Distinct anterior and posterior domains in the epithelial somite. (A) In a 12S (15 hours) embryo, MyoD (red) is expressed in nuclei in the posterior of newly formed somites and in adaxial cells of the anterior segmental plate. (B) In the anterior of a 14S (16 hours) embryo, myogenin labeling (red) is in the posterior and medial somite, whereas Pax7 (green) is restricted to anterior and lateral. (C) In a 24-hour embryo, Pax7-labeled nuclei (green) are on the external myotome surface, whereas myogenin labeled nuclei (red) are more medial. (D) Bodipy ceramide-stained Histone H2:GFP 12S embryo. (E) Cells from the embryo in D are outlined, anterior border cells (ABCs) are marked by green dots. (F-K) Selected time-lapse images of green, GFP-labeled nuclei. T, time in minutes from the commencement of analysis. An individual ABC is pseudo-coloured green and tracked through individual frames. (K) An additional ABC is pseudo-coloured yellow in the same medio-lateral position as an elongating posterior cell in blue. (A-K) Dorsal views, anterior to the top, the midline is in the center in A-C, at the right in D-K. Scale bars: 50 μm in C for A-C, and 50 μm in D for D-K.

Distinct fates of ABCs and posterior cells. (A-E) ABCs give rise to external cells. (A) The injected ABC (red). (B) Four hours later, this ABC had given rise to a cell on the external somite surface. (C) Ten hours after injection, 2 cells remain on the surface of the somite. (D) Both cells (red) are Pax7-positive [colocalisation of rhodamine and Pax7 (green) in the nucleus appears yellow] on the surface of the myotome (blue). (E) Transverse section of one labeled cell shown in D. (F-J) ABCs give rise to muscle. (F) The injected ABC (red). (G) Four hours later, this cell is on the external somite surface. (H) Ten hours after injection, this ABC formed an elongated fibre. (I) The elongated muscle fibre (red) is obliquely oriented, like fast fibres, Pax7-negative (green), and is superficial within the myotome (blue). (J) Transverse section of the embryo in I, the labeled cell is a lateral fast fibre. (K-O) Posterior cells elongate early. (K) The injected posterior cell. (L) Four hours later, the posterior cell had elongated, deep within the somite. (M) Ten hours after injection, the posterior cell had formed an elongated muscle fibre. (N) The elongated fibre (red) is Pax7-negative (green), and is deep within the myotome (blue). (O) Transverse section of the embryo in N, the labeled cell is a very medial fast fibre. (P-U) External cells persist into the larval stage. (P) The injected ABC (red). (Q) Ten hours after injection, this cell is on the external surface of the myotome. (R) Thirty-four hours after injection, this cell had generated multiple cells. (S) Fifty-seven hours after injection, all injected cells remain on the external surface, arrows show the position of sections shown in T,U. (T,U) The injected cell gave rise to two Pax7-positive (yellow) cells on the surface of the myotome (blue). A,F,K and P are dorsal views, anterior to the top, the notochord is at the right of the image. B-D,G-I,L-N and Q-S are lateral views, with anterior to the left and dorsal to the top. E,J,O,T,U are transverse sections with dorsal to the top. Scale bars: 50 μm in C for A-C,F-H,K-M,P-R; 50 μm in O for D,E,I,J,N,O; 25 μm in S; 25 μm in U for T,U.

Distinct waves of myogenesis and a model for myotome development. (A) A summary of the fates of injected ABCs and posterior cells at 24 hours. Some injected ABCs (green) have formed differentiated fast muscle fibres, all are between superficial slow fibres and the earlier formed, fast fibres. All posterior cells (blue) have differentiated into medial fast fibres. (B) Dorsal view of a 24-hour embryo labeled for Pax7 (green), Prox1 (red) and myosin (blue). Slow fibre nuclei (red, Prox1) are at the surface of the myotome (blue, myosin). Most Pax7-positive nuclei (green) are outside of the myotome, but some are within the slow (arrowhead) or fast (arrow) domains of the myotome. (C) Schematic showing the location of Pax7-positive nuclei that are within the myotome. The pax7-positive nuclei within the myotome (MyHC-positive domain) from 30 different somites (somites 9-11 in a 24-hour embryo) are shown. (D) Transverse section through a mid-trunk somite (S9-12) of a 24-hour embryo treated with BrdU from the 5S stage (11 hours 40 minutes) to 24 hours. BrdU-positive nuclei (red, arrowheads) are found throughout the myotome (MyHC, blue), except for the superficial slow nuclei (arrows) (Barresi et al., 2001). Insets show muscle nuclei labeled with MEF2 (green). (E) Transverse section through a mid-trunk somite (S9-12) of a 24-hour embryo treated with BrdU from the 20S stage (19 hours) to 24 hours. BrdU-positive nuclei in the myotome are found solely within the lateral fast myotome (arrowheads). (F-H) Fibres with a smaller cross-sectional area are found laterally at 48 hours (F), 72 hours (G) and 96 hours (H); superficial slow fibres were excluded from this analysis. Relative cross-sectional area is shown according to size quartiles: light-green fibres, smallest 25%; dark-red fibres, largest 25%. (I,J) Models of somite cell movements and fates, viewed from dorsal (the midline is to the right). (I) Posterior cells (blue) become the medial fast fibres and ABCs (green) move laterally, becoming external cells expressing Pax7. (J) External cells then move medially into the myotome to become lateral fast fibres. During this time, adaxial cells (red) are displaced laterally and become superficial slow muscle fibres. Scale bars: 50 μm in D,H.