Pharmacological Manipulation of Early Zebrafish Skeletal Development Shows an Important Role for Smad9 in Control of Skeletal Progenitor Populations

Abstract

Osteoporosis and other conditions associated with low bone density or quality are highly prevalent, are increasing as the population ages and with increased glucocorticoid use to treat conditions with elevated inflammation. There is an unmet need for therapeutics which can target skeletal precursors to induce osteoblast differentiation and osteogenesis. Genes associated with high bone mass represent interesting targets for manipulation, as they could offer ways to increase bone density. A damaging mutation in SMAD9 has recently been associated with high bone mass. Here we show that Smad9 labels groups of osteochondral precursor cells, which are not labelled by the other Regulatory Smads: Smad1 or Smad5. We show that Smad9+ cells are proliferative, and that the Smad9+ pocket expands following osteoblast ablation which induced osteoblast regeneration. We further show that treatment with retinoic acid, prednisolone, and dorsomorphin all alter Smad9 expression, consistent with the effects of these drugs on the skeletal system. Taken together these results demonstrate that Smad9+ cells represent an undifferentiated osteochondral precursor population, which can be manipulated by commonly used skeletal drugs. We conclude that Smad9 represents a target for future osteoanabolic therapies.

Fish Models of Induced Osteoporosis

“…Thus teleosts could be used for large screening assays aiming at identifying compound that could modulate sost expression toward an increased bone deposition. A similar example is associated with the modulation of smad9 expression in zebrafish ( Gregson et al, 2020 ; McDonald et al, 2021 ). These two examples highlight the evolutionary conservation of skeletal mechanisms from fish to human and reinforce the suitability of teleost models for target-based screening assays.…”

A primate-specific (GCC) repeat in SMAD9 undergoes natural selection in humans and harbors unambiguous genotypes in late-onset neurocognitive disorder.

“…SMAD9 is predominantly expressed in the brain and skeletal tissues [18, 29,30], and the protein encoded by this gene can translocate into the nucleus and affect the transcriptional regulation of target genes.…”

Osteoblast cell death triggers a pro-osteogenic inflammatory response regulated by reactive oxygen species and glucocorticoid signaling in zebrafish

“…Many cell populations have shown the potential to generate osteoblasts, summarized under the term skeletal stem cells in mammals (Serowoky et al, 2020). In zebrafish, osteoblasts self-renew by dedifferentiation and proliferation of mature osteoblasts (Geurtzen et al, 2014;Knopf et al, 2011;Sousa et al, 2011), but also become recruited from progenitor cell pools (Ando et al, 2017;Mcdonald et al, 2021). Notably, proliferation of osteoblasts could be observed in response to laser-assisted osteoblast ablation, albeit not frequently.…”