Figure 1.
Distribution of Myo/Nog cells in the human anterior segment.
Tissue sections through the anterior segment were stained with H&E (A) or double labeled with G8 and dendrimers to MyoD mRNA (MyoD m) or an antibody to Noggin (NOG) (B–P). The primary antibodies and colors of the fluorescent dendrimers and secondary antibodies are indicated in each photograph. Photographs are the merged images of DIC and fluorescence. Overlap of green and red appears yellow in merged images. Myo/Nog cells labeled for G8, MyoD mRNA and Noggin protein were present in the anterior (E–J), equatorial (K–M) and bow regions (N–P) of the lens, corneal epithelium (B), corneal stroma (C) and ciliary processes (D). AL = anterior lens, EL = equatorial lens, BR = bow region of the lens shown in the inset in A, CP = ciliary process, CE = corneal epithelium, S = corneal stroma. Bar = 135 µm in A and 9 µm in B–I.
Figure 2.
Distribution of Myo/Nog cells in human anterior lens tissue removed during cataract surgery.
A low magnification DIC image of anterior lens tissue fixed after capsulorhexis is shown in the inset in A. Tissue was double labeled with the G8 mAb and dendrimers to MyoD mRNA (MyoD m) or antibodies to MyoD protein (MyoD p), Noggin (NOG) or vimentin (VM). The primary antibodies and colors of the fluorescent dendrimers and secondary antibodies are indicated in each photograph. Unmerged images precede the merged images shown in C, F, I, L and Q. Overlap of green and red appears yellow in merged images. Nuclei were stained with Hoechst dye (HCT) (blue). Anterior lens tissue contained single, small groups or large clusters of Myo/Nog cells throughout the epithelium (A–L). Photographs in J-L illustrate a cluster of G8+ cells lying on the apical surface of lens epithelial cells. The underlying layer of nuclei in the same field is shown in the inset in J. G8+ cells were present along the cut edge of some (O) but not all samples (M). Vimentin staining was most intense at the periphery of the tissue (N and P). Tissue incubated with the E12 IgM or 2H3 IgG and their respective secondary antibodies lacked fluorescence (R and S). Bar = 5 mm in the inset in A and 9 µm in the other photomicrographs.
Table 1.
Myo/Nog cells in anterior lens tissue are immunoreactive for proteins found in skeletal muscle and lens tissue.
Figure 3.
Myo/Nog cells in human anterior lens tissue express muscle proteins.
Anterior lens tissue fixed after capsulorhexis was double labeled with the G8 mAb and dendrimers to MyoD mRNA (MyoD m) or antibodies to MyoD protein (MyoD p), Noggin (NOG), α-SMA, sarcomeric myosin heavy chain (MYOSIN), the skeletal muscle specific 12101 antigen (12101) and troponin T (TPNT). The primary antibodies and colors of the fluorescent dendrimers and secondary antibodies are indicated in each photograph. Overlap of green and red appears yellow in merged images. Nuclei were stained with Hoechst dye (blue). Panels I, L, U and V are quadruple merged images of DIC and fluorescence showing wrinkles in the capsule (arrow in I). G8+ cells co-stained for MyoD mRNA, Noggin and muscle proteins surrounded cell free areas of the capsule (A–V). Some Myo/Nog cells had migrated onto the capsule (arrows in V). Tissue incubated with the E12 or 2H3 mAbs, or an antiserum to LBX1 and their respective secondary antibodies, lacked fluorescence (W–Y). Bar = 9 µm.
Figure 4.
Effects of targeting Myo/Nog cells with the G8 mAb and complement in anterior lens explants.
Explants of lens tissue were incubated with the G8 mAb and complement (A, D–H), G8 only (B, I–M) or complement only (C). Five hours later, the tissue was double labeled with the G8 mAb (green) and TUNEL reagents (red). Nuclei were stained with Hoechst dye. G8+, but not G8- cells, were TUNEL+ following treatment with G8 and complement (A). G8+ cells were not TUNEL+ when treated with G8 or complement alone (B and C). Five days following ablation, explants were double labeled with antibodies to G8 and MyoD, α-SMA, sarcomeric myosin heavy chain (MYOSIN), filensin and cp49. The colors of the fluorescent secondary antibodies are indicated in each photograph. Overlap of green and red appears yellow in merged images. Ablation of Myo/Nog cells prevented the accumulation of G8+, MyoD+ and α-SMA+ cells, but not filensin+ and cp49+ cells (D–H). Both muscle and beaded filament proteins were detected in explants treated with the G8 mAb alone (I–M). Bar = 9 µm.
Table 2.
Effect of depleting Myo/Nog cells on the accumulation of cells immunoreactive for muscle and beaded filament proteins in anterior lens cultures.
Figure 5.
Effects of Myo/Nog cell depletion and TGF-β2 on wound healing in anterior lens explants.
A scratch wound was created in explants of anterior lens tissue. Cells had populated the capsule denuded of cells within 48 hours of wounding (A). The wound was also covered with cells following treatment with TGF-β2 (B). Few cells were present in the wound of an explant depleted of Myo/Nog cells (-M/N) (C). The combination of wounding, depletion of Myo/Nog cells and treatment with TGF-β2 resulted in a loss of most cells from the capsule (D). Addition of Noggin (NOG) prevented cell loss resulting from depletion of Myo/Nog cells and treatment with TGF-β2 (E) and promoted wound healing (F). Bar = 27 µm in A-C and F, and 135 µm in D and E.
Figure 6.
Effect of TGF-β on the accumulation of cells with muscle proteins following wounding and depletion of Myo/Nog cells.
Anterior lens explants were wounded and incubated in medium alone (untx) or medium containing TGF-β2 or −β1. Myo/Nog cells were ablated (abl) in some explants prior to wounding. Cells were double labeled with antibodies to α-SMA (green) and MyoD or sarcomeric myosin (red). Nuclei were stained with Hoechst dye. Photographs were taken of the wounded area. Explants incubated in the presence or absence of TGF-β2 or −β1 contained α-SMA+, MyoD+ and myosin+ cells (A–D, I and J). Following depletion of Myo/Nog cells, α-SMA+ cells were less prevalent in the wounds of untreated (E and F) and TGF-β2 treated explants (G and H) than those treated with TGF-β1 (K). No MyoD+ or myosin+ cells were observed in the wounds following Myo/Nog cell depletion and incubation in the presence (G, H, K and L) or absence (E and F) of TGF-β2 or −β1. Bar = 9 µm.
Table 3.
Effect of TGF-β on the accumulation of cells with muscle proteins in the presence and absence of Myo/Nog cells.