Lrp4 Regulates Initiation of Ureteric Budding and Is Crucial for Kidney Formation – A Mouse Model for Cenani-Lenz Syndrome

Background: Development of the kidney is initiated when the ureteric bud (UB) branches from the Wolffian duct and invades the overlying metanephric mesenchyme (MM) triggering the mesenchymal/epithelial interactions that are the basis of organ formation. Multiple signaling pathways must be integrated to ensure proper timing and location of the ureteric bud formation. Methods and Principal Findings: We have used gene targeting to create an Lrp4 null mouse line. The mutation results in early embryonic lethality with a subpenetrant phenotype of kidney agenesis. Ureteric budding is delayed with a failure to stimulate the metanephric mesenchyme in a timely manner, resulting in failure of cellular differentiation and resulting absence of kidney formation in the mouse as well as comparable malformations in humans with Cenani-Lenz syndrome. Conclusion: Lrp4 is a multi-functional receptor implicated in the regulation of several molecular pathways, including Wnt and Bmp signaling. Lrp4 2/2 mice show a delay in ureteric bud formation that results in unilateral or bilateral kidney agenesis. These data indicate that Lrp4 is a critical regulator of UB branching and lack of Lrp4 results in congenital kidne

Unilateral and bilateral kidney agenesis in LRP4 knockout mice.

<p>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).</p

Wnt Overexpression in the Ureteric Bud Leads to Kidney Agenesis.

<p>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.</p

Lrp4 binds the Bmp4 antagonist Gremlin1 <i>in vitro</i>.

<p>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 <i>in vitro</i> (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).</p

Expression of Lrp4 in the developing kidney.

<p>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.</p

Ureteric Budding is delayed in Lrp4 Mutants.

<p>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.</p

Pax2 signaling remains intact in the absence of Lrp4.

<p>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).</p

Expression of branching regulators in Lrp4 mutants.

<p>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.</p