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First published online 25 April 2006
doi: 10.1242/jcs.02954

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Elucidation of megalin/LRP2-dependent endocytic transport processes in the larval zebrafish pronephros

¹ Max Delbrueck Center (MDC) for Molecular Medicine, Robert-Roessle Str. 10, 13125 Berlin, Germany
² Department of Cardiology, The Charité - University Medical School of Berlin, Campus Buch and Campus Virchow Clinics, Berlin, Germany

View larger version (40K): [in a new window]   Fig. 1. Sequence comparison of zebrafish megalin/LRP2 and cubilin with human orthologs. (A) Aligned protein structures of human megalin/LRP2 and known parts of the zebrafish ortholog that include the transmembrane domain (TM, black vertical line) and the intracellular tail (horizontally striped pattern). In contrast to the extracellular domain, which is similar in sequence in all members of the LDL receptor gene family, the cytoplasmic domain of megalin/LRP2 is unique and highly conserved across species, as shown here for comparative analysis of the respective amino acid sequence in human and zebrafish. Sequence comparison of both orthologs of the TM and intracellular tail domains shows 49.6% sequence identity and 65.7% sequence similarity. (B) Sequence alignment between two regions of the intracellular tail, including one of the NPxY motif sites (black box) of human and zebrafish megalin/LRP2. (C) Aligned protein structures of cubilin from human and zebrafish. Cubilin domains are shown as gray boxes, EGF domains are black lines, and calcium-binding EGF-like domains are dark gray lines. Both orthologs display 49.6% sequence identity and 65.7% sequence similarity over the known portion of zebrafish cubilin. (D) Sequence comparison of a single cubilin domain from human and zebrafish orthologs.

View larger version (120K): [in a new window]   Fig. 2. Expression of megalin, cubilin, dab2, wt1 and pax2.1 in the developing pronephros. Dorsal view of wild-type embryos at 20 hpf (A-E), 24 hpf (F-K), 48 hpf (L-P), and 72 hpf (Q-U) analyzed for expression of megalin/lrp2, cubilin, dab2, wt1 and pax2.1. Combinatorial gene expression patterns are indicated in the model. Arrows indicate the tubular expression of megalin/lrp2 (Q), arrowheads point at the pronephros expression domain of pax2.1 (P,U). gl, glomerulus; np, nephric primordium; pd, pronephric duct; pt, pronephric tubule. Bar, 100 µm.

View larger version (58K): [in a new window]   Fig. 3. Co-expression of megalin/lrp2 with dab2 or cubilin within the developing pronephros. Dorsal view of wild-type embryos double-labeled by two-color in situ hybridization at 72 hpf. Single staining for megalin/lrp2 is red and for dab2 and cubilin is blue, overlapping expression results in a brownish color precipitate. Arrows indicate the non-overlapping megalin/lrp2 gene expression within the otic vesicle. Within the pronephric duct, expression of all three genes overlaps. Bar, 200 µm.

View larger version (54K): [in a new window]   Fig. 4. Renal clearance of tracers within the proximal pronephric duct is restricted to the megalin/LRP2 expression domain. Wild-type embryos (72 hpf) were injected with 70kDa-FD (green) and renal clearance of tracer into the pronephric duct tubular cells evaluated 1.5 hours later on whole mounts (A,C) or transversal sections (D,F) by confocal fluorescence microscopy. (B,C) Immunostaining for megalin/LRP2 (red) demonstrates that renal clearance of 70kDa-FD occurs within the expression domain of the receptor. (E,F) Transversal sections show apical localization of megalin/LRP2. Bars, 100 µm (A-C) and 5 µm (D-F).

View larger version (105K): [in a new window]   Fig. 5. Renal clearance of tracers occurs within the proximal pronephric duct. Wild-type embryos (72 hpf) were injected with 10kDa-RD (red), 70kDa-FD (green), Cy2-RAP (green) or 500kDa-FD (green), and renal clearance of tracer into the pronephric-duct tubular cells was evaluated 1.5 hours later on (A) whole mounts or (B-G) transversal sections by confocal fluorescence microscopy. There is no tubular uptake of the 500kDa-FD tracer (D). Partial colocalization of 70kDa-FD and Rab4 (red) can be seen in endosomes of tubular cells of the pronephric duct that were immunostained for the early endosomal marker Rab4 (E-G). White dotted lines demarcate the position of the pronephric ducts in (B-D) or the outline of the pronephric duct lumen (E-G). Bars, 5 µm.

View larger version (57K): [in a new window]   Fig. 6. Molecular characterization of the megalin/lrp2 knock-down. Wild-type embryos were injected with buffer (WT) or with two different megalin/lrp2 splice-variant MOs (megMO1 or megMO2). Embryo extracts were generated at 48 hpf and analyzed by (A) RT-PCR or (B) western blotting of membrane fractions. megMO1 and megMO2 resulted in aberrant splicing of the megalin/lrp2 transcript as evidenced by truncated PCR products of the (A) cDNA encoding the transmembrane and intracellular portions of the receptor and (B) the absence of megalin/LRP2 immunoreactivity compared to controls. (C) Morphology of wild-type, megalin/lrp2 and dab2 morphants at 72 hpf. No gross abnormalities of the different morphants were apparent.

View larger version (71K): [in a new window]   Fig. 7. Tubular-clearance defects in megalin/lrp2 morphants and quantification thereof. (A,E,I) Wild-type embryos, (B,C,F,G,K,L) megalin/lrp2 morphants, (D,H,M) dab2 morphants at 72 hpf were injected with 70kDa-FD (green). Renal clearance of tracer into the pronephric duct tubular cells was evaluated after fixation and immunostaining against megalin/LRP2 (red) of whole mounts by confocal fluorescence microscopy. Renal clearance of tracer occurs in (A,I) wild-type but not in (B,C,K,L) megalin/LRP2- and (D,M) Dab2-deficient embryos. Expression of megalin/LRP2 is unaffected in dab2 morphants (H). (N) Wild-type, megalin/lrp2 morphant, dab2 morphant and dab2 rescued embryos at 72 hpf were injected with the indicated fluorescent tracers and the number of embryos exhibiting tubular accumulation of tracers evaluated by fluorescence microscopy. Data of all animals injected are given in percent. 70kDa-FD: wild type, 45/55 embryos (81.8%); megMO1, 2/24 embryos (8.3%); megMO2, 3/29 embryos (10.3%), dab2MO, 3/29 embryos (10.3%); dab2 rescue, 26/48 embryos (54.2%). 10kDa-RD: wild type, 46/52 embryos (88.5%); megMO1, 4/27 embryos (14.8%); megMO2, 5/29 embryos (17.2%); dab2MO, 5/39 embryos (12.8%). Cy2-RAP: wild type 42/46 embryos (91.3%); megMO1, 3/25 embryos (12.0%); megMO2, 3/22 embryos (13.6%); dab2MO, 3/31 embryos (9.7%). Bar, 100 µm.

View larger version (91K): [in a new window]   Fig. 8. Lack of Cy2-RAP uptake in megalin/lrp2 morphants and of 70kDa-FD in dab2 morphants. Confocal fluorescence microscopy on (A) whole mounts and (B) transversal sections through the pronephic ducts of 72 hpf wild-type and megalin/lrp2 morphants injected with (A) Cy2-RAP or (B) dab2 morphants injected with 70kDa-FD. megalin/lrp2 morphants fail to clear Cy2-RAP from the pronephric duct (A). Loss of renal uptake of 70kDa-FD despite correctly localized megalin/LRP2 at the apical membrane of dab2 morphants (B). Bars, 100 µm (A) and 5 µm (B).

View larger version (99K): [in a new window]   Fig. 9. Loss of megalin/LRP2 and Dab2 abolishes Rab4-positive endosomes within the pronephric duct. Confocal fluorescence microscopy on transversal sections through the pronephic ducts of 72 hpf wild-type (A,E,I), megalin/lrp2 morphants (B,C,F,G,K,L), or dab2 morphants (D,H,M) injected with 70kDa-FD (A-D,I-M) and immunostained for the early endosomal marker Rab4 (E-M). megalin/lrp2 morphants (K,L) and dab2 morphants (M) are devoid of tubular Rab4-positive endosomes and lack uptake of 70kDa-FD compared with controls (I). Bar, 20 µm.

View larger version (85K): [in a new window]   Fig. 10. Lack of renal clearance and Rab4-positive early endosomes in hasm567 mutants. Transverse sections of 72 hpf wild-type embryos (A-C,K-L), noks305 mutants (D-F,N-P), and hasm567 mutants (G-I,Q-S) injected with 70kDa-FD (green) and immunostained for the early endosomal marker Rab4 (B,C,E,F,H,I) or for megalin/LRP2 (L,M,O,P,R,S). Whereas, noks305 mutants show a robust presence of Rab4-positive endocytic vesicles filled with the tracer (D-F), hasm567 mutants completely lack uptake of the tracers and Rab4-positive vesicles (G-I). The apical localization of the megalin/LRP2 receptor is not affected in noks305 and hasm567 mutants (O,P,R,S). Bars, 5 µm.

View larger version (45K): [in a new window]   Fig. 11. Mosaic clonal analysis of has/prkci function in tubular endocytic processes. Confocal fluorescence microscopy on whole mounts of wild-type host embryos (non-GFP) that contain pronephric duct clones of has morphant tubular cells [genetically marked with Tg(cldnB:GFP)] that were injected with 10kDa-RD. (C) Endocytic punctate vesicles filled with the tracer are present within has/prkci morphant cell clones (arrowhead) and wild-type host neighboring cells (arrow). Bar, 40 µm.