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First published online 1 November 2005
doi: 10.1242/jcs.02650

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Endocytosis of megalin by visceral endoderm cells requires the Dab2 adaptor protein

Division of Basic Sciences and Molecular and Cellular Biology Program, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, Seattle, WA, 98109, USA

View larger version (58K): [in a new window]   Fig. 1. Endocytosis is decreased in dab2-/- mutants. (A,B) EM analysis of the VE of (A) control (+/) and (B) dab2-/- (–/–) embryos at E6.5. Electron-dense vesicles are indicated by red arrows. (C) HRP uptake in control and dab2-/- embryos at E7.5. Mutant embryos showed reduced uptake of HRP compared with control embryos. (D-I) TR-Tf uptake in E6.5 and E7.5 embryos. (D) In E7.5 embryos, Tf uptake is reduced in dab2-/- embryos compared with control littermates. (E,G) Dark-field images of E6.5 wild-type (E) and mutant (G) embryos. (F,H) Fluorescent images of the same embryos show decreased uptake in the VE of the mutant embryo. (F',H') Higher magnification images of regions indicated in F, H show a reduction in endosomal labeling in the dab2-/- VE. (I) TR-Tf uptake is significantly decreased in dab2 mutant embryos (P<0.001). Fluorescent images were taken at the same exposure. The average pixel intensity was determined within a central region of each embryo and plotted as a scatter plot (closed circles). The mean intensity (open circle) and standard error (bars) were calculated for each genotype, and statistical significance was determined using the Mann-Whitney test. Bar, 2 µm. PE, parietal endoderm; BB, brush border; VE, visceral endoderm.

View larger version (72K): [in a new window]   Fig. 2. Receptor trafficking-defects in dab2 mutant VE. Immunohistochemistry and immunofluorescence on E6.5 embryos. (A-D) Dab2 immunostaining. Dab2 protein was detected in the VE of control embryos (A), at the apical surface as well as associated with both EEA1-positive and negative vesicles (A',B). Absence of Dab2 staining was used to identify dab2-/- embryos (C-D). EEA1 positive vesicles were reduced in mutant embryos (D). (E-H) Megalin immunostaining. Megalin was detected at the apical surface and associated with EEA1-positive vesicles in control embryos (E-F). In dab2-/- VE (G-H), megalin protein was restricted to the apical surface and little intracellular protein or colocalization with EEA1 was detectable (G-H). (I-L) Cubilin immunostaining. Cubilin was detected in the apical brush-border and in vesicles within the apical and basolateral cytoplasm of control embryos (I-J). In dab2-/- embryos, cubilin was retained at the apical membrane and was not detected in vesicles (K-L). Bars, 40 µm (A); 10 µm (A',B). A, apical; BL, basolateral; (+/), dab2+/+ or dab2+/–.

View larger version (53K): [in a new window]   Fig. 3. ARH is absent from the VE. (A-C) Whole-mount immunofluorescence for Dab2 and ARH of E6.5 wild-type embryos. (A) Dab2 protein was detected by indirect fluorescence in the VE surrounding the entire embryo. (B) ARH was absent in the VE, but was detected in the PE. (C) As shown in the merged image, Dab2 and ARH are expressed in distinct tissues of the E6.5 embryo. The magnified region shows the low extent of colocalization (as indicated by yellow pixels) between Dab2 and ARH.

View larger version (38K): [in a new window]   Fig. 4. Differential expression of dab2 splice forms. (A) Schematics of the two alternatively spliced forms of dab2, p96 and p67. (B) Western blot analysis using a Dab2-specific antibody revealed differential expression of the two Dab2 protein forms in E6.5 embryos, embryoid bodies (EBs), kidney lysates and fibroblasts (MEF and NIH3T3). (C) Isoform-specific RT-PCR was performed to compare expression levels of p67 and p96 in embryos and cells. A common 5' PCR primer was used for both isoforms (a), together with a 3' primer that bridged the p67-specific splice junction (b) or that recognized the p96-specific exon (c). PCR was performed on serial threefold dilutions of cDNAs generated by RT-PCR from embryos and NIH3T3 cells. p67 mRNA levels were higher in an E7.5 embryo, whereas p96 mRNA levels were higher in NIH3T3 cells. PCR for gapdh was used as a control. (D) Immunofluorescence was used to visualize Dab2 protein in the VE of wild-type embryos at E6.5. Dab2 protein was both diffuse as well as vesicle-associated, at both the apical (A) and to as lesser extent the basolateral (BL) surface.

View larger version (43K): [in a new window]   Fig. 5. Generation of dab2 isoform-specific knock-in mice. (A) The `plug-and-socket' targeting strategy was used to insert a cDNA encoding either the p67 or p96 isoform of Dab2 into the dab2 genomic locus (1). The first targeting construct (2) contained a loxP-flanked hygromycin selection cassette and two-thirds of the 3' end of a neomycin selection cassette. Homologous recombination was used to insert these sequences into the dab2 locus of AK7 embryonic stem (ES) cells. Targeted clones (3) were selected with hygromycin, identified by PCR and verified by Southern blotting. `Socket' ES cells were subsequently re-targeted to insert the p67 or p96 cDNA into the dab2 locus, reading continuously from coding exon 2. The second targeting construct (4) contained two-thirds of the 5' end of a neomycin selection cassette as the 3'-end recombination arm, and the dab2 genomic sequence as the 5'-end arm. Homologous recombination produced a complete neomycin selection cassette, enabling the use of neomycin resistance as a screen for properly recombined clones (5). These clones were verified by Southern blotting. Following excision of drug-resistance-markers by Cre-mediated recombination (6), ES cells were injected into C57BL/6 donor blastocysts. N, NcoI; B, BamHI; S, SmaI; R, EcoRI; A, ApaI. (B) Protein analysis of tail lysates from P10 offspring of heterozygous intercrosses confirmed expression of the correct targeted allele. Only the p96 isoform of Dab2 was detected in lysates from p96/p96 mice (lane 4), and only the p67 isoform was detected in lysates from p67/p67 mice (lane 9). (C-F) Subcellular localization of Dab2 protein was determined in kidney proximal tubule cells from wild-type (C), dab2-/- (D), p96/p96 (E), and p67/p67 (F) mice. Basal regions were cropped to delete nonspecific trapping of antibody. A; apical surface.

View larger version (14K): [in a new window]   Fig. 6. The p96 allele supports embryonic development. Offspring of p96/+ (A) or p67/+ (B) intercrosses were genotyped during development or at postnatal day 1 and/or 10. (A) p96 homozygotes were present at the expected rate at both stages during development. P10 pups were genotyped from females that were not simultaneously nursing a previous litter. From these females, p96/p96 pups were present at the expected mendelian ratio. (B) p67 homozygous embryos were present at expected mendelian rates at both E6.5 and E10.5, but by P1 and P10 there was a significant decline in the number of pups recovered (P=0.06 and P<0.001, respectively). Dashed line, expected recovery of homozygotes (25%); n value for each genotype is given within each bar.

View larger version (72K): [in a new window]   Fig. 7. Endocytosis in the VE of E6.5 embryos is rescued completely by p96 but only partially by p67. TR-Tf uptake was assayed on embryos from heterozygous p96/+ or p67/+ intercrosses. (A-D') Bright-field images show that (A) control and (C) p96/p96 embryos are phenotypically similar. Fluorescent microscopy revealed similar uptake levels in both embryos at low magnification (B,D) and similar endosomal labeling in both (B') control and (D') p96 homozygous embryos. (E) Assays were scored as previously described. p67/p67 homozygotes showed a significant decrease in endocytosis of TR-Tf when compared with control embryos (P<0.005).

View larger version (62K): [in a new window]   Fig. 8. p67/p67 embryos are developmentally delayed and show defective receptor trafficking in the visceral yolk sac (A) Images of control and p67/p67 embryos at E10.5 and E14.5 show that p67 homozygotes are consistently smaller and developmentally delayed compared with littermates. (B,C) Dab2 localization in the VYS of E12.5 dab2+/+ (B) and p67/p67 (C) embryos. Dab2 protein is more diffuse when only the p67 isoform is present. (D,E) Cubilin localization in the VYS of these same embryos. In the wild-type embryo (D), cubilin staining was strongest in the apical brush-border, but was also visible inside the cell in vesicles. In p67/p67 embryos (E), cubilin was concentrated on the apical surface, and only a few small vesicles inside the cell contained cubilin. (F) Model for Dab2 function in the transport of nutrients and morphogens in the VE and VYS. YSC, yolk sac cavity.