Reconstruction of the basement membrane in a cultured submandibular gland

In a rat submandibular rudiment on day 16, both laminin (LM) and type IV collagen (Col-IV) were found in all cases to colocalize not only in the basement membrane, but also in the rough endoplasmic reticulum of the epithelial cells, indicating that the synthesis of the components of basement membrane is greatly enhanced at this particular stage of extensive branch formation. Using the submandibular gland from a 16-day embryo, the model system was developed to determine the structural organization of the basement membrane. The pre-existing basement membrane was digested with collagenase and dispase, causing its complete disappearance. The subsequent gradual reconstruction of an authentic basement membrane was confirmed by electron microscopy and immunohistochemistry of LM and Col-IV. In the model system, this recovery started at 4 h of culture, and formation was complete by 8 h. During the recovery, thick bundles of actin filaments appeared transitionally in the basal cytoplasm. Electron microscopic analysis indicated two precursor structures, aggregated fuzzy fibers (type 1 extracellular matrix (ECM)) and 10-nm-thick strand piles (type 2 ECM), and an authentic basement membrane structure appeared during the course of membrane reconstruction. LM and Col-IV were always located together in these three structures. These observations clearly indicate that the precursors, containing LM, Col-IV and most likely heparan sulfate proteoglycan, appeared to form immediately following their secretion into the extracellular space, and assembled into the rigid structure of basement membrane within 8 h. The ultrastructural and immunohistochemical process of basement membrane reconstruction appeared to coincide closely with that of the glomerular basement membrane in developing kidney.(ABSTRACT TRUNCATED AT 250 WORDS)