Zebrafish trilobite identifies new roles for Strabismus in gastrulation and neuronal movements

Embryonic morphogenesis is driven by a suite of cell behaviours, including coordinated shape changes, cellular rearrangements and individual cell migrations, whose molecular determinants are largely unknown. In the zebrafish, Dani rerio, trilobite mutant embryos have defects in gastrulation movements and posterior migration of hindbrain neurons. Here, we have used positional cloning to demonstrate that trilobite mutations disrupt the transmembrane protein Strabismus (Stbm)/Van Gogh (Vang), previously associated with planar cell polarity (PCP) in Drosophila melanogaster, and PCP and canonical Wnt/beta-catenin signalling in vertebrates. Our genetic and molecular analyses argue that during gastrulation, trilobite interacts with the PCP pathway without affecting canonical Wnt signalling. Furthermore, trilobite may regulate neuronal migration independently of PCP molecules. We show that trilobite mediates polarization of distinct movement behaviours. During gastrulation convergence and extension movements, trilobite regulates mediolateral cell polarity underlying effective intercalation and directed dorsal migration at increasing velocities. In the hindbrain, trilobite controls effective migration of branchiomotor neurons towards posterior rhombomeres. Mosaic analyses show trilobite functions cell-autonomously and non-autonomously in gastrulae and the hindbrain. We propose Trilobite/Stbm mediates cellular interactions that confer directionality on distinct movements during vertebrate embryogenesis.     

Human neural tube defects: genetic causes and prevention

Neural tube defects (NTDs) are severe congenital malformations affecting 1-2 in 1,000 live births, whose etiology is multifactorial, involving environmental and genetic factors. NTDs arise as consequence of the failure of fusion of the neural tube early during embryogenesis. NTDs' pathogenesis has been linked to genes involved in folate metabolism, consistent with an epidemiologic evidence that 70% of NTDs can be prevented by maternal periconceptional supplementation. However, polymorphisms in such genes are not linked in all populations, suggesting that other genetic factors and environmental factors could be involved. Animal models have provided crucial mechanistic information and possible candidate genes to explain susceptibility to NTDs. A crucial role has been assigned to the planar cell polarity (PCP) pathway, a highly conserved, non-canonical Wnt-frizzled-dishevelled signaling cascade that plays a key role in establishing and maintaining polarity in the plane of the epithelium and in the process of convergent extension during gastrulation and neurulation in vertebrates. The Loop-tail (Lp) mouse that develops craniorachischisis carry missense mutations in the PCP core gene Vangl2, that is the mammalian homolog of the Drosophila Strabismus/Van gogh (Stbm/Vang). The presence of mutations in human VANGL1 and VANGL2 genes encourages us to extend the investigation to other PCP genes that, with VANGL, play an essential role in neurulation during development.     

Independent mutations in mouse Vangl2 that cause neural tube defects in looptail mice impair interaction with members of the Dishevelled family

Mammalian Vangl1 and Vangl2 are highly conserved membrane proteins that have evolved from a single ancestral protein Strabismus/Van Gogh found in Drosophila. Mutations in the Vangl2 gene cause a neural tube defect (craniorachischisis) characteristic of the looptail (Lp) mouse. Studies in model organisms indicate that Vangl proteins play a key developmental role in establishing planar cell polarity (PCP) and in regulating convergent extension (CE) movements during embryogenesis. The role of Vangl1 in these processes is virtually unknown, and the molecular function of Vangl1 and Vangl2 in PCP and CE is poorly understood. Using a yeast two-hybrid system, glutathione S-transferase pull-down and co-immunoprecipitation assays, we show that both mouse Vangl1 and Vangl2 physically interact with the three members of the cytoplasmic Dishevelled (Dvl) protein family. This interaction is shown to require both the predicted cytoplasmic C-terminal half of Vangl1/2 and a portion of the Dvl protein containing PDZ and DIX domains. In addition, we show that the two known Vangl2 loss-of-function mutations identified in two independent Lp alleles associated with neural tube defects impair binding to Dvl1, Dvl2, and Dvl3. These findings suggest a molecular mechanism for the neural tube defect seen in Lp mice. Our observations indicate that Vangl1 biochemical properties parallel those of Vangl2 and that Vangl1 might, therefore, participate in PCP and CE either in concert with Vangl2 or independently of Vangl2 in discrete cell types.     

Planar cell polarity proteins differentially regulate extracellular matrix organization and assembly during zebrafish gastrulation

Zebrafish gastrulation cell movements occur in the context of dynamic changes in extracellular matrix (ECM) organization and require the concerted action of planar cell polarity (PCP) proteins that regulate cell elongation and mediolateral alignment. Data obtained using Xenopus laevis gastrulae have shown that integrin–fibronectin interactions underlie the formation of polarized cell protrusions necessary for PCP and have implicated PCP proteins themselves as regulators of ECM. By contrast, the relationship between establishment of PCP and ECM assembly/remodeling during zebrafish gastrulation is unclear. We previously showed that zebrafish embryos carrying a null mutation in the four-pass transmembrane PCP protein vang-like 2 (vangl2) exhibit increased matrix metalloproteinase activity and decreased immunolabeling of fibronectin. These data implicated for the first time a core PCP protein in the regulation of pericellular proteolysis of ECM substrates and raised the question of whether other zebrafish PCP proteins also impact ECM organization. In Drosophila melanogaster, the cytoplasmic PCP protein Prickle binds Van Gogh and regulates its function. Here we report that similar to vangl2, loss of zebrafish prickle1a decreases fibronectin protein levels in gastrula embryos. We further show that Prickle1a physically binds Vangl2 and regulates both the subcellular distribution and total protein level of Vangl2. These data suggest that the ability of Prickle1a to impact fibronectin organization is at least partly due to effects on Vangl2. In contrast to loss of either Vangl2 or Prickle1a function, we find that glypican4 (a Wnt co-receptor) and frizzled7 mutant gastrula embryos with disrupted non-canonical Wnt signaling exhibit the opposite phenotype, namely increased fibronectin assembly. Our data show that glypican4 mutants do not have decreased proteolysis of ECM substrates, but instead have increased cell surface cadherin protein expression and increased intercellular adhesion. These data indicate that Wnt/Glypican4/Frizzled signaling regulates ECM assembly through effects on cadherin-mediated cell cohesion. Together, our results demonstrate that zebrafish Vangl2/Prickle1a and non-canonical Wnt/Frizzled signaling have opposing effects on ECM organization underlying PCP and gastrulation cell movements.     

Comparison of phenotypes between different vangl2 mutants demonstrates dominant effects of the Looptail mutation during hair cell development

Experiments utilizing the Looptail mutant mouse, which harbors a missense mutation in the vangl2 gene, have been essential for studies of planar polarity and linking the function of the core planar cell polarity proteins to other developmental signals. Originally described as having dominant phenotypic traits, the molecular interactions underlying the Looptail mutant phenotype are unclear because Vangl2 protein levels are significantly reduced or absent from mutant tissues. Here we introduce a vangl2 knockout mouse and directly compare the severity of the knockout and Looptail mutant phenotypes by intercrossing the two lines and assaying the planar polarity of inner ear hair cells. Overall the vangl2 knockout phenotype is milder than the phenotype of compound mutants carrying both the Looptail and vangl2 knockout alleles. In compound mutants a greater number of hair cells are affected and changes in the orientation of individual hair cells are greater when quantified. We further demonstrate in a heterologous cell system that the protein encoded by the Looptail mutation (Vangl2(S464N)) disrupts delivery of Vangl1 and Vangl2 proteins to the cell surface as a result of oligomer formation between Vangl1 and Vangl2(S464N), or Vangl2 and Vangl2(S464N), coupled to the intracellular retention of Vangl2(S464N). As a result, Vangl1 protein is missing from the apical cell surface of vestibular hair cells in Looptail mutants, but is retained at the apical cell surface of hair cells in vangl2 knockouts. Similarly the distribution of Prickle-like2, a putative Vangl2 interacting protein, is differentially affected in the two mutant lines. In summary, we provide evidence for a direct physical interaction between Vangl1 and Vangl2 through a combination of in vitro and in vivo approaches and propose that this interaction underlies the dominant phenotypic traits associated with the Looptail mutation.     

The PCP protein Vangl2 regulates migration of hindbrain motor neurons by acting in floor plate cells,and independently of cilia function

Vangl2, a core component of the Planar Cell Polarity pathway, is necessary for the caudal migration of Facial Branchiomotor (FBM) neurons in the vertebrate hindbrain. Studies in zebrafish suggest that vangl2 functions largely non-cell autonomously to regulate FBM neuron migration out of rhombomere 4 (r4), but the cell-type within which it acts is not known. Here, we demonstrate that vangl2 functions largely in floor plate cells to regulate caudal neuronal migration. Furthermore, FBM neurons fail to migrate caudally in the mouse Gli2 mutant that lacks the floor plate, suggesting an evolutionarily conserved role for this cell type in neuronal migration. Although hindbrain floor plate cilia are disorganized in vangl2 mutant embryos, cilia appear to be dispensable for neuronal migration. Notably, Vangl2 is enriched in the basolateral, but not apical, membranes of floor plate cells. Taken together, our data suggest strongly that Vangl2 regulates FBM neuron migration by acting in floor plate cells, independently of cilia function.     

Bbs8, together with the planar cell polarity protein Vangl2, is required to establish left-right asymmetry in zebrafish

Laterality defects such as situs inversus are not uncommonly encountered in humans, either in isolation or as part of another syndrome, but can have devastating developmental consequences. The events that break symmetry during early embryogenesis are highly conserved amongst vertebrates and involve the establishment of unidirectional flow by cilia within an organising centre such as the node in mammals or Kupffer's vesicle (KV) in teleosts. Disruption of this flow can lead to the failure to successfully establish left-right asymmetry. The correct apical-posterior cellular position of each node/KV cilium is critical for its optimal radial movement which serves to sweep fluid (and morphogens) in the same direction as its neighbours. Planar cell polarity (PCP) is an important conserved process that governs ciliary position and posterior tilt; however the underlying mechanism by which this occurs remains unclear. Here we show that Bbs8, a ciliary/basal body protein important for intraciliary/flagellar transport and the core PCP protein Vangl2 interact and are required for establishment and maintenance of left-right asymmetry during early embryogenesis in zebrafish. We discovered that loss of bbs8 and vangl2 results in laterality defects due to cilia disruption at the KV. We showed that perturbation of cell polarity following abrogation of vangl2 causes nuclear mislocalisation, implying defective centrosome/basal body migration and apical docking. Moreover, upon loss of bbs8 and vangl2, we observed defective actin organisation. These data suggest that bbs8 and vangl2 act synergistically on cell polarization to establish and maintain the appropriate length and number of cilia in the KV and thereby facilitate correct LR asymmetry.     

VANGL1 rare variants associated with neural tube defects affect convergent extension in zebrafish

In humans, rare non-synonymous variants in the planar cell polarity gene VANGL1 are associated with neural tube defects (NTDs). These variants were hypothesized to be pathogenic based mainly on genetic studies in a large cohort of NTD patients. In this study, we validate the potential pathogenic effect of these mutations in vivo by investigating their effect on convergent extension in zebrafish. Knocking down the expression of tri, the ortholog of Vangl2, using an antisense morpholino (MO), as shown previously, led to a defective convergent extension (CE) manifested by a shortened body axis and widened somites. Co-injection of the human VANGL1 with the tri-MO was able to partially rescue the tri-MO induced phenotype in zebrafish. In contrast, co-injection of two human VANGL1 variants, p.Val239Ile and p.Met328Thr, failed to rescue this phenotype. We next carried out overexpression studies where we measured the ability of the human VANGL1 alleles to induce a CE phenotype when injected at high doses in zebrafish embryos. While overexpressing the wild-type allele led to a severely defective CE, overexpression of either p.Val239Ile or p.Met328Thr variant failed to do so. Results from both tri-MO knockdown/rescue results and overexpression assays suggest that these two variants most likely represent “loss-of-function” alleles that affect protein function during embryonic development. Our study demonstrates a high degree of functional conservation of VANGL genes across evolution and provides a model system for studying potential variants identified in human NTDs.     

Wnt signalling: refocusing on Strabismus

Vertebrate homologues of the Strabismus/van Gogh (stbm/vang) gene have been implicated in patterning and morphogenesis during gastrulation. Recent work shows that stbm/vang is mutated in zebrafish trilobite mutants and that stbm/vang is required for morphogenesis but not patterning during zebrafish gastrulation.     

The wnt receptor ryk plays a role in Mammalian planar cell polarity signaling

Wnts are essential for a wide range of developmental processes, including cell growth, division, and differentiation. Some of these processes signal via the planar cell polarity (PCP) pathway, which is a β-catenin-independent Wnt signaling pathway. Previous studies have shown that Ryk, a member of the receptor tyrosine kinase family, can bind to Wnts. Ryk is required for normal axon guidance and neuronal differentiation during development. Here, we demonstrate that mammalian Ryk interacts with the Wnt/PCP pathway. In vitro analysis showed that the Wnt inhibitory factor domain of Ryk was necessary for Wnt binding. Detailed analysis of two vertebrate model organisms showed Ryk phenotypes consistent with PCP signaling. In zebrafish, gene knockdown using morpholinos revealed a genetic interaction between Ryk and Wnt11 during the PCP pathway-regulated process of embryo convergent extension. Ryk-deficient mouse embryos displayed disrupted polarity of stereociliary hair cells in the cochlea, a characteristic of disturbed PCP signaling. This PCP defect was also observed in mouse embryos that were double heterozygotes for Ryk and Looptail (containing a mutation in the core Wnt/PCP pathway gene Vangl2) but not in either of the single heterozygotes, suggesting a genetic interaction between Ryk and Vangl2. Co-immunoprecipitation studies demonstrated that RYK and VANGL2 proteins form a complex, whereas RYK also activated RhoA, a downstream effector of PCP signaling. Overall, our data suggest an important role for Ryk in Wnt/planar cell polarity signaling during vertebrate development via the Vangl2 signaling pathway, as demonstrated in the mouse cochlea.     

Multiple mechanisms mediate motor neuron migration in the zebrafish hindbrain

The transmembrane protein Van gogh‐like 2 (Vangl2) is a component of the noncanonical Wnt/Planar Cell Polarity (PCP) signaling pathway, and is required for tangential migration of facial branchiomotor neurons (FBMNs) from rhombomere 4 (r4) to r5‐r7 in the vertebrate hindbrain. Since vangl2 is expressed throughout the zebrafish hindbrain, it might also regulate motor neuron migration in other rhombomeres. We tested this hypothesis by examining whether migration of motor neurons out of r2 following ectopic hoxb1b expression was affected in vangl2^? (trilobite) mutants. Hoxb1b specifies r4 identity, and when ectopically expressed transforms r2 to an “r4‐like” compartment. Using time‐lapse imaging, we show that GFP‐expressing motor neurons in the r2/r3 region of a hoxb1b‐overexpressing wild‐type embryo migrate along the anterior‐posterior (AP) axis. Furthermore, these cells express prickle1b (pk1b), a Wnt/PCP gene that is specifically expressed in FBMNs and is essential for their migration. Importantly, GFP‐expressing motor neurons in the r2/r3 region of hoxb1b‐overexpressing trilobite mutants and pk1b morphants often migrate, even though FBMNs in r4 of the same embryos fail to migrate longitudinally (tangentially) into r6 and r7. These observations suggest that tangentially migrating motor neurons in the anterior hindbrain (r1‐r3) can use mechanisms that are independent of vangl2 and pk1b functions. Interestingly, analysis of tri; val double mutants also suggests a role for vangl2‐independent factors in neuronal migration, since the valentino mutation partially suppresses the trilobite mutant migration defect. Together, the hoxb1b and val experiments suggest that multiple mechanisms regulate motor neuron migration along the AP axis of the zebrafish hindbrain. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2010     

Wnt signaling gradients establish planar cell polarity by inducing Vangl2 phosphorylation through Ror2

It is fundamentally important that signaling gradients provide positional information to govern morphogenesis of multicellular organisms. Morphogen gradients can generate different cell types in specific spatial order at distinct threshold concentrations. However, it is largely unknown whether and how signaling gradients also control cell polarities by acting as global cues. Here, we show that Wnt signaling gradient provides directional information to a field of cells. Vangl2, a core component in planar cell polarity, forms Wnt-induced receptor complex with Ror2 to sense Wnt dosages. Wnts dose-dependently induce Vangl2 phosphorylation of serine/threonine residues and Vangl2 activities depend on its levels of phosphorylation. In the limb bud, Wnt5a signaling gradient controls limb elongation by establishing PCP in chondrocytes along the proximal-distal axis through regulating Vangl2 phosphorylation. Our studies have provided new insight to Robinow syndrome, Brachydactyly Type B1, and spinal bifida which are caused by mutations in human ROR2, WNT5A, or VANGL.     

Planar Cell Polarity Enables Posterior Localization of Nodal Cilia and Left-Right Axis Determination during Mouse and Xenopus Embryogenesis

Left-right asymmetry in vertebrates is initiated in an early embryonic structure called the ventral node in human and mouse, and the gastrocoel roof plate (GRP) in the frog. Within these structures, each epithelial cell bears a single motile cilium, and the concerted beating of these cilia produces a leftward fluid flow that is required to initiate left-right asymmetric gene expression. The leftward fluid flow is thought to result from the posterior tilt of the cilia, which protrude from near the posterior portion of each cell''s apical surface. The cells, therefore, display a morphological planar polarization. Planar cell polarity (PCP) is manifested as the coordinated, polarized orientation of cells within epithelial sheets, or as directional cell migration and intercalation during convergent extension. A set of evolutionarily conserved proteins regulates PCP. Here, we provide evidence that vertebrate PCP proteins regulate planar polarity in the mouse ventral node and in the Xenopus gastrocoel roof plate. Asymmetric anterior localization of VANGL1 and PRICKLE2 (PK2) in mouse ventral node cells indicates that these cells are planar polarized by a conserved molecular mechanism. A weakly penetrant Vangl1 mutant phenotype suggests that compromised Vangl1 function may be associated with left-right laterality defects. Stronger functional evidence comes from the Xenopus GRP, where we show that perturbation of VANGL2 protein function disrupts the posterior localization of motile cilia that is required for leftward fluid flow, and causes aberrant expression of the left side-specific gene Nodal. The observation of anterior-posterior PCP in the mouse and in Xenopus embryonic organizers reflects a strong evolutionary conservation of this mechanism that is important for body plan determination.     

Loss of membrane targeting of Vangl proteins causes neural tube defects

In the mouse, the loop-tail mutation (Lp) causes a very severe neural tube defect, which is caused by mutations in the Vangl2 gene. In mammals, Vangl1 and Vangl2 code for integral membrane proteins that assemble into asymmetrically distributed membrane complexes that establish planar cell polarity in epithelial cells and that regulate convergent extension movements during embryogenesis. To date, VANGL are the only genes in which mutations cause neural tube defects in humans. Three independently arising Lp alleles have been described for Vangl2: D255E, S464N, and R259L. Here we report a common mechanism for both the naturally occurring Lp (S464N) and a novel ENU-induced mutation Lp(m2Jus)(R259L). We show that the S464N and R259L variants stably expressed in polarized MDCK kidney cells fail to reach the plasma membrane, their site for biological function. The mutant variants are retained intracellularly in the endoplasmic reticulum, colocalizing with ER chaperone calreticulin. Furthermore, the mutants also show a dramatically reduced half-life of ～3 h, compared to ～22 h for the wild-type protein, and are rapidly degraded in a proteasome-dependent and MG132-sensitive fashion. Coexpressing individually the three known allelic Lp variants with the wild-type protein does not influence the localization of the WT at the plasma membrane, suggesting that the codominant nature of the Lp trait in vivo is due to haploid insufficiency caused by a partial loss of function in a gene dosage-dependent pathway, as opposed to a dominant negative phenotype. Our study provides a biochemical framework for the study of recently identified mutations in hVANGL1 and hVANGL2 in sporadic or familial cases of neural tube defects.     

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.     

Cloning and characterization of two cytoplasmic dynein intermediate chain genes in mouse and human

Cytoplasmic dynein is a large multisubunit microtubule-based motor protein, which mediates movement of numerous intracellular organelles. We report here the identification of the human homologue of cytoplasmic dynein intermediate chain 1 gene (DNCI1) located on human chromosome 7q21.3-q22.1. The mouse orthologue (Dnci1) was identified along with another highly related gene, Dnci2, and their RNA in situ expression patterns were examined during mouse embryogenesis. Dnci1 was found to have a highly restricted expression domain in the developing forebrain as well as the peripheral nervous system (PNS), while Dnci2 displayed a broad expression profile throughout the entire central nervous system and most of the PNS. A dynamic expression profile was also found for Dnci2 in the developing mouse limb bud. The data presented here provide a framework for the further analysis of the functional role of Dnci1 and Dnci2 in mouse and DNCI1 in human.     

Strabismus regulates asymmetric cell divisions and cell fate determination in the mouse brain

The planar cell polarity (PCP) pathway organizes the cytoskeleton and polarizes cells within embryonic tissue. We investigate the relationship between PCP signaling and cell fate determination during asymmetric division of neural progenitors (NPs) in mouse embryos. The cortex of Lp/Lp (Loop-tail) mice deficient in the essential PCP mediator Vangl2, homologue of Drosophila melanogaster Strabismus (Stbm), revealed precocious differentiation of neural progenitors into early-born neurons at the expense of late-born neurons and glia. Although Lp/Lp NPs were easily maintained in vitro, they showed premature differentiation and loss of asymmetric distribution of Leu-Gly-Asn–enriched protein (LGN)/partner of inscuteable (Pins), a regulator of mitotic spindle orientation. Furthermore, we observed a decreased frequency in asymmetric distribution of the LGN target nuclear mitotic apparatus protein (NuMa) in Lp/Lp cortical progenitors in vivo. This was accompanied by an increase in the number of vertical cleavage planes typically associated with equal daughter cell identities. These findings suggest that Stbm/Vangl2 functions to maintain cortical progenitors and regulates mitotic spindle orientation during asymmetric divisions in the vertebrate brain.     

The expression of gelatinase A (MMP-2) is required for normal development of zebrafish embryos

Gelatinase A, also called matrix metalloproteinase 2 (MMP-2), belongs to the matrix metalloproteinase (MMP) family. MMP-2 cleaves type IV collagen, denatured collagen (gelatin), and other extracellular matrix (ECM) components. MMP-2 has been reported to be involved in a number of biological and pathological processes, but previous studies have not indicated that its expression is essential for early embryogenesis. In the current study, we have utilized zebrafish as a developmental model to study the role of MMP-2 during embryogenesis. We have successfully isolated a zebrafish MMP-2 (zMMP-2) homologue showing over 80% identity and over 90% similarity to its human counterpart. In situ analysis showed that zMMP-2 was expressed as early as the one-cell stage implying a maternal origin during oogenesis, and embryos continued to express zMMP-2 through at least the 72-h stage of development. RT-PCR analysis confirmed the in situ expression pattern and gelatin zymography indicated that a metalloproteinase with the same gel mobility as vertebrate MMP-2 was present in zebrafish embryos. Injection of zMMP-2 antisense morpholino oligonucleotides into 1- to 4-cell embryos resulted in a truncated axis, monitored through 72 h of development indicating that this metalloproteinase plays an important role in zebrafish embryogenesis. Monpholino-induced alterations in development began to be observed at 12 h of embryogenesis based on morphological and axis marker studies. The results obtained in zebrafish are in contrast to murine knockout studies that indicate that MMP-2 does not have a major role in mouse embryogenesis.Edited by D. Tautz     

Planar polarization in embryonic epidermis orchestrates global asymmetric morphogenesis of hair follicles

Mammalian body hairs align along the anterior-posterior (A-P) axis and offer a striking but poorly understood example of global cell polarization, a phenomenon known as planar cell polarity (PCP). We have discovered that during embryogenesis, marked changes in cell shape and cytoskeletal polarization occur as nascent hair follicles become anteriorly angled, morphologically polarized and molecularly compartmentalized along the A-P axis. Hair follicle initiation coincides with asymmetric redistribution of Vangl2, Celsr1 and Fzd6 within the embryonic epidermal basal layer. Moreover, loss-of-function mutations in Vangl2 and Celsr1 show that they have an essential role in hair follicle polarization and orientation, which develop in part through non-autonomous mechanisms. Vangl2 and Celsr1 are both required for their planar localization in vivo, and physically associate in a complex in vitro. Finally, we provide in vitro evidence that homotypic intracellular interactions of Celsr1 are required to recruit Vangl2 and Fzd6 to sites of cell-cell contact.     

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.