Botulinum Toxin Induces Muscle Paralysis and Inhibits Bone Regeneration in Zebrafish

Abstract

Intramuscular administration of Botulinum toxin (BTx) has been associated with impaired osteogenesis in diverse conditions of bone formation (e.g., development, growth, and healing), yet the mechanisms of neuromuscular-bone crosstalk underlying these deficits have yet to be identified. Motivated by the emerging utility of zebrafish (Danio rerio) as a rapid, genetically tractable, and optically transparent model for human pathologies (as well as the potential to interrogate neuromuscular-mediated bone disorders in a simple model that bridges in vitro and more complex in vivo model systems), in this study we developed a model of BTx-induced muscle paralysis in adult zebrafish, and examined its effects on intramembranous ossification during tail fin regeneration. BTx administration induced rapid muscle paralysis in adult zebrafish in a manner that was dose-dependent, transient, and focal, mirroring the paralytic phenotype observed in animal and human studies. During fin regeneration, BTx impaired continued bone ray outgrowth, morphology, and patterning, indicating defects in early osteogenesis. Further, BTx significantly decreased mineralizing activity and crystalline mineral accumulation, suggesting delayed late-stage osteoblast differentiation and/or altered secondary bone apposition. Bone ray transection proximal to the amputation site focally inhibited bone outgrowth in the affected ray, implicating intra- and/or inter-ray nerves in this process. Taken together, these studies demonstrate the potential to interrogate pathological features of BTx-induced osteoanabolic dysfunction in the regenerating zebrafish fin, define the technological toolbox for detecting bone growth and mineralization deficits in this process, and suggest that pathways mediating neuromuscular regulation of osteogenesis may be conserved beyond established mammalian models of bone anabolic disorders.

Osteogenic programs during zebrafish fin regeneration

“…22 Using a combination of motor activity and behavioral assays, we showed that the paralytic effects of BTx in adult zebrafish were site-dependent, transient and focal, mimicking the paralysis observed in both animal and human studies. When subjected to tail fin regeneration, BTx impaired bone outgrowth, patterning and mineral accrual.…”

“…Using this technique, we found that birefringence was significantly decreased in the regenerated bone rays of BTx-treated fish. 22 More recently, by imaging the same fin using Rotopol microscopy and microCT, we showed that birefringence is highly correlated with TMD (Figure 2), except between joints (where there is additional birefringence from the inter-segment ligament 7 ). The development of a rapid (B2 Â to 10 Â faster than microCT), light-based modality for bone mineralization imaging during fin regeneration opens new opportunities for multi-scale and systems-based investigations in this system.…”

“…To overcome this challenge, we recently developed a novel approach to quantify bone mineral accrual during zebrafish fin regeneration using Rotopol microscopy. 22 In this technique, a custom microscope is used to acquire sequential images of the fin under a step-wise rotating polarizer. By applying the appropriate relations, 68 birefringence, transmittance and orientation can be independently computed on a pixel-by-pixel basis, directly enabling quantitative analysis.…”

“…Although the regenerative capacity of the fin depends greatly on the blastema, recent studies suggest that the redevelopment phase involves major pathways known to be involved in mammalian bone growth, 19,20 patterning 21 and mineralization. 22 Several excellent reviews have described the value of fin regeneration as a tractable model of regenerative biology, 18,23,24 as well as the broader utility of fish models for bone biomedical research. 5,8,10,24 However, an updated review of the potential applications for fin regeneration as a model of bone growth and mineralization has been lacking.…”

MicroCT-Based Phenomics in the Zebrafish Skeleton Reveals Virtues of Deep Phenotyping in a Distributed Organ System

“…Studies were conducted in mixed sex adult zebrafish. WT ARO and AB fish were obtained from Aquatic Research Organisms (Recidoro et al, 2014) and the Zebrafish International Resource Center (ZIRC, http://zebrafish.org), respectively. opallus b1071 (McMenamin et al, 2014) and pissarro utr8e1 (Quigley et al, 2004) were isolated in forward genetic screens.…”