Sonic hedgehog signaling defect in <i>Wdpcp^Cys40</i> mutant.

<p>(A–F) In-situ hybridization of E10.5 forelimbs shows <i>Wdpcp^Cys40</i> mutants with expanded expression of <i>Fgf4</i> in the AER (apical ectodermal ridge) (A, B) and <i>Gremlin</i> in the limb mesenchyme (C, D), but reduced expression of <i>Ptch1</i> (E, F and asterisk in F). In (A) and (B), black arrowheads indicate the span of the AER, and white arrowheads are the anterior and posterior bases of the limb bud. (G–J) <i>Wdpcp</i> deficiency rescued the severe defect phenotypes of <i>Smo</i>^−/− (G) and <i>Ptch1</i>^−/− (I) mutant embryos at E10.5 dpc. The <i>Wdpcp^Cys40/Cys40</i>;<i>Smo</i>^−/− (H) and <i>Wdpcp^Cys40/Cys40;Ptch1</i>^−/− (J) double homozygous mutant embryos collected at E10.5 dpc show more robust growth with better axial development and also more normal head and heart development. (K) Western blotting of Gli3 in tissue extracts obtained from the limb and neural tube shows a decrease of Gli3-R/Gli3-FL ratio in <i>Wdpcp^Cys40</i> mutant embryos. Scale bars, 200 µm in (A), 1 mm in (G). Scales are the same in (A–F) and (G–J).</p

Wdpcp is required for cilia recruitment of Sept2, Mks1, and Nphp1.

<p>(A–D) Ift88 is localized correctly to the basal body and ciliary tip in the rare cilium formed in <i>Wdpcp^Cys40</i> mutant MEFs. (E–H) Nphp1 is normally found at the transition zone (E, and arrowhead in F), but in <i>Wdpcp^Cys40</i> mutant MEFs, Nphp1 is mislocalized in the basal body (G, H). (I–L) Sept2 is normally found in the transition zone and sometimes in the ciliary axoneme in control MEFs (I, and arrowhead in J), but is absent from the cilium of <i>Wdpcp^Cys40</i> mutant MEF (K, L). (M–P) Mks1 is normally found in the transition zone of the cilium (M, N), but it is absent from the cilium of <i>Wdpcp^Cys40</i> mutant MEF (O, P). (Q–T) Wdpcp is localized to the transition zone in both control (Q, R) and <i>Mks1</i> mutant MEFs (S, T). All panels are at the same scales, and the scale bar in (A) is 2 µm.</p

<i>Wdpcp</i> mutation, <i>Wdpcp</i> cilia localization, and ciliary phenotypes in <i>Wdpcp^Cys40</i> mutants.

<p>(A) <i>Wdpcp</i> genomic and protein structure. (B) An A>G mutation in <i>Wdpcp^Cys40</i> mutant (red arrow) resulted in two abnormally spliced transcripts—one with exon 5 deleted and one with exons 5 and 8 deleted. (C) Exon 5 deletion generates premature stop, causing protein truncation. (D) IMCD3 cells immunostained with Wdpcp (green) and acetylated α-tubulin (red) antibodies showed Wdpcp localization in the axoneme and ring-like structure (arrowhead) at the cilia base. (E–H) Immunostaining of NIH-3T3 cells transfected with Wdpcp–FLAG showed colocalization of Wdpcp (red) and FLAG (green) in the cilia, with strong staining at the base of the cilium (acetylated α-tubulin, blue). (I–M) Immunostaining of MEFs showed Wdpcp (red, I: Wdpcp) localized in ring-like structure at the base of the cilia (green, I: anti-cetylated α-tubulin). Sept2 (green) showed colocalization with Wdpcp (red, panels L and M). The arrowheads (J–L) point to Wdpcp (J, L) and Sept2 staining (K, L). Note ring-like structure, better seen in 3D isosurface reconstruction (M) of same confocal images as in (J–L). (N, O) Acetylated α-tubulin antibody staining (red) of E15.5 wild-type kidney shows cilia (arrowheads in N), but few cilia were seen in the <i>Wdpcp^Cys40</i> mutant collecting duct (stained green with Dolichos Biflorus Agglutinin; arrowheads in O) and they were much shorter. (P, Q) Scanning EM of E10.5 embryo floor plate showed <i>Wdpcp^Cys40</i> mutant neural epithelium (Q) are less ciliated (see arrow) and lack microvilli as compared to control (P). (R) Percentage of ciliated cells is reduced in <i>Wdpcp^Cys40</i> mutant kidney epithelia and MEFs. Scale bars, 1 µm in (D), 2 µm in (E–L), 5 µm in (N, O), 2 µm in (P, Q).</p

Perturbation of membrane ruffling and focal adhesion contacts in <i>Wdpcp</i>-deficient cells.

<p>(A–H) Time-lapse imaging of wild-type (A–D) and <i>Wdpcp^Cys40</i> mutant (E–H) MEFs shown in 40 s intervals revealed the mutant MEFs have less membrane ruffling (arrowheads) and little or no membrane protrusive activity. In (E–H), the bottom-left arrow points to a long filopodial extension from a cell out of the field of view and top-left arrow points to the filopodial extension of the cell in the field of view that were immobile for the entire duration of the 320 s time-lapse sequence. (I–L) Immunostaining showed Wdpcp (green, J) is enriched at the cell cortex where actin filaments (phalloidin, K) insert into vinculin- (red, I) containing focal adhesions (arrow and arrowheads in L). (M, N) In <i>Wdpcp^Cys40</i> mutant MEFs (N), the vinculin-containing focal adhesions were smaller and more rounded as compared to wild-type MEFs (M). This was demonstrated by quantitative measurements of the major axis and roundness of vinculin-containing focal adhesions (O, P). (O, P) In wild-type MEFs (<i>n</i> = 279), the vinculin-containing focal contacts were more elongated (O) and less round (P), than <i>Wdpcp^Cys40</i> mutant MEFs (<i>n</i> = 206). The <i>p</i> values were calculated with student's <i>t</i> test. Scale bars, 10 µm in (A), 2 µm in (L), and 10 µm in (N). Scales are the same in (A–H); (I–L); and (M, N).</p

Ciliary defects in <i>wdpcp</i> morpholino knockdown embryos.

<p>(A–D) <i>wdpcp</i> morpholino knockdown causes pericardial edema (black arrow in B), pronephric tubule cyst (red arrows in B), severe hydrocephaly (arrow in D), and increased number of otoliths (red asterisks in D) as compared to control MO injected embryo (A). (E–L) Wdpcp antibody staining of the zebrafish embryo pronephric tubule showed punctate localization (red; arrows in F) along the ciliary axoneme (stained green with acetylated α-tubulin). Such staining is absent in <i>wdpcp</i> morphants, showing efficacy of the <i>wdpcp</i> MO knockdown (E). Cilia in proximal straight/distal early tubule (PST/DL) of <i>wdpcp</i> morphants are disorganized (K), as compared to that of control morphants (I) and uninjected embryos (G), while the distal late pronephric duct (DL/PD) showed little or no change compared to uninjected (H) or control MO (J) injected embryos. Scale bars, 2 µm in (E) and 10 µm (G). (E,F) and (G–L) are at the same scale.</p

<i>Wdpcp^Cys40</i> mutants show PCP defects and disrupted canonical Wnt signaling.

<p>(A–D) Scanning EM of cochlear hair cells showed chevron-shaped stereocilia pointing laterally in control (A, C), but in <i>Wdpcp^Cys40</i> mutants, the stereocilia point in varying directions (B, D), with misshappened stereocilia bundles. The kinocilia, normally positioned at the chevron tip, were mislocalized in <i>Wdpcp^Cys40</i> mutant. (E–H) Staining with phalloidin (Phal; E, F) and Vangl2 antibody (G, H) showed diminished Vangl2 expression in hair cells of <i>Wdpcp^Cys40</i> mutant cochlea, while Vangl2 in control exhibited the characteristic asymmetric expression pattern. (I, J) Transverse sections of E13.5 embryos showed base of the heart counterstained with nuclear fast red and immunostained with anti α-SMA antibody (blue) to visualize migrating cardiomyocytes. In wild-type heart (I), cardiomyocytes were observed in the outflow tract cushion (arrow), but in <i>Wdpcp^Cys40</i> mutants, cardiomyocytes were mostly absent in the cushion tissue (asterisk in J). (K, L) Cardiomyocytes in outflow cushion of wild-type embryos (K) visualized with MF20 immunostaining showed polarized cell morphology with distinct elongated finger-like projections (asterisks) aligned with direction of cell migration and projecting into forming outflow septum (arrow in K). In contrast, in <i>Wdpcp^Cys40</i> mutant embryos (L), the cardiomyocytes exhibited rounded morphology without obvious cell polarity, nor the elongated cell projections seen in wild-type embryos. (M, N) The heart of control embryo at E13.5 showed very weak BAT–lacZ expression at the base of the pulmonary trunk (M), while markedly elevated BAT–lacZ expression was observed in the same region in a <i>Wdpcp^Cys40</i> mutant embryo (N). (O) Quantitative real-time PCR analysis showed changes in expression level for noncanonical and canonical Wnt signaling pathway genes at the base of the outflow tract of <i>Wdpcp^Cys40</i> mutant embryos. Blue bars indicate elevated versus orange bars, indicating reduced expression levels. Standard errors and <i>p</i> values are shown. Scale bars, 5 µm in (A, B), 2 µm in (C, D), 50 µm in (E–H), 100 µm in (I–L), 1 mm in (M, N).</p

<i>Wdpcp</i> deficiency disrupts polarized cell migration.

<p>(A–D) In a wound healing assay, control MEFs (A) were well aligned with the direction of wound closure (indicated by white arrow). In contrast, <i>Wdpcp^Cys40</i> mutant MEFs (B) showed a disorganized distribution and with many long thin filopodial extensions. These differences in cell polarity were also reflected in their Golgi orientation—Golgi orientation is indicated by a white line drawn from the cell nucleus through the center of the Golgi (C, D). In wild-type MEFs, the Golgi was mostly situated at the cell's leading edge (C), aligned with the direction of wound closure (white arrow). However, in <i>Wdpcp^Cys40</i> mutant MEFs, Golgi orientation was randomized (D). (E) Quantitative analysis of Golgi orientation showed wild-type MEFs (<i>n</i>=117) with highly polarized arrangement of the Golgi well aligned with the direction of cell migration (94 of 117 cells with Golgi apparatus in 0–60° sector), while <i>Wdpcp^Cys40</i> mutant MEFs (<i>n</i>=161) showed random orientation of the Golgi apparatus (64 of 161 cells with Golgi in 0–60° sector) (see Materials and Methods). Scale bar, 20 µm in (B) and (D). Scales are the same in (A, B) and (C, D).</p

Wdpcp colocalizes with Sept2 and actin filaments.

<p>(A–D) Confocal imaging of control MEFs stained with phalloidin, and antibodies to Wdpcp and Sept2 showed Sept2 (red) and Wdpcp (green) are colocalized in actin stress fiber (phalloidin stained, blue) (examples denoted by arrow). This is better visualized in the magnified image shown in (E–H). (E–H) Magnified view of the region indicated by the asterisk-denoted arrow from (A–D) show colocalization of Wdpcp (green) and Sept2 (red) with actin filaments (blue). (I–L) Confocal imaging of <i>Wdpcp^Cys40</i> mutant MEFs showed only background fluorescence (green, panel J) with the Wdpcp antibody. However, Sept2 immunostaining remained robust (red, panel I), but there was no colocalizaiton with actin filament visualized with phalloidin staining (blue, panel K). The region indicated by the asterisk-denoted arrow is magnified in (M–P). Inset shown is magnified view of a region from (M), showing ‘c’ and ‘o’ shaped Sept2 immunostained structures. (M–P) Magnified view of the region marked by the asterisk-denoted arrow in (I–L). While actin (blue, panel O) and Sept2 filaments (red, panel M) can be observed, Sept2 is not colocalized with actin (P). Sept2 immunostaining delineated ‘c’ and ‘o’ shaped structures (M), which are better visualized in the further magnified view shown in the inset in (I). Scale bars, 20 µm in (A), 10 µm in (E), and 5 µm in inset image in (I). Scales are the same in (A–D), (I–L); (E–H), (M–P).</p

Developmental defects of neonatal <i>Wdpcp^Cys40</i> mutants.

<p>(A–I) Necropsy showed <i>Wdpcp^Cys40</i> neonatal mutants have a wide spectrum of defects including facial cleft and anophthalmia (arrowhead in A), polydactyly (B, C), intestinal cysts (D), duplex kidney (black arrowheads in E) with glomerular cysts (white arrows in E), outflow tract defect consisting of a single common trunk (asterisk in G), and cloaca septation defect with abnormal connection between the intestine and bladder (box region in I). (J, K, L, M) Episcopic fluorescence image capture (EFIC) histopathology showed a <i>Wdpcp^Cys40</i> mutant with an incomplete septum unevenly dividing the outflow tract into one large and one small chamber, indicating pulmonary atresia (PAtr; black arrow in K). Also observed was an atrioventricular septal defect (AVSD; asterisk in M). Shown in (J, L) are comparable views of a control heart. Abbreviations: P, pancreas; K, kidney in (D); P, pulmonary trunk in (F) and (J); LV, left ventricle; PAtr, Pulmonary atresia; AVSD, Atrioventricular septal defect. Scale bars, 1 mm in (B, C, D, E, F, H). Magnifications are the same in (F, G, J–M; H, I).</p