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First published online 15 February 2006
doi: 10.1242/dev.02291

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Differential effects of N-cadherin-mediated adhesion on the development of myotomal waves

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

View larger version (49K): [in a new window]   Fig. 1. Electroporation strategies used to transfect distinct somite domains. (A-D) Electroporation of pioneers (A), dermomyotome sheet (B), dorsomedial lip (C), and rostral and caudal lips (D, only rostral is depicted, the location of the positive electrode is changed to the opposite edge for caudal transfections). The shapes and placement of the electrodes are drawn in white. Arrows indicate the direction of the current; red blocks represent the targeted domains.

View larger version (94K): [in a new window]   Fig. 2. Expression of N-cadherin protein during development of the pioneer myoblasts. (A,E,G,I) N-cadherin; (B-D,F,H) overlay of desmin and N-cadherin. (A) A newly-formed somite in a 22ss embryo showing homogeneous expression of N-cadherin, including the medial region that generates pioneer myoblasts (P). Note enhanced immunoreactivity in the apical adherens junctions. (B) Somite 26 in a 28ss embryo showing the initial bending of desmin-positive pioneers (green) under the forming DM. Desmin-positive pioneers still in the epithelium (arrowhead), and the bending cells, no longer express N-cadherin. (C-E) Somite 23 in two 28ss embryos. Arrowheads indicate mesenchymal pioneers in the dissociated somite that are desmin+/N-cadherin- during migration (D,E). N-cadherin is enriched at the apical pole of DM cells (E, arrow). (F,G) Frontal section as in C-E, revealing desmin+/N-cadherin- pioneers (delimited by a thin white line) underneath the DM. Pioneers are predominantly located in the rostral domain of the segment. (H,I) Somite 22 in a 30ss embryo. Upon differentiation into myofibers, pioneers reexpress N-cadherin (arrowheads). DM, dermomyotome; EC, ectoderm; NT, neural tube; SC, somitocoele; Scl, sclerotome. Scale bar: 40 µm in A-C,H-I; 20 µm in D-G.

View larger version (90K): [in a new window]   Fig. 3. Expression of N-cadherin during the second wave of myotome development. (A,H,K) Desmin (green); (B,E,F,I,L) N-cadherin (red); (C,J,M) overlay of desmin and N-cadherin, nuclear Hoechst is blue. (D-G) Electroporation of GFP/DNA into a rostral DM lip (green in D and G) co-stained for N-cadherin (E-G). (A-G) Flank somites of E3 embryos. (A-C) The apical surface of the DM and desmin+ fibers express N-cadherin (arrowhead). (D-G) Semi-frontal section showing that the GFP+ epithelial cells in the rostral lip co-express N-cadherin. Note in F and G that, like in the DM sheet, N-cadherin is restricted to the apical aspect of the cells (arrows). (H-M) Transverse sections through flank somites at E4. (H-J) High magnification of the DML region showing the myotome and DML positive for N-cadherin. The latter displays strong apical N-cadherin staining (arrowhead). Cells within the SLD (delineated between arrows) express N-cadherin but are desmin negative; note that their cell and nuclear shapes are rounder than the elongated epithelial cells of the DML. (K-M) Dermal cells (D) downregulate N-cadherin expression to a basal level. Scale bar: 80 µm in A-C,K-M; 40 µm in D-E; 20 µm in F-I.

View larger version (83K): [in a new window]   Fig. 4. Differential allocation of N-cadherin to the apical daughters of perpendicularly dividing DM progenitors. (A) Transverse section showing the DM and underlying myotome (M) co-stained with nuclear Hoechst (blue) and N-cadherin (green). Cell divisions occur at the apical portion of the DM (arrowhead). (B-E) High magnifications of dividing cells stained with Hoechst (blue), N-cadherin (green) and -tubulin to label centrosomes (red). (B) A cell dividing parallel to the mediolateral axis of the DM. Both daughter cells retain N-cadherin staining at their apical aspects. (C,D) Perpendicularly dividing DM cells in which only the apical progeny remains N-cadherin positive. (E) Oblique mitosis showing a similar segregation of N-cadherin to the apical daughter cell. In contrast to the DM, myotomal cells express homogeneous N-cadherin on their surface. Panels in second and third rows show, separately, Hoechst staining of nuclei associated with -tubulin and N-cadherin with -tubulin. n, at least 60 mitoses were scored with equal N-cadherin distribution in progeny of all parallel mitoses, and apically localized N-cadherin in all perpendicular divisions. Scale bar: 33 µm in A; 10 µm in B-E.

View larger version (91K): [in a new window]   Fig. 5. Effects of N-cadherin-mediated adhesion on cell translocation and fate segregation of DM sheet progenitors. (A,E) Control GFP-treated somites (n=31/31 embryos). (B,G-I) cN390-GFP-treated somites (n=27/30 embryos). (C,F) Full-length N-cadherin-treated somites (n=27/30). (D) N-cadherin (CBR-)-treated somites (n=12/12). Desmin is red, GFP is green and Hoechst staining is blue in A-D. The blue products in G-I are in situ hybridizations for Alx4 (G,H) and Pax7 (I). Electroporations were performed in somite 22 of 28ss embryos (A-H). (A-D) Transverse sections two days after electroporation, showing (A) the distribution of control GFP-treated cells among both the dermis and myotome, (B) cN390-GFP-treated cells in the dermal domain only, and (C,D) wild-type N-cadherin or N-cadherin (CBR-)-treated cells in myotome only. (E,F) Whole mounts showing that wild-type N-cadherin-treated DM cells generate fibers (F), whereas GFP only-treated cells remain mesenchymal (E). (G,H) DM cells that received cN390-GFP relocate into the nascent dermis while maintaining expression of Alx4 (arrows). (I) Premature dissociation of DM cells induced by focal electroporation of cN390-GFP 10 hours after electroporating an epithelial somite. Dissociating cells downregulated Pax7 mRNA (arrowheads) when compared with similar cells still resident in the epithelium (arrows) or with untransfected cells. Scale bar: in A, 40 µm; in B, 30 µm; in C,D, 50 µm; in G,H, 60 µm; in I, 15 µm.

View larger version (125K): [in a new window]   Fig. 6. cN390 causes DML dissociation and lack of myotome colonization. (A-F) Control GFP; (G-L) cN390-GFP. Electroporations were directed to the DML of early dissociating somites 22-23 of 28ss embryos. GFP is green; desmin, red. (A-D) Five hours after transfection, labeled DML cells retain their epithelial structure as revealed in dorsal views (A,B) and transverse sections (C,D, arrowheads, n=15/17). (E) Thirty hours after electroporation, GFP-positive myofibers derived from the DML occupy the medial-most aspect of the myotome (arrowheads). The DML itself retains its epithelial structure (n=12/14). (F) Dermal stage showing a larger number of labeled DML-derived fibers in the desmin-positive myotome with a still epithelial DML. (G-J) Five hours after transfection, DML cells that received cN390-GFP have begun dissociating, as revealed in dorsal views (G,H, arrows) and transverse sections (I,J, arrowheads, n=8/10). (K) By 30 hours, cN390-GFP-treated cells have lost their epithelial morphology and have located subectodermally (arrows). None of them generated myofibers (n=8/11). (L) Dermal stage to further emphasize the dissociation of DML cells and the lack of myotomal colonization. Scale bars: 20 µm in C,D,I,J; 10 µm in E,F,K,L.

View larger version (78K): [in a new window]   Fig. 7. Electroporation of control GFP, cN390-GFP or wtN-cadherin to rostral and caudal lips of the DM. (A-D) Electroporations of control GFP (A,C) and cN390-GFP (B,D) to caudal (A,B) and rostral (C,D) DM lips to somites 22-23 of 28ss embryos. (E) Similar transfection of wtN-cadherin-GFP to a caudal lip. Embryos were re-incubated for 24 hours. In control-treated segments, the labeled lip cells generated fibers that elongated toward the opposite direction (arrows in A, whole-mount view, and in C, frontal section); a similar phenotype is obtained upon wtN-cadherin overexpression (whole-mount view, E, n=7/7). In cN390-GFP-treated lips (B,D), labeled cells lost their epithelial shape, remained in the inter-somitic region and did not generate fibers. Arrowheads indicate the inter-somitic spaces. (F-K) Similar electroporations as in A-D. Whole mounts (F,G) and frontal sections (H-K) were in situ hybridized with a Myod probe and stained for GFP. Each panel shows separately and in combination Myod transcripts and GFP expression. Both caudal and rostral lip cells treated with control GFP, co-express Myod (arrowheads in F, arrows in H,J) and generated myofibers (n=8/9). cN390-GFP-expressing cells (G,I,K) dissociated from the epithelium, do not express Myod (arrowheads in G, arrows in I,K) and did not generate myofibers (n=10/11). DM, dermomyotome; EC, ectoderm; IS, intersomite; R and C, rostral and caudal; Myo, myotome; Scl, sclerotome. Scale bars: in K, 10 µm for C,D; 20 µm for H-K.

View larger version (145K): [in a new window]   Fig. 8. N-cadherin is not required for the development of the earliest myotome composed of pioneer fibers. (A-D) Electroporation of control GFP to the medial region of newly-formed epithelial somites in 23-25ss embryos. (A) Five hours after transfection, the pioneer cells (arrowhead) express GFP, whereas the dorsal somite (delineated between arrows) is devoid of labeled cells (n=20/24). (B) Dorsal view of a somite 5 hours after transfection. Note the homogeneous rostral-to-caudal distribution of labeled cells. (C) Sixteen hours later, pioneers have dissociated, and are localized preferentially in the rostral half of the segment, forming a triangular shape (arrowhead); in addition, a few partial-length fibers that elongate rostrocaudally (arrow) and localize medially close to the neural tube (NT) are apparent (n=10/10). (D) Twenty-four hours after transfection, full-length myofibers formed (n=13/17). (E-G) Electroporation of cN390-GFP to the medial region of newly-formed epithelial somites in 23-25ss embryos. (E,F) Transverse (E) and dorsal (F) views 5 hours after transfection. Labeled cells are localized to the pioneer region (arrowhead) but not to the prospective DM and DML (between arrows, n=14/17). (G) Twenty-four hours after electroporation, full-length myofibers formed (n=15/16). (H,I) Electroporation of cN390-GFP to the medial region of newly-formed epithelial somites in young 15ss embryos. Twenty-four hours later myofibers had formed normally and already span a significant mediolateral extent of the segment (n=9/10 embryos). (J,K) Electroporation of wtN-cadherin-GFP to the medial region of newly-formed epithelial somites in 23-25ss embryos. As early as twenty hours post-transfection, continuous expression of the protein is compatible with the normal formation of myofibers. In addition, some labeled mesenchymal cells are still apparent in the lateral domain of the somite (arrowheads, n=8/8). H and J depict GFP+ cells on a phase contrast background, I and K are GFP only. Note in D,G,H and K that in controls and all experimental treatments, formation of the pioneer fibers occurs as a discrete process leaving no residual GFP labeling of the DML region. The labeled cells observed at 5 hours in the medial domain of panels B and F correspond to the labeled cells in the transverse sections in A and E, respectively (i.e. ventrally located with respect to the future DML). Scale bars: 20 µm for A,C,E.