Embryogenic Zebrafish Skeleton Development: High Resolution Micro-CT Imaging for Phenotype Identification

Legends for the videos

Video 1: Micro-CT view of the bones in the 30 dpf wild type zebrafish showing only the head and first vertebrae of the axial skeleton.

Pause 1: Note the 5 vertebrae bearing ribs, and between the vertebrae and the head, the Weberian apparatus (white arrow).

Pause 2: View from the dorsal surface of the head. The smooth and rounded large otoliths are the lapilli, the lentil-shaped bodies are the asterisci, and the sagittae cannot be seen.

The movie then zooms in to show the elongated teeth attached to the jaws (white arrows).

Video 2: FIB SEM in the SSV mode showing first the aligned stack of X images of a pair of hemi-cylindrical tail fin bones (lepidotrichia) in transverse section. The smooth high contrast objects are the dense mineralized bones. The pigment cells also produce high contrast due to the presence of heavy ions and stain. The pigment cells are enclosed inside a sack. After moving through the image stack, the dense contrasting materials are surface rendered to show their 3D structures.

Pause 1: the hemi-cylindrical shape of the bones can be recognized, and the pigment sack is seen to be highly elongated. Of particular interest is the ”grey” elongated granular structure just below the upper left hemi-cylinder (white arrow).

Pause 2: After 90° rotation, this grey elongated granular structure is seen to be located below the entire upper hemi-cylinder (white arrow).

Next is a view through the stack of images starting from the dorsal surface with a pause showing the elongated granular structure located between two smooth dark collagen bundles (actinotrichia) (white arrow).

Video 3. FIB SEM in the SSV mode of a demineralized fin tail bone (lepidotrichia) showing the stack of X images in the transverse section.

Pause 1: a cell and associated tissue (grey) overlain by the collagen fibrils isosurface rendered in yellow.

Collagen layer is rotated 90°.

Pause 2: transverse view through the demineralized bone section showing that the upper and lower surfaces are composed of aligned fibrils, whereas the center is dominated by disordered fibrils.

This is followed by a series of images from the stack starting with the dorsal surface and moving to the ventral surface. Note that the line in the center is an artifact.

Pause 3: end of the ordered zone.

Pause 4: end of the disordered zone.

Last sequence shows the ventral ordered layer.

 

Video 4: Micro-CT view of the head and upper axial skeleton of the nacre albino line at 30 dpf. By comparing to video 1 of the 30 dpf wild type it can be seen that micro-CT reveals all the bones, as well as the otoliths of the head and the vertebra. Many of these bones cannot be seen when stained by calcein. Note that the small dense fragments are from the ingested food.