INTRODUCTION Palatogenesis is a very important event in craniofacial development of the group of higher vertebrates known as amniotes. In alligator, rodent and human, the medial edge epithelia (MEE) of the paired palatal shelves that arise from the maxillary processes make contact to form a two-layered midline seam (Ferguson, 1988). Then, the epithelial seam disappears and the palate becomes confluent. In the avian embryo, palatal shelves form in similar fashion, but the MEE do not fuse and the palate does not become confluent. Later, the MEE stratify and keratinize to line the medial edge of the naturally cleft palate (Lillie, 1908). The formation and disappearance of the MEE seam have been the focus of many studies of the easily accessible rodent palate. Early investigations suggested that programmed cell death is the mechanism for the disappearance of the seam (Glucksmann, 1951; Saunders, 1966). However, this conclusion has been challenged by recent workers (Fitchett and Hay, 1989; Griffith and Hay, 1992; Shuler et al., 1992), who studied the rodent MEE in vivo and in vitro by transmission electron microscopy (TEM) and with lineage markers, such as carboxyfluorescein and DiI, to obtain definitive evidence that the MEE undergo epithelial- mesenchymal transformation (EMT) to produce fibroblasts that form the connective tissue that creates palatal shelf confluence (but see Carrette and Ferguson, 1992). Earlier studies of chicken palatogenesis suggested that failure of apoptosis in the MEE plays the causal role in preventing avian palatal fusion. This conclusion implies that a fundamental difference exists in the developmental potential of avian MEE as compared to other amniotes (Shah and Crawford, 1980; Koch and Smiley, 1981; Greene et al., 1983; Shah and Cheng, 1988). However, no cell lineage studies have been done in chicken. Ferguson and Honig (1985) induced chicken palate fusion in vitro and in vivo by lacerating the MEE of each palatal shelf. Because this surgical procedure not only removed epithelia, but 95 Development 125, 95-105 (1998) Printed in Great Britain © The Company of Biologists Limited 1998 DEV3737 Epithelial-mesenchymal transformation plays an important role in the disappearance of the midline line epithelial seam in rodent palate, leading to confluence of the palate. The aim of this study was to test the potential of the naturally cleft chicken palate to become confluent under the influence of growth factors, such as TGFβ3, which are known to promote epithelial-mesenchymal transformation. After labeling medial edge epithelia with carboxyfluorescein, palatal shelves (E8-9) with or without beak were dissected and cultured on agar gels. TGFβ1, TGFβ2 or TGFβ3 was added to the chemically defined medium. By 24 hours in culture, medial edge epithelia form adherent midline seams in all paired groups without intact beaks. After 72 hours, seams in the TGFβ3 groups disappear and palates become confluent due to epithelial- mesenchymal transformation, while seams remain mainly epithelial in control, TGFβ1 and TGFβ2 groups. Epithelium-derived mesenchymal cells are identified by carboxyfluorescein fluorescence with confocal microscopy and by membrane-bound carboxyfluorescein isolation bodies with electron microscopy. Labeled fibroblasts completely replace the labeled epithelia of origin in TGFβ3- treated palates without beaks. Single palates are unable to undergo transformation, and paired palatal shelves with intact beaks do not adhere or undergo transformation, even when treated with TGFβ3. Thus, physical contact of medial edge epithelia and formation of the midline seam are necessary for epithelial-mesenchymal transformation to be triggered. We conclude that there may be no fundamental difference in developmental potential of the medial edge epithelium for transformation to mesenchyme among reptiles, birds and mammals. The bird differs from other amniotes in having developed a beak and associated craniofacial structures that seemingly keep palatal processes separated in vivo. Even control medial edge epithelia partly transform to mesenchyme if placed in close contact. However, exogenous TGFβ3 is required to achieve complete confluence of the chicken palate. Key words: TGFβ, epithelial-mesenchymal transformation, medial edge epithelium, carboxyfluorescein, chicken palate development, craniofacial morphogenesis SUMMARY TGFβ3 promotes transformation of chicken palate medial edge epithelium to mesenchyme in vitro Dazhong Sun 1 , Charles R. Vanderburg 2 , Gregory S. Odierna 3 and Elizabeth D. Hay 1, * 1 Department of Cell Biology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA 2 Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114, USA 3 Harvard Dental School, 180 Longwood Avenue, Boston, MA 02115, USA *Author for correspondence (E-mail: ehay@warren.med.harvard.edu) Accepted 20 October 1997