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First published online 1 September 2004
doi: 10.1242/dev.01341

Development 131, 4787-4795 (2004)
Published by The Company of Biologists 2004
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Mouse amnionless, which is required for primitive streak assembly, mediates cell-surface localization and endocytic function of cubilin on visceral endoderm and kidney proximal tubules

Sharon Strope1,2, Roberta Rivi2, Thomas Metzger2, Katia Manova2 and Elizabeth Lacy1,2,*

1 Molecular Biology Graduate Program, Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021, USA
2 Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue, New York, NY 10021, USA



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  		Fig. 1. Organization of the pre-implantation and gastrulation-stage mouse embryo. (Left) An E3.5 blastocyst. The inner cell mass (ICM) gives rise to the epiblast lineages; the primitive endoderm (PrE) gives rise to parietal and visceral endoderms; the trophectoderm (TE) to the extra-embryonic ectoderm. (Right) An E6.5 gastrulation stage embryo with the organization of embryonic/extra-embryonic tissue layers in detail.

 


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  		Fig. 2. Amn is expressed in kidney proximal tubule (KPT) and in the epithelium of the small intestine. (A) Hybridization of the antisense Amn cDNA probe to a sagittal paraffin section of an E16.5 wild-type mouse embryo. The plane of section is shown in the schematic diagram on the left. Amn expression was detected in the intestinal epithelium and kidney. (B) In situ hybridization to adult kidney. Amn expression is restricted to the cortex and outer stripe of the outer medulla of the kidney. (C) The Amn protein is localized to the apical surface of proximal tubule (PT) cells. (D) Bright-field and (E) corresponding dark-field image of the adult small intestine. Amn expression is associated with intestinal villi. (F) The Amn protein is localized to the apical surface of the small intestine. (Inset) Staining with the preimmune antisera.

 


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  		Fig. 3. Amn is not required for cell proliferation or differentiation in fetal kidney and intestine. Cryosections from E16.5 Amn-/-{leftrightarrow}+/+ chimeras. (A,C) Two kidney sections and (B,D) two intestine sections stained with X-gal to identify wild-type (blue cells indicated by blue arrows) and Amn-/- cells (pink cells indicated by red arrows). (A) Amn-/- cells colonize the kidney and contribute to glomeruli (G) and tubules. (B) Amn-/- cells colonize the intestinal villi (V). (C) Section of a chimeric kidney containing ~100% Amn-/- cells with normal gross morphology, including properly formed glomeruli (G) and tubules. (D) A section of chimeric intestine containing ~100% Amn-/- cells with normal morphology and well-formed villi (V). (E) Chimeric kidney section stained with LTL. (Inset) Kidney cryosection from an E16.5 Amn+/-{leftrightarrow}+/+ chimera stained with LTL. (F) X-gal staining of section adjacent to (E) identifies Amn-/- cells in properly differentiated proximal tubules (PT). (Inset) X-gal staining of kidney cryosection from an E16.5 Amn+/-{leftrightarrow}+/+ chimera.

 


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  		Fig. 4. Cubn is not properly localized to the apical cell surface of the VE in amn. Immunohistochemical analyses of serial paraffin sections of an E7.5 wild-type embryo (A-C) and an E7.5 amn mutant (D-F), which were counterstained with Hematoxylin. The regions designated by brackets in A-F are shown at high magnification in a-f. (A,a) Megalin, (B,b) Amn and (C,c) Cubn co-localize to the apical cell surface (red asterisks) of the VE in the wild-type embryos. The basal cell surface faces the epiblast (red arrowheads). (D,d) Megalin staining of the apical cell surface of the VE in amn, identified by the absence of Amn antibody staining in the adjacent section (E,e). (F,f) Cubn appears cytoplasmic and not apical in amn. (a and d, inset) Lysozyme staining in wild-type (a) and amn mutant (d) embryos.

 


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  		Fig. 5. Cubn is not localized to the apical cell surface in Amn-deficient KPT. Immunohistochemical analyses of serial paraffin sections (A,D,G) from an adult chimeric kidney with high contribution of Amn-/- cells. Two sets of serial high magnification views are shown in B,E,H and C,F,I. (A-C) Cubn, (D-F) Amn and (G-I) megalin antibody staining of wild-type KPT (blue asterisks; WT) and Amn-deficient KPT (red asterisks; mut). A chimeric tubule (green asterisks; ch), which contains both wild-type and Amn-/- cells, is designated as well as a distal tubule (yellow asterisks; DT). Of significance, Cubn does not localize to the apical surface in Amn-deficient KPT but is observed in the cytoplasm (B,C).

 


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  		Fig. 6. Adult Amn-/-{leftrightarrow}+/+ chimeras excrete increased levels of Cubn-specific ligands in the urine. Western blotting analyses of urine samples normalized for creatinine from adult chimeric females (A) and males (B). Loading volumes were equivalent to 163 ng creatinine (albumin) or 2.45 µg creatinine (transferrin and lysozyme). Estimated percent chimerism is indicated by the triangle above the figures and ranged from ~0% to 70%. C57/BL6 (B6) and 129 SvJ (129) mice were used as controls. While albumin is a ligand for both megalin and Cubn, transferrin is a Cubn-specific ligand and lysozyme is a megalin-specific ligand. (C) Albumin excretion by female chimeras and controls was quantitated by an ELISA assay and normalized by creatinine levels. Adult Amn-/-{leftrightarrow}+/+ chimeras excrete elevated levels of albumin in the urine when compared with controls. (D) Transferrin excretion, which was quantitated by an ELISA assay and normalized by creatinine levels, was elevated in adult female Amn-/-{leftrightarrow}+/+ chimeras when compared with female controls.

 

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© The Company of Biologists Ltd 2004