Epidermal Differentiation During Ontogeny and After Hatching in the Snake Liasis fuscus (Pythonidae, Serpentes, Reptilia), With Emphasis on the Formation of the Shedding Complex L. Alibardi 1 * and M.B. Thompson 2 1 Dipartimento di Biologia evoluzionistica sperimentale, University of Bologna, 40126, Bologna, Italy 2 School of Biological Sciences and Wildlife Research Institute, University of Sydney, NSW 2006, Sydney, Australia ABSTRACT Differentiation and localization of keratin in the epidermis during embryonic development and up to 3 months posthatching in the Australian water py- thon, Liasis fuscus, was studied by ultrastructural and immunocytochemical methods. Scales arise from dome- like folds in the skin that produce tightly imbricating scales. The dermis of these scales is completely differ- entiated before any epidermal differentiation begins, with a loose dermis made of mesenchymal cells beneath the differentiating outer scale surface. At this stage (33) the embryo is still unpigmented and two layers of su- prabasal cells contain abundant glycogen. At Stage 34 (beginning of pigmentation) the first layers of cells be- neath the bilayered periderm (presumptive clear and oberhautchen layers) have not yet formed a shedding complex, within which prehatching shedding takes place. At Stage 35 the shedding complex, consisting of the clear and oberhautchen layers, is discernible. The clear layer contains a fine fibrous network that faces the underlying oberhautchen, where the spinulae initially contain a core of fibrous material and small beta-keratin packets. Differentiation continues at Stage 36 when the beta-layer forms and beta-keratin packets are deposited both on the fibrous core of the oberhautchen and within beta-cells. Mesos cells are produced from the germinal layer but remain undifferentiated. At Stage 37, before hatching, the beta-layer is compact, the mesos layer contains mesos granules, and cells of the alpha-layer are present but are not yet keratinized. They are still only partially differentiated a few hours after hatching, when a new shedding complex is forming underneath. Using antibodies against chick scale beta-keratin re- solved at high magnification with immunofluorescent or immunogold conjugates, we offer the first molecular confirmation that in snakes only the oberhautchen com- ponent of the shedding complex and the underlying beta cells contain beta-keratin. Initially, there is little im- munoreactivity in the small beta-packets of the ober- hautchen, but it increases after fusion with the under- lying cells to produce the syncytial beta layer. The beta- keratin packets coalesce with the tonofilaments, including those attached to desmosomes, which rapidly disappear in both oberhautchen and beta-cells as differ- entiation progresses. The labeling is low to absent in forming mesos-cells beneath the beta-layer. This study further supports the hypothesis that the shedding com- plex in lepidosaurian reptiles evolved after there was a segregation between alpha-keratogenic cells from beta- keratogenic cells during epidermal renewal. J. Morphol. 256:29 – 41, 2003. © 2003 Wiley-Liss, Inc. KEY WORDS: python; snake; embryogenesis; epidermis; beta-keratins; immunocytochemistry; ultrastructure; skin In snakes the epidermis is not continuously re- newed, as it is in other vertebrates, but cell multi- plication in the germinal layer takes place during limited periods called renewal phases (Maderson, 1965a, 1985; Landmann, 1979). During these peri- ods a new (inner) epidermal generation is produced beneath the old (outer) generation, which is later shed (Maderson et al., 1998). In contrast to most lizards, shedding in all snakes is a single event resulting in the whole skin being lost in one piece. Thus, snakes have a perfectly synchronized mecha- nism of shedding and are among the most special- ized vertebrates to renew their epidermis in cyclical phases. The specialized epidermis of snakes is formed by numerous layers called oberhautchen, beta, mesos, alpha, lacunar, and clear (Maderson, 1965a, 1985; Roth and Jones, 1967, 1970; Landmann, 1979, 1986). Morphological and biophysical studies sug- gest that the oberhautchen and beta-layers mainly synthesize beta-keratin, while the remaining layers synthesize alpha-keratin (Roth and Maderson, 1968; Contract grant sponsor: University of Bologna grant (segnali mole- colari dello sviluppo). *Correspondence to: L. Alibardi, Dipartimento di Biologia evoluzi- onistica sperimentale, University of Bologna, via Selmi 3, 40126, Bologna, Italy. E-mail: Alibardi@biblio.cib.unibo.it DOI: 10.1002/jmor.10071 JOURNAL OF MORPHOLOGY 256:29 – 41 (2003) © 2003 WILEY-LISS, INC.