Serial intravital imaging captures dynamic and functional endothelial remodeling with single-cell resolution.

Endothelial cells are important in the maintenance of healthy blood vessels and in the development of vascular diseases. However, the origin and dynamics of endothelial precursors and remodeling at the single-cell level have been difficult to study in vivo owing to technical limitations. Therefore, we aimed to develop a direct visual approach to track the fate and function of single endothelial cells over several days and weeks in the same vascular bed in vivo using multiphoton microscopy (MPM) of transgenic Cdh5-Confetti mice and the kidney glomerulus as a model. Individual cells of the vascular endothelial lineage were identified and tracked owing to their unique color combination, based on the random expression of cyan/green/yellow/red fluorescent proteins. Experimental hypertension, hyperglycemia, and laser-induced endothelial cell ablation rapidly increased the number of new glomerular endothelial cells that appeared in clusters of the same color, suggesting clonal cell remodeling by local precursors at the vascular pole. Furthermore, intravital MPM allowed the detection of distinct structural and functional alterations of proliferating endothelial cells. No circulating Cdh5-Confetti+ cells were found in the renal cortex. Moreover, the heart, lung, and kidneys showed more significant clonal endothelial cell expansion compared with the brain, pancreas, liver, and spleen. In summary, we have demonstrated that serial MPM of Cdh5-Confetti mice in vivo is a powerful technical advance to study endothelial remodeling and repair in the kidney and other organs under physiological and disease conditions.

Prkci is required for a non-autonomous signal that coordinates cell polarity during cavitation.

Polarized epithelia define boundaries, spaces, and cavities within organisms. Cavitation, a process by which multicellular hollow balls or tubes are produced, is typically associated with the formation of organized epithelia. In order for these epithelial layers to form, cells must ultimately establish a distinct apical-basal polarity. Atypical PKCs have been proposed to be required for apical-basal polarity in diverse species. Here we show that while cells null for the Prkci isozyme exhibit some polarity characteristics, they fail to properly segregate apical-basal proteins, form a coordinated ectodermal epithelium, or participate in normal cavitation. A failure to cavitate could be due to an overgrowth of interior cells or to an inability of interior cells to die. Null cells however, do not have a marked change in proliferation rate and are still capable of undergoing cell death, suggesting that alterations in these processes are not the predominant cause of the failed cavitation. Overexpression of BMP4 or EZRIN can partially rescue the phenotype possibly by promoting cell death, polarity, and differentiation. However, neither is sufficient to provide the required cues to generate a polarized epithelium and fully rescue cavitation. Interestingly, when wildtype and Prkci(-/-) ES cells are mixed together, a polarized ectodermal epithelium forms and cavitation is rescued, likely due to the ability of wildtype cells to produce non-autonomous polarity cues. We conclude that Prkci is not required for cells to respond to these cues, though it is required to produce them. Together these findings indicate that environmental cues can facilitate the formation of polarized epithelia and that cavitation requires the proper coordination of multiple basic cellular processes including proliferation, differentiation, cell death, and apical-basal polarization.

Iroquois Proteins Promote Skeletal Joint Formation by Maintaining Chondrocytes in an Immature State.

An early event in skeletal joint development is the specification of articular chondrocytes at the joint surface. Articular chondrocytes are distinct in producing lower levels of cartilage matrix and not being replaced by bone, yet how they acquire these properties remains poorly understood. Here, we show that two members of the Iroquois transcriptional repressor family, Irx7 and Irx5a, function to block chondrocyte maturation at the developing hyoid joint of zebrafish. These Irx factors suppress the production of cartilage matrix at the joint in part by preventing the activation of a col2a1a enhancer by Sox9a. Further, both zebrafish Irx7 and mouse IRX1 are able to repress cartilage matrix production in a murine chondrogenic cell line. Iroquois proteins may therefore have a conserved role in keeping chondrocytes in an immature state, with the lower levels of cartilage matrix produced by these immature cells contributing to joint flexibility.

Natural large-scale regeneration of rib cartilage in a mouse model.

The clinical need for methods to repair and regenerate large cartilage and bone lesions persists. One way to make new headway is to study skeletal regeneration when it occurs naturally. Cartilage repair is typically slow and incomplete. However, an exception to this observation can be found in the costal cartilages, where complete repair has been reported in humans but the cellular and molecular mechanisms have not yet been characterized. In this study, we establish a novel animal model for cartilage repair using the mouse rib costal cartilage. We then use this model to test the hypothesis that the perichondrium, the dense connective tissue that surrounds the cartilage, is a tissue essential for repair. Our results show that full replacement of the resected cartilage occurs quickly (within 1 to 2 months) and properly differentiates but that repair occurs only in the presence of the perichondrium. We then provide evidence that the rib perichondrium contains a special niche that houses chondrogenic progenitors that possess qualities particularly suited for mediating repair. Label-retaining cells can be found within the perichondrium that can give rise to new chondrocytes. Furthermore, the perichondrium proliferates and thickens during the healing period and when ectopically placed can generate new cartilage. In conclusion, we have successfully established a model for hyaline cartilage repair in the mouse rib, which should be useful for gaining a more detailed understanding of cartilage regeneration and ultimately for developing methods to improve cartilage and bone repair in other parts of the skeleton.