As the first step to understand the mechanism of pharyngeal cartilage development in the embryo of flounder, Paralichthys olivaceus, we studied the process of cartilage formation in the pharynx histologically, then analyzed the role of fibroblast growth factor (FGF) during the process. At hatching (56-62 hr post-fertilization), each mandible, hyoid and gill cartilage existed as a pair of blastemas composed of prechondroblasts. Paired blastemas fused at medial line at 1.5 days post-hatching, forming primordia. Labeling with 5-bromodeoxyuridine indicated that proliferation of prechondroblasts was highly active at this stage. Deposition of cartilage matrix began from the mandible and hyoid primordia at 2.5 days post-hatching, and at 4.0 days the basic patterning of the pharyngeal cartilage was complete.

Immunohistochemical analyses with an antiserum against 22.5 kD FGF (anti-22.5 kD FGF) isolated from swimbladder of red seabream, Pagrus major, revealed that the surface ectoderm, gut, lateral line blastema and otic vesicle show FGF-like immunoreactivity. Immunoreactivity signals persisted through the stages when the proliferation of prechondroblasts was active. In order to estimate possible function of 22.5 kD FGF, the blocking effect of anti-22.5 kD FGF towards pharyngeal cartilage formation was assayed using cranial explants dissected from hatched embryos. When incubated with anti-22.5 kD FGF, cartilaginous nodules formed bilaterally at the position of the original blastemas, while in the control treatment, incubation with normal rabbit IgG, cartilages connecting the two lateral sides were formed. Thus, development of pharyngeal cartilage was severely curtailed in the presence of anti-22.5 kD FGF. These results suggest the possibility that 22.5 kD FGF is produced in the pharynx and that it takes part in the regulation of pharyngeal cartilage development, possibly as a signal to stimulate the growth of prechondroblasts.