Fish larvae experience many environmental challenges during development such as variation in water velocity, food availability and predation. The rapid development of structures involved in feeding, respiration and swimming increases the chance of survival. It has been hypothesized that mechanical loading induced by muscle forces plays a role in prioritizing the development of these structures. Mechanical loading by muscle forces has been shown to affect larval and embryonic bone development in vertebrates, but these investigations were limited to the appendicular skeleton. To explore the role of mechanical load during chondrogenesis and osteogenesis of the cranial, axial and appendicular skeleton, we subjected zebrafish larvae to swim-training, which increases physical exercise levels and presumably also mechanical loads, from 5 until 14 days post fertilization. Here we show that an increased swimming activity accelerated growth, chondrogenesis and osteogenesis during larval development in zebrafish. Interestingly, swim-training accelerated both perichondral and intramembranous ossification. Furthermore, swim-training prioritized the formation of cartilage and bone structures in the head and tail region as well as the formation of elements in the anal and dorsal fins. This suggests that an increased swimming activity prioritized the development of structures which play an important role in swimming and thereby increasing the chance of survival in an environment where water velocity increases. Our study is the first to show that already during early zebrafish larval development, skeletal tissue in the cranial, axial and appendicular skeleton is competent to respond to swim-training due to increased water velocities. It demonstrates that changes in water flow conditions can result into significant spatio-temporal changes in skeletogenesis.

The proper formation, growth and maintenance of many bones depends on the mechanical loads generated by gravity and muscles. Mechanical loading by muscle forces does not only affect bone growth and maintenance in adult and juvenile vertebrates, but also affects larval and embryonic bone development. We have reviewed the current understanding of mechanotransduction in birds and mammals and compared it to teleosts. The major mechanosensing cells in the adult mammalian and avian skeleton are osteocytes. They are interconnected via cell processes and are contained within a canalicular network. Basal teleosts have osteocytes but their connectivity is questionable and the presence of a functional canalicular network is unlikely. Advanced teleosts have acellular bone and therefore lack osteocytes. Yet the skeleton of teleosts does show adaptive responses to changes in mechanical load. In these animals it is likely that osteoblasts, bone surface cells and chondrocytes act as mechanosensors. The factors expressed by osteocytes upon mechanical stimulation have been extensively investigated in vitro and in vivo in adult mammals and birds. Less is, however, known about the mechanotransduction pathway during embryonic bone development. The zebrafish presents new opportunities to analyze the mechanotransduction pathway during early (larval) bone formation due to the ex utero development and genetic analyses.

SummaryIn adult vertebrates, endurance training leads to physiological, metabolical and molecular adaptations which improve endurance performance. Only very few studies have focused on adaptive responses to endurance training during early vertebrate development, and molecular data is limited. Here, we explored the effect of swim-training on the transcriptome of the zebrafish during early development on a quantitative and spatial gene expression level. We subjected larval zebrafish from 5 to 14 dpf (days post fertilization) to swim-training and performed a whole genome microarray analysis of trained and control fish sampled at 10 dpf. In addition, we investigated if swim-training affected the expression of genes involved in muscle growth and structure with quantitative real-time PCR in trained and control fish sampled at 5 and 14 dpf. To obtain a general overview of the effects of swimtraining on the transcriptome, we selected 52 genes from the whole genome microarray analysis based on a number of criteria. In situ hybridization demonstrated that 15 genes were specifically expressed in the brain, muscle, kidneys, liver, pancreas or intestines. Thus, swim-training led to molecular changes already after 6 days of swim-training and in a variety of organ systems. In addition, the expression of slow fiber markers was increased after 10 days of swim-training, indicating that muscle can already shift towards a slow aerobic phenotype during zebrafish larval development. Taken together, this study demonstrates that significant changes occur, even at early stages, as an adaptive response to endurance training during early vertebrate development.

Summary In vitro and in vivo studies have shown that mechanical forces play an important role during development. The molecular mechanisms via which mechanical forces regulate development have been extensively investigated by in vitro studies. However, knowledge about the molecular pathways that mediate the effect of mechanical forces during development in vivo is limited. Previously, we showed that swim‐training increased maximum normalized curvatures in the caudal fin (suggesting that the caudal fin experienced increased mechanical loads) and prioritized the development of skeletal structures in the caudal fin. Therefore, we used the zebrafish caudal fin to explore the molecular link between an increased swimming activity and development in vivo. Whole genome microarray analysis of caudal fins of zebrafish subjected to swim‐training and control fish identified 46 genes which were up‐regulated with a fold change of 1.5 or larger at 10 dpf. Fourteen genes were expressed specifically in the following tissues in the caudal fin: the neural tube, the tissue surrounding the hypurals, the finfold, or muscle fibers. Subsequently, we identified two muscle specific genes, aste1 (asteroid homolog 1) and zgc:65811, which showed an increased expression specifically in the caudal fin in response to swim‐training. This makes these genes interesting candidate genes for further research on the molecular link between mechanical forces and caudal fin development. Our study is the first to investigate the molecular link between swim‐training and caudal fin development and offers a system that can provide a deeper understanding of the link between mechanical and molecular signals during development in vivo.