Retinoic acid‐induced alterations in the expression of growth factors in embryonic mouse palatal shelves

Abstract

Retinoic acid (RA) is teratogenic in many species, producing multiple malformations, including cleft palate. The effects of RA which lead to cleft palate vary depending on the stage of development exposed. After exposure of embryonic mice to RA on gestation day (GD) 10, abnormally small palatal shelves form. After exposure on GD 12 shelves of normal size form, but fail to fuse, as the medial cells proliferate and differentiate into a nasal‐like epithelium. Growth factors and their receptors play an important role in regulating development, and the expression of EGF receptors, EGF, TGF‐α, TGF‐β1, and TGF‐β2 has been reported in the mouse embryo. In a variety of cell types in culture, these growth factors are capable of regulating proliferation, differentiation, expression of matrix proteins, and other cellular events including epithelial‐mesenchymal transformations. The present study examines immunohistochemically the expression of EGF, TGF‐α, TGF‐β1, and TGF‐β2 in the control embryonic palatal shelves from GD 12 to 15 and the effects of RA treatment on GD 10 or 12 on their expression on GD 14 and 16. These growth factors were shown to have specific temporal and spatial expression in the palatal shelf. With advancing development the levels of TGF‐α decreased while the expression of EGF increased. TGF‐β2 localization became regional by GD 14–15, with higher levels found in epithelial cells and chondrogenic mesenchyme. TGF‐β1 occurred in epithelial and mesenchymal cells and distribution did not change substantially with advancing development. RA exposure altered the expression of TFG‐α, TGF‐β1, and TGF‐β2, but significant effects on EGF were not found. The effects on TGF‐α and TGF‐β1 expression were dependent on the gestational age exposed. Levels of TGF‐α on GD 14 decreased after RA exposure on GD 10, but increased after GD 12 exposure. TGF‐β1 expression in the mesenchyme was increased after exposure on GD 12, but was unaffected by RA on GD 10. After exposure on either day, the levels of TGF‐β2 increased in GD 14 nasal epithelial cells. Acting in concert, growth factors could regulate events critical to formation of the secondary palate, including cessation of medial epithelial cell proliferation, synthesis of extracellular matrix proteins in the mesenchyme, programmed cell death of medial epithelial peridermal cells, and transformation of basal epithelial medial cells to mesenchymal cells. The disruption in growth factor expression occurring after RA exposure appears to be important in the induction of cleft palate and the specific patterns of growth factor expression may determine whether clefting is due to abnormal differentiation of the me dial cells and/or to the formation of small shelves. Recent studies in our laboratory show that human palatal shelves express EGF, TGF‐α, and TGF‐β's in a pattern like that demonstrated for the embryonic mouse shelves. RA is a human teratogen, and cultured human embryonic palatal shelves respond to RA with altered differentiation of the medial cells. The mechanism through which RA induces cleft palate may be similar in mouse and human, and studies of the effects of RA on growth factor expression in the human shelf would provide further evidence of a common mechanism.