Anat Akiva

On the pathway of mineral deposition in larval zebrafish caudal fin bone

A poorly understood aspect of bone biomineralization concerns the mechanisms whereby ions are sequestered from the environment, concentrated, and deposited in the extracellular matrix. In this study, we follow mineral deposition in the caudal fin of the zebrafish larva in vivo. Using fluorescence and cryo-SEM-microscopy, in combination with Raman and XRF spectroscopy, we detect the presence of intracellular mineral particles located between bones, and in close association with blood vessels. Calcium-rich particles are also located away from the mineralized bone, and these are also in close association with blood vessels. These observations challenge the view that mineral formation is restricted to osteoblast cells juxtaposed to bone, or to the extracellular matrix. Our results, derived from observations performed in living animals, contribute a new perspective to the comprehensive mechanism of bone formation in vertebrates, from the blood to the bone. More broadly, these findings may shed light on bone mineralization processes in other vertebrates, including humans.

Simultaneous Raman microspectroscopy and fluorescence imaging of bone mineralization in living zebrafish larvae.

Confocal Raman microspectroscopy and fluorescence imaging are two well-established methods providing functional insight into the extracellular matrix and into living cells and tissues, respectively, down to single molecule detection. In living tissues, however, cells and extracellular matrix coexist and interact. To acquire information on this cell-matrix interaction, we developed a technique for colocalized, correlative multispectral tissue analysis by implementing high-sensitivity, wide-field fluorescence imaging on a confocal Raman microscope. As a proof of principle, we study early stages of bone formation in the zebrafish (Danio rerio) larvae because the zebrafish has emerged as a model organism to study vertebrate development. The newly formed bones were stained using a calcium fluorescent marker and the maturation process was imaged and chemically characterized in vivo. Results obtained from early stages of mineral deposition in the zebrafish fin bone unequivocally show the presence of hydrogen phosphate containing mineral phases in addition to the carbonated apatite mineral. The approach developed here opens significant opportunities in molecular imaging of metabolic activities, intracellular sensing, and trafficking as well as in vivo exploration of cell-tissue interfaces under (patho-)physiological conditions.

Transport of membrane-bound mineral particles in blood vessels during chicken embryonic bone development

During bone formation in embryos, large amounts of calcium and phosphate are taken up and transported to the site where solid mineral is first deposited. The initial mineral forms in vesicles inside osteoblasts and is deposited as a highly disordered calcium phosphate phase. The mineral is then translocated to the extracellular space where it penetrates the collagen matrix and crystallizes. To date little is known about the transport mechanisms of calcium and phosphate in the vascular system, especially when high transport rates are needed and the concentrations of these ions in the blood serum may exceed the solubility product of the mineral phase. Here we used a rapidly growing biological model, the chick embryo, to study the bone mineralization pathway taking advantage of the fact that large amounts of bone mineral constituents are transported. Cryo scanning electron microscopy together with cryo energy dispersive X-ray spectroscopy and focused-ion beam imaging in the serial surface view mode surprisingly reveal the presence of abundant vesicles containing small mineral particles in the lumen of the blood vessels. Morphologically similar vesicles are also found in the cells associated with bone formation. This observation directly implicates the vascular system in solid mineral distribution, as opposed to the transport of ions in solution. Mineral particle transport inside vesicles implies that far larger amounts of the bone mineral constituents can be transported through the vasculature, without the danger of ectopic precipitation. This introduces a new stage into the bone mineral formation pathway, with the first mineral being formed far from the bone itself.

Bone Mineralization Pathways during the Rapid Growth of Embryonic Chicken Long Bones

The uptake and transport of ions from the environment to the site of bone formation is only partially understood and, for the most part, based on disparate observations in different animals. Here we study different aspects of the biomineralization pathways in one system, the rapidly forming long bones of the chicken embryo. We mainly used cryo-fixation and cryo-electron imaging to preserve the often unstable mineral phases in the tissues. We show the presence of surprisingly large amounts of mineral particles located inside membrane-delineated vesicles in the bone forming tissue between the blood vessels and the forming bone surface. Some of these particles are also located inside mitochondrial networks. The surfaces of the forming bones in the extracellular space contain abundant aggregates of amorphous calcium phosphate particles, but these are not enveloped by vesicle membranes. In the bone resorbing region, osteoclasts also contain many particles in both mitochondrial networks and within vesicles. Some of these particles are present also between cells. These observations, together with the previously reported observation that CaP mineral particles inside membranes are present in blood vessels, leads us to the conclusion that important components of the bone mineralization pathways in rapidly forming chicken bone are dense phase mineral particles bound within membranes. It remains to be determined whether these mineral particles are transported to the site of bone formation in the solid state, fluid state or dissolve and re-precipitate.

Deposition of collagen type I onto skeletal endothelium reveals a new role for blood vessels in regulating bone morphology.

Recently, blood vessels have been implicated in the morphogenesis of various organs. The vasculature is also known to be essential for endochondral bone development, yet the underlying mechanism has remained elusive. We show that a unique composition of blood vessels facilitates the role of the endothelium in bone mineralization and morphogenesis. Immunostaining and electron microscopy showed that the endothelium in developing bones lacks basement membrane, which normally isolates the blood vessel from its surroundings. Further analysis revealed the presence of collagen type I on the endothelial wall of these vessels. Because collagen type I is the main component of the osteoid, we hypothesized that the bone vasculature guides the formation of the collagenous template and consequently of the mature bone. Indeed, some of the bone vessels were found to undergo mineralization. Moreover, the vascular pattern at each embryonic stage prefigured the mineral distribution pattern observed one day later. Finally, perturbation of vascular patterning by overexpressing Vegf in osteoblasts resulted in abnormal bone morphology, supporting a role for blood vessels in bone morphogenesis. These data reveal the unique composition of the endothelium in developing bones and indicate that vascular patterning plays a role in determining bone shape by forming a template for deposition of bone matrix. Highlighted article: Collagen I is deposited by osteoblasts onto endothelial cells within bone and serves as a template for mineralisation, with ossification thus spatially and temporally following vascular patterning.

Cryo-FIB-SEM serial milling and block face imaging: large volume structural analysis of biological tissues preserved close to their native state.

Many important biological questions can be addressed by studying in 3D large volumes of intact, cryo fixed hydrated tissues (⩾10,000μm3) at high resolution (5-20nm). This can be achieved using serial FIB milling and block face surface imaging under cryo conditions. Here we demonstrate the unique potential of the cryo-FIB-SEM approach using two extensively studied model systems; sea urchin embryos and the tail fin of zebrafish larvae. We focus in particular on the environment of mineral deposition sites. The cellular organelles, including mitochondria, Golgi, ER, nuclei and nuclear pores are made visible by the image contrast created by differences in surface potential of different biochemical components. Auto segmentation and/or volume rendering of the image stacks and 3D reconstruction of the skeleton and the cellular environment, provides a detailed view of the relative distribution in space of the tissue/cellular components, and thus of their interactions. Simultaneous acquisition of secondary and back-scattered electron images adds additional information. For example, a serial view of the zebrafish tail reveals the presence of electron dense mineral particles inside mitochondrial networks extending more than 20μm in depth in the block. Large volume imaging using cryo FIB SEM, as demonstrated here, can contribute significantly to the understanding of the structures and functions of diverse biological tissues.

Tracking the Early Events of Mineral Formation during Coral Development

1. Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel. 2. Marine Biology Department, The Leon H. Charney School of Marine Sciences, University of Haifa, Mt. Carmel, Haifa 31905, Israel. #. Present address: Laboratory of Materials and Interface Chemistry and Center of Multiscale Electron Microscopy, Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, The Netherlands.

Minerals in the pre-settled coral Stylophora pistillata crystallize via protein and ion changes

Aragonite skeletons in corals are key contributors to the storage of atmospheric CO2 worldwide. Hence, understanding coral biomineralization/calcification processes is crucial for evaluating and predicting the effect of environmental factors on this process. While coral biomineralization studies have focused on adult corals, the exact stage at which corals initiate mineralization remains enigmatic. Here, we show that minerals are first precipitated as amorphous calcium carbonate and small aragonite crystallites, in the pre-settled larva, which then evolve into the more mature aragonitic fibers characteristic of the stony coral skeleton. The process is accompanied by modulation of proteins and ions within these minerals. These findings may indicate an underlying bimodal regulation tactic adopted by the animal, with important ramification to its resilience or vulnerability toward a changing environment.Coral biomineralization is an important example of natural mineralization and understanding the process will aid biomineralization research. Here, the authors identify the precipitation of amorphous calcium carbonate and small aragonite crystals in pre-settled larva of Stylophora pistillata.