DEVELOPMENTAL DYNAMICS 228:337–357, 2003

ARTICLE

Developmental Morphology of the Axial Skeleton

of the Zebrafish, Danio rerio (Ostariophysi:
Cyprinidae)
Nathan C. Bird* and Paula M. Mabee*

Before our rapidly increasing knowledge of gene interactions can be connected with the morphologic defects in
mutant zebrafish, the normal course of skeletal development must be understood. Here, we describe the
developmental morphology of the axial skeleton of zebrafish and review it in relation to the morphology of related
species. The relative sequence of ossification in the skeleton is described. Two separate centers of development
were found in the axial skeleton (Weberian apparatus and caudal fin) in contrast to tetrapods, which have a single
anterior center. Slight variation was found in the overall relative timing of development. The extensive ichthyological
literature on teleost anatomy and recent genetic data form the basis for our review and interpretation of homologies
of various elements of the axial skeleton. Because homology forms the basis for all evolutionary comparisons, these data
are critical for integration in evo– devo studies. Developmental Dynamics 228:337–357, 2003. © 2003 Wiley-Liss, Inc.

Key words: zebrafish; axial skeleton; vertebra; fins; bone; cartilage; development; Weberian apparatus

Received 9 May 2003; Accepted 2 July 2003

INTRODUCTION
Questions regarding the relationship
between genes and morphology
are a driving force behind research
in evolution and development (Car-
roll et al., 2001). The zebrafish, Danio
rerio, is a well-established system for
genetic studies of development
(Nüsslein-Volhard and Dahm, 2002),
and because of its basal phyloge-
netic position within vertebrates, it is
a key species for evo– devo studies.
With thousands of identified mutants
(Haffter et al., 1996), and ease of
genetic manipulation and mu-
tagenesis, the zebrafish has moved
to the forefront as a vertebrate
model system. Yet for all of the mo-
lecular work, there has been no
comprehensive study on the devel-
opment and morphology of the ze-
brafish axial skeleton. A detailed
study of skeletal development, how-
ever, is crucial for interpretation of
skeletal mutations and gene expres-
sion patterns and ultimately for un-
derstanding the connections be-
tween genes and morphology.
The purpose of this study is to pro-
vide a detailed developmental an-
atomic reference and a review and
assessment of homologies of the ze-
brafish axial skeleton. The extensive
ichthyological literature on oto-
physan anatomy forms the basis for
our interpretation of homologies of
various elements of the axial skele-
ton. We present data on the devel-
opmental timing and origin of skele-
tal elements in the axial skeleton.
When paired with other morpho-
logic studies and genetic studies of
zebrafish development, a more
comprehensive understanding of
zebrafish morphogenesis will emerge.
BACKGROUND
The zebrafish, Danio rerio, is a small
freshwater cyprinid native to South-
eastern Asia (Barman, 1991). It is a
member of the Rasborinae clade
(Meyer et al., 1993) of the subfamily
Leuciscinae (Cavender and Coburn,
1992) in the family Cyprinidae, order

Department of Biology, University of South Dakota, Vermillion, South Dakota

N.C. Bird’s current address is Department of Biological Sciences, The George Washington University, 2023 G. Street, NW, Washington, DC
20052.
*Correspondence to: Nathan C. Bird, Department of Biological Sciences, The George Washington University, 2023 G. Street, NW,
Washington, DC 20052. E-mail: nbird@gwu.edu or Paula M. Mabee, Department of Biology, CL-185, University of South Dakota,
Vermillion, SD 57069. E-mail: pmabee@usd.edu
DOI 10.1002/dvdy.10387

© 2003 Wiley-Liss, Inc.

338 BIRD AND MABEE
Fig. 1. Phylogeny of Ostariophysi and Clupeomorpha. Characters associated with the Weberian apparatus are mapped on the lineage
in which they were modified. Adapted from Fink and Fink (1981, 1996).
Cypriniformes (Fig. 1). Its rapid devel-
opment, short generation time, em-
bryonic transparency, ease of mu-
tagenesis, and ongoing genomic
research has made it ideal for labo-
ratory study (Nüsslein-Volhard and
Dahm, 2002).
In recent years, a variety of signif-
icant studies on molecular aspects
of zebrafish skeletal development
have been published. Among these
are studies discussing the role of
chordin in skeletal development
(Fisher and Halpern, 1999), dlg3 in
vertebral ankylosis (König et al.,
1999), Gdf5 in median fins (Crotwell
et al., 2001), sox9 in cartilage differ-
entiation (Chiang et al., 2001), shh
and bmp2b in the caudal fin (Quint
et al., 2002), and endothelin-1 and
pbx4 in the larval head (Kimmel et
al., 2001).
Morphologic studies on skeleton of
D. rerio have had a scattered cov-
erage, including studies on the de-
velopment of the skull (Cubbage
and Mabee, 1996; Schilling and Kim-
mel, 1997), paired fins (Sordino et al.,
1995; Cubbage and Mabee, 1996;
Grandel and Schulte-Merker, 1998),
pharyngeal cartilages (Kimmel et
al., 1998), and developmental (Du
et al., 2001) and adult axial anatomy
(Sanger and McCune, 2002). These
descriptions are not comprehensive,
however, as various parts of the skel-
eton (e.g., intermuscular bones, dor-
sal and anal fins, and supraneurals)
are not included. A description of
developmental morphology of the
zebrafish axial skeleton has been
lacking.
The axial skeleton is derived from
somitic mesoderm (Gilbert, 2003).
The vertebral column develops from
sclerotome, a mesenchymal cell
population derived from the ventral
somite. Sclerotome cells that will
contribute to the vertebral column in
the zebrafish move to surround axial
midline structures and differentiate
as cartilage and bone (Morin-Ken-
sicki et al., 2002). Bones may de-
velop from a cartilage precursor
(cartilage bone) or directly from a
mesenchymal condensation (mem-
brane bone or dermal bone). Carti-
lage bones may ossify in two ways,
either endochondrally with ossifica-
tion beginning within the cartilage
model, or perichondrally with ossifi-
cation beginning in the perichon-
drium surrounding the cartilage
model (Patterson, 1977). Lower te-
leost fishes, including ostariophysans
(zebrafish), are reported to lack
acellular bone (Parenti, 1986).
In fishes, the axial skeleton includes
the vertebral column and associ-
ated median (unpaired) fins. The
vertebral column is regionalized into
precaudal (Fig. 2, green and red)
and caudal vertebrae (Fig. 2, or-
ange and purple). In ostariophysans,
the most anterior precaudal verte-
brae are regionalized as the Webe-
rian vertebrae. The Weberian verte-
brae in otophysans are so named for
the Weberian apparatus (Weber,
1820). In the following description,
we follow the ichthyological con-
vention for ostariophysans of using
the term “precaudal vertebrae” to
refer to those vertebrae posterior to
the Weberian vertebrae and ante-
rior to the caudal vertebrae. Precau-
dal vertebrae are composed of
centra, neural arches and spines,
parapophyses, and ribs. The caudal
vertebrae are regionalized termi-
 ZEBRAFISH SKELETON 339

Fig. 2. Generalized diagram of the zebrafish axial skeleton. Centra are black, the Weberian apparatus is green, supraneurals are light
blue, precaudal vertebrae are red, caudal vertebrae are orange, the caudal fin skeleton is purple, and the dorsal and anal fin
endoskeletons are blue.

Fig. 3. Sequence of development and os-

sification within the axial skeleton. Diamonds
indicate earliest appearance of skeletal el-
ements. Bar indicates presence in all speci-
mens. Blue indicates cartilage; red indicates
bone. In endochondral elements, purple in-
dicates earliest sign of ossification. Names of
skeletal elements are color coded by group
or region. Within labels, caudal fin elements
are blue, Weberian vertebral elements are
red, centra are dark yellow, anal fin is light
purple, vertebral arches and spines are light
blue, dorsal fin is purple, ribs are dark green,
parapophyses are yellow, supraneurals are
green, and intermusculars are black. For hy-
pural 4, the blue–red transition line between
the green diamond and bar indicates that,
in specimens with an early developing hy-
pural 4, ossification begins prior to the length
at which hypural 4 is found in all specimens.
340 BIRD AND MABEE
nally to support the caudal fin. Cau-
dal vertebrae are composed of
centra, neural arches and neural
spines, and hemal arches and he-
mal spines of vertebra 15 through
vertebra 28. The most posterior three
caudal vertebrae are modified to
support the caudal fin. The last pre-
caudal and/or the first caudal verte-
bra is frequently categorized as a
“transitional” vertebra, exhibiting
elongated unfused hemal arches or
parapophyses, drastically shortened
ribs, and absence of a hemal spine.
Associated with many vertebrae,
particularly the most anterior and
most posterior, are basidorsals and
basiventrals, paired cartilaginous
structures that give rise to neural
arches or hemal arches, respectively
(Gadow and Abbott, 1895). Basiven-
trals also give rise to parapophyses.
The associated median fins include
the dorsal, anal, and caudal fins,
their bony internal support elements
(radials) and external supports (fin
rays).
RESULTS
The length of first appearance of a
skeletal element is given in all de-
scriptions below, and it is repre-
sented in Figure 3 as a green dia-
mond. All measurements are given
millimeters standard length; noto-
chord length (NL) is given where ap-
propriate. For ease of interpretation,
a glossary is included. Those struc-
tures and terms included in the glos-
sary are used in headings or have
been bolded at first use below.
Centra
In D. rerio, centra are formed se-
quentially. Centra 3 and 4 form first
(Fig. 4A), as bone encircling the no-
tochord (3.5 mm NL). Additional
centra are added bidirectionally,
with the anterior two centra formed
by 4.1 mm NL. A second region of
development appears at 5.4 mm NL
with the ossification of the com-
pound centrum of preural 1 ⫹ ural 1.
Ossification proceeds bidirectionally
within the caudal fin: anteriorly to
preural 2 and 3 and then posteriorly
to ural 2. By 5.5 mm NL, all centra are
visible. Centra are concave dorso-
ventrally, as well as at both anterior
and posterior ends (Fig. 4D,E).
Weberian centra (vertebrae 1– 4) dorsals that extend dorsally and fuse
remain unfused in D. rerio through- medially to form the neural arch (Fig.
out development, although in many 5A). In D. rerio, the neural arches of
fishes, e.g., silurids and some cyprini- the Weberian vertebrae are first to
forms (Nelson, 1948; Alexander, develop, and they are visible as
1962; Roberts and Kottelat, 1984; small clusters of chondrocytes (4.3
Vandewalle et al., 1990), the Webe- mm NL), which extend dorsally by
rian vertebrae, usually the second means of membrane bone. Neural
and third centra, may develop as a arches of the first and second verte-
fused centrum. This fusion is hypoth- brae are modified into Weberian os-
esized to be the basal condition for sicles, the scaphium and claustrum,
Otophysi, with failure of fusion (as in respectively. Neural arches of the
D. rerio), a secondary condition preural vertebrae, including the
(Roberts and Kottelat, 1984). double arch of preural 2, also de-
The modal number of vertebrae in velop from bilaterally paired carti-
D. rerio, including the urostyle is 31: 4 lage basidorsals. On vertebrae 6 –28,
Weberian, 10 precaudal, 17 caudal the neural arches develop from bi-
(including the 3 caudal fin verte- laterally paired membranous bones.
brae). Our meristic counts (n ⫽ 50) Neural arches 6 –7 are the first mem-
are in agreement with Ferreri et al. branous neural arches visible (4.9
(2000), who found an average of 31 mm NL). Although most remaining
vertebrae in both wild and reared D. neural arches develop simulta-
rerio. neously, the more anterior arches
are longer and they fuse before
more posterior arches. Develop-
Neural Arches and Spines ment proceeds similar to basidorsal
The neural arches form from either arches, with the membrane bone
cartilage (endochondral), or mem- extending dorsally and fusing medi-
brane bone precursors, depending ally to form the neural arch.
on their position along the vertebral Neural spines develop as single
column (Figs. 2, 4C,D,E, 5A,F). Neural median dorsal extensions from the
arches of vertebrae 1–5 form from fused neural arches (5.1 mm NL, Fig.
bilaterally paired cartilaginous basi- 4D,E). All neural spines are dermal in
Fig. 4. Vertebral development, lateral views. A: Early development of anterior centra (5.1
mm NL). Centra 1 (c1), 2 (c2), 3 (c3), and 4 (c4) are visible as rings of bone encircling the
notochord. B: Precaudal vertebrae (6.3 mm). Ossification of the parapophysis (pop) of
centrum 10 (c10) and the relationship between parapophysis and rib. C: Caudal vertebrae
(8.9 mm). Development of neural prezygapophyses (nprez) and neural and hemal
postzygapophyses (npstz, hpstz) on centra 18 –20 (c18 –20) extending from a neural arch
(na) and hemal arch (ha). D: Typical precaudal vertebrae, with neural spines (ns), neural
pre- (nprez) and postzygapophyses (npstz), ribs (rib), and centra 10 –13 (c10 –13; 35.0 mm).
E: Typical caudal vertebrae, with neural spines (ns), hemal arches, hemal spines (hs), as
well as neural and hemal prezygapophyses (nprez, hprez) and postzygapophyses (npstz,
hpstz), and centra 23–27 (c23–27; 35.0 mm). Scale bars ⫽ 0.1 mm in A,C,D,E, 0.05 mm in B.
Fig. 5. Weberian apparatus development, lateral views. A: Early development on the
third and fourth vertebrae as cartilage basidorsals (bd) and basiventrals (bv; 4.7 mm NL).
B: Later development, with supraneurals (sn), neural arches (na), tripus (tr), os suspenso-
rium (os), and parapophyses (pop) present (5.6 mm). C: Continued development, with
scaphium (sc), intercalarium (in), and neural spine 5 (ns5) now present. Mesenchymal
condensation (asterisk) is visible dorsal to the third neural arch (5.9 mm). sn, supraneurals.
D: All structures of the Weberian apparatus present, including the claustrum (cl), scaphium
(sc), intercalarium (in), tripus (tr), neural arch 3 (na3), neural arch 4 (na4), neural spine 4
(ns4), and roofing cartilage (rc; 7.9 mm). The interossicular ligament (asterisk) is visible,
connecting the scaphium to the intercalarium and the intercalarium to the tripus. Rc,
roofing cartilage; sb, swim bladder. E: Ossification of supraneural 2 (sn2) and supraneural
3 (sn3) within the roofing cartilage (rc; 7.8 mm). The intercalarium is out of the plane of
focus. Neural arch 3 (na3), neural arch 4 (na4), neural spine 4 (ns4), scaphium (sc) and
claustrum (cl) are also visible. F: Full diagram of the adult Weberian apparatus (vertebrae
1–5 are shown), including scaphium (sc), intercalarium (in), tripus (tr), os suspensorium
(os), neural arch 3 (na3), neural arch 4 (na4), neural spine 4 (ns4), neural arch 5 (na5),
neural spine 5 (ns5), supraneural 5 (sn5), supraneural 2 (sn2), supraneural 3 (sn3), lateral
process 1 (lp1), lateral process 2 (lp2), rib 4 (rib4), rib 5 (rib5), and swim bladder (sb). Scale
bars ⫽ 0.1 mm in A–C,E,F, 0.5 mm in D.
 Fig. 5.
Fig. 6. Histologic frontal sections through
the Weberian apparatus (6.0 mm). A: First
vertebra (c1). Concha scaphium (con) has
fused with the first basidorsal (bd1). Roofing
cartilage (rc) can be seen dorsal to the neu-
ral tube (nt). B: Third vertebra (c3). The third
neural arch (na3) has become continuous
with the roofing cartilage (rc), completely en-
circling the neural tube (nt). The tripus (tr) is
seen lateral to centrum 3 (c3) and neural
Fig. 4. arch 3 (na3). Scale bars ⫽ 0.05 mm.
Fig. 7. Intermuscular bones, lateral views
(29.7 mm). A: Forked epineural, showing
medial (med) and lateral (lat) extensions.
Neural arches (na) and neural spines (ns)
can be seen in the background. B: Forked
epipleural, showing medial (med) and lat-
eral (lat) extensions. Hemal arches (ha)
and hemal spines (hs) can be seen in the
background. Scale bars ⫽ 0.5 mm.
342 BIRD AND MABEE
origin except for the neural spines of
the preural vertebrae, which de-
velop, at least in part, from carti-
lage. Neural spines form earlier on
anterior vertebrae.
Neural arches in the precaudal re-
gion are broad, with well-developed
prezygapophyses (Fig. 4D). Prezyg-
apophyses (neural prezygapophy-
ses) form as paired, anteromedially
directed processes from the neural
arches, and they are ligamentously
attached to the postzygapophyses
of the preceding vertebra (Fig. 4C).
The prezygapophyses are larger in
precaudal vertebrae than in caudal
vertebrae (Fig. 4D,E). The Weberian
associated vertebrae (vertebrae
1– 4) lack prezygapophyses. Verte-
bra 5 has a highly reduced prezyg-
apophysis, but vertebrae 6 and 7
are enlarged and appear to be liga-
mentously connected to a posterior
flange extending from the neural
spines of these vertebrae.
Postzygapophyses (neural postzyg-
apophyses) form as paired dorsome-
dially directed processes (6.2 mm) on
the posterior ends of centra (Fig.
4C,E). They are ligamentously at-
tached to the prezygapophyses of
the immediately posterior vertebra.
The postzygapophyses of precaudal
vertebrae are larger than those of
caudal vertebrae (Fig. 4D,E). Signifi-
cant variation in shape is found in the
precaudal region. The first postzyg-
apophysis is present on vertebra 5,
and they are found posteriorly
through preural 3 (6.2 mm). The devel-
opment of postzygapophyses is bidi-
rectional, with the first group visible on
vertebrae 11–23. Ventral postzyg-
apophyses develop slightly later (6.3
mm), but develop in the same man-
ner as neural postzygapophyses.
Neural spines are generally longer
in the precaudal region when com-
pared with the more posterior verte-
brae (Fig. 2), although their morphol-
ogies are similar. Neural spines form
immediately after fusion of the neu-
ral arches. Timing of fusion is variable
but proceeds in an anterior-to-pos-
terior direction.
Parapophyses and Ribs
Parapophyses are bony lateral ex-
tensions from centra that serve as
articulation points for ribs (Figs. 2,
4B,D). In D. rerio all parapophyses
develop endochondrally from ba-
siventrals (Fig. 4B). Parapophyses
are first visible as clusters of chondro-
cytes on the lateral surface of the
anterior ends of the precaudal ver-
tebrae (5.5 mm NL). Parapophyses
of the fifth and sixth vertebrae are
much larger compared with those of
more posterior precaudal verte-
brae. Ossification is first visible at the
center of the parapophysis (6.0
mm). The fifth parapophysis is always
the first to develop, with remaining
parapophyses added posteriorly. In
the adult, the fifth parapophysis is
thinner, anterior to posterior, relative
to the remaining parapophyses. A
small depression is visible in parap-
ophysis 5, to which the posterior
head of the fifth rib articulates. In
contrast, the parapophysis of the
sixth vertebra is broad. It is slightly
concave ventrolaterally, forming a
groove within which the sixth rib ar-
ticulates.
Ribs are intersegmental rod-
shaped bones serving to protect the
viscera (Figs. 2, 4B,D). In most fishes,
the first rib develops in association
with centrum 2. In D. rerio, centrum 2
lacks a rib, and the ribs on centra 3
and 4 are modified as part of the
Weberian apparatus (see below).
Typically 10 ribs are present (range
of 9 –11), with the rib-heads and par-
apophyses of the most anterior (V5)
and most posterior (V13–V15) verte-
brae showing distinct morphologies.
The fifth rib head is reduced in size
relative to the sixth, but maintains
slightly flared anterior and posterior
tips. The heads of the posterior ribs
(ribs 13 and 14) are reduced to a ball
shape; they loosely articulate with
their parapophyses. The parapophy-
ses are rounded and extend distally
to articulate with the ribs.
Ribs are first visible as slivers of
membranous bones distal to the
developing parapophyses (5.5 mm
NL). Ribs grow posteroventrally to
surround the viscera, as well as dor-
sally to articulate with the parap-
ophyses (Fig. 4B). The first non-We-
berian rib that develops is always
the rib of the fifth vertebra, with
remaining ribs added posteriorly. In
the adult, ribs 5–10 have broad and
flat heads (Fig. 4D). The ribs retain a
broad thin shape in their proximal
half and possess a thickened lat-
eral ridge that develops from the
initial thickened rod. At approxi-
mately half the overall rib length,
the morphology reverts to the typ-
ical rod-like shape as they curve
around the viscera and swim blad-
der. The last (and second-to-last in
some instances) rib either loses the
“flared” appearance of its proxi-
mal half, or it is highly reduced in
length.
Hemal Arches and Spines
The hemal arches characterize the
caudal vertebrae; they do not de-
velop on precaudal vertebrae (Fig.
4C,E). The caudal vertebrae range
in number from 14 to 16, with an av-
erage of 15 vertebrae (excluding
the vertebrae associated with the
caudal fin). Hemal arches develop
from ventrally projecting, bilaterally
paired, membranous ossifications
on anterior ends of caudal verte-
brae. These ossifications grow ven-
trally and fuse medially to form the
hemal arch. Hemal arches may be
fused medially at the smallest size at
which they are visible (5.4 mm NL).
Hemal spines develop as a result of
continued membranous ossification
of the hemal arches in the ventral
midline (5.5 mm).
The hemal arches and spines of
preural vertebrae, however, preform
in cartilage (5.2 mm NL). The hemal
arches are fused and the hemal
spine formed at formation; i.e., they
do not form as a result of fusion from
initially paired basiventrals. The he-
mal spines elongate by means of
continued cartilage growth, and
they later (6.0 mm) ossify.
Hemal arches are broad, with
small prezygapophyses (hemal
prezygapophyses). Prezygapophy-
ses form as anteriorly directed pro-
cesses (8.6 mm) from the bases of
hemal arches. They are ligamen-
tously attached to the postzyg-
apophyses of the preceding verte-
bra.
Postzygapophyses (hemal postzyg-
apophyses, Fig. 4C) form as ventrally
directed processes (6.3 mm) on the
posterior ends of centra. They are liga-
mentously attached to the prezyg-
apophyses of the immediately poste-
rior vertebra (Fig. 4C,E). The first ventral

postzygapophysis is found on the first
caudal vertebra (vertebra 15), and
they are found posteriorly through
preural 3 (6.2 mm). The addition of
postzygapophyses is bidirectional,
with the first formed on vertebra 19.
Ventral postzygapophyses develop
slightly later (6.3 mm) than neural
postzygapophyses.
Weberian Apparatus
The Weberian apparatus is a novel
adaptation of a large clade of teleost
fishes, the otophysans (Fink and Fink,
1996), which provides a route for the
transmission of sound/vibration from
the swim bladder to the inner ear
(Weber, 1820). Development and
anatomy of the inner ear (Haddon
and Lewis, 1996; Bang et al., 2001;
Bever and Fekete, 2002) and the spe-
cific role of the Weberian apparatus
in sound transmission (Popper and
Fay, 1999) have been reviewed previ-
ously. Otophysan teleosts (within the
larger clade Ostariophysi) include
members of Cypriniformes (e.g., ze-
brafish), Characiformes, Siluriformes,
and Gymnotiformes (Rosen and
Greenwood, 1970; Berra, 2001) (Fig.
1). This adaptation is considered a key
innovation facilitating the enormous
radiation of this group in freshwaters
worldwide (Berra, 2001). Otophysi (Fig.
1) comprise approximately 75% of all
freshwater fishes and 27% of all fishes
(⬃7,500 species; Helfman et al., 1997;
Berra, 2001). Rudiments of the Webe-
rian apparatus are found in the sister
taxon of otophysans, the Gonoryn-
ichiformes and in the Clupeomorpha
(Fig. 1; Rosen and Greenwood, 1970;
Fink and Fink, 1981; Grande and
Braun, 2002). Thus, the Weberian ap-
paratus is thought to have evolved
piece by piece, and only once all el-
ements were present did it function in
sound transmission (Grande and
Braun, 2002). The Weberian appara-
tus is modified but not lost in any oto-
physan.
The Weberian apparatus is tradi-
tionally subdivided into the Webe-
rian ossicles, the scaphium, claus-
trum, intercalarium, and tripus, and
the remaining bones, which include
the centra, neural arches and
spines, supraneurals. These bones
serve to connect the swim bladder
to the membranous labyrinth (inner
ear) of the fish by means of the ca-
vum sinus impar, allowing for greater
sensitivity to vibration and acute
hearing (Chardon and Vandewalle,
1997).
Weberian ossicles.
These include, from anterior to pos-
terior, the scaphium, claustrum, in-
tercalarium, and the tripus. The os
suspensorium, considered to be a
fifth ossicle (Vandewalle et al., 1990),
also will be addressed in this section.
The scaphium, intercalarium, and tri-
pus are connected by means of the
interossicular ligament.
Scaphium.
The scaphium is connected posteri-
orly to the manubrium of the interca-
larium by means of the interossicular
ligament (Fig. 5C–F). The concha of
the scaphium forms the posterolat-
eral wall of the sinus impar perilym-
phaticus, the connection to the
membranous labyrinths of the inner
ear (Radermaker et al., 1989). The
scaphium in D. rerio represents the
most fully developed otophysan
state: the scaphium possesses an ar-
ticular process that articulates with a
depression in centrum 1, an ascend-
ing process that extends posterodor-
sally, and a spoon-shaped concha
located anterior to these processes.
The basidorsal from which the
scaphium develops is first visible as a
small cartilage condensation on the
dorsal surface of centrum 1 (4.8 mm
NL, Fig. 5C). The ascending process
develops as a cartilaginous dorsal
extension from this basidorsal. The
articular process ossifies from the
original basidorsal cartilage. The
concha scaphium develops from a
separate anterior mesenchymal os-
sification that fuses with basidorsal 1
(5.2 mm NL). The scaphium begins
ossification at 5.3 mm NL.
In the adult zebrafish, the
scaphium is connected to the claus-
trum by means of a syndesmosis.
The concha of the scaphium does
not make contact with the exoccipi-
tal strut or other parts of the skull. The
concha is medially concave and
laterally convex. The dorsal surface
of the concha is thickened and
arch-shaped.
ZEBRAFISH SKELETON 343
Claustrum.
The claustrum is a bilaterally paired,
triangularly shaped bone embed-
ded in connective tissue dorsome-
dial to the scaphium (Fig. 5D–F). The
claustrum appears as a thin band of
cartilage, consisting of only a few
cells, immediately anterior to the an-
terodorsal edges of the roofing car-
tilage (5.5 mm). It develops in close
association with the roofing carti-
lage, and it is most easily distin-
guished in specimens around 7.0
mm. The claustrum extends antero-
ventrally through membrane ossifi-
cation to encircle the neural tube
(Fig. 6A). The claustrum does not en-
tirely preform in cartilage but instead
forms from only a sliver of cartilage
and primarily membrane bone. The
base of the claustrum articulates
with the medial surface of the
scaphium (Fig. 5F). The dorsal sur-
face of the claustrum is connected
to supraneural 2 by means of a syn-
chondrosis. Ossification begins at 6.3
mm.
Intercalarium.
The intercalarium is a rod-shaped
bone that originates from basidorsal
2 (Fig. 5C,D,F). It is connected ante-
riorly to the scaphium and posteriorly
to the anterior process of the tripus
by means of the interossicular liga-
ment. At most in otophysans, the in-
tercalarium is triradiate, with an an-
terior process, or manubrium,
projecting anterolaterally to lie be-
tween the scaphium and the tripus,
as well as ascending and articular
processes (Fig. 5F).
The intercalarium is first visible as a
small basidorsal cartilage mass on
the dorsal surface of centrum 2 (4.8
mm NL, Fig. 5C). The manubrium de-
velops as a membranous ossification
that extends laterally from the basi-
dorsal (5.1 mm NL). The ascending
and articular processes begin ossifi-
cation at 5.3 mm NL. The ascending
process develops as a dorsally pro-
jecting cartilaginous extension from
the basidorsal (5.1 mm NL). The as-
cending process is longer relative to
the manubrium in larvae (5.1–11.0
mm). By 13.0 mm, the ascending
process appears highly reduced in
size. The articular process ossifies
from the cartilage condensation
344 BIRD AND MABEE
and articulates with a groove in cen-
trum 2.
Tripus.
The tripus is the largest of the Webe-
rian ossicles (Fig. 5A,B,D,F). The tripus
is typically separated into three re-
gions: the anterior process, which
connects to the intercalarium; the
articular process, which articulates
with centrum 3; and the transforma-
tor process, which articulates with
the os suspensorium and the swim
bladder.
Development of the tripus begins
as a large basiventral cartilage
forming on the ventrolateral surface
of centrum 3 (4.7 mm NL, Fig. 5A).
The anterior and transformator pro-
cesses are formed by membranous
ossification, with the articular pro-
cess ossifying within the initial carti-
lage condensation. The interossicu-
lar ligaments do not contribute to
the developing tripus.
The transformator process ossifies
as a ventrally projecting rod-like os-
sification from basiventral 3 (5.1 mm
NL). As it grows, it becomes thick-
ened and it curves posteriorly such
that the tip lies medial to the os sus-
pensorium and is in contact with the
swim bladder (5.6 mm, Fig. 5B). Most
of the transformator process ap-
pears to be derived from the rib of
the third vertebra.
The anterior process develops as
an anteriorly directed membranous
extension from the body of the tripus
(6.2 mm). In the adult, the tip of the
anterior process is dorsal to lateral
process 2 at the level of centrum 2
and connects by means of the in-
terossicular ligament to the manu-
brium of the intercalarium.
From a dorsal view, the tripus is
wing-shaped, extending from verte-
bra 2 to vertebra 4 (Fig. 5F). Laterally,
the tripus is tilted, with a lowered
posterior hook-shaped tip (transfor-
mator process) positioned ventral to
centrum 4. The posterior tip is slender
and curves medially. The posterior
tip of the transformator process ar-
ticulates with the medial portion of
the os suspensorium, as well as with
the tunica externa of the camera
aerea Weberiana, or the anterior sur-
face of the anterior sac of the swim
bladder (Radermaker et al., 1989).
The surface of the tripus of D. rerio is
smooth, a shared, derived feature of
slender-bodied Danio; it lacks a per-
pendicular ridge found in deep-
bodied species of Danio (Sanger
and McCune, 2002).
Os suspensorium.
The os suspensorium is a slender
bone curving along the anterior sur-
face of the anterior sac of the swim
bladder (Fig. 5B,F). It articulates with
the tripus at the tripus–swim bladder
connection.
The os suspensorium begins devel-
opment as a separate anteriorly di-
rected membrane bone outgrowth
from the basiventral cartilage of ver-
tebra 4 (parapophysis 4; 5.4 mm NL).
By 5.6 mm, the os suspensorium has
extended anteriorly and has arched
ventrally over the swim bladder (Fig.
5B). Midsagittally, the os suspensoria
become tightly juxtaposed. A thin
line of fusion is evident in adults; the
bones are not easily separated. In
no specimen does the os suspenso-
rium develop from the fourth rib;
rather, they both extend from the
cartilage of parapophysis 4.
The upper body of the os suspen-
sorium has a large groove; this may
be an attachment site for tendons or
muscles associated with the swim
bladder (Evans, 1925; Alexander,
1962). The shape and size of this
groove varies within Danio (Sanger
and McCune, 2002).
Other elements of the
Weberian apparatus.
These include elements associated
with the anterior four vertebrae, in-
cluding neural arches and spines, su-
praneurals 2 and 3, the roofing car-
tilage, transverse processes, and the
centra. As in all ostariophysans (Fig.
1), the first four vertebral centra of D.
rerio are foreshortened relative to
the more posterior vertebrae (Fink
and Fink, 1981).
Vertebra 1. The first vertebra has
been modified as part of the Webe-
rian apparatus. Vertebra 1 is com-
posed of a compact centrum articu-
lating anteriorly with the basioccipital
of the skull, a short lateral process,
and a modified neural arch known as
the scaphium (Figs. 4A, 5F).
The centrum for vertebra 1 is first
visible at 5.0 mm NL (Fig. 4A). The
anterior portion is dorsoventrally nar-
rowed to articulate with a notch in
the basioccipital. A short bony lat-
eral process, which is an extended
parapophysis fused to the centrum,
is first visible as a small lateral bud
extending from the centrum (5.7
mm, Fig. 5F). The lateral process ex-
tends slightly, and it remains small in
the adult. The ventromedial surface
of the scaphium articulates with the
dorsal surface of centrum 1.
Vertebra 2. The second vertebra is
composed of a compact centrum
that is slightly longer than centrum 1,
an elongated lateral process, and
the intercalarium (Figs. 4A, 5C).
The centrum of vertebra 2 is first
visible at 5.0 mm NL (Fig. 4A). A long
and slender bony lateral process
(5.1 mm NL, Fig. 5F) extends from this
centrum, later curving posteriorly at
the distal tip. As with the lateral pro-
cess of the first vertebra, it is fused to
the centrum in the adult. The ventral
surface of the articular process of
the intercalarium articulates with a
depression in the dorsal surface of
the centrum.
Vertebra 3. The third vertebra is
composed of a shortened centrum
(4.6 mm NL, Figs. 4A, 5) that is slightly
larger than the centrum of vertebra
2, an enlarged neural arch, and the
tripus. Development of the third neu-
ral arch begins from large paired
basidorsal cartilage masses on the
dorsal surface of the centrum (4.3
mm NL, Fig. 5A). The masses extend
dorsally and medially (5.9 mm, Fig.
5B,C), and they become continuous
with the roofing cartilage (Fig. 6B).
Ossification begins at the center of
the neural arch (5.0 mm NL). The
basidorsals fuse midlaterally to form
the adult arch. A third neural spine
does not form. Parapophysis 3 de-
velops from a large basiventral car-
tilage on the ventrolateral surface of
the centrum, and together with the
third rib, it is modified as the tripus.
Vertebra 4. Vertebra 4 is com-
posed of a centrum (4.6 mm NL, Fig.
4A, 5), an enlarged neural arch and
neural spine, and a modified and
combined parapophysis/rib that
contributes to the os suspensorium

and lateral process/rib. A large neu-
ral arch develops on the dorsal sur-
face of centrum 4 in the same man-
ner as neural arch 3, and it is first
visible at 4.3 mm NL (Fig. 5A). A
large, broad, blade-like neural spine
develops as ossification proceeds
distally (5.1 mm NL, Fig. 5D–F). Carti-
lage from neural arch 4 is not con-
tinuous with the roofing cartilage.
Ossification of neural arch 4 begins
at the dorsal surface of the paired
arches (5.0 mm NL), and it proceeds
ventrally. Ossification of neural arch
4 and neural spine 4 is complete by
6.6 mm (Figs. 5B,D,E).
The first rib in the anterior–posterior
series develops from this centrum
(Fig. 5D,F) as a small membranous
posterior projection from the carti-
laginous parapophysis (5.6 mm). The
rib is a separate outgrowth from the
parapophysis, and it is not con-
nected to the os suspensorium (5.6
mm). Rib 4 is oriented such that it
projects anteriorly; all other ribs
project posteriorly. The rib lies lateral
to the tripus, and it becomes bifur-
cated distally (Fig. 5F). It is fully de-
veloped and ossified by 7.5 mm.
Supraneural 2.
Supraneural 2 is an ovoid bone lo-
cated within the midsagittal septum
dorsal to centrum 2 (Figs. 2, 5B,C,E,F).
Based on topology and shape,
namely that this structure resembles
the dorsal portion of a normal neural
arch, others have identified supra-
neural 2 as neural arch 2 (Ra-
maswami, 1955; Howes, 1978; May-
den, 1989).
Supraneural 2 is first visible as a sin-
gle cartilage condensation dorsal to
vertebra 2 (4.8 mm NL, Fig. 5B,C).
Supraneural 2 develops immediately
before supraneural 3 (5.1 mm NL).
The remaining supraneurals appear
successively; development of all su-
praneurals proceeds in an anterior
to posterior direction, as in other te-
leosts (Mabee, 1988). Supraneural 2
becomes surrounded by and be-
comes continuous with the roofing
cartilage. A midsagittal projection
marks the location of supraneural 2
within the cartilage mass. The early
ossification of supraneural 2 wraps
around the anterior tip of the roofing
cartilage/supraneural mass (6.7 mm,
Fig. 5E). Supraneural 2 is fully ossified
by 13.8 mm (Fig. 5F).
In contrast to other Danio (Sanger
and McCune, 2002), in D. rerio the
ventral margins of supraneural 2 are
not curved medially and the dorsal
margin is nearly horizontal and lacks
a medial ridge.
Supraneural 3.
Supraneural 3 is an irregularly
shaped bone located within the
midsagittal septum (Figs. 2, 5B,C,E,F).
It develops as a single cartilage con-
densation dorsal to the fourth neural
arch (5.1 mm NL, Fig. 5B,C), slightly
anterior to the fourth neural spine.
Like supraneural 2, it becomes sur-
rounded by and continuous with the
roofing cartilage (5.6 mm, Fig. 5D). A
dorsally elevated midsagittal ridge
marks its location within the roofing
cartilage (Fig. 5D,E). Ossification be-
gins on the surface of this dorsal el-
evation (6.7 mm, Fig. 5E) and pro-
ceeds dorsally as a thin sheet of
membrane bone and ventrally over
the surface of the cartilage. The dor-
sal surface of supraneural 3 projects
anteriorly in the adult. It is fully ossi-
fied by 14.5 mm (Fig. 5F). A fused
anterior margin of supraneural 3 is a
shared derived feature of all Danio
(Sanger and McCune, 2002). In con-
trast, the dorsal and posterior mar-
gins of Weberian supraneural 3 are
fused in D. rerio. In the slender-bod-
ied clade of Danio, which includes
D. rerio, supraneural 3 is short relative
to its tall primitive condition (Sanger
and McCune, 2002).
In some otophysans, supraneural 4
is retained (primitively present), but
in some cypriniforms such as Danio,
supraneural 4 is lost. Although Gayet
(1986) proposed that supraneural 3
might be formed by means of a fu-
sion of supraneural 3 and supraneu-
ral 4, we do not see a separate con-
densation for a fourth supraneural in
D. rerio. This finding is concordant
with the observations of Fink and Fink
(1996) for otophysans.
Roofing cartilage.
The roofing cartilage is a larval struc-
ture located dorsal to the neural
tube (5.6 mm) (Figs. 5D,E, 6). It is first
visible as strands of mesenchyme
forming amongst and thereby con-
necting supraneurals 2 and 3 and
ZEBRAFISH SKELETON 345
neural arch 3 (Fig. 5B,C). This matrix is
Alcian blue–positive before chon-
drocytes are visible. The roofing car-
tilage differentiates to surround su-
praneural 2, supraneural 3, and the
neural arch and spine of vertebra 3
(Figs. 5D,E, 6B). It closely approxi-
mates the neural arch and spine of
vertebra 4. The roofing cartilage
forms a synchondrosis with the claus-
trum. The roofing cartilage is contin-
uous with the supraneurals and neu-
ral arch 3 only during larval
development. Our observation of
the formation of the roofing carti-
lage independent from neural
arches 3 and 4 and supraneurals 2
and 3 is consistent with those of
Bogutskaya (1991) and Coburn and
Futey (1996) from a wide variety of
cyprinforms.
In the adult, the roofing cartilage
appears to de-differentiate to be-
come non–matrix-positive connec-
tive tissue. A remnant of the roofing
cartilage is retained as a connective
tissue mat connecting neural arches
3 and 4, the claustrum, and supran-
eurals 2 and 3. Typically, there is a
gap between the roofing cartilage
and exoccipital of the skull, al-
though in some specimens they ap-
pear fused.
Intermuscular Bones
Intermuscular bones, which are
unique to teleostean fishes (Patter-
son and Johnson, 1995; Gemballa
and Britz, 1998), are membranous os-
sifications that form in the myosepta
and attach ligamentously to neural
arches, centra, hemal arches, or ribs
(Fig. 7). Three sets of serially homolo-
gous intermusculars may be present
in teleosts and are classified in refer-
ence to the site of attachment
(Patterson and Johnson, 1995; Gem-
balla and Britz, 1998). From dorsal to
ventral, epineurals attach to neural
arches, epicentrals attach to cen-
tra, and epipleurals attach to hemal
arches or ribs. Epicentrals are not
present in D. rerio. Epineurals de-
velop in association with vertebra 5
through preural 2 (6.0 mm). They de-
velop and ossify predominantly from
posterior to anterior. Beginning at
approximately the precaudal– cau-
dal transition, each epineural devel-
ops an anterior fork (Fig. 7A). The first
346 BIRD AND MABEE
signs of forking are difficult to visual-
ize, but at 10.6 mm, both epineurals
and epipleurals appear to fork at
approximately vertebral level 17–18.
Epineurals associated with precau-
dal vertebrae are not forked. The
more anterior epineurals span ap-
proximately two precaudal centra,
and the more posterior epineurals
span approximately three caudal
centra.
In D. rerio, the most anterior epi-
pleural is associated with the first
complete hemal arch (the first cau-
dal vertebra). Epipleurals do not de-
velop in association with ribs. Devel-
opment of the epipleurals proceeds
in concert with the epineurals.
Epipleurals develop as slender
rods lateral to the vertebral column.
In forked epineurals and epipleurals,
it is the medial extension that is liga-
mentously attached to the verte-
brae (Fig. 7A,B).
Supraneurals
Supraneurals are dorsal median
structures located anterior to the
dorsal fin pterygiophores and poste-
rior to the skull (Figs. 2, 8). They are
slender skeletal rods that may be ho-
mologous to neural spines or radials
(Mabee, 1988). In D. rerio, supraneu-
rals range in number from 4 to 6, with
a mode of 5. These supraneurals are
numbered 5–9 (Fig. 2). In specimens
with six supraneurals, the “extra” is
positioned posterior to supraneural
9. The sole exception was a speci-
men in which the additional supra-
neural was located dorsal to the
roofing cartilage. In the (only) spec-
imen examined with four supraneu-
rals, the ninth supraneural was ab-
sent.
Supraneurals develop from small
clusters of chondrocytes dorsal to
the anterior precaudal neural spines
(Fig. 8A). Supraneurals develop from
anterior to posterior: supraneural 5 is
always the first visible (6.2 mm) fol-
lowed by supraneurals 6 –9 (6.4 mm,
6.6 mm, 6.6 mm, and 7.0 mm, re-
spectively). The supraneurals are ar-
ranged one per myomere, with the
most anterior located between the
neural spines of the fourth and fifth
vertebrae, and the most posterior
between the neural spines of the
eighth and ninth vertebrae. Supra-
neural 5 enlarges and broadens, tive fin. Additional radials develop
while supraneurals 6 –9 develop dor- anteriorly and posteriorly (Fig. 9A,C).
soventrally as small slender rods of Correlation between direction of
cartilage. Supraneural 5 is the first to development within the dorsal and
ossify, with ossification beginning anal fins is similar to those found in
later in more posterior supraneurals other fishes, and this finding is prob-
(supraneural 6 at 8.5 mm, supraneu- ably the ancestral condition for fin
ral 7 and 8 at 9.2 mm, and supran- positioning and patterning (Mabee
eural 9 at 9.8 mm). Supraneurals ex- et al., 2002). Later in development,
hibit perichondral ossification, with radials segment proximodistally into
first uptake of stain forming a ring two parts, a larger proximal radial
around the cluster of chondrocytes and smaller distal radial (5.7 mm, Fig.
(Fig. 8B). 10). The distal radials articulate di-
In the adult, the posterior four su- rectly with the lepidotrichia. Devel-
praneurals are arrowhead in shape, opment of dorsal fin radials typically
with a wide dorsal surface that begins with those radials located
tapers ventrally. Many have a proxi- between the neural spines of the
modistal groove in the lateral sur- 14th and 15th vertebrae, which cor-
face running the length of the supra- respond to the 4th and 5th radials in
neural (Fig. 8C). The shape of the first the adult (5.5 mm NL, Fig. 9A).
supraneural is variable, but it is al- Growth proceeds proximally toward
ways larger than supraneurals 6 –9 the vertebral column.
and generally exhibits an ovoid Ossification of the proximal radials
shape. proceeds proximally and distally
from the middle of the radial (7.0
Median Fins mm, Fig. 9B). In all but the anterior
two radials, a second center of ossi-
Dorsal fin. fication is located at the distal tip of
The dorsal fin is an unpaired median the proximal radial. Ossification gen-
fin located on the dorsal surface of erally follows the same order as
the body (Figs. 2, 9A–C). The dorsal chondrogenesis, i.e., radials 4 and 5
fin consists of endoskeletal supports, are the first to begin ossification. Dis-
or radials, and exoskeletal supports, tal radials ossify in the same order as
or fin rays. The radials, also termed proximal radials, but ossification is
“pterygiophores,” are rod-like inter- delayed relative to the proximal ra-
nal median skeletal supports of the dials (8.6 mm). Dorsal radials were
dorsal and anal fins. not found anterior to the neural
In D. rerio, initiation of develop- spine of vertebra 11 or posterior to
ment of the dorsal fin radials is first the neural spine of vertebra 18. The
visible as a central group of carti- modal number of dorsal fin radials
lage condensations at approxi- for the zebrafish is eight, with a range
mately the middle of the presump- of seven to nine (n ⫽ 50). It is at the
Fig. 8. Supraneurals, lateral views. A: Cartilage development of the anterior three supra-
neurals (sn5, sn6, and sn7; 6.6 mm). B: Early ossification of supraneural 9 (sn9; 12.2 mm).
Neural spine 8 (ns8) is visible anterior to supraneural 9. C: Adult morphology of supraneu-
rals, arrow points to groove (29.7 mm). Supraneural 8 (sn8) and supraneural 9 (sn9) are
visible. Scale bars ⫽ 0.1 mm in A, 0.05 in B,C.
Fig. 9. A–C: Development of the dorsal fin, lateral views. A: Early formation of proximal
radials (pr; 5.6 mm). B: Ossification within the proximal radials (pr) and distal radials (dr; 8.9
mm). Fin rays (fr) are visible attaching to distal radials. C: Adult dorsal fin (35.0 mm).
Proximal radials (pr), distal radials (dr), and fin rays (fr) are visible dorsal to the caudal
vertebrae, which include centra (c), neural arches (na) and neural spines (ns). D–F:
Development of the anal fin, lateral view. D: Early formation of proximal radials (pr; 5.6
mm). E: Ossification within the proximal radials (pr) and distal radials (dr; 5.6 mm). Fin rays
(fr) are visible attaching to distal radials. F: Adult anal fin (35.0 mm). Proximal radials (pr),
distal radials (dr), and fin rays (fr) are visible ventral to the caudal vertebrae, which include
centra (c), hemal arches (ha) and hemal spines (hs). Scale bars ⫽ 0.05 in A,D, 0.1 mm in
B,C,E,F.
Fig. 10. Segmentation of anal fin radials. A: Unsegmented anal fin radials (pr; 6.0 mm). B:
Segmentation of distal radial (dr) from the proximal radial (pr; 6.1 mm). C: Proximal (pr)
and distal radials (dr) have completely separated (6.5 mm). Scale bars ⫽ 0.05 mm in A–C.
 ZEBRAFISH SKELETON 347

posterior end of the fin that radials

are added or lost. Interdigitation
patterns (i.e., the relationship be-
tween the radials and neural spines)
of the dorsal fin are variable, and the
modal pattern is represented in Fig-
ure 2.
Fin rays (lepidotrichia) are bony,
bilaterally paired, segmented der-
mal ossifications extending distally
from the endoskeletal supports.
They develop around actinotrichia
as part of the dermal exoskeleton,
and they evolved in Osteichthyes
(Lauder and Liem, 1983). Dorsal fin
ray development mirrors that of the
radials, i.e., the rays associated
with the fourth and fifth radials are
first to development with the re-
maining added bidirectionally (5.6
mm). Rays usually number n ⫹ 2,
where n ⫽ radial number, with two
extra rays articulating with the first
radial. Development of the dermal
rays proceeds proximal to distal. As
the rays develop, they segment
proximodistally, and bifurcate dis-
tally (Fig. 9C).

Anal fin.
The anal fin is an unpaired median
fin located on the ventral surface of
Fig. 8.
the body, and it is delimited anteri-
Fig. 10. orly by the anus (Figs. 2, 9D–F). De-
velopment of anal fin radials begins
with those radials developing ante-

Fig. 9.
348 BIRD AND MABEE
rior and posterior to the hemal spine
of the 16th vertebra. These corre-
spond to the third and fourth anal
radials in the adult (5.1 mm NL, Fig.
9D). The radials develop as stacks of
chondrocytes near the ventral sur-
face of the body. Growth proceeds
proximally toward the vertebral col-
umn. Anal radials are added ante-
rior and posterior to the original radi-
als (Fig. 9D). Ossification proceeds
proximally and distally from the mid-
dle of the radial (7.0 mm, Fig. 9E),
and it is seen in the same order as
chondrogenesis. Similar to dorsal fin
radials, all but the anterior two radi-
als exhibit a second center of ossifi-
cation at the distal end of the prox-
imal radial (Fig. 9E). Shortly after the
onset of development, the radials
segment into proximal and distal
portions (5.5 mm, Fig. 10). Distal radi-
als ossify in the same order as proxi-
mal radials, but ossification is de-
layed relative to the proximal radials
(8.6 mm). Anal radials were not
found anterior to the hemal arch of
vertebra 14 (position of the anus) or
posterior to the neural spine of the
21st vertebra. The modal number of
anal fin radials for the D. rerio is 13,
with a range of 12–14 (n ⫽ 50). The
average interdigitation pattern is
represented in Figure 2.
Anal fin ray development mirrors
that of the radials, i.e., the rays asso-
ciated with the third and fourth radi-
als are first to development with the
remaining added bidirectionally (5.5
mm). As in the dorsal fin, rays usually
number n ⫹ 2, where n ⫽ radial num-
ber, with two extra rays articulating
with the first radial (Fig. 2).
Caudal fin.
The caudal fin is the first median fin
to show both endoskeletal and ex-
oskeletal development (Figs. 2, 3,
11). The endoskeleton of D. rerio is
composed of five hypurals, the par-
hypural, and hemal and neural
arches and spines of the second
and third preural vertebrae, one pair
of uroneurals, the neural arch of
preural 1, one epural, and the uro-
style. The urostyle is composed of the
compound centrum preural 1 ⫹ ural
1, which fuses in development to the
centrum of ural 2.
The five hypurals are expanded
and laterally flattened hemal arches
and spines that support the lepi-
dotrichia of the caudal fin. The hy-
purals form ventral to the posterior
tip of the dorsally flexed notochord
(Fig. 11A,B).
Development is first visible as the
independent condensations of the
parhypural and hypural 1 (3.8 mm
NL, Fig. 11A). The remaining hypurals
(2–5) are added posteriorly, with hy-
pural 2 developing at 4.0 mm NL,
hypural 3 at 4.4 mm NL, hypural 4 at
4.8 mm NL, and hypural 5 at 5.0 mm
NL (Fig. 11B,C–E). Hypural 2 be-
comes fused (is nonautogenous) to
the compound centrum of preural
1 ⫹ ural 1. The proximal cartilages of
the parhypural and hypural 1, which
abut the compound centrum of
preural 1 ⫹ ural 1, fuse proximally
(Fig. 11B). Ossification is first visible
near the proximal end of the hy-
purals, and it proceeds bidirection-
ally (Fig. 11C–E). The proximal and
distal tips are last to ossify. Ossifica-
tion proceeds first in hypurals 1–3 (5.0
mm NL), and by 5.1 mm NL hypural 4
has begun to ossify. Hypural 5 begins
ossification later (6.2 mm). The distal
tips of the hypurals remain as carti-
lage late in development, with all
elements fully ossified by 24 mm (Fig.
11E).
Danio rerio has three hypurals (hy-
purals 3–5) in the upper lobe (Fig.
11E), a derived condition of the slen-
der-bodied clade of Danio. Ances-
trally in Danio, the lower lobe of the
caudal fin is supported by the par-
hypural and hypurals 1 and 2, and
the upper lobe is supported by four
hypurals (3– 6). As noted by Sanger
and McCune (2002), the slender-
bodied clade of Danio has a de-
rived condition of only three hy-
purals in the upper lobe, for a total of
five hypurals.
The parhypural (Fig. 11) is derived
from the hemal arch and spine of
preural centrum 1. The parhypural is
defined as the most posterior hemal
arch with an open canal through
which major blood vessels (dorsal
aorta) and nerves pass. The cartilag-
inous precursor of the parhypural
develops ventral to the posterior tip
of the notochord as a stack of chon-
drocytes two to three layers in thick-
ness (3.8 mm NL, Fig. 11A). Ossifica-
tion of the parhypural proceeds in a
manner similar to hypurals, with the
center of ossification found near the
proximal end (5.1 mm NL). The par-
hypurapophyses, also termed “hy-
purapophyses” are a bilateral pair
of bony posterolateral extensions
from the parhypural (Fig. 11E). They
serve as the origin for the anterolat-
eral bundle of the hypochordal lon-
gitudinal muscle (Lundberg and
Baskin, 1969).
The caudal fin vertebrae, which
support the caudal fin rays, are
composed of preural 3, preural 2,
and the compound centrum of
preural 1 ⫹ ural 1, and ural 2 (collec-
tively known as the urostyle). The os-
sifications of preural 1 and ural 1 are
always fused (5.4 mm NL); they are
never visible as two independent os-
sification centers (Fig. 11C,D). Thus,
they are referred to as “the com-
pound centrum preural 1 ⫹ ural 1.”
The ossification of ural 2 is posterior to
the preural 1 ⫹ ural 1 ossification (Fig.
11D). Ural centrum 2 fuses to the
compound centrum of preural 1 ⫹
ural 1 at 7.3 mm.
Preural 2, or the penultimate ver-
tebra, may possess two neural
arches. This feature appears to be
highly variable. In specimens with
the doubled arch condition, they
develop from cartilaginous basidor-
sals. The anterior set of basidorsals is
first to develop (5.1 mm NL). While
the anterior neural arch is forming,
the posterior set of basidorsals devel-
ops (5.4 mm NL). Spines may de-
velop from the anterior and/or the
posterior arch (5.2 mm NL and 5.4
mm NL, respectively). Both arches
fuse midlaterally, from which large
neural spines develop in cartilage.
Preural centrum 2 is first visible as
bone encircling the flexing noto-
chord (5.5 mm, Fig. 11D). The hemal
arch develops from basiventrals (4.9
mm NL). A cartilaginous spine is
present shortly after the appear-
ance of the arch (5.0 mm NL). Ossi-
fication proceeds ventrally from the
base of the basiventrals (5.5 mm)
into the spine (5.6 mm).
The neural arch and spine of preu-
ral 3, or antepenultimate vertebra, is
also first visible as a small stack of
chondrocytes, and it appears
slightly later than those of preural 2
(5.2 mm NL, Fig. 11B). The neural
spines of preural 2 and preural 3 de-
velop as cartilaginous extensions of

the neural arches, and they begin
developing at 4.1mm NL and 5.0
mm NL, respectively (Fig. 11B). Preu-
ral centrum 3 is first visible as bone
encircling the flexing notochord (5.5
mm). The hemal arch (5.0 mm NL)
and spine (5.1 mm NL) develop in
the same manner as the hemal arch
of preural 2.
The urostyle (Fig. 11E) is the termi-
nal posterior vertebra, also known as
the ultimate vertebra. In D. rerio, as
in most otophysans, the urostyle is
compound, consisting of a fusion
between the first preural centrum
and two ural centra, as well as the
first pair of uroneurals (Fink and Fink,
1981, 1996). Hypural 2 is fused to the
urostyle (Fig. 11E). The urostyle devel-
ops similar to all other vertebrae,
with ossifications first seen at 5.4 mm
NL. As the notochord flexes, the sep-
arate ossifications extend to encom-
pass the entire posterior end of the
notochord to form the urostyle. The
neural arch of the urostyle is first vis-
ible as a small ossification on the dor-
sal surface of the notochord (5.7
mm).
Uroneurals are modified neural
arches of the ural vertebrae that
are located lateral and slightly
dorsal to the urostyle (Fig. 11D,E).
The single uroneural in D. rerio
forms as a bilateral pair of bone
slivers along the lateral surface of
the urostyle, extending from the
neural arch of preural 1 to the tip of
the notochord (5.8 mm). In the
adult, the uroneural is fused near
the proximal end of the urostyle. An
unmodified neural arch is first visi-
ble as membranous bone dorsal to
PU1 (Fig. 11D), directly above the
anterior end of the urostyle (Fig.
11E).
Epurals are homologs of the neu-
ral spines of ural centra that develop
slightly above the urostyle (Fig.
11B,D,E). A single epural develops in
D. rerio. It is first visible as a slender
cartilaginous rod parallel to the flex-
ing notochord (5.4 mm NL, Fig.
11B,E). As development proceeds,
the epural extends distally (posteri-
orly). Ossification of the epural be-
gins near the anterior tip of the
epural and proceeds posteriorly (6.3
mm, Fig. 11E).
Caudal fin rays.
Development of the caudal fin rays
begins as thickened membrane bone
extending from the distal tip of the
hypurals (4.8 mm NL; Fig. 11C–E). Adult
caudal fin rays are hypertrophied.
There are 19 principal caudal rays,
with 10 in the dorsal lobe and nine in
the ventral lobe. The dorsal lobe rays
articulate with hypurals 3–5. Ventral
principal rays articulate with the he-
mal spine of preural 2, as well as the
parhypural and hypurals 1–2. Six dor-
sal procurrent rays and five ventral
procurrent rays develop in D. rerio (6.2
mm). No hypertrophy was found in
the procurrent rays, but all varied in
length, with more anterior being
shorter. The average number of dorsal
procurrent rays was greater than the
ventral procurrent rays, with six dorsal
and five ventral. This is a primitive con-
dition for leuciscins. The procurrent
caudal rays in D. rerio are also not
hypertrophied, consistent with the
primitive condition in leuciscins (Cav-
ender and Coburn, 1992).
DISCUSSION
Phylogenetic Comparisons
Most aspects of the morphology of
the axial skeleton of D. rerio reflect the
basal (primitive) conditions for various
hierarchical phylogenetic levels: the
slender-bodied clade within Danio,
Danio, Leuciscinae, Cyprinidae, and
Otophysi (Coburn and Cavender,
1992; Sanger and McCune, 2002). For
example, D. rerio retains the shared
derived features for the slender-bod-
ied clade of Danio (Sanger and Mc-
Cune, 2002) such as a broad fourth
neural spine, a reduced first lateral
process, and a reduced supraneural
3. It also retains the shared derived
features for Danio such as a reduced
ascending process of the interca-
larium (Sanger and McCune, 2002),
and D. rerio retains the primitive rib
condition (thin and straight), among
other characters, for cyprinids
(Coburn and Cavender, 1992).
D. rerio shares nine of 13 derived
characters associated with the axial
skeleton of basal leuciscins (Coburn
and Cavender, 1992). It differs in
that the fourth rib is short and stout,
the fifth parapophysis is cuplike, and
the associated rib head is rounded
ZEBRAFISH SKELETON 349
and reduced, the third uroneural is
absent, and the double neural arch
of preural 2 is fused, but the neural
spines remain unfused. Relative to
the basal condition for otophysans,
the Weberian apparatus of D. rerio
has been modified (Chardon and
Vandewalle, 1997).1 Modifications
include: the concha scaphia do not
fit in a notch in either the basi- or
exoccipitals; the os suspensorium is
not a simple anterior limb; the supra-
neurals are never continuous with
neural arch 4 or the exoccipitals;
and the cartilaginous connection
between the claustrum and supran-
eural 2 is lost during development.
Regionalization and the Axial
Skeleton
The results of recent molecular ge-
netic and embryologic studies of the
developing zebrafish skeleton im-
pact the classic interpretations of
the homologies within the fish axial
skeleton. The genes involved in re-
gionalizing the tetrapod vertebral
column appear to be involved in re-
gionalizing at least part of the verte-
bral column in the zebrafish (Morin-
Kensicki et al., 2002). The vertebral
column of fishes is traditionally sepa-
rated into two general regions: the
precaudal and the caudal verte-
brae. The anterior expression border
of Hoxd12a may correspond to the
posterior end of the trunk and the
transition from rib-bearing to hemal
arch-bearing (caudal) vertebrae
(Morin-Kensicki et al., 2002; Fig. 12).
We further note that this boundary
may also correspond to the anus
and the anterior limit of the anal fin.
Molecular genetic studies of Hox
genes in fishes and other vertebrates
(Burke et al., 1995; Prince et al., 1998;
1
The basal condition involves (1) concha
scaphii fits into a notch in the basi- and exoc-
cipital; (2) all ossicles and claustrum present
and distinct; (3) scaphium and intercalarium
possess ascending and articular processes; (4)
tripus with large, crescent-shaped transforma-
tor; (5) fourth vertebra with recognizable par-
apophyses and ribs, the os suspensorium a sim-
ple anterior limb, free from anterior vertebrae;
(6) large supraneurals 2 and 3 articulating on,
or continuous through cartilage with the basi-
dorsals of the third and fourth vertebrae and
the exoccipitals; (7) a claustrum just above the
concha scaphii, continuous through cartilage
with supraneural 2.
350 BIRD AND MABEE

Morin-Kensicki et al., 2002) have re-
vealed the correlation of the rib-
bearing vertebrae (the “thoracic”
vertebrae in amniotes) with the an-
terior expression boundary of Hoxc6
(Fig. 12). In the zebrafish, this bound-
ary lies between vertebra 2 and ver-
tebra 3 (Morin-Kensicki et al., 2002;
Fig. 12). This finding is of significance
in interpretation of the homologies
of the Weberian apparatus. Specifi-
cally, whether the ribs of v3 and v4
contribute to the tripus and os sus-
pensorium, respectively, has been
debated (see below). The expres-

Fig. 12. Anterior expression boundaries of Hox genes in relation to myomeres and
vertebrae in zebrafish. Adapted from Morin-Kensicki et al. (2002, Fig. 7).

Fig. 13. Sequence of development and ossification within the Weberian apparatus.
Diamonds indicate earliest appearance of skeletal elements. Bar indicates presence in all
specimens. Blue indicates cartilage; red indicates bone. In endochondral elements, pur-
ple indicates earliest sign of ossification.

Fig. 11. Development of the caudal fin, lateral views. A: Early formation of parhypural
(phy) and hypural 1 (hy1) ventral to the notochord (no; 4.0 mm NL). B: Entire caudal fin
endoskeleton present, including all hypurals (hy1– hy5), hemal spines (hspu2 and hspu3)
and neural spines (nspu2 and nspu3) of the preural vertebrae, the parhypural (phy) and
the epural (ep; 5.6 mm). no, notochord. C: Early ossification of preural 1 ⫹ ural 1 vertebra
(pu1⫹u1), as well as hypural 1 (hy1), the parhypural (phy), and hypural 3 (hy3) on the
ventral surface of the flexing notochord (no; 5.4 mm NL). fr, fin ray. D: Ossification within the
caudal fin, including ural 2 (u2), the compound centrum composed of preural 1 (pu1) and
ural 1 (u1; 6.0 mm). Also visible and labeled are the centrum of preural 2 (pu2), epural
(ep), parhypural (phy), hypural 1 (hy1), hypural 3 (hy3), uroneural (uro) and neural arch
of the urostyle (pound sign). fr, fin ray. E: Adult caudal fin (35.0 mm). Urostyle (ust),
uroneural (uro), parhypural (phy), hypural 1 (hy1), hypural 2 (hy2), hypural 3 (hy3), the
epural (ep), and the centra of preural 2 (pu2) and preural 3 (pu3) are labeled. The
parhypurapophysis is marked by an asterisk, and the neural arch of the urostyle by a
Figure 11. pound sign. Scale bars ⫽ 0.05 mm in A, 0.1 mm in B–E.

sion of Hoxc6a and Hoxc6b in the
myomeres from which vertebrae 3
and 4 (as well as v5–13) are derived,
lends support to a rib homology for
these elements.
The precaudal vertebrae immedi-
ately posterior to the skull in fishes
are never termed “cervical” verte-
brae in the ichthyological or com-
parative vertebrate literature; this
term is reserved for those vertebrae
in amniotes (Hildebrand, 1995).
Morin-Kensicki et al. (2002), how-
ever, termed the first two vertebrae
in zebrafish cervical, because they
form anterior to Hoxc6 expression
and they lack ribs. The discovery of
genes unique to a “cervical” region
across vertebrate taxa might be in-
dicative of a deeper homology
among the most anterior vertebrae
than is evident from comparative
anatomy.
Hoxa9, -b9, and -c9 are expressed
at the end of the thoracic series in
mouse and chick (Burke et al., 1995),
and in zebrafish, the anterior expres-
sion boundary of the paralogues
(Hoxa9, -b9) marks the transition be-
tween Weberian and precaudal
vertebrae (Fig. 12). The thoracic–
lumbar boundary in amniotes is a
transition from rib-bearing to non–
rib-bearing vertebrae, but in ze-
brafish, the boundary is between
highly modified vertebrae (which
bear highly modified ribs) and typi-
cal precaudal (precaudal) fish ver-
tebrae (which also bear ribs). Thus,
this distinction may represent a
novel use for the Hox9 genes in oto-
physan teleosts.
The specialized vertebrae associ-
ated with the caudal fin are unlikely,
according to Morin-Kensicki et al.
(2002), to be regionalized by Hox
genes. Their results, as others (Kanki
and Ho, 1997; Griffin et al., 1998; Ahn
and Gibson, 1999; Kimelman and
Griffin, 2000), lend support to the
concept that the tail region devel-
ops with mechanisms that are dis-
tinct from those used for head and
trunk (Morin-Kensicki et al., 2002).
Homologies of the
Components of the Weberian
Apparatus
Homologies of the Weberian ossicles
(scaphium, claustrum, intercalarium,
tripus, os suspensorium), as well as
Weberian support structures (roofing
cartilage and supraneurals) are is-
sues of intense debate in the ichthy-
ological literature (Fink et al., 1984;
Gayet, 1986). The developmental
complexity of Weberian elements,
which involves variability and modi-
fication of the centra, neural arches
and spines, parapophyses, ribs, and
supraneurals of the first four verte-
brae, has made interpretation of
their homologies quite difficult. In
addition, individual bones may have
a compound origin and may de-
velop differently among species.
In the following section, we review
differences in Weberian ossicle de-
velopment among species and de-
scribe the bases for previous homol-
ogy assessments. Although it is not
possible to fully assess homology
given the narrow focus on zebrafish
development herein, we relate our
observations to previous assess-
ments of homology. Only through a
broad comparative phylogenetic
analysis of development, and, in our
view, with new molecular genetic
data, can the homologies of these
elements be better understood.
The scaphium may form from one or
two ossifications, depending on the
species. In D. rerio, the scaphium is de-
rived from basidorsal 1 and the concha
scaphium is formed from a separate
mesenchymal ossification. This “dual or-
igin” has been observed in many other
Cypriniformes (Matveiev, 1929; Watson,
1939; Kulshrestha, 1977; Bogutskaya,
1991; Coburn and Futey, 1996). On the
other hand, the concha may form as
an anterior ossification from basidorsal 1
(Radermaker et al., 1989; Vandewalle
et al., 1990; Fukushima et al., 1992; Ichiy-
anagi et al., 1993, 1996). Despite the
dual origin for the scaphium in some
species, it is generally accepted as the
homologue to the first neural arch (basi-
dorsal 1).
The homology of the claustrum
(Fig. 5F) has long been debated.
Berg (1940) and Chranilov (1927) de-
scribed the claustrum as a short-
ened neural spine of vertebra 1. The
facts, however, that, unlike the neu-
ral spines, the claustrum is paired
and forms in cartilage argue against
a homology with neural spine 1. Oth-
ers have described the claustrum as
homologous to dissociated ele-
ZEBRAFISH SKELETON 351
ments of the first neural arch (Sar-
bathi, 1932; Fink and Fink, 1981;
Sanger and McCune, 2002), also
termed “supradorsals” (Rosen and
Greenwood, 1970).
Based on developmental data,
Coburn and Futey (1996) proposed
that the claustrum is homologous to
supraneural 1, as did Gayet (1982,
1985) from paleontologic data. In six
species of cyprinids and catosto-
mids, Coburn and Futey (1996) ob-
served separate bilaterally paired
cartilage condensations for supra-
neural 2, which later coalesce; they
thus proposed that the bilaterally
paired claustral cartilages may be
homologues of supraneural 1. In
other cyprinids, however (Coburn
and Futey, 1996), and in D. rerio, su-
praneural 2 develops as a single car-
tilage condensation dorsal to verte-
bra 2 (Fig. 5B,C). Danio rerio
develops its Weberian supraneurals
(supraneurals 2 and 3) at a smaller
size (4.8 –5.1 mm NL) than other oto-
physans (7–12 mm) (Coburn and
Futey, 1996). This change in devel-
opmental timing, possibly unique to
the slender-bodied Danio clade
(Sanger and McCune, 2002), might
explain the difficulty in locating the
supraneural condensations before
roofing cartilage development.
Grande and De Pinna (in press)
proposed that the claustrum is the
homologue of the accessory neural
arch of clupeocephalans (clupeo-
morphs and salmoniforms), a hy-
pothesis supported by the observa-
tion of an accessory neural arch in
larval Chanos, a gonorhynchiform
(Fig. 1; Coburn and Chai, 2003).
Morin-Kensicki et al. (2002) found
that the first two somites (and
maybe part of a third) do not con-
tribute to the vertebral column in ze-
brafish but may contribute to the
head skeleton. It is possible that
these somites may contribute the
sclerotome that differentiates as the
claustrum and perhaps other struc-
tures such as the concha scaphium.
Determining the somite(s) of origin
for these structures can be ad-
dressed by future lineage-labeling
studies on zebrafish. The homo-
logues of these structures might then
be determined by lineage-labeling
of closely related fishes.
In D. rerio, we observed an initial
352 BIRD AND MABEE
small band of cartilage from which
significant membranous ossification
proceeded anteriorly to form the
body of the claustrum. In many cyp-
riniforms, the claustrum has been re-
ported as entirely membranous in or-
igin (Coburn and Futey, 1996),
although Coburn and Futey (1996)
also describe a cartilaginous claus-
trum in many other species. It thus
appears that the claustrum in closely
related species may have different
developmental origins. Future lin-
eage-labeling studies on different
species of fishes may clarify this issue.
The intercalarium (Fig. 5F), like the
scaphium, may be singular or com-
pound in origin among otophysans.
Specifically, the manubrium may
develop from a separate ossification
in the interossicular ligament which
later fuses to basidorsal 2 (Watson,
1939; Kulshrestha, 1977) or as a
membranous extension from the
basidorsal 2 (Vandewalle et al.,
1990; Bogutskaya, 1991; Fukushima
et al., 1992; Ichiyanagi et al., 1993,
1996). In D. rerio, the manubrium de-
velops as a simple anterolateral
membrane bone extension from
basidorsal 2; no interossicular ossifi-
cations were found in either whole-
mount or sectioned specimens. Like
the claustrum, the intercalarium
may have different developmental
origins in different species. Despite
this, intercalarium is generally ac-
cepted as the homologue of neural
arch 2.
The tripus (Fig. 5D) has been de-
scribed as homologous to the par-
apophysis of vertebra 3 (Chranilov,
1927; Berg, 1940; Vandewalle et al.,
1990; Fukushima et al., 1992; Ichi-
yanagi et al., 1993, 1996; Chardon
and Vandewalle, 1997). Others de-
scribe the tripus as a modification of
both the parapophysis and rib of
vertebra 3 (Rosen and Greenwood,
1970; Patterson, 1984; Coburn and
Futey, 1996). In D. rerio, the tripus de-
velops as a thickened, posteriorly
projecting ridge of bone from the
parapophysis (transformator pro-
cess). The remaining body of the tri-
pus is added by means of continued
anterior membrane bone extension
from the parapophysis. The transfor-
mator process is similar in shape to a
rib, but the other ribs in Danio de-
velop as separate ossifications that
grow toward and articulate with the
parapophyses; ribs never develop
out of parapophyses. On the other
hand, developmentally, the out-
growth (transformator) from the par-
apophysis more closely resembles a
lateral process than a rib. The ex-
pression of Hoxc6a and Hoxc6b in
the myomere from which vertebra 3
is derived, supports to a rib deriva-
tion of this element (see above).
The os suspensorium demonstrates
considerable morphologic and de-
velopmental variation among oto-
physans. That the homology of the
os suspensorium (Fig. 5F) is not re-
solved, thus, is not surprising. It has
been proposed as a homologue of
the rib of the fourth vertebra (Chra-
nilov, 1927; Berg, 1940; Soni et al.,
1978), the parapophysis (basiven-
tral) of the fourth vertebra (Watson,
1939; Vandewalle et al., 1990; Fuku-
shima et al., 1992; Ichiyanagi et al.,
1993, 1996), or a modification of
both the rib and parapophysis of the
fourth vertebra (Evans, 1925; Rosen
and Greenwood, 1970). In D. rerio,
the os suspensorium develops as a
thin, rod-shaped, anteroventrally di-
rected, membrane bone extension
from the parapophysis. The os sus-
pensorium is similar in shape to a rib,
but the other ribs in Danio develop
as separate ossifications that grow
toward and articulate with the par-
apophyses. Moreover, the expres-
sion of Hoxc6a and Hoxc6b in the
myomere from which vertebra 4 is
derived lends support to a rib contri-
bution to this element (see above).
However, developing as an out-
growth from the parapophysis, the
os suspensorium is more like a lateral
process than a rib. Thus, it is unclear
to what extent the fourth rib vs. the
parapophysis is involved in the de-
velopment of the os suspensorium.
The structure termed the “fourth
rib” is also continuous with both the
os suspensorium and the parap-
ophysis. It develops as posteriorly di-
rected membranous extension from
parapophysis 4 (Fig. 5B). None of the
other ribs are continuous with their
respective parapophyses; they de-
velop as discrete independent ele-
ments that grow to articulate with
parapophyses. Also, ribs typically
develop in an anterior to posterior
manner, but in D. rerio, rib 4 devel-
ops after rib 5. It is plausible that the
fourth rib and the os suspensorium
are modifications of the rib ⫹ parap-
ophysis of the fourth vertebra.
Variation
Although size (body length) is a bet-
ter predictor than age (days) of skel-
etal development in zebrafish and
other fishes, the size at which a spe-
cific skeletal element develops is
somewhat variable, ranging from 0
to 0.8 mm (Fig. 3), with an average
of 0.26. Highly variable elements
were not concentrated in a single
region; rather they were found
throughout the skeleton. In the cau-
dal fin vertebrae, for example, hy-
purals 4 and 5 vary 0.6 – 0.8 mm, but
hypurals 1 and 2 do not vary at all
(Fig. 3). In the Weberian apparatus,
supraneurals 2 and 3 vary 0.4 – 0.7
mm, but the intercalarium and
scaphium do not vary. Moreover,
skeletal elements that form late
(e.g., lateral process 1 and supra-
neural 7) are as likely as elements
that form early (e.g., os suspenso-
rium and tripus) to demonstrate low
levels of variation. We noted a lack
of variation in the timing of the re-
gions of earliest development along
the anterior–posterior body axis (We-
berian apparatus and caudal fin).
Although this finding may be due to
their functional importance, it re-
mains to be tested.
A relative sequence of appear-
ance of bones in the axial skeleton
may be derived from the smallest
specimen in which a skeletal ele-
ment is present (Fig. 3, green dia-
monds) or the size at which an ele-
ment is always present (Fig. 3, bars).
There is little variation in relative se-
quence within specific regions. For
example, within the caudal fin, the
hypural 1 and the parhypural always
develop before other bones within
the region. If the development of
other elements is delayed, they are
delayed together such that the rel-
ative sequence of development
within the caudal fin does not
change, e.g., hypural 2 is always
present before hypurals 3–5. The se-
quence of appearance of the ele-
ments in the Weberian apparatus is
similarly fixed (Fig. 13). Basidorsals 3
and 4 develop before basiventrals 3

and 4 (the tripus and os suspensorium).
This sequence is likely conserved
within cypriniforms, as noted by
Coburn and Futey (1996).
Patterns of Axial Skeleton
Development
An overall pattern of development
within the axial skeleton emerged
from our analysis of the relative tim-
ing of ossification events (Fig. 3). As
also noted by Du et al. (2001), devel-
opment within the axial skeleton be-
gins simultaneously within the Webe-
rian region (third and fourth centra)
and the caudal fin region (hypural 1
and parhypural; Figs. 2, 3). This find-
ing is notable in that the two regions
are at the anterior and posterior lim-
its of the axial skeleton. Two centers
of ossification within the vertebral
column are common among ostari-
ophyans (Emelianov, 1939). This may
be related to a possible difference in
molecular patterning mechanisms
between these two regions (Morin-
Kensicki et al., 2002).
Development proceeds posteri-
orly and anteriorly from the third and
fourth (Weberian) vertebrae, such
that caudal vertebrae (orange in
Fig. 2) are last to form. Development
of associated vertebral structures,
such as neural arches and spines,
mirrors this pattern. In contrast, the
vertebral column of amniotes devel-
ops from anterior to posterior (Arey,
1940; Rieppel, 1993). The supraneu-
rals develop from anterior to poste-
rior as in other fishes (Mabee, 1988).
Among the median fins, the cau-
dal fin develops first, followed by the
anal and finally the dorsal fin radials
and fin rays (Fig. 3). The caudal fin is
first to form in most fishes (Mabee et
al., 2002). Between the dorsal and
anal fins, the more posterior fin de-
velops before the more anterior fin.
Thus, in D. rerio, the anal fin, which is
slightly more posterior than the dor-
sal fin, develops first. The exoskeleton
and endoskeleton of both the dorsal
and anal fins develop bidirection-
ally, a copatterning similarity first
noted by Mabee et al. (2002). Bidi-
rectional development of these fins
appears to be the basal condition
for teleost fishes (Mabee et al.,
2002).
CONCLUSION
Our detailed data on the develop-
ment and anatomy of the zebrafish
axial skeleton and median fins pro-
vide a baseline against which ze-
brafish genetic and evo– devo com-
parisons may be made. As
expected, most of the features of
the axial skeleton of D. rerio repre-
sent the basal (primitive) conditions
for phylogenetic levels above the
species, and we identify the level at
which these characters arose. Ulti-
mately correlating the recency of
evolutionary origin of these skeletal
features with the type of genetic
network that underlies each, may
elucidate the nature of evo– devo
changes in genetics and morphol-
ogy.
Homologies of the Weberian os-
sicles, which are developmentally
complex, are difficult to draw with
certainty. New molecular genetic
data, such as that from Morin-Ken-
sicki et al. (2002), have provided a
new level of understanding for the
evolutionary origin of such struc-
tures. Our reviews of the evidence
for homology of the various ele-
ments will be a basis against which
the contribution of additional ge-
netic data may be assessed.
We describe skeletal develop-
ment using size as a measure of de-
velopmental stage. Size is more
highly correlated with stage of oste-
ological development than age. This
ichthyological convention will be
useful for zebrafish researchers as
they move toward using older (and
larger) specimens in their research.
The molecular developmental signif-
icance of the overall pattern of de-
velopment within the axial skeleton
(Fig. 3) is as of yet unknown. The de-
gree to which this sequence of ossi-
fication reflects phylogenetic history
can be understood through similar
comparative developmental studies
on related taxa, and the degree to
which it reflects functional con-
straints may be determined through
biomechanical analyses. The way in
which molecular developmental
genetics is tied to evolutionary and
functional constraints will represent
a new and high level of integration
in the biological sciences.
ZEBRAFISH SKELETON 353
EXPERIMENTAL PROCEDURES
Adult zebrafish were obtained from
a local pet supply company (Safari-
land, Sioux Falls, SD) and maintained
in 10-gallon aquaria at 28.5°C (⫾
1°C) with a 14-hr photoperiod. Al-
though wild-caught zebrafish would
be ideal for analysis, obtaining large
quantities of larvae from wild stocks
is difficult (A. McCune, personal
communication). Slight variability in
meristics may be found between
wild and reared zebrafish (Ferreri et
al., 2000), but overall skeletal devel-
opment is expected to be similar.
Aquaria were maintained following
general suggestions from the Ze-
brafish Book (Westerfield, 2000). In
preparation for spawning, tank bot-
toms were thoroughly siphoned, and
tank levels were restored with fresh
water. Spawning protocol followed
those outlined previously by Cub-
bage and Mabee (1996). Fry were
fed Paramecium multimicronuclea-
tum and newly hatched brine
shrimp. Fry were maintained at
28.5°C in a Fisher Scientific Low Tem-
perature Incubator (Model 307C) for
1 week after fertilization and trans-
ferred to aquaria.
Larvae from multiple spawns were
pooled to obtain a complete devel-
opmental series. The description of
axial skeleton development is based
on a developmental series of 1,579
individuals (30 used in histologic
analysis, remaining used in whole-
mount analysis) ranging from young
larvae at 3 days after fertilization to
adults (3 mm notochord length (NL)
to 35 mm standard length [SL]).
Specimens were fixed in 4% parafor-
maldehyde and transferred to 70%
ethanol for storage or staining.
Specimens were stained with Al-
cian blue for cartilage, enzymati-
cally cleared with trypsin, and differ-
entially stained with alizarin red for
bone following the method of
Dingerkus and Uhler (1977) with
modifications of Potthoff (1984) for
larval fishes. Other specimens were
stained only for bone, as exposure to
cartilage stain may inhibit alizarin
red uptake, which can make bones
appear clear.
For histologic analysis, specimens
were paraffin-embedded and sec-
tioned at 8 ␮m by using a Leica 2040
354 BIRD AND MABEE
autocut microtome. Large speci-
mens (⬎7 mm) were decalcified be-
fore embedding. Sections were
stained by using a Milligan’s
trichrome protocol (Presnell and
Schreibman, 1997) and analyzed by
using a Zeiss Axioplan 2 compound
microscope. Images were collected
as described below.
Specimens were initially scored for
the presence of bones as well as os-
sification within 1 week of staining.
Cartilaginous structures were re-
corded as present upon visualization
of Alcian blue staining of chondro-
cytes. The earliest sign of ossification,
based on uptake of alizarin red
staining of mineralized bone matrix,
was recorded as presence of that
bone. Although histologic sections
may, in some cases, show bones as
“present” before the clearing and
staining whole-mount procedure
shows their presence in smaller and
younger larvae (Clark and Smith,
1993), the relative sequence of ossi-
fication is expected to remain the
same. Thus, sections were only ob-
tained to address specific questions
of development within the axial skel-
eton, primarily in the Weberian ap-
paratus.
Specimens were examined by us-
ing a Wild M5 stereomicroscope at
magnifications of ⫻11–94, measured
using an ocular micrometer, and
their lengths recorded to the nearest
0.1 mm. Prenotochord flexion and
early flexion individuals were mea-
sured from the anterior end of the
upper jaw to posterior tip of noto-
chord. Notochord length is the stan-
dard measure of preflexion larval
fishes in ichthyology and fisheries sci-
ences (Ahlstrom and Moser, 1976;
Leis and Trnski, 1989). Flexion occurs
at approximately 5.5 mm, corre-
sponding to 9 –10 days after fertiliza-
tion (Cubbage and Mabee, 1996,
Appendix A for age vs. length
chart). Specimens with flexed noto-
chords (⬃5.6 mm and larger) were
measured from the anterior end of
the upper jaw to the posterior end of
the hypurals (standard length). Illus-
trations were drawn from images
collected by using a Dage MTI
3CCD videocamera. Images were
then processed by using a Power
Macintosh G3 computer equipped
with Scion 1.62c and Adobe Photo-
shop 5.0 software.
ACKNOWLEDGMENTS
We thank R. Britz, P. Crotwell, D.
Neufeld, and D. Swanson for their
comments on this manuscript and E.
Haberman for help with histology.
N.C.B. thanks SICB and USD for
Grants in Aid of Research, and
P.M.M. and N.C.B. thank USD EPS-
CoR and CoBRE (NIH P20 RR15567)
for additional funds.
GLOSSARY
Actinotrichia: Unsegmented der-
mal rays in the fin fold of larval fishes;
replaced by lepidotrichia.
Anal fin: Median unpaired fin on
the ventral surface of the body, pos-
terior to the anus.
Antepenultimate vertebra: Verte-
bra anterior to the penultimate ver-
tebra (preural 3 in Danio rerio).
Basidorsal: Bilaterally paired carti-
lage condensations located on the
dorsal half of a centrum. Compo-
nent of “arcualia” in basal acti-
nopterygians (Gadow and Abbott,
1895).
Basiventral: Bilaterally paired carti-
lage condensations located on the
ventral half of a centrum. Compo-
nent of “arcualia” in basal acti-
nopterygians (Gadow and Abbott,
1895).
Camera aerea Weberiana: Ante-
rior part of the swim bladder (Rader-
maker et al., 1989) that is in contact
with the os suspensorium and the tri-
pus. This term was created by (Chra-
nilov, 1927) for the anterior chamber
of the bipartite swim bladder.
Caudal fin: Tail fin; most posterior
median fin.
Caudal vertebrae: Posterior verte-
brae; the first caudal vertebra has a
completely fused hemal arch.
Centra: Central bodies of verte-
brae.
Claustrum: Commonly considered
the first Weberian ossicle.
Dorsal fin: Median fin(s) on the dor-
sal side of the body. Only a single
dorsal fin is present in zebrafish.
Endochondral ossification: Bone
formation through which preexisting
cartilage is replaced by bony matrix.
Endoskeletal/Endoskeleton: Bony
or cartilaginous internal frame of the
body.
Epicentrals: Intramuscular bones
that attach to centra (Patterson and
Johnson, 1995). First termed by
Owen (1866).
Epineurals: Intramuscular bones
that attach to neural spines (Patter-
son and Johnson, 1995). First termed
by Owen (1866).
Epipleurals: Intramuscular bones
that attach to hemal spines or ribs
(Patterson and Johnson, 1995). First
termed by Owen (1846).
Epural: Flattened median bone
posterior to the last neural spine, dor-
sal to the urostyle.
Exoskeletal/Exoskeleton: External
skeleton, including fin rays and
spines.
Fin ray: Lepidotrich. Bilaterally
paired, jointed, exoskeletal bony
rays supporting the external fins.
Hemal arch: Arch on the ventral
surface of a centrum. Characterizes
caudal vertebrae.
Hemal spine: Cartilage or mem-
branous median extensions of one
or both hemal arches. May also de-
velop autogenously and fuse to the
proximal tips of the developing he-
mal arches.
Hypurals: Laterally flattened me-
dian caudal endoskeletal supports
which articulate with the urostyle
and serve as attachments for the
caudal fin rays. Homologous to he-
mal spines of preural vertebra 1 and
the ural vertebrae.
Hypurapophysis: See Parhypurap-
ophysis.
Intercalarium: Third Weberian ossi-
cle. Modification of the neural
arches of the second vertebra. Con-
sists of three parts: the manubrium,
ascending process, and articulating
process.
Intermuscular bones: Long, ray-
free bones lateral to the vertebral
column. Include epineurals, epicen-
trals, and epipleurals (Owen, 1846,
1866; Patterson and Johnson, 1995).
Lateral process: Transverse pro-
cess. Modified parapophyses ex-
tending laterally from a centrum.
Typically fused to the centrum in
Danio rerio.
Manubrium: Anterior branch of
the intercalarium. Also termed “an-
terolateral process.”

Membranous ossification: Direct
ossification from mesenchymal pre-
cursors.
Neural arch: Bony arch on the dor-
sal surface of a centrum.
Neural spine: Cartilage or mem-
branous median extensions from
neural arches. May also develop au-
togenously and fuse to the develop-
ing arches.
Notochord length (NL): Measure-
ment from the anterior-most tip of
the jaw to the posterior-most tip of
the notochord.
Os suspensorium: Separate ossifi-
cation from the parapophysis of the
fourth vertebra; develops ventrally
to curve around the anterior head of
the swim bladder. Often termed a
fifth Weberian ossicle.
Ostariophysi: Monophyletic group,
including Gonorynchiformes, Cyprini-
formes, Characiformes, Siluriformes,
and Gymnotiformes (Fig. 1).
Otophysan: Fishes within the series
Otophysi, including Cypriniformes,
Characiformes, Siluriformes, and
Gymnotiformes (Fig. 1).
Parapophyses: Ventrally project-
ing transverse processes that serve
as a point of articulation for ribs.
Form from basiventrals.
Parhypural: Hemal arch and spine
of preural 1; most posterior arch
through which the dorsal aorta
passes.
Parhypurapophysis (hypurapophy-
sis): Lateral process off of the parhy-
pural that serves as attachment for
the hypochordal longitudinal muscle.
Penultimate vertebra: Vertebra
anterior to the urostyle (preural 2 in
Danio rerio).
Perichondral ossification: Ossifica-
tion type in which bone forms
around a cartilage model.
Postzygapophyses: Bony dorsal
(neural postzygapophyses) or ven-
tral (hemal postzygapophyses) ex-
tensions from the posterior end of a
centrum. They articulate with the
prezygapophyses of the immedi-
ately posterior centrum.
Precaudal vertebrae: Trunk or ab-
dominal vertebrae. Anterior verte-
bral region lacking hemal arches
and spines, bearing ribs and parap-
ophyses.
Preural vertebrae: Caudal verte-
brae anterior to the urostyle and
lack hypurals.
Prezygapophyses: Bony dorsal
(neural prezygapophyses) or ventral
(hemal prezygapophyses) exten-
sions from the anterior end of a neu-
ral or hemal arch. They articulate
with the postzygapophyses of the
preceding centrum.
Radial: Pterygiophore. Endoskel-
etal fin support of the dorsal or anal
fin, to which the fin rays attach. May
be unsegmented, bipartite (proxi-
mal and distal), or tripartite (proxi-
mal, middle, and distal).
Ribs: Pleural ribs, ventral ribs. Inter-
segmental bones that articulate
with parapophyses. They protect
and support internal organs.
Roofing cartilage: Cartilaginous
larval structure dorsal to the four an-
terior-most vertebrae, which forms
the connection between neural
arches and supraneurals in the We-
berian apparatus.
Scaphium: First Weberian ossicle.
Formed from a modification of the
neural arches of the first vertebra.
Consists of three parts (concha, as-
cending process, articulating pro-
cess).
Standard length (SL): Measure-
ment from the anterior-most tip of
the jaw to the posterior-most tip of
the hypurals.
Supraneural: Slender, median,
T-shaped or rod-like, bony or carti-
laginous elements that lie in the me-
dian skeletogenous septum be-
tween the cranium and the dorsal
fin.
Swim bladder: Gas bladder. Gas-
filled sac used for buoyancy.
Synchondrosis: Cartilaginous con-
nection between two bones.
Syndesmosis: Fibrous connective
tissue connection between two
bones.
Tripus: Fourth Weberian ossicle.
Modification of the parapophysis
and rib of the third vertebra. Consists
of three parts (ascending process,
articulating process, transformator
process).
Tunica externa: Fibrous outer layer
of the swim bladder.
Uroneurals: Paired endochondral
bones located above the urostyle;
homologs of neural arches of the
ural vertebrae.
Urostyle (terminal vertebra; ulti-
mate vertebra): Compound final
segment of the vertebral column. In
ZEBRAFISH SKELETON 355
zebrafish, it is composed of preural
vertebra 1 and ural centra 1 and 2.
Vertebra: Unit of the vertebral col-
umn; consists of a centrum, neural
arches and spine, and may include
hemal arches and spine, or parap-
ophyses and ribs.
Weberian apparatus: Series of ver-
tebral modified ossicles serving to
connect the swim bladder to the in-
ner ear.
Weberian vertebrae: Most ante-
rior vertebrae, specialized as the
Weberian apparatus (vertebrae
1– 4). Typically reduced in length
and size.
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