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The primordium of the mammalian adenohypophysis derived from Rathke’s pouch (RP) is known to be formed by oral ectoderm invagination. However, in the early phase of pituitary development, the detailed process by which the oral ectoderm develops into the adenohypophysis remains largely unknown. Using high-resolution non-radiolabeled in situ hybridization and the BrdU and TUNEL methods, we have examined the detailed expression pattern of factors involved in the formation of RP of chicken and the changes in the mitotic and apoptotic cell regions in RP. In the chicken embryo, Sonic hedgehog (Shh) mRNA was initially expressed in the stomodeal plate but not in the oral ectoderm. After prospective diencephalon had detached from the oral ectoderm, another Shh-expressing region appeared in the most rostral part of the recess. LIM homeobox gene 3 (Lhx3) mRNA first appeared in the anterior area of Rathke’s recess, and expression then spread to the caudal region. αGSU mRNA-expressing cells were observed at both ends of the Lhx3-expressing region, and thereafter the expression area moved to the posterior region. Furthermore, a close overlap was found between the proliferating region and Lhx3 mRNA-expressing area, and TUNEL-positive cells appeared in Seessel’s pouch derived from the foregut. Thus, the primordium of the pituitary gland corresponding to the Lhx3-expressing region is surrounded by the Shh-expressing region, which appears in two steps, and the mass growth and invagination of RP of chicken result from the coordination of the dorsal extension of the anterior region and the ventral extension of the posterior region of RP. [ABSTRACT FROM AUTHOR]