Whole-body replacement of larval myofibers generates permanent adult myofibers in zebrafish

Drastic increases in myofiber number and size are essential to support vertebrate post-embryonic growth. However, the collective cellular behaviors that enable these increases have remained elusive. Here, we created the palmuscle myofiber tagging and tracking system for in toto monitoring of the growth and fates of ~5000 fast myofibers in developing zebrafish larvae. Through live tracking of individual myofibers within the same individuals over extended periods, we found that many larval myofibers readily dissolved during development, enabling the on-site addition of new and more myofibers. Remarkably, whole-body surveillance of multicolor-barcoded myofibers further unveiled a gradual yet extensive elimination of larval myofiber populations, resulting in near-total replacement by late juvenile stages. The subsequently emerging adult myofibers are not only long-lasting, but also morphologically and functionally distinct from the larval populations. Furthermore, we determined that the elimination-replacement process is dependent on and driven by the autophagy pathway. Altogether, we propose that the whole-body replacement of larval myofibers is an inherent yet previously unnoticed process driving organismic muscle growth during vertebrate post-embryonic development.


The EMBO Journal
Uday Kumar et al 20 The EMBO Journal © The Author(s)

Figure EV2 .
Figure EV2.Individual deformed myofibers are fully eliminated and replaced.
(A-C) Time-lapse images of the same myotome over a 3-day period, showing the myofiber elimination and the replacement processes in a cross-sectional view (A) or as a 3D rendering (B, C).Blue dashed lines outline a deformed myofiber.Red dashed lines outline newborn myofibers.Neighboring myofibers are labeled with respective numbers.(D) Spatial distribution of deformed myofibers in the dorsal and ventral myotomes.A total of 98 dissolution events were captured and mapped.(E) Representative image of pax7b-positive muscle stem cells expressing mCherry.(F) Representative image showing pax7b-positive cells (yellow arrows) in close proximity to a deformed myofiber.(G) Cross-sectional view of a myotome showing pax7b-positive cells located either at the myotome's periphery (white arrows) or within the interstitial space (yellow arrows).(H, I) Quantification of myofiber composition within a myotome (H) and the interstitial pax7b-positive cells closely associated with either normal or deformed myofibers (I).Data from biological replicates are shown as mean ± standard deviation (H, I).n = number of myotome (H) or cells (I).Scale bar, 50 µm (A, E-G).

Figure EV3 .
Figure EV3.Pre-existing pectoral fin myofibers are eliminated and replaced by a de novo source.(A) The palmuscle-Multi and the myofiber:iCre#2 transgenic constructs.(B) Timeline of the tracking scheme.(C, D) Long-term tracking of the Tg(palmuscle-Multi; myofiber:iCre#2) double transgenic line in different anatomical regions, including the pectoral fin region (C) and the craniofacial region (D).No leaky Cre activity was observed.(E) The mylpfa:Brainbow1.0 L and myofiber:iCre#2 transgenic constructs.(F) Timeline of the treatment and tracking scheme.(G) Long-term tracking of the Tg(mylpfa:Brainbow1.0 L; myofiber:iCre#2) double transgenic line showed no leaky Cre activity.(H) Long-term time-lapse imaging of the same myofibers at 14, 21, and 28 dpf.White arrows highlight "disappearing myofibers".White dashed lines (bottom right, G and H) outline the bottom pectoral fin myofiber compartment, which becomes less visible at 28 dpf due to tissue thickening.(I) Quantification of tagged myofiber numbers.(J) Cross-sectional view of the pectoral fin myofibers showing both top and bottom layers at 14, 21 and 28 dpf.(K, L) Quantification of myofiber numbers (K) and myofiber areas (L).The entire top layer of fin myofibers were included in the quantification.Data from biological replicates are shown as mean ± standard deviation (K) or violin plots (solid lines, median; dashed lines, quartiles; L).Significance was examined by two-tailed Student's t-test (K) or two-tailed Mann-Whitney test (L).Percent differences and P values are shown above the horizontal lines for intergroup comparisons.n = number of animals (C, D, G, H, K) or myofibers (I, L).Scale bars, 50 µm (J) and 100 µm (C, D, G, H). dpf, days post-fertilization.

Figure EV4 .
Figure EV4.Rapid growth condition promotes myofiber elimination.(A)Timeline of larval growth manipulation and tracking scheme.SG, slow growth.FG, fast growth.(B, C) Larval growth under the SG and FG conditions as determined by standard length (B), and trunk surface area (C).(D, E) Long-term tracking of the Tg(palmuscle-Multi; myofiber:iCre#2) double transgenic line under SG (D) and FG (E) conditions.(F, G) Quantification of tagged myofiber numbers (F) and decreases in percentage (G).Gray dashed line highlights the standard length of 9 mm.(H) RT-qPCR analysis of whole-animal myofiber loss at 42 dpf under either SG or FG conditions.Data from biological replicates are shown as mean ± standard deviation (B, C) and mean ± standard error (H).Significance was examined by two-tailed Student's t-test.Percent differences and P values are shown above the horizontal lines for intergroup comparisons.n = number of animals (B-F) or biological replicates (H).m = number of myofibers (F).Scale bar, 100 µm (D, E). dpf, days post-fertilization.

Figure EV5 .
Figure EV5.Larval myofibers are functionally distinct from adult myofibers.(A) Illustration of zebrafish at larval and adult stages, reflecting their relative size differences.(B, C) RT-qPCR analyses of expression of the intermediate filament desma (B), and sarcomere myosin myhc4 (C) in whole animals (14 dpf) or dissected muscle tissues (1.7 years of age).(D) Cross-sectional image of the middle-trunk region of the animal at 16 mpf with tagged myofibers.F310 Ab stains fast myofibers.(E) Magnified view of the trunk region indicated in (D) by red dashed box.(F) Individual tagged myofibers from cross-sectional images shown in pseudocolor (left) and with respective ROI numbers (right).(G) Timeline of the treatment and tracking scheme.(H) Schematic drawing of the progressive shift in hyperplastic growth zone across different developmental stages.(I-K) Histological examinations of the tagged myofibers in the middle-trunk region from larvae to adult stage, showing growth zone shift from the periphery at 29 dpf (I), to the interstitial space at 43 dpf (J), and ultimately to the deep region of the myotome at 16 mpf (K).White dashed boxes outline the magnified regions shown to the right of each panel.F310 Ab stains fast myofibers.Data from biological replicates are shown as mean ± standard error (B, C).Significance was examined by two-tailed Student's t-test.Percent differences and P values are shown above the horizontal lines for intergroup comparisons.n = the number of biological replicates.Scale bars, 1 mm (D); 200 µm (E, I); 300 µm (J); 500 µm (K).dpf, days postfertilization; mpf, months post-fertilization.

Figure EV6 .
Figure EV6.Live monitoring of autophagic activation in deformed myofibers.(A) The transgenic construct for live monitoring of autophagic activation in myofibers.(B) Treatment with rapamycin activates autophagic response in myofibers.White arrows point to activated, mCherry-positive myofibers.Of note, without the treatment of rapamycin, all 6 dpf myofibers were both EGFP-and mCherry-positive.(C) Deformed myofibers contain abundant red puncta.White arrow points to a deformed myofiber.(D) Percentages of red and yellow puncta in three individual deformed myofibers.(E, F) RT-qPCR was performed to analyze expression of the autophagic genes atg5 (E) and atg7 (F) in whole animals (14 dpf) or dissociated myofibers (70 dpf and 1.7 years of age).(G) Timeline and three different thyroxine hormone (T4) treatment schemes.(H-J) RT-qPCR analysis of the whole-animal myofiber loss for each of the schemes.Data from biological replicates are shown as mean ± standard error (E, F, H, I, J).Significance was examined by two-tailed Student's t-test.Percent differences and P values are shown above the horizontal lines for intergroup comparisons.n = number of puncta (D) or biological replicates (E, F, H, I, J).Scale bars, 100 µm (B); 50 µm (C).dpf, days post-fertilization.