Epithelial Changes caused by Diffusible Mesenchymal Agents

Morphogenetic factors of the mesenchyme and an appropriate extracellular matrix are vital in epithelial branching (Takahashi, 1991). Takahashi demonstrated that the submandibular gland epithelium could branch when it was in the presence of matrigel, a reconstituted matrix of basement membrane components from EHS sarcoma- laminin, type IV collagen, heparan sulfate proteoglycan, nidogen and entactin (Takahashi, 1991). Each of these components are also found in the basement membrane of the mouse epithelial cells. Branching morphogenesis continued when the epithelium and matrigel were separated from the underlying mesenchyme with a porous filter. This demonstrated that diffusible morphogenetic factors from the mesenchyme had a large influence on epithelial branching. These experiments complement the interpretations of the mechanical aspect of the branching morphogenesis. Without the added component of the extracellular matrix, Matrigel, the cells did not undergo branching, demonstrating that the mesenchyme alone was not sufficient to cause full differentiation of the epithelium.

A further study by Nogawa showed that the epithelium could undergo branching morphogenesis without close proximity of the mesenchyme in experimental conditions. This could be done if the epidermal cells were cultured on Matrigel along with Epidermal Growth Factor (EGF) or Transforming Growth Factor-a (TGF-a) (Nogawa, 1991). This experiment also concluded that traction forces from the mesenchyme alone did not derive a normal branching pattern . The authors concluded that the mesenchyme provides the epithelium with the necessary basement membrane substratum and aids in accelerating the growth of the epithelium (Nogawa, 1991).

Heperan Sulfate proteoglycans

As seen before, heperan sulfate chains are a component of Matrigel. They have also been seen in embryonic tissues along cell surfaces and in extracellular spaces (Nakanishi et. al., 1993) . If these chains are removed, the differentiating epithelium does not form clefts and results in an elongated epithelial form. This is due to the possible interaction of heperan sulfate chains with collagen fibers to organize the collagen network. If a collagenase is added, collagen fibrils do not adhere to the basal lamina and again no clefts form. This helps support the fact that collagen III is responsible for initiating cleft formation of the submandibular gland in the 12 day old mouse. One form of heperan sulfate proteoglycan is syndecan which has been seen to be a connecting molecule (Nakanishi et. al., 1993). Syndecan is located at the basal regions of the lobules so it is a prime candidate for connecting cells of the mesenchyme to the basal lamina. Perhaps, it also connects collagens within the mesenchyme to form the collagen substratum.

Transforming Growth Factor b1

The TGF-b1 protein is a paracrine factor which can effect branching patterns (Gilbert, 1994). It is present in day 13 embryos in the mesenchyme as well as at the epithelial-mesenchymal interfaces during the branching of the submandibular and sublingual glands. It is also present in large amounts in day 14 embryos as well (Hardman, 1994). If there is an over-abundance of TGF-b1 during development, it will cause a cessation in branching and a reduced organ size as well as epithelial abnormalities (Hardman, 1994). This data suggests that TGF-b1has a role in regulating the branching morphogenesis within the mouse salivary gland. TGF-b1 may have an interactive role with collagen as well. There is often an elongation of the sublingual lobes and ducts with added TGF-b1. This phenotype is similar to that seen in a heperan sulfate proteoglycan mutant as well. TGF-b1 acts to increase the expression of matrix components like collagen and prevent the expression of metalloproteins which can degrade the matrix (Gilbert, 1994). The actions of the TGF-b1 proteins may result in the stabilization of the branches by maintaining an adequate collagen amount.

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