8a is expressed mainly in the trunk ectoderm (Fig. 2J).

Complementary to pax2/5/8a expression, pax2/5/8b was

detected in most of the internal portion of the trunk,

probably including some presumptive CNS cells (Fig. 2M).

These diffuse pax2/5/8 expression patterns are likely not

artifactual since different non-overlapping probes render

identical patterns, and the same probes reveal distinctive

tissue-specificity at later stages (Fig. 2K and data not

shown).

Mid-tailbud stage

At mid-tailbud stage, early broad gene expression

domains become refined and the expression of new CNS

ion in Oikopleura dioica incipient-tailbud (A,D,F,G,J,M,P) and mid-tailbud

ed, D–F), hox1 (light blue, G,H), hox1 + brachyury (I), pax2/5/8a (green,

central large image in each panel is a left lateral view, and the inset corner

levels of the dashed lines, viewed from the aspect of the label relative to the

insets, anterior is towards the left. Double detection of hox1 and brachyury

otochord (I). Inspection of different specimens reveals variability of the cell

insets in panel I). The degree of twisting of the trunk relative to the tail is

n of the expression of hox1 and engrailed (R) demonstrates the hox1 –en –

; comparable to panels H.h2 and L.l2 optical sections). Arrowheads label

nk). The position of notochord cells, sometimes in a different focal plane, is

ve to expression domains were routinely visualized by nuclear staining, as

and L, respectively. Schematized summaries of gene expression patterns are

yury expression (Bra) labels notochord (white) (Bassham and Postlethwait,

C. Canestro et al. / Developmental Biology 285 (2005) 298–315 303

markers appears. At this time, while otxb continues to

label a row of cells in the midline of the presumptive

anterior brain, pax6 expression is reduced to a single

domain (Figs. 2B,E,Q), apparently by down-regulation of

the more posterior of the two domains seen in incipient-

tailbud embryos. At mid-tailbud stage, otxa expression was

C. Canestro et al. / Developmental Biology 285 (2005) 298–315304

detected for the first time in the CNS, labeling asymmetri-

cally one cell in the right side and at least four cells in the

left side of the presumptive anterior brain (Fig. 2C). At

this stage, otxc expression was not yet detected within the

CNS.

The posterior CNS becomes subdivided in mid-tailbud

embryos, with the appearance of a hox1 –engrailed –hox1

nested pattern. Analysis of this nested pattern by single and

double in situ hybridization experiments, together with

notochord cell positioning by detection of brachyury

expression (Bassham and Postlethwait, 2000) and nuclear

staining (Figs. 2H,I,L,O,Q,R), revealed an anterior hox1

domain spanning approximately from the AP level of the

anterior tip of the notochord to the third notochord cell, a

central engrailed domain at the level of the fourth notochord

cell, and a posterior hox1 domain extending from the fifth to

seventh notochord cells (Figs. 2H,I). Analysis of several

dozen mid-tailbud embryos revealed slight variations in the

positions of cells forming the anterior hox1 domain with

respect to the tip of the notochord (Figs. 2H,I). At this stage,

frontal views of embryos probed for CNS markers revealed

an approximately 60- counterclockwise twist of the dorso-

ventral axis in the tail relative to the trunk, which causes the

posterior CNS to come to lie left of and somewhat dorsal to

the notochord (Fig. 2e1,h1,k1,l1). Double detection of

hox1 and brachyury expression showed that some of the

cells of the anterior hox1 domain come to lie in the midline

of the trunk, dorsal and sometimes anterior to the

notochord. We did not detect hox1 expression within the

notochord (Figs. 2H,I).

Although there is a gap between the posterior border of

the otxa + otxb expression domain and the hox1 expression

domain (see Figs. 2P,Q), we detected no pax2/5/8

expression specific for the CNS in that gap. This contrasts

with ascidians, in which pax2/5/8a expression fills the

otx–hox1 gap (Wada et al., 1998). In contrast, two other

tissues express Oikopleura pax2/5/8a in the trunk: a small

patch of epidermal cells in the prospective mouth region,

and a group of internal cells at the boundary between the

anterior brain and pharynx, rostral to the pax6 expression

domain (Fig. 2K, black, yellow arrowheads, respectively).

At this mid-tailbud stage, we could not discern the exact

anterior-most border of the anterior brain by morphology,

and fate mapping is needed to confirm whether individual

cells and their descendents in that region become part of

the anterior brain or part of the pharynx roof. Broad

expression of pax2/5/8b continues, restricted mainly to the

trunk (Fig. 2N).

Early and mid-hatchling stages

During early hatchling stages, larvacean embryos

rapidly elongate and organs related to movement, such

as the notochord, muscle cells, and CNS, are more mature

than the digestive system (Fig. 1D). Although the trans-

parency of living Oikopleura embryos allowed us to

distinguish much of the developing CNS from surrounding

tissues as early as incipient-tailbud stage (Figs. 1A–C), the

four main AP regions of the CNS—anterior brain (AB),

compact trunk nerve cord (TNC), caudal ganglion (CG),

and hollow tail nerve cord or spinal cord (SC)—do not

become obviously demarcated from each other until early

hatchling stages (Fig. 1D). This suggests that the primary

molecular definition of these regions is probably complete

before hatching, and therefore, the later expression of otx,

pax6, engrailed, and hox1 likely reflects secondary

functions related to cell fate maintenance or to specifica-

tion of neuron subpopulations, especially in the caudal

ganglion where motor neurons probably start to control tail

movements. Accordingly, while expression patterns of

these genes are largely continuous from mid-tailbud stage,

expression becomes restricted to specific cell subpopula-

tions (Fig. 3).

In early and mid-hatchlings, pax6 is expressed in the

anterior brain, outside the future sensory vesicle (Figs.

3A,B). A thin extension of expression reaches forward from

pax6-positive cells (rostral arrowhead in Fig. 3A), suggest-

ing the formation of neuronal processes. The internal pax2/

5/8a expression domain observed anterior to pax6 expres-

sion at mid-tailbud stage disappears by mid-hatchling stages

(data not shown).

Otxb expression narrows in hatchlings, from initially

spanning the entire presumptive anterior brain (Fig. 2A) to

comprising only a few cells (Fig. 3D). Interestingly, as otxb

expression declines in early hatchlings (Fig. 3D), the otxa

expression signal becomes intense (Fig. 3G), and for the

first time, otxc transcripts begin to accumulate in CNS cells

(Fig. 3J). This process, in which otxa + otxc expression

seems to replace otxb expression over time, is finished by

late hatchling stages when otxb transcription is almost

undetectable in the anterior brain (Fig. 3F).

During hatchling stages, the expression of the three otx

duplicates is restricted to the central portion of the

anterior brain, although there are differences in their

expression patterns; while some cells apparently co-

express multiple otx duplicates, other cells express only

one, and in some cases the expression patterns are asym-

metric (Figs. 3D,E,G,H,J,K).

In early hatchling stages, in addition to strong epidermal

hox1 expression at the trunk-tail transition (Fig. 4A, black

arrowheads), a row of hox1-expressing cells extends left of

the midline from anterior positions near the notochord tip

(within the trunk) caudally to at least the level of the fourth

notochord cell (Figs. 4A,B), probably spanning the

prospective posterior TNC and anterior caudal ganglion

(Fig. 1D). Additional hox1 signal with irregular shape and

thin diameter appears at the level of the fifth and sixth

notochord cells (Figs. 4A,B), and could mark caudal

cellular extensions from anterior hox1-expressing cells.

These observations broadly confirm and extend those for

hox1 in Seo et al. (2004). In early hatchlings, in addition to

the expression of hox1 in the anterior segment of the

caudal ganglion, we observed expression of engrailed (Fig.

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.

C. Canestro et al. / Developmental Biology 285 (2005) 298–315 305

4D), and, transiently, pax6 (Fig. 3A) posterior to the

notochord tip.

In mid-hatchling stages, while precursor cells of the

posterior TNC continue to express hox1, the prospective

caudal ganglion down-regulates hox1 expression (Fig. 4B).

We found no expression of hox1 in the spinal cord, although

punctate, non-epidermal hox1 and engrailed signal appears

broadly distributed along mid- and late-hatchling tails (Figs.

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.

C. Canestro et al. / Developmental Biology 285 (2005) 298–315306

4B,E,F, white arrowheads). Although this punctate pattern is

specific for these two genes, the pattern varies in different

animals.

Late-hatchling stage

In late hatchlings, the expression pattern of CNS

markers mapped to the four morphologically distinct

regions of the CNS, the anterior brain (AB), trunk nerve

cord (TNC), caudal ganglion, and spinal cord (Figs. 3–5),

and they divide the anterior brain into three subdivisions

(AB1–3) (Figs. 5A,B). AB1, the anterior subdivision, is

labeled by pax6 (Fig. 3C). The ciliary funnel and the

anterior extensions from AB1 that form the rostral paired

nerve n1 appear to be free of pax6 expression. AB2, the

central subdivision, is broadly marked by the expression of

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

ventral cells appear not to express pax6. In addition, the

statocyte expresses otxa, but not pax6 or otxc (Figs.

3C,I,L). AB3, the posterior subdivision, was free of any

pax6, otx, engrailed, pax2/5/8, and hox1 signal (Figs.

3C,F,I,L, 4C,F, and 5A). These three subdivisions of the

anterior brain broadly coincide with regions of the

Oikopleura brain previously called fore-, mid-, and hind-

brain, based on fine structure and nerve positions (Olsson

et al., 1990). Calling these regions AB1– 3 avoids

potentially misleading homologies with the vertebrate

brain (see Discussion).

In the posterior trunk of late hatchlings, the TNC bends

90- towards the caudal ganglion (Fig. 1F). Only the

posterior half of the TNC is labeled by hox1 expression

(Figs. 4C and 5A). Hox1 signal was also detected in the

sensory field that includes the epidermal Langerhans

mechanoreceptors (Fig. 4C), which are innervated by

axons from the caudal ganglion (Bone and Mackie,

1975). At this late stage, the caudal ganglion itself does

not express hox1, and among the genes we studied, only

engrailed expression appeared in the anterior part of the

caudal ganglion (Figs. 4C,F).

Discussion

Homologies and differences between larvacean and

ascidian CNS development

This study of gene expression in the developing

Oikopleura CNS reveals the molecular genetic region-

alization of the larvacean CNS, and allows comparison

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.

C. Canestro et al. / Developmental Biology 285 (2005) 298–315 307

with ascidians. The ascidian CNS has been divided into

four regions: the ‘‘brain’’ (which includes the sensory

vesicle housing the statocyte and ocellus), neck,

visceral ganglion, and spinal cord (Satoh, 2003). The

following section highlights evidence that defines

regional homologies in the CNS of two urochordate

classes, and discusses the differences observed along

the AP axis of the CNS, starting rostrally and moving

caudally.

The anterior CNS

The Oikopleura anterior brain and ascidian ‘‘brain’’ are

homologous. Expression of Oikopleura otxb and ascidian

otx demarcates the anterior CNS in cleavage stages

(Hinman and Degnan, 2000; Hudson and Lemaire, 2001;

Wada et al., 1996; Wada et al., 2004; and this study). Both

urochordate classes have two pax6 expression domains, the

posterior of which overlaps the expression of otx near the

C. Canestro et al. / Developmental Biology 285 (2005) 298–315308

anterior tip of the notochord (Figs. 2P and 5C.c,f) (Glardon

et al., 1997; Mazet et al., 2003). By the mid-tailbud stage

in Oikopleura, although the otxb posterior boundary does

not change relative to the notochord, pax6 is expressed

only in the former anterior domain, suggesting down-

regulation of pax6 expression in the posterior domain

(Figs. 2Q and 5C.d). Similarly, pax6 expression is absent

from the posterior part of the ascidian brain at mid-tailbud

stage (Glardon et al., 1997; Mazet et al., 2003). This

congruence of otx and pax6 expression patterns in the early

tailbud stages of Oikopleura and ascidians supports

homology of the larvacean anterior brain and the ascidian

‘‘brain’’.

Expression of otx and pax6 reveals differences between the

Oikopleura anterior brain and the ascidian ‘‘brain’’. Al-

though early expression of otx and pax6 is similar in the two

urochordate classes, at later stages, there are three major

differences. First, pax6, which is typically expressed in

photoreceptors (Callaerts et al., 1997), is expressed in the

photosensitive ocellus in the ascidian sensory vesicle

(Glardon et al., 1997), but not in that of Oikopleura, which

lacks pigmented photoreceptor cells. The second major

difference is that while pax6 is no longer expressed in the

ascidian ‘‘brain’’ by late larval stages (Mazet et al., 2003), in

Oikopleura, it continues to be expressed in the anterior brain

until at least the late hatchling stage (Figs. 3C and 5A,C.e).

This expression difference perhaps reflects the continuity of

embryonic and adult Oikopleura CNS patterning (see

discussion below), while the ascidian CNS is drastically

restructured during metamorphosis.

The third major difference is that the three Oikopleura

otx genes, in contrast to the single ascidian otx gene, show

‘‘phased’’ expression dynamics, differing in their temporal

and spatial expression patterns during CNS development.

These results suggest the hypothesis that the three otx

genes in Oikopleura have different developmental func-

tions. The broad and uniform expression of the single otx

gene described in the ascidian brain (Wada et al., 1996)

would mask potential multiple functions. Like Oikopleura,

tetrapods have three OTX paralogs (OTX1, OTX2, and

OTX5/CRX) due to independent duplications within the

vertebrate lineage. The vertebrate paralogs, like their

Oikopleura homologs, have also assumed separate early

and late developmental roles. First, the earliest expression

of Oikopleura otxb in the anterior neuroectoderm and

endoderm is comparable to the expression of OTX2 during

vertebrate gastrulation (Reichert and Simeone, 2001). And

second, the later appearance of Oikopleura otxa and otxb

expression in a reduced number of cells in the anterior

brain can be compared to the action of OTX1 and OTX2 in

specifying identity and fate of specific cell populations in

the vertebrate brain (Puelles et al., 2003; Puelles et al.,

2004). This parallelism between vertebrates and Oiko-

pleura provides an example of trans-homologs [genes

duplicated independently in different lineages from a

common ancestral pro-ortholog (Sharman, 1999)] converg-

ing on similar evolutionary outcomes. Functional analysis

of otx regulation in Oikopleura duplicates will test whether

this evolutionary convergence is due to parallel subfunc-

tionalization events in conserved regulatory modules (Force

et al., 1999) present in the last common ancestor of

vertebrates and urochordates, or whether phased otx

regulation has been gained independently in the two

chordate lineages.

Pax2/5/8 genes reveal differences between Oikopleura and

ascidian anterior CNS regions. In ascidians, the expres-

sion of pax2/5/8a in the gap between otx and hox1 domains

suggested homology with the vertebrate MHB (Wada et al.,

1998). Despite the expression similarities between Oiko-

pleura and ascidian pax2/5/8a and pax2/5/8b orthologs in

several domains (Mazet et al., 2005; Wada et al., 1998),

neither Oikopleura pax2/5/8 gene is expressed in the gap

between otx and hox1 domains. Therefore, pax2/5/8

expression data argue against the presence of an MHB

homolog in Oikopleura.

In mid-tailbud stages, ascidians lack pax2/5/8a expres-

sion similar to the Oikopleura pax2/5/8a internal domain at

the brain–pharynx border anterior to the pax6 domain

(Mazet et al., 2003; Wada et al., 1998). In amphioxus, pax2/

5/8 expression in the anterior cerebral vesicle has been

compared to vertebrate Pax2 expression in development of

the optic stalk and optic nerve (Fig. 5C.a) (Kozmik et al.,

1999; Krauss et al., 1991; Macdonald et al., 1997).

Additionally, it was proposed that pax2.1, in cooperation

with Fgf signaling, influences axon guidance and early

rostral midline development in the zebrafish forebrain

(Shanmugalingam et al., 2000). Because AB1, the rostral

subdivision of the anterior brain of Oikopleura, projects

axons rostrally via the paired nerves n1 and n2 to the ventral

organ and sensory receptors in the lips (Figs. 1I and 5B),

Oikopleura pax2/5/8a expression is consistent with a role in

axon guidance like its vertebrate homolog Pax2. Study of

more markers and gain- and loss-of-function experiments

are necessary to test these hypotheses for the role of the

Oikopleura pax2/5/8a.

Links between developmental expression patterns and the

adult larvacean brain. In contrast to ascidians, the

continuous transition of the Oikopleura CNS from hatch-

ling to adult provides direct links between embryonic gene

expression domains and the fine structure and function of

the adult Oikopleura brain (Olsson et al., 1990). Figs. 5A,B

show the major anatomical features of the anterior CNS in

the juvenile and adult Oikopleura. The rostral subdivision

of the anterior brain (AB1) labeled by pax6 expression

receives afferent pathways from anterior sensory cells,

including the ventral organ (via n1), and ciliated receptor

cells in the lips and pharynx (via n2) (Olsson et al., 1990;

and Fig. 5B). The efferent pathways described by Olsson et

al. (1990) may originate in the central and posterior

C. Canestro et al. / Developmental Biology 285 (2005) 298–315 309

subdivisions AB2 and AB3. AB2, which is labeled by pax6,

otxa, and otxc, probably includes three neurons that project

axons via n3, a postulated motor nerve carrying fibers to

the spiracle’s ciliary rings and to ventral epidermal cells

that might have a role in house secretion (Fig. 5B). The

posterior subdivision AB3, which does not express any of

the genes in this study during late development, sends one

process via the left n3 to the ciliary ring and another into

the TNC, probably towards the caudal ganglion. Therefore,

the AB1–3 subdivisions revealed by molecular markers

during development seem to correspond to different func-

tional areas of the brain in charge of integrating afferent

and efferent pathways.

The posterior CNS

The posterior urochordate CNS features a prominent

ganglion near the anterior tip of the notochord called the

caudal ganglion in Oikopleura and the visceral ganglion

in ascidians (Fig. 1) (Meinertzhagen et al., 2000). Variation

in gene expression and morphology among ascidian species,

however, hinders a straightforward assignment of homology

between larvacean and ascidian posterior CNS regions.

Larvaceans and ascidians share a two-domain expression

profile for hox1 (Fig. 5C.d,g): an anterior domain near the

anterior tip of the notochord [compare dorsal views of

Oikopleura in Fig. 4A to views of the ascidian in Fig. 5 of

Katsuyama et al. (1995)] and a posterior, transient domain in

the presumptive caudal/visceral ganglion and spinal cord

(Figs. 2P–Q and 5A,C) (Ikuta et al., 2004; Katsuyama et al.,

1995; Nagatomo and Fujiwara, 2003). In Oikopleura, we

can trace the anterior hox1 domain throughout development

to the posterior TNC. In ascidians, although the hox1

expression pattern is the same in different species, various

authors have differed in their interpretation of the fate of the

anterior domain, assigning it either to the neck or to the

visceral ganglion (Ikuta et al., 2004; Katsuyama et al., 1995;

Nagatomo and Fujiwara, 2003; Wada et al., 1998). This

controversy is probably due to the lack of a morphologically

distinguished neck in H. roretzi rather than a true fate

difference.

These expression data also indicate shared morphoge-

netic behaviors of larvacean and ascidian axial tissues. The

anterior hox1 expression domain shifts forward with

respect to the notochord during Oikopleura development,

as it does in ascidians (Katsuyama et al., 1995). This

domain shift probably explains the variability of the cell

positions of the anterior hox1 domain observed in

Oikopleura mid-tailbud stages as different individuals are

fixed at slightly different stages (Figs. 2H–I,R). In Ciona

intestinalis, neck precursor cells apparently move from

behind the tip of the notochord in tailbud embryos to an

anterior location in hatchlings, suggesting that the neck is

part of the visceral ganglion (Cole and Meinertzhagen,

2004; Meinertzhagen et al., 2000). This shift applies also to

H. roretzi pax2/5/8a-positive cells (Wada et al., 1998).

Therefore, despite the morphological differences between

the CNS of the two urochordate classes, similar morpho-

genetic processes seem to be conserved underlying the

development of the neural tube at the level of the anterior

tip of the notochord.

Engrailed expression in the posterior CNS is also similar

among urochordates. In Oikopleura, engrailed expression

begins at mid-tailbud stage between the two initial hox1

domains creating a hox1–engrailed–hox1 nested pattern

(Fig. 2R). Throughout development, engrailed expression

continues posterior to the anterior notochord tip, labeling the

anterior part of the caudal ganglion (Figs. 2L,O,R and 4D–

F). Expression of engrailed differs between the two

congeneric ascidian species in which it has been described.

In Ciona savignyi, a single bilateral pair of cells co-

expresses engrailed and pax2/5/8a (Jiang and Smith, 2002).

In the congener C. intestinalis, pax2/5/8a is also expressed

in a single pair of cells, but engrailed flanks the pax2/5/8

expression domain in an engrailed-pax2/5/8a-engrailed

nested pattern (Fig. 5C.g), in which the posterior engrailed

domain labels the anterior part of the visceral ganglion (Imai

et al., 2002). Although no direct comparison of hox1 and

engrailed expression has been published for any ascidian,

comparing the positions of hox1 and engrailed expression

domains with the notochord (Ikuta et al., 2004; Imai et al.,

2002; Nagatomo and Fujiwara, 2003) makes it likely that

the hox1 –engrailed –hox1 nested pattern is shared by

Oikopleura and at least some ascidians (Fig. 5C.g). The

fact that pax6 is expressed transiently at early hatchling

stages in the presumptive caudal/visceral ganglion approx-

imately at the same level as engrailed (Glardon et al.,

1997; Mazet et al., 2003; and our Fig. 3A) bolsters the

correspondence of larvacean and ascidian posterior

engrailed domains.

The similar expression of engrailed, pax6, and hox1

during development of the caudal ganglion in Oikopleura

and the visceral ganglion in ascidians suggests that these

two structures are homologous. The present expression data

are also consistent with the homology of the ascidian neck

and the posterior Oikopleura TNC. In agreement with the

interpretation that the ascidian neck is part of the visceral

ganglion (Meinertzhagen et al., 2000), we conclude that the

posterior TNC and the caudal ganglion derive from the same

embryonic CNS region labeled by the single hox1 domain at

incipient-tailbud stage (Fig. 2G). Because Oikopleura

engrailed expression remains in a constant position poste-

rior to the tip of the notochord, while the hox1 domain shifts

anteriorly, there may also be elongation or migration of

specific cells, or stationary cells turning on and off

expression, rather than simply a general anterior shift of

this entire part of the posterior CNS.

Homologies and differences between urochordate and

vertebrate CNS development

Our comparative analysis of Oikopleura and ascidian

urochordates has revealed homologies and differences in

C. Canestro et al. / Developmental Biology 285 (2005) 298–315310

developmental genetic pathways, which now can be

compared to other chordate Subphyla.

Which part of the vertebrate CNS is homologous to the

urochordate spinal cord?

In contrast to ascidians and vertebrates, Oikopleura and

cephalochordates (Glardon et al., 1998) lack pax6 expres-

sion along the length of their spinal cords. Because the

posterior Drosophila CNS expresses ey, which is the fly

ortholog of pax6 (Quiring et al., 1994), the most parsimo-

nious explanation for species-specific pax6 expression

patterns is that the last common ancestor of extant chordates

had pax6 expression in the spinal cord and it was

independently lost in the Oikopleura and cephalochordate

lineages.

The similar expression pattern between ascidian hox5

(Gionti et al., 1998) and the only Oikopleura central Hox-

subclass gene (called hox4, although equally related to

hox4/5/6/7) (Seo et al., 2004) suggests that these genes

may function to define the anterior boundary of the

urochordate spinal cord. Therefore, despite the variation

in pax6 expression patterns among chordates, we conclude

that the spinal cord of urochordates and vertebrates is

homologous.

Which part of the vertebrate CNS is homologous to the

urochordate TNC/neck and caudal ganglion/visceral

ganglion?

Since the caudal ganglion of Oikopleura and the

ascidian visceral ganglion are probably homologous, as

are the posterior TNC in Oikopleura and the ascidian

neck, and since these structures derive from a posterior

CNS region expressing hox1, we conclude that these

structures are homologous to at least part of the

vertebrate hindbrain (Fig. 5C.b,c). The presence of motor

function in the vertebrate hindbrain (Lumsden and

Krumlauf, 1996), the existence of motor neurons in the

caudal ganglion that coordinate muscular tail movements

in larvaceans (Bone, 1998), and the presence of motor

neurons in the neck and visceral ganglion of ascidians

(Katsuyama et al., 2005; Meinertzhagen et al., 2000;

Okada et al., 2002) are consistent with this proposed

homology; we will therefore refer to the posterior-TNC/

neck plus caudal/visceral ganglion as the ‘‘urochordate

hindbrain’’.

In ascidians, hox3 expression suggests that the anterior

limit of the visceral ganglion corresponds to the anterior

limit of r4 of the vertebrate hindbrain (Locascio et al.,

1999). Unexpectedly, despite other gene expression

similarities between the larvacean caudal ganglion and

the ascidian visceral ganglion (Fig. 5C.d,g), there does not

appear to be a hox3 ortholog in the Oikopleura genome

(Seo et al., 2004). Analysis of additional hindbrain

markers such as Kreisler and Krox20 could help us

understand the consequences of the loss of Oikopleura

hox3.

Which part of the vertebrate CNS is homologous to the

urochordate ‘‘anterior brain’’?

In addition to the ‘‘urochordate hindbrain’’, we designate

the anterior part of the urochordate CNS as ‘‘anterior brain’’,

rather than just ‘‘brain’’, because in vertebrates the term

‘‘brain’’ includes the forebrain, midbrain, and hindbrain.

Data from ascidians have led to conflicting interpreta-

tions concerning homologies between the ascidian ‘‘brain’’

and the vertebrate brain, sometimes because of differences

in morphology and gene expression among ascidian species

(Lemaire et al., 2002; Locascio et al., 1999; Meinertzhagen

and Okamura, 2001; Meinertzhagen et al., 2004; Satoh,

2003; Takahashi and Holland, 2004; Wada and Satoh,

2001). For example, the posterior part of the ascidian

‘‘brain’’ has inconsistently been proposed to be homologous

to (i) the vertebrate metencephalon [based on the co-

expression of Ci-fgf9/16/20 and Ci-engrailed (Meinertzha-

gen et al., 2004)]; (ii) the vertebrate midbrain [based on the

co-expression of Ci-otx and Ci-engrailed (Imai et al.,

2002)]; or (iii) the vertebrate forebrain [based on the

presence of Ci-otx expression (Hudson and Lemaire,

2001) and the absence of Ci-dmbx expression (Takahashi

and Holland, 2004)]. To address these conflicting conclu-

sions, we first integrated larvacean and ascidian CNS gene

expression patterns (see above), and now we compare that

result to vertebrate expression patterns.

In vertebrates, forebrain and midbrain are labeled by Otx

expression (Fig. 5C.b). The expression of Pax6 in two

domains, one in the posterior forebrain and one in the

anterior hindbrain, has been used to define the midbrain,

which develops in the intervening gap (‘‘pax6-gap’’) and is

regulated by Pax2 and Engrailed expression (Fig. 5C.b)

(Matsunaga et al., 2000; Scholpp et al., 2003; Schwarz et

al., 1999).

As in vertebrates, the prospective anterior brain of

urochordates is labeled by otx expression (Fig. 5C.c,f),

suggesting at first glance that the urochordate anterior brain

is homologous to the vertebrate forebrain + midbrain (Fig.

5C.b). In urochordates, however, the fact that the otx

domain is subdivided along the AP axis by two expression

domains of pax6 at early tailbud stage (Fig. 5C.c,f), leads to

an alternative interpretation. In this alternative, the anterior

pax6 domain in urochordates labels the homolog of the

vertebrate forebrain, while the posterior pax6 domain labels

the homolog of the anterior hindbrain, and the pax6-gap

could be the urochordate homolog of the vertebrate

midbrain. This interpretation, however, conflicts with two

facts. First, the pax6-gap of the urochordate anterior brain

fails to express the vertebrate midbrain markers pax2/5/8

and engrailed (Fig. 5C.b,c,f). And second, the posterior

expression domain of pax6 in the urochordate anterior brain

overlaps the otx expression domain, while Otx expression is

excluded from the vertebrate hindbrain (Fig. 5C.b,c,f).

Therefore, these data lead to the conclusion that the

urochordate anterior brain is homologous to the vertebrate

forebrain (Fig. 5C.b,c).

C. Canestro et al. / Developmental Biology 285 (2005) 298–315 311

New perspectives on chordate CNS evolution from an

Oikopleura point of view

Gbx gene loss, an ancestral event in urochordate evolution

In vertebrates, the action of Gbx excludes the posterior

boundary of Otx expression from the anterior hindbrain

labeled by Pax6 (Fig. 5C.b) (reviewed in Rhinn and Brand,

2001). We know that the boundary between Otx and Gbx

expression domains is crucial to define the limit between the

midbrain and the hindbrain in vertebrates because vertebrate

Gbx mutants show caudal expansion of Otx expression back

to the level of rhombomere 4. The finding that Gbx also

antagonizes Otx in Drosophila suggests that this gene

interaction is ancient in Bilateria (Hirth et al., 2003; Reichert

and Simeone, 2001). Interestingly, the apparent absence

of any Gbx homolog from the genome projects of C.

intestinalis (Wada et al., 2003) and C. savignyi (http://

www.broad.mit.edu/annotation/ciona/index.html), and the

failure to isolate a Gbx homolog from Oikopleura [despite

our unpublished efforts and the genome sequencing traces

(Edvardsen et al., 2005)], strongly suggest that the loss of

gbx is a urochordate synapomorphy. This gene loss might

have affected the evolution and regionalization of the

urochordate CNS, perhaps by permitting a caudal expansion

of the otx domain as observed in vertebrate Gbx mutants. A

caudal expansion of otx expression in an ancestral urochor-

date would be consistent with our comparative analysis of

Oikopleura and ascidian data, which shows that the

posterior boundary of otx expression overlaps with the

posterior pax6 expression domain in the anterior brain (see

discussion above and Fig. 5C.b,c,f). The study of additional

urochordate orthologs of genes upstream and downstream of

Gbx function in the vertebrate anterior hindbrain (e.g.,

Wnt1, Fgf8) will help assess the impact of the loss of gbx

from the urochordate genome.

Do urochordates have a homolog of the vertebrate

midbrain?

In the Oikopleura CNS, absence of engrailed and pax2/

5/8 expression anterior to the hox1 expression domain that

labels the hindbrain argues against the presence of midbrain

in the larvacean lineage. Thus, we conclude that the absence

of a midbrain in ascidians, recently proposed by T.

Takahashi and P.W.H. Holland (2004) based on the absence

of dmbx expression in the anterior brain, is not just a

peculiarity of the ascidian Class, resulting perhaps from its

drastic CNS metamorphosis, but is a synapomorphy of the

urochordate Subphylum.

Absence of evidence supporting the existence of a

midbrain in amphioxus (Holland et al., 1997) and ascidians

(Takahashi and Holland, 2004) led to the most parsimonious

conclusion that the absence of a distinct midbrain was the

ancestral condition for chordates, and that the midbrain is a

developmental innovation in the vertebrate lineage (Takaha-

shi and Holland, 2004). Although this conclusion continues

to be the most parsimonious explanation, our data on

Oikopleura CNS are also consistent with a different

hypothesis. The posterior overlap of otx and pax6 ex-

pression, the absence of gbx in urochordates, and the

variability or absence of the expression of orthologs of

vertebrate midbrain markers (such as engrailed and pax2/

5/8) in the urochordate anterior brain, suggest that the

midbrain was present in the last common ancestor of

extant chordates, but was modified or lost independently in

the urochordate and in the cephalochordate lineages. Study

of the developmental mechanisms underlying the posterior

subdivision of the anterior brain of Oikopleura (AB3)

could provide new data to better understand the evolution

of the urochordate brain, because AB3 is located just

anterior to the urochordate hindbrain, and its homology is

as yet uncertain due to the absence of expression of all

CNS markers so far analyzed.

Do urochordates have a midbrain–hindbrain organizer

(MHB)?

In vertebrates, the MHB forms at the border of Otx and

Gbx expression domains, and requires expression of Pax2,

Engrailed, Wnt11, and Fgf8 genes (reviewed in Rhinn and

Brand, 2001). The absence of a Gbx ortholog in urochor-

dates makes it difficult to recognize a putative MHB

organizer in this Subphylum. In Oikopleura, the absence

of engrailed and pax2/5/8 expression immediately posterior

to the otx expression domain argues against the presence of

an MHB organizer homolog in the larvacean CNS.

In ascidians, it was argued that because pax2/5/8

expression in the neck coincides with the gap between otx

and hox1 expression domains (Wada et al., 1998), and

because engrailed and fgf8/17/18 expression appears in the

anterior visceral ganglion (Imai et al., 2002), the neck region

of the ascidian CNS is homologous to the vertebrate

MHB (Imai et al., 2002; Jiang and Smith, 2002; Meinertz-

hagen et al., 2004; Satoh, 2003; Wada et al., 1998;

Wada and Satoh, 2001). Close inspection of Ci-pax2/5/8,

Ci-engrailed, and Ci-fgf8/17/18, however, reveals that they

are not co-expressed in C. intestinalis (Imai et al., 2002). If

these genes are not co-expressed, then they cannot have the

same genetic interactions their orthologs have in the

development of the vertebrate MHB. Even if Ci-fgf8/17/

18 signaling might provide organizer function in the

anterior part of the visceral ganglion in ascidians, that

function might not necessarily be homologous to the MHB

organizer specifically, because Fgf8 is also active in the r4

organizer in the hindbrain of the vertebrate Danio rerio

(Maves et al., 2002; Walshe et al., 2002). The interpretation

that Fgf expression in the ascidian CNS could be

homologous to the r4 organizer is compatible with our

definition of the ‘‘urochordate hindbrain’’, and with the

correspondence of the anterior part of the visceral ganglion

in ascidians with rhombomere 4 in vertebrates based on

expression of Ci-hox3 (Locascio et al., 1999).

Finally, the obvious differences in the expression patterns

of engrailed and pax2/5/8a between Oikopleura and

C. Canestro et al. / Developmental Biology 285 (2005) 298–315312

ascidians, and indeed among different ascidian species [i.e.,

even between the congeners C. savignyi and C. intestinalis

(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

the light of our data from Oikopleura and published results

in ascidians, and in the absence of any functional data about

the roles of engrailed, pax2/5/8, and fgf8/17/18 in ascidians,

we conclude that there is no convincing evidence for an

MHB homolog in urochordates. Characterization and func-

tional analysis of Fgf family members in Oikopleura and

ascidians would help to test whether organizer activity exists

in urochordates.

Alternative hypothesis for the function of ‘‘MHB genes’’ in

urochordates

Urochordate tailbud stage embryos may correspond to

much later developmental stages than the vertebrate gastrula

and neurula stages in which the MHB forms. In vertebrates,

many of the genes that are involved in the development of

the MHB are also expressed later in the hindbrain, where

they perform different functions than they do in the MHB

(Lumsden and Krumlauf, 1996). In the vertebrate hindbrain,

Pax2, Pax5, Pax8, En1, En2, Pax6, Hox1, Dmbx1, and Lim

transcription factors, in conjunction with graded FGF

signals, specify motor neurons and interneurons, and are

important for axon guidance (Burrill et al., 1997; Dasen et

al., 2003; Gavalas et al., 2003; Irving et al., 2002; Kawahara

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

amphioxus hindbrain, has been postulated to be related to

functions in neuron specification (Holland and Holland,

1999; Holland et al., 1997; Kozmik et al., 1999). The late

expression of ‘‘MHB genes’’ in the vertebrate and cepha-

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

neuron fate and axon guidance rather than an organizer

function.

This alternative hypothesis is consistent with several

facts. First, shortly after hatching, Oikopleura and ascidians

probably have functional motor neurons because they show

coordinated tail movement. Second, motor neurons confined

to the ascidian CNS neck region begin to be specified as

early as late-gastrula stage (Katsuyama et al., 2005; Okada

et al., 2002). And third, the differences in expression

patterns of ‘‘MHB genes’’ in tailbud embryos between

different ascidian species, and between Oikopleura and

ascidians could merely reflect species-specific modifications

of developmental time and embryonic position of specific

neurons in the urochordate hindbrain rather than fundamen-

tal differences in CNS regionalization. Taken together, these

considerations suggest that the expression of ‘‘MHB genes’’

described so far in Oikopleura and ascidians reflects

development of specific neurons rather than action in an

MHB organizer. Functional experiments will be necessary

to disprove this conclusion.

Acknowledgments

We are grateful to Skipper B. Young of the ‘‘Charming

Polly’’ for help in larvacean collecting. We thank F. Mazet

for sharing with us her unpublished results on the expression

of Ci-pax2/5/8b in Ciona intestinalis. We thank L. Maves

and W. Cresko for helpful suggestions and discussion, and

A. Amores for providing Hox and Engrailed primers. We

thank undergraduate researchers T. Siriphatnaboon and T.

Keopuhiwa for help with animal care. We appreciate the

work of two anonymous reviewers for helping to improve

the manuscript. Complete fosmid sequencing was per-

formed under the auspices of the U.S. Department of

Energy, Office of Biological and Environmental Research,

in the University of California, Lawrence Berkeley National

Laboratory (contract DE-AC03-76SF00098). This material

is based on work supported by the National Science

Foundation under Grant No. IBN-0345203 and an IGERT

grant in Evolution of Development DGE-9972830. Any

opinions, findings, and conclusions or recommendations

expressed in this material are those of the author(s) and do

not necessarily reflect the views of the National Science

Foundation. We thank the Spanish Ministry of Education,

Culture and Sports for support for CC (EX2002-0059).

Appendix A. Supplementary data

Supplementary data associated with this article can be

found, in the online version, at doi:10.1016/j.ydbio.2005.

06.039.

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