## 4 Discussion  
Mechanical forces shape living embryos starting from the fertilization of the egg and later, modulating cell division, differentiation processes, and structural remodeling [15]. We analyzed in vivo the effect of a static mechanical force, generated by a water column, on fertilized zebrafish eggs. Embryos grown in more than +4.4% HP were seriously damaged in the developmental processes whereas at +5.9% they died before 48 hpf. Zebrafish  
Figure 4: (**a**) Alican Blue/ARS staining highlights a decreased number of mineralized vertebral bodies (black arrow) and a delayed chondrogenic mineralization of lower jawbone (red arrow) in +3.4% HP embryos. Scale bar: 200 μm. (**b**) Vertebral mineralization rate of five days post fertilization embryos grown under different increased HP from 0% (CTR) up to 3.4%, evaluated as a ratio of the number of vertebral bodies positive for ARS staining along the length of the fish body (N.V./L). Embryos grown under +3.4% HP showed a statistically significant variation in N.V./L. (CTR vs. +3.4%, *** \(p<\) 0.001, \(-\)54%). embryos remained in the chorion membranes up to 48 hpf [12]. The embryos that died because of the treatment with 4.4% and 3.4% HP died during the first 48 h; this fact indicates that the increased hydrostatic pressure affects embryo health even in the presence of the chorion. Some evidence indicated that eggs and larvae may be susceptible to barotrauma injury, a direct consequence of hydrostatic pressure variation [6]. Eggs from other fish species have been shown to be prone to genetic damage 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]. The literature is consistent with our data where embryos died before 48 hpf. The mechanosensitivity of embryonic structures have also been reported for mammalian development, where hydraulic forces in the blastocel regulate embryo size and cell fate [16,17]. When we analyzed the mineralization rate, the vital embryos grown at +3.4% HP showed a significant delay in the mineralization of vertebral bodies and, less pronounced, head bones. It is well know that mechanical forces are necessary for normal bone maintenance and remodeling [18] so that in vivo bone tissue can be created and modeled basing on the mechanical stimuli that act on skeletal structures, but it is also widely accepted that mechanical stimulation is very influential on embryonic osteogenesis [16]. [PERSON] et al. demonstrated in embryonic chicks that the mechanical stimuli interact with cells thanks to \"mechanosensitive genes\" in order to generate a spatiotemporal regulation of the bone tissue [19]. Literature data give evidence that adult mesenchymal stem cells can be stimulated in vitro to bone differentiation and regeneration by intermittent pulses of hydrostatic pressure [20]. Nevertheless, during embryo development, the cells are proliferating and remodeling and the mechanical and biochemical crosstalk is much more complicated and delicate. Functions such as exocytosis, proliferation, apoptosis, ion fluxes, and cell shape can be modulated by hydraulic pressure [21]. High hydrostatic pressure (HHP) negatively interferes with cell membrane behavior, cellular shape, and protein synthesis in yeast [22] and in mammalian cells [23,24] At +3.4%, HP did not induce embryo mortality but negatively regulated development, as suggested by reduced embryo length (\(-2\%\)), even if not significant, and by the delayed bone mineralization. The neurocranium and the pharyngeal arches are ossified by an endochondral process, whereas vertebral bodies are formed by intramembranous ossification in a progressive cranio-caudal direction through notochord direct mineralization [25]. In our results, both types of mineralization seem to be affected by the increase in hydrostatic pressure.