Molecular cloning and expression of dead end homologue in chicken primordial germ cells

Abstract

Chicken primordial germ cells (PGCs) dynamically migrate towards the prospective gonadal area through the germinal crescent region and bloodstream at early embryonic stages. To date, chicken PGCs have been mainly identified by histochemical and immunohistochemical methods or by their morphological characteristics. However, their origin, migration and differentiation are not fully understood because of the lack of specific PGC molecular markers. Here, we have isolated the chicken dead end homologue (CDH) in order to clone its full-length cDNA with an open reading frame of 329 amino acids. The RNA-binding motif present in CDH at amino acids 54-133 was highly homologous to those in the dead end proteins of human, mouse and Xenopus. The temporal and spatial distribution of PGCs was also investigated by in situ hybridization (ISH) on whole-mount embryos with CDH cRNA as a probe. Chicken embryos from stage X to stage 20 were subjected to ISH. The hybridized samples were then sectioned to analyse the translocation of PGCs. CDH-positive cells could be counted from stage X to stage 4, with minimally 30 cells at the blastderm and approximately 260 cells at the germinal crescent. Thus, specific expression of CDH mRNA has been established in chicken PGCs located at the blastderm, germinal crescent and prospective gonadal area by ISH and reverse transcription/polymerase chain reaction. We conclude that isolated CDH is specifically expressed in chicken PGCs during embryogenesis.