Cell Polarity Gene Research Articles

Perturbation Of Gpsm2/Lgn Enhances Haematopoietic Stem Cell Function

A gain of function screen of candidate cell polarity genes identified the gene, Gpsm2 (G protein signaling modulator 2) also known as LGN or Pins, as an enhancer of haematopoietic stem cell (HSC) function. Mammalian Gpsm2 comprises an N-terminal domain composed of ten Leucine–Glycine–Asparagine (LGN) repeats within seven to eight tetratricopeptide motifs, which functions to bind NuMA (Nuclear Mitotic Apparatus) and a C-terminal domain containing four GoLoco repeats that modulates guanine nucleotide exchange via Gai interaction. In effect, Gpsm2 is an integral component that coordinates G protein signalling to mitotic spindle orientation during cell division. In multiple independent experiments, Gpsm2-transduced HSCs and cultured for 7 days in vitro compared to vector-transduced HSCs showed enhanced HSC repopulation out to 20 weeks-plus post transplant. At these time points, bone marrow and thymic DNA from multiple recipient mice were analysed via Southern blots with a GFP probe. This showed Gpsm2-transduced HSCs retained multipotency but that only one out of a minimum of 12 clones had undergone in vitro symmetrical expansion. Current limiting dilution assay will assess quantitatively this relative absence of HSC expansion. Although in vitro assays with Gpsm2-transduced bone marrow cells showed reduced colony formation and slightly attenuated cell proliferation, limiting dilution assays in secondary transplants showed Gpsm2-transduced HSCs maintained self-renewal ability together with a significant (p=0.009) 4-fold increase in HSC frequency compared to vector-transduced HSCs. This presumably reflects the importance of the interaction between Gpsm2-transduced HSCs within the niche in vivo. A mouse mutant lacking all GoLoco repeats of the C-terminal domain (LGN deltaC) is viable, where analyses of the neuroprogenitors of this mouse revealed altered asymmetrical versus symmetrical cell divisions without a detrimental effect on neuronal production (Konno et al. Nat Cell Biol 2008). A subsequent study of the role of LGN in epidermal development identified that a significant (>80%) knockdown of LGN via shRNA also altered asymmetrical cell divisions of epidermal stem-progenitor cells, however with a functional loss of skin barrier (Williams et al. Nature 2011). This defect could be partially rescued by the LGN N-terminal protein suggesting the LGN deltaC mouse still has functional, albeit, incomplete LGN/Gpsm2 function. We have analysed haematopoiesis in the LGN deltaC mouse. There were no significant differences in wild-type versus homozygous LGN deltaC LSK numbers, in vitro colony formation or in vivo CFU-spleen assays. However, limiting dilution assays showed a 7-fold increase (p=0.02) in HSC frequency in the LGN deltaC mouse, and competitive CD45.2/CD45.1 transplantations analysed at 16 weeks post transplant showed CD45.2 LGN deltaC HSCs reconstituted significantly (p=0.015) better compared to CD45.2 wild-type HSCs. On the premise that the Gpsm2 function of controlling somatic stem cell fate is conserved, we are investigating the hypothesise that the mechanism of perturbation of Gpsm2 enhancement of HSC function is through altered HSC asymmetric and symmetric divisional fate. Disclosures:No relevant conflicts of interest to declare.

Src42A modulates tumor invasion and cell death via Ben/dUev1a-mediated JNK activation in Drosophila

Loss of the cell polarity gene could cooperate with oncogenic Ras to drive tumor growth and invasion, which critically depends on the c-Jun N-terminal Kinase (JNK) signaling pathway in Drosophila. By performing a genetic screen, we have identified Src42A, the ortholog of mammalian Src, as a key modulator of both RasV12/lgl−/− triggered tumor invasion and loss of cell polarity gene-induced cell migration. Our genetic study further demonstrated that the Bendless (Ben)/dUev1a ubiquitin E2 complex is an essential regulator of Src42A-induced, JNK-mediated cell migration. Furthermore, we showed that ectopic Ben/dUev1a expression induced invasive cell migration along with increased MMP1 production in wing disc epithelia. Moreover, Ben/dUev1a could cooperate with RasV12 to promote tumor overgrowth and invasion. In addition, we found that the Ben/dUev1a complex is required for ectopic Src42A-triggered cell death and endogenous Src42A-dependent thorax closure. Our data not only provide a mechanistic insight into the role of Src in development and disease but also propose a potential oncogenic function for Ubc13 and Uev1a, the mammalian homologs of Ben and dUev1a.

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of <em>Drosophila melanogaster</em>

Drosophila has illuminated our understanding of the genetic basis of normal development and disease for the past several decades and today it continues to contribute immensely to our understanding of complex diseases 1-7. Progression of tumors from a benign to a metastatic state is a complex event 8 and has been modeled in Drosophila to help us better understand the genetic basis of this disease 9. Here I present a simple protocol to genetically induce, observe and then analyze the progression of tumors in Drosophila larvae. The tumor induction technique is based on the MARCM system 10 and exploits the cooperation between an activated oncogene, RasV12 and loss of cell polarity genes (scribbled, discs large and lethal giant larvae) to generate invasive tumors 9. I demonstrate how these tumors can be visualized in the intact larvae and then how these can be dissected out for further analysis. The simplified protocol presented here should make it possible for this technique to be utilized by investigators interested in understanding the role of a gene in tumor invasion.

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of <em>Drosophila melanogaster</em>

Drosophila has illuminated our understanding of the genetic basis of normal development and disease for the past several decades and today it continues to contribute immensely to our understanding of complex diseases (1-7). Progression of tumors from a benign to a metastatic state is a complex event (8) and has been modeled in Drosophila to help us better understand the genetic basis of this disease (9). Here I present a simple protocol to genetically induce, observe and then analyze the progression of tumors in Drosophila larvae. The tumor induction technique is based on the MARCM system (10) and exploits the cooperation between an activated oncogene, Ras(V12) and loss of cell polarity genes (scribbled, discs large and lethal giant larvae) to generate invasive tumors (9). I demonstrate how these tumors can be visualized in the intact larvae and then how these can be dissected out for further analysis. The simplified protocol presented here should make it possible for this technique to be utilized by investigators interested in understanding the role of a gene in tumor invasion.

Postnatal Refinement of Auditory Hair Cell Planar Polarity Deficits Occurs in the Absence of Vangl2

The distinctive planar polarity of auditory hair cells is evident in the polarized organization of the stereociliary bundle. Mutations in the core planar cell polarity gene Van Gogh-like 2 (Vangl2) result in hair cells that fail to properly orient their stereociliary bundles along the mediolateral axis of the cochlea. The severity of this phenotype is graded along the length of the cochlea, similar to the hair cell differentiation gradient, suggesting that an active refinement process corrects planar polarity phenotypes in Vangl2 knock-out (KO) mice. Because Vangl2 gene deletions are lethal, Vangl2 conditional knock-outs (CKOs) were generated to test this hypothesis. When crossed with Pax2-Cre, Vangl2 is deleted from the inner ear, yielding planar polarity phenotypes similar to Vangl2 KOs at late embryonic stages except that Vangl2 CKO mice are viable and do not have craniorachischisis like Vangl2 KOs. Quantification of planar polarity deficits through postnatal development demonstrates the activity of a Vangl2-independent refinement process that rescues the planar polarity phenotype within 10 d of birth. In contrast, the Pax2-Cre;Vangl2 CKO has profound changes in the shape and distribution of outer pillar cell and Deiters' cell phalangeal processes that are not corrected during the period of planar polarity refinement. Auditory brainstem response analyses of adult mice show a 10-15 dB shift in auditory threshold, and distortion product otoacoustic emission measurements indicate that this mild hearing deficit is of cochlear origin. Together, these data demonstrate a Vangl2-independent refinement mechanism that actively reorients auditory stereociliary bundles and reveals an unexpected role of Vangl2 during supporting cell morphogenesis.

Mutations in Planar Cell Polarity Gene SCRIB Are Associated with Spina Bifida

Neural tube defects (NTDs) (OMIM #182940) including anencephaly, spina bifida and craniorachischisis, are severe congenital malformations that affect 0.5–1 in 1,000 live births in the United States, with varying prevalence around the world. Mutations in planar cell polarity (PCP) genes are believed to cause a variety of NTDs in both mice and humans. SCRIB is a PCP-associated gene. Mice that are homozygous for the Scrib p.I285K and circletail (Crc) mutations, present with the most severe form of NTDs, namely craniorachischisis. A recent study reported that mutations in SCRIB were associated with craniorachischisis in humans, but whether SCRIB mutations contribute to increased spina bifida risk is still unknown. We sequenced the SCRIB gene in 192 infants with spina bifida and 190 healthy controls. Among the spina bifida patients, we identified five novel missense mutations that were predicted-to-be-deleterious by the PolyPhen software. Of these five mutations, three of them (p.P1043L, p.P1332L, p.L1520R) significantly affected the subcellular localization of SCRIB. In addition, we demonstrated that the craniorachischisis mouse line-90 mutation I285K, also affected SCRIB subcellular localization. In contrast, only one novel missense mutation (p.A1257T) was detected in control samples, and it was predicted to be benign. This study demonstrated that rare deleterious mutations of SCRIB may contribute to the multifactorial risk for human spina bifida.

WS4.6 MicroRNA-30s negatively regulate planar cell polarity genes in human cystic fibrosis bronchial epithelial cells

Objectives: Mucociliary clearance (MCC), abnormal in CF, is physiologically regulated by epithelial ion transport together with mechanical phenomenon such as cilia movement and cough. Planar Cell Polarity (PCP) is a tightly controlled protein network that drives ciliogenesis and cilia function. Cilia structure and function have been studies in CF. Although the majority of these studies showed no structural abnormality and a normal cilia beat frequency (CBF), it has also been showed that ciliary disorientation, rather than ultrastructural abnormalities or slow CBF, may occur secondary to lung inflammation and result in delayed MCC. We hypothesized that CF HBEs may display abnormalities in PCP network which could further impair coordinated cilia function in the plan of the epithelium. Methods: We profiled PCP genes expression by PCR in HBEs. By quantitative RT-PCR, we determined that expression of CELSR3 and Vangl-1 (Van-Gogh like 1) is down-regulated in CF (F508del/F508del) HBEs as compared to NL cells. In contrast, Fz3, Fz6, Pk1 and Vangl-2 genes expression is upregulated in CF cells. In silico analysis revealed that Vangl-1 and Vangl-2, Pk1, Dvl1, Dvl2, and CELSR3 are putative targets of miR-30 family. Expression of miR-30a, miR-30b, miR-30c, miR-30d and miR-30e is elevated is CF-HBEs. Knock-down of miR-30s expression in CF-HBEs significantly increased expression of CELSR3, Fz6, Vangl-1, Pk1, Dvl 1 and Dvl2 but did not modify expression of Fz3, Vangl-2 and Dvl3. Conclusion: Our results validate several PCP genes as miR-30s target genes and suggest that decreasing miR-30s expression in CF HBEs could modulate cilia function through restoration of PCP genes expression.

Abstract 3795: Vangl2 and Prickle1 expression, interaction and regulation of Wnt/PCP signaling in medulloblastoma and neuroblastoma.

Abstract Background /Aims: Wnt are a family of intercellular signaling factors that regulates a wide range of functions during embryonic development including cell proliferation, cell fate determination, differentiation, cell polarity and cell migration. They bind to Frizzled receptors and thereafter proceed via Dishevelled to several intracellular pathways known as the canonical and non-canonical signaling pathways. Activation of the canonical pathway results in an accumulation of cytoplasmic beta-catenin levels. Increased Wnt/beta-catenin signaling is associated with many forms of cancer, including the embryonic neural childhood tumors medulloblastoma and neuroblastoma. The non-canonical Wnt signaling pathways can inhibit the canonical signaling pathway, although the mechanism is still unclear. In this study, we investigate the roles of Prickle1 and Van Gogh-Like 2 (Vangl2), two planar cell polarity (PCP) genes in regulation of the cytoskeleton, Wnt/beta-catenin activity and tumorigenicity in neuroblastoma and medulloblastoma. Methods: mRNA and protein expression of human Prickle1, Vangl2 and beta-catenin were examined in neuroblastoma and medulloblastoma cell lines. siRNA and cDNA transfection were performed together with co-immunoprecipitation and immunocytochemistry. Results and conclusions: We show that Prickle1 and Vangl2 are expressed in neuroblastoma and medulloblastoma cell lines at different levels. Decreased Prickle1 expression correlates with increased levels of Wnt/beta-catenin in both medullo- and neuroblastoma cell lines, demonstrating a downregulation of PCP components and a possible activation of the canonical Wnt signaling pathway. Immunoprecipitation demonstrated that Prickle1 is present in complex with Vangl2. However, the exact mode of interaction between Prickle1 and Vangl2 remains to be eluciated. Microarray analysis revealed that low expressions of Prickle1 or Vangl2 are both associated significantly worse clinical outcome in neuroblastoma. Additionally, siRNA experiments revealed that knockdown of Prickle1 and Vangl2 increased cell proliferation of neuroblastoma cells. Taken together this indicate that the non-canonical Wnt pathway may be an interesting therapeutic target in embryanol neural childhood tumors. Citation Format: Cecilia H. Dyberg, Panagiotis Papachristou, Hugo Lagercrantz, John I Johnsen, Malin Wickström. Vangl2 and Prickle1 expression, interaction and regulation of Wnt/PCP signaling in medulloblastoma and neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3795. doi:10.1158/1538-7445.AM2013-3795

Planar cell polarity genes control the connectivity of enteric neurons

A highly complex network of intrinsic enteric neurons is required for the digestive and homeostatic functions of the gut. Nevertheless, the genetic and molecular mechanisms that regulate their assembly into functional neuronal circuits are currently unknown. Here we report that the planar cell polarity (PCP) genes Celsr3 and Fzd3 are required during murine embryogenesis to specifically control the guidance and growth of enteric neuronal projections relative to the longitudinal and radial gut axes. Ablation of these genes disrupts the normal organization of nascent neuronal projections, leading to subtle changes of axonal tract configuration in the mature enteric nervous system (ENS), but profound abnormalities in gastrointestinal motility. Our data argue that PCP-dependent modules of connectivity established at early stages of enteric neurogenesis control gastrointestinal function in adult animals and provide the first evidence that developmental deficits in ENS wiring may contribute to the pathogenesis of idiopathic bowel disorders.

Cancer driver–passenger distinction via sporadic human and dog cancer comparison: a proof-of-principle study with colorectal cancer

Herein we report a proof of principle study illustrating a novel dog-human comparison strategy that addresses a central aim of cancer research, namely cancer driver–passenger distinction. We previously demonstrated that sporadic canine colorectal cancers (CRCs) share similar molecular pathogenesis mechanisms as their human counterparts. In this study, we compared the genome-wide copy number abnormalities between 29 human- and 10 canine sporadic CRCs. This led to the identification of 73 driver candidate genes (DCGs), altered in both species and with 27 from the whole genome and 46 from dog-human genomic rearrangement breakpoint (GRB) regions, as well as 38 passenger candidate genes (PCGs), altered in humans only and located in GRB regions. We noted that DCGs significantly differ from PCGs in every analysis conducted to assess their cancer relevance and biological functions. Importantly, while PCGs are not enriched in any specific functions, DCGs possess significantly enhanced functionality closely associated with cell proliferation and death regulation, as well as with epithelial cell apicobasal polarity establishment/maintenance. These observations support the notion that, in sporadic CRCs of both species, cell polarity genes not only contribute in preventing cancer cell invasion and spreading, but also likely serve as tumor suppressors by modulating cell growth. This pilot study validates our novel strategy and has uncovered four new potential cell polarity and colorectal tumor suppressor genes (RASA3, NUPL1, DENND5A, and AVL9). Expansion of this study would make more driver-passenger distinctions for cancers with large genomic amplifications or deletions, and address key questions regarding the relationship between cancer pathogenesis and epithelial cell polarity control in mammals.

Copy number variation analysis implicates the cell polarity gene glypican 5 as a human spina bifida candidate gene

Neural tube defects (NTDs) are common birth defects of complex etiology. Family and population-based studies have confirmed a genetic component to NTDs. However, despite more than three decades of research, the genes involved in human NTDs remain largely unknown. We tested the hypothesis that rare copy number variants (CNVs), especially de novo germline CNVs, are a significant risk factor for NTDs. We used array-based comparative genomic hybridization (aCGH) to identify rare CNVs in 128 Caucasian and 61 Hispanic patients with non-syndromic lumbar-sacral myelomeningocele. We also performed aCGH analysis on the parents of affected individuals with rare CNVs where parental DNA was available (42 sets). Among the eight de novo CNVs that we identified, three generated copy number changes of entire genes. One large heterozygous deletion removed 27 genes, including PAX3, a known spina bifida-associated gene. A second CNV altered genes (PGPD8, ZC3H6) for which little is known regarding function or expression. A third heterozygous deletion removed GPC5 and part of GPC6, genes encoding glypicans. Glypicans are proteoglycans that modulate the activity of morphogens such as Sonic Hedgehog (SHH) and bone morphogenetic proteins (BMPs), both of which have been implicated in NTDs. Additionally, glypicans function in the planar cell polarity (PCP) pathway, and several PCP genes have been associated with NTDs. Here, we show that GPC5 orthologs are expressed in the neural tube, and that inhibiting their expression in frog and fish embryos results in NTDs. These results implicate GPC5 as a gene required for normal neural tube development.

A consideration of the evidence that genetic defects in planar cell polarity contribute to the etiology of human neural tube defects

A variety of human birth defects originate in failure of closure of the embryonic neural tube. The genetic cause of the most common nonsyndromic defects, spina bifida (SB) or anencephaly, is considered to be combinations of variants at multiple genes. The genes contributing to the etiology of neural tube closure defects (NTDs) are unknown. Mutations in planar cell polarity (PCP) genes in mice cause a variety of defects including the NTD, craniorachischisis, and sometimes SB or exencephaly (EX); they also demonstrate the role of digenic combinations of PCP mutants in NTDs. Recent studies have sought rare predicted-to-be-deleterious alterations (putative mutations) in coding sequence of PCP genes in human cases with various anomalies of the neural tube. This review summarizes the cumulative results of these studies according to a framework based on the embryopathogenesis of NTDs, and considers some of the insights from the approaches used and the limitations. Rare putative mutations in the PCP genes VANGL2, SCRIB, DACT1, and CELSR1 cumulatively contributed to over 20% of cases with craniorachischisis, a rare defect; no contributing variants were found for PRICKLE1 or PTK7. PCP rare putative mutations had a weaker role in myelomeningocele (SB), being found in approximately 6% of cases and cumulated across CELSR1, FUZ, FZD6, PRICKLE1, VANGL1, and VANGL2. These results demonstrate that PCP gene alterations contribute to the etiology of human NTDs. We recommend that future research should explore other types of PCP gene variant such as regulatory mutations and low frequency (1 to 5%) deleterious polymorphisms.

Planar cell polarity genes, Celsr1-3, in neural development

flamingo is among the 'core' planar cell-polarity genes, protein of which belongs to a unique cadherin subfamily. In contrast to the classic cadherins, composed of several extracellular cadherin repeats, one transmembrane domain and one cytoplasmic segment linked to catenin binding, Drosophila Flamingo has seven transmembrane segments and a cytoplasmic tail with no catenin-binding sequence. In Drosophila, Flamingo has pleotropic roles in controlling epithelial polarity and neuronal morphogenesis. Three mammalian orthologs of flamingo, Celsr1-3, are widely expressed in the nervous system. Recent work has shown that Celsr1-3 play important roles in neural development, such as in axon guidance, neuronal migration, and cilium polarity. Celsr1-3 single-gene knockout mice exhibit different phenotypes, but there are cooperative interactions among these genes.

Caudal-related Homeobox (Cdx) Protein-dependent Integration of Canonical Wnt Signaling on Paired-box 3 (Pax3) Neural Crest Enhancer

One of the earliest events in neural crest development takes place at the neural plate border and consists in the induction of Pax3 expression by posteriorizing Wnt·β-catenin signaling. The molecular mechanism of this regulation is not well understood, but several observations suggest a role for posteriorizing Cdx transcription factors (Cdx1/2/4) in this process. Cdx genes are known as integrators of posteriorizing signals from Wnt, retinoic acid, and FGF pathways. In this work, we report that Wnt-mediated regulation of murine Pax3 expression is indirect and involves Cdx proteins as intermediates. We show that Pax3 transcripts co-localize with Cdx proteins in the posterior neurectoderm and that neural Pax3 expression is reduced in Cdx1-null embryos. Using Wnt3a-treated P19 cells and neural crest-derived Neuro2a cells, we demonstrate that Pax3 expression is induced by the Wnt-Cdx pathway. Co-transfection analyses, electrophoretic mobility shift assays, chromatin immunoprecipitation, and transgenic studies further indicate that Cdx proteins operate via direct binding to an evolutionarily conserved neural crest enhancer of the Pax3 proximal promoter. Taken together, these results suggest a novel neural function for Cdx proteins within the gene regulatory network controlling neural crest development.

Functional interactions between Fat family cadherins in tissue morphogenesis and planar polarity

The atypical cadherin fat (ft) was originally discovered as a tumor suppressor in Drosophila and later shown to regulate a form of tissue patterning known as planar polarity. In mammals, four ft homologs have been identified (Fat1-4). Recently, we demonstrated that Fat4 plays a role in vertebrate planar polarity. Fat4 has the highest homology to ft, whereas other Fat family members are homologous to the second ft-like gene, ft2. Genetic studies in flies and mice imply significant functional differences between the two groups of Fat cadherins. Here, we demonstrate that Fat family proteins act both synergistically and antagonistically to influence multiple aspects of tissue morphogenesis. We find that Fat1 and Fat4 cooperate during mouse development to control renal tubular elongation, cochlear extension, cranial neural tube formation and patterning of outer hair cells in the cochlea. Similarly, Fat3 and Fat4 synergize to drive vertebral arch fusion at the dorsal midline during caudal vertebra morphogenesis. We provide evidence that these effects depend on conserved interactions with planar polarity signaling components. In flies, the transcriptional co-repressor Atrophin (Atro) physically interacts with Ft and acts as a component of Fat signaling for planar polarity. We find that the mammalian orthologs of atro, Atn1 and Atn2l, modulate Fat4 activity during vertebral arch fusion and renal tubular elongation, respectively. Moreover, Fat4 morphogenetic defects are enhanced by mutations in Vangl2, a 'core' planar cell polarity gene. These studies highlight the wide range and complexity of Fat activities and suggest that a Fat-Atrophin interaction is a conserved element of planar polarity signaling.

Planar polarity genes and inhibition of supernumerary neurites

Planar cell polarity (PCP) genes have recently emerged as important players in sculpting neuronal connections. The bipolar VC neurons display stereotypical differences in axon extension along the anterior-posterior (AP) body axis: VC1–3 and VC6 polarize along the AP axis while VC4 and VC5 polarize along the orthogonal left-right (LR) axis generated by the developing vulva. vang-1 and prkl-1, the worm orthologs of Van Gogh and Prickle, are required to restrict the polarity of neurite emergence to a specific tissue axis. vang-1 and prkl-1 loss results in ectopic VC4 and VC5 neurites extending inappropriately along the AP axis. Conversely, prkl-1 overexpression in VC neurons suppresses neurite formation. These findings suggest that a PCP-like pathway acts to silence or antagonize neuronal responses to polarity cues that would otherwise be permissive for neurite growth.

Role of the planar cell polarity gene CELSR1 in neural tube defects and caudal agenesis

Neural tube defects (NTDs), including anencephaly and spina bifida, have a complex etiology. Defects in the planar cell polarity (PCP) signaling pathway have been strongly associated with NTDs in animal models and human cohorts. In this genetic study, we examined the core PCP gene CELSR1 in NTDs and caudal agenesis cases to determine whether mutations at this gene predispose to these defects. We sequenced the coding region and the exon-intron junctions of CELSR1 in a cohort of 473 patients affected with various forms of open and closed NTDs (412) or caudal agenesis (61). Novel and rare variants (<1%) were genotyped in a cohort of 639 ethnically-matched individuals. The effect of novel missense mutations absent in controls and in public databases on protein function was predicted in silico. We identified in our cohort one nonsense mutation in exon 1 of CELSR1 that truncates the majority of the protein in one patient with NTD and one in-frame 12 bp deletion that removes a putative PKC phosphorylation "SSR" motif in one caudal agenesis patient. We also detected a total of 13 novel missense variants in 12 patients (11 NTDs and 1 caudal agenesis) that were predicted to be pathogenic in silico. We detected novel CELSR1 mutations predicted to be pathogenic in 2.9% of our NTD cohort and 3.3% of our caudal agenesis cohort. Our findings implicate CELSR1 as a risk factor for NTDs or caudal agenesis and provide additional evidence for a pathogenic role of PCP signaling in these malformations.

Planar cell polarity gene mutations in severe neural tube defects

Planar cell polarity gene mutations in severe neural tube defects

Mutations in the planar cell polarity genes CELSR1 and SCRIB are associated with the severe neural tube defect craniorachischisis.

Craniorachischisis (CRN) is a severe neural tube defect (NTD) resulting from failure to initiate closure, leaving the hindbrain and spinal neural tube entirely open. Clues to the genetic basis of this condition come from several mouse models, which harbor mutations in core members of the planar cell polarity (PCP) signaling pathway. Previous studies of humans with CRN failed to identify mutations in the core PCP genes, VANGL1 and VANGL2. Here, we analyzed other key PCP genes: CELSR1, PRICKLE1, PTK7, and SCRIB, with the finding of eight potentially causative mutations in both CELSR1 and SCRIB. Functional effects of these unique or rare human variants were evaluated using known protein-protein interactions as well as subcellular protein localization. While protein interactions were not affected, variants from five of the 36 patients exhibited a profound alteration in subcellular protein localization, with diminution or abolition of trafficking to the plasma membrane. Comparable effects were seen in the crash and spin cycle mouse Celsr1 mutants, and the line-90 mouse Scrib mutant. We conclude that missense variants in CELSR1 and SCRIB may represent a cause of CRN in humans, as in mice, with defective PCP protein trafficking to the plasma membrane a likely pathogenic mechanism.

Role of Spectraplakin in Drosophila Photoreceptor Morphogenesis

BackgroundCrumbs (Crb), a cell polarity gene, has been shown to provide a positional cue for the apical membrane domain and adherens junction during Drosophila photoreceptor morphogenesis. It has recently been found that stable microtubules in developing Drosophila photoreceptors were linked to Crb localization. Coordinated interactions between microtubule and actin cytoskeletons are involved in many polarized cellular processes. Since Spectraplakin is able to bind both microtubule and actin cytoskeletons, the role of Spectraplakin was analyzed in the regulations of apical Crb domain in developing Drosophila photoreceptors.Methodology/Principal FindingsThe localization pattern of Spectraplakin in developing pupal photoreceptors showed a unique intracellular distribution. Spectraplakin localized at rhabdomere terminal web which is at the basal side of the apical Crb or rhabdomere, and in between the adherens junctions. The spectraplakin mutant photoreceptors showed dramatic mislocalizations of Crb, adherens junctions, and the stable microtubules. This role of Spectraplakin in Crb and adherens junction regulation was further supported by spectraplakin's gain-of-function phenotype. Spectraplakin overexpression in photoreceptors caused a cell polarity defect including dramatic mislocalization of Crb, adherens junctions and the stable microtubules in the developing photoreceptors. Furthermore, a strong genetic interaction between spectraplakin and crb was found using a genetic modifier test.Conclusions/SignificanceIn summary, we found a unique localization of Spectraplakin in photoreceptors, and identified the role of spectraplakin in the regulation of the apical Crb domain and adherens junctions through genetic mutational analysis. Our data suggest that Spectraplakin, an actin-microtubule cross-linker, is essential in the apical and adherens junction controls during the photoreceptors morphogenesis.