Nishikawa A et al. (1992), Spatial, temporal, and hormonal regulation of e...

XB-ART-23791

Dev Biol 1992 May 01;1511:145-53. doi: 10.1016/0012-1606(92)90222-3.

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Spatial, temporal, and hormonal regulation of epidermal keratin expression during development of the frog, Xenopus laevis.

Nishikawa A , Shimizu-Nishikawa K , Miller L .

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To study the mechanism of hormone-induced keratin expression in the epidermis during Xenopus metamorphosis, a monospecific antibody was raised against a unique carboxy-terminal peptide of the 63-kDa keratin. Immunohistological analysis demonstrated that the onset of 63-kDa keratin expression showed distinct regional and temporal differences. The expression started at stage 54 in the hindlimb epidermis, at stage 57 in the head, and over 1 month later at stage 63 in the tail. The amount of 63-kDa keratin was further regulated during epidermal stratification and differentiation. The 63-kDa keratin was expressed first in basal epidermal cells before stratification began. The outer layer of the larval epidermis (periderm) did not express the 63-kDa keratin. As the cells moved out of basal layer, they stained more intensely with the anti-keratin antibody indicating that 63-kDa keratin synthesis is up-regulated during differentiation. Similar results were obtained with cultures of purified epidermal cells grown in high calcium conditions. Since we have shown that thyroid hormone (T3) induces 63-kDa keratin gene expression and hydrocortisone (HC) modulates T3 action we examined the effects of T3 and HC at the single cell level with the anti-keratin antibody. Immunostaining demonstrated that T3 alone and T3 plus HC increased the number of 63-kDa keratin-positive cells as well as the amount of 63-kDa keratin per cell. Unexpectedly these hormones had the same effects on head and tail epidermal cells even though the latter cells degenerate during metamorphosis. The major difference between tail and head cells was that the percentage 63-kDa keratin-producing cells was much greater in the head than in the tail.

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Species referenced: Xenopus laevis
Genes referenced: krt12.4 krt78.6 krt78.8 nrn1 slc4a3
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	FIG. 1. Specificity of the anti-63-kDa keratin antibody. Epidermal keratins from adult Xenop~s (lane l), stage 4’7 larval Xenqpus (lane 2), or human skin (lane 3) were separated by SDS-PAGE. Proteins were stained with Coomassie brilliant blue (A), or transferred to nitrocellulose paper and reacted with the anti-63-kDa keratin antibody (B), or the monoclonal antibody AE3 (C). Closed circles indicate the position of 63-kDa keratin and arrowheads indicate the type II keratins.
	FIG. 2. Immunohistological analysis of 63-kDa keratin expression during development. Cross sections of larval and adult skin were reacted with the anti-63-kDa keratin antibody (A-P) or with preimmune serum (Q). The three columns of photographs, from left to right, are from the head (A, D, G, J, and M), tail (B, E, H, K, and N), and hindlimh (C, F, I, L, and 0). The six rows, from the top to the bottom, are from stages 50 (A-C), 54 (D-F), 5’7 (G-I), 60 (J-L), 63 (M-O), and the adult skin (P, Q). pd, periderm; SC, stratum corneum; gl, skin glands; sp, epidermal spine. The arrows indicate the position of the basement membrane. The black spots in A, D, N, and 0 are melanocytes. The bar in Q represents 20 um.
	FIG. 3. Keratin synthesis patterns in the head and tail epidermis during development. Skin was dissected from (A) head or (B) tail regions of tadpoles of different stages and labeled with [a’S]amino acids for 3 hr (lane 1, stage 47; lane 2, stage 52; lane 3, stage 56; lane 4, stage 61; lane 5, stage 64). The cytoskeletal fractions were isolated, subjected to SDS-PAGE, and detected by fluorography. a, 63-kDa keratin; b, actin.
	FIG. 4. Differentiation-associated regulation of 63-kDa keratin expression in adult epidermal cells in vitro. Adult epidermal cells were cultured for 4 days in low calcium media (0.05 ti A, C) or high calcium media (0.3 mM: B, D). Epidermal cells were fixed with ethanol and reacted with (A, B) or without (C, D) the anti-keratin antibody. Large arrowheads show cells which express the 63-kDa keratin at high levels. Small arrows indicate cornified cells. The bar represents 20 Nrn.
	FIG. 5. Immunoblots of keratins from cultured adult epidermal cells. Adult epidermal cells were cultured for 4 days in low calcium media (panel A, lane 1, and panel B) or in high calcium media (panel A, lane 2, and panel C). The cytoskeletal fractions were extracted and separated by SDS-PAGE (A) or two-dimensional electrophoresis (B, C). For two-dimensional electrophoresis, the first dimension was NEPHGE, from left (acidic) to right (basic), and second dimension was SDS-PAGE.
	FIG. 6. T3-induction of 63-kDa keratin synthesis in head and tail epidermal cells in vitro. Larval epidermal cells from the head (A) and tail (B) were cultured for 4 days with no hormone (lane l), 10-r’ M T3 (lane 2), 10-9MT, (lane 3), lo-*MT, (lane 4), or 10m7MT, (lane 5). After labeling with [?S]amino acids for 3 hr, the cytoskeletal fractions were extracted, separated by SDS-PAGE and detected by fluorography. The relatively large amount of 63-kDa keratin in the control sample (A, lane 1) is due to overloading (see Fig. 3A). a, 63-kDa keratin; b, actin.
	FIG. 7. Hormonal regulation of 63-kDa keratin expression in head and tail epidermal cells in vitro. Larval epidermal cells from the head (A, B, and C) and tail (D, E, and F) were cultured for 4 days with no hormone (A and D), lo-*MT, (B and E) or lO-‘M T3 and 5 X IO-‘M HC (C and F). Epidermal cells were fixed with ethanol and reacted with the anti-keratin antibody. Closed triangles indicate cells which are stained weakly by the antibody. Open triangles indicate cells which are stained strongly by the antibody. The bar represent 20 pm.
	FIG. 8. The effects of hormones on 63-kDa keratin expression in vitro. Larval epidermal cells from the head were cultured for 4 days with no hormone (a), lo-*MT, (b), 5 X 10-“MHC (c), or 10-‘MT, plus 5 x 10-‘A4 HC (d). The cells were fixed with ethanol and reacted with the anti-keratin antibody. Epidermal cells were categorized according to the intensity of antibody staining; weak 63-kDa keratin expression (open boxes) and strong 63-kDa keratin expression (hatched boxes). The ordinate indicates the percentage of epidermal cells which stained with the anti-keratin antibody. The vertical line at the top of each box indicates the standard error (N = 10).