The epidermis is a stratified epithelium constantly replenished through the ability of keratinocytes in its basal layer to proliferate and self-renew. The epidermis arises from a single-cell layer ectoderm during embryogenesis. Large proliferative capacity is central to ectodermal cell and basal keratinocyte function. DP-1, a heterodimeric partner of E2F transcription factors, is highly expressed in the ectoderm and all epidermal layers during embryogenesis. To investigate the role of DP-1 in epidermal morphogenesis, we inhibited DP-1 activity through exogenous expression of a dominantnegative mutant (dnDP-1). Expression of the dnDP-1 mutant interferes with binding of E2F/DP-1 heterodimers to DNA and inhibits DNA replication, as well as cyclin A mRNA and protein expression. Chromatin immunoprecipitation analysis demonstrated that the cyclin A promoter is predominantly bound in proliferating keratinocytes by complexes containing E2F-3 and E2F-4. Thus, the mechanisms of decreased expression of cyclin A in the presence of dnDP-1 seem to involve inactivation of DP-1 complexes containing E2F-3 and E2F-4. To assess the consequences on epidermal morphogenesis of inhibiting DP-1 activity, we expressed dnDP-1 in rat epithelial keratinocytes in organotypic culture and observed that DP-1 inhibition negatively affected stratification of these cells. Likewise, expression of dnDP-1 in embryonic ectoderm explants produced extensive disorganization of subsequently formed epidermal basal and suprabasal layers, interfering with normal epidermal formation. We conclude that DP-1 activity is required for normal epidermal morphogenesis and ectoderm-toepidermis transition.The skin performs many functions essential for life. It prevents loss of water and electrolytes, provides a barrier against the external environment, and is an integral component of the endocrine, immune, and nervous systems (for review, see Refs. 1 and 2). Two layers form the skin, the outer epidermis and the inner dermis. The epidermis is a complex stratified squamous epithelium formed by one basal and several suprabasal keratinocyte layers (for review, see Ref.3). The epidermis is constantly subjected to mechanical and chemical insults; as a result, its upper layers are constantly shed and replenished by underlying keratinocytes. This process allows the removal of damaged cells without compromising barrier function. The basal layer contains undifferentiated keratinocytes with high proliferative capacity, which allows them to continuously replenish the suprabasal layers. The stem cells necessary for this process reside in various regions, including the basal layer, and bulge areas in the hair follicles (2, 4).Early in development, the embryo is covered by a single cell-layered ectoderm. In the mouse, as early as 9.5 days postcoitus (dpc), 1 the single-layered ectoderm begins to express epithelial markers such as keratins 8 and 18 (5). Between 9 and 12 dpc, the ectoderm undergoes stratification, producing a second transitory layer termed the periderm (for review, se...