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First published online August 4, 2003
doi: 10.1242/10.1242/dev.00667

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Coherent development of dermomyotome and dermis from the entire mediolateral extent of the dorsal somite

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

View larger version (221K): [in a new window]   Fig. 1. Successive stages in dermomyotome development at flank levels of the axis. (A-D) Phase contrast images. (A) T0. Epithelial somite stage with the prospective dermomyotome (DM) highlighted by broken lines. (B) T1. Initial formation of the DM following mesenchymalization of the sclerotome (Scl). At this stage the pioneer (P) myoblasts bend underneath the nascent DM. (C) T2. The DM of a fully dissociated somite in which the primary myotome (M) is well differentiated. Note the well defined medial and lateral edges (DML and VLL, respectively). (D) T3. The DM dissociates into dermis except for the DML and VLL, which still remain epithelial (demarcated by broken lines). See text for precise stages. DRG, dorsal root ganglion; NT, neural tube. Scale bar: 8 µm in A; 15 µm in B; 22 µm in C; 80 µm in D.

View larger version (18K): [in a new window]   Fig. 5. The overall mediolateral growth pattern of the DM between T0 and T2 is proportional. (A) Scheme illustrating how the positions of dye-labeled cells relative to the mediolateral aspect of the epithelium were measured at T0 and T2 (18 hours). See Materials and Methods for details. (B) The localization of the midpoint values at T2 relative to T0 was plotted for each injected somite. Experimental values at T2 occupy similar relative positions along the mediolateral axis as they did at T0 with a deviation not exceeding 15% (broken lines) from an expected linear correlation (unbroken line).

View larger version (58K): [in a new window]   Fig. 2. Cell proliferation in the epithelial somite and developing DM. (A,C) BrdU incorporation into S-phase nuclei following a 1 hour pulse. (A) Note the presence of BrdU-labeled cells throughout the dorsal aspect of the epithelial somite (T0). BrdU-positive figures are restricted to the basal half of the epithelial sheath (compare with B where total nuclei are evidenced by Hoechst staining). A population of medially located epithelial progenitors (*) are BrdU negative, corresponding to prospective pioneer myofibers. (C,D) At T2, BrdU incorporation occurs along the entire mediolateral extent of the DM with an apparent minimum in the ventrolateral region (white arrows indicate Hoechst-positive nuclei in D that are BrdU negative in C). (E) The percentage of BrdU-positive nuclei compared with total nuclei along the mediolateral extent of the dorsal epithelial somite (T0) and developing DM (T1 and T2). Quantifications were performed as described in Materials and Methods. Scale bar: 17 µm in A,B; 22 µm in C,D.

View larger version (91K): [in a new window]   Fig. 3. Morphogenetic changes during development of the DM. (A,B) General CM-DiI labeling of embryo sections and highlighting of the dorsal somite at T0 (A) and the DM at T2 (B). (A',B') High magnifications of the medial domains of the images in A,B to illustrate the change in cell morphology between T0 and T2. Note reduced magnitude of cellular packing and of the number of nuclear layers in the apicobasal extent of the DM epithelium (T2) when compared with T0. (C,D) Changes in nuclear density (upper panels) and epithelial thickness (lower panels) along the mediolateral aspect of the epithelia as monitored between T0/T1 (C) and T1/T2 (D). Scale bar: 12 µm in A; 25 µm in B; 4 µm in A',B'.

View larger version (123K): [in a new window]   Fig. 4. The position along the DM (T2) of dorsal epithelial somite cells (T0) labeled at distinct mediolateral positions with CM-DiI. (A,C,E) Dorsal view of whole segments labeled at T0 with CM-DiI. Labeling was directed to medial, central or lateral positions of the dorsal somites, respectively. (B,D,F) At T2, the corresponding embryos were sacrificed, serially sectioned and analysed. Representative sections are shown for each type of labeling. Note that the relative mediolateral position of labeled cells at T2 was equivalent to that at T0. NT, neural tube. Scale bar: 25 µm in B,D,F.

View larger version (107K): [in a new window]   Fig. 6. The dynamics of dissociation of DM into dermis and the contribution of the lateral half of the dorsal somite to dermis formation. (A-C) Hematoxylin-stained sections illustrate successive stages of dermis dissociation from the DM. At flank levels of the axis, dissociation begins by E3 from the central region of the epithelium (A) and progresses medially and laterally (B) until E4, when only the DML and VLL remain epithelial (C). (D) Dorsal view of a living embryo fragment shortly after CM-DiI labeling within the lateral half of a flank-level epithelial somite. (E) The same embryo was further incubated until T3 (E4), when a mesenchymal dermis (D) is already apparent. Representative transverse section shows that lateral injection gave rise to mesenchymal cells localized in a lateral domain of the dorsal dermis (red cells). The arrow indicates the border between somite and lateral plate mesoderm-derived dermis. Because the ectodermal indentation in this section is not significant, the precise localization of the border was determined from adjacent sections. At this stage, the myotome (green as revealed by desmin immunostaining) has already invaded the somatopleura. (F) DiI labeling of the dorsolateral portion of an epithelial somite performed at a cervical level of the axis (somite 10) and fixed at E4.5. Note labeling of the lateralmost dermis located dorsal to the ectodermal notch. Note as well in E and F the presence of CM-DiI+ cells within the desmin-immunoreactive myotome (M, see text for details). (G,H) A GFP-encoding vector was electroporated into the dorsolateral portion of an epithelial somite. Seven hours after transfection, GFP+ cells are apparent in the lateral domain of two successive segments (G). (H) At E4, GFP-positive cells (green) are encountered in the lateral domain of the dorsal dermis, in the VLL and in the lateral part of the desmin-positive myotome (red). Note that the VLL and accompanying myotome have entered the somatopleura, and the lateral dermis containing GFP+ cells remains dorsal, distinctly apart from the somatopleural-derived mesenchyme. Note as well the presence of a few fluorescent cells in the ventralmost sclerotome, likely to derive from transfection of the appropriate progenitors located slightly ventral to the dorsolateral epithelium fated to become DM. D, dermis, DML, dorsomedial lip, K, kidney primordium, NT, neural tube, Scl, sclerotome, VLL, ventrolateral lip. Scale bar: 45 µm in A-C; 30 µm in E; 45 µm in F; 70 µm in H.

View larger version (96K): [in a new window]   Fig. 7. The dermis arises from the entire mediolateral extent of the dorsal epithelium in a topographically restricted manner. Double DiI/DiO injections to lateral and medial regions of segments, respectively. (A) Dorsal view of a living embryo shortly after labeling at T0. (C) Dorsal view of a living embryo shortly after labeling at T1. Embryos shown in A and C were further incubated until T3 (E4) and cryosectioned. (B,D) Representative transverse sections of A and C, respectively, showing that medial as well as lateral injections contributed to dermal cells localized at medial and lateral positions. Also note at T3 the presence of DiI/DiO-positive cells within the myotome (M, see text for details). (E-G) Dermal cells derived from the lateral somite are included within the Sim-1-expressing domain of the dermis. (E) Expression of Sim-1 mRNA at E4 (T3) in the lateral third of the dermis (D) and myotome (M), the lateral aspect of the dermomyotome excluding the VLL itself (see also F), the kidney primordium and a band of cells in the ventral neural tube lateral to the floor plate. (F,G) Higher magnification of the same section showing that CM-DiI-labeled dermal cells (G, small arrows), which developed from injections in the lateral region of the epithelial somite, are included within the Sim-1-expressing domain of the dermis (F). Note as well the presence of additional labeled cells in the dermis localized dorsomedial to the Sim-1 territory (arrowhead in G). Note that the ectodermal notch (large arrow) corresponds to the limit between somite and LPM-derived dermis as reflected both by the ventral limit of Sim-1 expression within the dermis and by the ventral border of CM-DiI-labeled dermal cells. As expected, lateral injections of CM-DiI in the somite, which comprises the VLL, also give rise to labeled cells in the myotome (M). DML, dorsomedial lip; No, notochord; NT, neural tube; VLL, ventrolateral lip. Scale bar: 50 µm in B; 35 µm in D; 68 µm in E; 38 µm in F,G.

View larger version (58K): [in a new window]   Fig. 8. Sclerotomal origin of the dorsomedial mesenchyme. (A,B) The dorsomedial mesenchyme between DML and dorsal neural tube is not of dermomyotomal origin. (A) Labeling of the medial DM region including the DML at T1 with CM-DiI, gave rise at T3 to fluorescent cells in the myotome, in the dorsal dermis lateral to the DML (arrows) and in the DML itself, but not in the dorsomedial mesenchyme (*). (B) The dorsal half of a quail epithelial somite was grafted into the equivalent place of a chick counterpart at E2. At E5, dorsal somite derivatives reveal the quail marker including muscle (M), dorsal dermis (D) and the residual DML. By contrast, the dorsomedial mesenchyme (*) was of the host (chick) type like the sclerotome (Scl). (C) CM-DiI labeling of the sclerotome at T1 gave rise at T3 to fluorescent cells in the dorsomedial mesenchyme (*). DRG, dorsal root ganglion. Scale bar: 50 µm in A,B; 35 µm in C.

View larger version (38K): [in a new window]   Fig. 9. Coherent development of the DM epithelium followed by the myotome and dermis, from the entire mediolateral aspect of the dorsal somite. The entire mediolateral aspect of the dorsal epithelial somite (T0) proportionally grows to give rise to the DM epithelium (T1 and T2). Despite local differences in morphogenetic traits, active growth occurs in at least three consecutive domains (medial, central and lateral as illustrated by the green, blue and red colors, respectively). Further dissociation of the DM into dermis (T3) also comprises a contribution of epithelial precursors from all three domains. This coherent growth pattern also applies for the development of the myotome, which occurs by progressive intercalation of fibers along the mediolateral aspect of the segment, as they originate in all four DM lips and contribute fibers in a regionalized manner (see text for references). An exception to this uniform and regionalized contributions are the pioneer myofibers (yellow), which (despite of originating at the medial epithelial somite) finally span the entire mediolateral extent of the growing myotome (yellow circles). Initial dermis dissociation from the entire mediolateral DM already occurs, while the latter is largely confined to the epaxial domain. During later development, while the expanding myotome and accompanying VLL colonize the somatopleura to give rise to hypaxial muscles (intercostal and abdominal), the somite-derived dorsal dermis remains restricted to the epaxial domain. LPM, lateral plate mesoderm.