Fluorescence-activated cell sorting (FACS) of osteoblasts and osteoclasts for RNA sequencing in a medaka, <i>Oryzias latipes</i>
 (Temming &amp; Schlegel, 1846), osteoporosis model

Buettner, Anita; Sundaram, Sudha; Vyas, Himanshu; Yu, Tingsheng; Mathavan, Sinnakarupan; Winkler, Christoph

doi:10.1111/jai.13660

Cited by 7 publications

(9 citation statements)

References 14 publications

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“…The transgenic lines Tg[ctsk:DsRed] and Tg[sp7:EGFP] (DeLaurier et al, 2010) were used to isolate osteoclasts and osteoblasts, respectively. Protocol was based on methodologies outlined by Madel et al (2018) and Buettner et al (2015). Fish were euthanized and tissue samples harvested for isolation of cells.…”

Section: Cell Isolation and Sortingmentioning

confidence: 99%

Unique and non-redundant function of csf1r paralogues in regulation and evolution of post-embryonic development of the zebrafish

et al. 2020

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Section: Cell Isolation and Sortingmentioning

confidence: 99%

Unique and non-redundant function of csf1r paralogues in regulation and evolution of post-embryonic development of the zebrafish

et al. 2020

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“…For details, see ref. 21 . ( B ) Venn diagram showing number of significantly up-regulated genes in osteoblast progenitors and osteoblasts, at 10 dpf, 1 d after Rankl induction.…”

Section: Resultsmentioning

confidence: 99%

“…RNA libraries were prepared from FACS purified osteoblast progenitors ( col10a1 -positive), premature osteoblasts ( osx / sp7 ), and osteoclasts ( ctsk ) as described previously ( 21 ). Briefly, medaka Rankl+ ( rankl :HSE: cfp ) and Rankl− control larvae expressing osteoblast ( col10a1 :nuGFP; osx :mCherry) or osteoclast ( ctsk :mCherry) reporters were heat-shocked at 9 dpf for 2 h at 39 °C to induce Rankl expression.…”

Section: Methodsmentioning

confidence: 99%

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Cxcl9l and Cxcr3.2 regulate recruitment of osteoclast progenitors to bone matrix in a medaka osteoporosis model

Phan

Tan

Liu

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Bone homeostasis requires continuous remodeling of bone matrix to maintain structural integrity. This involves extensive communication between bone-forming osteoblasts and bone-resorbing osteoclasts to orchestrate balanced progenitor cell recruitment and activation. Only a few mediators controlling progenitor activation are known to date and have been targeted for intervention of bone disorders such as osteoporosis. To identify druggable pathways, we generated a medaka (Oryzias latipes) osteoporosis model, where inducible expression of receptor-activator of nuclear factor kappa-Β ligand (Rankl) leads to ectopic formation of osteoclasts and excessive bone resorption, which can be assessed by live imaging. Here we show that upon Rankl induction, osteoblast progenitors up-regulate expression of the chemokine ligand Cxcl9l. Ectopic expression of Cxcl9l recruits mpeg1-positive macrophages to bone matrix and triggers their differentiation into osteoclasts. We also demonstrate that the chemokine receptor Cxcr3.2 is expressed in a distinct subset of macrophages in the aorta-gonad-mesonephros (AGM). Live imaging revealed that upon Rankl induction, Cxcr3.2-positive macrophages get activated, migrate to bone matrix, and differentiate into osteoclasts. Importantly, mutations in cxcr3.2 prevent macrophage recruitment and osteoclast differentiation. Furthermore, Cxcr3.2 inhibition by the chemical antagonists AMG487 and NBI-74330 also reduced osteoclast recruitment and protected bone integrity against osteoporotic insult. Our data identify a mechanism for progenitor recruitment to bone resorption sites and Cxcl9l and Cxcr3.2 as potential druggable regulators of bone homeostasis and osteoporosis.

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“…Fluorescence-activated cell sorting, RNA sequencing, and bioinformatics analysis FACS and RNA extraction were performed using previously described protocols (24) with some modifications. RNA library construction, Illumina sequencing, and bioinformatics analysis were performed by Novogene (Singapore) following company procedures.…”

Section: Cell Quantification and Statisticsmentioning

confidence: 99%

Macrophages Switch to an Osteo‐Modulatory Profile Upon RANKL Induction in a Medaka (Oryzias latipes) Osteoporosis Model

et al. 2020

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In mammals, osteoclasts differentiate from macrophages in the monocyte lineage. Although many factors driving osteoclast formation are known, the detailed processes underlying precursor recruitment, differentiation, and interaction of macrophages with other cell types involved in bone remodeling are poorly understood. Using live imaging in a transgenic medaka osteoporosis model, where ectopic osteoclasts are induced by RANKL expression, we show that a subset of macrophages is recruited to bone matrix to physically interact with bone-forming osteoblast progenitors. These macrophages subsequently differentiate into cathepsin K-(ctsk-) positive osteoclasts. One day later, other macrophages are recruited to clear dying osteoclasts from resorbed bone by phagocytosis. To better understand the molecular changes underlying these dynamic processes, we performed transcriptome profiling of activated macrophages upon RANKL induction. This revealed an upregulation of several bone-related transcripts. Besides osteoclast markers, we unexpectedly also found expression of osteoblast-promoting signals in activated macrophages, suggesting a possible non-cell autonomous role in osteogenesis. Finally, we show that macrophage differentiation into osteoclasts is dependent on inflammatory signals. Medaka deficient for TNFα or treated with the TNFα-inhibitor pentoxifylline exhibited impaired macrophage recruitment and osteoclast differentiation. These results show the involvement of inflammatory signals and the dynamics of a distinct subset of macrophages during osteoclast formation.

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Fluorescence-activated cell sorting (FACS) of osteoblasts and osteoclasts for RNA sequencing in a medaka, Oryzias latipes (Temming & Schlegel, 1846), osteoporosis model

Cited by 7 publications

References 14 publications

Unique and non-redundant function of csf1r paralogues in regulation and evolution of post-embryonic development of the zebrafish

Unique and non-redundant function of csf1r paralogues in regulation and evolution of post-embryonic development of the zebrafish

Cxcl9l and Cxcr3.2 regulate recruitment of osteoclast progenitors to bone matrix in a medaka osteoporosis model

Macrophages Switch to an Osteo‐Modulatory Profile Upon RANKL Induction in a Medaka (Oryzias latipes) Osteoporosis Model

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