INTRODUCTION Transdifferentiation is a relatively rare phenomenon that denotes a switch in the phenotype of a cell that has acquired specific differentiated characteristics (Okada, 1991). The best known examples of experimentally induced transdifferentiation are changes in subclasses of neural crest- derived neurons upon transplantation (Anderson, 1989; Patterson, 1990; Okada, 1991), amphibian limb regeneration upon injury (Okada, 1991; Brockes, 1994) and lens regeneration upon lentectomy (Okada, 1991; Juric-Lekic et al., 1991; Hitchcock and Raymond, 1992). By contrast, smooth- to-skeletal muscle transdifferentiation in the esophagus takes place during normal mouse development (Patapoutian et al., 1995b) and does not need to be induced by experimental manipulations or injury. Therefore, esophageal transdifferentiation resembles the normally occurring switch in the phenotype of sympathetic neurons to sweat gland (Landis, 1990), and that of the iris sphincter muscle in the chicken (Volpe et al., 1993; Link and Nishi, 1998). Unlike the rest of the gastrointestinal tract, the muscularis externa of the entire cervical segment and the greater part of the thoracic segment of the adult mouse esophagus consists of striated muscle (Samarasinghe, 1972; Sang and Young, 1997). Smooth muscle cells begin to appear in the muscularis externa at the lower end of the thoracic segment, just proximal to the diaphragm, gradually replacing the striated muscle in a caudal direction. As a consequence, the entire muscularis externa in the caudal part of the abdominal esophageal segment is made up of smooth muscle cells (Samarasinghe, 1972). In contrast, during early stages of embryonic development, the outer layer of the entire mouse esophagus consists of differentiated smooth muscle cells (Patapoutian et al., 1995b; Sang and Young, 1997). Cells exhibiting striations are first detected in the outer layer of the most rostral parts of the esophagus at E15.5 (Sang and Young, 1997). The external muscle layer of the mouse esophagus undergoes smooth-to-skeletal muscle transdifferentiation in a craniocaudal direction, coexpressing both smooth (myosin light chain) and skeletal (myosin heavy chain) differentiation markers within individual cells (Patapoutian et al., 1995b). This process of transdifferentiation continues through the first 2 weeks of esophageal postnatal development (Patapoutian et al., 1995b), at which point the esophageal muscularis externa acquires its adult appearance. 1627 Development 127, 1627-1639 (2000) Printed in Great Britain © The Company of Biologists Limited 2000 DEV4327 Previously, coexpression of smooth and skeletal differentiation markers, but not myogenic regulatory factors (MRFs), was observed from E16.5 mouse fetuses in a small percentage of diaphragm level esophageal muscle cells, suggesting that MRFs are not involved in the process of initiation of developmentally programmed transdifferentiation in the esophagus. To investigate smooth-to-skeletal esophageal muscle transition, we analyzed Myf5nlacZ knock-in mice, MyoD-lacZ and myogenin-lacZ transgenic embryos with a panel of the antibodies reactive with myogenic regulatory factors (MRFs) and smooth and skeletal muscle markers. We observed that lacZ-expressing myogenic precursors were not detected in the esophagus before E15.5, arguing against the hypothesis that muscle precursor cells populate the esophagus at an earlier stage of development. Rather, the expression of the MRFs initiated in smooth muscle cells in the upper esophagus of E15.5 mouse embryos and was immediately followed by the expression of skeletal muscle markers. Moreover, transdifferentiation was markedly delayed or absent only in the absence of Myf5, suggesting that appropriate initiation and progression of smooth-to- skeletal muscle transdifferentiation is Myf5-dependent. Accordingly, the esophagus of Myf5 -/- :MyoD -/- embryos completely failed to undergo skeletal myogenesis and consisted entirely of smooth muscle. Lastly, extensive proliferation of muscularis precursor cells, without programmed cell death, occurred concomitantly with esophageal smooth-to-skeletal muscle transdifferentiation. Taken together, these results indicate that transdifferentiation is the fate of all smooth muscle cells in the upper esophagus and is normally initiated by Myf5. Key words: Myf5, MyoD, Esophagus, Transdifferentiation, Skeletal muscle SUMMARY Transdifferentiation of esophageal smooth to skeletal muscle is myogenic bHLH factor-dependent Boris Kablar 1 , Shahragim Tajbakhsh 2 and Michael A. Rudnicki 1, * 1 Institute for Molecular Biology and Biotechnology, McMaster University, Hamilton, Ontario, Canada L8S 4K1 2 Department of Molecular Biology, CNRS URA1947, Pasteur Institute, 25 rue du Dr Roux, 75724 Paris, Cedex 15, France *Author for correspondence (e-mail: rudnicki@mcmaster.ca) Accepted 4 February; published on WWW 21 March 2000