PMC

At E10.5 Lrp4 is expressed throughout the Wolffian duct and the ureteric bud. (a). At E11.5, Lrp4 is expressed in the ureteric bud and the pre-tubular aggregates (b). At E12.5 and E 14.5, Lrp4 expression is maintained in the ureteric bud and the renal vesicles (c and d, respectively). The Wolffian duct and ureteric bud are outlined by dotted lines; the arrow points to the early ureteric bud in (a) or the renal vesicles in (c), respectively.

Kidney agenesis in the Lrp4 knockout (b,d,e). Bilateral (b,d) or unilateral (e) kidney agenesis with rudimentary ureters (red arrows). The lower urinary and genital systems of males and females remain intact. Histological analysis (Hematoxylin-Eosin stain) does not reveal morphological defects in the kidneys that form in Lrp4 knockout animals (g) compared to the wild-type kidneys (f).

CT scan reveals a severely hypoplastic kidney on the right and mild hypoplasia on the left side (a–d). Both kidneys are ectopic with caudal and lateral shifts (a–d). Dynamic-static renal scintigraphy with Tc-99m DTPA suggest right kidney dysfunction (e). Global renal functional participation; right kidney 26% and left kidney 74%. Static renal cortical scintigraphy with Tc-99m DMSA background activity of radiopharmaceutical is higher than expected (f).

Lrp4 has been implicated in modulating the Bmp signaling pathway through binding of the Wnt and Bmp modulator Wise. Co-immunoprecipitation reveals Gremlin1 binding to Lrp4 in vitro (Panel A lane 4); we further confirmed the Lrp4 binding partners Wise, Dkk1 and SOST (Panel A, lanes 6, 10 and 12). The Wnt agonist R-spondin 2 did not interact with Lrp4 (Panel A lane 7 and 8). Transfection efficiency was confirmed by immunoblot analysis (Panel B).

Expression of a stabilized allele of β-catenin (Catnb^exon3flox) in the Wollfian duct using HoxB7Cre to activate transgene expression phenocopies the Lrp4 knockout phenotype with both uni- and bilateral kidney agenesis (a-c). The formation of the Wolffian duct and distal ureters as well as bladder and adrenal glands remained unaffected. The asterisks (a and b) indicate the position of regular kidneys. The arrows (b and c) indicate the predicted position of kidneys that have not formed.

Pax2 is expressed normally in the metanephric mesenchyme and the ureteric bud, indicated by the red arrows, at E10.5 in the wild type and Lrp4 knockout mice (a and b). At E11.5, Pax2 is expressed normally in both the ureteric bud and metanephric mesenchyme, the latter indicated by black arrows, of wild type (c) and Lrp4 knockout animals (d). However, the ureteric bud fails to invade the metanephric mesenchyme and does not undergo secondary branching in the Lrp4 knockout indicated by the yellow arrow (d). The black arrows indicate mesenchymal expression of Pax2, which is present in the wild-type, but subsequently lost in the knock-out kidney mesenchyme at E12.5 (e and f).

38 somite stage E10.5 embryos were stained with the epithelial markers Pax2 (red) and E-cadherin (green) to label the Wolffian duct and developing ureteric bud. Representative images of kidney pairs for two wild-type and knock-out animals are shown. In the wild-type (a–d), ureteric buds appear as expected while there is a frequent delay in ureteric bud outgrowth in the Lrp4 mutants (e,g,h). One Lrp4 mutant animal is shown with a unilateral outgrowth (f). In total, all 10 expected buds are formed at the 38 somite stage in the wild-type background while only 1 out of 8 predicted buds is present in the knock-out (Panel b). P values (Student's t-test) p<0.01 indicates significance.

Expression of c-Ret (a–f), GDNF (g–l) and Wnt11 (m–r) in E10.5 (a,b,g,h,m and n), E11.5 (c,d,i,j,o and p), and E12.5 (e,f,k,l,q,r) in wild-type (a,c,e,g,i,k,m,o, and q) and Lrp4 knockout (b,d,f,h,j,l,n,p and r) kidneys. C-Ret is expressed in the ureteric bud at basal levels in the Lrp4 knockout mice at E10.5 (a,b). At E11.5, the Lrp4 knockout ureteric bud fails to bifurcate or upregulate c-Ret expression at the tip of the ureteric bud (d) compared to wild-type embryos (c). At E12.5, the signal is greatly reduced in the knockout kidney (f). GDNF is expressed normally in the metanephric mesenchyme at both E10.5 and 11.5 in wild type and Lrp4 knockout animals (g–j). By E12.5, GDNF expression is completely lost from the Lrp4 knockout metanephric mesenchyme (k and l). Wnt11 is expressed normally at the tips of the ureteric bud at both E10.5 (m and n) and 11.5 (o and p) in Lrp4 mutants compared to wildtype. By E12.5 Wnt11 is absent from the ureteric bud of Lrp4 knockout animals (q and r). The Wolffian duct and ureteric bud are outlined by white dashed lines, mesenchyme (g, h, I, j) renal vesicles (e and q) and truncated ureteric bud (f, l, r) are indicated by black arrows.