Development 112, 207-219 (1991) Printed in Great Britain © The Company of Biologists Limited 1991 207 A chicken caudal homologue, CHox-cad, is expressed in the epiblast with posterior localization and in the early endodermal lineage AYALA FRUMKIN 1 , ZEHAVA RANGINI 2 , ADI BEN-YEHUDA 1 , YOSEF GRUENBAUM 2 and ABRAHAM FAINSOD 1 * 1 Department of Cellular Biochemistry, Hebrew Umversity-Hadassah Medical School, Em Kerem, Jerusalem 91010, Israel 2Department of Genetics, Hebrew University, Gtvat Ram, Jerusalem 91904, Israel * Author for correspondence Summary CHox-cad is a chicken homeobox gene whose homeo- domain is homologous to the Drosophila caudal and the murine Cdxl genes. Based on sequence analysis of a 2.5 kb CHox-cad cDNA clone, we deduced that the primary translation product consists of 248 ami no acids. Comparison between the cDNA and genomic clones revealed the presence of an intron within the CHox-cad homeodomain between amino acids 44 and 45. The onset of CHox-cad transcription correlates temporarily with the beginning of gastrulation. During primitive streak stages CHox-cad exhibits a caudally localized pattern of expression restricted to the epiblast and the primitive streak. At these stages, CHox-cad transcripts can also be detected in the definitive endoderm cells. Later in embryogenesis CHox-cad is expressed in the epithelial lining of the embryonic gut and yolk sac. After four days of chicken development, no CHox-cad transcripts could be detected. The early CHox-cad posterior expression in the germ layer undergoing gastrulation and its continu- ous expression in the early endodermal lineage raise the possibility of CHox-cad involvement in the establishment of the definitive endoderm. Key words: homeobox, caudal, chicken embryogenesis, cloning, in situ hybridization, endoderm, CHox-cad, cell lineage. Introduction Numerous homeobox-containing genes (homeobox genes) have been cloned from vertebrate genomes and, in several instances, the same or closely related genes have been cloned from different organisms (Scott et al. 1989; Graham et al. 1989; Duboule and Dolle", 1989). The interest in this vertebrate gene family arose as a result of the discovery of the homeobox sequence in Drosophila developmental genes (see Gehring, 1987a,/? for reviews), which was subsequently shown to have been conserved during evolution, in animals including vertebrates (McGinnis et al. 1984). The large number of vertebrate homeobox genes cloned to date are found in one of four major Hox clusters which, in mice and humans, contain 6-10 homeobox genes (Acampora et al. 1989; Graham et al. 1989; Duboule and Dolle\ 1989; Nj0lstad et al 1990; Fritz et al. 1989; Wedden et al. 1989). The organization of vertebrate homeobox genes in clusters is reminiscent of Drosophila homeobox gene organization. Comparison of sequence and genomic organization of the clusters in mouse and man led to the suggestion that the vertebrate Hox clusters arose as a result of duplication of an ancestral gene complex (Hart et al. 1987; Kappen et al. 1989; Schughart et al. 1989). The similarity between the Drosophila and vertebrate homeobox gene clusters extends beyond the arrange- ment of homeobox sequences in a gene complex. Comparison of vertebrate genes in the clusters to the Drosophila genes in the Antennapedia and Bithorax complexes revealed a colinear arrangement of cognate genes (Acampora et al. 1989; Duboule and Doll6, 1989; Graham et al. 1989). Furthermore, as in Drosophila, the order of the genes along the cluster represents the anteroposterior order of their rostral boundary of expression (Gaunt et al. 1988; Graham et al. 1989; Duboule and Dolle", 1989). The high degree of homology between the vertebrate homeobox sequences and their Drosophila counter- parts has resulted in the organization of the genes into families based on their homeodomain sequences (Scott et al. 1989). In some instances, a homeobox gene family contains several sequences from the same organism due to the duplication of the Hox complexes. The homology between cognate genes is not restricted to the homeo- box region but it extends throughout the genes