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: pk^pk, pk^M and pk^pk-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 pk^pk phenotype, affecting only the wing and notum, whereas mutations in the LIM- or PET-encoding domains result in pk^pk-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 pk^pk-sple phenotype described in this study (Rawls, 2003).

Although ommatidial polarity is not affected in individuals carrying the pk^pk1 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 pk^eq background (a genetic null that fails to complement pk^pk-sple alleles). However, Pk localization is disrupted in a stbm^6cn 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 stbm^6cn 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

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).

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 Scrb1^rw16 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 Scrb1^rw16 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).

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).

date revised: 20 November 2006

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