An ultrastructural analysis of cell and matrix differentiation during early limb development in Xenopus laevis

Early development of the hind limb of Xenopus (stages 44–48) has been analyzed at the level of ultrastructure with emphasis on differentiation of extracellular matrix components and intercellular contacts. By stages 44–45, mesenchyme is separated from prospective bud epithelium by numerous adepidermal granules in a subepithelial compartment (the lamina lucida), a continuous basal lamina and several layers of collagen (the basement lamella). Tricomplex stabilization of amphoteric phospholipid demonstrates that each adepidermal granule consists of several membranelike layers (electron-lucent band 25–30 Å; electron-dense band 20–40 Å), which are usually parallel to the basal surface of adjacent epithelial cells. Collagen fibrils are interconnected by filaments (35 Å in diameter) which stain with ruthenium red. Epithelial cells possess junctional complexes at their superficial borders, numerous desmosomes at apposing cell membranes and hemidesmosomes at their basal surface. Mesenchymal cells predominantly exhibit close contacts (100–150 Å separation) with few focal tight junctions at various areas of their surface. By stages 47–48, adepidermal granules are absent beneath bud epithelium and layers of collagen in the basement lamella lose filamentous cross-linking elements. Filopodia of mesenchymal cells penetrate the disorganized matrix and abut the basal lamina. Hemidesmosomes disappear at the basal surface of the epidermis and mesenchymal cells immediately subjacent to epithelium exhibit focal tight junctions and gap junctions at their lateral borders. These structural changes may be instrumental in the epitheliomesenchymal interactions of early limb development. Degradation of oriented collagenous lamellae permits direct association of mesenchymal cell surfaces (filopodia) with surface-associated products of epithelial cells (organized into the basal lamina). Development of structural pathways for intercellular ion and metabolite transport in mesenchyme may coordinate events specific to limb morphogenesis.