Xenopus Pkdcc1 and Pkdcc2 Are Two New Tyrosine Kinases Involved in the Regulation of JNK Dependent Wnt/PCP Signaling Pathway

Protein Kinase Domain Containing, Cytoplasmic (PKDCC) is a protein kinase which has been implicated in longitudinal bone growth through regulation of chondrocytes formation. Nevertheless, the mechanism by which this occurs remains unknown. Here, we identified two new members of the PKDCC family, Pkdcc1 and Pkdcc2 from Xenopus laevis. Interestingly, our knockdown experiments revealed that these two proteins are both involved on blastopore and neural tube closure during gastrula and neurula stages, respectively. In vertebrates, tissue polarity and cell movement observed during gastrulation and neural tube closure are controlled by Wnt/Planar Cell Polarity (PCP) molecular pathway. Our results showed that Pkdcc1 and Pkdcc2 promote the recruitment of Dvl to the plasma membrane. But surprisingly, they revealed different roles in the induction of a luciferase reporter under the control of Atf2 promoter. While Pkdcc1 induces Atf2 expression, Pkdcc2 does not, and furthermore inhibits its normal induction by Wnt11 and Wnt5a. Altogether our data show, for the first time, that members of the PKDCC family are involved in the regulation of JNK dependent Wnt/PCP signaling pathway.Fundacao Ciencia e Tecnologia - IP; IBB/CBME, LA [PTDC/BIA-BCM/69912/2006, Pest-OE/EQB/LA0023/2013]; FCTinfo:eu-repo/semantics/publishedVersio

Mapping of clouston hidrotic ectodermal dysplasia

Clouston hidrotic ectodermal dysplasia (BED) is an autosomal dominant skin disorder that is characterized by nail dystrophy, hair defects and palmoplantar hyperkeratosis. This condition has been described in families of various ethnic origins but is particularly common in the French Canadian population. Using linkage analysis in eight French Canadian families segregating HED, we mapped the HED gene to the pericentromeric region of chromosome 13q with a combined two-point lod score of 8.12 at zero recombination from the marker D13S175. Haplotype analysis allowed us to define D13S143 as the telomeric flanking marker for the HED candidate region. We tested five genes that map to this region, connexin 26, connexin 46, fibroblast growth factor 9, zinc-finger ZNF198 and alpha tubulin TUBA2, for involvement in HED by PCR-SSCP analysis. No mutation specific to HED was found in any of them suggesting that they most likely are not defective in this disease.To facilitate the identification of the HED gene, we constructed a radiation hybrid (RH) map of 48 loci surrounding the HED locus on chromosome 13q. This map integrates 3 genes (TUBA2, GJbeta2 and FGF-9) and 18 ESTs with 27 markers including 19 polymorphic loci. A major inconsistency in order involving a reversed interval of six loci was found between our RH map and a YAC contig established in the region. We used Fiber-FISH and FISH on interphase nuclei to confirm our order. To refine the localization of the HED gene, we isolated eight new chromosome 13q polymorphic (CA)n markers and used seven of them along with three others in genetic analysis of a multiethnic group of 29 HED families. We demonstrated genetic homogeneity in HED in four families of French, Spanish, African and Malaysian origins and showed evidence for a strong founder effect in families of French Canadian origin. Recombination mapping placed the HED gene in a 2.4 cM region flanked by D13S1828 proximally and D13S1830 distally. Multipoint linkage and linkage disequilibrium analyses finely mapped the HED gene at 0--0.08 cM telomeric to D13S1835. These studies will greatly facilitate the physical mapping and positional cloning of the HED gene

Update on the Role of the Non-Canonical Wnt/Planar Cell Polarity Pathway in Neural Tube Defects

Neural tube defects (NTDs), including spina bifida and anencephaly, represent the most severe and common malformations of the central nervous system affecting 0.7–3 per 1000 live births. They result from the failure of neural tube closure during the first few weeks of pregnancy. They have a complex etiology that implicate a large number of genetic and environmental factors that remain largely undetermined. Extensive studies in vertebrate models have strongly implicated the non-canonical Wnt/planar cell polarity (PCP) signaling pathway in the pathogenesis of NTDs. The defects in this pathway lead to a defective convergent extension that is a major morphogenetic process essential for neural tube elongation and subsequent closure. A large number of genetic studies in human NTDs have demonstrated an important role of PCP signaling in their etiology. However, the relative contribution of this pathway to this complex etiology awaits a better picture of the complete genetic architecture of these defects. The emergence of new genome technologies and bioinformatics pipelines, complemented with the powerful tool of animal models for variant interpretation as well as significant collaborative efforts, will help to dissect the complex genetics of NTDs. The ultimate goal is to develop better preventive and counseling strategies for families affected by these devastating conditions

Genetic Analysis of Disheveled 2 and Disheveled 3 in Human Neural Tube Defects

Neural tube defects are severe malformations affecting 1/1,000 live births. The planar cell polarity pathway controls the neural tube closure and has been implicated in the pathogenesis of neural tube defects both in animal models and human cohorts. In mouse disruption of Dvl2 alone (Dvl2(−/−)) or Dvl2 and Dvl3 (Dvl2(−/−); Dvl3(+/−), Dvl2(+/−); Dvl3(−/−)) results in incomplete neurulation, suggesting a role for Disheveled in neural tube closure. Disheveled is a multifunctional protein that is involved in both the canonical Wnt signaling and the noncanonical planar cell polarity pathway. In this study, we analyzed the role of the human orthologs DVL2 and DVL3 in a cohort of 473 patients with neural tube defects. Rare variants were genotyped in 639 ethnically matched controls. We identified seven rare missense mutations that were absent in all controls analyzed. Two of these mutations, p.Tyr667Cys and p.Ala53Val, identified in DVL2 were predicted to be detrimental in silico. Significantly, a 1-bp insertion (c.1801_1802insG) in exon 15 of DVL2 predicted to lead to the truncation of the protein was identified in a patient with a complex form of caudal agenesis. In summary, we demonstrate a possible role for rare variants in DVL2 gene as risk factors for neural tube defects. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12031-012-9871-9) contains supplementary material, which is available to authorized users

Connexin46 Mutations in Autosomal Dominant Congenital Cataract

Loci for autosomal dominant "zonular pulverulent" cataract have been mapped to chromosomes 1q (CZP1) and 13q (CZP3). Here we report genetic refinement of the CZP3 locus and identify underlying mutations in the gene for gap-junction protein alpha-3 (GJA3), or connexin46 (Cx46). Linkage analysis gave a significantly positive two-point LOD score (Z) at marker D13S175 (maximum Z [Zmax]=>7.0; maximum recombination frequency [thetamax] =0). Haplotyping indicated that CZP3 probably lies in the genetic interval D13S1236-D13S175-D13S1316-cen-13pter, close to GJA3. Sequencing of a genomic clone isolated from the CZP3 candidate region identified an open reading frame coding for a protein of 435 amino acids (47,435 D) that shared approximately 88% homology with rat Cx46. Mutation analysis of GJA3 in two families with CZP3 detected distinct sequence changes that were not present in a panel of 105 normal, unrelated individuals. In family B, an A-->G transition resulted in an asparagine-to-serine substitution at codon 63 (N63S) and introduced a novel MwoI restriction site. In family E, insertion of a C at nucleotide 1137 (1137insC) introduced a novel BstXI site, causing a frameshift at codon 380. Restriction analysis confirmed that the novel MwoI and BstXI sites cosegregated with the disease in families B and E, respectively. This study identifies GJA3 as the sixth member of the connexin gene family to be implicated in human disease, and it highlights the physiological importance of gap-junction communication in the development of a transparent eye lens.This work is supported by grants from the Wellcome Trust (043073, 053416), the National Institutes of Health (EY12284, EY11411), and the Medical Research Council of Canada (to G.R.). A.I. is supported by a grant from the Friends of Moorfields Eye Hospital, and D.M. is a Wellcome Prize Student (044573)

Novel mutations in Lrp6 orthologs in mouse and human neural tube defects affect a highly dosage-sensitive Wnt non-canonical planar cell polarity pathway

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Diagrammatic illustrations to show the hypothetical effects of compensatory changes in skull dimensions and brain shape when there is craniosynostosis or shortening of certain skull bones.

<p>These figures were created using Adobe Photoshop (<a href="http://www.adobe.com/products/photoshopfamily.html" target="_blank">http://www.adobe.com/products/photoshopfamily.html</a>) which enables manipulation of shapes with fixed points facilitating a simple representation of the distortion. a) Normal; the skull is represented by a black box and brain by a grey circle with reference points w, x, y and z. Asterisk – fixed reference point outside brain. b) Uniform rostrocaudal shortening (black arrows). There is a compensatory increase in height of the brain (white arrow). Lines xy, yz (yellow) and wy (blue) increase and angles wyz and xyz decrease c) Cranial base shortening (black arrow); asymmetrical shortening results in an axial tilting of the brain. There is also a compensatory increase in height (white arrow). Line xy and yz increases (yellow), wy shortens (blue) and angle wyx and xyz decrease. d) Caudal dorsoventral shortening (black arrow). There is a compensatory lengthening of the brain and rostral increase in height (white arrow). Line wy increase (blue), xy and yz shorten (yellow), angle xyz decreases e) cranial base and caudal dorsoventral shortening. When the cranial base and the caudal part of the skull are shortened the circular model brain becomes increasing ellipsoid and there is greater axial tilt with an increase in height rostrally (white arrow). The ventorostral part (olfactory bulbs) is displaced ventrally, the dorsocaudal part (occipital lobes) is displaced caudally and the ventrocaudal part (hindbrain) is displaced rostrally. Line yz shortens and angle xyz decreases. However distance to the external reference point (asterisk) increases (red dotted line). This illustration is offered as an explanation for the increase in line AE and BC in this study and also for the change in brain shape with increasing CM affectedness.</p

Significant variables identified in the statistical analysis for syringomyelia.

<p>F is the Analysis of Variance [ANOVA] test statistic. It assesses the ratio of between group variation to within group variation with higher values indicating the likelihood of a group effect.</p

Function values for the SM NO and SM YES groups.

<p>The SM YES dogs have slightly higher values of the function (more positive) and the SM NO dogs have slightly lower values. A few SM YES dogs can be seen to be outliers (*) with unusually high function values.</p