Myo/Nog Cells: The Jekylls and Hydes of the Lens

Abstract

Herein, we review a unique and versatile lineage composed of Myo/Nog cells that may be beneficial or detrimental depending on their environment and nature of the pathological stimuli they are exposed to. While we will focus on the lens, related Myo/Nog cell behaviors and functions in other tissues are integrated into the narrative of our research that spans over three decades, examines multiple species and progresses from early stages of embryonic development to aging adults. Myo/Nog cells were discovered in the embryonic epiblast by their co-expression of the skeletal muscle-specific transcription factor MyoD, the bone morphogenetic protein inhibitor Noggin and brain-specific angiogenesis inhibitor 1. They were tracked from the epiblast into the developing lens, revealing heterogeneity of cell types within this structure. Depletion of Myo/Nog cells in the epiblast results in eye malformations arising from the absence of Noggin. In the adult lens, Myo/Nog cells are the source of myofibroblasts whose contractions produce wrinkles in the capsule. Eliminating this population within the rabbit lens during cataract surgery reduces posterior capsule opacification to below clinically significant levels. Parallels are drawn between the therapeutic potential of targeting Myo/Nog cells to prevent fibrotic disease in the lens and other ocular tissues.

Keywords: BAI1; Myo/Nog; MyoD; Noggin; PCO; PVR; fibrosis; lens; myofibroblasts; retina.

Figure 1

Cells that co-express MyoD, Noggin and BAI1 in the embryonic epiblast differentiate into skeletal muscle in vitro. In situ hybridization and immunofluorescence localization revealed a small population of cells that co-expressed BAI1 (green) and MyoD mRNA (red) (B) and Noggin protein (red) (C) in the posterior/medial epiblast of chick embryos before the onset of gastrulation (red box in A). Nuclei are stained with Hoechst dye (blue). Labeling with the skeletal muscle-specific 12,101 mAb demonstrated that BAI1-positive Myo/Nog cells isolated from the epiblast differentiated into skeletal myofibers when cultured in serum-free medium (D). BAI1-negative epiblast cells were labeled with a mAb to cardiac muscle-specific troponin T (E). Addition of Noggin to the medium resulted in the synthesis of the 12,101 antigen in BAI1-negative cells (F). Bar = 13 µM.

Figure 2

Epiblast-derived Myo/Nog cells are integrated into the developing lens. Myo/Nog cells were fluorescently labeled with the BAI1 mAb in the epiblast and the embryos were incubated for 1–3 days. Sections were stained with Hoechst dye to label nuclei (blue). In the stage 7 embryo, Myo/Nog cells were found in the pre-lens placode (boxed area in (A) shown at high magnification in (B)). A stage 17 eye is shown in (C). The area within the red box is shown at higher magnification in (D,E). The white box delineates the enlargement in (F). Prelabeled Myo/Nog cells were found in the equatorial zone (EZ) of the lens (D) and among the differentiating primary lens fiber cells (arrow in (D)). BAI1+ and Noggin+ cells (green) co-expressed MyoD mRNA (red) throughout the lens (E,F). Overlap of green and red appears yellow in merged images. Bar = 135 µM in (A), 13 µM in (B) and (D–F) and 56 µM in (C).

Figure 3

Myo/Nog cells are required for normal eye development and migrate to wounds in the embryo. Myo/Nog cells were eliminated in the chick embryo epiblast by incubating the embryo in the BAI1 mAb and complement. Embryos were incubated for approximately 5 days. Some embryos received beads soaked in Noggin (Nog) a day after treatment. A normal PBS control embryo (CT) and an H&E-stained section of the eye are shown in (A,B). Depletion of Myo/Nog cells resulted in malformations of the body wall and eyes, including microphthalmia (arrow in (C)) and dysgenesis of the lens and retina (D). Addition of Noggin-soaked beads prevented malformations caused by Myo/Nog cell depletion (E,F). Embryos treated with the D4 mAb and complement to eliminate a separate population of cells in the central epiblast (blue box in (G)) stimulated the migration of BAI1-labeled Myo/Nog cells (green) from the posterior/medial epiblast (red box in (G)) to TUNEL+ cells (red) (H). The direction of migration is indicated by the arrow in H. A puncture wound was produced in the day-1 chick embryo lateral to the primitive streak (boxed area in (I)). BAI1+ cells (red) surrounded the wound (J). BAI1+ (green) cells containing MyoD mRNA (red) were present in niches in explants of fetal chick lens tissue (arrows in (K)). Within two days after plating, BAI1+/MyoD mRNA+ cells had migrated to the cut edge of the tissue (arrow in (L)). Bar = 5 mM in (A,C), 56 µM in (B,D), 2.5 mM in (E), 41 µM in (F) and 13 µM in (H–L).

Figure 4

Myo/Nog cells are present throughout the adult mouse eye. Sections of the adult mouse eye were stained with H&E (A) or double-labeled with antibodies to BAI1 (red) and Noggin (B–F,H) or fibrillin (green) (G). The overlap of red and green appears yellow in merged images. Nuclei were stained with Hoechst dye (blue). Arrows point to Myo/Nog cells in the equatorial (B) and bow regions (C) of the lens, the endothelial layer of the cornea (D), ciliary body (CB) (E) and on the zonules of Zinn (arrows in (F,G)). Each layer of the retina contained a small number of Myo/Nog cells (H). Bar = 55 µM in (A), 9 µM in (B–E,G,H) and 6.5 µM in (F).

Figure 5

Myo/Nog cells are the source of myofibroblasts and phagocytose dead cells in human anterior lens explant cultures. Anterior lens tissue was obtained from patients undergoing cataract surgery and cultured in serum-free medium. Myo/Nog cells double-labeled with antibodies to BAI1 (red) and Noggin, α-SMA and skeletal muscle-specific troponin T (green) surrounded wounds in the epithelium (A–C) and extended processes towards wrinkles in the capsule (arrow in (A)). They also migrated onto the capsule and synthesized α-SMA after producing a scratch wound in 2-day cultures (E). Cells filling the scratch wound in the presence of Myo/Nog cells are shown in the phase contrast photomigraph (F). Myo/Nog cells were depleted with the anti-BAI1 mAb and complement (-M/N) (G–J). Treatment of explants with TGF-β1 after Myo/Nog cell depletion contained α-SMA+/BAI1-cells (D). Scratch wounds contained fewer cells on the capsule after Myo/Nog cell depletion (G). Incubation with TGF-β2 in the absence of Myo/Nog cells resulted in detachment of most cells from the capsule (H). Addition of Noggin (Nog) to TGF-β2 treated cultures prevented detachment and promoted migration into the wound, but the cells did not express MyoD or α-SMA (I,J). Explants were treated on days 2 and 13 with the BAI1 mAb conjugated to 3DNA nanocarriers intercalated with doxorubicin (BAI1 Ab:3DNA:Dox) (K,M). TUNEL staining revealed apoptotic BAI1+ cells (K). BAI1+/α-SMA+ cells migrated into the scratch wound in 30-day control cultures treated with BAI1 Ab:3DNA lacking doxorubicin (L). BAI1+/Noggin+ were not present in explants treated with BAI1 Ab:3DNA:Dox (M). Anterior lens explants were labeled with lysosensor red dye and then treated with doxorubicin to induce apoptosis. Treated cells were added to untreated explant cultures. BAI1+ cells (green) phagocytosed cells prelabeled with lysosenser dye (red) and killed with doxorubicin (black) (arrow in N). Bar = 9 µM in (A–E,I,K–N), 27 µM in (F–I).

Figure 6

Myo/Nog cells differentiate into myofibroblasts in posterior capsule opacification (PCO) and proliferative vitreoretinopathy (PVR). Rabbit lenses were injected with balanced saline solution (BSS) (A,C) or BAI1 Ab:3DNA:Dox (B,D) during cataract surgery. Lenses were harvested 30 days later and sections were labeled with an antibody to SMA (green) (A–D). SMA and wrinkles (arrow) were abundant in lenses injected with BSS) (A). Depletion of Myo/Nog cells significantly reduced α-SMA+ myofibroblasts and wrinkles in the capsule (B,D). Human epiretinal membranes (ERM) removed from patients with PVR were sectioned and stained with H&E (E) or antibodies to BAI1 (red) and striated muscle myosin (green) (F). Overlap of red and green in merged images appears yellow. Nuclei were labeled with Hoechst dye (blue). Myosin+ myofibroblasts express BAI1 in human epiretinal membranes (F). PVR was induced in the mouse retina by injecting SF6 gas and human ARPE-19 cells into the vitreous. Sections were double labeled with antibodies to BAI1 (red) and α-SMA (green) (G,H). BAI1+ myofibroblasts were present in epiretinal membranes and throughout the retina (G,H) and overlaid areas of detachment (arrow in G). OPL = outer plexiform layer. Bar = 27 µM in (A–D), 9 µM in (E–H) and 135 µM in (G).

Figure 7

BAI1+ cells in human rhabdomyosarcoma cell lines and tumors express beaded filament proteins. Cultures of RC13 (A,B) and RD RMS cell lines (C,D) derived from alveolar and embryonal RMS tumors, respectively, were double labeled with antibodies to BAI1 (red) and filensin (Fil) or CP49 (green). Nuclei were stained with Hoechst dye (blue). Both cell lines contained cells that co-expressed BAI1 and beaded filaments (A–D). Tissue sections of alveolar (D,E) and embryonal RMS tumors (F,G) were double-labeled with the BAI1 mAb (green) and an anti-sense probe for filensin mRNA (red) (D) or an antibody to filensin (green) (D,F). Double labels also were performed with antibodies to filensin (red) and CP49 (green) (E,G). BAI1+ cells expressed filensin and filensin co-localized with CP49 (D–H). Bar = 9 µM.