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The third wave of myotome colonization by mitotically competent progenitors: regulating the balance between differentiation and proliferation during muscle development

Department of Anatomy and Cell Biology, Hebrew University-Hadassah Medical School, Jerusalem 91120, PO Box 12272, Israel

View larger version (116K): [in a new window]   Fig. 1. Mitotically competent cells within the myotome first appear at the extreme edges and later scatter throughout the entire muscle mass. (A) Frontal section through a cervical level of the axis of a 35-somite stage quail embryo showing the presence of thymidine-positive nuclei predominantly at the rostral and caudal extremities of the desmin-positive myotome. (B) Mitotically active cells in the nascent muscle of an E4 embryo are scattered throughout the entire structure. In both cases, short pulses of radiolabeled thymidine were delivered followed by immediate fixation thus reflecting intrinsic cell proliferation. D, dermis; M, myotome, S, sclerotome. Scale bar: 30 µm in A; 70 µm in B.

View larger version (99K): [in a new window]   Fig. 2. Mitotically active cells enter the myotome from the extreme rostral and caudal lips of the dermomyotome. Iontophoretic labeling with CM-DiI (red) of (A) the center of the rostral lip, (B) the medial aspect of the caudal lip, (C) the DML of dermomyotomes. Embryos were incubated for additional 20 hours and then pulsed with BrdU (green) for one hour prior to fixation. (A,B) Note the presence within the myotomes of double-labeled cells containing BrdU-immunoreactive nuclei surrounded by CM-DiI-positive material (arrows). The double labeling represents cells that entered from the respective lips and continued proliferating within the myotome. Myofibers spanning part of or the entire rostrocaudal length of the myotome are also apparent (arrowheads in A and C). One day after CM-DiI labeling, dye distribution adopts a dotted appearance in the cell membrane. BrdU-positive/CM-DiI-negative cells were also apparent as only a small number of lip cells were dye labeled. The presence of CM-DiI-positive cells that lack BrdU in their nuclei was evident as well; this finding was expected as a 1 hour pulse enables the detection of only a subset of the mitotic population given a cell cycle length of about 8 hours (Langman and Nelson, 1968; Summerbell et al., 1986). D, dermis; EC, ectoderm; Myo, myotome; Scl, sclerotome. Scale bar: 11 µm.

View larger version (95K): [in a new window]   Fig. 3. Expression of FREK mRNA in developing myotomes. (A) Whole-mount in situ hybridization of E3.5 embryo, revealing that expression of FREK is enriched in both rostral and caudal regions of the myotome (arrowheads) as well as in discrete stripes subjacent the DML epithelium (between arrows). (B) Sagittal section of a similar embryo, further showing the distribution to the extreme regions of the myotomes. (C) Frontal section through a cervical region of a 30-somite stage embryo again revealing intense FREK signal at the edges of the myotomes (arrowheads), whereas the rest of the myotome and the overlying dermomyotome show a fainter intensity of labeling (see also D). (D) Transverse section showing that FREK signal is particularly intense in myotomal cells localized towards the sclerotomal portion of the myotome, a position adopted by ingrowing second-wave myofibers relative to pre-existing pioneers that always remain apposed to the dermal side of the myotome. C, caudal; DM, dermomyotome; M, myotome; NT, neural tube; NO, notochord, R, rostral; Scl, sclerotome. Scale bar, 50 µm.

View larger version (100K): [in a new window]   Fig. 4. FREK (but not MyoD, Myf5 or FGF4) is expressed by proliferating cells of the third wave that enter the myotome. E3.5 quail embryos (45 somites approximately) were labeled with BrdU for 1 hour followed by fixation and in situ hybridization. Frontal sections were hybridized with probes to (A) FREK, (B) MyoD, (C) Myf5 and (D) FGF4, and then subjected to immunodetection of BrdU. Note that in all cases, BrdU-labeled nuclei are preferentially localized towards the rostral and caudal regions of the myotomes (small arrows in B-D; white arrowheads in A). In A, the BrdU-positive nuclei are a subset of FREK-containing cells (white arrowheads) that are localized in the myotome extremities. MyoD, Myf5 and FGF4 are enriched at this developmental time in the center of myotomes (central region marked by M). These contain the nuclei of differentiated myofibers that are BrdU negative. Rostral is towards the top. D, dermis; EC, ectoderm; M, center of myotome; Scl, sclerotome. Scale bar, 25 µm.

View larger version (94K): [in a new window]   Fig. 5. Effect of added FGF4 on the amount and topographical distribution of third-wave progenitors. (A-D) In situ hybridization for FREK of E3.5 quail embryos that received an implant of FGF4-coated beads at the 28-30-somite stage (A, treated side; B, contralateral side) or control beads (C). Note the preferential distribution of FREK signal to the rostral and caudal portions of the myotomes in B and C (arrowheads); in A, FREK transcripts are homogeneously dispersed in the vicinity of the implanted beads (area between arrows). (D) Frontal section at the level of the notochord (NO) of a FGF-treated embryo, showing the preferential distribution of cells to the extreme regions of the myotome (arrowheads) in the control side when compared with the uniform distribution of FREK mRNA in the treated side. (E) Thirty-somite stage embryos received FGF4-soaked beads as depicted above. One hour before fixation at E3.5 they were pulsed with BrdU. The number of BrdU-positive cells was counted in serial transverse sections starting from the rostral end to the caudal extreme of each myotome. Note that in the control side (blue), the majority of labeled cells are found close to the extreme edges of the myotome with very few in the center, whereas in FGF-treated embryos (red), their distribution is uniform along the myotome. Note also that the number of labeled cells is higher in most sections comprising the treated side (see Results for quantification). C, caudal; D, dermis; Scl, sclerotome; R, rostral. Scale bar: in D, 22 µm for D.

View larger version (8K): [in a new window]   Fig. 6. Age-dependent increase in the number of mitotically-active progenitors of the third wave. Single pulses of radiolabeled thymidine, 1 hour long, were delivered to embryos at different stages followed by immediate fixation. The number of cells with thymidine-positive grains over nuclei was scored per myotome, as described in Materials and Methods. Results represent the mean±s.d. of seven to 14 cervical myotomes per experimental point.

View larger version (8K): [in a new window]   Fig. 7. Age-dependent decrease in generation of post-mitotic, second-wave myofibers. Embryos at different ages starting after establishment of the pioneer myotome were pulsed for 6 hours with radiolabeled thymidine and then chased with unlabeled metabolite until fixation at E5 as described in Materials and Methods (protocol 1). Cells that continued dividing during the chase diluted the radioactive metabolite and became unlabeled. In contrast, cells that were mitotically active during the 6 hour labeling period and then withdraw from the cell cycle remained labeled and represent a subpopulation of the second wave. Note that the number of post-mitotic cells is maximal at the 30-32-somite stage in cervical regions of the axis and then progressively decreases until E3.5 (approximately 46 somite pairs). Results represent the mean±s.d. of 15 myotomes per experimental point.

View larger version (86K): [in a new window]   Fig. 8 MyoD is expressed in subsets of epithelial progenitors within rostral and caudal dermomyotome lips. (A,B) Quail embryos (35-somite stage) were hybridized with a MyoD probe. Some embryos also received a single, 1 hour pulse with BrdU after fixation, in situ hybridization and then immunodetection of BrdU (C,D). Note in A (frontal section) and B (sagittal section) that only some of the epithelial cells in both rostral and caudal dermomyotome lips contain MyoD-positive cells (arrowheads), while other cells are negative for the marker (arrows). In addition, C (frontal section) and D (sagittal section) reveal the presence of Myo-D-positive/BrdU-positive precursors (arrowheads, putative progenitors of the second wave) and also of Myo-D-negative/BrdU-positive epithelial cells (arrows, presumed third-wave cells). Rostral is towards the top. DM, dermomyotome; M, myotome; Scl, sclerotome. Scale bar: 25 µm in A,B; 18 µm in C,D.

View larger version (198K): [in a new window]   Fig. 9. Shh promotes MyoD expression in epithelial DML lips followed by enhanced myofiber differentiation. Control QT6 or Shh-expressing QT6 cells were grafted at the cervical level of 30-somite-stage embryos. The latter were further incubated for 10 hours (A-C) or 48 hours (D), pulsed with BrdU for 1 hour and in situ hybridized for MyoD or Myf5 in combination with immunodetection of proliferating cells. In control segments (B,C), MyoD is expressed in only a few cells of the extreme dermomyotome (DML) lips (see, for example, arrows in C). In contrast, most epithelial DML progenitors, which are adjacent to the Shh-expressing cells, express MyoD in A. The DML was highlighted by a thin line. Note that grafted control (B) as well as the Shh-expressing QT6 cells (A) produce MyoD. (D) Two days after grafting, the myotomes are much bigger in the operated side that received Shh when compared with the contralateral intact side or to QT6-grafted myotomes (not shown). These myotomes contain well-developed and organized myofibers expressing Myf5 (and also MyoD, not shown), and also reveal the presence of BrdU-positive cells. Note also the absence of dermis (D) in the operated side. Myo, myotome; NO, notochord; Scl, sclerotome. Scale bar: 15 µm in A-C; 40 µm in D.