Cellular and molecular characterization of a novel primary osteoblast culture from the vertebrate model organism Xenopus tropicalis

Abstract

Osteogenesis is the fundamental process by which bones are formed, maintained and regenerated. The osteoblasts deposit the bone mineralized matrix by secreting large amounts of extracellular proteins and by allowing the biochemical conditions for the nucleation of hydroxyapatite crystals. Normal bone formation requires a tight control of osteoblastic activity, and therefore, osteoblasts represent a major focus of interest in biomedical research. Several crucial features of osteogenesis can be readily recapitulated using murine, avian and fish primary and immortalized osteoblastic cultures. Here, we describe a novel and straightforward in vitro culture of primary osteoblasts from the amphibian Xenopus tropicalis, a major vertebrate model organism. X. tropicalis osteoblasts can readily be extracted from the frontoparietal bone of pre-metamorphosing tadpole skulls by series of gentle protease treatments. Such primary cultures efficiently proliferate and can conveniently be grown at room temperature, in the absence of CO2, on a variety of substrates. X. tropicalis primary osteoblasts express well-characterized genes known to be active during osteogenesis of teleost fish, chick, mouse and human. Upon differentiation, such cultures mineralize and activate DMP1, an osteocyte-specific gene. Importantly, X. tropicalis primary osteoblasts can be efficiently transfected and respond to the forced activation of the bone morphogenetic protein pathway by increasing their nuclear levels of phospho-Smad. Therefore, this novel primary culture is amenable to experimental manipulations and represents a valuable tool for improving our understanding of the complex network of molecular interactions that govern vertebrate bone formation.

Evolutionary repression of chondrogenic genes in the vertebrate osteoblast

“…Recent studies have revealed that osteoblasts of earlier-diverged clades, such as bony fishes and amphibians, express molecular markers normally associated with cartilage of later-diverged clades, such as mammals and birds [25][26][27][28]. A big surprise came when extremely high levels of col10a1 expression (again, THE definitive marker of mature chondrocytes in chick and mouse) were demonstrated in osteoblasts of both zebrafish and gar [25].…”

A Single Chance to Contact Multiple Targets: Distinct Osteocyte Morphotypes Shed Light on the Cellular Mechanism Ensuring the Robust Formation of Osteocytic Networks

“…To characterize in greater details such perpendicular processes, we took advantage of the fact that the developing X. tropicalis calvaria contains two mirror‐image osteocyte fields derived from osteoblasts lying onto the bone surfaces facing the brain or the skin (see Fig. A; Espinoza et al., ; Bertin et al., ). Because these two fields grow in opposite directions, the middle region of the calvaria is mostly devoid of osteocytes and cell processes (dotted line in Fig.…”

“…The Nieuwkoop and Faber developmental table was applied to X. tropicalis larvae and used to select fully mineralized calvaria at stage NF60 (Nieuwkoop and Faber, ; Espinoza et al., ). Xenopus tropicalis tadpoles were anaesthetized with tricaine, killed by decapitation (Close et al., ; Rhee et al., ), and the dissected calvaria were stripped from all superficial cells by a series of protease treatments, as previously described (Bertin et al., ). Calvaria were washed in Hank's Balanced Salt Solution 0.6× (Hyclone, Logan, UT), fixed in 4% PFA overnight at 4°C, and gently washed four times in PBS.…”

“…Here, we introduce the amphibian Xenopus tropicalis as a suitable vertebrate model to study osteocytes in their native context. We have recently shown that, in this species, developing calvaria highly enriched in osteocytes can be obtained by a series of protease treatments removing superficial cells (Bertin et al., ). Using high‐resolution imaging and three‐dimensional reconstructions, we characterize cell density, size, shape, volume, and morphology.…”

Discovery and modulation of acid-sensing ion channel modulating venom peptides

“…X. tropicalis and the Marsabit clawed frog X. borealis are widely used in many laboratories for genetic and developmental studies (114)(115)(116)(117)(118)(119). Although X. tropicalis is more widely used, X. borealis is more closely related to X. laevis (120).…”