Developmental morphology of the axial skeleton of the zebrafish, Danio rerio (Ostariophysi: Cyprinidae)

Abstract

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, 2003. © 2003 Wiley-Liss, Inc.

INTRODUCTION

Questions regarding the relationship between genes and morphology are a driving force behind research in evolution and development (Carroll 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 phylogenetic 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 mutagenesis, the zebrafish has moved to the forefront as a vertebrate model system. Yet for all of the molecular work, there has been no comprehensive study on the development and morphology of the zebrafish axial skeleton. A detailed study of skeletal development, however, is crucial for interpretation of skeletal mutations and gene expression patterns and ultimately for understanding the connections between genes and morphology.

The purpose of this study is to provide a detailed developmental anatomic reference and a review and assessment of homologies of the zebrafish axial skeleton. The extensive ichthyological literature on otophysan anatomy forms the basis for our interpretation of homologies of various elements of the axial skeleton. We present data on the developmental timing and origin of skeletal elements in the axial skeleton. When paired with other morphologic 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 Southeastern 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 Cypriniformes (Fig. 1). Its rapid development, short generation time, embryonic transparency, ease of mutagenesis, and ongoing genomic research has made it ideal for laboratory study (Nüsslein-Volhard and Dahm, 2002).

Figure 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).

In recent years, a variety of significant 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 differentiation (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 coverage, including studies on the development of the skull (Cubbage and Mabee, 1996; Schilling and Kimmel, 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 skeleton (e.g., intermuscular bones, dorsal 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-Kensicki et al., 2002). Bones may develop from a cartilage precursor (cartilage bone) or directly from a mesenchymal condensation (membrane bone or dermal bone). Cartilage bones may ossify in two ways, either endochondrally with ossification beginning within the cartilage model, or perichondrally with ossification beginning in the perichondrium surrounding the cartilage model (Patterson, 1977). Lower teleost fishes, including ostariophysans (zebrafish), are reported to lack acellular bone (Parenti, 1986).

In fishes, the axial skeleton includes the vertebral column and associated median (unpaired) fins. The vertebral column is regionalized into precaudal (Fig. 2, green and red) and caudal vertebrae (Fig. 2, orange and purple). In ostariophysans, the most anterior precaudal vertebrae are regionalized as the Weberian vertebrae. The Weberian vertebrae in otophysans are so named for the Weberian apparatus (Weber, 1820). In the following description, we follow the ichthyological convention for ostariophysans of using the term “precaudal vertebrae” to refer to those vertebrae posterior to the Weberian vertebrae and anterior to the caudal vertebrae. Precaudal vertebrae are composed of centra, neural arches and spines, parapophyses, and ribs. The caudal vertebrae are regionalized terminally to support the caudal fin. Caudal vertebrae are composed of centra, neural arches and neural spines, and hemal arches and hemal spines of vertebra 15 through vertebra 28. The most posterior three caudal vertebrae are modified to support the caudal fin. The last precaudal and/or the first caudal vertebra 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). Basiventrals 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).

Figure 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.

RESULTS

The length of first appearance of a skeletal element is given in all descriptions below, and it is represented in Figure 3 as a green diamond. All measurements are given millimeters standard length; notochord length (NL) is given where appropriate. For ease of interpretation, a glossary is included. Those structures and terms included in the glossary are used in headings or have been bolded at first use below.

Figure 3. Sequence of development and ossification within the axial skeleton. Diamonds indicate earliest appearance of skeletal elements. Bar indicates presence in all specimens. Blue indicates cartilage; red indicates bone. In endochondral elements, purple indicates 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 hypural 4, the blue–red transition line between the green diamond and bar indicates that, in specimens with an early developing hypural 4, ossification begins prior to the length at which hypural 4 is found in all specimens.

Centra

In D. rerio, centra are formed sequentially. Centra 3 and 4 form first (Fig. 4A), as bone encircling the notochord (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 compound 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 dorsoventrally, as well as at both anterior and posterior ends (Fig. 4D,E).

Figure 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.

Weberian centra (vertebrae 1–4) remain unfused in D. rerio throughout development, although in many fishes, e.g., silurids and some cypriniforms (Nelson, 1948; Alexander, 1962; Roberts and Kottelat, 1984; Vandewalle et al., 1990), the Weberian vertebrae, usually the second and third centra, may develop as a fused centrum. This fusion is hypothesized to be the basal condition for Otophysi, with failure of fusion (as in D. rerio), a secondary condition (Roberts and Kottelat, 1984).

The modal number of vertebrae in D. rerio, including the urostyle is 31: 4 Weberian, 10 precaudal, 17 caudal (including the 3 caudal fin vertebrae). Our meristic counts (n = 50) are in agreement with Ferreri et al. (2000), who found an average of 31 vertebrae in both wild and reared D. rerio.

Neural Arches and Spines

The neural arches form from either cartilage (endochondral), or membrane bone precursors, depending on their position along the vertebral column (Figs. 2, 4C,D,E, 5A,F). Neural arches of vertebrae 1–5 form from bilaterally paired cartilaginous basidorsals that extend dorsally and fuse medially to form the neural arch (Fig. 5A). In D. rerio, the neural arches of the Weberian vertebrae are first to develop, and they are visible as small clusters of chondrocytes (4.3 mm NL), which extend dorsally by means of membrane bone. Neural arches of the first and second vertebrae are modified into Weberian ossicles, the scaphium and claustrum, respectively. Neural arches of the preural vertebrae, including the double arch of preural 2, also develop from bilaterally paired cartilage basidorsals. On vertebrae 6–28, the neural arches develop from bilaterally paired membranous bones. Neural arches 6–7 are the first membranous neural arches visible (4.9 mm NL). Although most remaining neural arches develop simultaneously, the more anterior arches are longer and they fuse before more posterior arches. Development proceeds similar to basidorsal arches, with the membrane bone extending dorsally and fusing medially to form the neural arch.

Figure 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 suspensorium (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.

Neural spines develop as single median dorsal extensions from the fused neural arches (5.1 mm NL, Fig. 4D,E). All neural spines are dermal in origin except for the neural spines of the preural vertebrae, which develop, at least in part, from cartilage. Neural spines form earlier on anterior vertebrae.

Neural arches in the precaudal region are broad, with well-developed prezygapophyses (Fig. 4D). Prezygapophyses (neural prezygapophyses) 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. Vertebra 5 has a highly reduced prezygapophysis, but vertebrae 6 and 7 are enlarged and appear to be ligamentously connected to a posterior flange extending from the neural spines of these vertebrae.

Postzygapophyses (neural postzygapophyses) form as paired dorsomedially directed processes (6.2 mm) on the posterior ends of centra (Fig. 4C,E). They are ligamentously attached to the prezygapophyses of the immediately posterior vertebra. The postzygapophyses of precaudal vertebrae are larger than those of caudal vertebrae (Fig. 4D,E). Significant variation in shape is found in the precaudal region. The first postzygapophysis is present on vertebra 5, and they are found posteriorly through preural 3 (6.2 mm). The development of postzygapophyses is bidirectional, with the first group visible on vertebrae 11–23. Ventral postzygapophyses develop slightly later (6.3 mm), but develop in the same manner as neural postzygapophyses.

Neural spines are generally longer in the precaudal region when compared with the more posterior vertebrae (Fig. 2), although their morphologies are similar. Neural spines form immediately after fusion of the neural arches. Timing of fusion is variable but proceeds in an anterior-to-posterior direction.

Parapophyses and Ribs

Parapophyses are bony lateral extensions from centra that serve as articulation points for ribs (Figs. 2, 4B,D). In D. rerio all parapophyses develop endochondrally from basiventrals (Fig. 4B). Parapophyses are first visible as clusters of chondrocytes on the lateral surface of the anterior ends of the precaudal vertebrae (5.5 mm NL). Parapophyses of the fifth and sixth vertebrae are much larger compared with those of more posterior precaudal vertebrae. 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 parapophysis 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 articulates.

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 parapophyses of the most anterior (V5) and most posterior (V13–V15) vertebrae 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 parapophyses 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 dorsally to articulate with the parapophyses (Fig. 4B). The first non-Weberian 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 lateral ridge that develops from the initial thickened rod. At approximately half the overall rib length, the morphology reverts to the typical rod-like shape as they curve around the viscera and swim bladder. The last (and second-to-last in some instances) rib either loses the “flared” appearance of its proximal half, or it is highly reduced in length.

Hemal Arches and Spines

The hemal arches characterize the caudal vertebrae; they do not develop on precaudal vertebrae (Fig. 4C,E). The caudal vertebrae range in number from 14 to 16, with an average 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 vertebrae. These ossifications grow ventrally 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 hemal spines elongate by means of continued cartilage growth, and they later (6.0 mm) ossify.

Hemal arches are broad, with small prezygapophyses (hemal prezygapophyses). Prezygapophyses form as anteriorly directed processes (8.6 mm) from the bases of hemal arches. They are ligamentously attached to the postzygapophyses of the preceding vertebra.

Postzygapophyses (hemal postzygapophyses, Fig. 4C) form as ventrally directed processes (6.3 mm) on the posterior ends of centra. They are ligamentously attached to the prezygapophyses of the immediately posterior 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 specific role of the Weberian apparatus in sound transmission (Popper and Fay, 1999) have been reviewed previously. Otophysan teleosts (within the larger clade Ostariophysi) include members of Cypriniformes (e.g., zebrafish), 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 Weberian apparatus are found in the sister taxon of otophysans, the Gonorynichiformes and in the Clupeomorpha (Fig. 1; Rosen and Greenwood, 1970; Fink and Fink, 1981; Grande and Braun, 2002). Thus, the Weberian apparatus is thought to have evolved piece by piece, and only once all elements were present did it function in sound transmission (Grande and Braun, 2002). The Weberian apparatus is modified but not lost in any otophysan.

The Weberian apparatus is traditionally subdivided into the Weberian ossicles, the scaphium, claustrum, 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 cavum sinus impar, allowing for greater sensitivity to vibration and acute hearing (Chardon and Vandewalle, 1997).

Weberian ossicles.

These include, from anterior to posterior, the scaphium, claustrum, intercalarium, 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 tripus are connected by means of the interossicular ligament.

Scaphium.

The scaphium is connected posteriorly to the manubrium of the intercalarium by means of the interossicular ligament (Fig. 5C–F). The concha of the scaphium forms the posterolateral wall of the sinus impar perilymphaticus, 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 articular process that articulates with a depression in centrum 1, an ascending process that extends posterodorsally, 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 ossification 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 claustrum by means of a syndesmosis. The concha of the scaphium does not make contact with the exoccipital 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.

Claustrum.

The claustrum is a bilaterally paired, triangularly shaped bone embedded in connective tissue dorsomedial 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 anterodorsal edges of the roofing cartilage (5.5 mm). It develops in close association with the roofing cartilage, and it is most easily distinguished in specimens around 7.0 mm. The claustrum extends anteroventrally through membrane ossification to encircle the neural tube (Fig. 6A). The claustrum does not entirely 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 surface of the claustrum is connected to supraneural 2 by means of a synchondrosis. Ossification begins at 6.3 mm.

Figure 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 neural tube (nt). B: Third vertebra (c3). The third neural arch (na3) has become continuous with the roofing cartilage (rc), completely encircling the neural tube (nt). The tripus (tr) is seen lateral to centrum 3 (c3) and neural arch 3 (na3). Scale bars = 0.05 mm.

Intercalarium.

The intercalarium is a rod-shaped bone that originates from basidorsal 2 (Fig. 5C,D,F). It is connected anteriorly to the scaphium and posteriorly to the anterior process of the tripus by means of the interossicular ligament. At most in otophysans, the intercalarium is triradiate, with an anterior process, or manubrium, projecting anterolaterally to lie between 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 develops as a membranous ossification that extends laterally from the basidorsal (5.1 mm NL). The ascending and articular processes begin ossification at 5.3 mm NL. The ascending process develops as a dorsally projecting cartilaginous extension from the basidorsal (5.1 mm NL). The ascending 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 and articulates with a groove in centrum 2.

Tripus.

The tripus is the largest of the Weberian ossicles (Fig. 5A,B,D,F). The tripus is typically separated into three regions: the anterior process, which connects to the intercalarium; the articular process, which articulates with centrum 3; and the transformator 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 processes are formed by membranous ossification, with the articular process ossifying within the initial cartilage condensation. The interossicular ligaments do not contribute to the developing tripus.

The transformator process ossifies as a ventrally projecting rod-like ossification from basiventral 3 (5.1 mm NL). As it grows, it becomes thickened and it curves posteriorly such that the tip lies medial to the os suspensorium and is in contact with the swim bladder (5.6 mm, Fig. 5B). Most of the transformator process appears 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 interossicular ligament to the manubrium of the intercalarium.

From a dorsal view, the tripus is wing-shaped, extending from vertebra 2 to vertebra 4 (Fig. 5F). Laterally, the tripus is tilted, with a lowered posterior hook-shaped tip (transformator process) positioned ventral to centrum 4. The posterior tip is slender and curves medially. The posterior tip of the transformator process articulates with the medial portion of the os suspensorium, as well as with the tunica externa of the camera aerea Weberiana, or the anterior surface 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 perpendicular 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 surface 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 development as a separate anteriorly directed membrane bone outgrowth from the basiventral cartilage of vertebra 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 suspensorium develop from the fourth rib; rather, they both extend from the cartilage of parapophysis 4.

The upper body of the os suspensorium 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, including neural arches and spines, supraneurals 2 and 3, the roofing cartilage, 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 Weberian apparatus. Vertebra 1 is composed of a compact centrum articulating 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 narrowed to articulate with a notch in the basioccipital. A short bony lateral 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 extends 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 process 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 neural 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 develops from a large basiventral cartilage on the ventrolateral surface of the centrum, and together with the third rib, it is modified as the tripus.

Vertebra 4.

Vertebra 4 is composed 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 neural arch develops on the dorsal surface of centrum 4 in the same manner 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). Cartilage from neural arch 4 is not continuous 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 cartilaginous parapophysis (5.6 mm). The rib is a separate outgrowth from the parapophysis, and it is not connected 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 bifurcated distally (Fig. 5F). It is fully developed and ossified by 7.5 mm.

Supraneural 2.

Supraneural 2 is an ovoid bone located 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 supraneural 2 as neural arch 2 (Ramaswami, 1955; Howes, 1978; Mayden, 1989).

Supraneural 2 is first visible as a single 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 supraneurals proceeds in an anterior to posterior direction, as in other teleosts (Mabee, 1988). Supraneural 2 becomes surrounded by and becomes 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 condensation 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 surrounded 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 begins on the surface of this dorsal elevation (6.7 mm, Fig. 5E) and proceeds dorsally as a thin sheet of membrane bone and ventrally over the surface of the cartilage. The dorsal surface of supraneural 3 projects anteriorly in the adult. It is fully ossified 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 contrast, the dorsal and posterior margins of Weberian supraneural 3 are fused in D. rerio. In the slender-bodied 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 fusion of supraneural 3 and supraneural 4, we do not see a separate condensation 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 structure 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 connecting supraneurals 2 and 3 and neural arch 3 (Fig. 5B,C). This matrix is Alcian blue–positive before chondrocytes are visible. The roofing cartilage differentiates to surround supraneural 2, supraneural 3, and the neural arch and spine of vertebra 3 (Figs. 5D,E, 6B). It closely approximates the neural arch and spine of vertebra 4. The roofing cartilage forms a synchondrosis with the claustrum. The roofing cartilage is continuous with the supraneurals and neural arch 3 only during larval development. Our observation of the formation of the roofing cartilage 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 become non–matrix-positive connective tissue. A remnant of the roofing cartilage is retained as a connective tissue mat connecting neural arches 3 and 4, the claustrum, and supraneurals 2 and 3. Typically, there is a gap between the roofing cartilage and exoccipital of the skull, although in some specimens they appear fused.

Intermuscular Bones

Intermuscular bones, which are unique to teleostean fishes (Patterson and Johnson, 1995; Gemballa and Britz, 1998), are membranous ossifications that form in the myosepta and attach ligamentously to neural arches, centra, hemal arches, or ribs (Fig. 7). Three sets of serially homologous intermusculars may be present in teleosts and are classified in reference to the site of attachment (Patterson and Johnson, 1995; Gemballa and Britz, 1998). From dorsal to ventral, epineurals attach to neural arches, epicentrals attach to centra, and epipleurals attach to hemal arches or ribs. Epicentrals are not present in D. rerio. Epineurals develop in association with vertebra 5 through preural 2 (6.0 mm). They develop and ossify predominantly from posterior to anterior. Beginning at approximately the precaudal–caudal transition, each epineural develops an anterior fork (Fig. 7A). The first signs of forking are difficult to visualize, but at 10.6 mm, both epineurals and epipleurals appear to fork at approximately vertebral level 17–18. Epineurals associated with precaudal vertebrae are not forked. The more anterior epineurals span approximately two precaudal centra, and the more posterior epineurals span approximately three caudal centra.

Figure 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 lateral (lat) extensions. Hemal arches (ha) and hemal spines (hs) can be seen in the background. Scale bars = 0.5 mm.

In D. rerio, the most anterior epipleural is associated with the first complete hemal arch (the first caudal vertebra). Epipleurals do not develop in association with ribs. Development 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 ligamentously attached to the vertebrae (Fig. 7A,B).

Supraneurals

Supraneurals are dorsal median structures located anterior to the dorsal fin pterygiophores and posterior to the skull (Figs. 2, 8). They are slender skeletal rods that may be homologous to neural spines or radials (Mabee, 1988). In D. rerio, supraneurals 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 specimen in which the additional supraneural was located dorsal to the roofing cartilage. In the (only) specimen examined with four supraneurals, the ninth supraneural was absent.

Figure 8. Supraneurals, lateral views. A: Cartilage development of the anterior three supraneurals (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 supraneurals, 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.

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) followed by supraneurals 6–9 (6.4 mm, 6.6 mm, 6.6 mm, and 7.0 mm, respectively). The supraneurals are arranged 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. Supraneural 5 enlarges and broadens, while supraneurals 6–9 develop dorsoventrally as small slender rods of cartilage. Supraneural 5 is the first to ossify, with ossification beginning later in more posterior supraneurals (supraneural 6 at 8.5 mm, supraneural 7 and 8 at 9.2 mm, and supraneural 9 at 9.8 mm). Supraneurals exhibit perichondral ossification, with first uptake of stain forming a ring around the cluster of chondrocytes (Fig. 8B).

In the adult, the posterior four supraneurals are arrowhead in shape, with a wide dorsal surface that tapers ventrally. Many have a proximodistal groove in the lateral surface running the length of the supraneural (Fig. 8C). The shape of the first supraneural is variable, but it is always larger than supraneurals 6–9 and generally exhibits an ovoid shape.

Median Fins

Dorsal fin.

The dorsal fin is an unpaired median fin located on the dorsal surface of the body (Figs. 2, 9A–C). The dorsal fin consists of endoskeletal supports, or radials, and exoskeletal supports, or fin rays. The radials, also termed “pterygiophores,” are rod-like internal median skeletal supports of the dorsal and anal fins.

Figure 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.

In D. rerio, initiation of development of the dorsal fin radials is first visible as a central group of cartilage condensations at approximately the middle of the presumptive fin. Additional radials develop anteriorly and posteriorly (Fig. 9A,C). Correlation between direction of development within the dorsal and anal fins is similar to those found in other fishes, and this finding is probably the ancestral condition for fin positioning and patterning (Mabee et al., 2002). Later in development, radials segment proximodistally into two parts, a larger proximal radial and smaller distal radial (5.7 mm, Fig. 10). The distal radials articulate directly with the lepidotrichia. Development of dorsal fin radials typically begins with those radials located between the neural spines of the 14th and 15th vertebrae, which correspond to the 4th and 5th radials in the adult (5.5 mm NL, Fig. 9A). Growth proceeds proximally toward the vertebral column.

Figure 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.

Ossification of the proximal radials proceeds proximally and distally from the middle of the radial (7.0 mm, Fig. 9B). In all but the anterior two radials, a second center of ossification is located at the distal tip of the proximal radial. Ossification generally follows the same order as chondrogenesis, i.e., radials 4 and 5 are the first to begin ossification. Distal radials ossify in the same order as proximal radials, but ossification is delayed relative to the proximal radials (8.6 mm). Dorsal radials were not found anterior to the neural spine of vertebra 11 or posterior to the neural spine of vertebra 18. The modal number of dorsal fin radials for the zebrafish is eight, with a range of seven to nine (n = 50). It is at the posterior end of the fin that radials are added or lost. Interdigitation patterns (i.e., the relationship between the radials and neural spines) of the dorsal fin are variable, and the modal pattern is represented in Figure 2.

Fin rays (lepidotrichia) are bony, bilaterally paired, segmented dermal 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 remaining 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 distally (Fig. 9C).

Anal fin.

The anal fin is an unpaired median fin located on the ventral surface of the body, and it is delimited anteriorly by the anus (Figs. 2, 9D–F). Development of anal fin radials begins with those radials developing anterior and posterior to the hemal spine of the 16th vertebra. These correspond 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 surface of the body. Growth proceeds proximally toward the vertebral column. Anal radials are added anterior and posterior to the original radials (Fig. 9D). Ossification proceeds proximally and distally from the middle 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 radials exhibit a second center of ossification at the distal end of the proximal radial (Fig. 9E). Shortly after the onset of development, the radials segment into proximal and distal portions (5.5 mm, Fig. 10). Distal radials ossify in the same order as proximal radials, but ossification is delayed 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 associated with the third and fourth radials 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 number, 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 exoskeletal development (Figs. 2, 3, 11). The endoskeleton of D. rerio is composed of five hypurals, the parhypural, 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 urostyle. The urostyle is composed of the compound centrum preural 1 + ural 1, which fuses in development to the centrum of ural 2.

Figure 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 pound sign. Scale bars = 0.05 mm in A, 0.1 mm in B–E.

The five hypurals are expanded and laterally flattened hemal arches and spines that support the lepidotrichia of the caudal fin. The hypurals 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 hypural 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 becomes 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 hypurals, and it proceeds bidirectionally (Fig. 11C–E). The proximal and distal tips are last to ossify. Ossification 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 cartilage late in development, with all elements fully ossified by 24 mm (Fig. 11E).

Danio rerio has three hypurals (hypurals 3–5) in the upper lobe (Fig. 11E), a derived condition of the slender-bodied clade of Danio. Ancestrally in Danio, the lower lobe of the caudal fin is supported by the parhypural 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 derived condition of only three hypurals 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 cartilaginous precursor of the parhypural develops ventral to the posterior tip of the notochord as a stack of chondrocytes two to three layers in thickness (3.8 mm NL, Fig. 11A). Ossification 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 parhypurapophyses, also termed “hypurapophyses” are a bilateral pair of bony posterolateral extensions from the parhypural (Fig. 11E). They serve as the origin for the anterolateral bundle of the hypochordal longitudinal 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 (collectively known as the urostyle). The ossifications of preural 1 and ural 1 are always fused (5.4 mm NL); they are never visible as two independent ossification centers (Fig. 11C,D). Thus, they are referred to as “the compound 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 vertebra, may possess two neural arches. This feature appears to be highly variable. In specimens with the doubled arch condition, they develop from cartilaginous basidorsals. 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 develops (5.4 mm NL). Spines may develop 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 notochord (5.5 mm, Fig. 11D). The hemal arch develops from basiventrals (4.9 mm NL). A cartilaginous spine is present shortly after the appearance of the arch (5.0 mm NL). Ossification proceeds ventrally from the base of the basiventrals (5.5 mm) into the spine (5.6 mm).

The neural arch and spine of preural 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 develop as cartilaginous extensions of the neural arches, and they begin developing at 4.1mm NL and 5.0 mm NL, respectively (Fig. 11B). Preural 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 terminal 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 develops similar to all other vertebrae, with ossifications first seen at 5.4 mm NL. As the notochord flexes, the separate ossifications extend to encompass the entire posterior end of the notochord to form the urostyle. The neural arch of the urostyle is first visible as a small ossification on the dorsal 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 visible as membranous bone dorsal to PU1 (Fig. 11D), directly above the anterior end of the urostyle (Fig. 11E).

Epurals are homologs of the neural 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 flexing notochord (5.4 mm NL, Fig. 11B,E). As development proceeds, the epural extends distally (posteriorly). Ossification of the epural begins 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 hemal spine of preural 2, as well as the parhypural and hypurals 1–2. Six dorsal 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 condition for leuciscins. The procurrent caudal rays in D. rerio are also not hypertrophied, consistent with the primitive condition in leuciscins (Cavender 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-bodied clade of Danio (Sanger and McCune, 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 intercalarium (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 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 supraneurals are never continuous with neural arch 4 or the exoccipitals; and the cartilaginous connection between the claustrum and supraneural 2 is lost during development.

Regionalization and the Axial Skeleton

The results of recent molecular genetic and embryologic studies of the developing zebrafish skeleton impact the classic interpretations of the homologies within the fish axial skeleton. The genes involved in regionalizing the tetrapod vertebral column appear to be involved in regionalizing at least part of the vertebral column in the zebrafish (Morin-Kensicki et al., 2002). The vertebral column of fishes is traditionally separated into two general regions: the precaudal and the caudal vertebrae. 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.

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

Molecular genetic studies of Hox genes in fishes and other vertebrates (Burke et al., 1995; Prince et al., 1998; Morin-Kensicki et al., 2002) have revealed the correlation of the rib-bearing vertebrae (the “thoracic” vertebrae in amniotes) with the anterior expression boundary of Hoxc6 (Fig. 12). In the zebrafish, this boundary lies between vertebra 2 and vertebra 3 (Morin-Kensicki et al., 2002; Fig. 12). This finding is of significance in interpretation of the homologies of the Weberian apparatus. Specifically, whether the ribs of v3 and v4 contribute to the tripus and os suspensorium, respectively, has been debated (see below). The expression 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 immediately posterior to the skull in fishes are never termed “cervical” vertebrae in the ichthyological or comparative vertebrate literature; this term is reserved for those vertebrae in amniotes (Hildebrand, 1995). Morin-Kensicki et al. (2002), however, 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 indicative 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 expression boundary of the paralogues (Hoxa9, -b9) marks the transition between 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 zebrafish, the boundary is between highly modified vertebrae (which bear highly modified ribs) and typical precaudal (precaudal) fish vertebrae (which also bear ribs). Thus, this distinction may represent a novel use for the Hox9 genes in otophysan teleosts.

The specialized vertebrae associated 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 develops with mechanisms that are distinct 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 issues of intense debate in the ichthyological literature (Fink et al., 1984; Gayet, 1986). The developmental complexity of Weberian elements, which involves variability and modification of the centra, neural arches and spines, parapophyses, ribs, and supraneurals of the first four vertebrae, has made interpretation of their homologies quite difficult. In addition, individual bones may have a compound origin and may develop differently among species.

In the following section, we review differences in Weberian ossicle development among species and describe the bases for previous homology assessments. Although it is not possible to fully assess homology given the narrow focus on zebrafish development herein, we relate our observations to previous assessments 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 derived from basidorsal 1 and the concha scaphium is formed from a separate mesenchymal ossification. This “dual origin” 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; Ichiyanagi 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 (basidorsal 1).

The homology of the claustrum (Fig. 5F) has long been debated. Berg (1940) and Chranilov (1927) described the claustrum as a shortened neural spine of vertebra 1. The facts, however, that, unlike the neural spines, the claustrum is paired and forms in cartilage argue against a homology with neural spine 1. Others have described the claustrum as homologous to dissociated elements of the first neural arch (Sarbathi, 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 catostomids, Coburn and Futey (1996) observed separate bilaterally paired cartilage condensations for supraneural 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, supraneural 2 develops as a single cartilage condensation dorsal to vertebra 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 otophysans (7–12 mm) (Coburn and Futey, 1996). This change in developmental 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 (clupeomorphs and salmoniforms), a hypothesis supported by the observation 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 contribute to the vertebral column in zebrafish 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 structures such as the concha scaphium. Determining the somite(s) of origin for these structures can be addressed by future lineage-labeling studies on zebrafish. The homologues of these structures might then be determined by lineage-labeling of closely related fishes.

In D. rerio, we observed an initial small band of cartilage from which significant membranous ossification proceeded anteriorly to form the body of the claustrum. In many cypriniforms, the claustrum has been reported as entirely membranous in origin (Coburn and Futey, 1996), although Coburn and Futey (1996) also describe a cartilaginous claustrum in many other species. It thus appears that the claustrum in closely related species may have different developmental origins. Future lineage-labeling studies on different species of fishes may clarify this issue.

The intercalarium (Fig. 5F), like the scaphium, may be singular or compound 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 develops as a simple anterolateral membrane bone extension from basidorsal 2; no interossicular ossifications 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 accepted as the homologue of neural arch 2.

The tripus (Fig. 5D) has been described as homologous to the parapophysis of vertebra 3 (Chranilov, 1927; Berg, 1940; Vandewalle et al., 1990; Fukushima et al., 1992; Ichiyanagi et al., 1993, 1996; Chardon and Vandewalle, 1997). Others describe 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 develops as a thickened, posteriorly projecting ridge of bone from the parapophysis (transformator process). The remaining body of the tripus is added by means of continued anterior membrane bone extension from the parapophysis. The transformator process is similar in shape to a rib, but the other ribs in Danio develop as separate ossifications that grow toward and articulate with the parapophyses; ribs never develop out of parapophyses. On the other hand, developmentally, the outgrowth (transformator) from the parapophysis more closely resembles a lateral process than a rib. The expression of Hoxc6a and Hoxc6b in the myomere from which vertebra 3 is derived, supports to a rib derivation of this element (see above).

The os suspensorium demonstrates considerable morphologic and developmental variation among otophysans. That the homology of the os suspensorium (Fig. 5F) is not resolved, thus, is not surprising. It has been proposed as a homologue of the rib of the fourth vertebra (Chranilov, 1927; Berg, 1940; Soni et al., 1978), the parapophysis (basiventral) of the fourth vertebra (Watson, 1939; Vandewalle et al., 1990; Fukushima 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 directed, membrane bone extension from the parapophysis. The os suspensorium is similar in shape to a rib, but the other ribs in Danio develop as separate ossifications that grow toward and articulate with the parapophyses. Moreover, the expression of Hoxc6a and Hoxc6b in the myomere from which vertebra 4 is derived lends support to a rib contribution to this element (see above). However, developing as an outgrowth 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 development of the os suspensorium.

The structure termed the “fourth rib” is also continuous with both the os suspensorium and the parapophysis. It develops as posteriorly directed membranous extension from parapophysis 4 (Fig. 5B). None of the other ribs are continuous with their respective parapophyses; they develop as discrete independent elements that grow to articulate with parapophyses. Also, ribs typically develop in an anterior to posterior manner, but in D. rerio, rib 4 develops after rib 5. It is plausible that the fourth rib and the os suspensorium are modifications of the rib + parapophysis of the fourth vertebra.

Variation

Although size (body length) is a better predictor than age (days) of skeletal development in zebrafish and other fishes, the size at which a specific 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 caudal fin vertebrae, for example, hypurals 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 supraneural 7) are as likely as elements that form early (e.g., os suspensorium and tripus) to demonstrate low levels of variation. We noted a lack of variation in the timing of the regions of earliest development along the anterior–posterior body axis (Weberian apparatus and caudal fin). Although this finding may be due to their functional importance, it remains to be tested.

A relative sequence of appearance of bones in the axial skeleton may be derived from the smallest specimen in which a skeletal element is present (Fig. 3, green diamonds) or the size at which an element is always present (Fig. 3, bars). There is little variation in relative sequence 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 relative sequence of development within the caudal fin does not change, e.g., hypural 2 is always present before hypurals 3–5. The sequence of appearance of the elements 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).

Figure 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, purple indicates earliest sign of ossification.

Patterns of Axial Skeleton Development

An overall pattern of development within the axial skeleton emerged from our analysis of the relative timing of ossification events (Fig. 3). As also noted by Du et al. (2001), development within the axial skeleton begins simultaneously within the Weberian region (third and fourth centra) and the caudal fin region (hypural 1 and parhypural; Figs. 2, 3). This finding is notable in that the two regions are at the anterior and posterior limits of the axial skeleton. Two centers of ossification within the vertebral column are common among ostariophyans (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 posteriorly 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 develops from anterior to posterior (Arey, 1940; Rieppel, 1993). The supraneurals develop from anterior to posterior as in other fishes (Mabee, 1988).

Among the median fins, the caudal 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 develops before the more anterior fin. Thus, in D. rerio, the anal fin, which is slightly more posterior than the dorsal fin, develops first. The exoskeleton and endoskeleton of both the dorsal and anal fins develop bidirectionally, a copatterning similarity first noted by Mabee et al. (2002). Bidirectional development of these fins appears to be the basal condition for teleost fishes (Mabee et al., 2002).

CONCLUSION

Our detailed data on the development and anatomy of the zebrafish axial skeleton and median fins provide a baseline against which zebrafish genetic and evo–devo comparisons may be made. As expected, most of the features of the axial skeleton of D. rerio represent the basal (primitive) conditions for phylogenetic levels above the species, and we identify the level at which these characters arose. Ultimately 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 morphology.

Homologies of the Weberian ossicles, which are developmentally complex, are difficult to draw with certainty. New molecular genetic data, such as that from Morin-Kensicki et al. (2002), have provided a new level of understanding for the evolutionary origin of such structures. Our reviews of the evidence for homology of the various elements will be a basis against which the contribution of additional genetic data may be assessed.

We describe skeletal development using size as a measure of developmental stage. Size is more highly correlated with stage of osteological 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 significance of the overall pattern of development within the axial skeleton (Fig. 3) is as of yet unknown. The degree to which this sequence of ossification reflects phylogenetic history can be understood through similar comparative developmental studies on related taxa, and the degree to which it reflects functional constraints 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.

EXPERIMENTAL PROCEDURES

Adult zebrafish were obtained from a local pet supply company (Safariland, Sioux Falls, SD) and maintained in 10-gallon aquaria at 28.5°C (± 1°C) with a 14-hr photoperiod. Although 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 development is expected to be similar. Aquaria were maintained following general suggestions from the Zebrafish Book (Westerfield, 2000). In preparation for spawning, tank bottoms were thoroughly siphoned, and tank levels were restored with fresh water. Spawning protocol followed those outlined previously by Cubbage and Mabee (1996). Fry were fed Paramecium multimicronucleatum and newly hatched brine shrimp. Fry were maintained at 28.5°C in a Fisher Scientific Low Temperature Incubator (Model 307C) for 1 week after fertilization and transferred to aquaria.

Larvae from multiple spawns were pooled to obtain a complete developmental 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% paraformaldehyde and transferred to 70% ethanol for storage or staining.

Specimens were stained with Alcian blue for cartilage, enzymatically cleared with trypsin, and differentially 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 sectioned at 8 μm by using a Leica 2040 autocut microtome. Large specimens (>7 mm) were decalcified before 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 ossification within 1 week of staining. Cartilaginous structures were recorded as present upon visualization of Alcian blue staining of chondrocytes. 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 ossification is expected to remain the same. Thus, sections were only obtained to address specific questions of development within the axial skeleton, primarily in the Weberian apparatus.

Specimens were examined by using 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 measured from the anterior end of the upper jaw to posterior tip of notochord. Notochord length is the standard measure of preflexion larval fishes in ichthyology and fisheries sciences (Ahlstrom and Moser, 1976; Leis and Trnski, 1989). Flexion occurs at approximately 5.5 mm, corresponding to 9–10 days after fertilization (Cubbage and Mabee, 1996, Appendix A for age vs. length chart). Specimens with flexed notochords (∼5.6 mm and larger) were measured from the anterior end of the upper jaw to the posterior end of the hypurals (standard length). Illustrations 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 Photoshop 5.0 software.

GLOSSARY

Actinotrichia: Unsegmented dermal rays in the fin fold of larval fishes; replaced by lepidotrichia.

Anal fin: Median unpaired fin on the ventral surface of the body, posterior to the anus.

Antepenultimate vertebra: Vertebra anterior to the penultimate vertebra (preural 3 in Danio rerio).

Basidorsal: Bilaterally paired cartilage condensations located on the dorsal half of a centrum. Component of “arcualia” in basal actinopterygians (Gadow and Abbott, 1895).

Basiventral: Bilaterally paired cartilage condensations located on the ventral half of a centrum. Component of “arcualia” in basal actinopterygians (Gadow and Abbott, 1895).

Camera aerea Weberiana: Anterior part of the swim bladder (Radermaker et al., 1989) that is in contact with the os suspensorium and the tripus. This term was created by (Chranilov, 1927) for the anterior chamber of the bipartite swim bladder.

Caudal fin: Tail fin; most posterior median fin.

Caudal vertebrae: Posterior vertebrae; the first caudal vertebra has a completely fused hemal arch.

Centra: Central bodies of vertebrae.

Claustrum: Commonly considered the first Weberian ossicle.

Dorsal fin: Median fin(s) on the dorsal 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 (Patterson 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, dorsal 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 membranous median extensions of one or both hemal arches. May also develop autogenously and fuse to the proximal tips of the developing hemal arches.

Hypurals: Laterally flattened median caudal endoskeletal supports which articulate with the urostyle and serve as attachments for the caudal fin rays. Homologous to hemal spines of preural vertebra 1 and the ural vertebrae.

Hypurapophysis: See Parhypurapophysis.

Intercalarium: Third Weberian ossicle. Modification of the neural arches of the second vertebra. Consists of three parts: the manubrium, ascending process, and articulating process.

Intermuscular bones: Long, ray-free bones lateral to the vertebral column. Include epineurals, epicentrals, and epipleurals (Owen, 1846, 1866; Patterson and Johnson, 1995).

Lateral process: Transverse process. Modified parapophyses extending laterally from a centrum. Typically fused to the centrum in Danio rerio.

Manubrium: Anterior branch of the intercalarium. Also termed “anterolateral process.”

Membranous ossification: Direct ossification from mesenchymal precursors.

Neural arch: Bony arch on the dorsal surface of a centrum.

Neural spine: Cartilage or membranous median extensions from neural arches. May also develop autogenously and fuse to the developing arches.

Notochord length (NL): Measurement from the anterior-most tip of the jaw to the posterior-most tip of the notochord.

Os suspensorium: Separate ossification 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, Cypriniformes, Characiformes, Siluriformes, and Gymnotiformes (Fig. 1).

Otophysan: Fishes within the series Otophysi, including Cypriniformes, Characiformes, Siluriformes, and Gymnotiformes (Fig. 1).

Parapophyses: Ventrally projecting 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 (hypurapophysis): Lateral process off of the parhypural that serves as attachment for the hypochordal longitudinal muscle.

Penultimate vertebra: Vertebra anterior to the urostyle (preural 2 in Danio rerio).

Perichondral ossification: Ossification type in which bone forms around a cartilage model.

Postzygapophyses: Bony dorsal (neural postzygapophyses) or ventral (hemal postzygapophyses) extensions from the posterior end of a centrum. They articulate with the prezygapophyses of the immediately posterior centrum.

Precaudal vertebrae: Trunk or abdominal vertebrae. Anterior vertebral region lacking hemal arches and spines, bearing ribs and parapophyses.

Preural vertebrae: Caudal vertebrae anterior to the urostyle and lack hypurals.

Prezygapophyses: Bony dorsal (neural prezygapophyses) or ventral (hemal prezygapophyses) extensions from the anterior end of a neural or hemal arch. They articulate with the postzygapophyses of the preceding centrum.

Radial: Pterygiophore. Endoskeletal fin support of the dorsal or anal fin, to which the fin rays attach. May be unsegmented, bipartite (proximal and distal), or tripartite (proximal, middle, and distal).

Ribs: Pleural ribs, ventral ribs. Intersegmental bones that articulate with parapophyses. They protect and support internal organs.

Roofing cartilage: Cartilaginous larval structure dorsal to the four anterior-most vertebrae, which forms the connection between neural arches and supraneurals in the Weberian apparatus.

Scaphium: First Weberian ossicle. Formed from a modification of the neural arches of the first vertebra. Consists of three parts (concha, ascending process, articulating process).

Standard length (SL): Measurement 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 cartilaginous elements that lie in the median skeletogenous septum between the cranium and the dorsal fin.

Swim bladder: Gas bladder. Gas-filled sac used for buoyancy.

Synchondrosis: Cartilaginous connection 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; ultimate vertebra): Compound final segment of the vertebral column. In zebrafish, it is composed of preural vertebra 1 and ural centra 1 and 2.

Vertebra: Unit of the vertebral column; consists of a centrum, neural arches and spine, and may include hemal arches and spine, or parapophyses and ribs.

Weberian apparatus: Series of vertebral modified ossicles serving to connect the swim bladder to the inner ear.

Weberian vertebrae: Most anterior vertebrae, specialized as the Weberian apparatus (vertebrae 1–4). Typically reduced in length and size.

Acknowledgements

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 EPSCoR and CoBRE (NIH P20 RR15567) for additional funds.

• 1

  The basal condition involves (1) concha scaphii fits into a notch in the basi- and exoccipital; (2) all ossicles and claustrum present and distinct; (3) scaphium and intercalarium possess ascending and articular processes; (4) tripus with large, crescent-shaped transformator; (5) fourth vertebra with recognizable parapophyses and ribs, the os suspensorium a simple anterior limb, free from anterior vertebrae; (6) large supraneurals 2 and 3 articulating on, or continuous through cartilage with the basidorsals of the third and fourth vertebrae and the exoccipitals; (7) a claustrum just above the concha scaphii, continuous through cartilage with supraneural 2.