Van Gogh/strabismus: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Van Gogh

Synonyms - strabismus (stbm)

Cytological map position - 45A7--45A10

Function - unknown, but potentially a receptor or transmembrane signaling protein

Keywords - tissue polarity, eye, wing, leg

Symbol - Vang

FlyBase ID: FBgn0015838

Genetic map position - 2-61

Classification - novel protein

Cellular location - presumably transmembrane



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

Strabismus, is formally defined as "a disorder of vision due to a deviation from normal orientation of one or both eyes" and is the medical term for 'cross-eyed'. Mutants of Drosophila strabismus, now more properly termed Van Gogh (Vang), show a disrupted eye polarity, the relative orientation of ommatidia or photoreceptor units. Polarity in the Drosophila eye is manifested as a dorsoventral reflection of two chiral forms of the individual ommatidia. If one imagines the two forms were to resemble the face of a clock, one of them would appear in a normal orientation while the other would appear as its mirror opposite. These forms fall on opposite sides of a dorsoventral midline of mirror symmetry known as the equator. Polarity is established in the eye imaginal disc as cells adopt their fates and as the ommatidial precursors undergo coordinated rotation within the epithelium; the mechanisms that coordinate these early patterning events remain poorly understood. strabismus (stbm) is required to establish polarity in the eye, legs and bristles of Drosophila. Many stbm ommatidia are reversed anteroposteriorly and/or dorsoventrally. In stbm eye discs, ommatidial rotation is delayed and some ommatidial precursors initiate rotation in the wrong direction. Mosaic analysis indicates that stbm is ommatidium autonomous and required in most, if not all, photoreceptors within an ommatidium to establish normal polarity (Wolff, 1998).

Before describing research that investigates the role of stbm in eye polarity in greater depth, a slight digression will allow the presentation of information on the cellular and genetic basis for eye polarity. The Drosophila compound eye comprises approximately 800 hexagonal unit eyes, or ommatidia, packed in a smooth array. Each ommatidium is a precise assembly of 20 cells: a central core of 8 photoreceptor cells (R1-R8) and 12 non-neuronal support cells. The rhabdomeres, or light-sensitive organelles of the photoreceptors, are arranged in a characteristic asymmetric trapezoid and are identified by their positions within the trapezoid; R3's rhabdomere occupies the point of the trapezoid. Each eye contains two chiral forms (i.e. mirror image reflections) of the trapezoid, which fall on opposite sides of the equator (the aforementioned dorsal/ventral midline of mirror symmetry). Assembly of cells into ommatidial precursors, or preclusters, begins in the eye imaginal disc, the primordium of the adult eye. Recruitment of cells begins posterior to the morphogenetic furrow, a dynamic front of differentiation that progresses from posterior to anterior across the epithelium. Photoreceptor recruitment follows a characteristic sequence, starting with R8 and followed by the pairwise addition of R2/5, R3/4, R1/6 and finally by R7. The remaining cell types are recruited between late larval and mid-pupal development (Wolff, 1998 and references).

The group of 8 photoreceptor cells is initially bilaterally symmetrical across the anterior-posterior (A/P) axis. As development proceeds, morphological movements break the symmetry so that the ommatidium becomes polarized across this axis. These morphological movements are associated with the specification of distinct fates in two of the photoreceptor cells: R3 and R4. Within each R3/R4 pair, the cell that is located closest to the midline of the eye disc, the equatorial cell, adopts the R3 fate, and the more lateral cell, the polar cell, adopts the R4 fate (Fig. 1A). Evidence that the R3 and R4 cells differ first becomes apparent in the third instar eye disc when the bilateral symmetry of the photoreceptor precluster breaks down: R4 loses contact with R8 and its cell body becomes displaced relative to that of R3. The chirality of an ommatidium is therefore associated with the adoption of the R3 and R4 fates (Wolff, 1998 and references).

Two events contribute to the origin of the equator in the third instar eye disc. First, chiral forms are created as a consequence of R3 and R4 adopting their appropriate fates with respect to their dorsal or ventral location in the eye. Second, the ommatidial precursors in the dorsal and ventral halves of the eye rotate as units, 90° in opposite directions to one another. As a result of these patterning events, the adult eye displays global mirror symmetry. A number of genes are involved in setting up this polarity. frizzled (fz) encodes a protein with 7 transmembrane domains and is a member of the serpentine class of receptors. fz ommatidia display an assortment of disruptions in ommatidial polarity, including partial rotations, reversals on either their A/P, dorsal/ventral (D/V) or both A/P and D/V axes (Zheng, 1995). dishevelled (dsh) mutant eyes resemble fz eyes (Theisen, 1994). The Drosophila homolog of the p21 GTPase RhoA has recently been shown to be required for the generation of tissue polarity. The RhoA eye phenotype is similar to that of fz and dsh and genetic interactions suggest RhoA is a component of the signaling pathway mediated by Fz and Dsh (Strutt, 1997). In addition to their eye phenotypes, fz, dsh and RhoA mutant flies also show a variety of polarity defects in other epithelia and cell types. Finally, there are eye-specific genes, such as nemo and roulette, which carry out the rotation program (Choi, 1994) (Wolff, 1998 and references).

To assess ommatidial rotation, eye discs were stained with an antibody that recognizes the nuclear neuron-specific protein Elav, or with cobalt and lead sulfide, which highlights the apical surfaces of cells. The Elav-stained discs demonstrate that stbm does not play a role in recruiting photoreceptors into the assembling ommatidium, as photoreceptors are recruited in the normal sequence and with normal timing in mutant discs. Views of the apical surface of the eye discs indicate that ommatidial rotation normally begins approximately 5 rows posterior to the furrow and is complete by row 12. In stbm eye discs, the majority of preclusters are delayed in initiating rotation. As the photoreceptor preclusters mature, their tips become effaced from the apical surface of the disc so their orientation can no longer be evaluated at the apical surface. However, the cone cells, which lie on top of the photoreceptor cells in stereotypic positions, provide a useful compass by which to measure ommatidial orientation at the posterior of the third instar eye disc. Observations of cone cells in lead and cobalt sulfide-stained mutant eye discs reveal that the majority of ommatidial preclusters at the posterior of stbm eye discs have rotated no more than approximately 45° whereas wild-type precursors would have rotated 90° at an equivalent point in development. Of those ommatidia that do begin to rotate on schedule, most appear to initiate rotation in the correct direction with respect to their dorsal or ventral location within the epithelium, but some initiate rotation in the wrong direction. These preparations demonstrate that ommatidial orientation is disrupted in two ways during the initial stages of pattern formation: (1) ommatidial rotation is significantly delayed in stbm eye discs and (2) some ommatidial precursors initiate rotation in the wrong direction (Wolff, 1998).

The aberrant ommatidial forms seen in stbm mutant eyes can be accounted for by invoking defects in either chirality, rotational direction or both. To distinguish between these possibilities the enhancer trap line H123 was used, which is differentially expressed in R3 and R4 and therefore provides a molecular marker to differentiate between these two cell fates. H123 expression was examined in a null stbm allele and both reversals in specification of the R3 and R4 cell fates and misrotations were found. Four classes of preclusters are seen in stbm discs and these correlate with the predicted classes. Ommatidia were seen in which the R3 and R4 fates are correctly specified and rotation occurs in either the correct or incorrect direction, with respect to the R3 and R4 fates. In addition, ommatidia are seen in which the R3 and R4 fates are reversed and rotation occurs in either the correct or incorrect direction, with respect to the R3 and R4 fates. This analysis of H123 expression in stbm discs suggests that stbm may participate in imparting the R3 and R4 fates and it therefore follows that the phenotype results from a failure in fate specification rather than later patterning events involving placement of the R3 and R4 rhabdomeres (Wolff, 1998).

Lead sulfide-stained stbm eye discs were examined to assess cell contacts within the photoreceptor preclusters. In wild-type eye discs, the symmetry in the 8 cell precluster is lost when R4, the polar cell of the R3/R4 pair, loses contact with the central photoreceptor, R8. In some preclusters in stbm eye discs the equatorial cell loses contact with R8. These findings are consistent with the H123 results described above and suggest that stbm participates in the specification of the R3 and/or R4 fates. The fact that the R3 and R4 fates can be mis-specified in stbm ommatidia raises the possibility that the fates of the remaining symmetrical photoreceptor pairs, R2/R5 and R1/R6, are also reversed, such that the anterior and posterior faces of the trapezoid are inverted. Because molecular markers that distinguish between the members of these photoreceptor pairs do not exist, it is not possible to address this question directly. However, in wild-type ommatidia, R8's rhabdomere extends between R1 and R2, and only rarely between R5 and R6, suggesting R8 can recognize a difference between cells to the anterior (R1 and R2) and those to the posterior (R5 and R6). If the R3 fate is assigned to the cell occupying the point of the trapezoid, then in stbm ommatidia, R8 extends its rhabdomere between R1 and R2 in 95 out of 96 cases examined. These results are consistent with the notion that the anterior (R1, R2 and R3) and posterior (R4, R5, and R6) cell fates may be switched in stbm ommatidia, and that the entire ommatidium is 'backwards' (Wolff, 1998).

stbm could be directing ommatidial orientation by one of several mechanisms. It could act locally to coordinate the process within an ommatidium or between neighboring ommatidia, or it could provide a diffusible polarity signal across the entire disc. To distinguish between these possibilities, ommatidia located in and near clones of homozygous mutant stbm tissue were examined. Three results are noted: (1) it was found that clones anywhere in the disc are autonomous. Consistent with this observation, stbm RNA is found throughout the eye disc using stbm as a probe for in situ analysis. (2) The phenotype of genotypically mutant ommatidia lying along the border of the clone was examined. Genotypically mutant (as well as mosaic) ommatidia are not rescued by their wild-type neighbors - they misrotate, even when located next to genotypically wild-type ommatidia. (3) Genetically wild-type ommatidia are not affected by neighboring mutant tissue. Therefore, stbm acts autonomously within ommatidia; in other words, the presence or lack of Stbm function in one ommatidium does not affect the polarity of neighboring ommatidia. To establish if stbm acts in one specific cell to establish orientation of the entire ommatidium, a mosaic analysis was carried out of ommatidia displaying either mutant or normal orientation. 169 misoriented and 119 normally oriented mosaic ommatidia at clonal boundaries were randomly chosen and the genotype of photoreceptors within these ommatidia was scored. Misorientation or normal orientation does not correlate absolutely with the mutant genotype of any specific photoreceptor or group of photoreceptor cells. Specifically, the combination of stbm mutant and wild-type photoreceptors does not predict the orientation of the ommatidium. Rather, many of the photoreceptors make a contribution. However, some interesting biases have been noticed in the frequency with which certain photoreceptors are mutant. (1) Tthere is an over-representation of stbm mutant R3 cells in misoriented mosaic ommatidia: 87% of misoriented ommatidia have a mutant R3 cell. (2) stbm mutant R4 cells are under-represented: 26% of misoriented ommatidia and 21% of properly oriented mosaic ommatidia have a mutant R4 cell. This suggested two possibilities: (1) stbm is required in R3 for proper orientation or (2) stbm is required to specify the R4 cell fate. Mosaic ommatidia were analyzed in which the R3/R4 pair is mosaic (R3 + /R4 - or R3 - /R4 + ). A total of 144 such cases were found, 141 of which were of the R3 - /R4 + type. This implies that if one of the R3/R4 pair is mutant it will become an R3. Thus, Stbm is required for the R4 cell to establish its fate (Wolff, 1998).

Furthermore, the choice of the R3/R4 cell fate appears subsequently to influence the direction of ommatidial rotation. Consistent with this is the observation that of these 141 R3 - /R4 + ommatidia, 74% acquire an incorrect orientation while only 26% adopt the correct orientation. If both cells are mutant, 59% are incorrectly oriented and 41% are correctly oriented, as would be expected if orientation were random. However, while having R3 + and R4 + increases the probability of correct orientation, it is not sufficient: 73% are correctly oriented and 27% are incorrectly oriented. These data are consistent with the results obtained using the H123 marker. In summary, two conclusions can be drawn from the mosaic analysis. (1) Stbm is ommatidium autonomous and the presence of the normal gene product in any one cell is not sufficient to establish correct orientation; rather, it is required in many, if not all, photoreceptors. This result suggests that each photoreceptor makes some contribution to the orientation process which must be a highly coordinated effort between cells within an ommatidium. (2) Stbm appears to be important in specifying the fate of the R4 cell (Wolff, 1998).

It is tempting to speculate that Stbm acts as a transmembrane receptor or ligand to send or receive signals from adjacent cells, passing on cell fate and cell polarity decisions from cell to cell. In addition, the C-terminal PDZ domain-binding motif could interact with PDZ motif proteins such as Dishevelled, implicated in Wingless signaling and tissue polarity; Discs large, involved in junctional integrity, or Canoe, involved in Notch and Ras signaling. Because of the phenotypic similarities between stbm and other tissue polarity mutants, genetic interactions between stbm and other tissue polarity mutants were sought. No obvious enhancement or suppression of the stbm phenotype upon removal of one copy of prickle, spiny legs, frizzled, dishevelled or nemo is found in a stbm mutant background (Wolff, 1998). Nevertheless, another study has appeared showing a strong genetic interaction of stbm with frizzled and prickle, putting stbm right in the middle of a well characterized tissue polarity pathway (Taylor, 1998).

Strabismus requires Flamingo and Prickle function to regulate tissue polarity in the Drosophila eye

Tissue polarity in Drosophila is regulated by a number of genes that are thought to function in a complex, many of which interact genetically and/or physically, co-localize, and require other tissue polarity proteins for their localization. The enhancement of the strabismus tissue polarity phenotype by mutations in two other tissue polarity genes, flamingo and prickle, is reported. Flamingo is autonomously required for the establishment of ommatidial polarity. Its localization is dynamic throughout ommatidial development and is dependent on Frizzled and Notch. Flamingo and Strabismus co-localize for several rows posterior to the morphogenetic furrow and subsequently diverge. While neither of these proteins is required for the other's localization, Prickle localization is influenced by Strabismus function. The data suggest that Strabismus, Flamingo and Prickle function together to regulate the establishment of tissue polarity in the Drosophila eye (Rawls, 2003).

In an attempt to define more precisely the role of Stbm in the tissue polarity pathway, genetic interactions were identifed between stbm and two other tissue polarity genes, fmi and pk. Characterization of the fmi-stbm interaction reveals a requirement for Fmi in ommatidial polarity and a dynamic pattern of Fmi localization that depends on Fz and N. An antibody was raised against Stbm, its subcellular localization was characterized, and the localization of Fmi and Stbm was shown to differ in two ways: first, Fmi is enriched in R4, whereas Stbm is not, and second, Fmi, but not Stbm, is endocytosed. Characterization of the pk-stbm interaction shows that pk enhances the stbm phenotype and that Pk localization requires Stbm (Rawls, 2003).

Three alternatively spliced transcripts are encoded by the pk locus: pkpk, pkM and pkpk-sple. Although these three isoforms differ in the 5' region, they all contain the single PET and three LIM domains characteristic of the Pk protein. PET and LIM domains are thought to mediate protein-protein interactions. Isoform-specific mutations in the 5' region of the transcript result in the pkpk phenotype, affecting only the wing and notum, whereas mutations in the LIM- or PET-encoding domains result in pkpk-sple alleles, null alleles that affect the eye, legs and abdomen in addition to the wing and notum (Rawls, 2003).

The observation that Pk distribution is altered in a null stbm background suggests that its localization is, at least in part, dependent on Stbm. The possibility that Pk localization is mediated directly by Stbm has not yet been explored, but the PET and LIM domains are candidates for domain-specific interactions with Stbm. Disruption of these domains would result in genetic null alleles, consistent with the pkpk-sple phenotype described in this study (Rawls, 2003).

Although ommatidial polarity is not affected in individuals carrying the pkpk1 allele, this allele enhances the stbm eye phentoype. Functional redundancy could account for the ability of pk to enhance the stbm phenotype such that there is no phenotype when pk is knocked out but a reduction in pk gene dose can be detected by Stbm. Furthermore, the balance of Pk isoforms contributes to the establishment of tissue polarity. Perhaps this balance is also required for Stbm function (Rawls, 2003).

The observations that fmi and stbm have similar phenotypes, that they interact genetically and that their products colocalize, suggests that they may act in the same pathway to specify tissue polarity. To explore the possibility that Stbm and Fmi define a complex, both the localization of Fmi was investigated in a null stbm background and the localization of Stbm was investigated in EGUF-fmi eyes. In neither case was the localization affected, demonstrating that Stbm is not required for Fmi localization, nor is Fmi required for Stbm localization. Furthermore, no physical interaction has been detected between Fmi and Stbm using co-immunoprecipitation assays (Rawls, 2003).

In a deficiency screen, pk was identified as a dominant genetic modifier of stbm. The genetic interaction between stbm and pk may have its basis in a physical interaction that enhances or stabilizes these proteins at the R3/R4 boundary. To explore this possibility, Stbm localization was examined in a pk mutant background, and Pk localization in a stbm mutant background. Stbm localization does not appear to be affected in a pkeq background (a genetic null that fails to complement pkpk-sple alleles). However, Pk localization is disrupted in a stbm6cn null background. The distribution of Pk was characterized in wild-type eye imaginal discs; it is indistinguishable from that of Stbm. Pk is significantly reduced overall in the stbm6cn background. While some protein does accumulate at the boundary between R3 and R4, Pk is not detectable at the R8/R1/R7/R6 boundary. Physical interactions have not been demonstrated between either of these proteins, nor have genetic interactions between fmi and pk been shown. These data are consistent with the possibility that Stbm, Fmi and Pk may all function together in a complex (Rawls, 2003).

Cadherins, or Ca2+-dependent cell adhesion molecules, have traditionally been recognized for their role in adhesion and the resulting tumorous phenotype. Fmi, Fat (Ft) and Dachsous (Ds), members of a class of cadherins that contain a large number of extracellular cadherin domains (atypical cadherins), have recently been shown to contribute to the polarization of ommatidia. While the ability of cells to adhere to one another is clearly essential for the establishment of polarity within epithelia, recent work suggests the role of cadherins extends beyond adhesion (Rawls, 2003).

Several lines of evidence suggest atypical cadherins may be involved in signaling. For example, Ft is required in the haltere to inhibit DV signaling and ft mutants display haltere to wing transformations. In the fly eye, Ft and Ds have been proposed to be required for the transduction of a dorsal-ventral positional signal via cell-cell relay. In addition, gradients of Ds and Four-jointed (Fj) activity may regulate Ft to establish this dorsal-ventral cue. It has been suggested that the combined activities of Ds, Fj and Ft, which appear to be functionally conserved in the wing, leg and abdomen, constitute the 'elusive' factor `X' in the morphogen model for tissue polarity (Rawls, 2003).

The data described here are consistent with the notion that Fmi also plays a role in the intracellular signaling required for the establishment of tissue polarity. Given that Fmi is capable of mediating homophilic association between S2 cells, its role in signal transduction may be indirect and a consequence of a primary role in cell adhesion. However, fmi clones in the eye do not give rise to tumors, nor is the tissue grossly disrupted as has been noted in clones of genes that maintain the integrity of tissue [for example, epithelial phenotypes described for shg mutant embryos]. Therefore, it is possible that the primary role of fmi is not to maintain the integrity of tissue via cell adhesion, but rather to maintain sufficient contact between cells to mediate signaling, or even to signal directly (Rawls, 2003).

Ommatidial polarization is thought to rely heavily upon the proper specification of two photoreceptors: R3 and R4. Although these two photoreceptors are recruited into the growing ommatidium as a pair and they morphologically resemble one another in early stages of development, they have long been known to be distinct from one another based on their adoption of distinct sets of contacts early in development. Recent work on a number of tissue polarity genes provides genetic and molecular evidence that the complexes of tissue polarity proteins are not identical in photoreceptors R3 and R4. The asymmetric regulation of N by these complexes may ultimately lead to low levels of N activity in R3 and high levels in R4, the combination of which is thought to be essential for the specification of the R3 and R4 cell fates (Rawls, 2003).

Fmi has been shown to interact homophilically, and while current data do not establish that Fmi is present in both R3 and R4 at the junction between R3 and R4, in the model that follows, it is assumed that homophilic interactions between the extracellular cadherin domains of Fmi help to anchor Fmi in R3 and R4 on both sides of the R3/R4 interface. Furthermore, it is suggested that the intracellular tail of Fmi is involved in signaling, and that it signals through a complex that is made up of at least three proteins: Fmi, Diego (Diego localization depends on Fmi) and Dsh (Dsh co-localizes with Fmi). Dsh has also been shown to interact physically with two proteins required for R4 specification, N and Stbm and with Pk. Finally, stbm-pk genetic and protein localization data suggest Pk and Stbm may physically interact within a complex (Rawls, 2003).

In order to differentially affect signal transduction through the N pathway, the assembly and/or activity of proteins that set up polarity must be different in R3 and R4. The model presented below requires that Stbm and Pk be restricted to the R4 cell to properly modulate N signaling. Stbm has been shown to be restricted to R4 at the R3/R4 boundary; the subcellular location of Pk in the eye has not yet been determined (Rawls, 2003).

It is proposed that the direct interaction between N and Dsh blocks N signaling, and that the different subset of proteins bound to Dsh is the basis of the asymmetry of the complex. In the future photoreceptor R3, N binds Dsh (which is part of the Fmi/Diego/Dsh scaffold) thereby inhibiting N activity in R3. In the future R4 cell, where Stbm and perhaps Pk are localized, Fmi, Diego and Dsh also form a complex. However, in this case, the re-organization of the Fmi/Diego/Dsh complex to include Stbm and Pk bound to Dsh may prevent N from binding to Dsh, leading to high levels of N-mediated signaling in R4. Ultimately, these differences in gene activity in the R3 and R4 precursors direct the fate specification of these cells (Rawls, 2003).


GENE STRUCTURE

cDNA clone length - 3.4 kb

Bases in 5' UTR - 162

Exons - 1

Bases in 3' UTR - 1343


PROTEIN STRUCTURE

Amino Acids - 584

Structural Domains

stbm encodes a novel protein with a potential PDZ domain-binding motif and two possible transmembrane domains. Stbm protein reveals 4 hydrophobic stretches in the N-terminal third of the protein, the longest of which spans 29 amino acids, suggesting a possible transmembrane localization for the protein. The four C-terminal amino acids of the protein, ETSV, are of particular interest as they match a consensus sequence for PDZ domain-binding motifs (X S/T X V) (Wolff, 1998).

Sequence analysis of both vertebrate and invertebrate homologs indicates that stbm has been highly conserved throughout evolution. A search of translated nucleotide databases identifies a human EST with homology to Stbm. Complete sequence of this partial cDNA reveals that the C-terminal half is 55% identical and 72% similar to the fly homolog. The human EST was used to screen a mouse teratocarcinoma cDNA library. The longest cDNA identified (3.7 kb) was sequenced; sequence comparison shows that the mouse protein is 50% identical to the fly protein with the C terminus showing a greater degree of conservation. A C. elegans homolog was identified through a search of the C. elegans database; the Drosophila and C. elegans proteins are 33% identical. The four hydrophobic domains and the PDZ domain-binding motif are conserved among the four species (Wolff, 1998).


EVOLUTIONARY HOMOLOGS

Neural tube defects (NTDs) such as spina bifida and anencephaly are common congenital malformations in humans (1/1,000 births) that result from failure of the neural tube to close during embryogenesis. The etiology of NTDs is complex, with both genetic and environmental contributions -- the genetic component has been extensively studied with mouse models. Loop-tail (Lp) is a semidominant mutation on mouse chromosome 1. In the two known Lp alleles (Lp, Lpm1Jus), heterozygous mice exhibit a characteristic looped tail, and homozygous embryos show a completely open neural tube in the hindbrain and spinal region, a condition similar to the severe craniorachischisis defect in humans. Morphological and neural patterning studies indicate a role for the Lp gene product in controlling early morphogenesis and patterning of both axial midline structures and the developing neural plate. The 0.6-cM/0.7-megabase (Mb) Lp interval is delineated proximally by D1Mit113/Apoa2/Fcer1g and distally by Fcer1a/D1Mit149/Spna1 and contains a minimum of 17 transcription units. One of these genes, Ltap, encodes a homolog of Drosophila Strabismus/Van Gogh (Stbm/Vang), a component of the frizzled/dishevelled tissue polarity pathway. Ltap is expressed broadly in the neuroectoderm throughout early neurogenesis and is altered in two independent Lp alleles, identifying this gene as a strong candidate for Lp (Kibar, 2001).

The Ltap ORF encodes a protein of 521 amino acids that includes 4 predicted transmembrane (TM) domains, a PDZ-domain binding motif (XS/TXV) at the carboxy-terminus, a cluster of predicted PKC and CK2 phosphorylation sites near the amino-terminus, and putative membrane targeting signals near the N-terminus and downstream of TM4. These predicted features are shared by other mouse, fly and worm Stbm proteins, indicating possible functional conservation in this protein family (Kibar, 2001).

The signaling mechanisms that specify, guide and coordinate cell behavior during embryonic morphogenesis are poorly understood. A Xenopus homolog of the Drosophila planar cell polarity gene strabismus (stbm) participates in the regulation of convergent extension, a critical morphogenetic process required for the elongation of dorsal structures in vertebrate embryos. Overexpression of Xstbm, which is expressed broadly in early development and subsequently in the nervous system, causes severely shortened trunk structures; a similar phenotype results from inhibiting Xstbm translation using a morpholino antisense oligo. Experiments with Keller explants further demonstrate that Xstbm can regulate convergent extension in both dorsal mesoderm and neural tissue. The specification of dorsal tissues is not affected. The Xstbm phenotype resembles those obtained with several other molecules with roles in planar polarity signaling, including Dishevelled and Frizzled-7 and -8. Unlike these proteins, however, Stbm has little effect on conventional Wnt/ß-catenin signaling in either frog or fly assays. Thus these results strongly support the emerging hypothesis that a vertebrate analog of the planar polarity pathway governs convergent extension movements (Darken, 2002).

Although the evidence is now compelling that PCP or non-canonical Wnt signaling is required for convergent extension, it is not at all clear what this pathway actually regulates and how convergent extension is, in turn, affected. Even basic questions are unanswered: for example, it is not known when and where PCP signaling is required. Although it is generally assumed that the pathway operates in the intercalating cell population itself, this has not been rigorously demonstrated. Perhaps the most interesting question is whether PCP signals act during vertebrate morphogenesis to polarize cells, as they do in Drosophila. Since convergent extension depends on directional cell rearrangement (mediolateral intercalation must predominate over intercalation in other orientations for net change in tissue shape to occur), it is tempting to suppose that PCP signals might provide this directionality by imposing or maintaining polarized cell motility. In support of this hypothesis, it has been found that protrusive activity, which is normally largely restricted to the mediolateral ends of intercalating mesodermal cells, is randomly oriented in explants expressing dominant-negative Dsh (Darken, 2002 and references therein).

Xenopus Strabismus (Xstbm), a homolog of the Drosophila planar cell or tissue polarity gene, encodes four transmembrane domains in its N-terminal half and a PDZ-binding motif in its C-terminal region, a structure similar to Drosophila and mouse homologs. Xstbm is expressed strongly in the deep cells of the anterior neural plate and at lower levels in the posterior notochordal and neural regions during convergent extension. Overexpression of Xstbm inhibits convergent extension of mesodermal and neural tissues, as well as neural tube closure, without direct effects on tissue differentiation. Expression of Xstbm(DeltaPDZ-B), which lacks the PDZ-binding region of Xstbm, inhibits convergent extension when expressed alone but rescues the effect of overexpressing Xstbm, suggesting that Xstbm(DeltaPDZ-B) acts as a dominant negative and that both increase and decrease of Xstbm function from an optimum retards convergence and extension. Recordings show that cells expressing Xstbm or Xstbm(DeltaPDZ-B) fail to acquire the polarized protrusive activity underlying normal cell intercalation during convergent extension of both mesodermal and neural tissues and that this effect is population size-dependent. These results further characterize the role of Xstbm in regulating the cell polarity driving convergence and extension in Xenopus (Goto, 2002).

In mammals, an example of planar cell polarity (PCP) is the uniform orientation of the hair cell stereociliary bundles within the cochlea. The PCP pathway of Drosophila refers to a conserved signalling pathway that regulates the coordinated orientation of cells or structures within the plane of an epithelium. A mutation in Vangl2, a mammalian homolog of the Drosophila PCP gene Strabismus/Van Gogh, results in significant disruptions in the polarization of stereociliary bundles in mouse cochlea as a result of defects in the direction of movement and/or anchoring of the kinocilium within each hair cell. Similar, but less severe, defects are observed in animals containing a mutation in the LAP protein family gene Scrb1 (homologous with Drosophila scribble). Polarization defects in animals heterozygous for Vangl2 and Scrb1 are comparable to Vangl2 homozygotes, demonstrating genetic interactions between these genes in the regulation of PCP in mammals. These results demonstrate a role for the PCP pathway in planar polarization in mammals, and identify Scrb1 as a PCP gene (Montcouquiol, 2003).

During vertebrate gastrulation, mesodermal and ectodermal cells undergo convergent extension, a process characterized by prominent cellular rearrangements in which polarised cells intercalate along the medio-lateral axis leading to elongation of the antero-posterior axis. A noncanonical Wnt/Frizzled (Fz)/Dishevelled (Dsh) signalling pathway related to the planar-cell-polarity (PCP) pathway in flies, regulates convergent extension during vertebrate gastrulation. A zebrafish homolog of Drosophila prickle (pk), a gene that is implicated in the regulation of PCP, has been isolated and functionally characterized. Zebrafish pk1 is expressed maternally and in moving mesodermal precursors. Abrogation of Pk1 function by morpholino oligonucleotides leads to defective convergent extension movements, enhances the silberblick (slb)/wnt11 and pipetail (Ppt)/wnt5 phenotypes and suppresses the ability of Wnt11 to rescue the slb phenotype. Gain-of-function of Pk1 also inhibits convergent extension movements and enhances the slb phenotype, most likely caused by the ability of Pk1 to block the Fz7-dependent membrane localization of Dsh by downregulating levels of Dsh protein. Furthermore, pk1 is shown to interact genetically with trilobite (tri)/strabismus to mediate the caudally directed migration of cranial motor neurons and convergent extension. These results indicate that, during zebrafish gastrulation Pk1 acts, in part, through interaction with the noncanonical Wnt11/Wnt5 pathway to regulate convergent extension cell movements, but is unlikely to simply be a linear component of this pathway. In addition, Pk1 interacts with Tri to mediate posterior migration of branchiomotor neurons, probably independent of the noncanonical Wnt pathway (Carreira-Barbosa, 2003).

In the developing vertebrate hindbrain, the characteristic trajectory of the facial (nVII) motor nerve is generated by caudal migration of the nVII motor neurons. The nVII motor neurons originate in rhombomere (r) 4, and migrate caudally into r6 to form the facial motor nucleus. Using a transgenic zebrafish line that expresses green fluorescent protein (GFP) in the cranial motor neurons, two novel mutants, designated landlocked (llk) and off-road (ord), have been isolated that both show highly specific defects in the caudal migration of the nVII motor neurons. The landlocked locus contains the gene scribble1 (scrb1), and its zygotic expression is required for migration of the nVII motor neurons mainly in a non cell-autonomous manner. Taking advantage of the viability of the llk mutant embryos, it was found that maternal expression of scrb1 is required for convergent extension (CE) movements during gastrulation. Furthermore, a genetic interaction is seen between scrb1 and trilobite(tri)/strabismus(stbm) in CE. The dual roles of the scrb1 gene in both neuronal migration and CE provide a novel insight into the underlying mechanisms of cell movement in vertebrate development (Wada, 2005).

Although the results suggest that there is a genetic interaction between scrb1 and stbm, it is not known whether the PDZ domains of Scrb directly interact with the PDZ-binding domain of Stbm. In Drosophila, the second PDZ domain of Scrb interacts with Dlg via GUKH (guanylate kinase holder protein) to form a scaffolding complex at synaptic junctions. Furthermore, Dlg interacts with Stbm and this complex is required for plasma membrane formation in epithelial cells. These results suggest that Scrb, Stbm and Dlg may constitute a functional complex during the formation of membrane structures. If Tri/Stbm and Llk/Scrb1 form a functional complex, this complex would probably have two sites that associate with membranes: the transmembrane domain of Tri/Stbm and the LRR domain of Llk/Scrb1. Knock-down of Tri/Stbm with overexpression of Llk/Scrb1 leads to the most severe impairment of CE. These results indicate that Tri/Stbm may be required for localization of Llk/Scrb1 protein to the specific site of the membrane where they are anchored and function together. Release of membrane-associated Llk/Scrb1 from such positional constraint in the absence of Stbm may have more markedly perturbed the functional protein complexes controlling CE than simple overexpression of Scrb1 in the presence of Stbm (Wada, 2005).

The Scrb1rw16 protein, which has a single amino acid substitution in the first PDZ domain, has lower activity than the wild-type protein to rescue migration of the nVII motor neurons in the llk mutation. Similarly, overexpression of Scrb1rw16 induces CE defects to a lesser extent than that of wild-type Scrb1 protein. These results indicate that the first PDZ domain is also essential for Scrb1 activity. The first PDZ domain of Llk/Scrb1 may interact with another, as yet unidentified, component to establish a multi-protein complex required for its function (Wada, 2005).

The planar cell polarity (PCP) pathway is conserved throughout evolution, but it mediates distinct developmental processes. In Drosophila, members of the PCP pathway localize in a polarized fashion to specify the cellular polarity within the plane of the epithelium, perpendicular to the apicobasal axis of the cell. In Xenopus and zebrafish, several homologs of the components of the fly PCP pathway control convergent extension. Mammalian PCP homologs regulate both cell polarity and polarized extension in the cochlea in the mouse. Using mice with null mutations in two mammalian Dishevelled homologs, Dvl1 and Dvl2, it has been shown that during neurulation a homologous mammalian PCP pathway regulates concomitant lengthening and narrowing of the neural plate, a morphogenetic process defined as convergent extension. Dvl2 genetically interacts with Loop-tail, a point mutation in the mammalian PCP gene Vangl2, during neurulation. By generating Dvl2 BAC (bacterial artificial chromosome) transgenes and introducing different domain deletions and a point mutation identical to the dsh1 allele in fly, a high degree of conservation was demonstrated between Dvl function in mammalian convergent extension and the PCP pathway in fly. In the neuroepithelium of neurulating embryos, Dvl2 shows C-terminal DEP domain-dependent membrane localization, a pre-requisite for its involvement in convergent extension. Intriguing, the Loop-tail mutation that disrupts both convergent extension in the neuroepithelium and PCP in the cochlea does not disrupt Dvl2 membrane distribution in the neuroepithelium, in contrast to its drastic effect on Dvl2 localization in the cochlea (Wang, 2006).

Environmental and genetic aberrations lead to neural tube closure defects (NTDs) in 1 in every 1000 births. Mouse and frog models for these birth defects have suggested that Van Gogh-like 2 (Vangl2, also known as Strabismus) and other components of planar cell polarity (PCP) signalling control neurulation by promoting the convergence of neural progenitors to the midline. This study reports a novel role for PCP signalling during neurulation in zebrafish. Non-canonical Wnt/PCP signalling polarizes neural progenitors along the anterior-posterior axis. This polarity is transiently lost during cell division in the neural keel but is re-established as daughter cells reintegrate into the neuroepithelium. Loss of zebrafish Vangl2 (in trilobite mutants) abolishes the polarization of neural keel cells, disrupts re-intercalation of daughter cells into the neuroepithelium, and results in ectopic neural progenitor accumulations and NTDs. Remarkably, blocking cell division leads to rescue of trilobite neural tube morphogenesis despite persistent defects in convergence and extension. These results reveal a role for PCP signalling in coupling cell division and morphogenesis at neurulation and suggest a novel mechanism underlying NTDs (Ciruna, 2006).

Opposing Wnt pathways orient cell polarity during organogenesis

The orientation of asymmetric cell division contributes to the organization of cells within a tissue or organ. For example, mirror-image symmetry of the C. elegans vulva is achieved by the opposite division orientation of the vulval precursor cells (VPCs) flanking the axis of symmetry. This study characterized the molecular mechanisms contributing to this division pattern. Wnts MOM-2 and LIN-44 are expressed at the axis of symmetry and orient the VPCs toward the center. These Wnts act via Fz/LIN-17 and Ryk/LIN-18, which control beta-catenin localization and activate gene transcription. In addition, VPCs on both sides of the axis of symmetry possess a uniform underlying 'ground' polarity, established by the instructive activity of Wnt/EGL-20. EGL-20 establishes ground polarity via a novel type of signaling involving the Ror receptor tyrosine kinase CAM-1 and the planar cell polarity component Van Gogh/VANG-1. Thus, tissue polarity is determined by the integration of multiple Wnt pathways (Green, 2008).

These results describe the contributions of multiple Wnt pathways to the orientation of cell polarity in the C. elegans vulval epithelium. Because no factor required for the posterior orientation of P5.p or P7.p had previously been identified, this orientation was thought to be signaling independent or 'default'. However, when a new approach was used to reduce Wnt levels in a spatiotemporally controlled manner (overexpression of Ror/CAM-1, a Wnt sink), the VPCs displayed instead a randomized orientation, which is likely to be the true default. The posterior orientation seen in the absence of Fz/lin-17 and Ryk/lin-18 depends on the instructive activity of Wnt/EGL-20. This polarity is referred to as 'ground' polarity. In response to centrally located Wnt/MOM-2 (and possibly Wnt/LIN-44), the receptors Fz/LIN-17 and Ryk/LIN-18 orient P5.p and P7.p toward the center. This reorientation of P7.p, 'refined' polarity, provides the mirror-image symmetry required for a functional organ (Green, 2008).

That P7.p is oriented toward the center in wild-type worms suggests that Wnts LIN-44 and MOM-2 have a greater ability to affect P7.p orientation than does EGL-20. Although the posterior-anterior EGL-20 gradient reaches the VPCs, EGL-20 levels may be much lower here than the levels of Wnts secreted from the nearby AC. Indeed, it was found that local expression of egl-20 in the AC can overcome the effects of distally expressed egl-20. lin-44 is expressed in the tail in addition to the AC but has not been shown to have long-range activity. It is thus possible that this posterior source of lin-44 does not affect P7.p orientation and that LIN-44, in addition to MOM-2, acts as a central cue (Green, 2008).

LIN-17 and LIN-18 were previously reported to reorient P7.p and to reverse the AP pattern of nuclear TCF/POP-1 levels in P7.p daughters. This study extended knowledge of the signaling downstream of Fz/LIN-17 and Ryk/LIN-18 by showing that these receptors control the asymmetric localization of two β-catenins, SYS-1 and BAR-1, the first evidence that Ryk proteins regulate β-catenin. Although asymmetric localization of SYS-1 suggests involvement of the Wnt/β-catenin asymmetry pathway, disruption of pathway components either did not cause a P-Rvl phenotype (lit-1(rf)) or caused only a weakly penetrant P-Rvl phenotype [pop-1(RNAi), sys-1(rf), and wrm-1(rf)], making the function of the Wnt/β-catenin asymmetry pathway in refined polarity unclear. LIN-17 and LIN-18 were also shown to activate transcription in the proximal VPC daughters. Yet, this transcription is not required for P7.p reorientation, since transcriptional states observed by POPTOP, a reporter of Wnt target genes, do not always correspond with the morphological phenotype. Therefore, refined polarity may be largely independent of BAR-1 or the Wnt/β-catenin asymmetry pathway and instead be analagous to the spindle reorientation of the EMS cell during C. elegans embryogenesis, in which Wnt signaling affects the cytoskeleton independent of Wnt's effect on gene expression (Green, 2008).

What then, is the purpose of the Wnt/β-catenin asymmetry pathway in the VPCs? The weakly penetrant A-Rvl phenotype seen in wrm-1(rf) and lin-17(lf); lit-1(lf) worms, combined with the observation that EGL-20 regulates SYS-1 asymmetry, suggests that the Wnt/β-catenin asymmetry pathway functions in ground polarity. Therefore, both ground and refined polarity may converge on regulation of these components, although they are not absolutely required for refined polarity. Because the localization of Wnt/β-catenin asymmetry pathway components in ground polarity matches the reiterative pattern seen in most other asymmetric cell divisions in C. elegans, it is hypothesized that localization of these components is initially established as part of a global anterior-posterior polarity. It is likely that LIN-17 and LIN-18 overcome ground polarity by inhibiting the Wnt/β-catenin asymmetry pathway, a scenario consistent with the ability of lit-1(rf) to suppress lin-17(lf) and lin-18(lf) mutations (Green, 2008).

Remarkably, it is only by peeling back the layer of refined polarity that ground polarity can be observed and manipulated. By doing so, it was found that Wnt/EGL-20, expressed from a distant posterior source, imparts uniform AP polarity to the field of VPCs via a new pathway involving Van Gogh/vang-1, a core PCP pathway component. It is noteworthy that Fz is also a core PCP pathway component, yet it does not seem to be involved in EGL-20 signaling via VANG-1. This is not incompatible with other descriptions of PCP. For example, in the Drosophila wing, Van Gogh and Fz antagonize each other and cause wing hairs to orient in opposite directions. The molecular mechanism by which VANG-1 functions in ground polarity is unknown; however, regulation of SYS-1 by VANG-1 provides evidence that the pathway involving egl-20 and vang-1is associated with the Wnt/β-catenin asymmetry pathway (Green, 2008).

A major difference between VPC orientation in C. elegans and PCP in Drosophila is that no Wnt has been directly implicated in Drosophila PCP. Therefore, VPC orientation may be more similar to some forms of PCP in vertebrates. For example, Wnts are believed to act as permissive polarizing factors during vertebrate convergent extension. Also, VPC orientation is strikingly similar to hair cell orientation in the utricular epithelia of the mammalian inner ear, wherein hair cells flanking the axis of symmetry are oriented in opposite directions. In this system, both medial and lateral hair cells possess a uniform underlying polarity as evidenced by asymmetric localization of Prickle, a core PCP pathway component, to the medial side of cells in both populations. Van Gogh is required for proper Prickle asymmetry, perhaps similarly to the role of vang-1 in ground polarity of the VPCs. It is not understood how the position of the utricular axis of symmetry is determined, but the similarities between these two systems suggest that it may represent a local source of Wnt (Green, 2008).

By moving the source of EGL-20 from the posterior to the anterior side of P7.p and thereby reversing P7.p orientation, this study showed that EGL-20 acts as a directional cue. Although it is not presently clear if the pathway involving egl-20 and vang-1 is mechanistically similar to the PCP pathway described in Drosophila and vertebrates, the result nonetheless provides a long-sought example of a Wnt that acts instructively via a PCP pathway component. Detailed description of the subcellular localization of Van Gogh/VANG-1 and other PCP pathway components in the VPCs will be required to make meaningful comparisons between VPC orientation and established models of PCP (Green, 2008).

In addition to vang-1, a role of Ror/cam-1 in ground polarity was identified. The results provide the first evidence that Ror proteins interpret directional Wnt signals, as well as the first evidence that they interact with Van Gogh. Although a Xenopus Ror homolog, Xror2, was previously described to function in PCP during convergent extension, a recent report indicates that the involvement of Xror2 in convergent extension (CE) is actually via a different pathway. In response to Wnt5a, Xror2 activates JNK by a mechanism requiring Xror2 kinase activity. In contrast to Wnt5a/Xror2 signaling, Ror/CAM-1 function in ground polarity does not require JNK. Therefore, the ground polarity pathway involving Wnt/EGL-20, Ror/CAM-1, and Van Gogh/VANG-1 may be a new type of Wnt signaling (Green, 2008).

Using C. elegans vulva development as a model, this study showed that multiple coexisting Wnt pathways with distinct ligand specificities and signaling mechanisms act in concert to regulate the polarity of individual cells during their assembly into complex structures (Green, 2008).

Zebrafish colgate/hdac1 functions in the non-canonical Wnt pathway during axial extension and in Wnt-independent branchiomotor neuron migration

Vertebrate gastrulation involves the coordinated movements of populations of cells. These movements include cellular rearrangements in which cells polarize along their medio-lateral axes leading to cell intercalations that result in elongation of the body axis. Molecular analysis of this process has implicated the non-canonical Wnt/Frizzled signaling pathway that is similar to the planar cell polarity pathway (PCP) in Drosophila. This study describes a zebrafish mutant, colgate (col), which displays defects in the extension of the body axis and the migration of branchiomotor neurons. Activation of the non-canonical Wnt/PCP pathway in these mutant embryos by overexpressing ΔNdishevelled, rho kinase2 and van gogh-like protein 2 (vangl2) rescues the extension defects suggesting that col acts as a positive regulator of the non-canonical Wnt/PCP pathway. Further, col is shown to normally regulate the caudal migration of nVII facial hindbrain branchiomotor neurons; the mutant phenotype can be rescued by misexpression of vangl2 independent of the Wnt/PCP pathway. col locus was cloned and found to encode histone deacetylase1 (hdac1). hdac1 has been implicated in repressing the canonical Wnt pathway. This study demonstrates novel roles for zebrafish hdac1 in activating non-canonical Wnt/PCP signaling underlying axial extension and in promoting Wnt-independent caudal migration of a subset of hindbrain branchiomotor neurons (Nambiar, 2007).

Studies of col mutants have revealed novel functions of Hdac1 in major signaling pathways regulating embryonic development. However, precisely how Hdac1 functions in these pathways is not fully understood. In the canonical Wnt pathway, Hdac1 functions as a co-repressor with molecules such as Groucho and LEF1 in the nucleus. Studies in Drosophila and vertebrates have shown that Groucho, a canonical Wnt signaling pathway repressor, readily interacts with Hdac1 forming a repressor complex that remains tethered to the promoter of Wnt target genes. Data also indicates that the Wnt transcription factor LEF1 can act as a repressor in the presence of Hdac1. Activation of LEF-dependent target genes occurs when the increasing level of β-catenin in the nucleus is able to dissociate Hdac1 from LEF1 and itself bind to LEF1 to form a dimeric activator. Thus, Hdac1 appears to maintain Wnt target genes in a repressed state until replaced by activators such as β-catenin (Nambiar, 2007).

This study has shown that col/hdac1 regulates both the non-canonical Wnt/PCP pathway that controls CE movements as well as the pathway that mediates the caudal migration of hindbrain facial motor neurons. There are a number of possible ways in which Hdac1 functions in these pathways. For example, since Hdac1 regulates both pathways, it is conceivable then that Col/Hdac1 could act by regulating the transcription of vangl2 or its interacting proteins. vangl2 expression was examined in col mutants and there appeared to be no significant difference compared to wildtype siblings. Another possible scenario for the functioning of Col/Hdac1 in this context could be via an interaction with Vangl2 and its interacting proteins such as Pk and Scribble that act at the common branchpoint. Another possibility is that Col/Hdac1 regulates the transcription of other components of the Wnt/PCP pathway and/or the targets of Wnt/PCP pathway genes. In the latter case, additional interactions of Hdac1 with Wnt/PCP signaling-independent genes or components of the pathway that also regulate branchiomotor neuron migration are possible. Further studies exploring the function of col should reveal the molecular mechanism by which col/hdac1 affects the activities of the genes involved in the morphogenetic events that were described (Nambiar, 2007).

Non-canonical Wnt signaling regulates cell polarity in female reproductive tract development via van gogh-like 2

Wnt signaling effectors direct the development and adult remodeling of the female reproductive tract (FRT); however, the role of non-canonical Wnt signaling has not been explored in this tissue. The non-canonical Wnt signaling protein van gogh-like 2 is mutated in loop-tail (Lp) mutant mice (Vangl2Lp), which display defects in multiple tissues. Vangl2Lp mutant uterine epithelium displays altered cell polarity, concommitant with changes in cytoskeletal actin and scribble (scribbled, Scrb1) localization. The postnatal mutant phenotype is an exacerbation of that seen at birth, exhibiting more smooth muscle and reduced stromal mesenchyme. These data suggest that early changes in cell polarity have lasting consequences for FRT development. Furthermore, Vangl2 is required to restrict Scrb1 protein to the basolateral epithelial membrane in the neonatal uterus, and an accumulation of fibrillar-like structures observed by electron microscopy in Vangl2Lp mutant epithelium suggests that mislocalization of Scrb1 in mutants alters the composition of the apical face of the epithelium. Heterozygous and homozygous Vangl2Lp mutant postnatal tissues exhibit similar phenotypes and polarity defects and display a 50% reduction in Wnt7a levels, suggesting that the Vangl2Lp mutation acts dominantly in the FRT. These studies demonstrate that the establishment and maintenance of cell polarity through non-canonical Wnt signaling are required for FRT development (Vandenberg, 2009).

The PCP pathway instructs the planar orientation of ciliated cells in the Xenopus larval skin

Planar cell polarity (PCP) is a property of epithelial tissues where cellular structures coordinately orient along a two-dimensional plane lying orthogonal to the axis of apical-basal polarity. PCP is particularly striking in tissues where multiciliate cells generate a directed fluid flow, as seen, for example, in the ciliated epithelia lining the respiratory airways or the ventricles of the brain. To produce directed flow, ciliated cells orient along a common planar axis in a direction set by tissue patterning, but how this is achieved in any ciliated epithelium is unknown. This study shows that the planar orientation of Xenopus multiciliate cells is disrupted when components in the PCP-signaling pathway are altered non-cell-autonomously. Wild-type ciliated cells located at a mutant clone border reorient toward cells with low Vangl2 or high Frizzled activity and away from those with high Vangl2 activity. These results indicate that the PCP pathway provides directional non-cell-autonomous cues to orient ciliated cells as they differentiate, thus playing a critical role in establishing directed ciliary flow (Mitchell, 2009).

The roles of maternal Vangl2 and aPKC in Xenopus oocyte and embryo patterning

The Xenopus oocyte contains components of both the planar cell polarity and apical-basal polarity pathways, but their roles are not known. This study examined the distribution, interactions and functions of the maternal planar cell polarity core protein Vangl2 and the apical-basal complex component aPKC. Vangl2 is distributed in animally enriched islands in the subcortical cytoplasm in full-grown oocytes, where it interacts with a post-Golgi v-SNARE protein, VAMP1, and acetylated microtubules. Vangl2 is required for the stability of VAMP1 as well as for the maintenance of the stable microtubule architecture of the oocyte. Vangl2 interacts with atypical PKC, and both the acetylated microtubule cytoskeleton and the Vangl2-VAMP1 distribution are dependent on the presence of aPKC. aPKC and Vangl2 are required for the cell membrane asymmetry that is established during oocyte maturation, and for the asymmetrical distribution of maternal transcripts for the germ layer and dorsal/ventral determinants VegT and Wnt11. This study demonstrates the interaction and interdependence of Vangl2, VAMP1, aPKC and the stable microtubule cytoskeleton in the oocyte, shows that maternal Vangl2 and aPKC are required for specific oocyte asymmetries and vertebrate embryonic patterning, and points to the usefulness of the oocyte as a model to study the polarity problem (Cha, 2011).

Planar cell polarity defects and defective Vangl2 trafficking in mutants for the COPII gene Sec24b

Among the cellular properties that are essential for the organization of tissues during animal development, the importance of cell polarity in the plane of epithelial sheets has become increasingly clear in the past decades. Planar cell polarity (PCP) signaling in vertebrates has indispensable roles in many aspects of their development, in particular, controlling alignment of various types of epithelial cells. Disrupted PCP has been linked to developmental defects in animals and to human pathology. Neural tube closure defects (NTD) and disorganization of the mechanosensory cells of the organ of Corti are commonly known consequences of disturbed PCP signaling in mammals. A typical PCP phenotype exists in a mouse mutant for the Sec24b gene, including the severe NTD craniorachischisis, abnormal arrangement of outflow tract vessels and disturbed development of the cochlea. In addition, genetic interaction was observed between Sec24b and the known PCP gene, scribble. Sec24b is a component of the COPII coat protein complex that is part of the endoplasmic reticulum (ER)-derived transport vesicles. Sec24 isoforms are thought to be directly involved in cargo selection, and evidence is presented that Sec24b deficiency specifically affects transport of the PCP core protein Vangl2, based on experiments in embryos and in cultured primary cells (Wansleeben, 2010).

Vang-like protein 2 and Rac1 interact to regulate adherens junctions

The Wnt planar cell polarity (Wnt/PCP) pathway signals through small Rho-like GTPases to regulate the cytoskeleton. The core PCP proteins have been mapped to the Wnt/PCP pathway genetically, but the molecular mechanism of their action remains unknown. This study investigated the function of the mammalian PCP protein Vang-like protein 2 (Vangl2). RNAi knockdown of Vangl2 impaired cell-cell adhesion and cytoskeletal integrity in the epithelial cell lines HEK293T and MDCK. Similar effects were observed when Vangl2 was overexpressed in HEK293T, MDCK or C17.2 cells. The effects of Vangl2 overexpression could be blocked by knockdown of the small GTPase Rac1 or by dominant-negative Rac1. In itself, knockdown of Rac1 impaired cytoskeletal integrity and reduced cell-cell adhesion. Vangl2 bound and re-distributed Rac1 within the cells but did not alter Rac1 activity. Moreover, both transgenic mouse embryos overexpressing Vangl2 in neural stem cells and loop-tail Vangl2 loss-of-function embryos displayed impaired adherens junctions, a cytoskeletal unit essential for neural tube rigidity and neural tube closure. In vivo, Rac1 was re-distributed within the cells in a similar way to that observed in vitro. It is propose that Vangl2 affects cell adhesion and the cytoskeleton by recruiting Rac1 and targeting its activity in the cell to adherens junctions (Lindqvist, 2010).

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). This study has 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, in vitro evidence is provided that homotypic intracellular interactions of Celsr1 are required to recruit Vangl2 and Fzd6 to sites of cell-cell contact (Devenport, 2008).

Mitotic internalization of planar cell polarity proteins preserves tissue polarity

Planar cell polarity (PCP) is the collective polarization of cells along the epithelial plane, a process best understood in the terminally differentiated Drosophila wing. Proliferative tissues such as mammalian skin also show PCP, but the mechanisms that preserve tissue polarity during proliferation are not understood. During mitosis, asymmetrically distributed PCP components risk mislocalization or unequal inheritance, which could have profound consequences for the long-range propagation of polarity. This study shows that when mouse epidermal basal progenitors divide PCP components are selectively internalized into endosomes, which are inherited equally by daughter cells. Following mitosis, PCP proteins are recycled to the cell surface, where asymmetry is re-established by a process reliant on neighbouring PCP. A cytoplasmic dileucine motif governs mitotic internalization of atypical cadherin Celsr1, which recruits Vang2 and Fzd6 to endosomes. Moreover, embryos transgenic for a Celsr1 that cannot mitotically internalize exhibit perturbed hair-follicle angling, a hallmark of defective PCP. This underscores the physiological relevance and importance of this mechanism for regulating polarity during cell division (Devenport, 2011).

This study has identified mitotic internalization as a mechanism for maintaining global PCP in a proliferative tissue. It is proposed that internalization provides a mechanism to distribute asymmetrically localized PCP components equally to daughter cells and temporarily block cells from sending and receiving PCP signals while they round up and divide (Devenport, 2011).

In the absence of wild-type Celsr1 in cultured keratinocytes, Celsr1LLtoAA clearly blocked internalization of its PCP associates. However, this did not happen in the presence of wild-type Celsr1, where Celsr1LLtoAA transgenic embryos showed no obvious defects in Vangl2 inheritance. The fact that these mutant embryos nevertheless showed marked non-autonomous disruption of planar cell polarity underscores the importance of Celsr1's endocytic motif in the process, and indicates that at least one function of mitotic internalization is to modulate signalling (Devenport, 2011).

PCP components are thought to transmit polarity cues by interacting across plasma membranes, and in Drosophila Celsr1's homologue Fmi is critical for cell-to-cell polarity transmission. Taken together with the current findings, it is posited that Celsr1 internalization should prevent cells from both sending and receiving PCP signals while they divide, thereby helping to maintain global alignment of polarity in a proliferative tissue. When an internalization-defective Celsr1 is expressed, mitotic cells continue to signal and this aberrant directional information is propagated from cell to cell (Devenport, 2011).

Polarized cells need a mechanism to maintain polarity when they divide. Single-layered epithelial cells orient their mitotic spindles parallel to the substratum to ensure that daughter cells maintain the apical-basal polarity of their parent. Furthermore, many polarized cell types regulate spindle orientation to divide asymmetrically and generate cellular diversity. This study has found that mitotic internalization is a mechanism for polarized epithelial cells to maintain planar polarity while they divide. This is the first time that components of a common pathway have been shown to internalize specifically when cells divide (Devenport, 2011).

Despite its essential role in mouse epidermis, the mitotic internalization mechanism is not a universal feature of PCP. In Drosophila sensory-organ precursors, planar divisions with asymmetric daughter fates are oriented by cortically localized PCP proteins. Perhaps the difference is that basal epidermal cells do not seem to depend on PCP for asymmetric cell fates. However, a recent study of PCP in the dividing Drosophila wing blade also did not report internalization in mitotic cells. While highly conserved in vertebrates, Celsr1's internalization motif does not have a clear counterpart in Drosophila Fmi. It is at present unknown whether mitotic internalization is a conserved feature of PCP components in lower eukaryotes, and whether dividing cells have alternative mechanisms for preservation of tissue polarity (Devenport, 2011).

While future studies will be necessary to resolve this issue, the highly proliferative nature of basal cells poses a particular challenge to maintain PCP. It is tempting to speculate that other highly proliferative tissues might maintain PCP by employing a mitotic internalization mechanism similar to the one unearthed in this study. If so, the internalization process may have evolved in vertebrates to suit the specialized needs of highly regenerative tissues (Devenport, 2011).

Planar polarity pathway and Nance-Horan syndrome-like 1b have essential cell-autonomous functions in neuronal migration

Components of the planar cell polarity (PCP) pathway are required for the caudal tangential migration of facial branchiomotor (FBM) neurons, but how PCP signaling regulates this migration is not understood. In a forward genetic screen, a new gene was identified, nhsl1b, that is required for FBM neuron migration. nhsl1b encodes a WAVE-homology domain-containing protein related to human Nance-Horan syndrome (NHS) protein and Drosophila GUK-holder (Gukh), which have been shown to interact with components of the WAVE regulatory complex that controls cytoskeletal dynamics and with the polarity protein Scribble, respectively. Nhsl1b localizes to FBM neuron membrane protrusions and interacts physically and genetically with Scrib to control FBM neuron migration. Using chimeric analysis, it was shown that FBM neurons have two modes of migration: one involving interactions between the neurons and their planar-polarized environment, and an alternative, collective mode involving interactions between the neurons themselves. The first mode of migration requires the cell-autonomous functions of Nhsl1b and the PCP components Scrib and Vangl2 in addition to the non-autonomous functions of Scrib and Vangl2, which serve to polarize the epithelial cells in the environment of the migrating neurons. These results define a role for Nhsl1b as a neuronal effector of PCP signaling and indicate that proper FBM neuron migration is directly controlled by PCP signaling between the epithelium and the migrating neurons (Walsh, 2011).

Non-cell-autonomous planar cell polarity propagation in the auditory sensory epithelium of vertebrates

Sensory epithelia of the inner ear require a coordinated alignment of hair cell stereociliary bundles as an essential element of mechanoreceptive function. Hair cell bundle alignment is mediated by core planar cell polarity (PCP) proteins, such as Vangl2, that localize asymmetrically to the circumference of the cell near its apical surface. During early phases of cell orientation in the chicken basilar papilla (BP), Vangl2 is present at supporting cell junctions that lie orthogonal to the polarity axis. Several days later, there is a striking shift in the Vangl2 pattern associated with hair cells that reorient towards the distal (apical) end of the organ. How the localization of PCP proteins transmits planar polarity information across the developing sensory epithelium remains unclear. To address this question, the normal asymmetric localization of Vangl2 was disrupted by overexpressing Vangl2 in clusters of cells. The BP was infected with replication-competent retrovirus encoding Vangl2 prior to hair cell differentiation. Virus-infected cells showed normal development of individual stereociliary bundles, indicating that asymmetry was established at the cellular level. Yet, bundles were misoriented in ears infected with Vangl2 virus but not Wnt5a virus. Notably, Vangl2 misexpression did not randomize bundle orientations but rather generated larger variations around a normal mean angle. Cell clusters with excess Vangl2 could induce non-autonomous polarity disruptions in wild-type neighboring cells. Furthermore, there appears to be a directional bias in the propagation of bundle misorientation that is towards the abneural edge of the epithelium. Finally, regional bundle reorientation was inhibited by Vangl2 overexpression. In conclusion, ectopic Vangl2 protein causes inaccurate local propagation of polarity information, and Vangl2 acts in a non-cell-autonomous fashion in the sensory system of vertebrates (Sienknecht, 2011).

Mitotic spindle asymmetry: a Wnt/PCP-regulated mechanism generating asymmetrical division in cortical precursors

The regulation of asymmetric cell division (ACD) during corticogenesis is incompletely understood. This study documents that spindle-size asymmetry (SSA) between the two poles occurs during corticogenesis and parallels ACD. SSA appears at metaphase and is maintained throughout division, and it is necessary for proper neurogenesis. Imaging of spindle behavior and division outcome reveals that neurons preferentially arise from the larger-spindle pole. Mechanistically, SSA magnitude is controlled by Wnt7a and Vangl2, both members of the Wnt/planar cell polarity (PCP)-signaling pathway, and relayed to the cell cortex by P-ERM proteins. In vivo, Vangl2 and P-ERM downregulation promotes early cell-cycle exit and prevents the proper generation of late-born neurons. Thus, SSA is a core component of ACD that is conserved in invertebrates and vertebrates and plays a key role in the tight spatiotemporal control of self-renewal and differentiation during mammalian corticogenesis (Delaunay, 2014).


Van Gogh/strabismus: Regulation | Developmental Biology | Effects of Mutation | References

date revised: 10 February 2014

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