In Toto Imaging of Dynamic Osteoblast Behaviors in Regenerating Skeletal Bone

Abstract

SUMMARY Osteoblasts are matrix-depositing cells that can divide and heal bone injuries. Their deep tissue location and the slow progression of bone regeneration challenge attempts to capture osteoblast behaviors in live tissue at high spatiotemporal resolution. Here, we have developed an imaging platform to monitor and quantify individual and collective behaviors of osteoblasts in adult zebrafish scales, skeletal body armor discs that regenerate rapidly after loss. Using a panel of transgenic lines that visualize and manipulate osteoblasts, we find that a founder pool of osteoblasts emerges through de novo differentiation within one day of scale plucking. These osteoblasts undergo division events that are largely uniform in frequency and orientation to establish a primordium. Osteoblast proliferation dynamics diversify across the primordium by two days after injury, with cell divisions focused near, and with orientations parallel to, the scale periphery, occurring coincident with dynamic localization of fgf20a gene expression. In posterior scale regions, cell elongation events initiate in areas soon occupied by mineralized grooves called radii, beginning approximately 2 days postinjury, with patterned osteoblast death events accompanying maturation of these radii. By imaging at single-cell resolution, we detail acquisition of spatiotemporally distinct cell division, motility, and death dynamics within a founder osteoblast pool as bone regenerates.

The transcriptome of regenerating zebrafish scales identifies genes involved in human bone disease

“…We first confirmed that,regenerating scales show an increase in the number of sp7 positive osteoblasts compared to original scales formed during development (ontogenetic) as previously reported (Cox et al, 2018, de Vrieze et al, 2014) ( figure 1A). Alkaline phosphatase (ALP) staining showed that regenerating scales contained more ALP activity (figure 1B).…”

“…Following removal of the ontogenetic scale from the dermal socket initiating a wound healing inflammation phase, regeneration is initiated and a small mineralised scale plate can be observed as early as two days post-harvest (dph) (Bereiter-Hahn & Zylberberg, 1993, Richardson, Slanchev et al, 2013, Sire & Akimenko, 2004. Regenerating scales have a high density of osteoblasts at the posterior edge of the plate forming the leading growth plane whose dynamics can be tracked in toto (Cox, De Simone et al, 2018). These osteoblasts form a (hyposquamal) monolayer and deposit hydroxyapatite into a type I collagen rich matrix that can be resorbed by osteoclasts (de Vrieze, Sharif et al, 2011, Guellec & Zylberberg, 1998.…”

Zebrafish: A Resourceful Vertebrate Model to Investigate Skeletal Disorders

“…Tracing bone cells in vivo using transgenic lines in adult zebrafish is challenging due to tissue depth and complexity, but is possible in external structures such as fin rays or scales, which are easily accessible and suitable for regeneration studies (198,230,231). Indeed, the available panel of transgenic lines expressing fluorescent and photo-switchable reporter genes in bone cells is useful to trace regeneration in vivo (198). This strategy has clarified important biological aspects such as the cellular basis of integumentary bone regeneration.…”

Model systems for regeneration: zebrafish

“…In the case of adult zebrafish, in vivo imaging of regenerating internal organs can prove more difficult. Nevertheless, short-and long-term imaging of regeneration of external structures have been achieved using fluorescent light microscopy, as described for caudal fin neoangiogenesis (Xu et al, 2015), regeneration of the skin epidermis (Chen et al, 2016) or scale regeneration (Cox et al, 2018). Although more challenging, even internal structures can be imaged and followed through time, as has been shown for adult neural stem cells in homeostasis and during regeneration (Barbosa et al, 2015;Dray et al, 2015).…”