Immunocytochemical and electrophoretic distribution of cytokeratins in the regenerating epidermis of the lizardPodarcis muralis

Abstract

Using immunocytochemistry at light‐ and electron‐microscope levels, we studied the distribution of three monoclonal antibodies (AE1, AE2, AE3) specific for mammalian α‐keratins in regenerating lizard epidermis. We also characterized the keratins expressed during this process by immunoblotting after electrophoretic separation. The AE1 antibody is localized in the basal and suprabasal layers of prescaling and scaling epidermis. During the first stages of scale neogenesis, the AE1 antibody also marks the differentiating oberhautchen and β‐layer, but it disappears from these layers as they mature. This antibody does not stain the prekeratinized and keratinized outermost layers in the hinge region. The AE2 antibody labels the superficial wound epidermis, prekeratinizing and keratinized β‐ and α‐layers, but not basal and suprabasal cells. The AE3 antibody labels all living and keratinized epidermal layers, although AE3 immunoreactivity decreases and disappears as the β‐layer matures. The ultrastructural study shows that the AE2 and AE3, but not the AE1, antibodies specifically label small electron‐dense areas within the β‐layer, suggesting retention of α‐keratins. In the stages of tail regeneration examined, immunoblotting with the three antibodies used for the immunolocalization gives a pattern similar to that of the normal epidermis, except distally, where the process of scale differentiation begins. In this region, in addition to the keratin forms discovered in the normal and in proximal regenerating epidermis, an intense low molecular weight band at 40–41 kDa, positive to all three antibodies, is clearly detectable. Furthermore, in the distal region AE1 and AE3 antibodies, but not the AE2, recognize a weak band at 77–78 kDa not present in the normal and proximal epidermis. The localization and the possible role of the different keratins in the regenerating epidermis is discussed. J. Morphol. 246:179–191, 2000. © 2000 Wiley‐Liss, Inc.

Immunocytochemical and electrophoretic distribution of cytokeratins in the resting stage epidermis of the lizard Podarcis sicula

“…The present study shows that all epidermal layers of a lizard, including those containing β-keratin (oberhautchen and β-layer), contain α-keratin, which has cross-reactive epitopes with those of mammals (O'Guin et al,'87). This confirms previous ultrastructural studies that showed β-keratin packets mixed with keratin bundles in adult resting (Roth and Jones,'68;Alexander,'70;Maderson et al,'72;Landmann,'79), embryonic (Alibardi,'98a,b;Alibardi and Thompson,'99), and regenerating epidermis (Alibardi, '95, '98c;Alibardi et al, 2000). The deposition of β-keratin in pre-formed bundles of β-keratin has also been observed in chick scutate scales (Carver and Sawyer,'88;Shames et al,'88,'89), feathers (Haake et al,'84;Meyer and Baumgartner,'98), beak (Shames et al,'91), and tongue (Carver and Sawyer,'89).…”

Immunocytochemical and electrophoretic distribution of cytokeratins in the resting stage epidermis of the lizard Podarcis sicula

“…The present study shows that all epidermal layers of a lizard, including those containing β-keratin (oberhautchen and β-layer), contain α-keratin, which has cross-reactive epitopes with those of mammals (O'Guin et al,'87). This confirms previous ultrastructural studies that showed β-keratin packets mixed with keratin bundles in adult resting (Roth and Jones,'68;Alexander,'70;Maderson et al,'72;Landmann,'79), embryonic (Alibardi,'98a,b;Alibardi and Thompson,'99), and regenerating epidermis (Alibardi, '95, '98c;Alibardi et al, 2000). The deposition of β-keratin in pre-formed bundles of β-keratin has also been observed in chick scutate scales (Carver and Sawyer,'88;Shames et al,'88,'89), feathers (Haake et al,'84;Meyer and Baumgartner,'98), beak (Shames et al,'91), and tongue (Carver and Sawyer,'89).…”

Immunocytochemical analysis of the process of keratinization of the epidermis of snakes

“…However, the general pattern resembles that of lizards (Alibardi, 2000a;Alibardi et al, 2000, and indicates that acidic alpha-keratins are restricted to the most basal layers of the epidermis. The basic keratins seem more widely distributed, being found from the basal layer to the uppermost pre-keratinized layers and in the compact alpha-layer, but almost absent in the beta-layer.…”

“…The low immunoreactivity to AE1, AE2, AE3, may also be because thin (1±4 mm) plastic sections were used in the present study, and that proteases were not used for keratin detection, a method that normally enhances keratin visualization (Batti¯ora & Kopinski, 1986). However, the general pattern resembles that of lizards (Alibardi, 2000a;Alibardi et al, 2000, and indicates that acidic alpha-keratins are restricted to the most basal layers of the epidermis. The basic keratins seem more widely distributed, being found from the basal layer to the uppermost pre-keratinized layers and in the compact alpha-layer, but almost absent in the beta-layer.…”

Structure and functions of keratin proteins in simple, stratified, keratinized and cornified epithelia

“…Keratins in different vertebrates have similar amino acid sequences as inferred from the observation that epithelial tissues of various species of teleost fishes, amphibians, reptiles, birds, and marsupial and placental mammals crossreact with anti-human keratin antibodies (Gigi et al 1982;Fuchs & Marchuk, 1983;Groff et al 1997;Alibardi et al 2000). Unlike in other vertebrates, non-epithelial cells in teleost fishes also cross-react with anti-human keratin antibodies (Markl & Franke, 1988;Groff et al 1997;Schaffeld et al 2002a,b).…”

Immunocytochemical and electrophoretic distribution of cytokeratins in the resting stage epidermis of the lizard Podarcis sicula

“…The present study shows that all epidermal layers of a lizard, including those containing β-keratin (oberhautchen and β-layer), contain α-keratin, which has cross-reactive epitopes with those of mammals (O'Guin et al,'87). This confirms previous ultrastructural studies that showed β-keratin packets mixed with keratin bundles in adult resting (Roth and Jones,'68;Alexander,'70;Maderson et al,'72;Landmann,'79), embryonic (Alibardi,'98a,b;Alibardi and Thompson,'99), and regenerating epidermis (Alibardi, '95, '98c;Alibardi et al, 2000). The deposition of β-keratin in pre-formed bundles of β-keratin has also been observed in chick scutate scales (Carver and Sawyer,'88;Shames et al,'88,'89), feathers (Haake et al,'84;Meyer and Baumgartner,'98), beak (Shames et al,'91), and tongue (Carver and Sawyer,'89).…”

Immunocytochemical analysis of the process of keratinization of the epidermis of snakes

“…However, the general pattern resembles that of lizards (Alibardi, 2000a;Alibardi et al, 2000, and indicates that acidic alpha-keratins are restricted to the most basal layers of the epidermis. The basic keratins seem more widely distributed, being found from the basal layer to the uppermost pre-keratinized layers and in the compact alpha-layer, but almost absent in the beta-layer.…”

“…The low immunoreactivity to AE1, AE2, AE3, may also be because thin (1±4 mm) plastic sections were used in the present study, and that proteases were not used for keratin detection, a method that normally enhances keratin visualization (Batti¯ora & Kopinski, 1986). However, the general pattern resembles that of lizards (Alibardi, 2000a;Alibardi et al, 2000, and indicates that acidic alpha-keratins are restricted to the most basal layers of the epidermis. The basic keratins seem more widely distributed, being found from the basal layer to the uppermost pre-keratinized layers and in the compact alpha-layer, but almost absent in the beta-layer.…”

Structure and functions of keratin proteins in simple, stratified, keratinized and cornified epithelia

“…Keratins in different vertebrates have similar amino acid sequences as inferred from the observation that epithelial tissues of various species of teleost fishes, amphibians, reptiles, birds, and marsupial and placental mammals crossreact with anti-human keratin antibodies (Gigi et al 1982;Fuchs & Marchuk, 1983;Groff et al 1997;Alibardi et al 2000). Unlike in other vertebrates, non-epithelial cells in teleost fishes also cross-react with anti-human keratin antibodies (Markl & Franke, 1988;Groff et al 1997;Schaffeld et al 2002a,b).…”

Immunocytochemical and electrophoretic distribution of cytokeratins in the resting stage epidermis of the lizard Podarcis sicula

“…The present study shows that all epidermal layers of a lizard, including those containing β-keratin (oberhautchen and β-layer), contain α-keratin, which has cross-reactive epitopes with those of mammals (O'Guin et al,'87). This confirms previous ultrastructural studies that showed β-keratin packets mixed with keratin bundles in adult resting (Roth and Jones,'68;Alexander,'70;Maderson et al,'72;Landmann,'79), embryonic (Alibardi,'98a,b;Alibardi and Thompson,'99), and regenerating epidermis (Alibardi, '95, '98c;Alibardi et al, 2000). The deposition of β-keratin in pre-formed bundles of β-keratin has also been observed in chick scutate scales (Carver and Sawyer,'88;Shames et al,'88,'89), feathers (Haake et al,'84;Meyer and Baumgartner,'98), beak (Shames et al,'91), and tongue (Carver and Sawyer,'89).…”

Immunocytochemical analysis of the process of keratinization of the epidermis of snakes

“…However, the general pattern resembles that of lizards (Alibardi, 2000a;Alibardi et al, 2000, and indicates that acidic alpha-keratins are restricted to the most basal layers of the epidermis. The basic keratins seem more widely distributed, being found from the basal layer to the uppermost pre-keratinized layers and in the compact alpha-layer, but almost absent in the beta-layer.…”

“…The low immunoreactivity to AE1, AE2, AE3, may also be because thin (1±4 mm) plastic sections were used in the present study, and that proteases were not used for keratin detection, a method that normally enhances keratin visualization (Batti¯ora & Kopinski, 1986). However, the general pattern resembles that of lizards (Alibardi, 2000a;Alibardi et al, 2000, and indicates that acidic alpha-keratins are restricted to the most basal layers of the epidermis. The basic keratins seem more widely distributed, being found from the basal layer to the uppermost pre-keratinized layers and in the compact alpha-layer, but almost absent in the beta-layer.…”

Structure and functions of keratin proteins in simple, stratified, keratinized and cornified epithelia

“…Keratins in different vertebrates have similar amino acid sequences as inferred from the observation that epithelial tissues of various species of teleost fishes, amphibians, reptiles, birds, and marsupial and placental mammals crossreact with anti-human keratin antibodies (Gigi et al 1982;Fuchs & Marchuk, 1983;Groff et al 1997;Alibardi et al 2000). Unlike in other vertebrates, non-epithelial cells in teleost fishes also cross-react with anti-human keratin antibodies (Markl & Franke, 1988;Groff et al 1997;Schaffeld et al 2002a,b).…”