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.

Context: Oxidative stress and formation of advanced glycation end products (AGEs), due to glycation of proteins, lipids and nucleic acids are characteristic in diabetic patients. Palmatine, a protoberberine alkaloid bioactive isolated from Coscinium fenestratum (CF) stem extract, which previously has shown to possess antidiabetic and antioxidant properties and to be able to protect the kidney and liver in a STZ-diabetic induced rat model. Aims: To evaluate the in vitro and in vivo antioxidant and anti-AGE activity of palmatine. Methods: In vitro and in vivo studies were conducted to measure radical scavenging, reducing power, inhibition of lipid peroxidation, carbonyl trapping and metal ion chelation. In vitro antiglycation activity was done using bovine serum albumin-methylglyoxal (BSA-MGO), bovine serum albumin – glucose (BSA-GLU) and glycated hemoglobin. In vivo antioxidant and antiglycation activity were used to evaluate liver and kidney extracted from STZ-induced diabetic rat after treatment with palmatine. Results: The results showed palmatine blocked the formation of AGE as shown by the results of BSA-GLU, BSA-MGO, glycated hemoglobin and inhibited the free radicals generated by DPPH, nitric oxide, hydrogen peroxide, lipid peroxidation, FRAP and metal ion chelating. It was able to stimulate in vivo the activity of catalase, super oxide dismutase and glutathione peroxidase. Conclusions: Palmatine possess antioxidant and antiglycation properties, the mechanism of action seems to be via the blockage of free radical formation, decreasing of reactive carbonyl. Further research is ongoing to determine the effect of palmatine on glyoxalase 1 and aldose reductase pathway and interaction with receptor for AGE.

Nonmammalian vertebrates have the capacity of lifelong tooth replacement. In all vertebrates, tooth formation requires contact and interaction between the oral or pharyngeal epithelium and the underlying mesenchyme. To secure lifelong replacement, the presence of odontogenic stem cells has been postulated, particularly in the epithelial compartment. This study uses an advanced teleost fish species, the marine medaka Oryzias melastigma, a close relative to Oryzias latipes, to examine the expression and distribution of telomerase reverse transcriptase (Tert), the catalytic unit of telomerase, in developing pharyngeal teeth and to relate these data to the proliferative activity of the cells. The data are complemented by expression analysis of the pluripotency marker oct4 and bona fide stem cell marker lgr5. Tert distribution and tert expression in developing tooth germs show a dynamic spatiotemporal pattern. Tert is present first in the mesenchyme but is downregulated as the odontoblasts differentiate. In contrast, in the epithelial enamel organ, Tert is absent during early stages of tooth formation and upregulated first in ameloblasts. Later, Tert is expressed and immunolocalized throughout the entire inner enamel epithelium. The pattern of Tert distribution is largely mutually exclusive with that of proliferating cell nuclear antigen (PCNA) immunoreactivity: highly proliferative cells, as revealed by PCNA staining, are negative for Tert; conversely, PCNA-negative cells are Tert-positive. Only the early condensed mesenchyme is both Tert- and PCNA-positive. The absence of tert-positive cells in the epithelial compartment of early tooth germs is underscored by the absence of oct4- and lgr5-positive cells, suggesting ways other than stem cell involvement to secure continuous renewal.

During bone development and repair, osteoblasts get recruited to bone deposition sites. To identify the origin of recruited osteoblasts, cell lineage tracing using Cre/loxP recombination is commonly utilized. However, a confounding factor is the use of transgenic Cre drivers that do not accurately recapitulate endogenous gene expression or knock-in Cre drivers that alter endogenous protein activity or levels. Here, we describe a novel CRISPR/Cas9 homology-directed repair knock-in approach that allows efficient generation of Cre drivers controlled by the endogenous gene promoter. Also, a self-cleaving peptide preserves reading frame of the endogenous protein. Using this approach, we generated col10a1p2a-CreERT2 knock-in medaka and showed that tamoxifen-inducible CreERT2 efficiently recombined loxP sites in col10a1 cells. Similar knock-in efficiencies were obtained when two unrelated loci (osr1, col2a2) were targeted. Using live imaging, we traced the fate of col10a1 osteoblast progenitors during bone lesion repair in the medaka vertebral column. We show that col10a1 cells at neural arches represent a mobilizable cellular source for bone repair. Together, our study describes a novel strategy for precise cell lineage tracing via efficient and non-disruptive knock-in of Cre.

No abstract