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 lecular work, there has been no various elements of the axial skele-
comprehensive study on the devel- ton. We present data on the devel-
Questions regarding the relationship
opment and morphology of the ze- opmental timing and origin of skele-
between genes and morphology
brafish axial skeleton. A detailed tal elements in the axial skeleton.
are a driving force behind research
study of skeletal development, how- When paired with other morpho-
in evolution and development (Car-
ever, is crucial for interpretation of logic studies and genetic studies of
roll et al., 2001). The zebrafish, Danio
skeletal mutations and gene expres- zebrafish development, a more
rerio, is a well-established system for
sion patterns and ultimately for un- comprehensive understanding of
genetic studies of development
derstanding the connections be- zebrafish morphogenesis will emerge.
(Nüsslein-Volhard and Dahm, 2002),
and because of its basal phyloge- tween genes and morphology.
netic position within vertebrates, it is The purpose of this study is to pro- BACKGROUND
a key species for evo– devo studies. vide a detailed developmental an- The zebrafish, Danio rerio, is a small
With thousands of identified mutants atomic reference and a review and freshwater cyprinid native to South-
(Haffter et al., 1996), and ease of assessment of homologies of the ze- eastern Asia (Barman, 1991). It is a
genetic manipulation and mu- brafish axial skeleton. The extensive member of the Rasborinae clade
tagenesis, the zebrafish has moved ichthyological literature on oto- (Meyer et al., 1993) of the subfamily
to the forefront as a vertebrate physan anatomy forms the basis for Leuciscinae (Cavender and Coburn,
model system. Yet for all of the mo- our interpretation of homologies of 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: [email protected] or Paula M. Mabee, Department of Biology, CL-185, University of South Dakota,
Vermillion, SD 57069. E-mail: [email protected]
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- pharyngeal cartilages (Kimmel et model, or perichondrally with ossifi-
opment, short generation time, em- al., 1998), and developmental (Du cation beginning in the perichon-
bryonic transparency, ease of mu- et al., 2001) and adult axial anatomy drium surrounding the cartilage
tagenesis, and ongoing genomic (Sanger and McCune, 2002). These model (Patterson, 1977). Lower te-
research has made it ideal for labo- descriptions are not comprehensive, leost fishes, including ostariophysans
ratory study (Nüsslein-Volhard and however, as various parts of the skel- (zebrafish), are reported to lack
Dahm, 2002). eton (e.g., intermuscular bones, dor- acellular bone (Parenti, 1986).
In recent years, a variety of signif- sal and anal fins, and supraneurals) In fishes, the axial skeleton includes
icant studies on molecular aspects are not included. A description of the vertebral column and associ-
of zebrafish skeletal development developmental morphology of the ated median (unpaired) fins. The
have been published. Among these zebrafish axial skeleton has been vertebral column is regionalized into
are studies discussing the role of lacking. precaudal (Fig. 2, green and red)
chordin in skeletal development The axial skeleton is derived from and caudal vertebrae (Fig. 2, or-
(Fisher and Halpern, 1999), dlg3 in somitic mesoderm (Gilbert, 2003). ange and purple). In ostariophysans,
vertebral ankylosis (König et al., The vertebral column develops from the most anterior precaudal verte-
1999), Gdf5 in median fins (Crotwell sclerotome, a mesenchymal cell brae are regionalized as the Webe-
et al., 2001), sox9 in cartilage differ- population derived from the ventral rian vertebrae. The Weberian verte-
entiation (Chiang et al., 2001), shh somite. Sclerotome cells that will brae in otophysans are so named for
and bmp2b in the caudal fin (Quint contribute to the vertebral column in the Weberian apparatus (Weber,
et al., 2002), and endothelin-1 and the zebrafish move to surround axial 1820). In the following description,
pbx4 in the larval head (Kimmel et midline structures and differentiate we follow the ichthyological con-
al., 2001). as cartilage and bone (Morin-Ken- vention for ostariophysans of using
Morphologic studies on skeleton of sicki et al., 2002). Bones may de- the term “precaudal vertebrae” to
D. rerio have had a scattered cov- velop from a cartilage precursor refer to those vertebrae posterior to
erage, including studies on the de- (cartilage bone) or directly from a the Weberian vertebrae and ante-
velopment of the skull (Cubbage mesenchymal condensation (mem- rior to the caudal vertebrae. Precau-
and Mabee, 1996; Schilling and Kim- brane bone or dermal bone). Carti- dal vertebrae are composed of
mel, 1997), paired fins (Sordino et al., lage bones may ossify in two ways, centra, neural arches and spines,
1995; Cubbage and Mabee, 1996; either endochondrally with ossifica- parapophyses, and ribs. The caudal
Grandel and Schulte-Merker, 1998), tion beginning within the cartilage 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- Weberian centra (vertebrae 1– 4) dorsals that extend dorsally and fuse
dal vertebrae are composed of remain unfused in D. rerio through- medially to form the neural arch (Fig.
centra, neural arches and neural out development, although in many 5A). In D. rerio, the neural arches of
spines, and hemal arches and he- fishes, e.g., silurids and some cyprini- the Weberian vertebrae are first to
mal spines of vertebra 15 through forms (Nelson, 1948; Alexander, develop, and they are visible as
vertebra 28. The most posterior three 1962; Roberts and Kottelat, 1984; small clusters of chondrocytes (4.3
caudal vertebrae are modified to Vandewalle et al., 1990), the Webe- mm NL), which extend dorsally by
support the caudal fin. The last pre- rian vertebrae, usually the second means of membrane bone. Neural
caudal and/or the first caudal verte- and third centra, may develop as a arches of the first and second verte-
bra is frequently categorized as a fused centrum. This fusion is hypoth- brae are modified into Weberian os-
“transitional” vertebra, exhibiting esized to be the basal condition for sicles, the scaphium and claustrum,
elongated unfused hemal arches or Otophysi, with failure of fusion (as in respectively. Neural arches of the
parapophyses, drastically shortened D. rerio), a secondary condition preural vertebrae, including the
ribs, and absence of a hemal spine. (Roberts and Kottelat, 1984). double arch of preural 2, also de-
Associated with many vertebrae, The modal number of vertebrae in velop from bilaterally paired carti-
particularly the most anterior and D. rerio, including the urostyle is 31: 4 lage basidorsals. On vertebrae 6 –28,
most posterior, are basidorsals and Weberian, 10 precaudal, 17 caudal the neural arches develop from bi-
basiventrals, paired cartilaginous (including the 3 caudal fin verte- laterally paired membranous bones.
structures that give rise to neural brae). Our meristic counts (n ⫽ 50) Neural arches 6 –7 are the first mem-
arches or hemal arches, respectively are in agreement with Ferreri et al. branous neural arches visible (4.9
(Gadow and Abbott, 1895). Basiven- (2000), who found an average of 31 mm NL). Although most remaining
trals also give rise to parapophyses. vertebrae in both wild and reared D. neural arches develop simulta-
The associated median fins include rerio. neously, the more anterior arches
the dorsal, anal, and caudal fins, are longer and they fuse before
their bony internal support elements more posterior arches. Develop-
(radials) and external supports (fin
Neural Arches and Spines ment proceeds similar to basidorsal
rays). 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.
RESULTS on their position along the vertebral Neural spines develop as single
The length of first appearance of a column (Figs. 2, 4C,D,E, 5A,F). Neural median dorsal extensions from the
skeletal element is given in all de- arches of vertebrae 1–5 form from fused neural arches (5.1 mm NL, Fig.
scriptions below, and it is repre- bilaterally paired cartilaginous basi- 4D,E). All neural spines are dermal in
sented in Figure 3 as a green dia-
mond. All measurements are given
millimeters standard length; noto-
chord length (NL) is given where ap- Fig. 4. Vertebral development, lateral views. A: Early development of anterior centra (5.1
propriate. For ease of interpretation, mm NL). Centra 1 (c1), 2 (c2), 3 (c3), and 4 (c4) are visible as rings of bone encircling the
a glossary is included. Those struc- 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
tures and terms included in the glos- (8.9 mm). Development of neural prezygapophyses (nprez) and neural and hemal
sary are used in headings or have postzygapophyses (npstz, hpstz) on centra 18 –20 (c18 –20) extending from a neural arch
been bolded at first use below. (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).
Centra 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,
In D. rerio, centra are formed se-
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.
quentially. Centra 3 and 4 form first Fig. 5. Weberian apparatus development, lateral views. A: Early development on the
(Fig. 4A), as bone encircling the no- third and fourth vertebrae as cartilage basidorsals (bd) and basiventrals (bv; 4.7 mm NL).
tochord (3.5 mm NL). Additional B: Later development, with supraneurals (sn), neural arches (na), tripus (tr), os suspenso-
centra are added bidirectionally, 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
with the anterior two centra formed condensation (asterisk) is visible dorsal to the third neural arch (5.9 mm). sn, supraneurals.
by 4.1 mm NL. A second region of D: All structures of the Weberian apparatus present, including the claustrum (cl), scaphium
development appears at 5.4 mm NL (sc), intercalarium (in), tripus (tr), neural arch 3 (na3), neural arch 4 (na4), neural spine 4
with the ossification of the com- (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,
pound centrum of preural 1 ⫹ ural 1.
roofing cartilage; sb, swim bladder. E: Ossification of supraneural 2 (sn2) and supraneural
Ossification proceeds bidirectionally 3 (sn3) within the roofing cartilage (rc; 7.8 mm). The intercalarium is out of the plane of
within the caudal fin: anteriorly to focus. Neural arch 3 (na3), neural arch 4 (na4), neural spine 4 (ns4), scaphium (sc) and
preural 2 and 3 and then posteriorly claustrum (cl) are also visible. F: Full diagram of the adult Weberian apparatus (vertebrae
to ural 2. By 5.5 mm NL, all centra are 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),
visible. Centra are concave dorso- neural spine 5 (ns5), supraneural 5 (sn5), supraneural 2 (sn2), supraneural 3 (sn3), lateral
ventrally, as well as at both anterior process 1 (lp1), lateral process 2 (lp2), rib 4 (rib4), rib 5 (rib5), and swim bladder (sb). Scale
and posterior ends (Fig. 4D,E). 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 Fig. 7. Intermuscular bones, lateral views
fused with the first basidorsal (bd1). Roofing (29.7 mm). A: Forked epineural, showing
cartilage (rc) can be seen dorsal to the neu- medial (med) and lateral (lat) extensions.
ral tube (nt). B: Third vertebra (c3). The third Neural arches (na) and neural spines (ns)
neural arch (na3) has become continuous can be seen in the background. B: Forked
with the roofing cartilage (rc), completely en- epipleural, showing medial (med) and lat-
circling the neural tube (nt). The tripus (tr) is eral (lat) extensions. Hemal arches (ha)
seen lateral to centrum 3 (c3) and neural and hemal spines (hs) can be seen in the
Fig. 4. arch 3 (na3). Scale bars ⫽ 0.05 mm. background. Scale bars ⫽ 0.5 mm.
342 BIRD AND MABEE

origin except for the neural spines of 4B,D). In D. rerio all parapophyses half and possess a thickened lat-
the preural vertebrae, which de- develop endochondrally from ba- eral ridge that develops from the
velop, at least in part, from carti- siventrals (Fig. 4B). Parapophyses initial thickened rod. At approxi-
lage. Neural spines form earlier on are first visible as clusters of chondro- mately half the overall rib length,
anterior vertebrae. cytes on the lateral surface of the the morphology reverts to the typ-
Neural arches in the precaudal re- anterior ends of the precaudal ver- ical rod-like shape as they curve
gion are broad, with well-developed tebrae (5.5 mm NL). Parapophyses around the viscera and swim blad-
prezygapophyses (Fig. 4D). Prezyg- of the fifth and sixth vertebrae are der. The last (and second-to-last in
apophyses (neural prezygapophy- much larger compared with those of some instances) rib either loses the
ses) form as paired, anteromedially more posterior precaudal verte- “flared” appearance of its proxi-
directed processes from the neural brae. Ossification is first visible at the mal half, or it is highly reduced in
arches, and they are ligamentously center of the parapophysis (6.0 length.
attached to the postzygapophyses mm). The fifth parapophysis is always
of the preceding vertebra (Fig. 4C). the first to develop, with remaining
The prezygapophyses are larger in parapophyses added posteriorly. In Hemal Arches and Spines
precaudal vertebrae than in caudal the adult, the fifth parapophysis is The hemal arches characterize the
vertebrae (Fig. 4D,E). The Weberian thinner, anterior to posterior, relative caudal vertebrae; they do not de-
associated vertebrae (vertebrae to the remaining parapophyses. A velop on precaudal vertebrae (Fig.
1– 4) lack prezygapophyses. Verte- small depression is visible in parap- 4C,E). The caudal vertebrae range
bra 5 has a highly reduced prezyg- ophysis 5, to which the posterior in number from 14 to 16, with an av-
apophysis, but vertebrae 6 and 7 head of the fifth rib articulates. In erage of 15 vertebrae (excluding
are enlarged and appear to be liga- contrast, the parapophysis of the the vertebrae associated with the
mentously connected to a posterior sixth vertebra is broad. It is slightly caudal fin). Hemal arches develop
flange extending from the neural concave ventrolaterally, forming a from ventrally projecting, bilaterally
spines of these vertebrae. groove within which the sixth rib ar- paired, membranous ossifications
Postzygapophyses (neural postzyg- ticulates. on anterior ends of caudal verte-
apophyses) form as paired dorsome- Ribs are intersegmental rod- brae. These ossifications grow ven-
dially directed processes (6.2 mm) on shaped bones serving to protect the trally and fuse medially to form the
the posterior ends of centra (Fig. viscera (Figs. 2, 4B,D). In most fishes, hemal arch. Hemal arches may be
4C,E). They are ligamentously at- the first rib develops in association fused medially at the smallest size at
tached to the prezygapophyses of with centrum 2. In D. rerio, centrum 2 which they are visible (5.4 mm NL).
the immediately posterior vertebra. lacks a rib, and the ribs on centra 3 Hemal spines develop as a result of
The postzygapophyses of precaudal and 4 are modified as part of the continued membranous ossification
vertebrae are larger than those of Weberian apparatus (see below). of the hemal arches in the ventral
caudal vertebrae (Fig. 4D,E). Signifi- Typically 10 ribs are present (range midline (5.5 mm).
cant variation in shape is found in the of 9 –11), with the rib-heads and par- The hemal arches and spines of
precaudal region. The first postzyg- apophyses of the most anterior (V5) preural vertebrae, however, preform
apophysis is present on vertebra 5, and most posterior (V13–V15) verte- in cartilage (5.2 mm NL). The hemal
and they are found posteriorly brae showing distinct morphologies. arches are fused and the hemal
through preural 3 (6.2 mm). The devel- The fifth rib head is reduced in size spine formed at formation; i.e., they
opment of postzygapophyses is bidi- relative to the sixth, but maintains do not form as a result of fusion from
rectional, with the first group visible on slightly flared anterior and posterior initially paired basiventrals. The he-
vertebrae 11–23. Ventral postzyg- tips. The heads of the posterior ribs mal spines elongate by means of
apophyses develop slightly later (6.3 (ribs 13 and 14) are reduced to a ball continued cartilage growth, and
mm), but develop in the same man- shape; they loosely articulate with they later (6.0 mm) ossify.
ner as neural postzygapophyses. their parapophyses. The parapophy- Hemal arches are broad, with
Neural spines are generally longer ses are rounded and extend distally small prezygapophyses (hemal
in the precaudal region when com- to articulate with the ribs. prezygapophyses). Prezygapophy-
pared with the more posterior verte- Ribs are first visible as slivers of ses form as anteriorly directed pro-
brae (Fig. 2), although their morphol- membranous bones distal to the cesses (8.6 mm) from the bases of
ogies are similar. Neural spines form developing parapophyses (5.5 mm hemal arches. They are ligamen-
immediately after fusion of the neu- NL). Ribs grow posteroventrally to tously attached to the postzyg-
ral arches. Timing of fusion is variable surround the viscera, as well as dor- apophyses of the preceding verte-
but proceeds in an anterior-to-pos- sally to articulate with the parap- bra.
terior direction. ophyses (Fig. 4B). The first non-We- Postzygapophyses (hemal postzyg-
berian rib that develops is always apophyses, Fig. 4C) form as ventrally
the rib of the fifth vertebra, with directed processes (6.3 mm) on the
Parapophyses and Ribs remaining ribs added posteriorly. In posterior ends of centra. They are liga-
Parapophyses are bony lateral ex- the adult, ribs 5–10 have broad and mentously attached to the prezyg-
tensions from centra that serve as flat heads (Fig. 4D). The ribs retain a apophyses of the immediately poste-
articulation points for ribs (Figs. 2, broad thin shape in their proximal rior vertebra (Fig. 4C,E). The first ventral
 ZEBRAFISH SKELETON 343

postzygapophysis is found on the first ear) of the fish by means of the ca- Claustrum.
caudal vertebra (vertebra 15), and vum sinus impar, allowing for greater
The claustrum is a bilaterally paired,
they are found posteriorly through sensitivity to vibration and acute
triangularly shaped bone embed-
preural 3 (6.2 mm). The addition of hearing (Chardon and Vandewalle,
ded in connective tissue dorsome-
postzygapophyses is bidirectional, 1997).
dial to the scaphium (Fig. 5D–F). The
with the first formed on vertebra 19.
claustrum appears as a thin band of
Ventral postzygapophyses develop Weberian ossicles. cartilage, consisting of only a few
slightly later (6.3 mm) than neural
These include, from anterior to pos- cells, immediately anterior to the an-
postzygapophyses.
terior, the scaphium, claustrum, in- terodorsal edges of the roofing car-
tercalarium, and the tripus. The os tilage (5.5 mm). It develops in close
Weberian Apparatus suspensorium, considered to be a association with the roofing carti-
fifth ossicle (Vandewalle et al., 1990), lage, and it is most easily distin-
The Weberian apparatus is a novel
also will be addressed in this section. guished in specimens around 7.0
adaptation of a large clade of teleost
The scaphium, intercalarium, and tri- mm. The claustrum extends antero-
fishes, the otophysans (Fink and Fink,
pus are connected by means of the ventrally through membrane ossifi-
1996), which provides a route for the
interossicular ligament. cation to encircle the neural tube
transmission of sound/vibration from
(Fig. 6A). The claustrum does not en-
the swim bladder to the inner ear
Scaphium. tirely preform in cartilage but instead
(Weber, 1820). Development and
forms from only a sliver of cartilage
anatomy of the inner ear (Haddon The scaphium is connected posteri- and primarily membrane bone. The
and Lewis, 1996; Bang et al., 2001; orly to the manubrium of the interca- base of the claustrum articulates
Bever and Fekete, 2002) and the spe- larium by means of the interossicular with the medial surface of the
cific role of the Weberian apparatus ligament (Fig. 5C–F). The concha of scaphium (Fig. 5F). The dorsal sur-
in sound transmission (Popper and the scaphium forms the posterolat- face of the claustrum is connected
Fay, 1999) have been reviewed previ- eral wall of the sinus impar perilym- to supraneural 2 by means of a syn-
ously. Otophysan teleosts (within the phaticus, the connection to the chondrosis. Ossification begins at 6.3
larger clade Ostariophysi) include membranous labyrinths of the inner mm.
members of Cypriniformes (e.g., ze- ear (Radermaker et al., 1989). The
brafish), Characiformes, Siluriformes, scaphium in D. rerio represents the Intercalarium.
and Gymnotiformes (Rosen and most fully developed otophysan
Greenwood, 1970; Berra, 2001) (Fig. The intercalarium is a rod-shaped
state: the scaphium possesses an ar-
1). This adaptation is considered a key bone that originates from basidorsal
ticular process that articulates with a
innovation facilitating the enormous 2 (Fig. 5C,D,F). It is connected ante-
depression in centrum 1, an ascend-
radiation of this group in freshwaters riorly to the scaphium and posteriorly
ing process that extends posterodor-
worldwide (Berra, 2001). Otophysi (Fig. to the anterior process of the tripus
sally, and a spoon-shaped concha
1) comprise approximately 75% of all by means of the interossicular liga-
located anterior to these processes.
freshwater fishes and 27% of all fishes ment. At most in otophysans, the in-
The basidorsal from which the
(⬃7,500 species; Helfman et al., 1997; tercalarium is triradiate, with an an-
scaphium develops is first visible as a
Berra, 2001). Rudiments of the Webe- terior process, or manubrium,
small cartilage condensation on the
rian apparatus are found in the sister projecting anterolaterally to lie be-
dorsal surface of centrum 1 (4.8 mm tween the scaphium and the tripus,
taxon of otophysans, the Gonoryn-
ichiformes and in the Clupeomorpha NL, Fig. 5C). The ascending process as well as ascending and articular
(Fig. 1; Rosen and Greenwood, 1970; develops as a cartilaginous dorsal processes (Fig. 5F).
Fink and Fink, 1981; Grande and extension from this basidorsal. The The intercalarium is first visible as a
Braun, 2002). Thus, the Weberian ap- articular process ossifies from the small basidorsal cartilage mass on
paratus is thought to have evolved original basidorsal cartilage. The the dorsal surface of centrum 2 (4.8
piece by piece, and only once all el- concha scaphium develops from a mm NL, Fig. 5C). The manubrium de-
ements were present did it function in separate anterior mesenchymal os- velops as a membranous ossification
sound transmission (Grande and sification that fuses with basidorsal 1 that extends laterally from the basi-
Braun, 2002). The Weberian appara- (5.2 mm NL). The scaphium begins dorsal (5.1 mm NL). The ascending
tus is modified but not lost in any oto- ossification at 5.3 mm NL. and articular processes begin ossifi-
physan. In the adult zebrafish, the cation at 5.3 mm NL. The ascending
The Weberian apparatus is tradi- scaphium is connected to the claus- process develops as a dorsally pro-
tionally subdivided into the Webe- trum by means of a syndesmosis. jecting cartilaginous extension from
rian ossicles, the scaphium, claus- The concha of the scaphium does the basidorsal (5.1 mm NL). The as-
trum, intercalarium, and tripus, and not make contact with the exoccipi- cending process is longer relative to
the remaining bones, which include tal strut or other parts of the skull. The the manubrium in larvae (5.1–11.0
the centra, neural arches and concha is medially concave and mm). By 13.0 mm, the ascending
spines, supraneurals. These bones laterally convex. The dorsal surface process appears highly reduced in
serve to connect the swim bladder of the concha is thickened and size. The articular process ossifies
to the membranous labyrinth (inner arch-shaped. from the cartilage condensation
344 BIRD AND MABEE

and articulates with a groove in cen- smooth, a shared, derived feature of anterior portion is dorsoventrally nar-
trum 2. slender-bodied Danio; it lacks a per- rowed to articulate with a notch in
pendicular ridge found in deep- the basioccipital. A short bony lat-
Tripus. bodied species of Danio (Sanger eral process, which is an extended
The tripus is the largest of the Webe- and McCune, 2002). parapophysis fused to the centrum,
rian ossicles (Fig. 5A,B,D,F). The tripus is first visible as a small lateral bud
is typically separated into three re- Os suspensorium. extending from the centrum (5.7
gions: the anterior process, which The os suspensorium is a slender mm, Fig. 5F). The lateral process ex-
connects to the intercalarium; the bone curving along the anterior sur- tends slightly, and it remains small in
articular process, which articulates face of the anterior sac of the swim the adult. The ventromedial surface
with centrum 3; and the transforma- bladder (Fig. 5B,F). It articulates with of the scaphium articulates with the
tor process, which articulates with the tripus at the tripus–swim bladder dorsal surface of centrum 1.
the os suspensorium and the swim connection.
bladder. The os suspensorium begins devel- Vertebra 2. The second vertebra is
Development of the tripus begins opment as a separate anteriorly di- composed of a compact centrum
as a large basiventral cartilage rected membrane bone outgrowth that is slightly longer than centrum 1,
forming on the ventrolateral surface from the basiventral cartilage of ver- an elongated lateral process, and
of centrum 3 (4.7 mm NL, Fig. 5A). tebra 4 (parapophysis 4; 5.4 mm NL). the intercalarium (Figs. 4A, 5C).
The anterior and transformator pro- By 5.6 mm, the os suspensorium has The centrum of vertebra 2 is first
cesses are formed by membranous extended anteriorly and has arched visible at 5.0 mm NL (Fig. 4A). A long
ossification, with the articular pro- ventrally over the swim bladder (Fig. and slender bony lateral process
cess ossifying within the initial carti- 5B). Midsagittally, the os suspensoria (5.1 mm NL, Fig. 5F) extends from this
lage condensation. The interossicu- become tightly juxtaposed. A thin centrum, later curving posteriorly at
lar ligaments do not contribute to line of fusion is evident in adults; the the distal tip. As with the lateral pro-
the developing tripus. bones are not easily separated. In cess of the first vertebra, it is fused to
The transformator process ossifies no specimen does the os suspenso- the centrum in the adult. The ventral
as a ventrally projecting rod-like os- rium develop from the fourth rib; surface of the articular process of
sification from basiventral 3 (5.1 mm rather, they both extend from the the intercalarium articulates with a
NL). As it grows, it becomes thick- cartilage of parapophysis 4. depression in the dorsal surface of
ened and it curves posteriorly such The upper body of the os suspen- the centrum.
that the tip lies medial to the os sus- sorium has a large groove; this may
pensorium and is in contact with the be an attachment site for tendons or
swim bladder (5.6 mm, Fig. 5B). Most Vertebra 3. The third vertebra is
muscles associated with the swim
of the transformator process ap- composed of a shortened centrum
bladder (Evans, 1925; Alexander,
pears to be derived from the rib of (4.6 mm NL, Figs. 4A, 5) that is slightly
1962). The shape and size of this
the third vertebra. larger than the centrum of vertebra
groove varies within Danio (Sanger
The anterior process develops as 2, an enlarged neural arch, and the
and McCune, 2002).
an anteriorly directed membranous tripus. Development of the third neu-
extension from the body of the tripus Other elements of the ral arch begins from large paired
(6.2 mm). In the adult, the tip of the basidorsal cartilage masses on the
Weberian apparatus.
anterior process is dorsal to lateral dorsal surface of the centrum (4.3
process 2 at the level of centrum 2 These include elements associated mm NL, Fig. 5A). The masses extend
and connects by means of the in- with the anterior four vertebrae, in- dorsally and medially (5.9 mm, Fig.
terossicular ligament to the manu- cluding neural arches and spines, su- 5B,C), and they become continuous
brium of the intercalarium. praneurals 2 and 3, the roofing car- with the roofing cartilage (Fig. 6B).
From a dorsal view, the tripus is tilage, transverse processes, and the Ossification begins at the center of
wing-shaped, extending from verte- centra. As in all ostariophysans (Fig. the neural arch (5.0 mm NL). The
bra 2 to vertebra 4 (Fig. 5F). Laterally, 1), the first four vertebral centra of D. basidorsals fuse midlaterally to form
the tripus is tilted, with a lowered rerio are foreshortened relative to the adult arch. A third neural spine
posterior hook-shaped tip (transfor- the more posterior vertebrae (Fink does not form. Parapophysis 3 de-
mator process) positioned ventral to and Fink, 1981). velops from a large basiventral car-
centrum 4. The posterior tip is slender Vertebra 1. The first vertebra has tilage on the ventrolateral surface of
and curves medially. The posterior been modified as part of the Webe- the centrum, and together with the
tip of the transformator process ar- rian apparatus. Vertebra 1 is com- third rib, it is modified as the tripus.
ticulates with the medial portion of posed of a compact centrum articu-
the os suspensorium, as well as with lating anteriorly with the basioccipital Vertebra 4. Vertebra 4 is com-
the tunica externa of the camera of the skull, a short lateral process, posed of a centrum (4.6 mm NL, Fig.
aerea Weberiana, or the anterior sur- and a modified neural arch known as 4A, 5), an enlarged neural arch and
face of the anterior sac of the swim the scaphium (Figs. 4A, 5F). neural spine, and a modified and
bladder (Radermaker et al., 1989). The centrum for vertebra 1 is first combined parapophysis/rib that
The surface of the tripus of D. rerio is visible at 5.0 mm NL (Fig. 4A). The contributes to the os suspensorium
 ZEBRAFISH SKELETON 345

and lateral process/rib. A large neu- Fig. 5E). Supraneural 2 is fully ossified neural arch 3 (Fig. 5B,C). This matrix is
ral arch develops on the dorsal sur- by 13.8 mm (Fig. 5F). Alcian blue–positive before chon-
face of centrum 4 in the same man- In contrast to other Danio (Sanger drocytes are visible. The roofing car-
ner as neural arch 3, and it is first and McCune, 2002), in D. rerio the tilage differentiates to surround su-
visible at 4.3 mm NL (Fig. 5A). A ventral margins of supraneural 2 are praneural 2, supraneural 3, and the
large, broad, blade-like neural spine not curved medially and the dorsal neural arch and spine of vertebra 3
develops as ossification proceeds margin is nearly horizontal and lacks (Figs. 5D,E, 6B). It closely approxi-
distally (5.1 mm NL, Fig. 5D–F). Carti- a medial ridge. mates the neural arch and spine of
lage from neural arch 4 is not con- vertebra 4. The roofing cartilage
Supraneural 3. forms a synchondrosis with the claus-
tinuous with the roofing cartilage.
Ossification of neural arch 4 begins Supraneural 3 is an irregularly trum. The roofing cartilage is contin-
at the dorsal surface of the paired shaped bone located within the uous with the supraneurals and neu-
arches (5.0 mm NL), and it proceeds midsagittal septum (Figs. 2, 5B,C,E,F). ral arch 3 only during larval
ventrally. Ossification of neural arch It develops as a single cartilage con- development. Our observation of
4 and neural spine 4 is complete by densation dorsal to the fourth neural the formation of the roofing carti-
6.6 mm (Figs. 5B,D,E). arch (5.1 mm NL, Fig. 5B,C), slightly lage independent from neural
The first rib in the anterior–posterior anterior to the fourth neural spine. arches 3 and 4 and supraneurals 2
series develops from this centrum Like supraneural 2, it becomes sur- and 3 is consistent with those of
(Fig. 5D,F) as a small membranous rounded by and continuous with the Bogutskaya (1991) and Coburn and
posterior projection from the carti- roofing cartilage (5.6 mm, Fig. 5D). A Futey (1996) from a wide variety of
laginous parapophysis (5.6 mm). The dorsally elevated midsagittal ridge cyprinforms.
rib is a separate outgrowth from the marks its location within the roofing In the adult, the roofing cartilage
parapophysis, and it is not con- cartilage (Fig. 5D,E). Ossification be- appears to de-differentiate to be-
nected to the os suspensorium (5.6 gins on the surface of this dorsal el- come non–matrix-positive connec-
mm). Rib 4 is oriented such that it evation (6.7 mm, Fig. 5E) and pro- tive tissue. A remnant of the roofing
projects anteriorly; all other ribs ceeds dorsally as a thin sheet of cartilage is retained as a connective
project posteriorly. The rib lies lateral membrane bone and ventrally over tissue mat connecting neural arches
to the tripus, and it becomes bifur- the surface of the cartilage. The dor- 3 and 4, the claustrum, and supran-
cated distally (Fig. 5F). It is fully de- sal surface of supraneural 3 projects eurals 2 and 3. Typically, there is a
veloped and ossified by 7.5 mm. anteriorly in the adult. It is fully ossi- gap between the roofing cartilage
fied by 14.5 mm (Fig. 5F). A fused and exoccipital of the skull, al-
Supraneural 2. anterior margin of supraneural 3 is a though in some specimens they ap-
shared derived feature of all Danio pear fused.
Supraneural 2 is an ovoid bone lo- (Sanger and McCune, 2002). In con-
cated within the midsagittal septum trast, the dorsal and posterior mar-
dorsal to centrum 2 (Figs. 2, 5B,C,E,F). gins of Weberian supraneural 3 are Intermuscular Bones
Based on topology and shape, fused in D. rerio. In the slender-bod- Intermuscular bones, which are
namely that this structure resembles ied clade of Danio, which includes unique to teleostean fishes (Patter-
the dorsal portion of a normal neural D. rerio, supraneural 3 is short relative son and Johnson, 1995; Gemballa
arch, others have identified supra- to its tall primitive condition (Sanger and Britz, 1998), are membranous os-
neural 2 as neural arch 2 (Ra- and McCune, 2002). sifications that form in the myosepta
maswami, 1955; Howes, 1978; May- In some otophysans, supraneural 4 and attach ligamentously to neural
den, 1989). is retained (primitively present), but arches, centra, hemal arches, or ribs
Supraneural 2 is first visible as a sin- in some cypriniforms such as Danio, (Fig. 7). Three sets of serially homolo-
gle cartilage condensation dorsal to supraneural 4 is lost. Although Gayet gous intermusculars may be present
vertebra 2 (4.8 mm NL, Fig. 5B,C). (1986) proposed that supraneural 3 in teleosts and are classified in refer-
Supraneural 2 develops immediately might be formed by means of a fu- ence to the site of attachment
before supraneural 3 (5.1 mm NL). sion of supraneural 3 and supraneu- (Patterson and Johnson, 1995; Gem-
The remaining supraneurals appear ral 4, we do not see a separate con- balla and Britz, 1998). From dorsal to
successively; development of all su- densation for a fourth supraneural in ventral, epineurals attach to neural
praneurals proceeds in an anterior D. rerio. This finding is concordant arches, epicentrals attach to cen-
to posterior direction, as in other te- with the observations of Fink and Fink tra, and epipleurals attach to hemal
leosts (Mabee, 1988). Supraneural 2 (1996) for otophysans. arches or ribs. Epicentrals are not
becomes surrounded by and be- present in D. rerio. Epineurals de-
comes continuous with the roofing Roofing cartilage. velop in association with vertebra 5
cartilage. A midsagittal projection The roofing cartilage is a larval struc- through preural 2 (6.0 mm). They de-
marks the location of supraneural 2 ture located dorsal to the neural velop and ossify predominantly from
within the cartilage mass. The early tube (5.6 mm) (Figs. 5D,E, 6). It is first posterior to anterior. Beginning at
ossification of supraneural 2 wraps visible as strands of mesenchyme approximately the precaudal– cau-
around the anterior tip of the roofing forming amongst and thereby con- dal transition, each epineural devel-
cartilage/supraneural mass (6.7 mm, necting supraneurals 2 and 3 and ops an anterior fork (Fig. 7A). The first
346 BIRD AND MABEE

signs of forking are difficult to visual- neural 5 enlarges and broadens, tive fin. Additional radials develop
ize, but at 10.6 mm, both epineurals while supraneurals 6 –9 develop dor- anteriorly and posteriorly (Fig. 9A,C).
and epipleurals appear to fork at soventrally as small slender rods of Correlation between direction of
approximately vertebral level 17–18. cartilage. Supraneural 5 is the first to development within the dorsal and
Epineurals associated with precau- ossify, with ossification beginning anal fins is similar to those found in
dal vertebrae are not forked. The later in more posterior supraneurals other fishes, and this finding is prob-
more anterior epineurals span ap- (supraneural 6 at 8.5 mm, supraneu- ably the ancestral condition for fin
proximately two precaudal centra, ral 7 and 8 at 9.2 mm, and supran- positioning and patterning (Mabee
and the more posterior epineurals eural 9 at 9.8 mm). Supraneurals ex- et al., 2002). Later in development,
span approximately three caudal hibit perichondral ossification, with radials segment proximodistally into
centra. first uptake of stain forming a ring two parts, a larger proximal radial
In D. rerio, the most anterior epi- around the cluster of chondrocytes and smaller distal radial (5.7 mm, Fig.
pleural is associated with the first (Fig. 8B). 10). The distal radials articulate di-
complete hemal arch (the first cau- In the adult, the posterior four su- rectly with the lepidotrichia. Devel-
dal vertebra). Epipleurals do not de- praneurals are arrowhead in shape, opment of dorsal fin radials typically
velop in association with ribs. Devel- with a wide dorsal surface that begins with those radials located
opment of the epipleurals proceeds tapers ventrally. Many have a proxi- between the neural spines of the
in concert with the epineurals. modistal groove in the lateral sur- 14th and 15th vertebrae, which cor-
Epipleurals develop as slender face running the length of the supra- respond to the 4th and 5th radials in
rods lateral to the vertebral column. neural (Fig. 8C). The shape of the first the adult (5.5 mm NL, Fig. 9A).
In forked epineurals and epipleurals, supraneural is variable, but it is al- Growth proceeds proximally toward
it is the medial extension that is liga- ways larger than supraneurals 6 –9 the vertebral column.
mentously attached to the verte- and generally exhibits an ovoid Ossification of the proximal radials
brae (Fig. 7A,B). shape. proceeds proximally and distally
from the middle of the radial (7.0
Median Fins mm, Fig. 9B). In all but the anterior
Supraneurals two radials, a second center of ossi-
Dorsal fin. fication is located at the distal tip of
Supraneurals are dorsal median
structures located anterior to the The dorsal fin is an unpaired median the proximal radial. Ossification gen-
dorsal fin pterygiophores and poste- fin located on the dorsal surface of erally follows the same order as
rior to the skull (Figs. 2, 8). They are the body (Figs. 2, 9A–C). The dorsal chondrogenesis, i.e., radials 4 and 5
slender skeletal rods that may be ho- fin consists of endoskeletal supports, are the first to begin ossification. Dis-
mologous to neural spines or radials or radials, and exoskeletal supports, tal radials ossify in the same order as
(Mabee, 1988). In D. rerio, supraneu- or fin rays. The radials, also termed proximal radials, but ossification is
rals range in number from 4 to 6, with “pterygiophores,” are rod-like inter- delayed relative to the proximal ra-
a mode of 5. These supraneurals are nal median skeletal supports of the dials (8.6 mm). Dorsal radials were
numbered 5–9 (Fig. 2). In specimens dorsal and anal fins. not found anterior to the neural
with six supraneurals, the “extra” is In D. rerio, initiation of develop- spine of vertebra 11 or posterior to
positioned posterior to supraneural ment of the dorsal fin radials is first the neural spine of vertebra 18. The
9. The sole exception was a speci- visible as a central group of carti- modal number of dorsal fin radials
men in which the additional supra- lage condensations at approxi- for the zebrafish is eight, with a range
neural was located dorsal to the mately the middle of the presump- of seven to nine (n ⫽ 50). It is at the
roofing cartilage. In the (only) spec-
imen examined with four supraneu-
rals, the ninth supraneural was ab-
sent. Fig. 8. Supraneurals, lateral views. A: Cartilage development of the anterior three supra-
Supraneurals develop from small neurals (sn5, sn6, and sn7; 6.6 mm). B: Early ossification of supraneural 9 (sn9; 12.2 mm).
clusters of chondrocytes dorsal to Neural spine 8 (ns8) is visible anterior to supraneural 9. C: Adult morphology of supraneu-
the anterior precaudal neural spines 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. 8A). Supraneurals develop from Fig. 9. A–C: Development of the dorsal fin, lateral views. A: Early formation of proximal
anterior to posterior: supraneural 5 is radials (pr; 5.6 mm). B: Ossification within the proximal radials (pr) and distal radials (dr; 8.9
always the first visible (6.2 mm) fol- mm). Fin rays (fr) are visible attaching to distal radials. C: Adult dorsal fin (35.0 mm).
lowed by supraneurals 6 –9 (6.4 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:
6.6 mm, 6.6 mm, and 7.0 mm, re-
Development of the anal fin, lateral view. D: Early formation of proximal radials (pr; 5.6
spectively). The supraneurals are ar- mm). E: Ossification within the proximal radials (pr) and distal radials (dr; 5.6 mm). Fin rays
ranged one per myomere, with the (fr) are visible attaching to distal radials. F: Adult anal fin (35.0 mm). Proximal radials (pr),
most anterior located between the distal radials (dr), and fin rays (fr) are visible ventral to the caudal vertebrae, which include
neural spines of the fourth and fifth centra (c), hemal arches (ha) and hemal spines (hs). Scale bars ⫽ 0.05 in A,D, 0.1 mm in
B,C,E,F.
vertebrae, and the most posterior Fig. 10. Segmentation of anal fin radials. A: Unsegmented anal fin radials (pr; 6.0 mm). B:
between the neural spines of the Segmentation of distal radial (dr) from the proximal radial (pr; 6.1 mm). C: Proximal (pr)
eighth and ninth vertebrae. Supra- 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 and spines that support the lepi- center of ossification found near the
of the 16th vertebra. These corre- dotrichia of the caudal fin. The hy- proximal end (5.1 mm NL). The par-
spond to the third and fourth anal purals form ventral to the posterior hypurapophyses, also termed “hy-
radials in the adult (5.1 mm NL, Fig. tip of the dorsally flexed notochord purapophyses” are a bilateral pair
9D). The radials develop as stacks of (Fig. 11A,B). of bony posterolateral extensions
chondrocytes near the ventral sur- Development is first visible as the from the parhypural (Fig. 11E). They
face of the body. Growth proceeds independent condensations of the serve as the origin for the anterolat-
proximally toward the vertebral col- parhypural and hypural 1 (3.8 mm eral bundle of the hypochordal lon-
umn. Anal radials are added ante- NL, Fig. 11A). The remaining hypurals gitudinal muscle (Lundberg and
rior and posterior to the original radi- (2–5) are added posteriorly, with hy- Baskin, 1969).
als (Fig. 9D). Ossification proceeds pural 2 developing at 4.0 mm NL, The caudal fin vertebrae, which
proximally and distally from the mid- hypural 3 at 4.4 mm NL, hypural 4 at support the caudal fin rays, are
dle of the radial (7.0 mm, Fig. 9E), 4.8 mm NL, and hypural 5 at 5.0 mm composed of preural 3, preural 2,
and it is seen in the same order as NL (Fig. 11B,C–E). Hypural 2 be- and the compound centrum of
chondrogenesis. Similar to dorsal fin comes fused (is nonautogenous) to preural 1 ⫹ ural 1, and ural 2 (collec-
radials, all but the anterior two radi- the compound centrum of preural tively known as the urostyle). The os-
als exhibit a second center of ossifi- 1 ⫹ ural 1. The proximal cartilages of sifications of preural 1 and ural 1 are
cation at the distal end of the prox- the parhypural and hypural 1, which always fused (5.4 mm NL); they are
imal radial (Fig. 9E). Shortly after the abut the compound centrum of never visible as two independent os-
onset of development, the radials preural 1 ⫹ ural 1, fuse proximally sification centers (Fig. 11C,D). Thus,
segment into proximal and distal (Fig. 11B). Ossification is first visible they are referred to as “the com-
portions (5.5 mm, Fig. 10). Distal radi- near the proximal end of the hy- pound centrum preural 1 ⫹ ural 1.”
als ossify in the same order as proxi- purals, and it proceeds bidirection- The ossification of ural 2 is posterior to
mal radials, but ossification is de- ally (Fig. 11C–E). The proximal and the preural 1 ⫹ ural 1 ossification (Fig.
layed relative to the proximal radials distal tips are last to ossify. Ossifica- 11D). Ural centrum 2 fuses to the
(8.6 mm). Anal radials were not tion proceeds first in hypurals 1–3 (5.0 compound centrum of preural 1 ⫹
found anterior to the hemal arch of mm NL), and by 5.1 mm NL hypural 4 ural 1 at 7.3 mm.
vertebra 14 (position of the anus) or has begun to ossify. Hypural 5 begins Preural 2, or the penultimate ver-
posterior to the neural spine of the ossification later (6.2 mm). The distal tebra, may possess two neural
21st vertebra. The modal number of tips of the hypurals remain as carti- arches. This feature appears to be
anal fin radials for the D. rerio is 13, lage late in development, with all highly variable. In specimens with
with a range of 12–14 (n ⫽ 50). The elements fully ossified by 24 mm (Fig. the doubled arch condition, they
average interdigitation pattern is 11E). develop from cartilaginous basidor-
represented in Figure 2. Danio rerio has three hypurals (hy- sals. The anterior set of basidorsals is
Anal fin ray development mirrors purals 3–5) in the upper lobe (Fig. first to develop (5.1 mm NL). While
that of the radials, i.e., the rays asso- 11E), a derived condition of the slen- the anterior neural arch is forming,
ciated with the third and fourth radi- der-bodied clade of Danio. Ances- the posterior set of basidorsals devel-
als are first to development with the trally in Danio, the lower lobe of the ops (5.4 mm NL). Spines may de-
remaining added bidirectionally (5.5 caudal fin is supported by the par- velop from the anterior and/or the
mm). As in the dorsal fin, rays usually hypural and hypurals 1 and 2, and posterior arch (5.2 mm NL and 5.4
number n ⫹ 2, where n ⫽ radial num- the upper lobe is supported by four mm NL, respectively). Both arches
ber, with two extra rays articulating hypurals (3– 6). As noted by Sanger fuse midlaterally, from which large
with the first radial (Fig. 2). and McCune (2002), the slender- neural spines develop in cartilage.
bodied clade of Danio has a de- Preural centrum 2 is first visible as
Caudal fin. rived condition of only three hy- bone encircling the flexing noto-
The caudal fin is the first median fin purals in the upper lobe, for a total of chord (5.5 mm, Fig. 11D). The hemal
to show both endoskeletal and ex- five hypurals. arch develops from basiventrals (4.9
oskeletal development (Figs. 2, 3, The parhypural (Fig. 11) is derived mm NL). A cartilaginous spine is
11). The endoskeleton of D. rerio is from the hemal arch and spine of present shortly after the appear-
composed of five hypurals, the par- preural centrum 1. The parhypural is ance of the arch (5.0 mm NL). Ossi-
hypural, and hemal and neural defined as the most posterior hemal fication proceeds ventrally from the
arches and spines of the second arch with an open canal through base of the basiventrals (5.5 mm)
and third preural vertebrae, one pair which major blood vessels (dorsal into the spine (5.6 mm).
of uroneurals, the neural arch of aorta) and nerves pass. The cartilag- The neural arch and spine of preu-
preural 1, one epural, and the uro- inous precursor of the parhypural ral 3, or antepenultimate vertebra, is
style. The urostyle is composed of the develops ventral to the posterior tip also first visible as a small stack of
compound centrum preural 1 ⫹ ural of the notochord as a stack of chon- chondrocytes, and it appears
1, which fuses in development to the drocytes two to three layers in thick- slightly later than those of preural 2
centrum of ural 2. ness (3.8 mm NL, Fig. 11A). Ossifica- (5.2 mm NL, Fig. 11B). The neural
The five hypurals are expanded tion of the parhypural proceeds in a spines of preural 2 and preural 3 de-
and laterally flattened hemal arches manner similar to hypurals, with the velop as cartilaginous extensions of
 ZEBRAFISH SKELETON 349

the neural arches, and they begin Caudal fin rays. and reduced, the third uroneural is
developing at 4.1mm NL and 5.0 absent, and the double neural arch
Development of the caudal fin rays
mm NL, respectively (Fig. 11B). Preu- of preural 2 is fused, but the neural
begins as thickened membrane bone
ral centrum 3 is first visible as bone spines remain unfused. Relative to
extending from the distal tip of the
encircling the flexing notochord (5.5 the basal condition for otophysans,
hypurals (4.8 mm NL; Fig. 11C–E). Adult
mm). The hemal arch (5.0 mm NL) the Weberian apparatus of D. rerio
caudal fin rays are hypertrophied.
and spine (5.1 mm NL) develop in has been modified (Chardon and
There are 19 principal caudal rays,
the same manner as the hemal arch Vandewalle, 1997).1 Modifications
with 10 in the dorsal lobe and nine in
of preural 2. include: the concha scaphia do not
the ventral lobe. The dorsal lobe rays
The urostyle (Fig. 11E) is the termi- fit in a notch in either the basi- or
articulate with hypurals 3–5. Ventral
nal posterior vertebra, also known as exoccipitals; the os suspensorium is
principal rays articulate with the he-
the ultimate vertebra. In D. rerio, as not a simple anterior limb; the supra-
mal spine of preural 2, as well as the
neurals are never continuous with
in most otophysans, the urostyle is parhypural and hypurals 1–2. Six dor-
neural arch 4 or the exoccipitals;
compound, consisting of a fusion sal procurrent rays and five ventral
and the cartilaginous connection
between the first preural centrum procurrent rays develop in D. rerio (6.2
between the claustrum and supran-
and two ural centra, as well as the mm). No hypertrophy was found in
eural 2 is lost during development.
first pair of uroneurals (Fink and Fink, the procurrent rays, but all varied in
1981, 1996). Hypural 2 is fused to the length, with more anterior being
urostyle (Fig. 11E). The urostyle devel- shorter. The average number of dorsal Regionalization and the Axial
ops similar to all other vertebrae, procurrent rays was greater than the Skeleton
with ossifications first seen at 5.4 mm ventral procurrent rays, with six dorsal The results of recent molecular ge-
NL. As the notochord flexes, the sep- and five ventral. This is a primitive con- netic and embryologic studies of the
arate ossifications extend to encom- dition for leuciscins. The procurrent developing zebrafish skeleton im-
pass the entire posterior end of the caudal rays in D. rerio are also not pact the classic interpretations of
notochord to form the urostyle. The hypertrophied, consistent with the the homologies within the fish axial
neural arch of the urostyle is first vis- primitive condition in leuciscins (Cav- skeleton. The genes involved in re-
ible as a small ossification on the dor- ender and Coburn, 1992). gionalizing the tetrapod vertebral
sal surface of the notochord (5.7 column appear to be involved in re-
mm). DISCUSSION gionalizing at least part of the verte-
Uroneurals are modified neural bral column in the zebrafish (Morin-
arches of the ural vertebrae that
Phylogenetic Comparisons Kensicki et al., 2002). The vertebral
are located lateral and slightly Most aspects of the morphology of column of fishes is traditionally sepa-
dorsal to the urostyle (Fig. 11D,E). the axial skeleton of D. rerio reflect the rated into two general regions: the
The single uroneural in D. rerio basal (primitive) conditions for various precaudal and the caudal verte-
forms as a bilateral pair of bone hierarchical phylogenetic levels: the brae. The anterior expression border
slivers along the lateral surface of slender-bodied clade within Danio, of Hoxd12a may correspond to the
the urostyle, extending from the Danio, Leuciscinae, Cyprinidae, and posterior end of the trunk and the
Otophysi (Coburn and Cavender, transition from rib-bearing to hemal
neural arch of preural 1 to the tip of
1992; Sanger and McCune, 2002). For arch-bearing (caudal) vertebrae
the notochord (5.8 mm). In the
example, D. rerio retains the shared (Morin-Kensicki et al., 2002; Fig. 12).
adult, the uroneural is fused near
derived features for the slender-bod- We further note that this boundary
the proximal end of the urostyle. An
ied clade of Danio (Sanger and Mc- may also correspond to the anus
unmodified neural arch is first visi-
Cune, 2002) such as a broad fourth and the anterior limit of the anal fin.
ble as membranous bone dorsal to Molecular genetic studies of Hox
neural spine, a reduced first lateral
PU1 (Fig. 11D), directly above the genes in fishes and other vertebrates
process, and a reduced supraneural
anterior end of the urostyle (Fig. (Burke et al., 1995; Prince et al., 1998;
3. It also retains the shared derived
11E).
features for Danio such as a reduced
Epurals are homologs of the neu-
ascending process of the interca- 1
ral spines of ural centra that develop larium (Sanger and McCune, 2002),
The basal condition involves (1) concha
slightly above the urostyle (Fig. scaphii fits into a notch in the basi- and exoc-
and D. rerio retains the primitive rib cipital; (2) all ossicles and claustrum present
11B,D,E). A single epural develops in condition (thin and straight), among and distinct; (3) scaphium and intercalarium
D. rerio. It is first visible as a slender other characters, for cyprinids possess ascending and articular processes; (4)
cartilaginous rod parallel to the flex- (Coburn and Cavender, 1992). tripus with large, crescent-shaped transforma-
tor; (5) fourth vertebra with recognizable par-
ing notochord (5.4 mm NL, Fig. D. rerio shares nine of 13 derived apophyses and ribs, the os suspensorium a sim-
11B,E). As development proceeds, characters associated with the axial ple anterior limb, free from anterior vertebrae;
the epural extends distally (posteri- skeleton of basal leuciscins (Coburn (6) large supraneurals 2 and 3 articulating on,
orly). Ossification of the epural be- and Cavender, 1992). It differs in or continuous through cartilage with the basi-
dorsals of the third and fourth vertebrae and
gins near the anterior tip of the that the fourth rib is short and stout, the exoccipitals; (7) a claustrum just above the
epural and proceeds posteriorly (6.3 the fifth parapophysis is cuplike, and concha scaphii, continuous through cartilage
mm, Fig. 11E). the associated rib head is rounded with supraneural 2.
350 BIRD AND MABEE

Morin-Kensicki et al., 2002) have re- (Fig. 12). In the zebrafish, this bound- of the Weberian apparatus. Specifi-
vealed the correlation of the rib- ary lies between vertebra 2 and ver- cally, whether the ribs of v3 and v4
bearing vertebrae (the “thoracic” tebra 3 (Morin-Kensicki et al., 2002; contribute to the tripus and os sus-
vertebrae in amniotes) with the an- Fig. 12). This finding is of significance pensorium, respectively, has been
terior expression boundary of Hoxc6 in interpretation of the homologies 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.
 ZEBRAFISH SKELETON 351

sion of Hoxc6a and Hoxc6b in the tripus, os suspensorium), as well as ments of the first neural arch (Sar-
myomeres from which vertebrae 3 Weberian support structures (roofing bathi, 1932; Fink and Fink, 1981;
and 4 (as well as v5–13) are derived, cartilage and supraneurals) are is- Sanger and McCune, 2002), also
lends support to a rib homology for sues of intense debate in the ichthy- termed “supradorsals” (Rosen and
these elements. ological literature (Fink et al., 1984; Greenwood, 1970).
The precaudal vertebrae immedi- Gayet, 1986). The developmental Based on developmental data,
ately posterior to the skull in fishes complexity of Weberian elements, Coburn and Futey (1996) proposed
are never termed “cervical” verte- which involves variability and modi- that the claustrum is homologous to
brae in the ichthyological or com- fication of the centra, neural arches supraneural 1, as did Gayet (1982,
parative vertebrate literature; this and spines, parapophyses, ribs, and 1985) from paleontologic data. In six
term is reserved for those vertebrae supraneurals of the first four verte- species of cyprinids and catosto-
in amniotes (Hildebrand, 1995). brae, has made interpretation of mids, Coburn and Futey (1996) ob-
Morin-Kensicki et al. (2002), how- their homologies quite difficult. In served separate bilaterally paired
ever, termed the first two vertebrae addition, individual bones may have cartilage condensations for supra-
in zebrafish cervical, because they a compound origin and may de- neural 2, which later coalesce; they
form anterior to Hoxc6 expression velop differently among species. thus proposed that the bilaterally
and they lack ribs. The discovery of In the following section, we review paired claustral cartilages may be
genes unique to a “cervical” region differences in Weberian ossicle de- homologues of supraneural 1. In
across vertebrate taxa might be in- velopment among species and de- other cyprinids, however (Coburn
dicative of a deeper homology scribe the bases for previous homol- and Futey, 1996), and in D. rerio, su-
among the most anterior vertebrae ogy assessments. Although it is not praneural 2 develops as a single car-
than is evident from comparative possible to fully assess homology tilage condensation dorsal to verte-
anatomy. given the narrow focus on zebrafish bra 2 (Fig. 5B,C). Danio rerio
Hoxa9, -b9, and -c9 are expressed development herein, we relate our develops its Weberian supraneurals
at the end of the thoracic series in observations to previous assess- (supraneurals 2 and 3) at a smaller
mouse and chick (Burke et al., 1995), ments of homology. Only through a size (4.8 –5.1 mm NL) than other oto-
and in zebrafish, the anterior expres- broad comparative phylogenetic physans (7–12 mm) (Coburn and
sion boundary of the paralogues analysis of development, and, in our Futey, 1996). This change in devel-
(Hoxa9, -b9) marks the transition be- view, with new molecular genetic opmental timing, possibly unique to
tween Weberian and precaudal data, can the homologies of these the slender-bodied Danio clade
vertebrae (Fig. 12). The thoracic– elements be better understood. (Sanger and McCune, 2002), might
lumbar boundary in amniotes is a The scaphium may form from one or explain the difficulty in locating the
transition from rib-bearing to non– two ossifications, depending on the supraneural condensations before
rib-bearing vertebrae, but in ze- species. In D. rerio, the scaphium is de- roofing cartilage development.
brafish, the boundary is between rived from basidorsal 1 and the concha Grande and De Pinna (in press)
highly modified vertebrae (which scaphium is formed from a separate proposed that the claustrum is the
bear highly modified ribs) and typi- mesenchymal ossification. This “dual or- homologue of the accessory neural
cal precaudal (precaudal) fish ver- igin” has been observed in many other arch of clupeocephalans (clupeo-
tebrae (which also bear ribs). Thus, Cypriniformes (Matveiev, 1929; Watson, morphs and salmoniforms), a hy-
this distinction may represent a 1939; Kulshrestha, 1977; Bogutskaya, pothesis supported by the observa-
novel use for the Hox9 genes in oto- 1991; Coburn and Futey, 1996). On the tion of an accessory neural arch in
physan teleosts. other hand, the concha may form as larval Chanos, a gonorhynchiform
The specialized vertebrae associ- an anterior ossification from basidorsal 1 (Fig. 1; Coburn and Chai, 2003).
ated with the caudal fin are unlikely, (Radermaker et al., 1989; Vandewalle Morin-Kensicki et al. (2002) found
according to Morin-Kensicki et al. et al., 1990; Fukushima et al., 1992; Ichiy- that the first two somites (and
(2002), to be regionalized by Hox anagi et al., 1993, 1996). Despite the maybe part of a third) do not con-
genes. Their results, as others (Kanki dual origin for the scaphium in some tribute to the vertebral column in ze-
and Ho, 1997; Griffin et al., 1998; Ahn species, it is generally accepted as the brafish but may contribute to the
and Gibson, 1999; Kimelman and homologue to the first neural arch (basi- head skeleton. It is possible that
Griffin, 2000), lend support to the dorsal 1). these somites may contribute the
concept that the tail region devel- The homology of the claustrum sclerotome that differentiates as the
ops with mechanisms that are dis- (Fig. 5F) has long been debated. claustrum and perhaps other struc-
tinct from those used for head and Berg (1940) and Chranilov (1927) de- tures such as the concha scaphium.
trunk (Morin-Kensicki et al., 2002). scribed the claustrum as a short- Determining the somite(s) of origin
ened neural spine of vertebra 1. The for these structures can be ad-
Homologies of the facts, however, that, unlike the neu- dressed by future lineage-labeling
Components of the Weberian ral spines, the claustrum is paired studies on zebrafish. The homo-
and forms in cartilage argue against logues of these structures might then
Apparatus a homology with neural spine 1. Oth- be determined by lineage-labeling
Homologies of the Weberian ossicles ers have described the claustrum as of closely related fishes.
(scaphium, claustrum, intercalarium, homologous to dissociated ele- In D. rerio, we observed an initial
352 BIRD AND MABEE

small band of cartilage from which grow toward and articulate with the ops after rib 5. It is plausible that the
significant membranous ossification parapophyses; ribs never develop fourth rib and the os suspensorium
proceeded anteriorly to form the out of parapophyses. On the other are modifications of the rib ⫹ parap-
body of the claustrum. In many cyp- hand, developmentally, the out- ophysis of the fourth vertebra.
riniforms, the claustrum has been re- growth (transformator) from the par-
ported as entirely membranous in or- apophysis more closely resembles a
igin (Coburn and Futey, 1996), lateral process than a rib. The ex- Variation
although Coburn and Futey (1996) pression of Hoxc6a and Hoxc6b in Although size (body length) is a bet-
also describe a cartilaginous claus- the myomere from which vertebra 3 ter predictor than age (days) of skel-
trum in many other species. It thus is derived, supports to a rib deriva- etal development in zebrafish and
appears that the claustrum in closely tion of this element (see above). other fishes, the size at which a spe-
related species may have different The os suspensorium demonstrates cific skeletal element develops is
developmental origins. Future lin- considerable morphologic and de- somewhat variable, ranging from 0
eage-labeling studies on different velopmental variation among oto- to 0.8 mm (Fig. 3), with an average
species of fishes may clarify this issue. physans. That the homology of the of 0.26. Highly variable elements
The intercalarium (Fig. 5F), like the os suspensorium (Fig. 5F) is not re- were not concentrated in a single
scaphium, may be singular or com- solved, thus, is not surprising. It has region; rather they were found
pound in origin among otophysans. been proposed as a homologue of throughout the skeleton. In the cau-
Specifically, the manubrium may the rib of the fourth vertebra (Chra- dal fin vertebrae, for example, hy-
develop from a separate ossification nilov, 1927; Berg, 1940; Soni et al., purals 4 and 5 vary 0.6 – 0.8 mm, but
in the interossicular ligament which 1978), the parapophysis (basiven- hypurals 1 and 2 do not vary at all
later fuses to basidorsal 2 (Watson, tral) of the fourth vertebra (Watson, (Fig. 3). In the Weberian apparatus,
1939; Kulshrestha, 1977) or as a 1939; Vandewalle et al., 1990; Fuku- supraneurals 2 and 3 vary 0.4 – 0.7
membranous extension from the shima et al., 1992; Ichiyanagi et al., mm, but the intercalarium and
basidorsal 2 (Vandewalle et al., 1993, 1996), or a modification of scaphium do not vary. Moreover,
1990; Bogutskaya, 1991; Fukushima both the rib and parapophysis of the skeletal elements that form late
et al., 1992; Ichiyanagi et al., 1993, fourth vertebra (Evans, 1925; Rosen (e.g., lateral process 1 and supra-
1996). In D. rerio, the manubrium de- and Greenwood, 1970). In D. rerio, neural 7) are as likely as elements
velops as a simple anterolateral the os suspensorium develops as a that form early (e.g., os suspenso-
membrane bone extension from thin, rod-shaped, anteroventrally di- rium and tripus) to demonstrate low
basidorsal 2; no interossicular ossifi- rected, membrane bone extension levels of variation. We noted a lack
cations were found in either whole- from the parapophysis. The os sus- of variation in the timing of the re-
mount or sectioned specimens. Like pensorium is similar in shape to a rib, gions of earliest development along
the claustrum, the intercalarium but the other ribs in Danio develop the anterior–posterior body axis (We-
may have different developmental as separate ossifications that grow berian apparatus and caudal fin).
origins in different species. Despite toward and articulate with the par- Although this finding may be due to
this, intercalarium is generally ac- apophyses. Moreover, the expres- their functional importance, it re-
cepted as the homologue of neural sion of Hoxc6a and Hoxc6b in the mains to be tested.
arch 2. myomere from which vertebra 4 is A relative sequence of appear-
The tripus (Fig. 5D) has been de- derived lends support to a rib contri- ance of bones in the axial skeleton
scribed as homologous to the par- bution to this element (see above). may be derived from the smallest
apophysis of vertebra 3 (Chranilov, However, developing as an out- specimen in which a skeletal ele-
1927; Berg, 1940; Vandewalle et al., growth from the parapophysis, the ment is present (Fig. 3, green dia-
1990; Fukushima et al., 1992; Ichi- os suspensorium is more like a lateral monds) or the size at which an ele-
yanagi et al., 1993, 1996; Chardon process than a rib. Thus, it is unclear ment is always present (Fig. 3, bars).
and Vandewalle, 1997). Others de- to what extent the fourth rib vs. the There is little variation in relative se-
scribe the tripus as a modification of parapophysis is involved in the de- quence within specific regions. For
both the parapophysis and rib of velopment of the os suspensorium. example, within the caudal fin, the
vertebra 3 (Rosen and Greenwood, The structure termed the “fourth hypural 1 and the parhypural always
1970; Patterson, 1984; Coburn and rib” is also continuous with both the develop before other bones within
Futey, 1996). In D. rerio, the tripus de- os suspensorium and the parap- the region. If the development of
velops as a thickened, posteriorly ophysis. It develops as posteriorly di- other elements is delayed, they are
projecting ridge of bone from the rected membranous extension from delayed together such that the rel-
parapophysis (transformator pro- parapophysis 4 (Fig. 5B). None of the ative sequence of development
cess). The remaining body of the tri- other ribs are continuous with their within the caudal fin does not
pus is added by means of continued respective parapophyses; they de- change, e.g., hypural 2 is always
anterior membrane bone extension velop as discrete independent ele- present before hypurals 3–5. The se-
from the parapophysis. The transfor- ments that grow to articulate with quence of appearance of the ele-
mator process is similar in shape to a parapophyses. Also, ribs typically ments in the Weberian apparatus is
rib, but the other ribs in Danio de- develop in an anterior to posterior similarly fixed (Fig. 13). Basidorsals 3
velop as separate ossifications that manner, but in D. rerio, rib 4 devel- and 4 develop before basiventrals 3
 ZEBRAFISH SKELETON 353

and 4 (the tripus and os suspensorium). CONCLUSION EXPERIMENTAL PROCEDURES

This sequence is likely conserved
Our detailed data on the develop- Adult zebrafish were obtained from
within cypriniforms, as noted by
ment and anatomy of the zebrafish a local pet supply company (Safari-
Coburn and Futey (1996).
axial skeleton and median fins pro- land, Sioux Falls, SD) and maintained
vide a baseline against which ze- in 10-gallon aquaria at 28.5°C (⫾
Patterns of Axial Skeleton brafish genetic and evo– devo com- 1°C) with a 14-hr photoperiod. Al-
parisons may be made. As though wild-caught zebrafish would
Development
expected, most of the features of be ideal for analysis, obtaining large
An overall pattern of development the axial skeleton of D. rerio repre- quantities of larvae from wild stocks
within the axial skeleton emerged sent the basal (primitive) conditions is difficult (A. McCune, personal
from our analysis of the relative tim- for phylogenetic levels above the communication). Slight variability in
ing of ossification events (Fig. 3). As species, and we identify the level at meristics may be found between
also noted by Du et al. (2001), devel- which these characters arose. Ulti- wild and reared zebrafish (Ferreri et
opment within the axial skeleton be- mately correlating the recency of al., 2000), but overall skeletal devel-
gins simultaneously within the Webe- evolutionary origin of these skeletal opment is expected to be similar.
rian region (third and fourth centra) features with the type of genetic Aquaria were maintained following
and the caudal fin region (hypural 1 network that underlies each, may general suggestions from the Ze-
and parhypural; Figs. 2, 3). This find- elucidate the nature of evo– devo brafish Book (Westerfield, 2000). In
ing is notable in that the two regions changes in genetics and morphol- preparation for spawning, tank bot-
are at the anterior and posterior lim- ogy. toms were thoroughly siphoned, and
its of the axial skeleton. Two centers tank levels were restored with fresh
Homologies of the Weberian os-
of ossification within the vertebral water. Spawning protocol followed
sicles, which are developmentally
column are common among ostari- those outlined previously by Cub-
complex, are difficult to draw with
ophyans (Emelianov, 1939). This may bage and Mabee (1996). Fry were
certainty. New molecular genetic
be related to a possible difference in fed Paramecium multimicronuclea-
data, such as that from Morin-Ken-
molecular patterning mechanisms tum and newly hatched brine
sicki et al. (2002), have provided a
between these two regions (Morin- shrimp. Fry were maintained at
new level of understanding for the
Kensicki et al., 2002). 28.5°C in a Fisher Scientific Low Tem-
evolutionary origin of such struc-
Development proceeds posteri- perature Incubator (Model 307C) for
tures. Our reviews of the evidence
orly and anteriorly from the third and 1 week after fertilization and trans-
for homology of the various ele-
fourth (Weberian) vertebrae, such ferred to aquaria.
ments will be a basis against which
that caudal vertebrae (orange in Larvae from multiple spawns were
the contribution of additional ge-
Fig. 2) are last to form. Development pooled to obtain a complete devel-
of associated vertebral structures, netic data may be assessed.
opmental series. The description of
such as neural arches and spines, We describe skeletal develop-
axial skeleton development is based
mirrors this pattern. In contrast, the ment using size as a measure of de-
on a developmental series of 1,579
vertebral column of amniotes devel- velopmental stage. Size is more individuals (30 used in histologic
ops from anterior to posterior (Arey, highly correlated with stage of oste- analysis, remaining used in whole-
1940; Rieppel, 1993). The supraneu- ological development than age. This mount analysis) ranging from young
rals develop from anterior to poste- ichthyological convention will be larvae at 3 days after fertilization to
rior as in other fishes (Mabee, 1988). useful for zebrafish researchers as adults (3 mm notochord length (NL)
Among the median fins, the cau- they move toward using older (and to 35 mm standard length [SL]).
dal fin develops first, followed by the larger) specimens in their research. Specimens were fixed in 4% parafor-
anal and finally the dorsal fin radials The molecular developmental signif- maldehyde and transferred to 70%
and fin rays (Fig. 3). The caudal fin is icance of the overall pattern of de- ethanol for storage or staining.
first to form in most fishes (Mabee et velopment within the axial skeleton Specimens were stained with Al-
al., 2002). Between the dorsal and (Fig. 3) is as of yet unknown. The de- cian blue for cartilage, enzymati-
anal fins, the more posterior fin de- gree to which this sequence of ossi- cally cleared with trypsin, and differ-
velops before the more anterior fin. fication reflects phylogenetic history entially stained with alizarin red for
Thus, in D. rerio, the anal fin, which is can be understood through similar bone following the method of
slightly more posterior than the dor- comparative developmental studies Dingerkus and Uhler (1977) with
sal fin, develops first. The exoskeleton on related taxa, and the degree to modifications of Potthoff (1984) for
and endoskeleton of both the dorsal which it reflects functional con- larval fishes. Other specimens were
and anal fins develop bidirection- straints may be determined through stained only for bone, as exposure to
ally, a copatterning similarity first biomechanical analyses. The way in cartilage stain may inhibit alizarin
noted by Mabee et al. (2002). Bidi- which molecular developmental red uptake, which can make bones
rectional development of these fins genetics is tied to evolutionary and appear clear.
appears to be the basal condition functional constraints will represent For histologic analysis, specimens
for teleost fishes (Mabee et al., a new and high level of integration were paraffin-embedded and sec-
2002). in the biological sciences. tioned at 8 ␮m by using a Leica 2040
354 BIRD AND MABEE

autocut microtome. Large speci- with Scion 1.62c and Adobe Photo- Endoskeletal/Endoskeleton: Bony
mens (⬎7 mm) were decalcified be- shop 5.0 software. or cartilaginous internal frame of the
fore embedding. Sections were body.
stained by using a Milligan’s Epicentrals: Intramuscular bones
trichrome protocol (Presnell and ACKNOWLEDGMENTS that attach to centra (Patterson and
Schreibman, 1997) and analyzed by We thank R. Britz, P. Crotwell, D. Johnson, 1995). First termed by
using a Zeiss Axioplan 2 compound Neufeld, and D. Swanson for their Owen (1866).
microscope. Images were collected comments on this manuscript and E. Epineurals: Intramuscular bones
as described below. Haberman for help with histology. that attach to neural spines (Patter-
Specimens were initially scored for N.C.B. thanks SICB and USD for son and Johnson, 1995). First termed
the presence of bones as well as os- Grants in Aid of Research, and by Owen (1866).
sification within 1 week of staining. P.M.M. and N.C.B. thank USD EPS- Epipleurals: Intramuscular bones
Cartilaginous structures were re- CoR and CoBRE (NIH P20 RR15567) that attach to hemal spines or ribs
corded as present upon visualization for additional funds. (Patterson and Johnson, 1995). First
of Alcian blue staining of chondro- termed by Owen (1846).
cytes. The earliest sign of ossification, Epural: Flattened median bone
based on uptake of alizarin red GLOSSARY posterior to the last neural spine, dor-
staining of mineralized bone matrix, Actinotrichia: Unsegmented der- sal to the urostyle.
was recorded as presence of that mal rays in the fin fold of larval fishes; Exoskeletal/Exoskeleton: External
bone. Although histologic sections replaced by lepidotrichia. skeleton, including fin rays and
may, in some cases, show bones as Anal fin: Median unpaired fin on spines.
“present” before the clearing and the ventral surface of the body, pos- Fin ray: Lepidotrich. Bilaterally
staining whole-mount procedure terior to the anus. paired, jointed, exoskeletal bony
shows their presence in smaller and Antepenultimate vertebra: Verte- rays supporting the external fins.
younger larvae (Clark and Smith, bra anterior to the penultimate ver- Hemal arch: Arch on the ventral
tebra (preural 3 in Danio rerio). surface of a centrum. Characterizes
1993), the relative sequence of ossi-
Basidorsal: Bilaterally paired carti- caudal vertebrae.
fication is expected to remain the
lage condensations located on the Hemal spine: Cartilage or mem-
same. Thus, sections were only ob-
branous median extensions of one
tained to address specific questions dorsal half of a centrum. Compo-
or both hemal arches. May also de-
of development within the axial skel- nent of “arcualia” in basal acti-
velop autogenously and fuse to the
eton, primarily in the Weberian ap- nopterygians (Gadow and Abbott,
proximal tips of the developing he-
paratus. 1895).
mal arches.
Specimens were examined by us- Basiventral: Bilaterally paired carti-
Hypurals: Laterally flattened me-
ing a Wild M5 stereomicroscope at lage condensations located on the
dian caudal endoskeletal supports
magnifications of ⫻11–94, measured ventral half of a centrum. Compo-
which articulate with the urostyle
using an ocular micrometer, and nent of “arcualia” in basal acti-
and serve as attachments for the
their lengths recorded to the nearest nopterygians (Gadow and Abbott,
caudal fin rays. Homologous to he-
0.1 mm. Prenotochord flexion and 1895). mal spines of preural vertebra 1 and
early flexion individuals were mea- Camera aerea Weberiana: Ante- the ural vertebrae.
sured from the anterior end of the rior part of the swim bladder (Rader- Hypurapophysis: See Parhypurap-
upper jaw to posterior tip of noto- maker et al., 1989) that is in contact ophysis.
chord. Notochord length is the stan- with the os suspensorium and the tri- Intercalarium: Third Weberian ossi-
dard measure of preflexion larval pus. This term was created by (Chra- cle. Modification of the neural
fishes in ichthyology and fisheries sci- nilov, 1927) for the anterior chamber arches of the second vertebra. Con-
ences (Ahlstrom and Moser, 1976; of the bipartite swim bladder. sists of three parts: the manubrium,
Leis and Trnski, 1989). Flexion occurs Caudal fin: Tail fin; most posterior ascending process, and articulating
at approximately 5.5 mm, corre- median fin. process.
sponding to 9 –10 days after fertiliza- Caudal vertebrae: Posterior verte- Intermuscular bones: Long, ray-
tion (Cubbage and Mabee, 1996, brae; the first caudal vertebra has a free bones lateral to the vertebral
Appendix A for age vs. length completely fused hemal arch. column. Include epineurals, epicen-
chart). Specimens with flexed noto- Centra: Central bodies of verte- trals, and epipleurals (Owen, 1846,
chords (⬃5.6 mm and larger) were brae. 1866; Patterson and Johnson, 1995).
measured from the anterior end of Claustrum: Commonly considered Lateral process: Transverse pro-
the upper jaw to the posterior end of the first Weberian ossicle. cess. Modified parapophyses ex-
the hypurals (standard length). Illus- Dorsal fin: Median fin(s) on the dor- tending laterally from a centrum.
trations were drawn from images sal side of the body. Only a single Typically fused to the centrum in
collected by using a Dage MTI dorsal fin is present in zebrafish. Danio rerio.
3CCD videocamera. Images were Endochondral ossification: Bone Manubrium: Anterior branch of
then processed by using a Power formation through which preexisting the intercalarium. Also termed “an-
Macintosh G3 computer equipped cartilage is replaced by bony matrix. terolateral process.”
 ZEBRAFISH SKELETON 355

Membranous ossification: Direct Prezygapophyses: Bony dorsal zebrafish, it is composed of preural

ossification from mesenchymal pre- (neural prezygapophyses) or ventral vertebra 1 and ural centra 1 and 2.
cursors. (hemal prezygapophyses) exten- Vertebra: Unit of the vertebral col-
Neural arch: Bony arch on the dor- sions from the anterior end of a neu- umn; consists of a centrum, neural
sal surface of a centrum. ral or hemal arch. They articulate arches and spine, and may include
Neural spine: Cartilage or mem- with the postzygapophyses of the hemal arches and spine, or parap-
branous median extensions from preceding centrum. ophyses and ribs.
neural arches. May also develop au- Radial: Pterygiophore. Endoskel- Weberian apparatus: Series of ver-
togenously and fuse to the develop- etal fin support of the dorsal or anal tebral modified ossicles serving to
ing arches. fin, to which the fin rays attach. May connect the swim bladder to the in-
Notochord length (NL): Measure- be unsegmented, bipartite (proxi- ner ear.
ment from the anterior-most tip of mal and distal), or tripartite (proxi- Weberian vertebrae: Most ante-
the jaw to the posterior-most tip of mal, middle, and distal). rior vertebrae, specialized as the
the notochord. Ribs: Pleural ribs, ventral ribs. Inter- Weberian apparatus (vertebrae
Os suspensorium: Separate ossifi- segmental bones that articulate 1– 4). Typically reduced in length
cation from the parapophysis of the with parapophyses. They protect and size.
fourth vertebra; develops ventrally and support internal organs.
to curve around the anterior head of Roofing cartilage: Cartilaginous
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