Planar Cell Polarization during Development: 14 (Advances in Developmental Biology)

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  1. Planar cell polarity in development and disease
  2. ZFIN Lab: Ciruna Lab
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VL1 and PK2 co-localize in node cells and form crescents pointed toward the anterior F , yellow. Motile cilia can be visualized above the plane of VL1 or PK2 localization not shown. As nodes were imaged from the ventral side, left side of the embryo is on the right side of each panel and right side of the embryo is on the left side of each panel.

To investigate the potential functional requirement for PCP signaling in vertebrate left-right asymmetry, we generated a Vangl1 mutant mouse using BayGenomics Vangl1 gene-trap ES cells, as has been done by another group [26] Fig. We similarly refer to the allele as Vangl1gt. Also as previously reported, we found that the two Vangl genes, Vangl1 and Vangl2 , genetically interact to control PCP.

Planar cell polarity in development and disease

S1 , S2 , not present in either of the Vangl heterozygotes. S3 , suggesting that alternative splicing creates small amounts of functional Vangl1 transcripts and protein that are sufficient to suppress the homozygous null phenotype in the majority of individuals. Embryos with these phenotypes were never recovered among heterozygous siblings, or from crosses between wild-type, BL6 or Ola animals.

DORSAL VENTRAL AXIS FORMATION IN DROSOPHILA(PART-4) -CSIR NET -DEVELOPMENTAL BIOLOGY

In addition, despite advancing to the 20—25 somite stage and maintaining viability up to E9. The turning of mouse embryos is a process that converts the embryo from lordotic to fetal position at the 8- to somite stage of development [28]. Turning defects are often associated with aberrant L—R patterning in the mouse ventral node [1] , [2] , [5]. Therefore, abnormal Pitx2 expression appears to correlate closely with aberrant turning.

However, striking polarized distribution of VANGL1 and PK2 proteins in central node cells suggests that PCP signaling establishes anterior-posterior polarity and that this A—P polarity may be required for determining left-right asymmetry. The mis-expression of Pitx2 and the turning defect suggest that Vangl1 might regulate L-R asymmetry establishment in the mouse ventral node. B' and D' are the boxed regions from B and D and are at the same magnification. S4 , while little or no Vangl1 expression could be detected not shown. Morpholinos were injected at the eight-cell stage, and the affected cells traced with co-injected RFP, thereby knocking down Vangl2 expression within a subset of cells in both the deep and superficial mesoderm on the dorsal side.

Injected embryos gastrulated normally, while the ciliated epithelium within the GRPs contained small cell clones with knocked down Vangl2 adjacent to wild-type, uninjected cells Fig. In GRP cells, motile cilia normally relocate from a predominantly middle to a predominantly posterior position, and this posterior positioning is required to produce leftward flow [8]. In GRP cells with knocked down Vangl2, however, the distribution of cilia positioning is more central compared to the wild type distribution. This difference is statistically significant Fig.

Together, these results indicate that PCP in the frog GRP regulates the posterior positioning of motile cilia required for leftward flow. GRP cilia both inside injected and outside uninjected the clone were scored based on the location in GRP cells that were divided into three equal zones anterior, middle and posterior.

Shown are data from a minimum of 3 GRPs where — injected and uninjected cells were scored. RFP red marks the position of the injected clone, cilia green are stained with the acetylated-tubulin antibody, and cell boundaries blue are stained with an antibody to ZO Anterior is indicated by direction of the arrow in B, C, E, F. To determine whether perturbing the posterior localization of motile cilia in the GRP affects left-right lateralization, we examined the expression of Xenopus nodal Xnr1—Xenopus nodal related 1 mRNA in the Vangl2 morpholino treated embryos.

Xnr1 is an early marker of lateralization that is normally expressed in the left lateral plate mesoderm LPM at late neurulation stage [8]. This result is similar to those previously reported upon knock-down of ciliary dynein heavy chain genes dnah9 and dnah5 or inhibition of the directional flow with methyl cellulose [8] , [9]. It contrasts with results obtained in mice, in which disruption of genes important for L—R determination results in increased right and bilateral but not absent expression of markers such as nodal or Pitx2 [2].

It is important to note that our Vangl2 MO treated embryos did not display any signs of convergent extension defects, probably because Vangl2 was knocked-down in only approximately one of eight cells, and these intermingled with wild-type cells within the deep layer of the mesoderm during intercalation.

Our results demonstrate that the PCP gene Vangl2 is required not only for posterior positioning of motile cilia in the GRP, but also for correct lateralization of Xenopus embryos. Sidedness of staining was scored for at least 60 embryos for each condition. Data from one experiment are shown.

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Xnr1 staining is evident on the left B right C or both D sides of the midline. Black arrowheads indicate staining.

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Our results from mouse and frog, taken together, suggest a conserved function for PCP in establishing L-R asymmetry in vertebrates. In mouse, at least two polarity proteins, VANGL1 and PK2, become asymmetrically distributed along the A—P axis in the node, most likely prior to L—R asymmetry determination, by localizing to anterior cortical domains in central node cells.

We suggest that the very early establishment of PCP in the ventral node and GRP triggers the proper posterior localization of motile cilia that is required for efficient leftward nodal flow [2] , [10] , [30]. Indeed, frog GRPs in which PCP is perturbed by knockdown or overexpression of Vangl2 show substantially less posterior bias of cilia positioning, and these embryos fail to correctly lateralize.

Results of other studies predict that this would impair directional fluid flow produced by nodal cilia [2] , [30]. Consistent with these results, in another report, Dishevelled Dvl compound mutant mice lacking 5 of 6 copies of the three Dvl genes also show L—R laterality defects, and motile cilia in the node are mislocalized [31].

DVL proteins are implicated in establishing the asymmetric cortical domains of PCP proteins [17] , and have also been shown to be required for proper basal body and therefore cilia localization [32]. It is not clear from these results in which capacity DVL proteins function in the ventral node. Based on the conserved localization of PK, VANG and DVL proteins in nodal epithelium, with PK2 and VANGL1 localized opposite to DVL2, one would predict that PCP proteins operate through asymmetric cortical domains to establish molecular asymmetry by a mechanism similar to that described in flies [14] , [15] , which would, in turn, affect placement of cilia and thus regulate morphologic planar polarity.

The functional importance of VANG in regulating PCP in the Xenopus GRP also highlights the likelihood that the evolutionarily conserved mouse and frog nodal structures share this mechanistic feature required for embryonic lateralization [10]. The signals that operate upstream of the core PCP proteins to provide anterior-posterior directionality to the planar polarization of the ventral node or GRP are unknown.

Similarly, the mechanism by which the anterior-posteriorly organized core proteins control the posterior localization of motile cilia is unknown. However, our results indicate that anterior-posterior PCP is important for establishment of vertebrate L—R asymmetry. Genotyping of embryos extracted at E9. The precise beta-Geo insertion site was mapped by PCR to nucleotide position of the third intron. The genotype of the obtained founder animals was further confirmed by Southern blotting not shown. The animals had highly curled tails and females had imperforate vaginas.

This region was also cloned into the pET28a vector to produce histidine-tagged VANGL1 fragment, which was used for affinity purification of the antiserum. Protein lysates were made 24 hours after transfection in a hypotonic buffer containing 10 mM Tris, pH 7. The lysates were prepared from dissected sensory epithelia using hypotonic buffer and were analyzed by Western blotting either before or after endoglycosidaseF treatment Roche, Indianapolis, IN.


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Embryos were washed 3—4 times, 30 minutes each wash, in PBT and then mounted for confocal imaging. Xenopus embryos were obtained by in vitro fertilization using standard protocols. A synthetic Vangl2 mRNA 1—5 ng [29] was injected into one dorsal animal blastomere of eight-cell stage Xenopus embryos [36]. In some experiments, cilium location, based on centrin-RFP foci, was scored as being in the anterior, middle or posterior third of the cell in relation to the blastopore in each embryo.

If centrin-RFP foci were not clearly evident, cilia were only scored if the entire cilium lay within a single one-third of the cell. In other experiments, cilium location was scored using acetylated tubulin, and cilia were only scored if the entire cilium lay within a single one-third of the cell. Data were gathered from 80— GRP cells marked with tracer as well as neighboring uninjected cells as a control, from at least 3 different embryos. A morpholino Genetools directed against Vangl2 initiation codon, as described previously [29] , or a standard control morpholino [39] , [40] was injected 20—30 ng; a concentration previously shown to produce a strong phenotype without toxicity into one dorsal animal blastomere of eight-cell stage embryos in order to target the developing GRP [8].

Cilia localization was analyzed as above for Vangl2 RNA injections. At stage 20—22, embryos were fixed and probed with an antisense probe directed against Xenopus nodal-related1 Xnr1 , to mark the lateral plate mesoderm, using standard in-situ hybridization protocols [41]. Following labeling, experiments were blinded to the scorer and embryos were scored as having Xnr1 expression on the left side, right side, both sides or staining absent.

These antibodies were generated in our labs. Louis, MO. Actin was stained with Alexaconjugated phalloidin Invitrogen, CA. Vangl1 mutant mice. Remaining three exons and introns of the Vangl1 gene are not drawn but are indicated with an arrow after exon 4.

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C—F E C—F Only double heterozygotes are stained with X-Gal, showing the distribution of VangL1 protein, and they have severe craniorachischisis. B—E Cell organization in the basal and apical regions of cochlea was similar in the mutants and in the wild-type animals. To do so, we prepared mRNA and protein samples from cerebellum and found that VL1 protein is abundantly expressed in this adult tissue not shown.

Example from two litters is shown E, E'. Staining of all embryos was performed at the same time and under identical conditions. After imaging, the dissected GRPs were cut in half and imaged from in cross-section. Lysates containing over-expressed proteins were analyzed by Western blots and compared to lysates of untransfected HeLa cells — or to vector-transfected cells vector. Western blots were probed with the affinity-purified antibody to VL1 protein or with the antibody to GFP protein.

Blots were also probed for tubulin as a loading control. Protein of the predicted size is detected after endoglycosidaseF treatment of lysates, demonstrating that glycosylation is the main reason for the observed discrepancy in size. We thank Ljiljana Milenkovic for numerous discussions, comments on the manuscript and technical guidance throughout the course of this work; Eszter Vladar and Alison McGuigan for discussions and comments on the manuscript: Klara Fekete for help with genotyping; Jeremy F.

Reiter for technical advice on mounting of the node-stage embryos; and members of the Axelrod and Scott laboratories for continuous encouragement.

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Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract 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. Introduction In the mouse, left-right asymmetry has been proposed to be controlled by fluid flow in the node propelled by clockwise movement of motile nodal cilia [1] — [5].

Results We hypothesized that PCP may be required to position the nodal cilia in the posterior of each cell. Download: PPT. Figure 1. Figure 2. Figure 3. J Cell Sci , Pt 20 , Development , 17 , Curr Biol , 16 13 , Dev Cell , 10 2 , Bioessays , 27 12 , Strutt D Organ shape: controlling oriented cell division. Curr Biol , 15 18 , RR Strutt D Mathematical modeling of planar polarity. Dev Cell , 8 2 , Advances in Developmental Biology , 14, Development , 4 , Strutt D Frizzled signalling and cell polarisation in Drosophila and vertebrates. Development , 19 , Curr Biol , 13 16 , Development , 13 , Dev Cell , 3 6 , Curr Biol , 12 11 , RR Strutt DI The asymmetric subcellular localisation of components of the planar polarity pathway..

Semin Cell Dev Biol , 13 3 , Curr Biol , 12 10 , Curr Biol , 11 13 , RR Strutt DI Asymmetric localization of frizzled and the establishment of cell polarity in the Drosophila wing.. Mol Cell , 7 2 , Dev Biol , 2 , Curr Biol , 10 16 , Curr Biol , 9 23 , Curr Opin Genet Dev , 9 4 , Genes Dev , 13 10 , Cell , 94 1 , Nature , , Mech Dev , 58 , Development , 12 , Mech Dev , 46 1 ,