Cystatin immunoreactivity in cornifying layers of the epidermis suggests a role in the formation of the epidermal barrier in amniotes.

The presence and localization of cystatin, a cysteine protease inhibitor involved in barrier formation in human and mice epidermis, has been studied in the epidermis of piscine and terrestrial vertebrates using a mouse monoclonal antibody. Cystatin has been localized by Immunostaining in the pre-corneous and corneous layers of monotreme, marsupial and placental mammals, and sparsely in the thin corneous layer of birds. Cystatin-immunolabeling is present in the pre-corneous and corneous layer of crocodilian and turtle epidermis, in the alpha-corneous layer and likely also in the beta-corneous layer of the epidermis in lizards, snakes and the tuatara. In keratinocytes of the pre-corneous (transitional) layers the protein initially shows a peripheral distribution that becomes compacted in mature corneocytes. The protein is not detected using the antibody in the epidermis of cyclostome, teleosts, sarcopterigian fish, and in amphibians. The study concludes that while in fish and amphibians cystatin is absent or however uncertainly localized in the epidermis, the protein instead appears present in the more external pre-corneous and corneous layers of amniotes. This special regionalization suggests a specific role of cystatin in the formation of the corneous epidermal barrier and regulation of desquamation originally evolved in the terrestrial environment.