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The roles of cell migration and myofiber intercalation in patterning formation of the postmitotic myotome

Nitza Kahane^*, Yuval Cinnamon^* and Chaya Kalcheim

Department of Anatomy and Cell Biology, Hebrew University–Hadassah Medical School, Jerusalem 91120, PO Box 12272, Israel
^* These authors contributed equally to this study

View larger version (62K): [in a new window]   Fig. 1. Dynamic expression of desmin immunoreactivity during development of the pioneer myotome. Confocal analysis of whole somites of an embryo aged 31-somite pairs. Consecutive panels illustrate somitic levels A, 29; B, 27; C, 25; D, 23; E, 21; and F, 18. Open arrowheads indicate mesenchymal cells that delaminate from the epithelium. Growing fibers are delimited between arrow and white arrowheads. DML, dorsomedial lip; C, caudal; L, lateral; M, medial; R, rostral.

View larger version (73K): [in a new window]   Fig. 2. Pioneer myoblasts migrate from caudal regions of the medial epithelial somite to the rostral edge prior to generating myofibers. The medial wall of epithelial somites at interlimb levels of the axis was focally labeled with CM-DiI approaching the caudal tip of the segment (A,A' time 0). Six (B,B') and 12 (C,C') hours after injection, mesenchymal myoblasts (open arrowheads) have relocated towards the rostral half of the somite. (D,D') Eighteen hours after labeling, differentiating fibers (between arrows and white arrowheads) are detected that are attached to the rostral pole and elongate caudally. Mesenchymal myoblasts located laterally always begun differentiating later than medial ones (see also Fig. 1). (A-D) Dye labeling on top of the phase contrast image. (A'-D') Dye-labeled fibers only. C, caudal; L, lateral; M, medial; R, rostral; NT, neural tube.

View larger version (163K): [in a new window]   Fig. 3. The primary myotome derived from the medial somite extends throughout the whole segment and pioneer fibers localize superficially. (A) Microinjection of CM-DiI to the center of the DMQ of an epithelial flank-level somite. (B) Formation of dye-labeled fibers that span the entire rostrocaudal and mediolateral extents of a segment. (C) Transverse section counterstained for desmin reveals the dorsoventral distribution of pioneer fibers (arrows pointing to red cells), which localize from the DML to the VLL and occupy a superficial position (towards the DM epithelium) within the desmin-positive myotome (green). C, caudal; L, lateral; M, medial; R, rostral; DM, dermomyotome; DRG, dorsal root ganglion; EC, ectoderm; Myo, myotome; NT, neural tube; Scl, sclerotome. Scale bar: 10 µm.

View larger version (128K): [in a new window]   Fig. 4. Pioneer myofibers fuse with younger cells during development of multinucleated intercostal muscles. Frontal sections through a E8 quail embryo pulse-chased with thymidine and counterstained for desmin (brown reaction product) as described in the Materials and Methods. (A) Low magnification to show localization of intercostal muscles (M) between successive ribs (R). Arrows indicate clearly distinguishable fibers containing nuclei with thymidine grains. (B-D) Higher magnifications illustrating several parallel fibers each containing multiple nuclei stained with Hematoxylin (light blue). Arrowheads indicate thymidine-positive pioneer nuclei which are an integral part of the multinucleated fiber. Scale bar: 100 µm in A; 22 µm in B-D.

View larger version (80K): [in a new window]   Fig. 5. Growth of the myotome occurs by even cell intercalation all along its dorsoventral extent and newly added fibers localize medially abutting the sclerotome. (A) Transverse section showing the homogeneous entry of labeled progenitors (newly added postmitotic fibers) along the entire dorsoventral extent of the desmin-positive myotome. Note the localization of labeled nuclei to the medial (sclerotomal) side of the myotome (arrowheads). The left side of the section shows the result of a DML ablation performed 1 day earlier. Note that both the DML and the dorsalmost part of the myotome are missing (dorsal to the long arrow). Nevertheless, the remaining myotome is an active structure growing by a similar, homogeneous addition of new cells all along the dorsoventral extent. (B) Quantification of the distribution of second-wave fibers that entered the myotome during an approximate period of 6 hours (two pulses of radiolabeled thymidine, each available to the embryo for 3 hours) (Kahane et al., 1998a; Kahane et al., 1998b). The proportion of newly added cells was counted in serial frontal sections, as described in the Materials and Methods. Calculated values (mean±s.d., n=6 somites) for six contiguous regions per segment are: 17.5±3.17; 18.2±3.8; 18.7±8.6; 12.2±1.63; 19.8±6.7; and 13.5±4.0. D, dermis; DA, dorsal aorta; DML, dorsomedial lip; M, myotome; NT, neural tube; NO, notochord; Scl, sclerotome. Scale bar: 30 µm.

View larger version (59K): [in a new window]   Fig. 6. The dorsalmost region of the desmin-positive myotome contains unit-length myofibers, with myofiber generation primarily beginning from the extreme edges of the dermomyotome. (A-C) The dorsomedial-most desmin-positive fibers detected in the myotome are anchored to both extremes of the segment. The central nucleus-containing area is less stained and no partial fibers can be visualized approaching the DML. DiI applied to the rostral extreme of a segment (asterisk) diffused along the fibers until reaching their caudal end (arrows in B,C). Co-localization of DiI and desmin staining define that dorsalmost fibers are of full length and highlight the absence of partial-length fibers. (D) Elongating fibers (between arrows and arrowheads) emerge from an extreme lip adjacent to the intersomitic region (IS, and broken line separating adjacent segments). Notably, unit length fibers had broad end-feet attached to the edges, whereas growing fibers demonstrated tapered attachments.

View larger version (34K): [in a new window]   Fig. 7. The formation of the primary myotome by pioneer myoblasts. Successive stages in the formation of the early myotome (first wave, red color) represented as a function of somitic level in a 28-somite embryo. (A) Pioneer myoblasts originate along the entire R-C extent of the dorsomedial wall of epithelial somites. (B) During somite dissociation, pioneer myoblasts bend underneath the forming DML, delaminate and begin migrating to rostral portions of the somite (curved arrow). White mesenchymal cells represent sclerotome (Scl). (C) Migrating mesenchymal myoblasts give rise to a transient triangular pattern. (D) The first growing myofibers differentiate apposed to the DML. The direction of myofiber formation proceeds from R to C (red arrows) and from M to L. Mesenchymal cells are located lateral to the differentiating fibers. The overall pattern of the forming myotome is still triangular. (E) All fibers have reached the caudal and lateral edges, giving rise to the early myotome (rectangular pattern), which is attached to the R and C lips of the dermomyotome. These are mononucleated fibers whose nuclei concentrate at the center (black dots). The second wave of myotome colonization (not shown) begins at segmental levels D/E. The drawings represented in the figure depict characteristic phases, yet the processes of myoblast delamination, migration and fiber generation are continuous and overlapping over discrete segmental levels. Hence, segments are found that reveal simultaneously delaminating and migrating cells, or migrating and differentiating cells. In C-E, only the four dermomyotome (DM) lips (DML, VLL, R and C) were left. The dorsal layer of the DM has been removed to appreciate the underlying myoblasts. Intersecting arrows represent coordinates for orientation: R, rostral; C, caudal; M, medial; L, lateral; D, dorsal; V, ventral.

View larger version (32K): [in a new window]   Fig. 8. Even growth of the myotome by progressive cell intercalation as opposed to incremental cell addition. (A,B) Simultaneous expansion of the myotome in the dorsoventral and transverse planes is accounted for by two successive myoblast waves first wave of pioneers in red and second wave in green). (A) Even pattern of myotome growth in the DM to VL orientation by progressive cell intercalation. Green arrows illustrate the contribution of the second wave stemming from all four lips of the DM. Fibers are directly generated from all along the rostral and caudal lips. By contrast, cells from along the DML and VLL first delaminate into the sub-lip domain (1), mesenchymal myoblasts then migrate longitudinally to one of the extreme edges (2) and generate myofibers (3) upon joining their rostral and caudal counterparts. Myofiber elongation occurs in a direction that is parallel to pre-existing pioneer fibers (red) and newly added cells intercalate among adjacent pioneers. This results in an even pattern of dorsoventral growth (arrows below the scheme) with young and old fibers evenly spread. (B) Growth in the transverse plane occurs in a superficial (DM) to deep (Scl) direction. Black arrows indicate the direction of myotome growth, leaving the pioneer fibers (red dots) superficially localized apposed to the DM. (C) Incremental growth of the myotome driven by the DML and VLL (small arrows) as proposed by Ordahl et al. (Ordahl et al., 2000). The oldest fibers are of unit length (dark green) and localize to the center of the myotome. Further growth occurs both medially and laterally (large arrows in which the green gradient represents relative myofiber age). Accordingly, cells from the DML and VLL delaminate into an intermediate domain where they begin differentiating bi-directionally in situ with no prior longitudinal relocation. This growth domain is therefore assumed to contain partial-length fibers in a staggered configuration subjacent to both medial and lateral edges, respectively.