Fig. 1. Characterisation of laminin matrix assembly in the myotome and its interaction with myotomal cells. (A-D) A Myf5^nlacZ/+ embryo at E9.5 processed for whole-mount immunostaining for ß-gal (A,C) and laminin (A-D; polyclonal antibody). Confocal imaging showing a sagittal plane of the myotome in caudal (A,B) and interlimb (C,D) somites. z-series projection of: (A) 10x1 µm, (B) 6x1 µm (starting 1 µm ventral to last section in A) and (C,D) 24x1 µm optical sections. Shortly after the myotome (white cells in A) begins to form, a laminin matrix appears under these cells (B). In an interlimb somite of the same embryo, more cells have entered the myotome (C) and the laminin matrix accompanies this growth (C,D). (E-M) Transverse (E-G), sagittal (H-J) and longitudinal (K-M) cryostat sections of interlimb myotomes from wild-type embryos at E9.5-10.0. (E-G) Immunoreactivity for the 5 (E), 1 (F) and ß1 (G) laminin chains delimits the dermomyotome dorsally under the surface ectoderm (arrowheads), and at the myotome-sclerotome interface (arrows). Myf5-expressing myotomal cells are frequently seen in contact with the myotomal laminin matrix (arrows in F,G). (H-J) Confocal imaging of the myotome in a thick (30 µm) sagittal cryostat section immunostained for Myf5 and myogenin. Three optical sections are shown: (H) immediately below the dermomyotome; (I) deeper into the myotome, near the myotome-sclerotome interface; and (J) showing the medioventral limit of the myotome immediately before entering the sclerotome. Myf5-positive cells are observed in the dermomyotomal lips. Closest to the dermomyotome (H), only a few Myf5-positive cells are detected in the myotome, while many myogenin-positive nuclei are centrally aligned. Numerous Myf5-positive cells are observed close to the sclerotome (I,J), most of these (arrows in I) located rostral and caudal to the central myogenin-positive nuclei. A few nuclei in the central myotome stain for both Myf5 and myogenin (arrowheads in I,J). (K-M) Longitudinal sections (plane shown in J) show the relative position of differentiating cells within the myotome. (K) Young, Myf5-positive, myogenin-negative cells are located rostral or caudal to the centrally located myogenin-positive cells. (L) Fully elongated myocytes expressing myosin (long red cells; between brackets) are located closest to the dermomyotome, while the myogenin-positive cells (red nuclei, no cytoplasmic extensions) located at the interface with the sclerotome are myosin-negative (arrowheads). They are located centrally, close to discontinuous laminin (arrowheads). A continuous sheet of laminin delimits the rostral and caudal areas of the myotome (arrows). (M) Below the fully elongated myocytes (between brackets), a few elongating myocytes, the extremities of which are desmin labelled, contact the laminin matrix at their tips (arrows). ep, epaxial; hyp, hypaxial; nt, neural tube; scl, sclerotome; lam, laminin chain; laminin, immunoreactivity with polyclonal antibody; myog, myogenin. Scale bars: 50 µm in A-D,H-M; 100 µm in E-G.