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.

Quantitative histomorphometry is the current gold standard for objective measurement of nerve architecture and its components. Many methods still in use rely heavily upon manual techniques that are prohibitively time consuming, predisposing to operator fatigue, sampling error, and overall limited reproducibility. More recently, investigators have attempted to combine the speed of automated morphometry with the accuracy of manual and semi-automated methods. Systematic refinements in binary imaging analysis techniques combined with an algorithmic approach allow for more exhaustive characterization of nerve parameters in the surgically relevant injury paradigms of regeneration following crush, transection, and nerve gap injuries. The binary imaging method introduced here uses multiple bitplanes to achieve reproducible, high throughput quantitative assessment of peripheral nerve. Number of myelinated axons, myelinated fiber diameter, myelin thickness, fiber distributions, myelinated fiber density, and neural debris can be quantitatively evaluated with stratification of raw data by nerve component. Results of this semi-automated method are validated by comparing values against those obtained with manual techniques. The use of this approach results in more rapid, accurate, and complete assessment of myelinated axons than manual techniques.

Embryonic tissues may provide clues about mechanisms required for tissue reassembly and regeneration, but few studies have utilized primary embryonic tissue to study tissue assembly. To test the capacity of tissue fragments to regenerate, we cultured fragments of embryonic day 13 (E13) mouse submandibular gland (SMG) epithelium and found that fragments as small as a quarter-bud retain the ability to branch. Further, we found that completely dissociated SMG epithelial cells self-organize into structures that undergo significant branching. Investigation into the mechanisms involved in tissue self-assembly demonstrated that inhibition of beta(1) integrin prevents cell aggregation, while inhibition of E-cadherin hinders aggregate compaction. Immunostaining showed that the cellular architecture and expression patterns of E-cadherin, beta-catenin, and actin in the reassembled aggregates mirror those seen in intact glands. Adding SMG mesenchymal cells to the epithelial cell cultures facilitates branching and morphological differentiation. Quantitative real-time RT-PCR indicated that the aggregates express the differentiation markers aquaporin-5 (AQP5), prolactin-inducible protein (PIP), and SMG protein C (SMGC). Together, these data show that dissociated SMG epithelial cells self-organize and undergo branching morphogenesis to form tissues with structural features and differentiation markers characteristic of the intact gland. These findings provide insights into self-assembly and branching that will facilitate future regeneration strategies in the salivary gland and other organs.