Planar Cell Polarity Signaling Pathway Research Articles

The Wingless planar cell polarity pathway is essential for optimal activity-dependent synaptic plasticity.

From fly to man, the Wingless (Wg)/Wnt signaling molecule is essential for both the stability and plasticity of the nervous system. The Drosophila neuromuscular junction (NMJ) has proven to be a useful system for deciphering the role of Wg in directing activity-dependent synaptic plasticity (ADSP), which, in the motoneuron, has been shown to be dependent on both the canonical and the noncanonical calcium Wg pathways. Here we show that the noncanonical planar cell polarity (PCP) pathway is an essential component of the Wg signaling system controlling plasticity at the motoneuron synapse. We present evidence that disturbing the PCP pathway leads to a perturbation in ADSP. We first show that a PCP-specific allele of disheveled (dsh) affects the de novo synaptic structures produced during ADSP. We then show that the Rho GTPases downstream of Dsh in the PCP pathway are also involved in regulating the morphological changes that take place after repeated stimulation. Finally, we show that Jun kinase is essential for this phenomenon, whereas we found no indication of the involvement of the transcription factor complex AP1 (Jun/Fos). This work shows the involvement of the neuronal PCP signaling pathway in supporting ADSP. Because we find that AP1 mutants can perform ADSP adequately, we hypothesize that, upon Wg activation, the Rho GTPases and Jun kinase are involved locally at the synapse, in instructing cytoskeletal dynamics responsible for the appearance of the morphological changes occurring during ADSP.

Integrated proteogenomic characterization of glioblastoma evolution

The evolutionary trajectory of glioblastoma (GBM) is a multifaceted biological process that extends beyond genetic alterations alone. Here, we perform an integrative proteogenomic analysis of 123 longitudinal glioblastoma pairs and identify a highly proliferative cellular state at diagnosis and replacement by activation of neuronal transition and synaptogenic pathways in recurrent tumors. Proteomic and phosphoproteomic analyses reveal that the molecular transition to neuronal state at recurrence is marked by post-translational activation of the wingless-related integration site (WNT)/ planar cell polarity (PCP) signaling pathway and BRAF protein kinase. Consistently, multi-omic analysis of patient-derived xenograft (PDX) models mirror similar patterns of evolutionary trajectory. Inhibition of B-raf proto-oncogene (BRAF) kinase impairs both neuronal transition and migration capability of recurrent tumor cells, phenotypic hallmarks of post-therapy progression. Combinatorial treatment of temozolomide (TMZ) with BRAF inhibitor, vemurafenib, significantly extends the survival of PDX models. This study provides comprehensive insights into the biological mechanisms of glioblastoma evolution and treatment resistance, highlighting promising therapeutic strategies for clinical intervention.

A framework for Frizzled-G protein coupling and implications to the PCP signaling pathways

The ten Frizzled receptors (FZDs) are essential in Wnt signaling and play important roles in embryonic development and tumorigenesis. Among these, FZD6 is closely associated with lens development. Understanding FZD activation mechanism is key to unlock these emerging targets. Here we present the cryo-EM structures of FZD6 and FZD3 which are known to relay non-canonical planar cell polarity (PCP) signaling pathways as well as FZD1 in their G protein-coupled states and in the apo inactive states, respectively. Comparison of the three inactive/active pairs unveiled a shared activation framework among all ten FZDs. Mutagenesis along with imaging and functional analysis on the human lens epithelial tissues suggested potential crosstalk between the G-protein coupling of FZD6 and the PCP signaling pathways. Together, this study provides an integrated understanding of FZD structure and function, and lays the foundation for developing therapeutic modulators to activate or inhibit FZD signaling for a range of disorders including cancers and cataracts.

A complex of Wnt/planar cell polarity signaling components Vangl1 and Fzd7 drives glioblastoma multiforme malignant properties

Targeting common oncogenic drivers of glioblastoma multiforme (GBM) in patients has remained largely ineffective, raising the possibility that alternative pathways may contribute to tumor aggressiveness. Here we demonstrate that Vangl1 and Fzd7, components of the non-canonical Wnt planar cell polarity (Wnt/PCP) signaling pathway, promote GBM malignancy by driving cellular proliferation, migration, and invasiveness, and engage Rho GTPases to promote cytoskeletal rearrangements and actin dynamics in migrating GBM cells. Mechanistically, we uncover the existence of a novel Vangl1/Fzd7 complex at the leading edge of migrating GBM cells and propose that this complex is critical for the recruitment of downstream effectors to promote tumor progression. Moreover, we observe that depletion of FZD7 results in a striking suppression of tumor growth and latency and extends overall survival in an intracranial mouse xenograft model. Our observations support a novel mechanism by which Wnt/PCP components Vangl1 and Fzd7 form a complex at the leading edge of migratory GBM cells to engage downstream effectors that promote actin cytoskeletal rearrangements dynamics. Our findings suggest that interference with Wnt/PCP pathway function may offer a novel therapeutic strategy for patients diagnosed with GBM.

Molecular mechanism of core planar cell polarity complex function elucidated with single-molecule methods.

The planar cell polarity (PCP) signaling pathway polarizes epithelial cells along an axis parallel to the epithelial sheet in order to generate front-back asymmetry at the tissue level. Failures in PCP signaling underlie developmental defects and diseases such as neural tube and heart defects, deafness and contribute to cancer. Six core PCP proteins assemble into large, asymmetric complexes at cell-cell junctions, with Vang and Prickle recruited to one side of the cell-cell junction while Frizzled and Dishevelled are recruited to the other. How this asymmetric assembly process occurs is not known. Genetic evidence suggests that the process of symmetry breaking at PCP clusters is coupled to cluster formation. To test how this occurs, we developed a method to count the number of each of the six core proteins in individual PCP complexes. Specifically, by analyzing bleaching traces obtained with a high frame rate in live Drosophila wing cells imaged using total internal reflection microscopy, we quantify the intensity of single fluorophores in each complex and thereby calculate the molecular number in the corresponding PCP complex. Using this approach, we find that the molecular size distribution of PCP complexes follows an exponential function, indicating a single underlying growth mechanism. We report that specifically, the number of Vang molecules in PCP complexes increases from 15 hours after puparium formation until reaching a steady state around 28 hours. Besides, we measure the asymmetry as a function of cluster size and test the dependence of the observed relationship by introducing gain and loss of function mutations that perturb cluster assembly. In conclusion, our findings provide a detailed understanding of how the PCP mechanism generates stable polarization during embryonic development.

Sequential action of JNK genes establishes the embryonic left-right axis.

ABSTRACTThe establishment of the left-right axis is crucial for the placement, morphogenesis and function of internal organs. Left-right specification is proposed to be dependent on cilia-driven fluid flow in the embryonic node. Planar cell polarity (PCP) signalling is crucial for patterning of nodal cilia, yet downstream effectors driving this process remain elusive. We have examined the role of the JNK gene family, a proposed downstream component of PCP signalling, in the development and function of the zebrafish node. We show jnk1 and jnk2 specify length of nodal cilia, generate flow in the node and restrict southpaw to the left lateral plate mesoderm. Moreover, loss of asymmetric southpaw expression does not result in disturbances to asymmetric organ placement, supporting a model in which nodal flow may be dispensable for organ laterality. Later, jnk3 is required to restrict pitx2c expression to the left side and permit correct endodermal organ placement. This work uncovers multiple roles for the JNK gene family acting at different points during left-right axis establishment. It highlights extensive redundancy and indicates JNK activity is distinct from the PCP signalling pathway.

Vangl2, a Core Component of the WNT/PCP Pathway, Regulates Adult Hippocampal Neurogenesis and Age-Related Decline in Cognitive Flexibility.

Decline in episodic memory is one of the hallmarks of aging and represents one of the most important health problems facing Western societies. A key structure in episodic memory is the hippocampal formation and the dentate gyrus in particular, as the continuous production of new dentate granule neurons in this brain region was found to play a crucial role in memory and age-related decline in memory. As such, understanding the molecular processes that regulate the relationship between adult neurogenesis and aging of memory function holds great therapeutic potential. Recently, we found that Vang-Gogh like 2 (Vangl2), a core component of the Planar Cell Polarity (PCP) signaling pathway, is enriched in the dentate gyrus of adult mice. In this context, we sought to evaluate the involvement of this member of the Wnt/PCP pathway in both adult neurogenesis and memory abilities in adult and middle-aged mice. Using a heterozygous mouse model carrying a dominant-negative mutation in the Vangl2 gene, called Looptail (Vangl2Lp), we show that alteration in Vangl2 expression decreases the survival of adult-born granule cells and advances the onset of a decrease in cognitive flexibility. The inability of mutant mice to erase old irrelevant information to the benefit of new relevant ones highlights a key role of Vangl2 in interference-based forgetting. Taken together, our findings show that Vangl2 activity may constitute an interesting target to prevent age-related decline in hippocampal plasticity and memory.

Wnt5a Regulates Junctional Function of Sertoli cells Through PCP-mediated Effects on mTORC1 and mTORC2.

The blood-testis barrier (BTB) and apical ectoplasmic specialization (ES), which are synchronized through the crosstalk of Sertoli cells and Sertoli germ cells, are required for spermatogenesis and sperm release. Here, we show that Wnt5a, a noncanonical Wnt signaling pathway ligand, is predominately expressed in both the BTB and apical ES and has a specific expression pattern during the seminiferous epithelium cycle. We employed siRNA to knockdown Wnt5a expression in testis and Sertoli cells, and then identified elongated spermatids that lost their polarity and were embedded in the seminiferous epithelium. Moreover, phagosomes were found near the tubule lumen. These defects were due to BTB and apical ES disruption. We also verified that the expression level and/or location of BTB-associated proteins, actin binding proteins (ABPs), and F-actin was changed after Wnt5a knockdown in vivo and in vitro. Additionally, we demonstrated that Wnt5a regulated actin dynamics through Ror2-mediated mTORC1 and mTORC2. This study clarified the molecular mechanism of Wnt5a in Sertoli cell junctions through the planar cell polarity (PCP) signaling pathway. Our findings could provide an experimental basis for the clinical diagnosis and treatment of male infertility caused by Sertoli cell junction impairment.

Grainyhead-like transcription factors: guardians of the skin barrier.

There has been selective pressure to maintain a skin barrier since terrestrial animals evolved 360 million years ago. These animals acquired an unique integumentary system with a keratinized, stratified, squamous epithelium surface barrier. The barrier protects against dehydration and entry of microbes and toxins. The skin barrier centres on the stratum corneum layer of the epidermis and consists of cornified envelopes cemented by the intercorneocyte lipid matrix. Multiple components of the barrier undergo cross-linking by transglutaminase (TGM) enzymes, while keratins provide additional mechanical strength. Cellular tight junctions also are crucial for barrier integrity. The grainyhead-like (GRHL) transcription factors regulate the formation and maintenance of the integument in diverse species. GRHL3 is essential for formation of the skin barrier during embryonic development, whereas GRHL1 maintains the skin barrier postnatally. This is achieved by transactivation of Tgm1 and Tgm5, respectively. In addition to its barrier function, GRHL3 plays key roles in wound repair and as an epidermal tumour suppressor. In its former role, GRHL3 activates the planar cell polarity signalling pathway to mediate wound healing by providing directional migration cues. In squamous epithelium, GRHL3 regulates the balance between proliferation and differentiation, and its loss induces squamous cell carcinoma (SCC). In the skin, this is mediated through increased expression of MIR21, which reduces the expression levels of GRHL3 and its direct target, PTEN, leading to activation of the PI3K-AKT signalling pathway. These data position the GRHL family as master regulators of epidermal homeostasis across a vast gulf of evolutionary history.

MiR-92 Family Members Form a Cluster Required for Notochord Tubulogenesis in Urochordate Ciona savignyi.

MicroRNAs are frequently clustered in the genome and polycistronically transcribed, regulating targeted genes in diverse signaling pathways. The miR-17-92 cluster is a typical miRNA cluster, playing crucial roles in the organogenesis and homeostasis of physiological processes in vertebrates. Here, we identified three miRNAs (csa-miR-92a, csa-miR-92b, and csa-miR-92c) that belonged to the miR-92 family and formed a miRNA cluster in the genome of a urochordate marine ascidian Ciona savignyi. Except for miR-92a and miR-92b, other homologs of the vertebrate miR-17-92 cluster members could not be identified in the Ciona genome. We further found that the mature sequences of urochordate miR-92 family members were highly conserved compared with the vertebrate species. The expression pattern revealed that three miR-92 family members had consistent expression levels in adult tissues and were predominantly expressed in heart and muscle tissue. We further showed that, at the embryonic and larval stages, csa-miR-92c was expressed in the notochord of embryos during 18–31 h post fertilization (hpf) by in situ hybridization. Knockout of csa-miR-92c resulted in the disorganization of notochord cells and the block of lumen coalescence in the notochord. Fibroblast growth factor (FGF), mitogen-activated protein kinase (MAPK), and wingless/integrated (Wnt)/planar cell polarity (PCP) signaling pathways might be involved in the regulatory processes, since a large number of core genes of these pathways were the predicted target genes of the miR-92 family. Taken together, we identified a miR-92 cluster in urochordate Ciona and revealed the expression patterns and the regulatory roles of its members in organogenesis. Our results provide expression and phylogenetic data on the understanding of the miR-92 miRNA cluster’s function during evolution.

Myelomeningocele genotype-phenotype correlation findings in cilia, HH, PCP, and WNT signaling pathways.

Myelomeningocele (MMC) is the most severe and frequent type of spina bifida. Its etiology remains poorly understood. The Hedgehog (Hh), Wnt, and planar cell polarity (PCP) signaling pathways are essential for normal tube closure, needing a structural-functional cilium for its adequate function. The present study aimed to investigate the impact of different gene variants (GV) from those pathways on MMC genotype-subphenotype correlations. The study comprised 500 MMC trios and 500 controls, from 16 Telethon centers of 16 Mexican states. Thirty-four GVs of 29 genes from cilia, Hh, PCP, and Wnt pathways, were analyzed, by an Illumina on design microarray. The total sample (T-MMC) was stratified in High-MMC (H-MMC) when thoracic and Low-MMC (L-MMC) when lumbar-sacral vertebrae affected. STATA/SE-12.1 and PLINK software were used for allelic association, TDT, and gene-gene interaction (GGI) analyses, considering p value <.01 as statistically significant differences (SSD). Association analysis showed SSD for COBL-rs10230120, DVL2-rs2074216, PLCB4-rs6077510 GVs in T-MMC and L-MMC, and VANGL2-rs120886448 in T-MMC and H-MMC, and INVS-rs7024375 exclusively in L-MMC. TDT assay showed SSD preferential transmissions of C2CD3-rs826058 in H-MMC, and LRP5-rs3736228, and BBS2-rs1373 in L-MMC. Statistically significant GGI was observed in four in T-MMC, four completely different in L-MMC, and one in H-MMC. Interestingly, no one repeated in subphenotypes. Our results support an association of GVs in Hh, Wnt, PCP, and cilia pathways, with MMC occurrence location, although further validation is needed. Furthermore, present results show a distinctive panel of gene-variants in H-MMC and LMMC subphenotypes, suggesting a feasible genotype-phenotype correlation.

SHROOM3 is downstream of the planar cell polarity pathway and loss-of-function results in congenital heart defects

Congenital heart disease (CHD) is the most common birth defect, and the leading cause of death due to birth defects, yet causative molecular mechanisms remain mostly unknown. We previously implicated a novel CHD candidate gene, SHROOM3, in a patient with CHD. Using a Shroom3 gene trap knockout mouse (Shroom3gt/gt) we demonstrate that SHROOM3 is downstream of the noncanonical Wnt planar cell polarity signaling pathway (PCP) and loss-of-function causes cardiac defects. We demonstrate Shroom3 expression within cardiomyocytes of the ventricles and interventricular septum from E10.5 onward, as well as within cardiac neural crest cells and second heart field cells that populate the cardiac outflow tract. We demonstrate that Shroom3gt/gt mice exhibit variable penetrance of a spectrum of CHDs that include ventricular septal defects, double outlet right ventricle, and thin left ventricular myocardium. This CHD spectrum phenocopies what is observed with disrupted PCP. We show that during cardiac development SHROOM3 interacts physically and genetically with, and is downstream of, key PCP signaling component Dishevelled 2. Within Shroom3gt/gt hearts we demonstrate disrupted terminal PCP components, actomyosin cytoskeleton, cardiomyocyte polarity, organization, proliferation and morphology. Together, these data demonstrate SHROOM3 functions during cardiac development as an actomyosin cytoskeleton effector downstream of PCP signaling, revealing SHROOM3’s novel role in cardiac development and CHD.

Noncanonical Wnt planar cell polarity signaling in lung development and disease.

The planar cell polarity (PCP) signaling pathway is a potent developmental regulator of directional cell behaviors such as migration, asymmetric division and morphological polarization that are critical for shaping the body axis and the complex three-dimensional architecture of tissues and organs. PCP is considered a noncanonical Wnt pathway due to the involvement of Wnt ligands and Frizzled family receptors in the absence of the beta-catenin driven gene expression observed in the canonical Wnt cascade. At the heart of the PCP mechanism are protein complexes capable of generating molecular asymmetries within cells along a tissue-wide axis that are translated into polarized actin and microtubule cytoskeletal dynamics. PCP has emerged as an important regulator of developmental, homeostatic and disease processes in the respiratory system. It acts along other signaling pathways to create the elaborately branched structure of the lung by controlling the directional protrusive movements of cells during branching morphogenesis. PCP operates in the airway epithelium to establish and maintain the orientation of respiratory cilia along the airway axis for anatomically directed mucociliary clearance. It also regulates the establishment of the pulmonary vasculature. In adult tissues, PCP dysfunction has been linked to a variety of chronic lung diseases such as cystic fibrosis, chronic obstructive pulmonary disease, and idiopathic pulmonary arterial hypertension, stemming chiefly from the breakdown of proper tissue structure and function and aberrant cell migration during regenerative wound healing. A better understanding of these (impaired) PCP mechanisms is needed to fully harness the therapeutic opportunities of targeting PCP in chronic lung diseases.

Polymorphisms and association of FAT1 gene with wool quality traits in Chinese Merino sheep

FAT atypical cadherin 1 (FAT1) is a member of the atypical cadherin superfamily and is involved in the planar cell polarity signalling pathway which regulates hair follicle morphogenesis, cycling, and orientation. The purpose of this study was to investigate the sheep FAT1 gene expression, polymorphisms and its association with wool quality traits in Chinese Merino sheep. Quantitative real-time PCR analysis showed that the FAT1 mRNA was differentially expressed in the adult skin of Chinese Merino and Kazak sheep. Seven SNPs (termed SNPs 1–7) were identified in exon 2 of sheep FAT1 gene by Sanger sequencing. SNPs 2 and 7 (novel SNPs) were significantly associated with wool crimp (P &amp;lt; 0.05). SNPs 4 and 5 (rs161528993 and rs161528992) were significantly associated with wool fibre length (P &amp;lt; 0.05). SNP 7 was highly significantly associated with average wool fibre diameter (P &amp;lt; 0.01). Similarly, FAT1 haplotypes were significantly associated with wool crimp (P &amp;lt; 0.05), and the haplotypes H1–H3 and H5 were significantly associated with higher wool crimp (P &amp;lt; 0.05). Bioinformatics analysis showed that the wool quality trait-associated SNPs (SNPs 2, 4, 5 and 7) might affect the pre-mRNA splicing by creating or abolishing the binding sites for serine/arginine-rich proteins, and in addition, SNPs 2 and 4 might alter the FAT1 mRNA secondary structure. Our results suggest that FAT1 influences wool quality traits and its SNPs 2, 4, 5 and 7 might be useful markers for marker-assisted selection and sheep breeding.

Prickle isoforms determine handedness of helical morphogenesis.

Subcellular asymmetry directed by the planar cell polarity (PCP) signaling pathway orients numerous morphogenetic events in both invertebrates and vertebrates. Here, we describe a morphogenetic movement in which the intertwined socket and shaft cells of the Drosophila anterior wing margin mechanosensory bristles undergo PCP-directed apical rotation, inducing twisting that results in a helical structure of defined chirality. We show that the Frizzled/Vang PCP signaling module coordinates polarity among and between bristles and surrounding cells to direct this rotation. Furthermore, we show that dynamic interplay between two isoforms of the Prickle protein determines right- or left-handed bristle morphogenesis. We provide evidence that, Frizzled/Vang signaling couples to the Fat/Dachsous PCP directional signal in opposite directions depending on whether Pkpk or Pksple predominates. Dynamic interplay between Pk isoforms is likely to be an important determinant of PCP outcomes in diverse contexts. Similar mechanisms may orient other lateralizing morphogenetic processes.

PLEKHA4/kramer Attenuates Dishevelled Ubiquitination to Modulate Wnt and Planar Cell Polarity Signaling

SUMMARYWnt signaling pathways direct key physiological decisions in development. Here, we establish a role for a pleckstrin homology domain-containing protein, PLEKHA4, as a modulator of signaling strength in Wnt receiving cells. PLEKHA4 oligomerizes into clusters at PI(4,5)P2-rich regions of the plasma membrane and recruits the Cullin-3 (CUL3) E3 ubiquitin ligase substrate adaptor Kelch-like protein 12 (KLHL12) to these assemblies. This recruitment decreases CUL3-KLHL12-mediated polyubiquitination of Dishevelled, a central intermediate in canonical and non-canonical Wnt signaling. Knock down of PLEKHA4 in mammalian cells demonstrates that PLEKHA4 positively regulates canonical and non-canonical Wnt signaling via these effects on the Dishevelled polyubiquitination machinery. In vivo knockout of the Drosophila melanogaster PLEKHA4 homolog, kramer, selectively affects the non-canonical, planar cell polarity (PCP) signaling pathway. We propose that PLEKHA4 tunes the sensitivities of cells toward the stimulation of Wnt or PCP signaling by sequestering a key E3 ligase adaptor controlling Dishevelled polyubiquitination within PI(4,5) P2-rich plasma membrane clusters.

Overexpression of Grainyhead-like 3 causes spina bifida and interacts genetically with mutant alleles of Grhl2 and Vangl2 in mice.

The genetic basis of human neural tube defects (NTDs), such as anencephaly and spina bifida (SB), is complex and heterogeneous. Grainyhead-like genes represent candidates for involvement in NTDs based on the presence of SB and exencephaly in mice carrying loss-of-function alleles of Grhl2 or Grhl3. We found that reinstatement of Grhl3 expression, by bacterial artificial chromosome (BAC)-mediated transgenesis, prevents SB in Grhl3-null embryos, as in the Grhl3 hypomorphic curly tail strain. Notably, however, further increase in expression of Grhl3 causes highly penetrant SB. Grhl3 overexpression recapitulates the spinal NTD phenotype of loss-of-function embryos, although the underlying mechanism differs. However, it does not phenocopy other defects of Grhl3-null embryos such as abnormal axial curvature, cranial NTDs (exencephaly) or skin barrier defects, the latter being rescued by the Grhl3-transgene. Grhl2 and Grhl3 can form homodimers and heterodimers, suggesting a possible model in which defects arising from overexpression of Grhl3 result from sequestration of Grhl2 in heterodimers, mimicking Grhl2 loss of function. This hypothesis predicts that increased abundance of Grhl2 would have an ameliorating effect in Grhl3 overexpressing embryo. Instead, we observed a striking additive genetic interaction between Grhl2 and Grhl3 gain-of-function alleles. Severe SB arose in embryos in which both genes were expressed at moderately elevated levels that individually do not cause NTDs. Furthermore, moderate Grhl3 overexpression also interacted with the Vangl2Lp allele to cause SB, demonstrating genetic interaction with the planar cell polarity signalling pathway that is implicated in mouse and human NTDs.

Disruption of Planar Cell Polarity Pathway Attributable to Valproic Acid-Induced Congenital Heart Disease through Hdac3 Participation in Mice.

Background:Valproic acid (VPA) exposure during pregnancy has been proven to contribute to congenital heart disease (CHD). Our previous findings implied that disruption of planar cell polarity (PCP) signaling pathway in cardiomyocytes might be a factor for the cardiac teratogenesis of VPA. In addition, the teratogenic ability of VPA is positively correlated to its histone deacetylase (HDAC) inhibition activity. This study aimed to investigate the effect of the VPA on cardiac morphogenesis, HDAC1/2/3, and PCP key genes (Vangl2/Scrib/Rac1), subsequently screening out the specific HDACs regulating PCP pathway.Methods:VPA was administered to pregnant C57BL mice at 700 mg/kg intraperitoneally on embryonic day 10.5. Dams were sacrificed on E15.5, and death/absorption rates of embryos were evaluated. Embryonic hearts were observed by hematoxylin-eosin staining to identify cardiac abnormalities. H9C2 cells (undifferentiated rat cardiomyoblasts) were transfected with Hdac1/2/3 specific small interfering RNA (siRNA). Based on the results of siRNA transfection, cells were transfected with Hdac3 expression plasmid and subsequently mock-treated or treated with 8.0 mmol/L VPA. Hdac1/2/3 as well as Vangl2/Scrib/Rac1 mRNA and protein levels were determined by real-time quantitative polymerase chain reaction and Western blotting, respectively. Total HDAC activity was detected by colorimetric assay.Results:VPA could induce CHD (P < 0.001) and inhibit mRNA or protein expression of Hdac1/2/3 as well as Vangl2/Scrib in fetal hearts, in association with total Hdac activity repression (all P < 0.05). In vitro, Hdac3 inhibition could significantly decrease Vangl2/Scrib expression (P < 0.01), while knockdown of Hdac1/2 had no influence (P > 0.05); VPA exposure dramatically decreased the expression of Vanlg2/Scrib together with Hdac activity (P < 0.01), while overexpression of Hdac3 could rescue the VPA-induced inhibition (P > 0.05).Conclusion:VPA could inhibit Hdac1/2/3, Vangl2/Scrib, or total Hdac activity both in vitro and in vivo and Hdac3 might participate in the process of VPA-induced cardiac developmental anomalies.

Association mapping reveals candidate loci for resistance and anaemic response to an emerging temperature-driven parasitic disease in a wild salmonid fish.

Even though parasitic infections are often costly or deadly for the host, we know very little which genes influence parasite susceptibility and disease severity. Proliferative kidney disease is an emerging and, at elevated water temperatures, potentially deadly disease of salmonid fishes that is caused by the myxozoan parasite Tetracapsuloides bryosalmonae. By screening >7.6K SNPs in 255 wild brown trout (Salmo trutta) and combining association mapping and Random Forest approaches, we identified several candidate genes for both the parasite resistance (inverse of relative parasite load; RPL) and the severe anaemic response to the parasite. The strongest RPL-associated SNP mapped to a noncoding region of the congeneric Atlantic salmon (S.salar) chromosome 10, whereas the second strongest RPL-associated SNP mapped to an intronic region of PRICKLE2 gene, which is a part of the planar cell polarity signalling pathway involved in kidney development. The top SNP associated with anaemia mapped to the intron of the putative PRKAG2 gene. The human ortholog of this gene has been associated with haematocrit and other blood-related traits, making it a prime candidate influencing parasite-triggered anaemia in brown trout. Our findings demonstrate the power of association mapping to pinpoint genomic regions and potential causative genes underlying climate change-driven parasitic disease resistance and severity. Furthermore, this work illustrates the first steps towards dissecting genotype-phenotype links in a wild fish population using closely related genome information.

FijiWingsPolarity: An open source toolkit for semi-automated detection of cell polarity

ABSTRACTEpithelial cells are defined by apical-basal and planar cell polarity (PCP) signaling, the latter of which establishes an orthogonal plane of polarity in the epithelial sheet. PCP signaling is required for normal cell migration, differentiation, stem cell generation and tissue repair, and defects in PCP have been associated with developmental abnormalities, neuropathologies and cancers. While the molecular mechanism of PCP is incompletely understood, the deepest insights have come from Drosophila, where PCP is manifest in hairs and bristles across the adult cuticle and organization of the ommatidia in the eye. Fly wing cells are marked by actin-rich trichome structures produced at the distal edge of each cell in the developing wing epithelium and in a mature wing the trichomes orient collectively in the distal direction. Genetic screens have identified key PCP signaling pathway components that disrupt trichome orientation, which has been measured manually in a tedious and error prone process. Here we describe a set of image processing and pattern-recognition macros that can quantify trichome arrangements in micrographs and mark these directly by color, arrow or colored arrow to indicate trichome location, length and orientation. Nearest neighbor calculations are made to exploit local differences in orientation to better and more reliably detect and highlight local defects in trichome polarity. We demonstrate the use of these tools on trichomes in adult wing preps and on actin-rich developing trichomes in pupal wing epithelia stained with phalloidin. FijiWingsPolarity is freely available and will be of interest to a broad community of fly geneticists studying the effect of gene function on PCP.