The PTK7 and ROR2 Protein Receptors Interact in the Vertebrate WNT/Planar Cell Polarity (PCP) Pathway

The non-canonical WNT/planar cell polarity (WNT/PCP) pathway plays important roles in morphogenetic processes in vertebrates. Among WNT/PCP components, protein tyrosine kinase 7 (PTK7) is a tyrosine kinase receptor with poorly defined functions lacking catalytic activity. Here we show that PTK7 associates with receptor tyrosine kinase-like orphan receptor 2 (ROR2) to form a heterodimeric complex in mammalian cells. We demonstrate that PTK7 and ROR2 physically and functionally interact with the non-canonical WNT5A ligand, leading to JNK activation and cell movements. In the Xenopus embryo, Ptk7 functionally interacts with Ror2 to regulate protocadherin papc expression and morphogenesis. Furthermore, we show that Ptk7 is required for papc activation induced by Wnt5a. Interestingly, we find that Wnt5a stimulates the release of the tagged Ptk7 intracellular domain, which can translocate into the nucleus and activate papc expression. This study reveals novel molecular mechanisms of action of PTK7 in non-canonical WNT/PCP signaling that may promote cell and tissue movements.     

Identification and characterization of a novel chemically induced allele at the planar cell polarity gene Vangl2

Planar cell polarity (PCP) signaling controls a number of morphogenetic processes including convergent extension during gastrulation and neural tube formation. Defects in this pathway cause neural tube defects (NTD), the most common malformations of the central nervous system. The Looptail (Lp) mutant mouse was the first mammalian mutant implicating a PCP gene (Vangl2) in the pathogenesis of NTD. We report on a novel chemically induced mutant allele at Vangl2 called Curly Bob that causes a missense mutation p.Ile268Asn (I268N) in the Vangl2 protein. This mutant segregates in a semi-dominant fashion with heterozygote mice displaying a looped tail appearance, bobbing head, and a circling behavior. Homozygote mutant embryos suffer from a severe form of NTD called craniorachischisis, severe PCP defects in the inner hair cells of the cochlea and posterior cristae, and display a distinct defect in retinal axon guidance. This mutant genetically interacts with the Lp allele (Vangl2 ^S464N) in neural tube development and inner ear hair cell polarity. The Vangl2^I268N protein variant is expressed at very low levels in affected neural and retinal tissues of mutant homozygote embryos. Biochemical studies show that Vangl2^I268N exhibits impaired targeting to the plasma membrane and accumulates in the endoplasmic reticulum. The Vangl2^I268N variant no longer physically interacts with its PCP partner DVL3 and has a reduced protein half-life. This mutant provides an important model for dissecting the role of Vangl2 in the development of the neural tube, establishment of polarity of sensory cells of the auditory and vestibular systems, and retinal axon guidance.

     

Genetic interactions between planar cell polarity genes cause diverse neural tube defects in mice

Neural tube defects (NTDs) are among the commonest and most severe forms of developmental defect, characterized by disruption of the early embryonic events of central nervous system formation. NTDs have long been known to exhibit a strong genetic dependence, yet the identity of the genetic determinants remains largely undiscovered. Initiation of neural tube closure is disrupted in mice homozygous for mutations in planar cell polarity (PCP) pathway genes, providing a strong link between NTDs and PCP signaling. Recently, missense gene variants have been identified in PCP genes in humans with NTDs, although the range of phenotypes is greater than in the mouse mutants. In addition, the sequence variants detected in affected humans are heterozygous, and can often be detected in unaffected individuals. It has been suggested that interactions between multiple heterozygous gene mutations cause the NTDs in humans. To determine the phenotypes produced in double heterozygotes, we bred mice with all three pairwise combinations of Vangl2^Lp, Scrib^Crc and Celsr1^Crsh mutations, the most intensively studied PCP mutants. The majority of double-mutant embryos had open NTDs, with the range of phenotypes including anencephaly and spina bifida, therefore reflecting the defects observed in humans. Strikingly, even on a uniform genetic background, variability in the penetrance and severity of the mutant phenotypes was observed between the different double-heterozygote combinations. Phenotypically, Celsr1^Crsh;Vangl2^Lp;Scrib^Crc triply heterozygous mutants were no more severe than doubly heterozygous or singly homozygous mutants. We propose that some of the variation between double-mutant phenotypes could be attributed to the nature of the protein disruption in each allele: whereas Scrib^Crc is a null mutant and produces no Scrib protein, Celsr1^Crsh and Vangl2^Lp homozygotes both express mutant proteins, consistent with dominant effects. The variable outcomes of these genetic interactions are of direct relevance to human patients and emphasize the importance of performing comprehensive genetic screens in humans.KEY WORDS: Neural tube defects, Planar cell polarity, Genetic interactions, Craniorachischisis, Multiple heterozygosity     

Screening for modulators of spermine tolerance identifies Sky1, the SR protein kinase of Saccharomyces cerevisiae, as a regulator of polyamine transport and ion homeostasis

Although most cells are capable of transporting polyamines, the mechanism that regulates polyamine transport in eukaryotes is still largely unknown. Using a genetic screen for clones capable of restoring spermine sensitivity to spermine-tolerant mutants of Saccharomyces cerevisiae, we have demonstrated that Sky1p, a recently identified SR protein kinase, is a key regulator of polyamine transport. Yeast cells deleted for SKY1 developed tolerance to toxic levels of spermine, while overexpression of Sky1p in wild-type cells increased their sensitivity to spermine. Expression of the wild-type Sky1p but not of a catalytically inactive mutant restored sensitivity to spermine. SKY1 disruption results in dramatically reduced uptake of spermine, spermidine, and putrescine. In addition to spermine tolerance, sky1Δ cells exhibit increased tolerance to lithium and sodium ions but somewhat increased sensitivity to osmotic shock. The observed halotolerance suggests potential regulatory interaction between the transport of polyamines and inorganic ions, as suggested in the case of the Ptk2p, a recently described regulator of polyamine transport. We demonstrate that these two kinases act in two different signaling pathways. While deletion or overexpression of SKY1 did not significantly affect Pma1p activity, the ability of overexpressed Sky1p, Ptk1p, and Ptk2p to increase sensitivity to LiCl depends on the integrity of PPZ1 but not of ENA1.     

SEC14 and Spectrin Domains 1 (Sestd1) and Dapper Antagonist of Catenin 1 (Dact1) Scaffold Proteins Cooperatively Regulate the Van Gogh-like 2 (Vangl2) Four-pass Transmembrane Protein and Planar Cell Polarity (PCP) Pathway during Embryonic Development in Mice

The planar cell polarity (PCP) pathway is a conserved non-canonical (β-catenin-independent) branch of Wnt signaling crucial to embryogenesis, during which it regulates cell polarity and polarized cell movements. Disruption of PCP components in mice, including Vangl2 and Dact1, results in defective neural tube closure and other developmental defects. Here, we show that Sestd1 is a novel binding partner of Vangl2 and Dact1. The Sestd1-Dact1 interface is formed by circumscribed regions of Sestd1 (the carboxyl-terminal region) and Dact1 (the amino-terminal region). Remarkably, we show that loss of Sestd1 precisely phenocopies loss of Dact1 during embryogenesis in mice, leading to a spectrum of birth malformations, including neural tube defects, a shortened and/or curly tail, no genital tubercle, blind-ended colons, hydronephrotic kidneys, and no bladder. Moreover, as with Dact1, a knock-out mutation at the Sestd1 locus exhibits reciprocal genetic rescue interactions during development with a semidominant mutation at the Vangl2 locus. Consistent with this, examination of Wnt pathway activities in Sestd1 mutant mouse embryonic tissue reveals disrupted PCP pathway biochemistry similar to that characterized in Dact1 mutant embryos. The Sestd1 protein is a divergent member of the Trio family of GTPase regulatory proteins that lacks a guanine nucleotide exchange factor domain. Nonetheless, in cell-based assays the Sestd1-Dact1 interaction can induce Rho GTPase activation. Together, our data indicate that Sestd1 cooperates with Dact1 in Vangl2 regulation and in the PCP pathway during mammalian embryonic development.     

An ENU-mutagenesis screen in the mouse: identification of novel developmental gene functions

Background

Mutagenesis screens in the mouse have been proven useful for the identification of novel gene functions and generation of interesting mutant alleles. Here we describe a phenotype-based screen for recessive mutations affecting embryonic development.

Methodology/Principal Findings

Mice were mutagenized with N-ethyl-N-nitrosurea (ENU) and following incrossing the offspring, embryos were analyzed at embryonic day 10.5. Mutant phenotypes that arose in our screen include cardiac and nuchal edema, neural tube defects, situs inversus of the heart, posterior truncation and the absence of limbs and lungs. We isolated amongst others novel mutant alleles for Dll1, Ptprb, Plexin-B2, Fgf10, Wnt3a, Ncx1, Scrib(Scrib, Scribbled homolog [Drosophila]) and Sec24b. We found both nonsense alleles leading to severe protein truncations and mutants with single-amino acid substitutions that are informative at a molecular level. Novel findings include an ectopic neural tube in our Dll1 mutant and lung defects in the planar cell polarity mutants for Sec24b and Scrib.

Conclusions/Significance

Using a forward genetics approach, we have generated a number of novel mutant alleles that are linked to disturbed morphogenesis during development.     

Loss of all three calreticulins, CRT1, CRT2 and CRT3, causes enhanced sensitivity to water stress in Arabidopsis

Key message

The calreticulin triple knockout mutant shows growth defects in response to abiotic stress.

Abstract

The endoplasmic reticulum (ER) is an essential organelle that is responsible for the folding and maturation of proteins. During ER stress, unfolded protein aggregates accumulate in the cell, leading to the unfolded protein response (UPR). The UPR up-regulates the expression of ER-stress-responsive genes encoding calreticulin (CRT), an ER-localized Ca²⁺-binding protein. To understand the function of plant CRTs, we generated a triple knockout mutant, t123, which lacks CRT1, CRT2 and CRT3 and examined the roles of calreticulins in abiotic stress tolerance. A triple knockout mutant increased sensitivity to water stress which implies that calreticulins are involved in the Arabidopsis response to water stress. We identified that the cyclophilin AtCYP21-2, which is located in the ER, was specifically enhanced in the t123 mutants. Seed germination of the atcyp21-1 mutant was retarded by water stress. Taken together, these results suggest that regulatory proteins that serve to protect plants from water stress are folded properly in part with the help of calreticulins. The AtCYP21-2 may also participate in this protein-folding process in association with calreticulins.     

Planar Cell Polarity Signaling in Collective Cell Movements During Morphogenesis and Disease

Collective and directed cell movements are crucial for diverse developmental processes in the animal kingdom, but they are also involved in wound repair and disease. During these processes groups of cells are oriented within the tissue plane, which is referred to as planar cell polarity (PCP). This requires a tight regulation that is in part conducted by the PCP pathway. Although this pathway was initially characterized in flies, subsequent studies in vertebrates revealed a set of conserved core factors but also effector molecules and signal modulators, which build the fundamental PCP machinery. The PCP pathway in Drosophila regulates several developmental processes involving collective cell movements such as border cell migration during oogenesis, ommatidial rotation during eye development, and embryonic dorsal closure. During vertebrate embryogenesis, PCP signaling also controls collective and directed cell movements including convergent extension during gastrulation, neural tube closure, neural crest cell migration, or heart morphogenesis. Similarly, PCP signaling is linked to processes such as wound repair, and cancer invasion and metastasis in adults. As a consequence, disruption of PCP signaling leads to pathological conditions. In this review, we will summarize recent findings about the role of PCP signaling in collective cell movements in flies and vertebrates. In addition, we will focus on how studies in Drosophila have been relevant to our understanding of the PCP molecular machinery and will describe several developmental defects and human disorders in which PCP signaling is compromised. Therefore, new discoveries about the contribution of this pathway to collective cell movements could provide new potential diagnostic and therapeutic targets for these disorders.     

The Wnt Coreceptor Ryk Regulates Wnt/Planar Cell Polarity by Modulating the Degradation of the Core Planar Cell Polarity Component Vangl2

The Wnt signaling pathways control many critical developmental and adult physiological processes. In vertebrates, one fundamentally important function of Wnts is to provide directional information by regulating the evolutionarily conserved planar cell polarity (PCP) pathway during embryonic morphogenesis. However, despite the critical roles of Wnts and PCP in vertebrate development and disease, little is known about the molecular mechanisms underlying Wnt regulation of PCP. Here, we have found that the receptor-like tyrosine kinase (Ryk), a Wnt5a-binding protein required in axon guidance, regulates PCP signaling. We show that Ryk interacts with Vangl2 genetically and biochemically, and such interaction is potentiated by Wnt5a. Loss of Ryk in a Vangl2^+/− background results in classic PCP defects, including open neural tube, misalignment of sensory hair cells in the inner ear, and shortened long bones in the limbs. Complete loss of both Ryk and Vangl2 results in more severe phenotypes that resemble the Wnt5a^−/− mutant in many aspects such as shortened anterior-posterior body axis, limb, and frontonasal process. Our data identify the Wnt5a-binding protein Ryk as a general regulator of the mammalian Wnt/PCP signaling pathway. We show that Ryk transduces Wnt5a signaling by forming a complex with Vangl2 and that Ryk regulates PCP by at least in part promoting Vangl2 stability. As human mutations in WNT5A and VANGL2 are found to cause Robinow syndrome and neural tube defects, respectively, our results further suggest that human mutations in RYK may also be involved in these diseases.     

Murine Dishevelled 3 Functions in Redundant Pathways with Dishevelled 1 and 2 in Normal Cardiac Outflow Tract,Cochlea, and Neural Tube Development

Dishevelled (Dvl) proteins are important signaling components of both the canonical β-catenin/Wnt pathway, which controls cell proliferation and patterning, and the planar cell polarity (PCP) pathway, which coordinates cell polarity within a sheet of cells and also directs convergent extension cell (CE) movements that produce narrowing and elongation of the tissue. Three mammalian Dvl genes have been identified and the developmental roles of Dvl1 and Dvl2 were previously determined. Here, we identify the functions of Dvl3 in development and provide evidence of functional redundancy among the three murine Dvls. Dvl3 ^−/− mice died perinatally with cardiac outflow tract abnormalities, including double outlet right ventricle and persistent truncus arteriosis. These mutants also displayed a misorientated stereocilia in the organ of Corti, a phenotype that was enhanced with the additional loss of a single allele of the PCP component Vangl2/Ltap (LtapLp/+). Although neurulation appeared normal in both Dvl3 ^−/− and LtapLp/+ mutants, Dvl3 ^+/−;LtapLp/+ combined mutants displayed incomplete neural tube closure. Importantly, we show that many of the roles of Dvl3 are also shared by Dvl1 and Dvl2. More severe phenotypes were observed in Dvl3 mutants with the deficiency of another Dvl, and increasing Dvl dosage genetically with Dvl transgenes demonstrated the ability of Dvls to compensate for each other to enable normal development. Interestingly, global canonical Wnt signaling appeared largely unaffected in the double Dvl mutants, suggesting that low Dvl levels are sufficient for functional canonical Wnt signals. In summary, we demonstrate that Dvl3 is required for cardiac outflow tract development and describe its importance in the PCP pathway during neurulation and cochlea development. Finally, we establish several developmental processes in which the three Dvls are functionally redundant.     

An FGF3-BMP Signaling Axis Regulates Caudal Neural Tube Closure,Neural Crest Specification and Anterior-Posterior Axis Extension

During vertebrate axis extension, adjacent tissue layers undergo profound morphological changes: within the neuroepithelium, neural tube closure and neural crest formation are occurring, while within the paraxial mesoderm somites are segmenting from the presomitic mesoderm (PSM). Little is known about the signals between these tissues that regulate their coordinated morphogenesis. Here, we analyze the posterior axis truncation of mouse Fgf3 null homozygotes and demonstrate that the earliest role of PSM-derived FGF3 is to regulate BMP signals in the adjacent neuroepithelium. FGF3 loss causes elevated BMP signals leading to increased neuroepithelium proliferation, delay in neural tube closure and premature neural crest specification. We demonstrate that elevated BMP4 depletes PSM progenitors in vitro, phenocopying the Fgf3 mutant, suggesting that excessive BMP signals cause the Fgf3 axis defect. To test this in vivo we increased BMP signaling in Fgf3 mutants by removing one copy of Noggin, which encodes a BMP antagonist. In such mutants, all parameters of the Fgf3 phenotype were exacerbated: neural tube closure delay, premature neural crest specification, and premature axis termination. Conversely, genetically decreasing BMP signaling in Fgf3 mutants, via loss of BMP receptor activity, alleviates morphological defects. Aberrant apoptosis is observed in the Fgf3 mutant tailbud. However, we demonstrate that cell death does not cause the Fgf3 phenotype: blocking apoptosis via deletion of pro-apoptotic genes surprisingly increases all Fgf3 defects including causing spina bifida. We demonstrate that this counterintuitive consequence of blocking apoptosis is caused by the increased survival of BMP-producing cells in the neuroepithelium. Thus, we show that FGF3 in the caudal vertebrate embryo regulates BMP signaling in the neuroepithelium, which in turn regulates neural tube closure, neural crest specification and axis termination. Uncovering this FGF3-BMP signaling axis is a major advance toward understanding how these tissue layers interact during axis extension with important implications in human disease.     

Circletail, a new mouse mutant with severe neural tube defects: chromosomal localization and interaction with the loop-tail mutation.

Circletail (Crc) is a new mouse mutant that exhibits a severe form of neural tube defect, craniorachischisis, in which almost the entire neural tube fails to close. This phenotype is seen in very few other mutants, the best characterized of which is loop-tail (Ltap(Lp), referred to hereafter as Lp). We tested the possibility of allelism between Lp and Crc by intercrossing Lp/+ and Crc/+mice. A proportion of double heterozygotes (Lp/+,Crc/+) exhibit craniorachischisis, revealing failure of complementation. However, genetic analysis shows that Crc is not linked to the markers that flank the Lp locus and cannot, therefore, be an allele of Lp. A genome-wide scan has localized the Crc gene to a region of 8.8 cM on central chromosome 15. Partial penetrance of the craniorachischisis phenotype in Crc/+,Lp/+double heterozygotes suggests the existence of a third, unlinked genetic locus that influences the interaction between Crc and Lp.     

The mouse Wnt/PCP protein Vangl2 is necessary for migration of facial branchiomotor neurons, and functions independently of Dishevelled

During development, facial branchiomotor (FBM) neurons, which innervate muscles in the vertebrate head, migrate caudally and radially within the brainstem to form a motor nucleus at the pial surface. Several components of the Wnt/planar cell polarity (PCP) pathway, including the transmembrane protein Vangl2, regulate caudal migration of FBM neurons in zebrafish, but their roles in neuronal migration in mouse have not been investigated in detail. Therefore, we analyzed FBM neuron migration in mouse looptail (Lp) mutants, in which Vangl2 is inactivated. In Vangl2(Lp/+) and Vangl2(Lp/Lp) embryos, FBM neurons failed to migrate caudally from rhombomere (r) 4 into r6. Although caudal migration was largely blocked, many FBM neurons underwent normal radial migration to the pial surface of the neural tube. In addition, hindbrain patterning and FBM progenitor specification were intact, and FBM neurons did not transfate into other non-migratory neuron types, indicating a specific effect on caudal migration. Since loss-of-function in some zebrafish Wnt/PCP genes does not affect caudal migration of FBM neurons, we tested whether this was also the case in mouse. Embryos null for Ptk7, a regulator of PCP signaling, had severe defects in caudal migration of FBM neurons. However, FBM neurons migrated normally in Dishevelled (Dvl) 1/2 double mutants, and in zebrafish embryos with disrupted Dvl signaling, suggesting that Dvl function is essentially dispensable for FBM neuron caudal migration. Consistent with this, loss of Dvl2 function in Vangl2(Lp/+) embryos did not exacerbate the Vangl2(Lp/+) neuronal migration phenotype. These data indicate that caudal migration of FBM neurons is regulated by multiple components of the Wnt/PCP pathway, but, importantly, may not require Dishevelled function. Interestingly, genetic-interaction experiments suggest that rostral FBM neuron migration, which is normally suppressed, depends upon Dvl function.     

The GPSM2/LGN GoLoco motifs are essential for hearing

The planar cell polarity (PCP) pathway is responsible for polarizing and orienting cochlear hair cells during development through movement of a primary cilium, the kinocilium. GPSM2/LGN, a mitotic spindle-orienting protein associated with deafness in humans, is a PCP effector involved in kinocilium migration. Here, we link human and mouse truncating mutations in the GPSM2/LGN gene, both leading to hearing loss. The human variant, p.(Trp326*), was identified by targeted genomic enrichment of genes associated with deafness, followed by massively parallel sequencing. Lgn ^ΔC mice, with a targeted deletion truncating the C-terminal GoLoco motifs, are profoundly deaf and show misorientation of the hair bundle and severe malformations in stereocilia shape that deteriorates over time. Full-length protein levels are greatly reduced in mutant mice, with upregulated mRNA levels. The truncated Lgn ^ΔC allele is translated in vitro, suggesting that mutant mice may have partially functioning Lgn. Gαi and aPKC, known to function in the same pathway as Lgn, are dependent on Lgn for proper localization. The polarization of core PCP proteins is not affected in Lgn mutants; however, Lgn and Gαi are misoriented in a PCP mutant, supporting the role of Lgn as a PCP effector. The kinocilium, previously shown to be dependent on Lgn for robust localization, is essential for proper localization of Lgn, as well as Gαi and aPKC, suggesting that cilium function plays a role in positioning of apical proteins. Taken together, our data provide a mechanism for the loss of hearing found in human patients with GPSM2/LGN variants.     

Acquisition of rifabutin resistance by a rifampicin resistant mutant of Mycobacterium tuberculosis involves an unusual spectrum of mutations and elevated frequency

Background

Mutations in a small region of the rpoB gene are responsible for most rifamycin resistance in Mycobacterium tuberculosis. In this study we have sequentially generated resistant strains to first rifampicin and then rifabutin. Portions of the rpoB gene were sequenced from 131 randomly selected mutants. Second round selection resulted in a changed frequency of specific mutations.

Methods

Mycobacterium tuberculosis (strain Mtb72) rifamycin resistant mutants were selected in vitro with either rifampicin or rifabutin. One mutant R190 (rpoB S522L) selected with rifampicin had a rifampicin MIC of 32 μg/ml but remained sensitive to rifabutin (MIC<0.8 μg/ml). This mutant was subjected to a second round of selection with rifabutin.

Results

All 105 first round resistant mutants derived from the parent strain (Mtb72) screened acquired mutations within the 81 bp rpoB hotspot. When the rifampicin resistant but rifabutin sensitive S522L mutant was subjected to a second round of selection, single additional rpoB mutations were identified in 24 (92%) of 26 second round mutants studied, but 14 (54%) of these strains contained mutations outside the 81 bp hotspot (codons 144, 146, 148, 505). Additionally, spontaneous rifabutin resistant mutants were produced at >10 times the frequency by the S522L mutant than the parent strain.

Conclusion

First round selection of mutation S522L with rifampicin increased the frequency and changed the spectrum of mutations identified after selection with rifabutin.