VEGF signaling activates the matrix metalloproteinases, MmpL7 and MmpL5 at the sites of active skeletal growth and MmpL7 regulates skeletal elongation.

Organisms can uptake minerals, shape them in different forms and generate teeth, skeletons or shells that support and protect them. Mineral uptake, trafficking and nucleation are tightly regulated by the biomineralizing cells through networks of specialized proteins. Specifically, matrix metalloproteases (MMPs) digest various extracellular substrates and allow for mineralization in the vertebrates' teeth and bones, but little is known about their role in invertebrates' systems. The sea urchin embryo provides an excellent invertebrate model for genetic and molecular studies of biomineralization. MMP inhibition prevents the growth of the calcite spicules of the sea urchin larval skeleton, however, the molecular mechanisms and genes that underlie this response are not well understood. Here we study the spatial expression and regulation of two membrane type MMPs that were found to be occluded in the sea urchin spicules, Pl-MmpL7 and Pl-MmpL5, and investigate the function of Pl-MmpL7 in skeletogenesis. The inhibition of MMPs does not change the volume of the calcium vesicles in the skeletogenic cells. The expression of Pl-MmpL7 and Pl-MmpL5 is regulated by the Vascular Endothelial Growth Factor (VEGF) signaling, from the time of skeleton initiation and on. The expression of these genes is localized to the subsets of skeletogenic cells where active spicule growth occurs throughout skeletogenesis. Downregulation of Pl-MmpL7 expression delays the growth of the skeletal rods and in some cases, strongly perturbs skeletal shape. The localized expression of Pl-MmpL7 and Pl-MmpL5 to the active growth zone and the effect of Pl-MmpL7 perturbations on skeletal growth, suggest that these genes are essential for normal spicule elongation in the sea urchin embryo.

Calcium-vesicles perform active diffusion in the sea urchin embryo during larval biomineralization.

Biomineralization is the process by which organisms use minerals to harden their tissues and provide them with physical support. Biomineralizing cells concentrate the mineral in vesicles that they secret into a dedicated compartment where crystallization occurs. The dynamics of vesicle motion and the molecular mechanisms that control it, are not well understood. Sea urchin larval skeletogenesis provides an excellent platform for investigating the kinetics of mineral-bearing vesicles. Here we used lattice light-sheet microscopy to study the three-dimensional (3D) dynamics of calcium-bearing vesicles in the cells of normal sea urchin embryos and of embryos where skeletogenesis is blocked through the inhibition of Vascular Endothelial Growth Factor Receptor (VEGFR). We developed computational tools for displaying 3D-volumetric movies and for automatically quantifying vesicle dynamics. Our findings imply that calcium vesicles perform an active diffusion motion in both, calcifying (skeletogenic) and non-calcifying (ectodermal) cells of the embryo. The diffusion coefficient and vesicle speed are larger in the mesenchymal skeletogenic cells compared to the epithelial ectodermal cells. These differences are possibly due to the distinct mechanical properties of the two tissues, demonstrated by the enhanced f-actin accumulation and myosinII activity in the ectodermal cells compared to the skeletogenic cells. Vesicle motion is not directed toward the biomineralization compartment, but the vesicles slow down when they approach it, and probably bind for mineral deposition. VEGFR inhibition leads to an increase of vesicle volume but hardly changes vesicle kinetics and doesn't affect f-actin accumulation and myosinII activity. Thus, calcium vesicles perform an active diffusion motion in the cells of the sea urchin embryo, with diffusion length and speed that inversely correlate with the strength of the actomyosin network. Overall, our studies provide an unprecedented view of calcium vesicle 3D-dynamics and point toward cytoskeleton remodeling as an important effector of the motion of mineral-bearing vesicles.

The biological regulation of sea urchin larval skeletogenesis - From genes to biomineralized tissue.

Biomineralization is the process in which soft organic tissues use minerals to produce shells, skeletons and teeth for various functions such as protection and physical support. The ability of the cells to control the time and place of crystal nucleation as well as crystal orientation and stiffness is far beyond the state-of-the art of human technologies. Thus, understanding the biological control of biomineralization will promote our understanding of embryo development as well as provide novel approaches for material engineering. Sea urchin larval skeletogenesis offers an excellent platform for functional analyses of both the molecular control system and mineral uptake and deposition. Here we describe the current understanding of the genetic, molecular and cellular processes that underlie sea urchin larval skeletogenesis. We portray the regulatory genes that define the specification of the skeletogenic cells and drive the various morphogenetic processes that occur in the skeletogenic lineage, including: epithelial to mesenchymal transition, cell migration, spicule cavity formation and mineral deposition into the spicule cavity. We describe recent characterizations of the size, motion and mineral concentration of the calcium-bearing vesicles in the skeletogenic cells. We review the distinct specification states within the skeletogenic lineage that drive localized skeletal growth at the tips of the spicules. Finally, we discuss the surprising similarity between the regulatory network and cellular processes that drive sea urchin skeletogenesis and those that control vertebrate vascularization. Overall, we illustrate the novel insights on the biological regulation and evolution of biomineralization, gained from studies of the sea urchin larval skeletogenesis.

Measuring Process Dynamics and Nuclear Migration for Clones of Neural Progenitor Cells.

Neural stem and progenitor cells (NPCs) generate processes that extend from the cell body in a dynamic manner. The NPC nucleus migrates along these processes with patterns believed to be tightly coupled to mechanisms of cell cycle regulation and cell fate determination. Here, we describe a new segmentation and tracking approach that allows NPC processes and nuclei to be reliably tracked across multiple rounds of cell division in phase-contrast microscopy images. Results are presented for mouse adult and embryonic NPCs from hundreds of clones, or lineage trees, containing tens of thousands of cells and millions of segmentations. New visualization approaches allow the NPC nuclear and process features to be effectively visualized for an entire clone. Significant differences in process and nuclear dynamics were found among type A and type C adult NPCs, and also between embryonic NPCs cultured from the anterior and posterior cerebral cortex.

Computational Image Analysis Reveals Intrinsic Multigenerational Differences between Anterior and Posterior Cerebral Cortex Neural Progenitor Cells.

Time-lapse microscopy can capture patterns of development through multiple divisions for an entire clone of proliferating cells. Images are taken every few minutes over many days, generating data too vast to process completely by hand. Computational analysis of this data can benefit from occasional human guidance. Here we combine improved automated algorithms with minimized human validation to produce fully corrected segmentation, tracking, and lineaging results with dramatic reduction in effort. A web-based viewer provides access to data and results. The improved approach allows efficient analysis of large numbers of clones. Using this method, we studied populations of progenitor cells derived from the anterior and posterior embryonic mouse cerebral cortex, each growing in a standardized culture environment. Progenitors from the anterior cortex were smaller, less motile, and produced smaller clones compared to those from the posterior cortex, demonstrating cell-intrinsic differences that may contribute to the areal organization of the cerebral cortex.

Segmentation of occluded hematopoietic stem cells from tracking.

Image sequences of live proliferating cells often contain visual ambiguities that are difficult even for human domain experts to resolve. Here we present a new approach to analyzing image sequences that capture the development of clones of hematopoietic stem cells (HSCs) from live cell time lapse microscopy. The HSCs cannot survive long term imaging unless they are cultured together with a secondary cell type, OP9 stromal cells. The HSCs frequently disappear under the OP9 cell layer, making segmentation difficult or impossible from a single image frame, even for a human domain expert. We have developed a new approach to the segmentation of HSCs that captures these occluded cells. Starting with an a priori segmentation that uses a Monte Carlo technique to estimate the number of cells in a clump of touching cells, we proceed to track and lineage the image data. Following user validation of the lineage information, an a posteriori resegmentation step utilizing tracking results delineates the HSCs occluded by the OP9 layer. Resegmentation has been applied to 3031 occluded segmentations from 77 tracks, correctly recovering over 84% of the occluded segmentations.

Axonal transport analysis using Multitemporal Association Tracking.

Multitemporal Association Tracking (MAT) is a new graph-based method for multitarget tracking in biological applications that reduces the error rate and implementation complexity compared to approaches based on bipartite matching. The data association problem is solved over a window of future detection data using a graph-based cost function that approximates the Bayesian a posteriori association probability. MAT has been applied to hundreds of image sequences, tracking organelle and vesicles to quantify the deficiencies in axonal transport that can accompany neurodegenerative disorders such as Huntington's Disease and Multiple Sclerosis and to quantify changes in transport in response to therapeutic interventions.