Int. J. Dev. Biol.

61: 655-664 (2017)

doi: 10.1387/ijdb.170258jg
www.intjdevbiol.com

Origin and evolution of the chordate central nervous system:

insights from amphioxus genoarchitecture
BEATRIZ ALBUIXECH-CRESPO1, CARLOS HERRERA-ÚBEDA1, GEMMA MARFANY1, MANUEL IRIMIA*,2,3
and JORDI GARCIA-FERNÀNDEZ*,1
1
Dept. Genetics, Microbiology and Statistics and Institute of Biomedicine (IBUB), Faculty of Biology, University of
Barcelona, 2EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), The Barcelona
Institute for Science and Technology, Barcelona, Spain and 3Universitat Pompeu Fabra (UPF), Barcelona, Spain

ABSTRACT The vertebrate brain is arguably the most complex anatomical and functional structure
in nature. During embryonic development, the central nervous system (CNS) undergoes a series of
morphogenetic processes that eventually obscure the major axes of the early neural plate to our
perception. Notwithstanding this complexity, the “genoarchitecture” of the developing neural tube
brings into light homologous regions between brains of different vertebrate species, acting as a
molecular barcode of each particular domain.Those homologous regions and their topological inter-
relations constitute the ancestral, deeply conserved, bauplan of the vertebrate brain. Remarkably,
although simpler, the cephalochordate amphioxus shares multiple features of this bauplan, serving
as a privileged reference point to understand the origins of the vertebrate brain. Here, we review
the development of the chordate CNS in view of the latest morphological and genoarchitectonic
data from amphioxus. This comparison reveals that the amphioxus CNS is far from simple and
provides unique insights into the structure of the vertebrate CNS and its evolutionary origins. In
particular, we summarize recent research in amphioxus and vertebrates that has challenged views
on the major partitions of the vertebrate brain, proposing a novel organization of the chordate CNS
bauplan that better reflects developmental and evolutionary data.

KEY WORDS: bauplan, brain partition, DiMes, vertebrate CNS, cephalochordate

Introduction been, and surely will be, the subject of study from very different per-
spectives by many researchers. Currently, there are large amounts
Homology is a fundamental concept in biology and one of the of gene expression data that can be used as topological markers in
key pillars of comparative embryology. Therefore, it is not surprising the developing vertebrate CNS. As all vertebrate neural tubes are
that since its early years, Evo-Devo researchers have discussed homologous and develop under similar principles, it is generally
whether anatomy or gene expression are better indicators of “strict” assumed that gene expression patterns, which provide positional
homology (“the same organ in different animals under every variety information and give insights into the specification and maintenance
of form and function”, Owen 1843, pp.379). These controversies of brain derivatives, are, overall, good markers of homology (Morona
started soon after developmental gene expression data from single et al., 2011, 2016). However, comparisons between vertebrate and
genes were first compared between two species (e.g., Abouihef et non-vertebrate CNS are more challenging, as similarity of (relative)
al., 1997, Wray and Abouihef 1998), and still persist in the postge- gene expression patterns are evaluated between highly diverse,
nomic era (Tschopp & Tabin, 2016), when entire transcriptomes and often quite dissimilar, anatomical structures, without a clear
can be compared.
It is not surprising, then, that searching for homologies, diver- Abbreviations used in this paper: AP, anteroposterior; ARCH, archencephalic protagma,
gences, and innovations in the most complex and fascinating structure CNS, central nervous system; DEU, deuteroencephalic protagma; DI, diencephalon;
in nature – the central nervous system (CNS) of vertebrates – has DiMes, DiMesenphalic primordium; MB, midbrain.

*Address correspondence to: Manuel Irimia. Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain.
E-mail: [email protected] http://orcid.org/0000-0002-2179-2567 or
Jordi Garcia-Fernàndez. Dep. Genetics, Microbiology and Statistics and Institute of Biomedicine (IBUB), Faculty of Biology, University of Barcelona. Av. Diagonal
643, 08028 Barcelona, Spain. E-mail: [email protected] - http://www.ub.edu/genetica/evo-devoen/garciare.htm http://orcid.org/0000-0001-5677-5970

Submitted: 10 October, 2017; Accepted: 30 October, 2017.

ISSN: Online 1696-3547, Print 0214-6282

© 2017 UPV/EHU Press
Printed in Spain
656 B. Albuixech-Crespo

A C
notochord neural tube dorsal fin corpuscles of
myomeres caudal fin de Quatrefages

neural tube

postanal tail
anus notochord
oral cirri atrium atrio pore
hepatic caecum
gill slit gonads

B D rostral

ampulla caudalis
filum terminale
anterior vesicle

nerves

frontal eye
intercalated anterior part intermediate part posterior part
region of spinal cord of spinal cord of spinal cord neuropore

cerebral
vesicle
lamellar
body

intermediate
pharyngeal region gut region postanal tail zone
rostrum buccal region
velar region

atrial region / atrial nervous system

gonadal region

Fig. 1. Amphioxus anatomy and neural system. (A) Schematic representation of an adult amphioxus, indicating major anatomical structures. (B) In-
nervation of the adult amphioxus, dorsal nerves 1, 2, 3… (n1, n2, n3…), infundibular organ (io), notochord (chd), Kölliker’s pit (Köp), Joseph cells (cJo),
nucleus of Rohde (nRo), Rohde cell (cRo), Hatschek’s pit (Hp), inner labial muscle (ilm), external labial muscle (elm), buccal cavity (bc), velar sphincter
muscle (vsm), pterygeal muscle (pm), gonad (gon), organ of Hesse (oHe), trapezius muscle of atriocoelomic funnel (tm), atriopore sphincter muscle
(atsm), anal sphincter muscle (ansm). (C) Detail of innervation in the most anterior tip, velar plexus (vp), inner buccal plexus (ibp), outer buccal plexus
(obp), first cell of Rohde (arrow), Hatschek’s pit (white arrowhead), anastomosis between nerves 1 and 2 (open arrowhead), two of the neuromuscular
contact zones (black arrowheads). (D) Schematic representation of the neural tube (dorsal view, anterior is up). (B,C) Reproduced with slight modifica-
tions from Witch and Lacalli (2005, © 2008 Canadian Science Publishing or its licensors, reproduced with permission). (D) Adapted from Lacalli (1994).
Anterior is to the right and dorsal is up in (A,B,C).

homologous topological reference. Hence, the study of amphioxus, embryologists and, since 1980’s, also of molecular biologists. First
which has a centralized CNS that develops from a neural plate that described by Peter Simon Pallas as a molluscan slug in 1774 (Pallas,
is unambiguously homologous to that of vertebrates, has been 1774), later classified as a close relative of agnathan vertebrates,
particularly useful to shed light into how the CNS of vertebrates has hagfish and lampreys (Costa, 1834), its phylogenetically privileged
originated and evolved. position is now thought to be that of being the most basal-branching
Here, after an introduction to the key phylogenetic position of extant chordate (Delsuc et al., 2006). In this Special Issue of the
the amphioxus in the path to vertebrates, we review classic and Int. J. Dev. Biol., readers can learn about the fascinating quest of
recent studies on vertebrates and cephalochordates to provide a several research groups to collect, get to reproducing in captivity,
comparative view of the development and morphology of their CNS. and work with amphioxus embryos, first using large amounts of
We begin by setting the conceptual framework of our comparisons: radioactive 32P probes, then performing many PCRs, and from strug-
comparative neuroanatomy and the prosomeric model. We will then gling for genome sequencing funding to, of late, trying all available
review recent results suggesting a novel ontogenetic organization and possible gene modification protocols (see Holland, this issue).
of vertebrate brain regions as inferred from comparative studies Cephalochordates share the chordate phylum with the Olfac-
with amphioxus, and, finally, we will discuss the impact of this tores (Urochordata and Vertebrata) (Delsuc et al., 2006), with an
organization on our understanding of key adult brain structures in estimated time of divergence from the last common ancestor at
cephalochordates. around 550 million years ago (Blair and Hedges, 2005; Delsuc et
al., 2008). The main characteristic of chordates is the presence of a
The amphioxus: key to understanding chordate and notochord, which extends along much of the body; in the particular
vertebrate transitions case of cephalochordates, the notochord seems to extend beyond
the most anterior tip of the neural tube (Fig. 1A), although this
For centuries, amphioxus has attracted the attention of zoologists, anterior-most part has been suggested to be homologous to the
 Amphioxus CNS genoarchitecture 657

prechordal plate (see below). The very name of amphioxus is in 2010; Puelles and Rubenstein, 2015). This model proposes that the
fact an allegory of its shape (amphis = both, oxys = sharp). Unlike vertebrate CNS is composed of basic units of neural development,
vertebrates, the amphioxus notochord is maintained throughout the neuromeres, whose organizational principles are identifiable in
life and is the only skeletal structure present in the adult body. The the longitudinal and transversal axes across all studied vertebrate
hollow neural tube, which is dorsal to the notochord, is another of species (Fig. 2 B-B’’). The prosomeric model facilitates comparisons
the morphological characteristics shared with vertebrates, in ad- across vertebrate species, since neuromeres refer to the ‘intrinsic’
dition to the gill slits in the pharynx, the myomeres (or segmental axes, as established in the early neural plate, instead of the ‘extrinsic’
muscle “bricks”), and the post-anal tail (Garcia-Fernàndez and axes that had been used in other neuroanatomic approximations
Benito-Gutiérrez, 2009) (Kuhlenbeck, 1967; Puelles and Rubenstein, 1993), and which are
Not only has its prototypical body plan (bauplan) and phyloge- greatly affected by the complex morphogenetic events undergone
netic position enticed evolutionary biologists, but also its genome by the neural tube upon closure (Fig. 2).
is highly remarkable. One of the first genes cloned in
amphioxus (Holland et al., 1992) supported the notion
that the amphioxus had a prototypical genome with A rp
B
respect to the vertebrate – namely human – genome.
fp
In 1994, after the cloning of the single amphioxus Hox
gene cluster (Garcia-Fernàndez and Holland, 1994), rp R
Peter Holland together with one of the co-authors here C
(back when they were both very young) and other A/R P/C
rp
colleagues, proposed that the origin of vertebrates
was linked to the double duplication of an ancestral A’ B’
fp
genome that was very similar to the present amphioxus
genome, the so-called 2R hypothesis (Holland et al., rp
1994). Susumu Ohno already noticed the potential of
gene duplication in evolution years before, based on
earlier measures of genome size, and even proposed
than the genome was duplicated somewhere in the path
A’’ D rp ap ap rp R
C

to vertebrates (Ohno, 1970). But it was not until 2005,

with the publication of the first urochordate genome V fp

(Dehal and Boore, 2005), and definitely in 2008, with B’’

the publication of the amphioxus genome sequence MB
PT
draft (Putman et al., 2008), that this hypothesis was Th
corroborated: about 95% of the human genome could A’’’ D rp rp
T
PTh
ZLI p1 IsO
p2
be traced back to a double polyploidisation of an ap ap p3
amphioxus-like genome. In these 14 years, the idea V fp ANR
PHy
hp1
D

that those new genes were instrumental to the morpho- THy hp2
SP
Rh
logical innovations of vertebrates shifted subtly from
emphasizing additional protein-coding sequences to the R
relevance of duplication and innovation of regulatory
sequences, through the Duplication, Degeneration,
A’’’’ D ap
rp
ap
SC

and Complementation model (the DDC model, Force

et al., 1999), or the Duplication, Degeneration, and V fp C
Innovation model of regulatory DNA (the DDI model,
Jiménez-Delgado et al., 2009). More recently, the link
Fig. 2. Chordate neurulation and vertebrate central nervous system development.
between genome duplication and epigenetic changes The vertebrate (A) and amphioxus (A’) neural plate (dorsal view) fold (A’’, A’’’) to form
(Acemel et al., 2016) also supports the view that the 2R the neural tube (A’’’’) similarly, generating a hollow nerve tube that closes dorsally by the
events at the origin and early evolution of vertebrates roofplate (rp). (B) Schematic lateral views of bending of the vertebrate neural tube as a
were instrumental to the path that led to us, humans. result of differential growth, resulting in an arching of its internal axis (red dotted line). Some
In this review, we will show how amphioxus has also neuromeres are indicated as the regionalization of the neural tube proceeds (grey dotted
illuminated the origin and evolution of the vertebrate lines). The forebrain is subdivided into secondary prosencephalon (SP) with peduncular
CNS, arguably the most paramount and defining organ and terminal prosomeres (hp1 and hp2, respectively), which include the hypothalamic
of our clade. region, telencephalon (T), and the optic vesicles, and Diencephalon (D), with prosomeres
1, 2, and 3 (p1–p3), which are represented by the pretectum (PT), thalamus (Th), and
prethalamus (PTh), respectively. More caudally, midbrain (MB), romboencephalon(Rh),
Conceptual framework: the prosomeric model and spinal cord (SC) regions are identified (for simplicity, their respective neuromeric
components are not depicted). The position of the secondary organizers zona limitans
A major breakthrough in our understanding of the intrathalamica (ZLI) and the isthmic organizer (IsO) are represented by blue spikes.
bauplan of vertebrate CNS came with the publication Lateral view. Dark grey represents floorplate (fp) in all schemes; terminal hypothalamic
of the prosomeric model by Puelles and Rubenstein prosomere (Thy), peduncular hypothalamic prosomere (PHy). A/R, anterior/rostral; P/C,
(Puelles and Rubenstein, 1993;Puelles, 1995; Puelles, posterior/caudal; D, dorsal; V, ventral. Adapted from Albuixech-Crespo et al., (2017).
658 B. Albuixech-Crespo

These primary axes are easy to identify in the early neural plate components: the prethalamus (p3), the thalamus (p2) and the
(Fig. 2A). The anteroposterior (AP) axis overlaps the longitudinal pretectum (p1), in a rostrocaudal order (Puelles and Rubenstein,
axis, whereas the mediolateral axis will correspond to the dorso- 1993; Puelles, 1995) (Fig. 2).
ventral (DV) axis after neural tube closure, in which the neural Many of these subdivisions are orchestrated by the action of
plate midline and border will become the neural tube floor and the secondary organizers (blue dashes in Fig. 2B’’). An organizer
roof, respectively. Interestingly, this layout results in an anatomical is a cellular domain that releases particular regulatory morpho-
singularity in the neural plate, since the presumptive basal (ventral) gens that act upon its neighboring tissues. Three main secondary
plate, alar (dorsal) plate and roof run concentrically across the floor AP organizers have been described for vertebrates, the anterior
in the anterior and posterior tips of the neural plate (Fig. 2A, B-B’’). neural ridge (ANR), located in the most rostral part of the neural
As development progresses, the AP axis bends due to the differ- tube; the zona limitans intrathalamica (ZLI), located between the
ential growth of different territories of the neural tube (Fig. 2 B-B’’). prethalamus and the thalamus, and the isthmic organizer (IsO),
Despite this bending, each neuromere is established according to which develops in the boundary between the midbrain and the
its position with respect to the AP axis (Fig. 2B). Moreover, the DV rhombencephalon. Each of these organizers releases a specific
axis maintains its orthogonal relationship with the AP axis. Thus, combination of morphogens that influences the development of the
the prosomeric model organically defines the basic developmental surrounding structures. In particular, the ANR expresses Fgf8, which
units of a tissue whose morphology is highly variable and plastic, is essential for the correct regionalization of the prosencephalon
assisting comparisons between neuroanatomies of equivalent and necessary for the correct formation of the telencephalon, a
bauplans (Puelles and Rubenstein, 1993; Puelles, 1995; Puelles dorsal outgrowth of the secondary prosencehalon (Kiecker and
and Rubenstein, 2015). Lumsden, 2012; Vieira et al., 2010). The reference morphogen in
Moreover, an additional crucial aid for comparative neuroanatomy the ZLI is Shh, which is crucial for the differential specification of
is provided by gene expression patterns. During the development the thalamus (Crossley et al., 1996; Chi et al., 2003; Vieira et al.,
of the CNS, different populations of neural progenitors are estab- 2005; Hirata, 2006; Vue et al., 2009). Regarding the IsO, Fgf8 acts
lished based on their molecular codes. The pool of transcriptionally again as morphogen, but in this case together with Wnt1. This lat-
active genes is different for each particular group of cells, and the ter region can be identified from very early stages of development
dynamics of these specification processes eventually result into by the apposition of the expression patterns of Otx2 and Gbx2,
positional identities in the neuroepithelium, what is known as neu- which act antagonistically and are crucial to define the position
ral patterning. This relationship between specific spatiotemporal and function of the IsO (Hidalgo-Sánchez et al., 2005; Kiecker
gene expression patterns and the positional identity of the neural and Lumsden, 2012).
progenitors is the basis for the genoarchitecture. This concept has Interestingly, the organizers and their associated genetic net-
been exploited by Evo-Devo approaches, facilitating comparisons works have been conserved since the origin of vertebrates (Osorio
of developing vertebrate brains and allowing direct homology as- et al., 2005). However, the extent of evolutionary conservation is
signments (Ferran et al., 2007; Ferran et al., 2009; Medina et al., under debate when larger phylogenetic distances are considered,
2011; Puelles and Ferran, 2012). Similar approaches have also namely between vertebrates and other chordates or other deutero-
been undertaken to compare domains between more distantly stomes (e.g. hemichordates). Several articles in recent years have
related species. In particular, similarity of relative gene expression discussed the presence/absence and homology/convergence of
patterns along the AP axis have received much attention, reaching secondary organizers and their potential functionality as bona fide
the conclusion that a subset of genes have likely maintained their organizers (see Holland et al., 1997; Kozmik et al., 1999; Shimeld,
relative AP positions since the last common ancestor of Bilateria 1999; Lowe et al., 2003; Takahashi and Holland, 2004; Lowe et
(Lowe et al., 2003; Castro et al., 2006; Hirth, 2010; Irimia et al., al., 2006; Denes et al., 2007; Hirth, 2010; Steinmetz et al., 2011;
2010). However, as mentioned above, these inferences are often Pani et al., 2012; Arendt et al., 2015; Yao et al., 2016, for engaging
obscured by unavoidable problems encountered when comparing discussions and controversies). As expected, one of the clades at
highly divergent CNSs. the center of these controversies is the cephalochordate amphioxus.

Secondary anteroposterior organizers: major players in The amphioxus CNS: challenging major subdivisions
vertebrate brain development and evolution of the vertebrate brain

The vertebrate CNS becomes regionalized along its AP axis very The amphioxus CNS is composed of a neural tube placed dor-
early in development. At the neural plate stage, expression patterns sally to the notochord (Fig. 1). Rostrally, the notochord continues
of genes such as Otx2, Gbx2, Fezf, Irx or Pax6 start establishing in appearance beyond the extension of the neural tube, although
molecular subdivisions (Hidalgo-Sánchez et al., 2005; Rodríguez- the anterior-most tip of the notochord has been proposed to be a
Seguel et al., 2009). As development proceeds, the neural tube homolog of the vertebrate prechordal plate (Albuixech-Crespo et
closes, bends, and the classical general AP subdivisions become al., 2017). A subtle widening in the anterior part of the neural tube,
morphologically apparent: the prosencephalon (which will be further which corresponds to the cerebral vesicle, is barely noticeable in
subdivided into secondary prosencephalon and diencephalon), the adult, although this swelling is much more obvious at larval
the midbrain, the rhombencephalon and the spinal cord. All these stages. Beyond this, there are neither anatomically recognizable
large AP domains will progressively regionalize and subdivide into neuromeres nor other morphological landmarks in the amphioxus
neuromeres. For example, the rhombencephalon will metamerize neural tube to easily identify a segmental organization besides
into 11 rhombomeres (the neuromeres of the rhombencephalon), the visible dorsal nerves arranged in series (Fig. 1). However,
and the diencephalon into 3 prosomeres, with distinctive alar examination at the histological and cellular level reveals visible
 Amphioxus CNS genoarchitecture 659

histoarchitectonic differences in the AP and DV axes in larvae as These differential histoarchitectonic arrangements imply that
well as in adults, which reflect differences or subdivisions between there must be a subjacent molecular regionalization of the amphi-
regions along the neural tube (Wicht and Lacalli, 2005) (Figs. 1 oxus CNS. In fact, many genes involved in neural development
and 2; for detailed descriptions and cyto- and histo-architecture in vertebrates are also expressed in cephalochordate neural de-
of the amphioxus nervous system, see Lacalli (this issue, and velopment. Literature that describes, compares and establishes
references therein)). correspondences between expression patterns of genes involved

A HyPTh DiMes RhSp

alar compartment

basal compartment
floor plate
basal compartment
Rostral-HyPTh
alar compartment

Interm-HyPTh
S1 S2 S3 S4 S5 S6

Caudal-HyPTh
ARCH DEU

B C
neuropore anterodorsal nerve lamellar body

ARCH DEU

frontal eye

balance infundibular neuropile PMC

rostral
organ organ
nerve

B’
posterior intermediate
anterior vesicle vesicle region
DiMes

HyPTh RhSp

C’
MB
PT

B’’ DI MB Rh Sp T PTh
Th

PHy

hp2
hp1 p3 p2 p1 THy
HB

Otx Gbx
SC
Fezf Irx B

Fig. 3. Ontogenetic correspondences and proposed homologies between cephalochordates and vertebrates CNS. (A) Summary of all identified
anteroposterior and dorsoventral partitions of the neural plate of amphioxus, dorsal-view scheme, anterior is to the left. Hypothalamo-prethalamic pri-
mordium (HyPTh), DiMesencephalic primordium (DiMEs), Rhombencephalo-Spinal primordium (RhSp). S1-S6 indicates the relative position of the six
most anterior somites. (B-B’’) Topological comparison of major molecular subdivisions between an ancestor with a bipartite brain (B), cephalochordates
(B’) and vertebrates (B’’). Archencephalic prototagma (ARCH), deuteroencephalic prototagma (DEU). The amphioxus DiMEs seems to be homologous
to the thalamus (p2), pretectecum (p1) and midbrain (MB), whereas the diencephalon (DI) is neither an ontogenetic nor an evolutionary unit, as the
prethalamus (p3) is derived from a vertebrate-specific subdivision of the HyPTh. Gene expression domains of relevant genes (Otx, Gbx, Fezf and IrxB)
that mark the three main subdivisions of the chordate brain are indicated below. (C-C’) Correspondence of the three main subdivisions (colour coded) of
the neural plate to the larval stages of amphioxus (C) and a representative neural tube of vertebrates (C’). Blue spikes indicate the secondary organizers
IsO (left) and ZLI (right) in C’. Primary motor center (PMC), hindbrain (HB). Adapted from Albuixech-Crespo et al., (2017).
660 B. Albuixech-Crespo

in vertebrate and amphioxus neural regionalization is abundant. pretectum and midbrain should be considered an evolutionary
Very recently, some of us with our collaborators (Albuixech-Crespo elaboration of an ancestral, perhaps amphioxus-like, DiMes region.
et al., 2017) mapped the expression of 48 genes whose orthologs The hypothesis that the diencephalon is neither an evolutionary
in vertebrates have a well-established morphological interpretation nor an ontogenetic primordial subdivision of the vertebrate brain
within the prosomeric model. These mappings were done at a single was further supported by knock-out and knock-down experiments
developmental stage of amphioxus (7-somite neurula stage), allow- in mouse and zebrafish (Albuixech-Crespo et al., 2017). Patterning
ing homochronic comparisons of gene expression patterns. With of the DiMes-like territory in vertebrates occurs under the control
these data, the authors proposed a genoarchitectonic model for of the secondary organizers ZLI and IsO, which, despite ongoing
amphioxus consistent with the prosomeric model (Fig. 3). As previ- debate, seem to be absent in amphioxus (Shimeld, 1999; Pani et
ously suggested (Castro et al., 2006), this model proposes that the al., 2012). Interfering with the vertebrate ZLI turns the thalamic
incipient amphioxus neural tube is molecularly divided into a rostral region into a pretectum-like territory, ablating the IsO abolishes
archencephalic (ARCH) domain and a caudal deuterencephalic the midbrain and expands the pretectum territory, and a quadruple
(DEU) domain from very early stages, similarly to vertebrates. This morpholino in zebrafish that alters both ZLI and IsO transforms
division is highlighted by the abutting expression domains of Otx those three DiMes regions of vertebrates (thalamus, pretectum
and Gbx. Furthermore, the amphioxus ARCH has two major sub- and midbrain) into a more molecularly homogeneous structure
divisions, which we termed Hypothalamo-Prethalamic primordium that resembles the amphioxus DiMes region (Fig. 4). Therefore,
(HyPTh; further subdivided into Rostral-HyPTh, Interm-HyPTh and these results suggest a close relationship between the gain or loss
Caudal-HyPTh) and Di-Mesencephalic primordium (DiMes) (Fig. of secondary organizers during evolution and the origin or loss of
3B), whose boundary is defined by the abutting expression of Fez specific partitions in the brain among the major chordate groups.
on the most caudal HyPTh domain (Caudal-HyPTh) with that or the
Irx genes in the DiMes. In contrast, and as mentioned above, the Amphioxus genoarchitectonic model provides novel
vertebrate ARCH is traditionally divided into three main regions, insights into the ontogeny of adult structures and their
the secondary prosencehalon (hypothalamus plus telencephalon), homology with vertebrates
the diencephalon (pretectum, thalamus and prethalamus) and
the midbrain. Remarkably, considering its topological position Our genoarchitectonic model of the developing amphioxus
relative to several markers as Fez and Irx, the Caudal-HyPTh neural tube also sheds light into the ontogenetic origins of several
may be homologous to the vertebrate prethalamus. In addition, larval and adult brain structures, and their possible evolutionary
the small amphioxus DiMes, consisting of two rows of cells along relationships with vertebrate derivatives. Ontogenetic assignments
the AP axis at the mid-neurula stage, seems to be homologous to between adult, larval and embryonic structures and domains
the thalamus, pretectum and midbrain all together. Therefore, the have many limitations in amphioxus. First, the lack of cell trac-
last two observations, together with multiple lines of experimental ing data on the formation of neural derivatives hampers direct
embryological evidence (Gardner and Barald, 1991; Martinez et extrapolations of adult neural populations from early stages of
al., 1991; Bally-Cuif et al., 1992; Bloch-Gallego et al., 1996; Vieira development. Second, there is no information about the possible
et al., 2005; Vue et al., 2009; Hirata, 2006), profoundly challenge migration processes that the progenitor populations of the terminal
textbook subdivisions of the vertebrate brain: (i) the diencephalon derivatives of the adult neural tube experience. Third, there are
loses its coherence as a single entity, since it has neither evolution- no robust continuous reference landmarks. Two main references
ary nor developmental support, and (ii) the vertebrate thalamus, have been used in the literature to infer correspondences among

A PTh
zli
Th PT MB
iso
Rh

Fig. 4. Experimental disruption of

WT vertebrate secondary organizers
-ZLI -ISO recapitulates the amphioxus brain.
(A) Schematic representation of major
B iso C zli neural compartments (PTh,Th, PT, MB,
PTh PT* MB Rh PTh Th PT* Rh and Rh) around DiMes, delimited by
the secondary organizers (ZLI and IsO:
red lines) in wild type (WT) vertebrates;
solid gray indicates Pax6 expression.
(B) Abrogation of the ZLI turns the
D PTh
zli
Th PT MB
iso
Rh
thalamic region into a pretectum-like
territory (PT*). (C) Genetic depletion
of the IsO abolishes the midbrain
and expands the pretectum territory
WT
(PT*). (D) Wild type and (E) quadruple
-ZLI morpholino of otx1a, otx2, eng2a,
-ISO
and eng2b in zebrafish eliminates ZLI
E PTh PT* Rh and IsO, and thus the whole DiMes
loses its subdivisions and mimics the
uniform amphioxus DiMes. Adapted
4MO from Albuixech-Crespo et al., (2017).
 Amphioxus CNS genoarchitecture 661

neural structures. One is the position of these structures respect visual center that is Otx- and Pax4/6-positive, located in the most
to the myomeres or somites in adults or embryos, respectively. posterior region of the cerebral vesicle in amphioxus larvae, and in
Unfortunately, however, it has not yet been proven whether the the caudal prosencephalon in lampreys (Suzuki et al., 2014) (Fig.
position and the size of myomeres are fixed or permanent in rela- 2A). In the latter study, Suzuki and colleagues suggested that the
tion to the neural tube, hampering ontogenetic assignments. The mesencephalic optic projections (which are retino-tectal, Pax6-
other one is innervation, which presents concordances with the negative and Otx2-positive) are secondarily developed, specific
myomeric asymmetry endorsed by the myoseptal position of the to vertebrates, and implicated in vision image formation. In con-
neural roots in the neural tube of the adult amphioxus. However, sequence, the retino-pretectal primary projections, which appear
these are difficult to extrapolate to early embryos. in earlier stages of lamprey larvae, would be more similar to the
Given these caveats, extrapolations on the correspondence ancestral state in vertebrates projecting to a visual Pax6-positive
of the neural derivatives in the adult amphioxus with respect the center, as it occurs in amphioxus. Lacalli (1996) identified the visual
progenitor territories in the neural plate stage during development center of amphioxus tracking the projections from the frontal eye
must come from meticulous correlations that include information for through electronic microscopy, and named it tectum, as a refer-
both its topological position and the combination of gene expres- ence of homology to the midbrain in vertebrates. However, other
sion patterns during neural ontogeny, always keeping in mind that authors concluded that cephalochordates lack a proper homolog to
gene expression patterns could be very dynamic and that they do the tectal region, based on the absence of expression of Dmbx in
not provide deep lineage information if considered independently. the amphioxus neural tube (Takahashi and Holland, 2004), which
Keeping these limitations in mind, we revise here previous literature is expressed in the midbrain of vertebrates. Our genoarchitectonic
and propose some ontogenetic correspondences for important model of the neural plate of amphioxus described above sheds light
terminal derivatives of the anterior CNS of amphioxus, and discuss into the evolutionary common origin for the midbrain, thalamus and
their implications to assign vertebrate homologies pretectum of vertebrates from a DiMes-like territory that was likely
ancestrally involved in visual processing. Based on these data, it
Frontal eye and visual system is tempting to speculate that a DiMes/pretectum-like function was
One of the most visible structures of the amphioxus neural tube ancestral, and that the origin of the midbrain as an independent
is the frontal eye, which can be observed from early larval stages. unit could have been linked to the origin of the vision with image
The topological position of the frontal eye suggests that it arises formation present in vertebrates, but not in amphioxus.
from the most anterior part of the neural plate (Lacalli, 1996), the
acroterminal domain (Fig. 3), as it occurs in vertebrates (McMahon Balance organ and circadian clock
and Bradley, 1990; Puelles et al., 2012; Puelles and Rubenstein, The topology and morphology of cells associated with the
2015; Albuixech-Crespo et al., 2017). The genoarchitecture of this circadian clock and the balance organ in the adult amphioxus,
area in the neural plate suggests its involvement in the development which are embedded in the ventral commissure and associated to
of this photoreceptive structure. Several genes expressed very early the lamellar body (a photoreceptor organ) (Figs. 1D and 3C), as
in amphioxus development are involved in eye structures in a wide well as immunoreactive to GABA, led some authors to propose its
number of bilaterians, such as Rx, Six3/6 and Lhx2/9 (Zuber et al., homology to the mammalian suprachiasmatic nucleus (Anadón et
2003; Kamijyo et al., 2015; Albuixech-Crespo et al., 2017), even al., 1998; Castro and Becerra, 2015). The suprachiasmatic nucleus
though the development of the amphioxus frontal eye becomes in vertebrates is a derivative of the alar part of the most rostral
apparent only later in development by the emergence of the cells hypothalamic territory (Puelles and Rubenstein, 2015), which is
with dendrites and cilia that protrude from the neuropore and by the Nkx2.2-positive. Also, in vertebrates, Lhx1 (also known as Lim1)
presence of the pigment cup. Remarkably, at larval stages, when is involved in the establishment and maintenance of this circadian
the frontal eye is evident, the resulting cellular types that form it rhythm in the suprachiasmatic nucleus (Hatori et al., 2014). In am-
maintain a characteristic genoarchitecture. Both in vertebrates and phioxus, the genoarchitecture of the most rostral part of the neural
cephalochordates Gi and c-Opsin are expressed in photoreceptors, plate is coincident, being Nkx2.2- and Lhx1/5-positive, further sup-
whereas Mitf and Pax2/5/8 appear in pigment cells (Vopalensky et porting the similarity between the two structures. However, the fact
al., 2012). In vertebrates, two eye regions develop as a result of that vertebrate suprachiasmatic nucleus arises from the alar part of
the splitting of the morphogenetic eye field by the action of signals the hypothalamus and the amphioxus balance organ is ventral in
coming from the prechordal mesoderm (Zuber et al., 2003), while origin casts doubts on the homology between the two derivatives.
in amphioxus it stays as a single medial structure.
The amphioxus frontal eye innervates the neuropile through Infundibular organ
serotonergic neurons (Vopalensky et al., 2012). This neuropile As mentioned above, the infundibular organ is located in the
develops immediately caudal to the infundibular organ, which marks most anterior part of the floor plate of the neural tube (Fig. 3C)
the transition between the anterior vesicle and the posterior region and secretes Reissner’s fiber to the central canal of the neural
of the cerebral vesicle. It projects to the primary motor center (PMC), tube (Olsson et al., 1994; López-Avalos et al., 1997). Its functions
located more caudally, in the region immediately posterior to the have not been characterized in amphioxus, although it seems to
limit between the 1st and the 2nd myomeres (Fig. 3C). Part of this be involved in axon guidance and establishment of commissures
region expresses Pax4/6 in larval stages (Suzuki et al., 2014) and, (Lehmann and Naumann, 2005). Both the amphioxus infundibular
topologically, it seems to correspond to the Pax4/6-positive DiMes organ and the vertebrate subcommissural and flexural organs
region described in our genoarchitectonic model (Albuixech-Crespo (which produce Reissner’s fiber in vertebrates) are formed near
et al., 2017). Interestingly, the frontal eye of amphioxus as well developing commissures (López-Avalos et al., 1997). The sub-
as the paired eyes of the lamprey larva project to a photoreceptor commissural organ develops in the pretectum (p1) and is the
662 B. Albuixech-Crespo

primary and permanent producer of Reissner’s fiber in vertebrates. de Excelencia Severo Ochoa 2013-2017’, SEV-2012-0208 to CRG. We
Based on these observations, some authors have proposed its would also like to acknowledge the support of the CERCA Programme
correspondence with the amphioxus infundibular organ (Olsson, / Generalitat de Catalunya. CH-B holds an FPI fellowship. The authors
1955; Lichtenfeld et al., 1999). In contrast, the flexural organ is wish to thank José Luis Ferrán and Ignacio Maeso for comments and
fruitful discussions.
located in the most rostral floor plate of vertebrates. Therefore,
given the topological position of the amphioxus infundibular organ,
a probable derivative of the HyPTh, it seems more likely that, if
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