Enhanced contrast synchrotron X-ray microtomography for describing skeleton-associated soft tissue defects in zebrafish mutants

Abstract

Detailed histological analyses are desirable for zebrafish mutants that are models for human skeletal diseases, but traditional histological techniques are limited to two-dimensional thin sections with orientations highly dependent on careful sample preparation. On the other hand, techniques that provide three-dimensional (3D) datasets including µCT scanning are typically limited to visualizing the bony skeleton and lack histological resolution. We combined diffusible iodine-based contrast enhancement (DICE) and propagation phase-contrast synchrotron radiation micro-computed tomography (PPC-SRµCT) to image late larval and juvenile zebrafish, obtaining high-quality 3D virtual histology datasets of the mineralized skeleton and surrounding soft tissues. To demonstrate this technique, we used virtual histological thin sections and 3D segmentation to qualitatively and quantitatively compare wild-type zebrafish and nkx3.2-/- mutants to characterize novel soft-tissue phenotypes in the muscles and tendons of the jaw and ligaments of the Weberian apparatus, as well as the sinus perilymphaticus associated with the inner ear. We could observe disrupted fiber organization and tendons of the adductor mandibulae and protractor hyoideus muscles associated with the jaws, and show that despite this, the overall muscle volumes appeared unaffected. Ligaments associated with the malformed Weberian ossicles were mostly absent in nkx3.2-/- mutants, and the sinus perilymphaticus was severely constricted or absent as a result of the fused exoccipital and basioccipital elements. These soft-tissue phenotypes have implications for the physiology of nkx3.2-/- zebrafish, and demonstrate the promise of DICE-PPC-SRµCT for histopathological investigations of bone-associated soft tissues in small-fish skeletal disease models and developmental studies more broadly.

The overlooked first intercostal ligament: Does it help to stabilize the Weberian apparatus?

“…Juvenile zebrafish ( Danio rerio , AB line) were obtained at 1mpf and 2mpf (8.5mm and 24.0mm SL, respectively). Following euthanasia using an overdose of MS-222 (300mg/L) and fixation in 4% paraformaldehyde, diffusible iodine-based contrast enhancement and propagation phase-contrast synchrotron radiation micro-computed tomography (DICE-PPC-SRµCT) was performed as previously described by Leyhr et al (2023), at BM05 of the European Synchrotron Radiation Facility - Extremely Brilliant Source (ESRF-EBS) in France, and with voxel sizes of 0.727µm and 3µm. Reconstructed jpeg2000 image stacks were imported into VGStudio MAX (v3.5.1) for manual segmentation and rendering.…”

“…Chardon and Vandewalle (1997) mention a “suspensor” ligament, however based on location, connectivity, shape (thick and crescent-shaped) and composition (collagen), they likely described the triple ligament. The suspensor ligament has also been described in Gobio (Vandewalle, 1974), Barbus (Vandewalle, 1989), zebrafish (Bird et al, 2020b; Leyhr et al, 2023), Botia (Alexander, 1964a) and more broadly across cypriniforms (Alexander, 1962; Bird et al 2020a).…”

“…In contrast, effects in Weberian vertebrae appear less severe, which aligns with the limited defects seen in the patterning of presumptive Weberian somites with regard to muscle fiber and dermomyotome patterning when compared to more posterior somites (Windner et al 2012). Recently, the role of nkx3.2 in Weberian apparatus development was examined, with null mutants revealing substantial defects in the axial skeleton (Waldmann et al, 2021) as well as in both the mineralized skeletal and soft tissue elements of the vertebral region of the Weberian apparatus (Leyhr et al 2023).…”

The sensory shark: high-quality morphological, genomic and transcriptomic data for the small-spotted catsharkScyliorhinus caniculareveal the molecular bases of sensory organ evolution in jawed vertebrates

“…Iodine staining and synchrotron scanning of the head of a S. canicula hatchling (8cm total length) were performed as previously described (Leyhr et al 2023). Briefly, the head was fixed in 4% paraformaldehyde, rinsed in 1x phosphate-buffered saline, and gradually dehydrated in 25%, 50%, 75%, and 100% ethanol.…”

Staining and resin embedding of wholeDaphniamagna samples for micro-CT imaging enabling 3D visualization of cells, tissues, and organs of various thicknesses

“…Micro-computed tomography (micro-CT) is increasingly recognized as a valuable imaging technique for generating three-dimensional datasets that enable 3D visualization, and qualitative and quantitative analysis of biological samples. Imaging of whole, intact samples allows detailed investigation of overall morphology and cellular structures is especially useful for evaluation of microanatomy and phenotypes in various model organisms [1][2][3][4][5]. Daphnia magna is a keystone branchiopod crustacean (order Cladocera) in freshwater lotic ecosystems worldwide and is an established model in ecology and evolution [6,7].…”