Identification of skeletal deformities towards deep phenotyping of zebrafish (Danio rerio) connective tissue disease models

Background/Introduction: Skeletal deformities in teleost fish have already been extensively described in studies on Atlantic salmon and zebrafish. Nevertheless, a toolset encompassing thorough identification and description of vertebral column deformities to study zebrafish models for human diseases with skeletal involvement is currently lacking. Purpose: A detailed characterization of skeletal deformities by identifying, describing and quantifying the anomalies will facilitate the development of a reliable deep-phenotyping tool. This tool can be used to establish data matrices by scoring anomalies present in different zebrafish models, which can be used to quantitatively distinguish mild and severe phenotypes. The ability to determine phenotypic severity in disease models is extremely valuable for proper translation towards human diseases, but also to reveal candidate modifier genes that contribute to intrafamilial skeletal variability. Methods: Zebrafish, 13 col1a1amh13/+, 13 col1a1adc124/+, 11 col1a2mh15/+ and 27 WT siblings, were fixed, and made translucent with a mixture of 4% formalin, Triton X-100 and potassium hydroxide (KOH). Subsequently, whole mount bone staining was performed with an Alizarin red S/KOH solution, followed by clearing in a glycerol series. Observations of the skeleton were made using a binocular microscope (Leica M165FC) with a fluorescent unit and equipped with a Leica DFC 450 C camera. Results: In total, 15 skeletal deformity types were identified and defined: (i) fusion, (ii) compression, (iii) vertical shift of the vertebra, (iv) fractures, (v) curvy ribs), (vi) extra intramembranous bone on the arches and spines (associated elements)and vertebral centra, (vii) bent associated elements, (viii) double associated elements, (ix) detached associated elements, (x) notochord tissue mineralization, (xi) intervertebral ligament mineralization, (xii) lordosis, (xii) kyphosis, (xiii) scoliosis and (xiv) torsion of the vertebral column around the central axis. Conclusion(s): Deep phenotyping of zebrafish models for skeletal disease will lead to better understanding of expressed phenotypes and of the underlying mechanisms and may lead to identifying new therapeutic targets