Wdpcp, a PCP Protein Required for Ciliogenesis, Regulates Directional Cell Migration and Cell Polarity by Direct Modulation of the Actin Cytoskeleton

Planar cell polarity (PCP) regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet-Biedl/Meckel-Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin cytoskeleton to regulate cell polarity and directional cell migration

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Disruption of Mks1 localization to the mother centriole causes cilia defects and developmental malformations in Meckel-Gruber syndrome

Meckel-Gruber syndrome (MKS) is a recessive disorder resulting in multiple birth defects that are associated with mutations affecting ciliogenesis. We recovered a mouse mutant with a mutation in the Mks1 gene (Mks1del64-323) that caused a 260-amino-acid deletion spanning nine amino acids in the B9 domain, a protein motif with unknown function conserved in two other basal body proteins. We showed that, in wild-type cells, Mks1 was localized to the mother centriole from which the cilium was generated. However, in mutant Mks1del64-323 cells, Mks1 was not localized to the centriole, even though it maintained a punctate distribution. Resembling MKS patients, Mks1 mutants had craniofacial defects, polydactyly, congenital heart defects, polycystic kidneys and randomized left-right patterning. These defects reflected disturbance of functions subserved by motile and non-motile cilia. In the kidney, glomerular and tubule cysts were observed along with short cilia, and cilia were reduced in number to a near-complete loss. Underlying the left-right patterning defects were fewer and shorter nodal cilia, and analysis with fluorescent beads showed no directional flow at the embryonic node. In the cochlea, the stereocilia were mal-patterned, with the kinocilia being abnormally positioned. Together, these defects suggested disruption of planar cell polarity, which is known to regulate node, kidney and cochlea development. In addition, we also showed that Shh signaling was disrupted. Thus, in the neural tube, the floor plate was not specified posteriorly even as expression of the Shh mediator Gli2 increased. By contrast, the Shh signaling domain was expanded in the anterior neural tube and anterior limb bud, consistent with reduced Gli3-repressor (Gli3R) function. The latter probably accounted for the preaxial digit duplication exhibited by the Mks1del64-323 mutants. Overall, these findings indicate that centriole localization of Mks1 is required for ciliogenesis of motile and non-motile cilia, but not for centriole assembly. On the basis of these results, we hypothesize a role for the B9 domain in mother centriole targeting, a possibility that warrants further future investigations

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

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