The organizing center located at the midbrain-hindbrain boundary (MHB)patterns the midbrain and hindbrain primordia of the neural plate. Studies in several vertebrates showed that the interface between cells expressing Otx and Gbx transcription factors marks the location in the neural plate where the organizer forms, but it is unclear how this location is set up. Using mutant analyses and shield ablation experiments in zebrafish, we find that axial mesendoderm, as a candidate tissue, has only a minor role in positioning the MHB. Instead, the blastoderm margin of the gastrula embryo acts as a source of signal(s) involved in this process. We demonstrate that positioning of the MHB organizer is tightly linked to overall neuroectodermal posteriorization, and specifically depends on Wnt8 signaling emanating from lateral mesendodermal precursors. Wnt8 is required for the initial subdivision of the neuroectoderm,including onset of posterior gbx1 expression and establishment of the posterior border of otx2 expression. Cell transplantation experiments further show that Wnt8 signaling acts directly and non-cell-autonomously. Consistent with these findings, a GFP-Wnt8 fusion protein travels from donor cells through early neural plate tissue. Our findings argue that graded Wnt8 activity mediates overall neuroectodermal posteriorization and thus determines the location of the MHB organizer.

Integrins are cellular adhesion receptors that mediate signaling and play key roles in the development of multicellular organisms. However, their role in the cellular events leading to myotome formation is completely unknown. Here, we describe the expression patterns of the ␣1, ␣4, ␣5, ␣6, and ␣7 integrin subunits in the mouse myotome and correlate them with the expression of several differentiation markers. Our results indicate that these integrin subunits may be differentially involved in the various phases of myogenic determination and differentiation. A detailed characterization of the myogenic cell types expressing the ␣4 and ␣6 subunits showed a regionalization of the myotome and dermomyotome based on cell-adhesion properties. We conclude that ␣6␤1 may be an early marker of epaxial myogenic progenitor cells. In contrast, ␣4␤1 is up-regulated in the intercalated myotome after myocyte differentiation. Furthermore, ␣4␤1 is expressed in the hypaxial dermomyotome and is maintained by early hypaxial myogenic progenitor cells colonizing the myotome. Developmental Dynamics 231:402-415, 2004.

We addressed the potential role of cell-laminin interactions during epaxial myotome formation in the mouse embryo. Assembly of the myotomal laminin matrix occurs as epaxial myogenic precursor cells enter the myotome. Most Myf5-positive and myogeninnegative myogenic precursor cells localise near assembled laminin, while myogenin-expressing cells are located either away from this matrix or in areas where it is being assembled. In Myf5 nlacZ/nlacZ (Myf5-null) embryos, laminin, collagen type IV and perlecan are present extracellularly near myogenic precursor cells, but do not form a basement membrane and cells are not contained in the myotomal compartment. Unlike wild-type myogenic precursor cells, Myf5-null cells do not express the ␣6␤1 integrin, a laminin receptor, suggesting that integrin ␣6␤1-laminin interactions are required for myotomal laminin matrix assembly. Blocking ␣6␤1-laminin binding in cultured wild-type mouse embryo explants resulted in dispersion of Myf5-positive cells, a phenotype also seen in Myf5 nlacZ/nlacZ embryos. Furthermore, inhibition of ␣6␤1 resulted in an increase in Myf5 protein and ectopic myogenin expression in dermomyotomal cells, suggesting that ␣6␤1-laminin interactions normally repress myogenesis in the dermomyotome. We conclude that Myf5 is required for maintaining ␣6␤1 expression on myogenic precursor cells, and that ␣6␤1 is necessary for myotomal laminin matrix assembly and cell guidance into the myotome. Engagement of laminin by ␣6␤1 also plays a role in maintaining the undifferentiated state of cells in the dermomyotome prior to their entry into the myotome.