## 1 Introduction  
Zebrafish embryos are widely used for developmental studies, in particular on bone formation and tissue mineralization, focusing on vertebral and head bone formation [1]. Zebrafish embryo osteogenesis is characterized by two different types of ossification: the endochondral ossification, that takes place from a cartilaginous scaffold, and the intramembranous ossification, which is a direct ossification operated by mesenchymal stem cell precursors [1]. In vivo and in vitro studies have demonstrated that mechanical forces, as well as chemical signals, are crucial for the differentiation and behavior of bone cells. For example, osteoblast function and RANKL expression can be modulated by mechanical forces [2]. The absence of gravity conditions modulates calcium metabolism and induces osteoporosis in astronauts [3]. In addition, osteoclasts, the bone resorbing cells, have been indicated as responsive to mechanical forces [4]. It has been suggested that bone cellspossess mechanosensors which transduce mechanical signals to chemical ones. Recently, a family of ion-channels named Piezo 1/2 were reported to have a role in bone development and mechano-stimulation of bone metabolism [5].  
Few data have been provided about the role of mechanical forces on embryo development in vivo, but some evidence indicates that fish eggs and larvae may be susceptible to barotrauma injury, a direct consequence of hydrostatic pressure variation [6]. Eggs from different fish species have been shown to be prone to genetic damages when exposed to hydrostatic pressure excess, driving developmental block and early death [7; 8].  
In addition, during embryonic development, the fragility of the internal organs as well as the early vascular network makes the larva more susceptible to barotrauma injury than juvenile fish [9].  
Different types of mechanical forces are able to modulate zebrafish embryonic osteogenesis. Previous experiments into increased mechanical pressure on zebrafish embryos have been performed by [PERSON] et al. [10], in which five days post fertilization (dpf) embryos were treated with hypergravity (3 g) using a large diameter centrifuge up to 9 dpf; similar experiments were performed by [PERSON] et al. [11], which exposed 3 dpf embryos to 3 g and 6 g for 48 h. [PERSON]'s study reported that embryo morphology is unchanged after a short hypergravity treatment, but highlighted small but detrimental changes in the extracellular matrix of the cartilaginous tissue with altered chondrocyte maturation [11].  
Both hypergravity and hydrostatic pressure (HP) can be applied to zebrafish embryos to verify their effect on osteogenesis: hypergravity is modeled by centrifugation using centrifugal forces to generate up to 20 g, whereas the hydrostatic pressure is an astratice for which can be easily modulated by varying the height of the water column above animals kept at the bottom.  
The aim of this study was to evaluate zebrafish embryo osteogenesis under a gradient of hydrostatic pressure. The evaluation in vivo of whether external physical forces are able to modulate the mineralization process may elucidate the mechanisms of human bone pathological conditions such as osteoporosis.