Volume 18, Issue 2, Pages 243-252 (February 2016) Intravital Imaging Reveals Ghost Fibers as Architectural Units Guiding Myogenic Progenitors during Regeneration  Micah T. Webster, Uri Manor, Jennifer Lippincott-Schwartz, Chen-Ming Fan  Cell Stem Cell  Volume 18, Issue 2, Pages 243-252 (February 2016) DOI: 10.1016/j.stem.2015.11.005 Copyright © 2016 Elsevier Inc. Terms and Conditions

Cell Stem Cell 2016 18, 243-252DOI: (10.1016/j.stem.2015.11.005) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Characterizing the SC Lineage during Regeneration by IVM (A) Diagram of the TA muscle (top, pink). Uninjured myofiber (middle, pink with red lines for sarcomeres) is shown, along with SCs (green cells with light blue nuclei) between the basal lamina (dark blue) and sarcolemma (myonuclei in white) and ghost fiber following myofiber injury (bottom, SC contained within remaining basal lamina). Longitudinal axis = x, radial axes = y and z. (B) IVM of eYFP+ SC (green) on myofiber (sarcomere SHG, red); arrows, bipolar projections of the SC. (C) Time course of eYFP+ (black circles) or H2B-GFP+ (4 dpi green circle) cell density in the TA at indicated dpi; ∗p < 0.05, t test; error bars = SEM. (D) Radar plots representing percentage (wedge length) of angles (15° bins) of eYFP+ cell long axes relative to the x and y axes (top row) and x and z axes (bottom row); ≥40 cells per time point; dashed line = 45°. (E) eYFP+ SCs (∗) at beginning (red) and end (green) of 369 min IVM session; sarcomere SHG, white; minutes elapsed, upper right. (F) eYFP+ MPs (green) following injury (dpi at top); arrows, ghost fiber collagen SHG, red. Images were chosen to illustrate cell alignment within ghost fibers, though many more cells are present in 3 dpi ghost fibers compared to earlier time points. (G) H2B-GFP+ nuclei (green) at 4 dpi within ghost fibers (red, arrows); brackets, short stretches of well-aligned nuclei. Planes of perspective are at bottom left for each image; scale bars, 50 μm. Cell Stem Cell 2016 18, 243-252DOI: (10.1016/j.stem.2015.11.005) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 MP Divisions Align with the Ghost Fiber’s Longitudinal Axis (A and B) IVM time points of (A) eYFP+ and (B) H2B-GFP+ MPs dividing (anaphase marked by arrowheads) in a ghost fiber (red) parallel to the longitudinal, x axis (dashed white line); minutes elapsed, upper right. (C) Division frequencies of MPs at indicated dpi. Frequency is represented as the number of divisions per total labeled cells in the IVM field per 1 hr period; ≥120 cells per day; 3 dpi versus uninjured, 1 dpi, and 4 dpi; ∗p < 0.05, t test; error bars = SEM. (D) 3 dpi H2B-GFP+ MP undergoing division in the xy plane. Division angle (θ) of yellow dashed lines is measured relative to x axis (dashed white line). (E) Radar plot representing percentage (wedge length) of 1 dpi to 4 dpi H2B-GFP+ cell division angles (15° bins) relative to the x and y axes; 156 divisions; dashed line = 45°. (F) As in (D) but division is viewed in the xz plane. (G) Radar plots as in (E) but division angles are measured relative to the x and z axes. (H) IVM of 1 dpi time points of H2B-GFP+ cell migrating prior to division (∗); minutes elapsed, upper left; dashed white line, spatial reference. Scale bars, 10 μm in (A), (B), (D), and (F); 25 μm in (H). Cell Stem Cell 2016 18, 243-252DOI: (10.1016/j.stem.2015.11.005) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 MPs Migrate Bi-directionally along the Longitudinal, x Axis of Ghost Fibers All data are from 1 to 3 dpi eYFP+ MPs by IVM. (A) Percentage of MPs migrating at given dpi; ≥135 cells per day. (B) MP migratory track displacements (μm) in the x axis at given dpi; ≥114 cells per day; red dash = mean. (C) 2 dpi IVM time points of MP (green) migrating next to uninjured fiber (sarcomere SHG, red). Leading edge bifurcates in two places (arrows). Minutes elapsed, bottom right. (D) 3 dpi IVM time points of MP (green) traversing the y axis. Leading edge projects bi-directionally (arrows) along the x axis and then stabilizes as the cell continues migrating along the x axis. Minutes elapsed, bottom right. (E) Example of lateral protrusions. 1 dpi IVM of eYFP+ MP with defined leading edge (arrowhead) and lateral protrusion development (arrows); minutes elapsed, bottom left. (F) Average lateral protrusions at a given dpi; 15 cells per day. (G) Quartile distribution showing duration (min) of MP lateral protrusions at a given dpi; 15 cells per day. (H) Average MP migration speed (μm/hr) at a given dpi; ≥100 cells per day. (I) Average total migratory track displacements (μm) for MPs at a given dpi; ≥100 cells per day. (J) Quartile distribution of MP track straightness at a given dpi; ≥100 cells per day. (K and L) Projected MP migration tracks at 3 dpi from xz and yz perspectives, respectively. Track color denotes passage of time during 1 hr (starting at blue, ending at red). (M) 3 dpi IVM image of MPs (green) distributed within ghost fiber, adjacent to uninjured fiber (sarcomere SHG). (N and O) Percentages of cells with either positive (+ve) displacement (right, black bar) or negative (–ve) displacement (left, gray bar) in the x axis during 1 hr migration at (N) given dpi; ≥90 cells per day; or (O) 3 days post needle track (NT) injury either at (NT) or distal to (∼500 μm) the NT site; ≥60 cells per site. (P) IVM time course of SCs (∗) on uninjured fibers (white) and MPs 3 d after NT injury at 0 min (red), 27 min (green), 60 min (cyan), and merged. Examples of migrating MPs in injury site (arrowheads) relative to SCs (∗) that do not change position on uninjured fibers are shown. ∗p < 0.05 throughout, t test; error bars = SEM for (A), (H), (I), (N), and (O) and SD for (F); scale bars, 50 μm (C), (D), (K–M), and (P); 25 μm in (E). Cell Stem Cell 2016 18, 243-252DOI: (10.1016/j.stem.2015.11.005) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Ghost Fibers Govern MP Migration and Division and Organization of Regenerated Muscle (A) Diagram of TA muscle showing surgical rotation of a 1 mm3 muscle block (thick black square; lines, fiber orientation). The rotated region, R, is surrounded by a margin of interstitial space, I, within the muscle, M. (B) 3 days post rotation IVM time points from region R of 2 MPs (arrowhead and arrow) at 0 min (green), 60 min (red), and merged, migrating along ghost fiber (collagen SHG, yellow arrow) in the y axis. (C) 3 days post rotation IVM of MPs (green) in region I, below region R (white dashed line). Some MPs form clusters (∗) in region I at the ends of severed fibers. (D) Average MP migratory track displacements (μm) in each axis 3 days after cardiotoxin (CTX) injury or rotation injury in regions R and I; ≥44 cells per injury; ∗p < 0.05, t test; error bars = SEM. (E and F) 3 days post rotation division angles relative to x and y axes (left) and y and z axes (right) of MPs located in (E) region R and (F) region I; 12 cells per region; red dashed line = 45°. (G) Longitudinal section of 14 days post rotation muscle probed with anti-eYFP, anti-MHC, and anti-Laminin and counterstained with DAPI. Newly regenerated fibers (eYFP+ MHC+ Laminin+) in the rotated region turn toward one another (arrows) or do not fuse (arrowheads) with large fibers in the interstitial space; enlarged region of identified cells is shown at top right. (H and I) Models for a ghost fiber's effect on MP behavior, starting from uninjured myofiber (pink with sarcomeres, red) with attached SCs (green cells with light blue nuclei). (H) SCs become activated MPs that begin migrating and dividing at 1 dpi along the inner surface of a ghost fiber (dark blue outline). Throughout 2 and 3 dpi, MPs increase division and bi-directional migration along the ghost fiber’s longitudinal axis, decrease lateral protrusions, and become evenly distributed along the inner surface of the ghost fiber prior to forming nascent myotubes at 4 dpi. (I) MPs that migrate from damaged ghost fibers enter interstitial ECM (webbed black lines) where their division angles and migratory paths become random, causing branched and disorganized regenerated muscle. Scale bars, 50 μm in (B) and (C); 100 μm in (G). Cell Stem Cell 2016 18, 243-252DOI: (10.1016/j.stem.2015.11.005) Copyright © 2016 Elsevier Inc. Terms and Conditions