Anat Embryol (2004) 208:109–120 DOI 10.1007/s00429-003-0376-8 ORIGINAL ARTICLE G. Melkonian · J. L. Wang · J. Chung · N. Munoz · P. Talbot CD44 and tenascin play critical roles in growth and vascular development of the chick chorioallantoic membrane and are targets of cigarette smoke Accepted: 27 November 2003 / Published online: 30 March 2004  Springer-Verlag 2004 Abstract Chorioallantoic membranes (CAMs) were used to determine which extracellular matrix molecules play essential roles in growth and vascular development in vivo and whether expression of critical molecules is af- fected by cigarette smoke exposure. Treatment of CAMs on day 5 of development with antibodies to CD44 or tenascin, but not to other matrix molecules, inhibited CAM growth and affected various aspects of blood vessel development including normal growth and branching of vessels, migration of vessels, and formation and differ- entiation of the capillary plexus. DNA synthesis was in- hibited by antibodies to both C44 and tenascin which probably accounted for many of the phenotypic changes observed in treated CAMs. CD44 was located on all cells in day 5 CAMs, and tenascin, while present throughout the CAM, was especially abundant around large, non- migratory mesodermal blood vessels and endodermal cells that were positioned away from the direction of blood vessel migration. These data suggest that while tenascin is required for normal blood vessel migration, high levels of tenascin inhibit migration. The different distributions of CD44 and tenascin in CAMs and the observation that antibodies to either CD44 or tenascin produced similar phenotypes indicate that CD44 and te- nascin were not functionally redundant. Mainstream smoke solutions, which produce a phenotype similar to that seen with anti-tenascin and anti-CD44, inhibited ex- pression of CD44 mRNA and increased tenascin mRNA expression. 3-Ethylpyridine, a chemical in cigarette smoke that produced changes in CAM development similar to anti-CD44 and anti-tenascin treatment, also increased tenascin mRNA expression, but did not affect CD44. Together these data show that tenascin and CD44 play critical roles in early growth and vascular develop- ment of the CAM and support the idea that 3-ethylpyri- dine in mainstream smoke impairs CAM growth and vascular development by targeting expression of tenascin. 3-Ethylpyridine is generally regarded as safe and is used in many consumer products including food and tobacco. Keywords Angiogenesis · Extracellular matrix · 3-ethylpyridine Introduction Tissue growth and the formation of new vasculature are vital processes in developing animals. Growth occurs more rapidly during embryonic development than at any other time in life. Tissue growth depends on rapid de- velopment of blood vessels, which may occur by vascu- larization, the formation of new vessels from angioblasts, or by angiogenesis, formation of new vessels from pre- existing vessels (Carmeliet 2000; Risau 1997). In vivo, growth of new tissue and vasculature occurs within the context of a complex, spatially organized extracellular matrix, and molecules in the matrix often play important roles in regulating both growth and vascular development (Li et al. 2003; Raines 2000; Su 2000). Given the im- portance of growth and blood vessel formation during normal development (Flamme et al. 1997; Wilting et al. 1995), in cycling female reproductive organs (Augustin et al. 1995; Findlay 1986), in wound healing (Martins-Green and Hanafusa 1997), and in the progression of several diseases including cancer (Folkman and Shing 1992), there is interest in identifying the molecules that regulate these processes, as they may provide tools for ultimately controlling both wanted and unwanted growth of new tissue and vasculature (Folkman 1996; Kornowski et al. 2000; Rosengart et al. 1999). The chick chorioallantoic membrane (CAM) is a well- established model that can be used to study both growth and vascular development in vivo (Melkonian et al. 2001; Ribatti et al. 1996). The CAM begins forming 4–5 days after fertilization by fusion of the allantois and chorion G. Melkonian · J. L. Wang · J. Chung · N. Munoz · P. Talbot ( ) ) Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521, USA e-mail: talbot@citrus.ucr.edu Tel.: +1-909-7873768 Fax: +1-909-7874286