Abstract :
[en] Zebrafish (Danio rerio) are increasingly used for physiological, genetic and developmental studies. Understanding the consequences of altered gravity on bone development and on general physiology in an entire organism remains to date incomplete. We used altered drug treatment and gravity experiments to evaluate their effects specifically on bone formation and more generally on whole genome gene expression. We started treatments at 5 days post-fertilization (dpf) and analyze early modifications in gene expression after 1 day using microarrays and the consequences on bone formation after 5 days using specific staining. We performed chemical treatments (Parathyroid Hormone, Vitamin D3), exposure to three different microgravity simulation devices (Clinostat, Random Positioning Machine and Rotating Wall Vessel) and finally exposure to hypergravity and "relative microgravity" in the Large Diameter Centrifuge.
By combining morphometric tools with an objective scoring system for the state of development for each element in the head skeleton, and specific gene expression analysis, we confirmed and characterized in detail the decrease or increase of bone formation caused by a 5 day treatment (from 5dpf to 10dpf) with, respectively parathyroid hormone (PTH) or vitamin D3 (VitD3). Microarray transcriptome analysis after 24 hours treatment reveals a general effect on physiology upon VitD3 treatment, while PTH causes more specifically developmental effects.
The microgravity simulators used were the 2D clinostat, random positioning machine and rotating wall vessel. Only clinorotation caused a significant decrease of bone formation when applied between 5 to 10dpf. This effect was not due to stress, as assessed by measuring cortisol levels in treated larvae. The two other devices caused no effect, or a slight acceleration of ossification. Gene expression results after one day in simulated microgravity indicate that musculo-skeletal, cardiavascular, and nuclear receptor systems are affected, however often in opposite directions in clinorotation compared to the two other devices. Based on the effects on bone formation and on the biological functions found to be affected, we conclude that clinorotation is the most appropriate method to simulate microgravity on ground when using free-swimming organisms such as zebrafish larvae.
Hypergravity (3g from 5dpf to 9 dpf) exposure results in a significantly larger head and a significant increase in bone formation for a subset of the cranial bones. Gene expression analysis after 24hrs at 3g revealed differential expression of genes involved in the development and function of the skeletal, muscular, nervous, endocrine and cardiovascular systems. Finally, we propose a novel type of experimental approach, the "Reduced Gravity Paradigm", by keeping the developing larvae at 3g hypergravity for the first 5 days before returning them to 1g for one additional day. 5 days exposure to 3g during these early stages also caused increased bone formation, while gene expression analysis revealed a central network of regulatory genes (hes5, sox10, lgals3bp, egr1, edn1, fos, fosb, klf2, gadd45ba and socs3a) whose expression was consistently affected by the transition from hyper- to normal gravity.
