Dynamics of the Zebrafish Skeleton in Three Dimensions During Juvenile and Adult Development

Abstract

Zebrafish are a valuable model for normal vertebrate skeletogenesis and the study of myriad bone disorders. Bones grow, ossify and change shape throughout the zebrafish lifetime, and 3D technologies allow us to examine skeletogenic processes in detail through late developmental stages. To facilitate analysis of shape, orientation and tissue density of skeletal elements throughout ontogeny and adulthood, we generated a high-resolution skeletal reference dataset of wild-type zebrafish development. Using microCT technology, we produced 3D models of the skeletons of individuals ranging from 12 to 25 mm standard length (SL). We analyzed the dynamics of skeletal density and volume as they increase during juvenile and adult growth. Our resource allows anatomical comparisons between meristic units within an individual-e.g., we show that the vertebral canal width increases posteriorly along the spine. Further, structures may be compared between individuals at different body sizes: we highlight the shape changes that the lower jaw undergoes as fish mature from juvenile to adult. We show that even reproductively mature adult zebrafish (17-25 mm SL) continue to undergo substantial changes in skeletal morphology and composition with continued adult growth. We provide a segmented model of the adult skull and a series of interactive 3D PDFs at a range of key stages. These resources allow changes in the skeleton to be assessed quantitatively and qualitatively through late stages of development, and can serve as anatomical references for both research and education.

Keywords: juvenile development; microcomputed tomographic (micro-CT); skeletal anatomy; skeletogenesis; zebrafish.

FIGURE 1

Cross sections of microCT scans visualized in 3D Slicer. (A), Surface rendering of 24 mm SL adult fish with lateral (top) and dorsal (bottom) views with coronal (blue), transverse (green), and sagittal (red) axes indicated. (B), Coronal cross section image. (C), Transverse cross section image. (D), Sagittal cross section image. (E), Quantification of vertebral canal width of 3 individual 24 mm SL adult fish; each individual differentiated with different shapes (circle, square, triangle). Inset shows higher resolution image through vertebrate (corresponding to the boxed detail in panel D). Red arrow indicates a canal from which interior width was measured. Scale bars, 2 mm.

FIGURE 2

Increasing skeletal density and volume with linear growth. (A), Relative density renderings of skeletons from zebrafish at four different sizes (12, 16, 20, and 24 mm SL). Warmer colors indicate higher density regions. (B), Average density of zebrafish skeleton along the body length of individual zebrafish at four sizes. (C). Volume renderings of zebrafish at four sizes. Scale bars, 2 mm.

FIGURE 3

Quantified skeletal density and volume relative to linear growth. (A), Bone density relative to SL. (B), Total bone volume relative to SL. Bone volume calculated from all cross sections of scan.

FIGURE 4

Anatomy of the craniofacial skeleton. (A), Volume rendering of the skull of a 12 mm SL zebrafish and (B), a 24 mm SL zebrafish. (C), Lateral view of skull of 24 mm SL zebrafish with segmented bones (left). Cross section of lateral view reveals some internal elements (right). b, basibrachials; ba, branchial arches; boc, basioccipital; d, dentary; dm, dermatocranium; ec, ectopterygoid; eoc, exocciptal; en, entopterygoid; h, hyoid; hm, hyomandibula; io, infraorbital; iop, interopercle; k, kinethmoid; le, lateral ethmoid; m, maxilla; mpt, metapterygoid; op, opercle; os, orbitosphenoid; pj, pharyngeal jaws; pm, premaxilla; pop, preopercle; ps, parasphenoid, q, quadrate; sc, supracleithrum; soc, supraoccipital; sop, subopercle, su, supraorbital. Scale bars, 1 mm.

FIGURE 5

Shape change of the lower jaw and caudal vertebrae. (A), Segmented lower jaws from 12, 16, 20, and 24 mm SL individuals, viewed from the ventral perspective. In the largest individuals, note the pronounced anguloarticular prominence (arrow) and posterior end of lower jaw (arrowhead). (B), Segmented first three caudal vertebrae from 12, 16, 20 and 24 mm SL individuals, viewed from a lateral perspective. Scale bars, 0.5 mm.