Visualization of basement membranes by a nidogen-based fluorescent reporter in mice.

Basement membranes (BMs) are thin, sheet-like extracellular structures that cover the basal side of epithelial and endothelial tissues and provide structural and functional support to adjacent cell layers. The molecular structure of BMs is a fine meshwork that incorporates specialized extracellular matrix proteins. Recently, live visualization of BMs in invertebrates demonstrated that their structure is flexible and dynamically rearranged during cell differentiation and organogenesis. However, the BM dynamics in mammalian tissues remain to be elucidated. We developed a mammalian BM imaging probe based on nidogen-1, a major BM-specific protein. Recombinant human nidogen-1 fused with an enhanced green fluorescent protein (Nid1-EGFP) retains its ability to bind to other BM proteins, such as laminin, type IV collagen, and perlecan, in a solid-phase binding assay. When added to the culture medium of embryoid bodies derived from mouse ES cells, recombinant Nid1-EGFP accumulated in the BM zone of embryoid bodies, and BMs were visualized in vitro. For in vivo BM imaging, a knock-in reporter mouse line expressing human nidogen-1 fused to the red fluorescent protein mCherry (R26-CAG-Nid1-mCherry) was generated. R26-CAG-Nid1-mCherry showed fluorescently labeled BMs in early embryos and adult tissues, such as the epidermis, intestine, and skeletal muscles, whereas BM fluorescence was unclear in several other tissues, such as the lung and heart. In the retina, Nid1-mCherry fluorescence visualized the BMs of vascular endothelium and pericytes. In the developing retina, Nid1-mCherry fluorescence labeled the BM of the major central vessels; however, the BM fluorescence were hardly observed in the peripheral growing tips of the vascular network, despite the presence of endothelial BM. Time-lapse observation of the retinal vascular BM after photobleaching revealed gradual recovery of Nid1-mCherry fluorescence, suggesting the turnover of BM components in developing retinal blood vessels. To the best of our knowledge, this is the first demonstration of in vivo BM imaging using a genetically engineered mammalian model. Although R26-CAG-Nid1-mCherry has some limitations as an in vivo BM imaging model, it has potential applications in the study of BM dynamics during mammalian embryogenesis, tissue regeneration, and pathogenesis.

Migration of glial cells differentiated from neurosphere-forming neural stem/progenitor cells depends on the stiffness of the chemically cross-linked collagen gel substrate.

Substrate stiffness affects cell migration and spreading. Our study revealed that the stiffness of the cell-adhesive substrate affected the migration pattern of neural cells. We observed the migration of neural cells differentiated from neurosphere-forming neural stem/progenitor cells (NSPCs) on collagen gels with various degrees of stiffness achieved by chemical cross-linking. Both glial and neuronal cells broadly spread and migrated when stiff collagen gels (G'=5.5kPa, G″=0.2kPa) were used as the substrate. In contrast, the migration of glial cells was suppressed within the limited area on the soft collagen gels (G'=0.8kPa, G″=0.2kPa). Filopodia were rarely observed in glial cells on the soft collagen gels. Analysis of the intercellular distance between the closest neural cells after differentiation from NSPCs indicated that glial cells more broadly spread on the stiff collagen gels than on the soft gels. Immunocytochemical analysis showed that most of the migrated cells were glial cells, suggesting that migration of glial cells was dependent on the stiffness of substrate.