Characterizing slc8a4b as an Osteoblast Marker in Danio rerio

Abstract

The solute carrier family 8 member 4b (slc8a4b) is an emerging marker for osteoblasts in zebrafish, enabling further study of healthy bone development, bone regeneration, and skeletal diseases. To guide future use of slc8a4b as an osteoblast marker, this study aims to identify which osteoblast functions slc8a4b expression is most closely associated with, describe where in developing bone tissue slc8a4b is expressed, and demonstrate the utility of slc8a4b to demarcate cells poorly captured by other markers. The analysis of pre-existing single-cell RNA-sequencing (scRNA-seq) data showed osteoblasts expressing slc8a4b had similar transcriptional profiles as cells marked by other osteoblast markers but reduced expression of matrix synthesis genes. The spatial expression of slc8a4b in the 3 days post fertilization (dpf) opercle and cleithrum was visualized using Hybridization Chain Reaction RNA Fluorescence in situ hybridization (HCR RNA-FISH). These images revealed more centralized expression of slc8a4b compared to runx2b and sp7 in the 3dpf opercle and compared to runx2b in the dorsal portion of the 3dpf cleithrum. HCR images of tnfrsf11a (RANK), recently identified as an important osteogenic regulator, showed substantial overlap in its spatial expression with slc8a4b suggesting slc8a4b’s potential use in future studies of tnfrsf11a. The utility of slc8a4b as a marker was reinforced by the scRNA-seq data that showed the slc8a4b expression to be high and specific within osteoblasts, more osteoblasts expressing slc8a4b than any other non-collagen osteoblast marker, and substantial co-expression of slc8a4b in cells expressing other osteoblast markers. This specificity to osteoblasts, similarity and overlap with other markers, and large number of expressing cells, makes slc8a4b an excellent tool for future osteoblast research in zebrafish.