Figure 1. Development of a 4D imaging... | Rockefeller University Press

Figure 1.

$Development of a 4D imaging method for visualizing basement membrane dynamics. (A) Schematic model of BM molecular structure and fluorescent protein insertion site in the collagen IV α2 chain. (B) Genotypes of pups from eGFP-Col4a2 het × eGFP-Col4a2 het crosses. Sample sizes in Table 1. (C) Appearance of 8-wk-old wild-type and eGFP-Col4a2 homo mice. (D) Immunofluorescence image of E15.5 embryos from eGFP-Col4a2 mice stained for eGFP. Scale bar: 3 mm. (E–H) Immunofluorescence images of adult tissues from eGFP-Col4a2 mice. (E) In P56 adult telogen dorsal skin from het mice, eGFP (green) colocalized with COL4A2 (magenta) in BMs of epidermis (white arrowheads), follicle epithelium (yellow arrowheads), and vessel-like tissues (open arrowheads). (F) In homo mice, eGFP (green) colocalizes similarly with collagen IV (magenta) and was also found in arrector pili muscles (arrows). The same field of view is presented in Fig. S1 I. (G) In adult kidneys, eGFP (green) was detected in Bouman’s capsules (closed arrowheads), the mesangial matrix (arrows), and collecting ducts (open arrowheads). (H) In adult brain, eGFP (green) localized to capillaries (closed arrowheads) and fractone-like structures (open arrowheads). DAPI (blue) = nuclear counterstaining. Scale bars: 50 μm. (I) Fluorescence images of cultured dorsal skin explants from E12.5 embryos of eGFP-Col4a2 mice. Magnified areas show primary (closed arrowheads) and secondary (open arrowheads) hair follicles. Scale bars: 200 μm. (J) Snapshot images of the 3D maximum projection of cultured E12.5 skin explants from eGFP-Col4a2 mice. Closed arrowheads indicate ring-like accumulations of the eGFP-COL4A2 signal at the neck regions of hair follicles. Hair follicles, indicated by closed arrowheads in the left panel, grow vertically from the plane of observation. The dashed rectangle indicates a follicle growing horizontally. The snapshot images in the right panels were obtained from a separate scan of the follicle outlined by the dashed rectangle in the 3D time-lapse maximum projection shown in the main image. The open arrowheads indicate vessel-like structures. Scale bars: 50 μm.$

Development of a 4D imaging method for visualizing basement membrane dynamics. (A) Schematic model of BM molecular structure and fluorescent protein insertion site in the collagen IV α2 chain. (B) Genotypes of pups from eGFP-Col4a2 het × eGFP-Col4a2 het crosses. Sample sizes in Table 1. (C) Appearance of 8-wk-old wild-type and eGFP-Col4a2 homo mice. (D) Immunofluorescence image of E15.5 embryos from eGFP-Col4a2 mice stained for eGFP. Scale bar: 3 mm. (E–H) Immunofluorescence images of adult tissues from eGFP-Col4a2 mice. (E) In P56 adult telogen dorsal skin from het mice, eGFP (green) colocalized with COL4A2 (magenta) in BMs of epidermis (white arrowheads), follicle epithelium (yellow arrowheads), and vessel-like tissues (open arrowheads). (F) In homo mice, eGFP (green) colocalizes similarly with collagen IV (magenta) and was also found in arrector pili muscles (arrows). The same field of view is presented in Fig. S1 I. (G) In adult kidneys, eGFP (green) was detected in Bouman’s capsules (closed arrowheads), the mesangial matrix (arrows), and collecting ducts (open arrowheads). (H) In adult brain, eGFP (green) localized to capillaries (closed arrowheads) and fractone-like structures (open arrowheads). DAPI (blue) = nuclear counterstaining. Scale bars: 50 μm. (I) Fluorescence images of cultured dorsal skin explants from E12.5 embryos of eGFP-Col4a2 mice. Magnified areas show primary (closed arrowheads) and secondary (open arrowheads) hair follicles. Scale bars: 200 μm. (J) Snapshot images of the 3D maximum projection of cultured E12.5 skin explants from eGFP-Col4a2 mice. Closed arrowheads indicate ring-like accumulations of the eGFP-COL4A2 signal at the neck regions of hair follicles. Hair follicles, indicated by closed arrowheads in the left panel, grow vertically from the plane of observation. The dashed rectangle indicates a follicle growing horizontally. The snapshot images in the right panels were obtained from a separate scan of the follicle outlined by the dashed rectangle in the 3D time-lapse maximum projection shown in the main image. The open arrowheads indicate vessel-like structures. Scale bars: 50 μm.

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