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First published online 19 January 2005
doi: 10.1242/dev.01617

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Lineage analysis of the avian dermomyotome sheet reveals the existence of single cells with both dermal and muscle progenitor fates

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

View larger version (70K): [in a new window]   Fig. 1. The DM sheet gives rise to derivatives that colonize both the dermis and the myotome. (A) Five hours after electroporation, GFP-expressing cells (green) are located in the epithelial DM sheet. (B,C) At E5, GFP-labeled cells are present in the desmin-positive myotome (red) intermingled among desmin+ fibers. Labeled cells are also present in the dermis. At this stage, the dermo-1 mRNA signal (blue reaction product) is predominantly expressed in the superficial domain of the young dermis close to the ectoderm, whereas GFP-expressing cells are widespread throughout the entire dermis. (C) High magnification of the insert in B. Note that the GFP-labeled cells in myotome and dermis are located at corresponding mediolateral regions, further substantiating the proposed coherent mode of DM development (Ben-Yair et al., 2003). D, dermis; DM, dermomyotome; M, myotome; NT, neural tube. Scale bar: 20 µm in A; 180 µm in B; 50 µm in C.

View larger version (104K): [in a new window]   Fig. 2. DM-sheet-derived cells that enter the myotome are mitotically competent progenitors that retain PAX7 expression. (A-D) Sequential expression of PAX7 immunoreactive protein to developing somites. (A) E2; (B) E3; (C) E3.5; (D) E5. See text for details. (E) DM-derived cells traced by labeling with DiI co-express PAX7 in the myotome. Time zero (t=0) image, showing a dorsal view of a somite at E2.5 immediately following labeling with DiI to the central DM sheet. The four panels below illustrate sections through the myotome of E4 embryos expressing nuclear PAX7 (green) surrounded by membrane-associated DiI (red). (F-I) PAX7-positive cells in the myotome are mitotically active. E4 embryos were pulsed for 1 hour with BrdU, fixed and processed for PAX7 and BrdU immunolabeling. (F) PAX7. (G) BrdU. (H,I) Overlay between the images in F and G. Yellow cells co-express both PAX7 and BrdU. See text for details. D, dermis; DM, dermomyotome; M, myotome; NT, neural tube. Scale bar: 25 µm in A,I; 44 µm in B,C; 165 µm in D; 5 µm in E (sections); 37 µm in F-H.

View larger version (93K): [in a new window]   Fig. 3. Expression of Pax3 and Frek mRNAs by mesenchymal cells in the myotome. (A) Cells expressing Pax3 mRNA (blue, arrowheads) intermingle but do not overlap with desmin-positive fibers (brown) in the E4 myotome. At this stage, Pax3 is also transcribed in a DM-derived intermediate domain located between myotome and forming dermis (see Fig. 4 and corresponding text for details). (B) Co-expression of PAX7 immunoreactivity (brown) and Frek mRNA (blue) in mesenchymal cells within the myotome. D, dermis; ID, intermediate domain; M, myotome; NT, neural tube. Scale bar: 30 µm in A; 23 µm in B.

View larger version (144K): [in a new window]   Fig. 4. Sequential dissociation of the epithelial DM and formation of its derivatives as revealed by morphology and Pax7 mRNA expression. Transverse sections in-situ hybridized with PAX7 (blue) and immunolabeled with desmin (brown). (A) The epithelial DM at E3 expresses PAX7, but the underlying myotome is devoid of PAX7+ cells. (B) At E3.5 the central DM partially dissociates into a compact cell aggregate (ID) that retains PAX7 expression. Few PAX7+ cells become apparent in the myotome but no dermis is yet apparent. (C) By E4 three distinct domains are clearly visible, the desmin+ myotome containing PAX7+ cells, the compact ID, which expresses PAX7, and the beginning of a PAX7-negative dermis composed of scattered mesenchymal cells. See also a similar expression pattern of PAX3 between the myotome and dermis in Fig. 3A. (D) At E5 the ID domain has almost disappeared and most PAX7+ cells are in the myotome intermingled among desmin+ fibers and also located medial to them. A well developed dermis is present. D, dermis; DM, dermomyotome; ID, intermediate domain; M, myotome; NT, neural tube. Scale bar: 40 µm in A; 55 µm in B; 60 µm in C; 120 µm in D.

View larger version (138K): [in a new window]   Fig. 5. The distribution of clonal progeny from the central DM sheet. Transverse sections of segments transfected with a GFP-encoding DNA into single cells followed by double desmin (red) and GFP (green) immunolabelings. (A) Time zero control. Six hours after transfection, a single cell is apparent spanning the apico-basal thickness of the epithelium. (B-D) Twenty-four hours after transfection, labeled progeny is localized to the ID and myotome (B), to the myotome only (C) or to the ID only (D). Each panel represents the overlay of two to three serial sections. (E-G) Sequences of five serial sections, each showing the distribution of clonal progeny 48 hours after transfection. (E1-E5) Labeled cells in both myotome and dermis. (F1-F5) Progeny restricted to dermis. (G1-G5) GFP+ descendants localized to the myotome. In all cases, GFP+ cells in myotome are desmin-negative. GFP+ cells are marked by white arrowheads. Yellow labeling represents blood cell autofluorescence. D, dermis; DM, dermomyotome; ID, intermediate domain; M, myotome; NT, neural tube. Scale bar: 40 µm in A; 50 µm in B-D; 70 µm in E-F; 60 µm in G.

View larger version (36K): [in a new window]   Fig. 6. Quantification of the sequential distribution of clonal progeny from the central DM sheet. (A,B) The proportion of total transfected cells localized in the myotome as PMPs, in dermis and in the ID at 24 (A) and 48 (B) hours after transfection. (C,D) Composition of the clones deriving from single DM progenitors at 24 and 48 hours after transfection, respectively. (E) Detailed distribution and size of clonal progeny (see text for details).

View larger version (16K): [in a new window]   Fig. 9. Sequential segregation of lineages from founder cells in the DM sheet. Schematic representation of the stage-dependent generation of restricted progeny from the DM epithelium. DM precursors act as stem cells with self-renewal ability in the epithelium. They express homogeneously both PAX3 and PAX7, divide with an orientation parallel to the apico-basal thickness of the epithelium, and give rise to progeny that remain confined to the DM and contribute to its mediolateral growth as long as the epithelium lasts. The DM epithelium begins its dissociation into the ID, which reveals an aggregated conformation. Most ID precursors either self-renew for one day and/or segregate asymmetrically into ID and PMPs or ID and dermis (intermediate restricted progeny). Finally, a significant proportion of ID cells generate both PMP and dermis at E5, suggesting that these still keep a stem-like character (see text for details).

View larger version (63K): [in a new window]   Fig. 7. A shift in the plane of cell divisions occurs in the DM prior to complete dissociation. (A-C) Frequency histograms of the orientation of mitotic spindles in the DM at stages HH16-20 of development. An angle of 0° represents a fully parallel orientation of mitosis vis-à-vis the mediolateral plane of the DM epithelium, whereas an angle of 90° reflects perpendicular cell divisions. (D) Quantification of the orientations of cell divisions as a function of embryonic stage. All measurements concerned the flank level of the axis. (E-G) Transverse sections of embryos aged E2.5, E3 and E3.5, respectively, double-labeled with -tubulin (red dots) and Hoechst nuclear stain (blue). Note in E and F the epithelial structure of the DM. In G the central portion of the epithelium is dissociating. Dividing cells are enclosed in inserts and high magnifications of the inserts highlight the planes of cell division. Note that in addition to centrosome-specific staining, -tubulin immunolabeling is also apparent throughout the cytoplasm of tissues such as myotome and ectoderm. DM, dermomyotome; M, myotome, NT, neural tube. Scale bar: 12 µm in E,F; 10 µm in G; 6 µm in E,G (inserts); 7 µm in F.

View larger version (36K): [in a new window]   Fig. 8. Fate map of the DM. Schematic representation of the derivatives emanating from the different regions of the DM epithelium. All four lips (blue) give rise to myofibers as part of the second wave of myotome colonization that follows establishment of the early pioneer fibers. In addition, the rostral and caudal lips also generate the first mitotically active muscle progenitors that we termed `third wave' (Kahane et al., 2001) (PMP, red). The DM sheet (orange) dissociates and its progeny colonizes the two opposite anlage, the subectodermal space to give rise to dermis (yellow arrow) and the myotome to give rise to mitotic muscle progenitors (red arrow). Hence, the proliferating PMPs derive from two distinct sources: first from the rostral and caudal DM lips (Kahane et al., 2001) and later from the DM sheet (this paper).

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