Zebrafish Bone and General Physiology Are Differently Affected by Hormones or Changes in Gravity

Abstract

Teleost fish such as zebrafish (Danio rerio) are increasingly used for physiological, genetic and developmental studies. Our understanding of the physiological consequences of altered gravity in an entire organism is still incomplete. We used altered gravity and drug treatment experiments to evaluate their effects specifically on bone formation and more generally on whole genome gene expression. 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 10 dpf) of, 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. 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 24 hrs 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.

Effects of microgravity simulation on zebrafish transcriptomes and bone physiology—exposure starting at 5 days post fertilization

“…In the cartilage skeleton, no changes in morphology due to sim-μg were observed ( Figure 1c,d ). This absence of major effects on head cartilage formation during this period was previously also shown in hypergravity, 9 indicating that the cranial cartilage morphology is not affected by mechanical constraints, at least past a certain stage. In this context, it is interesting to note that also inhibition of the Fgf or Bmp signaling pathways in zebrafish larvae older than 2 dpf did not affect cartilage formation.…”

Effects of microgravity simulation on zebrafish transcriptomes and bone physiology—exposure starting at 5 days post fertilization

“…In the cartilage skeleton, no changes in morphology due to sim-μg were observed ( Figure 1c,d ). This absence of major effects on head cartilage formation during this period was previously also shown in hypergravity, 9 indicating that the cranial cartilage morphology is not affected by mechanical constraints, at least past a certain stage. In this context, it is interesting to note that also inhibition of the Fgf or Bmp signaling pathways in zebrafish larvae older than 2 dpf did not affect cartilage formation.…”

Epigenetic and physiological alterations in zebrafish subjected to hypergravity

“…These zebrafish exhibited morphometric changes, including an enlarged head and increased cranial bone formation. These findings supported that hypergravity can induce consistent effects on the developmental processes in zebrafish [87].…”

Epigenetic and physiological alterations in zebrafish subjected to hypergravity

“…These zebrafish exhibited morphometric changes, including an enlarged head and increased cranial bone formation. These findings supported that hypergravity can induce consistent effects on the developmental processes in zebrafish (Aceto et al, 2015).…”

Effects of microgravity simulation on zebrafish transcriptomes and bone physiology—exposure starting at 5 days post fertilization

“…In the cartilage skeleton, no changes in morphology due to sim-μg were observed ( Figure 1c,d ). This absence of major effects on head cartilage formation during this period was previously also shown in hypergravity, 9 indicating that the cranial cartilage morphology is not affected by mechanical constraints, at least past a certain stage. In this context, it is interesting to note that also inhibition of the Fgf or Bmp signaling pathways in zebrafish larvae older than 2 dpf did not affect cartilage formation.…”

Epigenetic and physiological alterations in zebrafish subjected to hypergravity

“…These zebrafish exhibited morphometric changes, including an enlarged head and increased cranial bone formation. These findings supported that hypergravity can induce consistent effects on the developmental processes in zebrafish [87].…”

Epigenetic and physiological alterations in zebrafish subjected to hypergravity

“…These zebrafish exhibited morphometric changes, including an enlarged head and increased cranial bone formation. These findings supported that hypergravity can induce consistent effects on the developmental processes in zebrafish (Aceto et al, 2015).…”

Effects of microgravity simulation on zebrafish transcriptomes and bone physiology—exposure starting at 5 days post fertilization

“…In the cartilage skeleton, no changes in morphology due to sim-μg were observed ( Figure 1c,d ). This absence of major effects on head cartilage formation during this period was previously also shown in hypergravity, 9 indicating that the cranial cartilage morphology is not affected by mechanical constraints, at least past a certain stage. In this context, it is interesting to note that also inhibition of the Fgf or Bmp signaling pathways in zebrafish larvae older than 2 dpf did not affect cartilage formation.…”

Epigenetic and physiological alterations in zebrafish subjected to hypergravity

“…These zebrafish exhibited morphometric changes, including an enlarged head and increased cranial bone formation. These findings supported that hypergravity can induce consistent effects on the developmental processes in zebrafish [87].…”

Epigenetic and physiological alterations in zebrafish subjected to hypergravity

“…These zebrafish exhibited morphometric changes, including an enlarged head and increased cranial bone formation. These findings supported that hypergravity can induce consistent effects on the developmental processes in zebrafish (Aceto et al, 2015).…”