Abstract The lymphatic endothelium has mostly been thought to be derived by sprouting from specialized veins. Recently it has been shown that mice deficient for the homeobox transcription factor Prox1 are practically devoid of lymphatics. We have studied the expression of Prox1 mRNA and protein in chick embryos and human fetuses. In the chick, Prox1 is expressed in specific com- partments of all germ layers. In the ectoderm, it is found in the neural tube, trigeminal, spinal and sympathetic ganglia and the retina, and also in placodal structures such as the lens, olfactory, otic, facial, glossopharyngeal and vagal placodes, and the apical ectodermal ridge. In the endoderm, Prox1 is a marker of hepatocytes, bile duct and pancreatic epithelium. In the mesoderm, weak expression is observed in cardiomyocytes, and strong ex- pression in lymphatic endothelium. Identical expression domains are found in 19-week-old human fetuses. In day 6.5 chick embryos, there are several sites of contact of lymphatics with the jugular vein, which has a mixed en- dothelium of Prox1-positive and -negative cells. The on- ly non-lymphatic endothelial cells expressing Prox1 are found on the concave side of the cardiac valves. To fur- ther analyse development of lymphatics, we studied ear- ly chick embryos and observed scattered Prox1-positive cells in the dermatome, giving rise to Prox1-positive lymphatic networks during subsequent development. Furthermore, the anlagen of the posterior lymph sacs and the paired thoracic duct can already be observed in day-4 chick embryos. Our studies show that lymphatics devel- op much earlier than previously described, and they mostly do not seem to be derived by sprouting from veins. In contrast, lymphangioblasts are present in the deep and superficial compartments of the early meso- derm, independently giving rise to the deep and superfi- cial lymphatics. Keywords Chick embryo · Human fetus · Angiogenesis · Lymphangiogenesis · Endothelium Introduction Studies on the development of the lymphatic system have always been hampered by the non-existence of spe- cific markers of the lymphatic endothelium. Only in re- cent years, molecules have been identified with high specificity for lymphatic endothelial cells. Among these, the vascular endothelial growth factor receptor-3 (VEGFR-3; flt-4) is a marker of lymphatics in differenti- ated tissues (Enholm et al. 1998; Taipale et al. 1999), but is additionally expressed in the endothelium of blood vessels during early embryonic development (Kaipainen et al. 1995; Wilting et al. 1997). Mice deficient for the VEGFR-3 gene die of cardiovascular malformations dur- ing early development (Dumont et al. 1998). The ligands of VEGFR-3, VEGF-C and -D, are capable of inducing lymphangiogenesis in normal tissues and in tumors (Jeltsch et al. 1997; Oh et al. 1997; Achen et al. 1998; Papoutsi et al. 2000; Skobe et al. 2001; Mandriota et al. 2001). New lymphatic endothelial markers have been de- scribed recently, such as LYVE-1, a hyaluronan receptor (Banerji et al. 1999), and podoplanin, a mucoprotein expressed in podocytes and lymphatic endothelium (Breiteneder-Geleff et al. 1999). However, studies on the expression of these molecules during embryonic develop- ment have not been performed yet. A small number of M. Rodriguez-Niedenführ · M. Papoutsi · B. Christ · J. Wilting ( ✉ ) Anatomisches Institut der Albert-Ludwigs-Universität Freiburg, Albertstrasse 17, 79104 Freiburg, Germany e-mail: wilting@uni-freiburg.de Tel.: +49-761-2035087, Fax: +49-761-2035091 K.H. Nicolaides Harris Birthright Research Centre for Fetal Medicine, King’s College Hospital, London, UK C.S. von Kaisenberg Universitäts-Frauenklinik, Michaelisstrasse 16, 24105 Kiel, Germany S.I. Tomarev Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institute of Health, Bethesda, MD 20892-2730, USA Anat Embryol (2001) 204:399–406 © Springer-Verlag 2001 ORIGINAL ARTICLE Marc Rodriguez-Niedenführ · Maria Papoutsi Bodo Christ · Kypros H. Nicolaides Constantin S. von Kaisenberg · Stanislav I. Tomarev Jörg Wilting Prox1 is a marker of ectodermal placodes, endodermal compartments, lymphatic endothelium and lymphangioblasts Accepted: 9 August 2001