Three‐dimensional characterization of osteocyte volumes at multiple scales, and its relationship with bone biology and genome evolution in ray‐finned fishes

Abstract

Osteocytes, cells embedded within the bone mineral matrix, inform on key aspects of vertebrate biology. In particular, a relationship between volumes of the osteocytes and bone growth and/or genome size has been proposed for several tetrapod lineages. However, the variation in osteocyte volume across different scales is poorly characterized and mostly relies on incomplete, two‐dimensional information. In this study, we characterize the variation of osteocyte volumes in ray‐finned fishes (Actinopterygii), a clade including more than half of modern vertebrate species in which osteocyte biology is poorly known. We use X‐ray synchrotron micro‐computed tomography (SRµCT) to achieve a three‐dimensional visualization of osteocyte lacunae and direct measurement of their size (volumes). Our specimen sample is designed to characterize variation in osteocyte lacuna morphology at three scales: within a bone, among the bones of one individual and among species. At the intra‐bone scale, we find that osteocyte lacunae vary noticeably in size between zones of organized and woven bone (being up to six times larger in woven bone), and across cyclical bone deposition. This is probably explained by differences in bone deposition rate, with larger osteocyte lacunae contained in bone that deposits faster. Osteocyte lacuna volumes vary 3.5‐fold among the bones of an individual, and this cannot readily be explained by variation in bone growth rate or other currently observable factors. Finally, we find that genome size provides the best explanation of variation in osteocyte lacuna volume among species: actinopterygian taxa with larger genomes (polyploid taxa in particular) have larger osteocyte lacunae (with a ninefold variation in median osteocyte volume being measured). Our findings corroborate previous two‐dimensional studies in tetrapods that also observed similar patterns of intra‐individual variation and found a correlation with genome size. This opens new perspectives for further studies on bone evolution, physiology and palaeogenomics in actinopterygians, and vertebrates as a whole.

Bone metabolism and evolutionary origin of osteocytes: Novel application of FIB-SEM tomography

“…This is not unusual, as several studies have found that cell volume can fluctuate intra-and interspecifically (3,18,19,21). The morphology of the lacunae in Tremataspis is typical "spindle-shaped" cells that have been documented in 3D in teleosts (21), chick (57), mouse (58), and human osteocyte lacunae (59)(60)(61). In contrast, the Bothriolepis lacunae differed, from traditional spindle-shaped lacunae, to small round lacunae (fig.…”

“…Osteocyte shape is an important measure, as it is relevant to their mechano-sensing function (58,63), and some studies claim that particular osteocyte shapes are more sensitive than others (64). Osteocyte shape may also be related to bone type, with lacunae in organized (e.g., lamellar or parallel-fibered) bone being more elongate and regularly shaped than in woven bone (21,65). On the other hand, the large disparity in osteocyte size is relevant to paleontological genome estimation (5,6,21,66).…”

“…The most novel data, however, are those of the canalicular processes, which have previously only been imaged in 2D in fossil material (16,17) and only captured in 3D as noncontinuous fragments in modern material (21). Imaging and quantifying of the LCN in 3D have been challenging even in modern specimens effectively being done for the first time in mice (54).…”

“…However, despite the high-resolution quality of the TEM and SEM images, the visualizations are two-dimensional (2D) and are thus less informative concerning details of spatial arrangement and connectivity of the cell lacunae. Synchrotron micro-computed tomography (CT) studies carried out on early fossil vertebrates and modern taxa (18,19,21,22) have enhanced our knowledge (18,19,21) about the density, spatial organization, and arrangement of bone cell lacunae in fossil bone as well as the lacunar volume. However, even the highest-resolution scans (0.678 m voxel size) are usually not able to show the morphology and arrangement of the canaliculi in detail.…”

“…However, even the highest-resolution scans (0.678 m voxel size) are usually not able to show the morphology and arrangement of the canaliculi in detail. In the few cases where canaliculi are visible from synchrotron CT, resolution is not sufficient to illustrate their connections, branching, and spatial arrangement in enough detail (21). In Fig.…”