Fragile X Messenger Ribonucleoprotein 1 (FMR1), a novel inhibitor of osteoblast/osteocyte differentiation, regulates bone formation, mass, and strength in young and aged male and female mice.

Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene mutations lead to fragile X syndrome, cognitive disorders, and, in some individuals, scoliosis and craniofacial abnormalities. Four-month-old (mo) male mice with deletion of the FMR1 gene exhibit a mild increase in cortical and cancellous femoral bone mass. However, consequences of absence of FMR1 in bone of young/aged male/female mice and the cellular basis of the skeletal phenotype remain unknown. We found that absence of FMR1 results in improved bone properties with higher bone mineral density in both sexes and in 2- and 9-mo mice. The cancellous bone mass is higher only in females, whereas, cortical bone mass is higher in 2- and 9-mo males, but higher in 2- and lower in 9-mo female FMR1-knockout mice. Furthermore, male bones show higher biomechanical properties at 2mo, and females at both ages. Absence of FMR1 increases osteoblast/mineralization/bone formation and osteocyte dendricity/gene expression in vivo/ex vivo/in vitro, without affecting osteoclasts in vivo/ex vivo. Thus, FMR1 is a novel osteoblast/osteocyte differentiation inhibitor, and its absence leads to age-, site- and sex-dependent higher bone mass/strength.

Sex-specific differences in direct osteoclastic versus indirect osteoblastic effects underlay the low bone mass of Pannexin1 deletion in TRAP-expressing cells in mice.

Pannexin1 (Panx1) is a hemichannel-forming protein that participates in the communication of cells with the extracellular space. To characterize the role of osteoclastic Panx1 on bone, Panx1fl/fl;TRAP-Cre (Panx1ΔOc) mice were generated, and compared to Panx1fl/fl littermates at 6 weeks of age. Total and femoral BMD was ~20% lower in females and males whereas spinal BMD was lower only in female Panx1ΔOc mice. µCT analyses showed that cortical bone of the femoral mid-diaphysis was not altered in Panx1ΔOc mice. In contrast, cancellous bone in the distal femur and lumbar vertebra was significantly decreased in both female and male Panx1ΔOc mice compared to Panx1fl/fl controls and was associated with higher osteoclast activity in female Panx1ΔOc mice, with no changes in the males. On the other hand, vertebral bone formation was decreased for both sexes, resulting from lower mineral apposition rate in the females and lower mineralizing surface in the males. Consistent with an osteoclastic effect in female Panx1ΔOc mice, osteoclast differentiation with RANKL/M-CSF and osteoclast bone resorbing activity in vitro were higher in female, but not male, Panx1ΔOc mice, compared to Panx1fl/fl littermates. Surprisingly, although Panx1 expression was normal in bone marrow stromal-derived osteoblasts from male and female Panx1ΔOc mice, mineral deposition by male (but not female) Panx1ΔOc osteoblasts was lower than controls, and it was reduced in male Panx1fl/fl osteoblasts when conditioned media prepared from male Panx1ΔOc osteoclast cultures was added to the cell culture media. Thus, deletion of Panx1 in TRAP-expressing cells in female mice leads to low bone mass primarily through a cell autonomous effect in osteoclast activity. In contrast, our evidence suggests that changes in the osteoclast secretome drive reduced osteoblast function in male Panx1ΔOc mice, resulting in low bone mass.

The Sonozotz project: Assembling an echolocation call library for bats in a megadiverse country.

Bat acoustic libraries are important tools that assemble echolocation calls to allow the comparison and discrimination to confirm species identifications. The Sonozotz project represents the first nation-wide library of bat echolocation calls for a megadiverse country. It was assembled following a standardized recording protocol that aimed to cover different recording habitats, recording techniques, and call variation inherent to individuals. The Sonozotz project included 69 species of echolocating bats, a high species richness that represents 50% of bat species found in the country. We include recommendations on how the database can be used and how the sampling methods can be potentially replicated in countries with similar environmental and geographic conditions. To our knowledge, this represents the most exhaustive effort to date to document and compile the diversity of bat echolocation calls for a megadiverse country. This database will be useful to address a range of ecological questions including the effects of anthropogenic activities on bat communities through the analysis of bat sound.

MLO-Y4 osteocytic cell clones express distinct gene expression patterns characteristic of different stages of osteocyte differentiation / Clones de las células osteocíticas MLO-Y4 tienen diferentes patrones de expresión génica característicos de diferentes estadíos de diferenciación osteocítica

Osteocytes are the most abundant bone cell and are formed when osteoblasts become embedded in the bone matrix. Through changes in gene expression and paracrine effects, osteocytes regulate the number of osteoblasts, bone forming cells, and osteoclasts, bone resorbing cells, which are needed to maintain bone mass. MLO-Y4 is the better characterized osteocytic cell line; however, lacks expression of sclerostin, the product of the SOST gene, which is fundamental for osteocyte function and blocks bone formation. With the objective to isolate MLO-Y4 clones with different gene expression profiles, we performed cultures at very low density of MLO-Y4 cells stably transfected with nuclear green fluorescent protein (MLOnGFP). Cell morphology was visualized under a fluorescence microscope. Once the cells reached 80% confluency, RNA was extracted and quantitative real time PCR was performed. Clones exhibit different sizes and morphology, with some cells showing a spindle-like shape and others with abundant projections and a star-like shape. Gene expression also differed among clones. However, none of the clones examined expressed SOST. We conclude that the MLO-nGFP clones constitute a useful tool to study osteocyte differentiation and the role of osteocytes in the control of bone formation and resorption in vitro. (AU)

Los osteocitos son las células más abundantes del hueso y se forman cuando los osteoblastos se encuentran rodeados de matriz ósea. A través de cambios en la expresión génica y efectos paracrinos, los osteocitos controlan el número de osteoblastos que forman el hueso, y osteoclastos que resorben el hueso, células necesarias para mantener la masa ósea. Las células MLO-Y4 son la línea celular osteocítica más investigada; sin embargo, no expresan esclerostina, el pro esclerostina, el producto del gen SOST que bloquea la formación ósea y es indispensable para la función de los osteocitos. Con el objetivo de aislar clones de las células MLO-Y4 con diferentes perfiles de expresión génica, realizamos cultivos a muy baja densidad de las células transfectadas en forma estable con proteína verde fluorescente nuclear (MLO-nGFP). La morfología celular fue evaluada utilizando un microscopio de fluorescencia. Una vez que las células alcanzaron el 80% de confluencia, el ARN fue extraído y analizado por PCR cuantitativa en tiempo real. Las células de los diferentes clones tienen diferentes tamaños y morfología, algunas células son fusiformes y otras con proyecciones citoplasmáticas abundantes y en forma de estrella. La expresión de los genes también varió en los distintos clones. Sin embargo, ninguno de ellos expresó SOST. En conclusión, los clones de las células MLO-nGFP constituyen una herramienta útil para estudiar la diferenciación de los osteocitos y el rol de estas células en el control de la formación y resorción ósea in vitro. (AU)