No abstract

Gene targeting was used to create a null allele at the epidermal growth factor receptor locus (Egfr). The phenotype was dependent on genetic background. EGFR deficiency on a CF-1 background resulted in peri-implantation death due to degeneration of the inner cell mass. On a 129/Sv background, homozygous mutants died at mid-gestation due to placental defects; on a CD-1 background, the mutants lived for up to 3 weeks and showed abnormalities in skin, kidney, brain, liver, and gastrointestinal tract. The multiple abnormalities associated with EGFR deficiency indicate that the receptor is involved in a wide range of cellular activities.

Abstract. Epidermal differentiation is characterized by a series of coordinated morphological and biochemical changes which result in a highly specialized, highly organized, stratified squamous epithelium. Among the specific markers expressed in differentiating epidermis are (a) two early spinous cell proteins, keratins 1 and 10 (K1 and K10); and (b) two later granular cell proteins, filaggrin and a cornified envelope precursor (CE). In vitro, epidermal basal cells are selectively cultured in 0.05 mM Ca 2÷ medium, and terminal differentiation is induced when the Ca 2÷ concentration is increased to 1 mM. However, only a small fraction of the cells express the markers K1, K10, CE, or filaggrin in the higher Ca 2÷ medium. To explore the factors required for marker expression, cultured epidermal cells were exposed to intermediate Ca 2÷ concentrations and extracts were analyzed using specific antibody and nucleic acid probes for the four markers of interest. These studies revealed that marker expression was enhanced at a restricted concentration of Ca 2÷ in the medium of 0.10-0.16 mM. At this Ca 2÷ concentration, both protein and mRNA levels for each marker were substantially increased, whereas at higher or lower Ca 2÷ concentrations they were diminished or undetected. The percentage of ceils expressing each marker was increased two-to threefold in the permissive Ca 2÷ medium as determined by immunofluorescence analysis. This optimal level of Ca 2÷ was required both to initiate and sustain marker expression. At the permissive Ca 2÷ concentration, expression of the markers was sequential and similar to the order of appearance in vivo. K1 was expressed within 8-12 h and K10 was expressed in the ensuing 12-24-h period. CE and filaggrin were expressed in the subsequent 24 h. Inhibition of K1 expression by cycloheximide suggested that an inducible protein was involved. Other investigators have determined that a shallow Ca ~÷ gradient exists in epidermis, where the basal cells and spinous cells are in a Ca 2÷ environment substantially below serum Ca 2÷ levels. These in vitro results suggest that the Ca 2÷ environment is a fundamental regulator of expression of epidermal differentiation markers and provide an explanation for the existence of the Ca 2÷ gradient in vivo. MAMMALIAN epidermis has been a useful model to analyze tissue differentiation. The major products of epidermal differentiation are keratin intermediate filaments. Characteristic structural differences identify two major families of keratin intermediate filaments. The type I subclass includes at least 12 proteins with molecular masses of 40-60 kD and an acidic isoelectric point; the type II subclass is composed of proteins with molecular masses of 50-70 kD and a neutral to basic isoelectric point. This classification is functionally significant since epithelial cells express type I and II keratins in specific pairs (Eichner et al., 1984;Hanukoglu and Fuchs, 1983; for review see Steinert and Roop, 1988). The predominant pair expressed in mouse basal epidermal c...

Protocols for preparing and culturing primary keratinocytes from newborn and adult mouse epidermis have evolved over the past 35 years. This protocol is now routinely applied to mice of various genetic backgrounds for in vitro studies of signaling pathways in differentiation and cell transformation, and for assessing the in vivo phenotype of altered keratinocytes in grafts of cells on immunodeficient mice. Crucial in the development and application of the procedure was the observation that keratinocytes proliferate in media of low calcium concentration, but rapidly commit to differentiation at calcium concentrations >0.07 mM after the initial attachment period. Preparing primary keratinocytes from ten newborn mice requires 2-3 h of hands-on time. Related procedures are also provided: preparing immature hair follicle buds, developing dermal hair follicles and fibroblasts from newborn mice, preparing primary keratinocytes from adult mice and grafting cell mixtures on athymic nude mice.