Witten & Hall (2021) The Ancient Segmented Active and Permanent Notochord

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Witten & Hall: The Ancient, Segmented, Active and Permanent Notochord
Notochord Evolution and the Fossil Record
The truly ancient notochord provides the name for our own
phylum – Chordata – and connects vertebrates with tunicates
and cephalochordates and perhaps with more ancestral chor-
date ancestors (Barry 1841, Kowalevsky 1866, Hejnol & Love
2014, Annona et al. 2015).
We are all aware that chordates have a notochord during
their embryonic development and that the notochord forms the
primary embryonic skeleton of the antero-posterior body axis.
Less well known is that, with the exception of birds, almost
all adult vertebrates preserve the notochord throughout life
as a continuous rod, in the joints between vertebral bodies or
inside the vertebral centra. Commonly, between the vertebral
bodies the notochord expands and forms the inner part of the
intervertebral joint. In mammals this forms the nucleus pulposus
(Leeson & Leeson 1958, Grotmol et al. 2003, Shapiro & Risbut
2010, Choi & Harfe 2011). In birds it is not the notochord but
the rostral part of the sclerotome that contributes cells to the
intervertebral disk (Bruggeman et al. 2012). Within the vertebral
centra, the notochord typically does not extend beyond the
diameter it had in the embryo but becomes constricted in a
narrow notochord canal. The notochord canal can completely
disappear in amniotes but also in other vertebrates including
some teleosts (Schaeffer 1967). A persistent notochord canal is
a rare anatomical variation in humans and thus perhaps also
in other mammals (Christopherson et al. 1999).
In palaeontology the notochord is a well-used reference
structure to identify and position organs associated with it,
such as the dorsal nerve cord and dorsal muscle blocks; in a
study by John Maisey concerning the braincase of the Upper
Devonian shark Cladodoides wildungensis, the notochord is
referenced 42 times (Maisey 2005). A major difficulty arises
Ancient Fishes and their Living Relatives: a Tribute to John G. Maisey. Alan Pradel, John S. S. Denton & Philippe Janvier (eds.): pp. 215-224, 5 figs.
© 2021 by Verlag Dr. Friedrich Pfeil, München, Germany – ISBN 978-3-89937-269-4
The Ancient, Segmented, Active
and Permanent Notochord
P. Eckhard Witten* and Brian K. Hall**
* Department of Biology, Research Group ‘Evolutionary Developmental
Biology’, Ghent University, B-9000 Gent, Belgium;
peckhardwitten@aol.com (corresponding author)
** Department of Biology, Life Sciences Building. Dalhousie University,
Halifax, NS, B3H 4R2, Canada; bkh@dal.ca
The discovery of the notochord as a feature uniting all chor-
dates arose from comparative embryological studies in the 19th
century. This discovery established cephalochordates, ascidians
(tunicates) and vertebrates as chordates. Hemichordates, initially
included as chordates, came to be excluded when it was recog-
nized that the hemichordate stomochord is not homologous with
the notochord. This chapter examines the history of the search
for chordates and early vertebrates in the fossil record, where
the notochord is usually poorly preserved or not preserved at
all. This discussion includes analysis of candidate vertebrates in
the fossil record. The close resemblance between the notochord
and notochordal cells (chordoblasts) and cartilage and cartilage
cells (chondroblasts) has complicated interpretations of the
nature of the notochord and of its origin – whether in the first
Abstract
in identifying chordates in the fossil record if the notochord is
poorly preserved. Although the notochord withstands decay
better than other soft tissues in taphonomy experiments (Briggs
1995, Raff et al. 2006), normally the skeletal tissues of verte-
brates leave few records as fossils unless they are mineralised
(Johanson et al. 2012, Trueman 2013). As the notochord,
along with cartilage and connective tissue, preserves poorly,
considerable lengths have to be gone to confirm the presence
of a notochord. Philippe Janvier pointed out that for fossils that
are preserved as either soft-tissue imprints or minute skeletal
fragments, it is sometimes difficult for palaeontologists to tell
which of them are reliable vertebrate remains and which merely
reflect our idea of an ancestral vertebrate (Janvier 2015).
Another question one may ask is whether the notochord
is a kind of cartilage. Alternatively, based on its appearance
in the course of deuterostome evolution, is cartilage a type of
notochord? No cartilage is found in echinoderms or tunicates
but pharyngeal slits in hemichordates and cephalochordates
are supported by acellular cartilage. Cartilage is also found
in other invertebrate phyla but all invertebrate cartilages lack
type II collagen (Cole & Hall 2004, Rychel & Swalla 2007).
Notochord and the cartilages of all jawed vertebrates possess
type II collagen. Hagfish and lampreys express type II collagen,
prominently in the notochord and in limited amounts in some
cartilages. Wright et al. (2001) point out that type II collagen
is the major matrix protein of cartilages in jawed vertebrates.
The common ancestor of craniates likely had cartilage with-
out type II collagen, but had type II collagen in the notochord. As
the notochord is phylogenetically older than vertebrate cartilage,
notochord may have acquired the ability to synthesize and deposit
type II collagen before cartilage in which case “cartilage-type
collagen” should be named “notochord-type collagen.” Either
way, a close relationship is evident. Both the inner layer of the
perinotochordal sheath and cartilaginous extracellular matrices
contain a closely similar range of glycosaminoglycans and proteo-
glycans, including aggrecan, chondroitin sulphate and keratan
sulphate. Collagen types II, IX and X (discussed in Section 9)
are also found in the matrices of both tissues (Ruggeri 1972,
chordate or in an earlier invertebrate ancestor – whether that
ancestor was segmented or not – and whether the notochord
is segmented. It has long been known that the anlagen of
vertebral centra in teleosts are formed by segmented mineralisa-
tion of the notochord sheath. Recent studies on zebrafish and
medaka embryos have uncovered the molecular mechanisms of
a somite-independent notochord-driven segmentation process
that establishes vertebral centra and intervertebral spaces. The
fact that segmented mineralisation of the notochord sheath is
not restricted to teleosts and evolved much earlier requires a
new interpretation of the origin of vertebral bodies. Paleontology
and developmental biology have reinforced one another in the
search to understand the nature and evolutionary origin of the
notochord and the vertebral column.

216
Mathews 1975, Linsenmayer et al. 1986, Schmitz 1995, Cole
& Hall 2004, Stemple 2005a). Furthermore, Sox-9, the major
transcription factor for cartilage development is also required
for notochord development; Sox9 and Col2a1 are co-expressed
in the notochord (Ng et al. 1997, Zhao et al. 1997).
Brachyury, a transcription factor from the T-box gene fam-
ily characterizes the notochord of all chordates including the
most basal chordates. Brachyury is sufficient and required to
initiate notochord development. Brachyury is not expressed in
the hemichordate stomochord consistent with the assumed
non-homology of notochord and stomochord. Brachyury also
is a cartilage gene capable of initiating chondrogenic dif-
ferentiation of C3H10T1/2 cells in vitro and in vivo (Hoffman
et al. 2002, Hall 2015); Capellini et al. 2008, Satoh et al.
2012, Annona et al. 2015). Brachyury expression in cells of
the nucleus pulposus in adult mammals (Risbud & Shapiro
2011), provides additional evidence for the notochordal origin
of the nucleus pulposus. Chordomas, which are notochordal
tumours specifically express Brachyury. Chordomas are rare and
known to originate from notochordal cells in adult mammals.
Notochordal remnants found in the spinal column of mice are
suspected to be a source for the onset of chordomas (Tamplin
2009). Reports about chordomas also exist from rats, dogs
and cats (Hunter et al. 2003). In humans the frequency is
0.08/100 000 individuals in the USA (McMaster et al. 2001).
The low incidence has been said to reflect resistance of the
notochord to invasion by malignant tumours (Schroyens et al.
1991) but most if not all chordomas arise from transformation
of notochordal cells themselves. Chordomas have features of
cartilage and notochord (hence the name) and so represent
what have been called intermediate tissues (Hall 2015). Indeed,
a gradient of tissues extending from connective tissue to car-
tilage has been identified and many cartilage types shown to
consist of large chondrocytes with little or almost no extracellular
matrix (Beresford 1981, Benjamin 1989, 1990, Hall & Witten
2007, Witten et al. 2010). Chordomas are a neoplastic transi-
tion between notochord and cartilage, and as such represent
a transformation series.
A debate about the evolutionary relationship between
notochord and cartilage based on biochemical constituents
and genes naturally takes place in the realm of neontology
and evolutionary developmental biology. Paleontology is highly
specialised in analysing structures. As we will see below, if a
putative notochord is preserved as a midline structure there is
room for interpretation and debate to whether the structure
is really a notochord. Debates about the presence of a noto-
chord influence our ideas on the evolution of early chordates
as well as the evolution of early vertebrates. Especially in the
oldest fossils, such as those in the Early and Middle Cambrian,
identification of the notochord is difficult. Analysis of 114
specimens from the Middle Cambrian Burgess Shale led Con-
way Morris & Caron (2012) to interpret Pikaea gracilens as a
stem chordate, not because of unequivocal identification of a
notochord, but on the basis of some 100 segmented sigmoidal
myomeres (muscle blocks) in each individual. Identification of
a notochord was extremely tentative. The preserved structure
may have been (i) a notochord (their interpretation, in no small
part because of the presence of other chordate characters);
(ii) a protonotochord or (iii) a non-homologous structure, the
result of independent evolution and convergence with chordates.
Preservation of the notochord is better in two Early Cambrian
species – Cathaymyrus diadexus and C. haikoensis – from the
Chengjiang formation in Yunnan Province, China (530 Ma).
They were classified as cephalochordates on the basis of the
identification of a notochord and the presence of segmented
myomeres (Shu et al. 1996, 1999, 2003, Luo et al. 2001).
So, given that these are Early Cambrian in age, a notochord
in Pikaea would not be inconsistent with when the notochord
and the chordates appear to have originated.
Vertebrate Candidates: Jamoytius kerwoodi,
Conodonts and the “Tully Monster”
On the basis of two specimens, White (1946) named an Early
Silurian fossil Jamoytius kerwoodi (in honour of the English
palaeontologists, James Alan Moy-Thomas [1908-1944]) and
described it as the most basal vertebrate then known. White
came to this conclusion despite the absence of most vertebrate or
even chordate characters in these two specimens. Now assumed
to represent its own order of jawless vertebrates – †Jamoytii-
formes – Jamoytius was considered a vertebrate because of a
dorsal midline structure that was interpreted as a notochord,
but it could have been intestine or part of a branchial basket
(Sansom et al., 2010). Early vertebrates, like early chordates,
can be enigmatic.
Conodonts are another enigmatic group of fossils that
have fuelled the debate about the origin of vertebrates, also
because recognition of a notochord is so difficult. Conodonts
first appeared in the Cambrian 485 Ma and disappeared in
association with the Triassic-Jurassic extinction event 200 Ma.
Conodonts have been identified as plants, annelids, molluscs,
basal chordates or even vertebrates. Derek Briggs, on the basis
of the identification of segmented muscles, fins with fin rays, and
a notochord, classified conodonts as chordates (Briggs 1992).
Almost twenty years later, Turner et al. (2010) concluded that
conodonts possess insufficient characters to classify them as
vertebrates, craniates or jawed vertebrates, and that they may
not even be chordates. The “conodont debate” exemplifies the
difficulties in recognizing a notochord, for example to decide if
a midline structure is a dorsal notochord or a ventral annelid
gut (Blieck et al. 2010).
This is especially problematic when preserved mineralised
elements bear no obvious resemblance to any known vertebrate
skeletal elements. In the absence of a notochord and in the
absence of virtually any vertebrate characters the conodont
discussion focussed on tooth-shaped mineralised microfossils,
known as conodont elements (Turner et al. 2010). Scott (1934)
considered these to be essentially identical to the jaw apparatus
of annelid worms. From recent detailed analyses, we know that
conodont elements are unrelated to vertebrate dermal skeletal
elements, either to teeth (absence of dentine and enamel) or to
jaws (absence of bone) (Turner et al. 2010, Murdock et al. 2014,
Hall 2015, Donoghue & Rücklin 2016). They remain enigmatic.
Recognition and identification of the notochord is also central
in the debate about another vertebrate candidate the “Tully
Monster” Tullimonstrum gregarium, a soft-bodied organism
from the late Carboniferous Mazon Creek biota, 309-307 Ma.
Previously identified as a nemertean worm, polychaete worm,
gastropod, arthropod or conodont, an analysis of 1200 speci-
mens by McCoy et al. (2016) claimed evidence for the presence
of a notochord, cartilaginous vertebral bodies, gill pouches, and
multiple tooth rows around the mouth. These characters coupled
with a detailed phylogenetic analysis led McCoy and colleagues
to classify Tullimonstrum as a vertebrate and a stem lamprey.
This assignment of the “Tully Monster” to the vertebrate clade is
rejected by Lauren Sallan, Philippe Janvier and their colleagues
(Sallan et al. 2017). They argue that the light coloured stain
(the hypothetical notochord) extends anterior to the supposed
eye bars, which is never the case in any vertebrate, and further,
that by removing the notochord from the equation, other traits
of the fossil are not easily interpreted as vertebrate characters.
Similarly, the earliest craniates (named ostracoderms by Edward
Cope in 1889) are known only from their mineralised dermal
skeletons, and again, we have minimal data about their internal
anatomy, skeletal or otherwise (Maisey 2000). Ostracoderms
are not a monophyletic group but a polyphyletic assemblage
of six extinct jawless vertebrates. Even the two well-recognized
groups – heterostracans and cephalaspids – are not natural
groups. The six recognized groups are galeaspids, pituriaspids,
osteostracans, pteraspidomorphs, thelodonts and anaspids
(Janiver 2015).
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Witten & Hall: The Ancient, Segmented, Active and Permanent Notochord
Not only palaeontologists but developmental biologists take
a keen interest in the notochord because the notochord is an
important organizer (the primary organizer) in early develop-
ment (Anderson & Stern 2016). Sonic hedgehog signalling
(Shh) from the notochord induces the formation of somites
from paraxial mesoderm (Christ & Ordahl 1995, Fleming et
al. 2001). In amphibians, somites fail to differentiate without
the notochord (Malacinski & Youn 1982). In chick embryos,
information for segmentation is restricted to the most medial
presomitic mesoderm cells (Freitas et al. 2001). These cells give
rise to the vertebral bodies of each vertebra by migrating into
the perinotochordal space in response to signals, such as Shh
and Noggin from the notochord (Muller et al. 1996, McMahon
et al. 1998, Christ et al. 2004).
Later, when it comes to the development of the skeleton of
the vertebral column, the notochord has been regarded as a
rather passive scaffold that eventually disappears (Willams 1908).
This established the view that patterning information for the
vertebral column is provided only by the somites that give rise
to the sclerotomal mesenchyme (Shapiro 1992, Brand-Saberi
et al. 1996). Next, sclerotome-derived cells develop into the
cartilages of the haemal and neural arches. Expansion of the
cartilaginous bases of the haemal and neural arches around
the notochord was proposed as the cellular process that led
to the evolutionary origin of the vertebral centra (Ota et al.
2011). In extant amniotes, each vertebral body forms from a
solid block of cartilage that encompasses the vertebral centrum
and arches. The arches and centra form as vertebral bodies
from the continuous cartilaginous anlage by endochondral
bone formation, but from separate ossification centres for each
vertebra. Evidently, so we believe, the notochord disappears,
together with the cartilage during this endochondral bone for-
mation, without the notochord having been actively involved
in vertebral body formation.
Our knowledge on which this scenario rests derives from
detailed studies of early stages of vertebral body formation in
mouse and chicken embryos (Scaal 2016). The conservation
of early developmental stages in vertebrates suggests that the
chicken and mouse mode of vertebral body development is a
valid model for all vertebrates (Nikaido et al. 2002). But re-
member, although mammals and birds are tetrapods, should
we expect them to represent vertebral development in other
tetrapods (urodele or anuran amphibians, reptiles), in other
bony fishes or in cartilaginous fishes?
This accepted view of a passive function of the notochord in
vertebral column development has been challenged by recent
studies, primarily using zebrafish (Danio rerio) and Japanese
medaka (Oryzias latipes), two small and readily maintained
teleost species that have emerged as models in developmental
biology and biomedical research. Indeed, the zebrafish and
medaka mode of vertebral body development – as is now known
to be generally the case in teleost fish – is quite different from
the mouse and chicken model. There is increasing evidence
that not only the somites but also the notochord contains
patterning information and that the notochord establishes the
anlagen of vertebral centra. Patterning of the notochord is now
viewed as independent and perhaps even prior to patterning
of the somites.
Our “new knowledge” should not come as a total surprise;
two highly reputed scientists re-ported the same mode of estab-
lishing vertebral body anlagen – mineralisation of the notochord
Fig. 1. Mineralisation of the teleost notochord sheath in the Japanese medaka Oryzias latipes,
shown at 15 days post-fertilization (dpf) using Alizarin red S whole mount staining for calcium.
In the caudal part of the developing vertebral column, mineralisation of the notochord sheath
(NCS) establishes the anlagen of the vertebral centra (red), which is clearly visible when viewed
at high magnification (compound microscope, × 400). Huxley and Kölliker may have made
similar observations that led to their publications in 1859 about notochord sheath mineralisation
in the three-spined stickleback Gasterosteus aculeatus and in the European eel Anguilla an-
guilla, respectively. The left panel shows a wild type medaka. In the location of the intervertebral
spaces, the notochord sheath (NCS) and the notochord epithelium (NCE) are thickening. Inside
large vacuolated notochord cells (VNC) are visible, the spinal cord (SC) is visible dorsal to the
notochord. After the notochord sheath becomes mineralised the first intramembranous bone
(IMB) is deposited onto the mineralised notochord sheath, starting at the basis of the haemal
arches (HA) and neural arches (NA). The right panel shows an osterix/sp7 mutant medaka with
non-functional sclerotome-derived osteoblasts. Vertebral arches and perinotochordal bone are
missing but the development of the vertebral centra anlagen by mineralisation of the notochord
sheath is unaffected. See Yu et al. (2017) for the in-depth analysis of this osterix mutant pheno-
type. Scale bar = 25 µm
Chordal Mineralisation versus Perichordal Ossification
Huxley‘s and Kölliker‘s observations concerning vertebral body
development in the largest group of vertebrates (teleosts) were
not ignored by the palaeontology, ichthyology and zoological
communities (Laerm 1979, 1982, Lauder 1980, Arratia et al.
2001).
Laerm (1982) informs us that “shortly after the formation
of the notochord and its sheaths, a calcified ring, the chorda-
centrum, forms in the fibrous sheath of the notochord in each
prospective vertebral segment.” This is now known as the dual
segmentation model in which the segmental patterns of the
neural and haemal arches are somite-derived, while the verte-
bral segments are notochord-derived. Indeed, within teleost
vertebrae the mode of forming vertebral bodies is remarkably
consistent (Lauder 1980). Mineralisation of the notochord sheath
has been shown in Atlantic herring (Clupea harengus), northern
pike (Esox lucius), Atlantic salmon (Salmo salar) and Zebrafish
The Old/New Notochord: Segmented and Active
sheath – for basal (eels) and advanced (sticklebacks) teleosts
and in the same year (Huxley 1859, Kölliker 1859). Thomas
H. Huxley, who published his observations on vertebral column
development in the three-spined stickleback Gasterosteus acu-
leatus informs us that:
“In the greater part of its extent it [the notochord] was
enclosed neither in cartilage nor in bone – though bony rings,
the rudiments of the centra of the vertebrae, were developed
in the wall of the notochord throughout the rest of the body.”
(Huxley 1859, p. 39).
Albert Kölliker published his studies on vertebral column devel-
opment in the European eel Anguilla anguilla:
“In the Leptocephali [transparent eel larva] the sheath of
the chorda ossifies without having been transformed into car-
tilage; and the same seems to hold good for the other osseous
fishes.” (Kölliker 1859, p. 217).
One would assume that this understanding of the mode of tel-
eost vertebral body development would have become textbook
knowledge (Fig. 1). Not so. One reason is perhaps that thirty-
six years later, Hans Gadow and Elizabeth Caroline Abbott in
their influential and extensive treatise “On the evolution of the
vertebral column of fishes” do not mention Huxley‘s and Köl-
liker‘s observations (Gadow & Abbott 1895). Edwin S. Goodrich
relied on Gadow & Abbott (1895) for his leading textbook
(Goodrich 1909). Ever since, textbooks have perpetuated the
statement that vertebral body precursors are made from carti-
lage. Cartilage cells that form the bases of the haemal and
neural arches. Cells that derive from sclerotomal mesenchyme
establish the anlagen of vertebral centra (Kriwet & Pfaff 2019).

218
(Danio rerio) (Francois 1966, Schaeffer 1967, Grotmol et al.
2003, 2005, Nordvik et al. 2005, Nordvig 2007, Bensimion-
Brito et al. 2012b).
Developmental biology only started to take full notice of
the “non-textbook” model of the development of teleost ver-
tebral bodies once zebrafish and Japanese medaka became
established as model organisms (Figs. 1, 2). In the early days
of zebrafish research – over 20 years ago – the phenotype of
the fused somite mutant zebrafish could have changed the
paradigm. In this mutant, somites are fused, with the conse-
quence that haemal and neural arches are fused, malformed
or fail to form (van Eeden et al. 1996). Nevertheless, vertebral
body centra develop separately, which is a strong indication of
somite-independent patterning of the vertebral column. The
opposite phenotype, which also indicates independence, is seen
in zebrafish, medaka and salmon with fused vertebral centra.
Fusion may be a part of normal development (Bensimon-Brito
et al. 2012a, Hall & Witten 2019) or it may be pathological
(Takeuchi 1966, Witten et al. 2006). Fusion affects the centra
only; the arches remain separated.
There is, however, more evidence that patterning and
development of vertebral body centra are independent from
somite development (Fleming et al. 2004). Ablation of the
notochord in urodele and chicken embryos results in complete
fusion of cartilaginous vertebral body anlagen. Notochord
ablation removes vertebral body identity, but not arch identity
(Hall 1977, Fleming et al. 2001). Ablation experiments also
revealed the importance of dorsal root ganglia for segmentation
of haemal arches but not for segmentation of vertebral bodies
(Hall 1977, Senthinathan et al. 2012). Fleming et al. (2004)
presented a study according to which ablation of notochord
cells prevented the formation of vertebral centra in zebrafish.
Pogoda et al. (2018) further narrowed down the responsible
cell type and showed that targeted ablation of cells from the
notochord epithelium prevents the formation of vertebral centra
anlagen. According to Fleming et al. (2004) isolated, but intact,
notochords still produce mineralised anlagen of vertebral centra.
More evidence for the somite-independent, but notochord-
dependent, formation of vertebral centra anlagen is provided by
a study of the osterix /sp7-/- mutant in the Japanese medaka
by Yu et al. (2017). In these mutants, sclerotome-derived pre-
osteoblasts fail to differentiate into functional osteoblasts. The
consequence is the failure of haemal arches to form. Still, the
notochord forms mineralised segments in the notochord sheath,
which, essentially is the extracellular matrix of the notochord
(Figs. 1, 3). Pogoda et al. (2018) showed that osterix/sp7-positive
osteoblasts are indeed only located outside the notochord and
are recruited into the mineralised areas of the notochordal
sheath. Thus, notochord mineralisation occurs: (i) prior to
bone formation, (ii) within the notochord sheath, and (iii) is
osterix / sp7-independent. These studies confirm Joshua Laerm’s
conclusion from his studies on fossil and extant osteichthyans:
“In living teleosts, the perichordal tissue results from differentia-
tion of sclerotome cells. The chordal calcification forms in the
acellular fibrous sheath of the notochord and apparently does
so without the participation of sclerotome cells. This suggests
that separate developmental mechanisms may be responsible
for chordal calcifications and perichordal ossifications.” (Laerm
1979, p. 481).
Fig. 2. Oryzias latipes 15 dpf, cross section through the developing vertebral column and noto-
chord. The notochord sheath (NCS) has a thin inner elastic membrane (IEM) and a prominent
external elastic membrane (EEM). The external elastic membrane delimits the notochord sheath.
Intramembranous bone (IMB) that forms around the mineralised notochord sheath is visible in
connection with the development of the neural arches (NA). The cells of the notochord epithelium
(NCE) are clearly distinct from the vacuolated notochord cells (VNC) in the centre. Another name
for notochord epithelial cells is chordoblasts (Ward et al. 2018, Pogoda et al. 2018). Whether
the notochord epithelium constitutes a true epithelium was discussed by Jurand (1962). Schmitz
(1995) emphasises that the notochord epithelium in yellow perch (Perca flavescens) is indeed a
stratified squamous epithelium with cells that are interconnected by desmosomes and gap junc-
tions. Ellis et al. (2013) showed that the vacuoles of the cells in the centre of the notochord are
lysosome-related organelles whose formation and maintenance requir es late endosomal trafficking
regulated by the vacuole-specific Rab32a and H+-ATPase-dependent acidification. Toluidine blue
staining. Scale bar = 20 µm.
Segmentation Without the Segmentation Clock?
For amniotes, it is widely accepted that vertebral development,
and thus development of the vertebral column, is based on
somites and the “segmentation clock,” which is a molecular
oscillator that controls the timing (periodicity) of somite forma-
tion under the control of Notch, Wnt and Fgf gene pathways
(Giudicelli & Lewis 2004). It was assumed that the same cel-
lular basis and timing mechanism operated in teleosts such as
zebrafish (Schröter & Oates 2010).
In tetrapods such as chickens and mice, mutations that
disrupt the clock and the patterning of individual somites have
dramatic effects on the formation of vertebral bodies (White
et al. 2003). A somite-dependent basis for the establishment
of vertebral body anlagen also was proposed for zebrafish
and medaka and so for teleosts in general (Renn et al. 2013,
Inohaya et al. 2007). Lleras Forero et al. (2018) took a closer
look at zebrafish mutants with disturbed somitogenesis. They
analysed Tbx6 mutants, previously described by van Eeden et
al. (1996) as fused somite mutants (fss) on the basis of the
mutant phenotype. Despite the severely disturbed somitogenesis
shown by van Eeden et al. (1996), these mutants maintain
separate vertebral body centra. Indeed, disturbed somitogenesis
in zebrafish has no affect on the generation of vertebral centra.
Taking the model further Forero et al. (2018) analysed the
genes her1 and her7 that are central to the operation of the
somitic clock. Mutations for each of these clock genes had
no affect on separation of the vertebral centra. Even in triple
mutant zebrafish (her1-/-, her7-/-, tbx6-/-), vertebral centra
remained separated, which strongly suggests that, in zebrafish,
the segmental generation of vertebral body anlagen is somite-
independent and autonomous to the cells producing the centra.
This does not, however, exclude an influence of somites and the
somitic clock on the formation of functional vertebral centra;
functional vertebral bodies require co-ordinated development
of vertebral centra and arches.
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Mineralisation of the Notochord Sheath:
from Inside Out or from Outside In?
Data about mineralisation of the notochord sheath cannot be
obtained easily because mineralisation is weak and the sheath
is too thin to be visualised by microCT, unless synchrotron
based microCT is used (Bruneel & Witten 2015, Keating et
al. 2018) (Figs. 1-3).
As we know from teleost development, the mineralised
notochord sheath quickly becomes surrounded by intramem-
branous bone after which distinguishing between the bone and
the mineralised notochord sheath is very challenging (Figs. 4, 5).
Obviously, data from the fossil record about notochord sheath
mineralisation in early developmental stages are difficult to
obtain. John Maisey has addressed the problem of ontogenetic
data that are essentially unavailable from the fossil record and
for practical purposes are also not available for most extant
species (Maisey 1988). We have more data about bone that
forms directly around the notochord sheath – from all basal
actinopterygian and sarcopterygian groups including basal
tetrapods – than we have data about a mineralised notochord
sheath (Gardiner 1983). In neontology, histological studies of
the skeleton routinely decalcify specimens and thus lose data
about notochord sheath mineralisation. Moreover, conventional
whole-mount-staining for bone and cartilage removes mineral
from the notochord sheath and so produces false negative
results. This artefact has been known for thirty years (Vande-
walle et al. 1988, Springer & Johnson 2000, Bird & Mabee
2003) but it was not until the acid-free staining protocol of
Walker & Kimmel (2007) became popular within the zebrafish
research community that notochord sheath mineralisation was
re-recognised (Bensimon-Brito et al. 2016).
In teleosts, subdivision of the notochord sheath into miner-
alised (vertebral centra anlagen) and non-mineralised (prospec-
tive intervertebral spaces) segments is preceded by changes
in the shape and orientation of notochordal epithelial cells.
The first indication of segmentation is proliferation of cells of
the notochord epithelium in prospective intervertebral spaces,
resulting in thickening of the col2-based notochord sheath
(Hay 1895, Schauinsland 1903, Laerm 1976, Nordvik et al.
2005). As in cartilage, the collagen gene col9a2 is associated
with col2a in the notochord sheath extracellular matrix. Con-
sequently, the distinctive expression of col9a2 in the locations
of future intervertebral spaces indicates increased notochord
sheath production (Pogoda et al. 2018). Notochord sheath
mineralisation, which occurs in zebrafish without increased
col9a2 expression, is retinoic acid-dependent. Overexpression
of retinoic acid causes loss of the non-mineralised intervertebral
spaces in the notochord sheath (Spoorendonk et al. 2008),
blocking retinoic acid-signaling prevents notochordal sheath
mineralisation (Pogoda et al. 2018).
Osteoblasts control the process of mineralising bone matrix
by secreting pyrophosphate, which is a strong mineralisation
inhibitor. Secretion of pyrophosphate depends on ectonu-
cleoside pyrophosphatase/phosphodiesterase 1 (enpp1) and its
antagonist ectonucleoside triphosphate/diphosphohydrolase 5
(entpd5), which is a mineralisation promoter. Like osteoblasts,
the cells of the zebrafish notochord epithelium express entpd5
at sites of notochord mineralisation (Lleras Foreo et al. 2018,
Pogoda et al. 2018). In addition, secreted pyrophosphate must
be removed to make mineralisation possible. To accomplish
this, osteoblasts produce alkaline phosphatase (ALP), which
functions as a pyrophosphatase. Like osteoblasts, the cells
of the notochordal epithelium produce alkaline phosphatase;
expression is detected in a segmented pattern at sites of no-
tochordal sheath mineralisation, shown for Atlantic salmon
by Grotmol et al. (2003) and then for zebrafish by Bensimon-
Brito et al. (2012a). In addition, Bensimon-Brito et al. (2012b)
demonstrated segmented expression of osteocalcin (the most
abundant non-collagenous bone protein) in the notochord
epithelium.
The studies on zebrafish and medaka refute the hypothesis
that mineralisation of the notochordal sheath is facilitated by
osteoblasts from outside the notochord. We now know that in
zebrafish and medaka, and thus most likely in other teleosts,
notochord mineralisation starts before osteoblasts accumulate
around the notochord (Fig. 3). Mineralisation is controlled by the
notochordal epithelium, a process that is osteoblast-independent
as shown by Yu et al. (2017, Fig. 1). Notochordal epithelium
mineralises its own sheath in a segmented fashion, creating
the anlagen of the vertebral centra.
Witten & Hall: The Ancient, Segmented, Active and Permanent Notochord
Fig. 3. Transmission electron microscopical image of the notochord sheath of a zebrafish (Danio
rerio) at 10 dpf. The principal components – notochord epithelium (NCE), inner elastic membrane
(IEM), type 2 collagen based notochord sheath (NCS) and external elastic membrane (EEM)
characterise the notochord of vertebrates in all classes. It is clearly evident that the notochord
sheath starts to mineralise prior to bone formation around the notochord sheath. Abutting cell
membranes of vacuolated cells show numerous caveolae, which are submicroscopic cup-shaped
plasma membrane pits that can buffer tension in cells that undergo high levels of mechanical
stress (Lim et al., 2017). TEM preparation, postfixed with osmium tetroxide, contrasted with uranyl
acetate and lead citrate. See Bensimon-Brito et al. (2012b) for further details about notochord
sheath mineralisation in zebrafish.
The Resegmentation Debate
That vertebral bodies develop after a resegmentation of cells
from adjacent somites (Remak 1855) is a well accepted model
for amniotes although it has also been denied for this group
(Verbout 1976). The resegmentation model corroborates other
evidence that tetrapod vertebral centra derive from somites.
Resegmentation establishes a clearly defined spatial and de-
velopmental relationship between arches and vertebral centra,
resulting in a one-to one-relationship of arches and centra and
the long-recognized intersegmental position of vertebral bod-
ies; the intersegmental position of vertebral bodies in teleosts
has been viewed as evidence for resegmentation in teleosts
(reviewed by Lauder 1980).
Nonetheless, the adult centrum in different teleost groups
can have a variable anterior to posterior position in relation to
the vertebral arches (Schaeffer 1967, Lauder 1980). Morin-
Kensicki et al. (2002) showed experimentally that somite-derived
sclerotome cells in zebrafish do not strictly contribute to the
development of intersegmental vertebral bodies (as would be
predicted by the resegmentation hypothesis). Schaeffer (1967),
Laerm (1976) and Lauder (1980) are unequivocal about the
absence of sclerotomal resegmentation in osteichthyans. Wake
& Lawson (1973) did not find any evidence for resegmentation
of sclerotome in salamanders and extended the absence of
resegmentation to all non-amniotes. David Wake points out

220
(Gadow & Abbott 1895). If we follow this hypothesis or any
of its variants and if we consider that early osteichthyans lack
vertebral centra, the conclusion must be that vertebral centra
evolved independent in different classes of vertebrates (Arratia
et al. 2001). A different view is expressed by Gardiner (1983),
who emphasizes that consideration of the vertebral centrum
must begin with the origin and structure of the notochord and
its sheath, which in form and function is identical and thus
homologous in all vertebrates.
As we recognise the notochord as much more active than
commonly acknowledged we may define earlier events in devel-
opment as the start of vertebral body formation, events earlier
than considered in the framework of the arcualia hypothesis.
Each of the following key events could, for example, be defined
as the start of vertebral body development:
(1) Notochord signalling to subdivide the presomitic mesoderm
into somites.
(2) Subdivision of the notochord into intravertebral notochord
and intervertebral spaces.
(3) Expansion of the notochord establishes the intervertebral
joints.
(4) Signalling (brachyur y) by the notochord induces the dif-
ferentiation of skeletal precursor cells.
(5) Cartilaginous condensation of vertebral arch anlagen.
(6) Mineralisation of the notochord sheath.
(7) Formation of cartilage or bone around the notochord
(8) Mineralisation of cartilage or bone formation around the
notochord?
Classical embryology considers (5) – the condensation of somitic
chondrogenic cells around the notochord – as the start of
vertebral body development. Analysis of the fossil record must
rely on (8), the pattern of mineralisation of notochord, cartilage
or bone. We just started to reconsider the faint segmented
mineralisation of the notochord sheath (6), as a key step that
defines vertebral body anlagen.
Mineralisation of the notochord sheath is not a derived
character of teleosts. In fact, mineralisation of the notochord
sheath is a pre-teleostean feature (Patterson 1968). The earliest
recorded actinopterygian vertebral centra in Haplolepis from
the Carboniferous were made from mineralised notochord
sheath (Patterson 1968, Gardiner 1984). The Triassic chon-
drosteans Pholidopleurids and Turseodus had opposed dorsal
and ventral half-rings as vertebral centra based on notochord
sheath mineralisation (Patterson 1968, Schaeffer 1967). In
teleosts this pattern is no longer a character found in adults.
Separate dorsal and ventral mineralisation of the notochord
sheath occurs only during development before the ring-shaped
notochord sheath mineralisation is complete and before the
mineralised notochord sheath is encircled by membrane bone
(Bensimon-Brito et al. 2012b). In rhipidistians such as Rhizo-
dopsis, Megalichthys, Ectosteohachis and Strepsodus, vertebral
centra are made from membrane bone around the notochord
(Gardiner 1984). If centra formation went through the same
developmental steps as in teleosts, there would have been a
faint mineralised notochord sheath as substrate for the bone.
In the light of clear evidence that a mineralised notochord is
the anlage of the teleost vertebral centrum, our view about the
significance of this character may have to be revised. We know
that the notochord sheath can mineralise in chimeras (Francois
1966), in urodeles (Danto et al. 2019) and in mammalian
embryos (Willams 1908). Notochord sheath mineralisation is a
elasmobranch character and also known from the fossil record
of this group (Daniels 1934, Maisey 2008). Obviously, the
Fig. 5. Von Ebner’s unsurpassed detailed description of the noto-
chord as forming part of the inter vertebral joint in the freshwater pike
Esox. According to the von Ebner’s figure legend the magnification
is 40 ×. This would translate into 300 × 412 µm original size of the
depicted area. The principle components are found in all teleosts
although in miniaturised species such as zebrafish or medaka
identification of particular elements can become more difficult. Von
Ebner is best known for the discovery of a somite subdivision (von
Ebner’s fissure) that fuelled the resegmentation hypothesis (von
Ebner 1888). English terms have been added to the original figure
from von Ebner (1896) by PEW.
Fig. 4. Sagittal section through the vertebral column of a juvenile (15 mm standard length)
zebrafish. As in mammals the notochord is extended in the intervertebral spaces and functions
to support the intervertebral joint (Leeson & Leeson 1958). The broad notochord sheath (NCS)
and the broad external elastic membrane (EEM) are important inner parts of the intervertebral
ligament that connect vertebral centra. Notice the extension of the notochord epithelium (NCE)
and the orientation of cell nuclei, perpendicular to the animal axis. The latter has been described
for Atlantic salmon (Salmo salar) (Grotmol et al. 2003). In the centre of the notochord, vacuolated
notochord cells (VNC) condense and acquire the character of a fibrous connective tissues. The cells
form the notochord strand (NSD) contains keratan sulphate and can mineralise, for example in
sturgeons (Schmitz 1998, Leprevost et al. 2017). The condensation of the vacuolated cells makes
space for large secondary extracellular vacuoles (SEV) that also develop in the mammalian nucleus
pulposus (Trout et al. 1982). Intramembranous bone (IMB) has formed around the mineralised
notochord sheath. Figure provided by Arianna Martini, Toluidine blue staining. Scale bar = 50 µm.
/
Ancient Fishes and their Living Relatives: a Tribute to John G. Maisey

221
Witten & Hall: The Ancient, Segmented, Active and Permanent Notochord
notochord sheath of vertebrates has the capacity to mineralise
but, of course, for reasons discussed here, we do not have suf-
ficient data to allow us to conclude how common notochord
mineralisation is outside the actinopterygians. Nonetheless, as
vertebrates from all classes follow the first five steps (1-5) of
vertebral body development in the list above, then steps 1 to
5 should be considered as homologous. Homology includes
‘homology of processes’ as discussed by Gilbert et al. (1996)
and Hall (2003) but also includes homology of the structures
that develop.
Next vertebral centra formation continues around the
notochord sheath in essentially one of two ways: (a) by direct
formation of membranous bone or (b) by the formation of
cartilage that subsequently mineralises. It is interesting that
col10a is co-expressed with osterix expression by the osteoblast
population that produces the bone around the notochord sheath
in medaka (Renn et al. 2013, Seeman et al., 2015). In mam-
mals col10a is typically expressed in mineralising cartilage but
absent from osteoblasts. Col10a expression in this osteoblast
population may reflect the dual nature of these cells that
can produce bone or mineralising cartilage. If so, differences
concerning the involvement of cartilage or bone in centrum
formation are the consequence of descent with modifications
and not characters that evolved independent in different lines
of vertebrates.
Whether vertebral centra initially materialise as cartilage,
bone or mineralised notochord sheath is likely largely a func-
tion of speed of development. Wake & Wake (2000) compared
vertebral body development between gymnophiones and mam-
mals and concluded that the observed major differences can be
attributed to vastly different developmental rates. Further, large
parts of the vertebral centrum develop in response to mechanical
load. As Joshua Laerm put it: “It is possible to suggest that a
centrum is a centrum is a centrum, reflecting the generalised
adaptive response of sclerotome tissue to functional demands
for vertebral consolidation” (Laerm 1979, p. 482).
Notochord and somites as developmental modules
Recent studies on zebrafish and medaka have revealed develop-
mental mechanisms by which the notochord mineralises its own
sheath independently from the somites and the somitic clock.
These studies strongly support all previous findings that show
that vertebral centra and arches are developmental modules.
Modularity of the two systems has been long recognized for all
gnathostomes including mammals (Strudel 1953, Detwiler &
Holtzer 1956, DeClercq et al. 2017, 2018, Hall 1977, Lauder
1980, Hautier et al. 2010, de Azevedo et al. 2012, Hall 2015,
Yu et al. 2017, Ward et al. 2018). Nature provides numerous
examples that show this modularity. Diplospondyly (two centra
per segment) is an obvious one (Hay 1895, Lauder 1980) but
the complete range encompasses aspondyly, monospondyly,
diplospondyly and polyspondyly (Zhang 2009).
Regionalisation of the vertebral column also commonly
requires uncoupling of arch and centra development. Much
evidence was contributed by the ablation experiments, mutants
and developmental studies discussed above (reviewed by Hall
2015). Hautier et al. (2010) tested the patterns of events in the
sequences of development of arches and centra in the brown-
throated sloth Bradypus variegatus and identified significant
modularity in this mammal. Modularity between arches and
centra is widely accepted and in conflict with the also widely
accepted arcualia hypothesis. This conflict is however often
not addressed. Monsoro-Burq et al. (1994), for example,
communicate in the same paragraph that vertebrae derive
form somites and that the notochord induces the somites and
induces sclerotomal cells to form cartilage. Indeed, the fact
that vertebral body anlagen can arise independent from the
arches has the potential to refute the arcualia hypothesis that
views ventral cartilaginous arch elements as representing the
first elements of vertebral bodies (Ota et al. 2011, 2014).
The modularity of centra and arches triggers the question
of whether the notochord or somites were the first segmented
system. The lack of somites in basal chordates such as tuni-
cates, led Claudio Stern to suggest that the notochord may
have been the first segmented structure (Stern 1990). Ward
et al. (2018) and Peskin et al. (2020) argue that both somites
and notochord are important for the segmented development
of vertebral bodies, somites taking the lead in amniotes and
the notochord taking the lead in teleosts. This again raises the
question of how vertebral body development was determined in
early gnathostomes (Ward et al. 2018). Independently of whether
we will ever have an answer to this question, the rediscovery
of the active segmented notochord and the new knowledge
about the mechanisms by which the notochord establishes
vertebral centra anlagen in teleosts should encourage us to
revisit notochord development in non-teleost vertebrate. This
in turn may influence how we interpret fossils. In the words of
John Maisey: “The history of the skeletal systems in vertebrates
can best be understood through the reciprocal illumination that
comes through a combined paleontological and developmental
biological approach” (Maisey 1986).
Acknowledgements
PEW acknowledges funding from the European Union’s Horizon 2020
research and innovation programme under the Marie Skłodowska-
Curie grant agreement No. 766347. Notochord figures taken at the
lab of PEW at Ghent University derive form collaborative research
with the lab of Christoph Winkler (University of Singapore) and from
the PhD projects of Anabela Bensimon-Brito and Arianna Martini.
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Ancient Fishes and their Living Relatives: a Tribute to John G. Maisey

Alan Pradel, Alan Pradel,
John S. S. Denton,John S. S. Denton,
and Philippe Janvierand Philippe Janvier
(editors)(editors)
Verlag Dr. Friedrich Pfeil
Ancient Fishes
and their Living Relatives:
a Tribute to
John G. Maisey
Ancient Fishes
and their Living Relatives:
a Tribute to
John G. Maisey

5
Contents
Alan Pradel, John S. S. Denton and Philippe Janvier:
Preface by the editors ................................................................................................................................................. 7
Introduction
Maria da Gloria P. de Car valho:
John G. Maisey – a Biographical Sketch ..................................................................................................................... 9
Analysis and methodology
Gloria Arratia, Hans-Peter Schultze, Soledad Gouiric-Cavalli and Claudio Quezada-Romegialli:
The intriguing †Atacamichthys fish from the Middle Jurassic of Chile – an amiiform or a teleosteomorph? .............. 19
Jose Xavier-Neto and Ismar de Souza Carvalho:
Paleontological treasures among commonplace fossils: a paradigm to study evolutionary innovation ........................ 37
John S. S. Denton and Eric W. Goolsby:
Influence analysis of fossil chondrichthyan taxa ......................................................................................................... 49
Kevin K. Duclos, Terry C. Grande and Richard Cloutier:
Modularity of the Weberian apparatus in the zebrafish using micro-CT technology
and 3-D geometric morphometrics ............................................................................................................................. 59
Juan Liu:
You are how you look: potential utility of quantitative body shape analysis in classification
of Eocene cypriniforms ................................................................................................................................................ 71
Descriptive Anatomy and Development
Carole J. Burrow and Jan L. den Blaauwen:
Endoskeletal tissues of acanthodians (stem Chondrichthyes) ...................................................................................... 81
Allison W. Bronson:
A three-dimensionally preserved stethacanthid cranium and endocast
from the Late Mississippian Fayetteville Shale (Arkansas, USA) ................................................................................. 93
Friedrich H. Pfeil:
The new family Mesiteiidae (Chondrichthyes, Orectolobiformes), based on Mesiteia emiliae Kramberger, 1884.
A contribution to the Upper Cretaceous (early Cenomanian) shark fauna from Lebanon ........................................... 101
Alan Pradel, Richard P. Dearden , Antoine Cuckovic, Rohan Mansuit and Philippe Janvier:
The visceral skeleton and its relation to the head circulatory system of both a fossil,
the Carboniferous Iniopera, and a modern, Callorhinchus milii holocephalan (Chondrichthyes) .................................. 183
Michael I. Coates, Kristen Tietjen, Zerina Johanson, Matt Friedman and Stephanie Sang:
The cranium of Helodus simplex (Agassiz, 1838) revised ........................................................................................... 193
Zerina Johanson, Charlie Underwood, Michael I. Coates, Vincent Fernandez, Brett Clark and Moya M. Smith:
The stem-holocephalan Helodus (Chondrichthyes; Holocephali)
and the evolution of modern chimaeroid dentitions .................................................................................................... 205
P. Eckhard Witten and Brian K. Hall:
The Ancient, Segmented, Active and Permanent Notochord ....................................................................................... 215
Ann Huysseune:
The distribution of post-mandibular teeth in extant vertebrates revisited:
co-evolution of pharyngeal pouches and teeth? .......................................................................................................... 225
Stratigraphy and Biogeography
Rafael M. Lindoso and Ismar de S. Carvalho:
The Cretaceous fishes of Brazil: a paleobiogeographic perspective ............................................................................ 233
John A. Long, Victoria Thomson, Carole J. Burrow and Susan Turner:
Fossil chondrichthyan remains from the Middle Devonian Kevington Creek Formation,
South Blue Range, Victoria ......................................................................................................................................... 239
Hans-Peter Schultze, James Bullecks, Linda K. Soar and James W. Hagadorn:
Devonian fish from Colorado’s Dyer Formation and the appearance of Carboniferous faunas
in the Famennian ........................................................................................................................................................ 247
Contents

ISBN 978-3-89937-269-4
Knowledge of fossil sharks (chondrichthyans) has ad-
vanced tremendously over the past decade, giving scien-
tists a window into a historically understudied branch of
the evolutionary tree of fishes, and revealing anatomies
and ecologies just as diverse and fascinating as those
of bony fishes (osteichthyans). This volume assembles
cutting-edge research on the biology, anatomy, and
evolution of sharks and bony fishes, featuring works
by paleobiologists and associated researchers from 11
countries, spanning topics from taxonomy to statistical
methodology, in honor of Professor John G. Maisey,
for his pioneering work on Paleozoic chondrichthyan
anatomy, taxonomy, and paleobiogeography over his
half-century career at the American Museum of Natu-
ral History. With an introduction and 16 chapters, this
volume erects two new families and two new genera,
and provides 160 figures and illustrations, and 29
plates, including the most comprehensive collection of
high-resolution images of a rare fossil shark held pre-
dominantly in private collections.
John G. Maisey (right) and the publisher visiting
Neuschwanstein castle in Bavaria on Nov. 11, 2014