Cell and fibronectin dynamics during branching morphogenesis

Branching morphogenesis is a dynamic developmental process shared by many organs, but the mechanisms that reorganize cells during branching morphogenesis are not well understood. We hypothesized that extensive cell rearrangements are involved, and investigated cell migration using two-color confocal time-lapse microscopy to image cell and extracellular-matrix dynamics in developing salivary glands. We labeled submandibular salivary gland (SMG) epithelial cells with green fluorescent protein and matrix with fluorescent fibronectin. Surprisingly, we observed substantial, rapid and relatively random migration of individual epithelial cells during branching morphogenesis. We predicted that cell migration would decrease after formation of acini and, indeed, found that rapid cell movements do not occur in SMG from newborn mice. However, in embryonic SMG epithelial cells, we observed an absence of choreographed cell migration, indicating that patterned cell migration alone cannot explain the highly ordered process of branching morphogenesis. We therefore hypothesized a role for directional fibronection assembly in branching. Washout and pulse-chase experiments revealed that older fibronectin accumulates at the base of the clefts and translocates inwards as a wedge, with newer fibronectin assembling behind it. These findings identify a new mechanism for branching morphogenesis involving directional fibronectin translocation superimposed on individual cell dynamics.