A preliminary description of the anatomy and development of the zebrafish axial skeleton

Although the zebrafish, Danio rerio, is used extensively as a model organism for genetic and developmental studies, accurate descriptions of axial skeleton development are lacking. In fact, a detailed description of axial skeleton development has not been made for any cyprinid fish, though some elements have been described generally in the more inclusive group, the Ostariophysi (Howes 1980; Fink & Fink 1981). Using whole mount bone and cartilage staining we are collecting such baseline data for zebrafish. Specifically we are describing the development of the vertebrae (centra, neural and hemal arches, neural and hemal spines, and ribs), the Weberian apparatus (a complex structure consisting of bones modified from the first four vertebrae), and the median fins. Modal numbers and axial positions of all skeletal elements and a summary of their sequence and timing will also be a result of this study. These data are critical for appropriate timing of in situ studies, tests of gene function, and analysis of late-stage mutant skeletal phenotypes. In order to reduce confusion and foster progress between the areas of developmental and evolutionary biology, it is important to use uniform and informative terminology that is "standard" for a field. Herein we present an outline and preliminary description of the components of the axial skeleton, choosing ichthyological terms that are considered standard because they convey information on homology, the continuity of information from ancestor to descendant. The uniform use of a common terminology by the zebrafish community will facilitate comparisons among vertebrate taxa in an evolutionary context.

Methods

Adult zebrafish were obtained from a local wholesale company (Bailey Wholesale Tropical Fish, San Diego, CA) and maintained in ten gallon aquaria at 28.5°C (±1.0°C) with a 14 hr photoperiod. Aquaria were maintained and adults spawned following general suggestions from The Zebrafish Book (Westerfield 1995).

The description of axial development is based on a developmental series of individuals from very young larvae (3.0 mm) to adults (35.0 mm). The earliest sign of ossification, based on uptake of Alizarin red which stains mineralized bone matrix, was recorded as presence of that bone. The nonmineralized osteoid of some bones is visible before stain uptake, and this was also recorded as the presence of bone. Although histological sections would identify bones as "present" before (in smaller and younger larvae) than the clearing and staining whole mount procedure, based on previous data (Clark and Smith, '93), the relative sequence of ossification is expected to be the same.

Specimens were examined using a Wild M5 stereo microscope at magnifications of 11-94X, measured using an ocular micrometer, and recorded to the nearest 0.1 mm. Pre-notochord flexion and early flexion individuals were measured from the anterior end of the upper jaw to posterior tip of notochord (notochord length, NL). Notochord length is the standard measure of preflexion larval fishes in ichthyology and fisheries sciences (Ahlstrom et al. 1976; Leis & Trnski 1989). Flexion occurred at about 5.5 mm of length, and approximately 9-10 days post-fertilization. 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, SL). Illustrations were traced from images collected by a videocamera and computer. In the description of individual bones, we report the size of the smallest specimen in which an ossification is present. Due to variation in timing, however, the ossification may not be present in all specimens of that size. Throughout the text, notochord length (NL) is designated where appropriate; all undesignated lengths are standard length (SL).

Outline and preliminary description

General development of ostariophysan fishes can be found in Fink and Fink (1981). Development of specific ostariophysan fishes can be found in Howes (1978), Howes (1980), and Weitzman (1962).

I. Vertebrae: Maximally, a vertebra is composed of a centrum, a pair of neural arches, a neural spine, a pair of hemal arches, and a hemal spine.

A. Precaudal vertebrae: Precaudal vertebrae are comprised of the most anterior one of two regionalized groups of vertebrae in fishes. The precaudal vertebrae are characterized as lacking fused hemal arches and possessing ribs. The modal number of precaudal vertebrae is 13; Ferreri (2000) gives a range of 12-14.

Weberian apparatus: The Weberian apparatus is a complex group of bones that have been modified from the anteriormost four vertebrae. They are, from anterior to posterior, the claustrum, scaphium, intercalarium, and tripus. These bones connect the swimbladder (gas bladder) to the inner ear of the fish, allowing for greater sensitivity to vibration and acute hearing. The claustrum is a modified neural spine of the first vertebra. The scaphium is modified a neural arch of the first vertebra. The intercalarium is a modified neural arch of the second vertebra, the tripus is a modified parapophysis of the third vertebra, and the os suspensorium is a medial extension off of the elongated rib of the fourth vertebra.

The Weberian apparatus is unique to otophysian teleosts which include members of Cypriniformes (minnows, e.g. zebrafish), Characiformes (characins), Siluriformes (catfishes), and Gymnotiformes (South American electric fishes) (Moyle and Cech 2000). However, the Weberian apparatus has been described for other cyprinids including the Indian Major Carp, Labeo rohita (Kulshrestha 1977); the Bream, Abramis brama; the Roach, Rutilus rutilus; the Bleak, Alburnus alburnus; Macrochirichthys macrochirus (Howes 1979) and the fan-tail Goldfish, Carassius auratus (Bogutskaya 1991). General development of the Weberian apparatus within Cypriniformes has also been described (Ramaswami 1955; Mehta 1983-1984; Coburn and Futey 1996).

Precaudal vertebrae posterior to the Weberian apparatus (sometimes termed the "pre-haemal" region (Ferreri et al. 2000): These 9 vertebrae are all rib-bearing.

B. Caudal vertebrae: Caudal vertebrae comprise the posterior group of vertebrae in fishes. The first (most anterior) caudal vertebra is defined as having a complete (fused) hemal arch; they lack ribs. The modal number of caudal vertebrae (including the specialized vertebrae of the caudal fin is 18, Ferreri (2000) gives a range of 15-19. Sometimes termed the "haemal" vertebrae.

II. Caudal fin (tail fin): The caudal fin is the most posterior median fin. It is composed of a complex of three modified centra and modified neural and hemal arches and spines. These structures are given specialized names and include the following:

A. Specialized centra/vertebrae:

• Preural 2 (PU2): This vertebra is anterior to preural centrum 1 and is sometimes termed the "antepenultimate" vertebra.
• Preural 1 (PU1): This vertebra is anterior to the urostyle and is sometimes termed the "penultimate" vertebra.
• Urostyle: This centrum may be formed via the fusion of the terminal centrum and half-centrum. It is sometimes termed the "ultimate" vertebra.

B. Specialized neural arches and spines:

• Uroneurals: Modified neural arches that develop lateral and slightly posterior to the urostyle.
• Epurals: Modified neural spines that develop slightly above the urostyle. In the zebrafish there is one epural, located above the urostyle. It develops as a slender cartilaginous rod that ossifies later in development.

C. Specialized hemal arches and spines:

• Hypurals: The hypurals are expanded and laterally flattened hemal arches and spines. They support the lepidotrichia of the caudal fin. The hypurals form ventral to the posterior tip of the dorsally-flexed notochord. In zebrafish condensations of the hypurals begin forming at approximately 6 dpf (4.1 mm NL, notochord length).
• Parhypural: Hemal arch and spine of preural centrum 1. The parhypural is defined as the most posterior hemal arch with an open canal through which the ventral aorta passes. The parhypurapophyses (also "hypurapophyses") are the paired bony lateral extensions off the hemal arch of the parhypural. They serve as origin for the anterolateral bundle of the hypochordal longitudinal muscle (Lundberg and Baskin 1969).

III. Neural arches and spines: The neural arches form as bilaterally-paired cartilaginous condensations on the dorsal half of each centrum. In development these condensations grow dorsally and typically fuse mid-laterally, forming the neural arch, which encircles the neural tube. The neural spine is formed as a single dorsal extension of the fused neural arch. The neural arches and spines, are variously modified at the anterior (Weberian apparatus) and posterior (caudal fin) extremes of the fish as described.

IV. Hemal arches and spines: The hemal arches form as bilaterally-paired cartilaginous condensations on the ventral half of the caudal centra. In development these condensations grown ventrally and typically fuse mid-laterally, forming the hemal arch, which encircles the ventral aorta. The hemal spine is formed as a single ventral extension of the fused hemal arch. The hemal arches and spines of the caudal fin centra (above) are variously modified.

V. Fin rays (exoskeletal fin supports):

• Actinotrichia: Actinotrichia are strengthening fibers of elastoidin, which are laid down by the adjacent ectoderm in the developing finfolds of fishes (Bouvet 1974). Stiffening rays composed of elastoidin evolved first in the median finfold of cephalochordates, and they are retained in basal gnathostomes (Goodrich 1930).
• Lepidotrichia: Lepidotrichia are bony, bilaterally-paired, segmented fin rays. These develop around actinotrichia as part of the dermal exoskeleton; they evolved in Osteichthyes (Lauder and Liem 1983). Segmentation of lepidotrichia does not occur until after they have completely formed. Caudal fin lepidotrichia are neural crest in origin in zebrafish (Smith et al. 1994).

VI. Radials: Radials (also "pterygiophores") are rod-like median skeletal supports of the fin rays (lepidotrichia) of the dorsal and anal fins. In zebrafish, development of these structures begins as a central group of cartilage condensations at approximately the middle of the presumptive fin. Later in development radials segment proximo-distally into two parts, the larger proximal and smaller distal radials. The distal radials articulate directly with the lepidotrichia.

• Dorsal Fin radials: The modal number of dorsal fin radials for the zebrafish is 8; Ferreri (2000) gives a range of 7-9. Dorsal fin radials begin forming at ~12 dpf (5.6 mm SL).
• Anal Fin radials: The modal number of anal fin radials for the zebrafish is 13; Ferreri (2000) gives a range of 11-15. Anal fin radials begin forming at ~11 dpf (5.4 mm SL, standard length).

VII. Supraneurals: Supraneurals are median structures that are located anterior to the dorsal fin pterygiophores and posterior to the skull. They are slender skeletal rods that may be homologous to neural spines or radials (Mabee 1988). Also termed "predorsals" in perciform fishes.

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