BIOLOGICAL OVERVIEW

dachsous encodes a protein that controls imaginal disc morphogenesis in Drosophila and is a member of the cadherin superfamily. Two loci, dachsous(ds) and fat(ft), both members of the cadherin superfamily, have long been known to play important roles during imaginal disc development and morphogenesis. The genetic interaction between ds and ft suggested that these two genes might function in the same pathway. A spontaneous mutation at the ds locus, ds1 was discovered in 1917 by Calvin Bridges. The first recessive and dominant mutations of ft, known as ft1 and Gull, respectively, were isolated two years later; similarities between their phenotypes and that of ds1is the basis for the model suggesting that ds and ft function in the same genetic pathway. Consistent with this model, ds1 was shown by Mohr in 1929 to suppress the Gull phenotype, since one copy of ds1 causes a weak suppression of Gull, and two copies cause a strong suppression (Clark, 1995 and references).

Dachsous and Fat differ from the classic cadherins, and from another cadherin protein of Drosophila, Shotgun, mainly due to the much larger number of extracellular cadherin domains in Ds and Fat. In addition, Shotgun and Fat have EGF- and laminin A G-domain-like repeats in their extracellular domains that are absent in Ds and the classic cadherins. Based on the structures of Ds and Fat, and based on their genetic interaction, a model is presented which accounts for the physical interaction between Ds and Fat. It is proposed that Ds and Fat mediate cell-cell adhesion by homo- and heterophilic interaction of the cadherin domains and transmit signals regulating morphogenesis and cell proliferation via their cytoplasmic domains to the cell interior and nucleus. The morphogenetic signals transmitted by Ds and Ft are not necessarily the same, although the two proteins might cooperate. Only Ft (but not Ds), mediates signals controlling cell proliferation through its specific extracellular EGF-like and laminin A G-domain-like repeats, which act as receptors. Both of these processes, (1) control of cell proliferation and (2) morphogenesis, are intimately linked by coupled equilibria between homophilic and heterophilic associatons of the Ds and Fat cadherins. The likelyhood that Dachsous forms a heterodimeric association with Fat on adjacent cells is used to explain the suppression of Gull phenotype by dachsous mutation (Clark, 1995).

Flies mutant for dachsous exhibit a tissue polarity phenotype. At least some of the tissue polarity phenotypes associated with ds resemble those seen in frizzled pathway mutants and seem unlikely to be due to an effect on cell adhesion. It has been known for some time that the function of ds relates to tissue polarity. Held (1986) found that ds mutations disrupt the polarity of bristles on the legs and that they cause leg joint abnormalities that are similar to those produced by fz and spiny legs. The overall morphological abnormalities caused by ds mutations are greater on the leg than in the wing, but it appears that the effects on the wing and leg may be parallel (Adler, 1998).

Dachsous acts in the frizzled pathway where it affects signaling involved in the formation of proper wing hair polarity. dachsous clones disrupt the polarity of neighboring wild-type cells, suggesting the possibility that dachsous affects the intercellular signaling function of frizzled. The function of the frizzled pathway is essential for the formation of a wing with normal distally pointing hairs. This pathway is thought to be composed of both an intercellular signaling system and an intracellular signal transduction system. The cell autonomous function of this pathway leads to prehair initiation being restricted to the vicinity of the distal vertex. Mutations in genes such as dishevelled, inturned, fuzzy and RhoA produce a tissue polarity phenotype by interfering with the intracellular transduction of the fz signal. Dachsous is not required for the transduction of the fz signal and consistent with this conclusion, it does not cause a polarity pattern that is typical of either a lack of or reduction in fz pathway function. Instead, ds appears to cause a tissue polarity phenotype by altering the direction of fz signaling. The correspondence between the direction of hair polarity (distal), the subcellular location for prehair initiation (in the vicinity of the distal vertex of the cell) and the direction of fz domineering nonautonomy (distal) as seen in wild-type wings is maintained in regions of ds wings, but notably, with reversed polarity. In such wing regions, proximally pointed hairs are formed in the vicinity of the proximal vertex, and fz clones display proximal domineering nonautonomy. This has led to the conclusion that the abnormal hair polarity in ds wings is a consequence of the abnormal direction of fz signaling and that ds cells in these regions are responding normally to an abnormal signal (Adler, 1998).

Two models have been proposed to account for the role of Fz in tissue polarity and to explain the distal domineering nonautonomy of fz clones (Adler, 1997). The cell-by-cell signaling model suggests that the binding of ligand at one side of a cell leads to the Fz receptor becoming unevenly activated across the cell. This leads to both prehair initiation and the relaying of the signal being localized at the distal edge of cells, which leads to hair polarity being coincident with the direction of signaling. In this model the domineering distal nonautonomy of fz clones was ascribed to a failure to receive the signal in cells distal to the clone. The secondary signal model suggests that the Fz receptor is activated in a gradient fashion along the proximal/distal axis of the wing by a long range gradient of a morphogen ligand. Fz activation leads to the proportional production of a secondary signal, which acts locally to polarize cells. In this model the nonautonomy of fz clones is due to a failure of clone cells to produce the secondary signal. A group of proximal cells presumably form the source for the gradient of Fz ligand. Either of these models can easily accommodate the observation that there are regions in a ds wing where the direction of fz signaling is reversed. dachsous mutations could result in a change in the fate of cells that then serve either as ectopic sources of the gradient morphogen or could alter locations that initiate cell-by-cell signaling. Such models might also be able to explain the altered wing shape and wing vein pattern as a consequence of a population of cells with altered cell fate. However, this hypothesis does not however, explain the need for ds function for tissue polarity development in all regions of the wing, as has been shown by an analysis of ds clones. Nor does it explain the enhanced domineering nonautonomy of fz that is observed in ds mutant wings. Therefore, a hypothesis is preferred wherein ds mutations produce their phenotypic effects by altering the function of the fz pathway in all regions of the wing. It is possible that fz signaling takes place at the adherens junction and that ds mutations alter the structure or composition of the junction in a way that alters fz signaling. For example, ds could promote the assembly of an Fz receptor complex at the junction. An 'incomplete' complex formed in a ds mutant might be unstable, leading to aberrant signaling (Adler, 1998).

Planar cell polarity in the Drosophila eye is directed by graded Four-jointed and Dachsous expression

Planar cell polarity (PCP) occurs when the cells of an epithelium are polarized along a common axis lying in the epithelial plane. During the development of PCP, cells respond to long-range directional signals that specify the axis of polarization. It has been proposed that with respect to Drosophila eye morphogenesis a crucial step in this process is the establishment of graded expression of the cadherin Dachsous (Ds) and the Golgi-associated protein Four-jointed (Fj). These gradients have been proposed to specify the direction of polarization by producing an activity gradient of the cadherin Fat within each ommatidium. In this report, the key predictions of this model were tested and confirmed by altering the patterns of Fj, Ds and Fat expression. It was shown that the gradients of Fj and Ds expression provide partially redundant positional information essential for specifying the polarization axis. It was further demonstrated that reversing the Fj and Ds gradients can lead to reversal of the axis of polarization. Finally, it was shown that an ectopic gradient of Fat expression can re-orient PCP in the eye. In contrast to the eye, the endogenous gradients of Fj and Ds expression do not play a major role in directing PCP in the wing. Thus, this study reveals that the two tissues use different strategies to orient their PCP (Simon, 2004).

The development of organized PCP requires cells to polarize in response to directional signals within the plane of the epithelium. The apparent absence of local cues has suggested that cells orient their polarity in response to long-range diffusible signaling molecules that form gradients across the tissue. It has been proposed that the role of the diffusible signals, such as Wingless produced at the poles of the eye disc, is to drive graded transcription of Ds and Fj. In this model, the resulting Ds and Fj protein gradients then regulate the function of the cadherin Ft, resulting in a Ft activity gradient, which in turn controls the pattern of Fz competition within each ommatidium. Crucial tests of the model have been precluded by an inability to alter the patterns of Ds and Ft expression. This study has analyzed the effects of altering Fj, Ds and Ft expression in the eye, and provides evidence supporting crucial features of the model. Most importantly, it has been demonstrated that the Fj and Ds expression gradients provide redundant directional information that together orient PCP. Furthermore, the data shows that it is the combination of both gradients that provides the robust directional cues needed to support the perfect fidelity of polarization in wild-type eyes. In addition, it has been shown that graded Ft expression can direct the pattern of ommatidial polarity, thus providing support for the role of Ft as a graded regulator of Fz signaling acting under the control of the Fj and Ds gradients (Simon, 2004).

In the proposed model, the consistent equatorial bias of Fz signaling results from more effective Ft action in each equatorial R3/4 precursor cell when compared with its adjacent polar counterpart. Since this Ft difference results from the action of the Fj and Ds gradients, a key question is how these gradients could control the level of Ft function. Important insight into this issue has come from studies of the wing that suggest that Ft and Ds form a complex in which the localization of Ft on the surface of one cell is promoted by binding to Ds on the surface of the neighboring cell. The dependence of Ft plasma membrane localization on Ds may account for the requirement for Ds function during planar polarization in the eye, even when sufficient directional cues are provided by the Fj expression gradient (Simon, 2004).

The existence of Ds:Ft intercellular dimers suggests several mechanisms by which Ds might regulate Ft. One simple possibility is that Ds merely controls the accumulation of Ft on the surface of the neighboring cell. Thus, the relatively higher level of Ds in the polar R3/R4 precursor, which results from the polar gradient of Ds expression, would lead to the accumulation of more Ft on the bordering surface of the equatorial cell. This would result in an asymmetry in Ft protein levels precisely along the border between the precursor cells where Fz/PCP competition occurs. Although no such gradient has been observed, it would certainly be very subtle and perhaps undetectable. A second possibility is that Ds binding to Ft regulates Ft activity rather than localization. A third possibility is that Ds could participate with Ft in binding to the extracellular domain of a downstream target (Simon, 2004).

Fj appears to play a more limited role than Ds during planar polarization of the eye. Unlike Ds, which both contributes a directional signal through its graded expression and plays an essential role in the interpretation of directional cues, Fj appears only to participate in PCP establishment via the directional information provided by its graded expression. This more limited role can be seen in the observations that either the absence or the ubiquitous expression of Fj yields equivalent phenotypes, and does not grossly disrupt the pattern of polarization unless the Ds gradient has been replaced with ubiquitous expression. How might graded Fj fulfill this role? One possibility is that Fj may regulate the ability of Ft and Ds to productively interact with each other. Thus, the higher expression of Fj in the equatorial cell of each ommatidium leads to more Ft:Ds dimers being formed with Ft in the equatorial cell than in the opposite orientation. Since Fj appears to function in the Golgi, this regulation may involve the direct modification of Ft or Ds (Simon, 2004).

It is important to note that one aspect of the data reported here requires reconsideration of a feature of a previous model. In previous work, it was proposed that Fj acts upstream of Ds, perhaps by modifying the Ds activity gradient. This placement was based on genetic experiments showing that strong differences in Fj activity between R3/R4 precursor cells can direct ommatidial polarization only when Ds is present. The identification of an essential gradient-independent function for Ds clearly complicates the interpretation of these epistasis experiments. As a result, it is no longer possible to infer whether the information provided by the Fj expression gradient acts upstream of Ds to modify the information provided by the Ds gradient. An equally plausible possibility is that Fj regulates the function of the Ds:Ft complexes by modifying Ft rather than Ds function (Simon, 2004).

The work presented here was designed to test specific predictions of the model proposed in an earlier study. However, alternate roles for Ft function have also been proposed. In one model, Ft regulates the production of an unidentified long-range signal that is secreted at the equator and that directly controls eye polarity. The existence of such an unidentified patterning signal, often called Factor X, has been invoked frequently to explain the 'domineering nonautonomy' phenomenon seen in both the wing and the eye near clones of cells lacking function of PCP genes such as Fz. In the alternate model, the role of Ft is to prevent production of this factor everywhere in the eye except at the equator where Ft activity is proposed to be inhibited by unspecified mechanisms, presumably involving Ds. An important distinction between the two models relates to the predicted effects of graded Ft expression. In the model, graded Ft activity provides the key PCP directional cues, and thus ectopic Ft expression gradients are predicted to have the potential to orient ommatidial polarity. In an alternate model, gradients of Ft activity do not provide directional cues. Instead, it is the lack of Ft activity in a sharp zone at the equator that leads to the production of the unidentified patterning factor. As a result, this second model predicts that subtle gradients of Ft expression should not orient polarity, especially in the polar regions of the eye where Ft activity is proposed to be uninhibited. Thus, the data presented in this report demonstrating the orienting ability of Ft expression gradients presents a challenge to this alternate model. In addition, the need for Factor X, whose putative existence has been a common feature of PCP models in both the wing and eye, has been challenged recently on both experimental and theoretical grounds. These reports suggested that domineering nonautonomy results from the tendency of neighboring cells to align their polarization rather than the existence of an additional polarizing signal (Simon, 2004).

The key roles of Ft and the Fj and Ds expression gradients in the eye naturally raised the question of whether similar mechanisms are used to provide directional cues in other tissues, such as the wing. That such conservation might exist was suggested by the existence of gradients of Fj and Ds in the wing. Additionally, it has been demonstrated recently that ectopic gradients of Ft and Ds expression in the wing can produce re-orientation of polarity in the wing. Given the redundant nature of the directional cues provided by the Fj and Ds gradients in the eye, the most rigorous way to evaluate the roles of the Ds and Fj expression gradients in the wing was to examine the consequences of removing the directional information of both gradients simultaneously. When this was done, the resulting wings displayed almost completely normal polarity. Thus, the Ds and Fj expression gradients do not play a major role in orienting PCP in most of the wing blade. One possibility is that there are additional directional signals that act redundantly with the Ds and Fj gradients. Another possibility is that these gradients exist for reasons unrelated to PCP. For example, they may serve to regulate the function of Ft as a regulator of cellular proliferation. Possible support for such a role comes from the observation that flies in which both graded Fj and Ds expression has been replaced with ubiquitous expression survive to adulthood at reduced frequencies, and often display defects in the size and shape of their legs, wings and eyes (Simon, 2004).

The dispensability of the Fj and Ds gradients of expression during the polarization of the wing indicates that there must be currently unidentified directional cues directing wing PCP. Despite their mysterious nature, it is likely that their mode of action will involve the Ds:Ft complex. This inference can be drawn from the observation that animals lacking Ds function, or clones of cells lacking Ft or Ds activity, have substantial PCP defects in the wing. Importantly, clones of ft mutant cells in the wing appear not to read directional cues and instead align their polarity with that of their neighbors. Thus, whatever the nature of the unidentified signals, they appear not to function effectively in the absence of Ds and Ft. Since neither Ft nor Ds is directly required for the Fz PCP signaling at cell-cell junctions, the dependence of these unidentified signals on Ds and Ft suggests that they may act by asymmetrically modifying the action of the Ds:Ft complexes at cell-cell junctions engaged in PCP signaling. Thus, the elegant regulation of polarity in the eye by graded Fj and Ds expression may represent only one of a number of ways to modulate the action of Ft. Further analysis of the mechanisms by which Ft and Ds regulate the pattern of Fz/PCP signaling will undoubtedly aid in the identification of these unknown signals and their mode of action (Simon, 2004).

GENE STRUCTURE

Exons - 12

PROTEIN STRUCTURE

Amino Acids - 3503

Structural Domains and Evolutionary Homologs

The protein begins with a signal sequence. The N-terminal extracellular domain includes 27 tandemly repeated domains of about 110 amino acids each that are similar in sequence to those found in all other members of the cadherin superfamily. In particular, most of the key amino acids of the consensus sequence of cadherin domains and their putative Ca2+ binding sites are conserved in the Ds protein. Only a short stretch of amino acids separates the last cadherin domain from the transmembrane domain. This is in contrast to the Fat protein, in which, after the last of its 34 cadherin domains, the extracellular portion includes five EGF-like repeats, the last being flanked by two G-domain-like repeats of laminin A. The lack of EGF-like or laminin A G-domain-like repeats, may account for the observation that mutations in ds lead exclusively to defects in morphogenesis and do not affect the control of cell proliferation as do strong fat alleles. The cytoplasmic domain of the Ds protein has sequence similarity to that of the classic vertebrate cadherins, such as E-cadherin. This sequence, which corresponds to the beta-catenin-binding region in classic cadherins, is interreputed in the cytoplasmic domain of both Ds and Ft. The intervening peptide between the two conserved regions substantially accounts for the larger cytoplasmic domain of Ds, as compared with that of vertebrate cadherins (Clark, 1995)

FAT, a new member of the human cadherin super-family, has been isolated from the T-leukemia cell line J6. The predicted protein closely resembles the Drosophila tumor suppressor fat, which is essential for controlling cell proliferation during Drosophila development. The gene has the potential to encode a large transmembrane protein of nearly 4600 residues with 34 tandem cadherin repeats, five EGF-like repeats, and a laminin A-G domain. The cytoplasmic sequence contains two domains with distant homology to the cadherin catenin-binding region. Northern blotting analysis of J6 mRNA demonstrate full-length, approximately 15-kb, FAT message in addition to several 5'-truncated transcripts. In addition to its presence in J6 cells, in situ hybridization reveals FAT mRNA expression in epithelia and in some mesenchymal compartments. Furthermore, higher levels of expression are observed in fetal tissue (as opposed to adult tissue), suggesting that its expression may be developmentally regulated in these tissues. FAT shows homologies with a number of proteins important in developmental decisions and cell:cell communication and is the first fat-like protein reported in vertebrates. The gene encoding FAT was located by in situ hybridization on chromosome 4q34-q35. It is proposed that this family of molecules is likely to be important in mammalian developmental processes and cell communication (Dunne, 1995).

The expression during rat embryogenesis of the protocadherin fat, the murine homolog of a Drosophila tumour suppressor gene, has been defined. As previously described for human fat, the sequence encodes a large protocadherin with 34 cadherin repeats, five epidermal growth factor (EGF)-like repeats containing a single laminin A-G domain and a putative transmembrane portion followed by a cytoplasmic sequence. This cytoplasmic sequence shows homology to the beta-catenin binding regions of classical cadherin cytoplasmic tails and also ends with a PDZ domain-binding motif. In situ hybridization studies at E15 show that fat is predominately expressed in fetal epithelial cell layers and in the CNS, although expression is also seen in tongue musculature and condensing cartilage. Within the CNS, expression is seen in the germinal regions and in areas of developing cortex, and this neural expression pattern is also seen at later embryonic (E18) and postnatal stages. At E18 fat expression is seen in the brain throughout the entire neuraxis in the cells next to the ventricles, with more extensive labelling within the regions of the cortex containing the germinal or ventricular zones adjacent to the lateral ventricles. No labelling is seen in adult tissues except in the CNS, where the remnant of the germinal zones, as well as the dentate gyrus, continue to express fat (Ponassi, 1999).

date revised: 12 April 1998  

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