Cornish JA et al. (1992), Xlcaax-1 is localized to the basolateral membra...

XB-ART-23955

Dev Biol 1992 Mar 01;1501:108-20. doi: 10.1016/0012-1606(92)90011-5.

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Xlcaax-1 is localized to the basolateral membrane of kidney tubule and other polarized epithelia during Xenopus development.

Cornish JA , Kloc M , Decker GL , Reddy BA , Etkin LD .

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Xlcaax-1 is a novel, maternally expressed, 110-kDa, CAAX box containing protein that undergoes isoprenylation and palmitoylation through which it associates with the plasma membrane. We report here the cellular and subcellular localization of the xlcaax-1 protein during development of Xenopus laevis. Whole-mount immunocytochemistry and immunoperoxidase staining of tissue sections show that during development the xlcaax-1 protein accumulation is coincident with the differentiation of the epidermis, pronephros, and mesonephros. In the pronephros and mesonephros the xlcaax-1 protein is localized to the basolateral membrane of differentiated tubule epithelial cells. Thus, the xlcaax-1 protein serves as a marker for tubule formation and polarization during Xenopus kidney development. Xlcaax-1 may also be used as a marker for the functional differentiation of the epidermis and the epidermally derived portions of the lens and some cranial nerves. Western blot analysis shows that in the adult the xlcaax-1 protein is most abundant in kidney. Immunogold EM analysis shows that the xlcaax-1 protein is highly enriched in the basal infoldings of the basolateral membrane of the epithelial cells in adult kidney distal tubules. In addition, immunoperoxidase staining of tissue sections detected low levels of xlcaax-1 protein in the epithelial cells of skin, urinary bladder, gall bladder, and parietal glands of the stomach. The localization pattern of xlcaax-1 suggests that the protein may function in association with an ion transport channel or pump.

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Species referenced: Xenopus laevis
Genes referenced: palm3 uqcc6
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	FIG. 1. Developmental Western. Protein extracts from differentstaged embryos were run on 7.5% SDS-PAGE gels, blotted to nylon membrane, and stained with xlcaax-1 antisera. The numbers at the top of each lane indicate the stage number, the arrow indicates the IlO-kDa major band and the bar represents the 98-kDa marker: (A) 100 pg of protein loaded per lane; (B) 250 Kg of protein loaded per lane.
	FIG. 2. In situ hybridization. (A) Section of a stage 26 embryo stained with Azur B. (B) Adjacent section of stage 26 embryo hybridized with xlcaax-1 anti-sense RNA probe showing specific epidermal hybridization. (C) Section of pronephros of stage 46 embryo stained with Azur B. (D) Adjacent section of stage 46 embryo hybridized with xlcaax-1 anti-sense RNA probe showing specific hybridization in the pronephros. There was no specific hybridization with control, sense RNA probes (data not shown). Magnification (Magn.) of A and B is 100X. Magn. of C and D is 250x
	FIG. 3. Whole-mount immunocytochemistry of embryos. (A) Skin dissected from stage 25 whole-mount embryo labeled with antigen affinity purified L13 pAb (see Materials and Methods). (B) Skin dissected from control stage 25 whole-mount embryos labeled with antigen affinity-purified preimmune sera. (C) Stage 41 whole-mount embryo stained with xlcaax-1 pAb. The arrows indicate the specific staining of the pronephric tubule. (D) Control stage 41 whole-mount embryo. Arrows indicate pronephric area with no specific staining. (E) Higher magnification of stage 41 pronephros. al, ascending limb; act, anterior convoluted tubule; dl, descending limb, w, wolffian duct. (Parts of pronephros from Tytler, 1933). Magn. of A and B is 1000~. Magn. of C and D is 25X. Magn. of E is 60X.
	FIG. 4. Accumulation of xlcaax-1 protein in differentiated skin, pronephros and mesonephros. Immunocytochemistry of embryonic tissue sections stained with the L13 pAb. (A) Stage 15/16 embryos, arrows indicate undifferentiated outer ectoderm. (B) Stage 23/24 embryo, arrows indicate differentiated epidermis with basolateral xlcaax-1 staining. (C) Stage 29136 embryo, arrows indicate pronephric tubules in initial stages of differentiation. (D) Stage 35 embryo, arrows indicate differentiated pronephric tubules. (E) Stage 47/48 embryo, arrows indicate undifferentiated mesonephric tissue, open arrows indicate the pronephric ducts. (F) Stage 49 embryo showing specific xlcaax-1 staining in the tubules, open arrow indicates the pronephric duct, curved arrow indicates the distal tubule, arrow indicates the proximal tubule, s, skin. Magn. is 500X.
	FIG. 5. Xlcaax-1 protein is found in epidermally derived tissues. (A) Sagittal section of stage 49 embryo stained with the xlcaax-1 pAb, the roman numerals indicate and are located above the ganglia of cranial nerves V/VII, VIII, and IX/X, m, medulla, n, notochord. (B) Section from same embryo as in A, arrows indicate the optic nerve (cranial nerve II) which does not stain with the L13 pAb. (C) Section from same embryo as in A, arrow indicates spinal ganglia which does not stain, SC, spinal cord. (D) Stage 43 embryo stained with the xlcaax-1 pAb, arrow indicates the olfactory nerve (cranial nerve I), oe, olfactory epithelium, t, telencephalon. (E) Stage 43 control embryo, arrows indicate olfactory nerve. (F) Stage 49 cranial ganglia IX/X, arrows indicate the area stained with xlcaax-1 pAb. (G) Control stage 49 cranial ganglia IX/X. (H) Stage 30 embryo, arrows indicate lens primordia, s, skin, ov, optic vesicle. (I) Stage 37/38 embryo, arrows indicate regions of the lens which stain with xlcaax-1 pAb, 1, lens, oc, optic cup, s, skin. Magn. of A is 100X. Magn. of B, C, D, and E is 400X. Magn. of F, G, H, and I is 500X.
	FIG. 6. Western blot of adult tissues, Xenopus tissues culture cells, and bacterially produced protein. Protein extracts were run on a 7.5% SDS-PAGE gel, transferred to nylon membrane, and stained with L13 anti-sera. B, bacterially produced protein; x, Xtc tissue culture cells; k3, k2 tissue culture cells; ub, urinary bladder; k, kidney; s, skin; 1, lung; o, ovary; od, oviduct; li, liver; h, heart; p, pancreas; m, muscle; br, brain; e, eye; g, gut; sp, spleen; gl, gall bladder. The arrow indicates the llO-kDa major band and the bar indicates the location of the 9%kDa marker. There is 100 pg of protein loaded per lane.
	FIG. 7. Xlcaax-1 staining in the distal tubule of the kidney. (A) Section of adult kidney stained with L13 pAb, box indicates region shown in C. (B) Control section of adult kidney. (C) High magn. of boxed area in A, arrows indicate the basolateral membrane, box indicates region shown in Fig. 8. d, distal tubule; p, proximal tubule; g, glomerulus; n, nucleus; lu, lumen. Magn. of A and B is 160~. Magn. of C is 1000X.
	FIG. 8. Electron microscopy of distal tubule. (A) Preimmune, Protein A purified IgC control. (B) L13 pAb specific binding over the basal infoldings of the plasma membranes of distal tubules. (C) High magnification showing localization to the cytoplasmic aspect of the basal plasma membrane infoldings. Magn. of A and B is 14,909~. Magn. of C is 49,799x.
	FIG. 9. Xlcaax-1 localization in adult tissues. (A) Section of skin stained with L13 pAb, arrows show the region of the plasma membranes, Kl, keratinized layer; bl, basal layer. (B) Adjacent, control section of skin. (C) Section of urinary bladder, arrows indicate basolateral xlcaax-1 staining, lu, lumen. (D) Adjacent, control section of urinary bladder. (E) Section of stomach parietal glands stained with L13 pAb, arrows indicate the region of the lateral membranes. (F) Adjacent, control section of stomach parietal glands. Magn. of A and B is 1000X. Magn. of C and D is 1000X. Magn. of E and F is 500X.