LTP is impaired in <i>Lrp4</i>^{<i>ECD/ECD</i>} but not in <i>Lrp4</i>^{<i>ΔICD/ΔICD</i>} mice.

<p><b>A</b>: <i>Upper Panel</i>, Sample traces before and 40 min after theta-burst stimulation (TBS); <i>Lower Panel</i>, Results of experiments from <i>Lrp4</i>^{<i>ECD/ECD</i>} mice compared to their <i>Lrp4</i>^{<i>WT-KI/WT-KI</i>} controls. TBS induced on average a 37.63 ± 7.88% increase in <i>Lrp4</i>^{<i>WT-KI/WT-KI</i>} control slices (open squares, n = 16, N = 5), but only 14.83 ± 3.39% LTP in slices from the <i>Lrp4</i>^{<i>ECD/ECD</i>} mice (black triangles, n = 10, N = 3). <b>B</b>: Unpaired t-test was used to compare each sample for LTP calculated 40–60 min after theta-burst. Values are the means of the normalized fEPSP slopes. * denotes significance, p = 0.0387. <b>C:</b><i>Upper Panel</i>, Sample traces before and 40 min after TBS. <i>Lower Panel</i>, Results of experiments from <i>Lrp4</i>^{<i>ΔICD/ΔICD</i>} mice compared to their <i>Lrp4</i>^{<i>WT-KI/WT-KI</i>} controls. <i>Lrp4</i>^{<i>WT-KI/WT-KI</i>} slices were recorded on consecutive days and used as internal controls and pooled together. TBS induced a 37.63 ± 7.88% LTP in <i>Lrp4</i>^{<i>WT-KI/WT-KI</i>} control slices (open squares, n = 16, N = 5), and 32.42 ± 6.27% LTP in slices from the <i>Lrp4</i>^{<i>ΔICD/ΔICD</i>} mice (gray filled rhombus, n = 11, N = 5). <b>D</b>: There was no significant difference in LTP between <i>Lrp4</i>^{<i>WT-KI/WT-KI</i>}<i>and Lrp4</i>^{<i>ΔICD/ΔICD</i>} mice (p = 0.63). <b>E.</b> Input-output curves calculated as a function of fiber volley amplitude to the slopes of fEPSP’s. Average peak amplitudes for <i>Lrp4</i>^{<i>WT-KI/WT-KI</i>}, <i>Lrp4</i>^{<i>ECD/ECD</i>} and <i>Lrp4</i>^{<i>ΔICD/ΔICD</i>} slices used in the experiments were 1.68 ± 0.15 mV, 1.27 ± 0.25 mV, and 1.50 ± 0.20 mV), respectively, and were not significantly different from each other. (One-way ANOVA, F = 1.124, p = 0.33.) <b>F:</b> Theta-burst analysis or <b>G:</b> paired pulse ratios (n = 8, N = 3 for each) did not reveal any significant differences between <i>Lrp4</i>^{<i>WT-KI/WT-KI</i>} and <i>Lrp4</i>^{<i>ECD/ECD</i>} (two-way ANOVA, F(3,56) = 0.46, p = 0.71). N = number of animals.</p

Limb and bone structure of different <i>Lrp4</i> KI mutants.

<p><b>A</b>: Illustration of the different Lrp4 protein products of all KI mutants. Panels are aligned to paw images in B and C to indicate genotypes. <b>B</b>: Ventral view of fore and hind limbs of <i>Lrp4</i> KI mutants. Homozygous mutant mice for each allelic variant (<i>KI/KI</i>) and compound mutant mice that carry one allelic variant and one KO allele (<i>KO/KI</i>) are shown. Note that there are strong defects in the limb pattering of <i>Lrp4</i>^{<i>ECD/ECD</i>}, intermediate defects in <i>Lrp4</i>^{<i>ΔICD/ΔIC</i>}, and only mild defects in <i>Lrp4</i>^{<i>LDLR-ICD/LDLR-ICD</i>} (red arrows). <b>C</b>: Ventral view of alizarin red (stains bones) and alcian blue (stains cartilage) of different <i>Lrp4</i> KI mutants. A WT-KI allele (2^nd panel in A and B) was generated to control for the lack of introns in the ICD-cassette in the other KI mutants. Black arrowheads: ectopic bone or bony fusion; red arrowheads: soft-tissue fusion. (modified from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116701#pone.0116701.ref022" target="_blank">22</a>]).</p

Normal brain development in <i>Lrp4</i>^{<i>ECD/ECD</i>} and <i>Lrp4</i>^<i>-/-</i> mice.

<p><b>A-D</b>: Sagittal slices of the <i>Lrp4</i>^{<i>ECD/ECD</i>} (A,C) and wild type (<i>Lrp4</i>^<i>+/+</i> B,D) mouse cerebellum labeled with NeuN (green), Brn1 (red) and DAPI (blue). Brn1 and NeuN are commonly used markers to label neurons. ML = molecular layer, PL = Purkinje cell layer, and GCL = granule cell layer are clearly distinguishable and not different in the cerebellum of <i>Lrp4</i>^{<i>ECD/ECD</i>} and <i>Lrp4</i>^<i>+/+</i> adult mice (>2 months). <b>E-H</b>: Coronal sections of <i>Lrp4</i>^{<i>ECD/ECD</i>} (E,F) and <i>Lrp4</i>^<i>+/+</i> (G,H) brains showing hippocampus (E,G) and somatosensory cortex (F,H). Slices are labeled for NeuN and DAPI to visualize normal cortical lamination (layers I-VI). I-N: Coronal sections of E18.5 <i>Lrp4</i>^<i>-/-</i> brains compared to their wild type litter mates. Brn1 (I,J) and GFAP (K,L) immunoreactivity in the cortex and hippocampus and Tbr1 plus NeuN double labeling in the cortex are illustrated. Scale bars = 200 μm (A-H), 400 μm (I-L), 100 μm (M,N).</p

Hypoplastic and Hypofunctional Kidney in Human Lrp4 Mutations.

<p>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).</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

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

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