Phenotyping of <i>Pkd1l1</i>^{<i>tm1/tm1</i>} Mutants.

<p>(A) Schematic diagram of PKD1L1 and PKD2 showing protein domains and the nature of the <i>Pkd1l1</i>^<i>rks</i> and <i>Pkd2</i>^<i>lrm4</i> point mutations. The double headed red arrow denotes the site of interaction between PKD1L1 and PKD2. PKD—Polycystic Kidney Disease; REJ—Receptor for Egg Jelly; GPS—G-protein Coupled Receptor Proteolytic Site; PLAT—Polycystin-1, Liopoxygenase, Alpha-Toxin. (B) <i>Pkd1l1</i>^{<i>tm1/tm1</i>} and sibling control showing reversed and normal situs, respectively. White arrows indicate stomach position. (C) Heart-stomach discordance (H-S Disc.) in <i>Pkd1l1</i>^{<i>tm1/tm1</i>}, <i>Dnah11</i>^<i>iv/iv</i> and <i>Pkd1l1</i>^{<i>rks/rks</i>} mutants scored at E13.5. Normally, the heart apex and stomach are positioned to the left. H-S Disc. is defined as the heart apex and stomach being on opposite sides. ns—not significant; *—p<0.05; **—p<0.001, Fisher’s Exact Test applied. (D-F) Lung situs assessed at E13.5 for embryos of the indicated genotypes with the ratio of lung lobes between left and right sides given. The percentage and total numbers of embryos showing each phenotype are indicated in <i>(F)</i>. (G-P) Expression patterns of <i>Nodal</i>, <i>Pitx2</i>, and <i>Lefty1/2</i> in embryos at E8.5 of the indicated genotypes, with the percentage number of embryos exhibiting each phenotype and the total number given. Embryos exhibiting bilateral marker expression are further categorized by whether they show equal or biased expression between the left and right sides. The inset in <i>(M)</i> shows a <i>Pkd1l1</i>^{<i>tm1/tm1</i>} embryo with bilateral <i>Pitx2</i> expression but with a right-sided bias. Arrowheads in <i>(N)</i> and <i>(O)</i> indicate midline <i>Lefty1</i> expression. <i>t</i> is shorthand for <i>Pkd1l1</i>^<i>tm1</i>. (Q-R) Sonic hedgehog (<i>Shh</i>) expression in the node (n) and notochord (nc) at E8.5.</p

The Genetic Relationship between <i>Pkd1l1</i>, <i>Pkd2</i>, and Cilia.

<p>(A-F) <i>Cerl2</i> (<i>A-C</i>) and <i>Nodal</i> (<i>D-F</i>) expression at the node of <i>Pkd1l1</i>^{<i>tm1/tm1</i>} and control embryos. Quantitation of <i>in situ</i> signal reveals expression of both genes to be more symmetrical in mutant embryos (<i>C</i>, <i>F</i>). *—p<0.05, unpaired <i>t</i>-test applied. Error bars represent 95% confidence intervals. (G-H) Lung situs <i>(G)</i> (assessed at E13.5) and <i>Pitx2</i> expression <i>(H)</i> (assessed at E8.5) for embryos of the indicated genotypes, with the percentage of embryos exhibiting each phenotype and the total number given.</p

Cilia and PKD2 Localization and Function.

<p>(A-E) PKD2 localization in nodal cilia of embryos of the indicated genotype. Staining was divided into categories and quantitation is given in (<i>A</i>). In <i>(A)</i>, all genotypes are statistically significantly different from each other (p<0.001) except for <i>Pkd2</i>^{<i>+/lrm4</i>} and <i>Pkd1l1</i>^<i>+/tm1</i> which are statistically not significantly different.</p

The Relationship Between Nodal Flow and <i>Pkd1l1/Pkd2</i> Function.

<p>(A-C) Nodal flow in embryos of indicated genotypes was examined at the 1–3 somite stages by means of PIV analysis. Flow was normal in <i>Pkd1l1</i>^{<i>tm1/tm1</i>} mutants and wild-type controls but was absent in <i>Dnah11</i>^<i>iv/iv</i> mutants. Black arrowheads denote the direction and speed of flow at that position while the false coloring indicates the direction and magnitude of the flow. Red indicates leftward and blue rightward fluid movements. (D) Lung situs (assessed at E13.5) and <i>Pitx2</i> expression (assessed at E8.5) for embryos of the indicated genotypes, with the percentage of embryos exhibiting each phenotype and the total number given. (E) <i>Pitx2</i> expression for <i>Pkd1l1</i>^{<i>tm1/tm1</i>}, <i>Dnah11</i>^<i>iv/iv</i> and control embryos for each of the 1–7 somite stages. The onset of <i>Pitx2</i> expression is delayed in <i>Dnah11</i>^<i>iv/iv</i> mutants but not in <i>Pkd1l1</i>^{<i>tm1/tm1</i>} embryos.</p

Destabilization of a PKD Domain by the <i>Pkd1l1</i>^<i>rks</i> Mutation.

<p>(A-C) Structure of human PKD1 PKD domain 1 (<i>A</i>) and models of mouse PKD1L1 PKD domain 2; wild-type (<i>B</i>) or <i>rks</i>-mutated (<i>C</i>). Domains are largely composed of β-sheets (block arrows). The aspartic acid mutated in <i>Pkd1l1</i>^<i>rks</i>, or its equivalent in PKD1, is shown in space-fill. The asterisks denote loss of secondary structure in the <i>rks</i>-mutated domain. (D) SRCD spectroscopy of mouse PKD1L1 PKD domain 2 for wild-type and <i>rks</i>-mutated domains. Spectra are consistent with decreased stability (decreased secondary structure) in mutated domains. (E) Thermal denaturation analysis of PKD1L1 PKD domain 2: a reduced melting temperature (Tm) of 56.4°C is evident in the <i>rks</i>-mutated domain; in wild-type controls a Tm of 68.6°C is detected.</p

Multi-repression Model for L-R Asymmetry Determination in Crown Cells.

<p>(A) Schematic of a 3 ss flat-mounted mouse embryo showing somites (yellow), and node (blue). (B) Pictorial representation of the multi-repression model in which flow represses <i>Pkd1l1</i> on the left side, resulting in the derepression of <i>Pkd2</i>, inhibition of <i>Cerl2</i> and, as a result, higher NODAL activity on the left. (C-E) Predictions of the multi-repression model in various genetic mutants including the impact on the crown cell genetic pathway as well as the predicted LPM Nodal cascade activity.</p

Flow-Induced Ca²⁺ Signaling Depends on PKD1L1.

<p>(A-B) <i>Pkd1</i>^<i>+/+</i> <i>(A)</i> and <i>Pkd1</i>^{<i>–/–</i>}<i>(B)</i> cells were transfected with vector-GFP alone, PKD1L1-GFP or PKD1L1^rks-GFP. Successfully transfected cells had green fluorescence (GFP), and the entire cell population was observed by DIC. After baseline Ca²⁺ level was taken, fluid-shear stress was applied to cells (arrow). Numbers indicate time in seconds (s). Color bars indicate Ca²⁺ level (pseudocoloured), where black-purple and yellow-red represent low and high Ca²⁺ levels, respectively (C-D) Quantitation from independent experiments of <i>Pkd1</i>^<i>+/+</i> <i>(C)</i> and <i>Pkd1</i>^{<i>–/–</i>}<i>(D)</i> cells was averaged and plotted in line graphs. Within the same cell population, successfully transfected (GFP+) and non-transfected (GFP-) cells were analyzed separately. Arrows indicate the start of fluid-shear stress. Time is indicated in seconds (s). (E) Statistical analysis was done by analyzing the peak changes of intracellular Ca²⁺. While vector-GFP is used as a negative control, non-transfected cells (GFP-) were also used as an internal control. n = 150 cells for each group in three independent transfections. *—p<0.05.</p