InteractiveFly: GeneBrief

starry night : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - starry night

Synonyms - Flamingo

Cytological map position - 47B4-7

Function - surface receptor

Keywords - tissue polarity

Symbol - stan

FlyBase ID: FBgn0024836

Genetic map position -

Classification - Cadherin-related 7TM protein

Cellular location - surface

NCBI link: Entrez Gene
stan orthologs: Biolitmine
Recent literature
Wang, Y., Wang, H., Li, X. and Li, Y. (2015). Epithelial microRNA-9a regulates dendrite growth through Fmi-Gq signaling in Drosophila sensory neurons. Dev Neurobiol [Epub ahead of print]. PubMed ID: 26016469
microRNA-9 (miR-9) is highly expressed in the nervous system across species and plays essential roles in neurogenesis and axon growth; however, little is known about the mechanisms that link miR-9a with dendrite growth. Using an in vivo model of Drosophila class I dendrite arborization (da) neurons, miR-9a, a Drosophila homolog of mammalian miR-9a, was shown to downregulate the cadherin protein Flamingo (Fmi) thereby attenuating dendrite development in a non-cell autonomous manner. In miR-9a knockout mutants, the dendrite length of a sensory neuron ddaE was significantly increased. Intriguingly, miR-9a is specifically expressed in epithelial cells but not in neurons, thus the expression of epithelial but not neuronal Fmi is greatly elevated in miR-9a mutants. In contrast, overexpression of Fmi in the neuron resulted in a reduction in dendrite growth, suggesting that neuronal Fmi plays a suppressive role in dendrite growth, and that increased epithelial Fmi might promote dendrite growth by competitively binding to neuronal Fmi. Fmi has been proposed as a G protein-coupled receptor (GPCR). Neuronal G protein Galphaq (Gq), but not Go, may function downstream of Fmi to negatively regulate dendrite growth. Taken together, these results reveal a novel function of miR-9a in dendrite morphogenesis. Moreover, it is suggested that Gq might mediate the intercellular signal of Fmi in neurons to suppress dendrite growth. These findings provide novel insights into the complex regulatory mechanisms of microRNAs in dendrite development, and further reveal the interplay between the different components of Fmi, functioning in cadherin adhesion and GPCR signalling.
Strutt, H., Langton, P. F., Pearson, N., McMillan, K. J., Strutt, D. and Cullen, P. J. (2019). Retromer controls planar polarity protein levels and asymmetric localization at intercellular iunctions. Curr Biol 29(3): 484-491. PubMed ID: 30661800
The coordinated polarization of cells in the plane of a tissue, termed planar polarity, is a characteristic feature of epithelial tissues. In the fly wing, trichome positioning is dependent on the core planar polarity proteins adopting asymmetric subcellular localizations at apical junctions, where they form intercellular complexes that link neighboring cells. Specifically, the seven-pass transmembrane protein Frizzled and the cytoplasmic proteins Dishevelled and Diego localize to distal cell ends, the four-pass transmembrane protein Strabismus and the cytoplasmic protein Prickle localize proximally, and the seven-pass transmembrane spanning atypical cadherin Flamingo localizes both proximally and distally. To establish asymmetry, these core proteins are sorted from an initially uniform distribution; however, the mechanisms underlying this polarized trafficking remain poorly understood. This study describes the identification of retromer, a master controller of endosomal recycling, as a key component regulating core planar polarity protein localization in Drosophila. Through generation of mutants, it was verified that loss of the retromer-associated Snx27 cargo adaptor, but notably not components of the Wash complex, reduces junctional levels of the core proteins Flamingo and Strabismus in the developing wing. It was established that Snx27 directly associates with Flamingo via its C-terminal PDZ binding motif, and Snx27 was shown to be essential for normal Flamingo trafficking. it is concluded that Wash-independent retromer function and the Snx27 cargo adaptor are important components in the endosomal recycling of Flamingo and Strabismus back to the plasma membrane and thus contribute to the establishment and maintenance of planar polarization.
Li, R., Liang, Y., Zheng, S., He, Q. and Yang, L. (2019). The atypical cadherin flamingo determines the competence of neurons for activity-dependent fine-scale topography. Mol Brain 12(1): 109. PubMed ID: 31823796
The topographic projection of afferent terminals into two-dimensional maps is essential for sensory systems to encode the locations of sensory stimuli. In vertebrates, guidance cues are critical for establishing a coarse topographic map, while neuronal activity directs fine-scale topography between adjacent afferent terminals. However, the molecular mechanism underlying activity-dependent fine-scale topography is not well known. Studies in the Drosophila visual system have demonstrated that cell-adhesion molecules direct fine-scale topography, but whether or not these molecules are involved in activity-dependent fine-scale topography remains to be determined. Previously it was reported that the nociceptors in Drosophila larvae form an activity-dependent fine-scale topographic system. The establishment of this system is instructed by the level of neuronal activity in individual nociceptors. This study shows that the atypical cadherin Flamingo (Fmi) is required for establishing the nociceptor topographic map. The topographic defect caused by loss of fmi was found to be epistatic to the inhibition of neuronal activity and the overexpression of the activity-regulated gene Trim9. These results suggest that Fmi and neuronal activity interact to regulate fine-scale topography. This study provides a link between neuronal activity and the cell-adhesion molecule in the establishment of fine-scale topography.
Arguello, J. R., Abuin, L., Armida, J., Mika, K., Chai, P. C. and Benton, R. (2021). Targeted molecular profiling of rare olfactory sensory neurons identifies fate, wiring and functional determinants. Elife 10. PubMed ID: 33666172
Determining the molecular properties of neurons is essential to understand their development, function and evolution. Using Targeted DamID (TaDa), RNA polymerase II occupancy and chromatin accessibility were characterized in selected Ionotropic receptor (Ir)-expressing olfactory sensory neurons in Drosophila. Although individual populations represent a minute fraction of cells, TaDa is sufficiently sensitive and specific to identify the expected receptor genes. Unique Ir expression is not consistently associated with differences in chromatin accessibility, but rather to distinct transcription factor profiles. Genes that are heterogeneously-expressed across populations are enriched for neurodevelopmental factors, and functions for the POU-domain protein Pdm3 were identified as a genetic switch of Ir neuron fate, and the atypical cadherin Flamingo in segregation of neurons into discrete glomeruli. Together this study reveals the effectiveness of TaDa in profiling rare neural populations, identifies new roles for a transcription factor and a neuronal guidance molecule, and provides valuable datasets for future exploration.
Takechi, H., Hakeda-Suzuki, S., Nitta, Y., Ishiwata, Y., Iwanaga, R., Sato, M., Sugie, A. and Suzuki, T. (2021). Glial insulin regulates cooperative or antagonistic Golden goal/Flamingo interactions during photoreceptor axon guidance. Elife 10. PubMed ID: 33666170
Transmembrane protein Golden goal (Gogo) interacts with atypical cadherin Flamingo to direct R8 photoreceptor axons in the Drosophila visual system. However, the precise mechanisms underlying Gogo regulation during columnar- and layer-specific R8 axon targeting are unknown. These studies demonstrated that the insulin secreted from surface and cortex glia switches the phosphorylation status of Gogo, thereby regulating its two distinct functions. Non-phosphorylated Gogo mediates the initial recognition of the glial protrusion in the center of the medulla column, whereas phosphorylated Gogo suppresses radial filopodia extension by counteracting Flamingo to maintain a one axon to one column ratio. Later, Gogo expression ceases during the midpupal stage, thus allowing R8 filopodia to extend vertically into the M3 layer. These results demonstrate that the long- and short-range signaling between the glia and R8 axon growth cones regulates growth cone dynamics in a stepwise manner, and thus shape the entire organization of the visual system.
Mieszczanek, J., Strutt, H., Rutherford, T. J., Strutt, D., Bienz, M. and Gammons, M. V. (2022). Selective function of the PDZ domain of Dishevelled in noncanonical Wnt signalling J Cell Sci 135(11). PubMed ID: 35542970
Dishevelled is a cytoplasmic hub that transduces Wnt signals to cytoplasmic effectors, which can be broadly characterised as canonical (β-catenin dependent) and noncanonical, to specify cell fates and behaviours during development. To transduce canonical Wnt signals, Dishevelled binds to the intracellular face of Frizzled through its DEP domain and polymerises through its DIX domain to assemble dynamic signalosomes. Dishevelled also contains a PDZ domain, whose function remains controversial. This study used genome editing to delete the PDZ domain-encoding region from Drosophila dishevelled. Canonical Wingless signalling is entirely normal in these deletion mutants; however, they show defects in multiple contexts controlled by noncanonical Wnt signalling, such as planar polarity. This study used nuclear magnetic resonance spectroscopy to identify bona fide PDZ-binding motifs at the C termini of different polarity proteins. Although deletions of these motifs proved aphenotypic in adults, changes were detected in the proximodistal distribution of the polarity protein Flamingo (also known as Starry night) in pupal wings that suggest a modulatory role of these motifs in polarity signalling. New genetic evidence is provided that planar polarity relies on the DEP-dependent recruitment of Dishevelled to the plasma membrane by Frizzled.

Starry night (Stan), also known as Flamingo (Fmi), is a protein involved in the establishment of tissue polarity. Described almost simultaneously by three research groups (Usui, 1999; Lu, 1999 and Chae, 1999), Starry night possesses a huge protocadherin domain containing nine cadherin motifs, four EGF-like motifs, and two laminin G motifs. The dependence of proper Starry night localization on Frizzled (Fz) activity suggests that Stan functions downstream of Fz in controlling planar polarity. Stan protein is localized in specific domains of the plasma membranes in polarizing cells and controls planar cellular polarity. During a restricted interval prior to prehair outgrowth, Stan distribution is polarized along the proximal distal (P/D) axis; Stan molecules are present predominantly at proximal and distal cell boundaries rather than at anterior and posterior ones. Stan mutation disrupts the orientation of the axis of cell division of sensory organ precursors. stan phenotypes and biology call attention to the relationship between establisment of the axis of cell division and the resulting tissue polarity trait. It also ties together two heretofore disparate pathways: the asymmetric cell division trait in which Numb functions as a downstream factor, and the Frizzled pathway for the establisment of tissue polarity (Usui, 1999; Lu, 1999, and Chae, 1999).

In an attempt to pursue functional relationships between stan and previously discovered tissue polarity genes, a study was carried out to see whether Stan distribution is altered in various polarity mutants, particularly in fz complete loss-of-function mutants. In the total absence of Fz protein (fzD21/fzK21) Stan is not redistributed, as it normally is, toward the proximal/distal (P/D) cellular boundaries at 24 or 30 hr after puparium formation (APF). At the onset of prehair formation (30 hr APF), bright staining at cell boundaries is greatly reduced in length, and the fragmented signals are not necessarily restricted to the P/D boundaries, indicating that generation of the normal Stan pattern is strongly dependent on Fz. Residual boundary signals become even less prominent at later stages, leaving only fine dots both in the cytoplasm and along cell borders. Along the apicobasal cell axis, these intracellular particles are present from near the apical surface to the basolateral level. dsh1 (an allele of dishevelled) is a genetic null allele for planar polarity. Wings with this mutation also show a decrease in intensity of Stan staining at cell boundaries, and the distribution appears to be much less polarized than that in wild type. Mutations of other genes involved in tissue polarity do not necessarily disrupt the Stan distribution. For example, in mutant cells of the multiple wing hair (mwh) gene, which is currently considered to be further downstream in the tissue polarity pathway, Stan molecules are present predominantly at P/D boundaries. Therefore, Fz and one downstream component, Dsh, are thought to be necessary to accomplish the normal distribution of Stan (Usui, 1999).

In the preceding experiment, Stan distribution was studied in pupal wings where all the cells had lost fz expression. To dissect how Fz-dependent intercellular communication controls Stan localization, two approaches were adopted to juxtapose cells with different fz expression levels and see how those conditions affect Stan distribution. One approach generated fz mutant clones, and the other expressed fz in a gradient fashion. Clones were made of cells homozygous for a strong fz allele, fzR52, which produces a truncated polypeptide at a very low level. Strikingly, the fz mutant cells appear to decide where to localize Stan molecules in a neighbor-dependent manner. Along clone borders, Stan accumulates at almost all the interfaces between fz+ (fzR52/+ or +/+) and fzR52/fzR52 cells, whether the interface is a P/D cell boundary or not. In contrast, Stan is never localized intensely at boundaries between outermost mutant cells; in other words, those outermost mutant cells always restrict the distribution of Stan to contact sites with fz+ cells. These results indicate that every cell has a system to monitor fz expression levels across each boundary and that if there is an imbalance across a certain boundary, the cell deposits Stan molecules preferentially at this particular cell-cell contact site (Usui, 1999).

Inner mutant cells that do not contact the clone borders display fuzzy Stan signals both at interfaces between themselves and in the cytoplasm, and the distribution of the boundary signals is not polarized. These abnormal patterns are reminiscent of those in wings of the fz null mutant. This observation of the fz clone confirms that the fz gene is necessary to concentrate Stan to cell-cell boundaries and, in addition, to bias the distribution of Stan toward P/D boundaries (Usui, 1999).

Several recessive stan alleles have been isolated on the basis of the wing hair polarity phenotype seen in a wing in an FLP/FRT based F1 screen. Thus, it is clear that the presence of wild-type neighboring cells do not rescue all of the mutant cells in a clone. Several of the tissue polarity genes display domineering nonautonomy in wing clones -- that is, the presence of mutant cells in a clone alters the development of wild-type cells that are near the clone. To see if stan clones also display domineering nonautonomy, mosaic wings were generated where stan clones were marked with the hair marker pwn. Several different alleles were tested including the putative null allele stan24, the recessive lethal allele stan21 and the recessive viable allele stan3, and similar results were obtained. In all cases, the majority of clones behaved cell autonomously. Further, the extent of domineering nonautonomy for those clones scored as showing such nonautonomy was typically much weaker than seen with fz or Vang (Van Gogh). It is concluded from these experiments that stan principally functions cell autonomously (Chae, 1999).

For an in vivo assay for fz pathway function, the domineering non-autonomy of fz clones was used. To do this, fzR52 strb clones were induced in stan3 wings. In a wild-type wing, more than 80% of fz clones show distal domineering non-autonomy. That is, cells distal (and in part anterior/posterior) but not proximal to the clone show altered polarity that extends to cells that do not border the clone. fzR52 strb clones were induced in regions of stan3 wings, where the polarity was consistent enough to be able to score the clones for domineering non-autonomy. Out of 54 clones, forty two clones behave cell autonomously and only 12 clones showed evidence of domineering non-autonomy. Further, the extent of domineering non-autonomy in these 12 clones was modest. Thus, stan appears to be a suppressor of the domineering non-autonomy of fz. That there remains some fz domineering non-autonomy in stan3 wings may reflect the fact that stan3 is not a null allele. The ability of a stan mutation to suppress this fz phenotype argues that stan is downstream of fz and is required for the cell non-autonomous function of the fz pathway (Chae, 1999).

As a second in vivo assay for fz pathway function, the ability of a gradient of fz expression, with its highest point near the distal tip of the wing, was used to reverse the normal distal polarity of wing hairs. This result argues that cells can 'sense' the fz activity of neighboring cells and respond to this information. The production of a region of reversed polarity is likely to require both cell non-autonomous (e.g., a fz-dependent intercellular signal) and cell autonomous functions (e.g., transduction of the fz-dependent signal). stan3 was found to completely block the ability of a gradient of fz expression to reorganize wing hair polarity. Hence it is concluded that stan functions downstream of fz and is required either for the cells to be able to sense the fz activity of neighboring cells or to respond to this information (Chae, 1999).

The overexpression of fz just prior to prehair initiation causes the formation of large numbers of multiple hair cells that are a phenocopy of the in-like mutations. This fz gain-of-function phenotype has been used as a test to identify genes that are downstream of and required for the transduction of the fz signal. The function of the dsh gene, which is thought to function downstream of fz, is indeed required for this phenocopy. However, the function of several other tissue polarity genes, pk, ds and Vang, is not required. To determine if stan is required for the transduction of the fz signal, stan;hs-fz flies were constructed and fz expression was induced just prior to prehair initiation. The stan3 does not block the ability of fz overexpression to induce cells to form multiple hairs. Rather, it appears to slightly enhance the ability of fz overexpression to induce multiple hair cells (Chae, 1999).

Advantage was taken of the GAL4-UAS system to appose cells with different levels of fz expression and an examination was carried out of how the distribution of Stan was altered. The patched (ptc)-GAL4 driver was used to generate a short-range gradient of fz expression along the A/P axis of the wing imaginal disc: this ectopic expression made the wing hairs point from high to low levels of Fz. In contrast to the normal zigzag patterns of Stan at P/D cell boundaries, Stan was concentrated primarily at A/P cell boundaries in the presence of the Fz gradient along the A/P axis. This result shows that under this experimental condition, juxtaposition of cells with different Fz levels is sufficient to accumulate Stan at the interface (Usui, 1999).

Effects of fz overexpression are known to be suppressed by mutations in the dsh gene, which acts downstream of fz; this suggests that fz overexpression mimics activation of the signaling pathway. Given that levels of fz gene expression correlate with those of Fz protein activity, the results of the gradient expression and the clonal analyses allow two conclusions to be drawn: (1) a cell is able to communicate with its neighbors to monitor the amplitude of Fz activity across each cell-cell boundary; (2) enrichment of Stan at one boundary is a hallmark of a difference of Fz activity in between the two juxtaposed cells (Usui, 1999).

Results described so far suggest a model showing (1) that in the wild-type wing, Fz activity is made unequal across every P/D cell boundary; (2) that this difference causes bilateral assembly of Stan molecules at the P/D boundary, and (3) that those assembled Stan molecules play an important role in initiating prehair formation in the vicinity of the P/D boundary. However, bilateral distribution of Stan per se does not explain how the distal edge, not the proximal one, is selected to reorganize the cytoskeleton to form a prehair. An experiment of stan overexpression, described next, provides an insight into how Stan breaks cellular symmetry. As is also the case with fz, not only loss-of-function mutations but also overexpression of stan could disrupt planar polarity. By using a number of GAL4 drivers, the effects of overproduction of Stan has been compared with those of overproduction of Fz. The most striking result is obtained when a gradient of Stan is induced with the ptc-Gal4 driver. The phenotype induced by the Stan gradient presents a sharp contrast to that caused by the Fz gradient, that is, hairs are oriented toward the highest point of Stan levels. Cells within the gradient tend to accumulate overproduced Stan molecules in cytoplasm. This polarization along the Stan gradient requires the Stan ectodomain, because gradient production of the DeltaEX form has no effect on the hair polarity (Usui, 1999).

These results strongly suggest that when cells with equivalent P/D positional information are given the graded expression of stan, they perceive the difference in the expression level across the cell boundaries and direct hairs toward their neighbors, which produce higher levels of Stan. If overproduced Stan molecules activate a hypothetical downstream signaling cascade, whether they are located in plasma membranes or in cytoplasm, it follows that wing hairs point toward cells with stronger activity of Stan. This assumption permits a model that explains generation of distally oriented hairs in the wild-type wing, where neither Fz nor Stan is distributed in a gradient fashion along the P/D axis. In this model, the distal side of the P/D boundary, that is, the proximal edge of every cell, has a stronger Stan activity in spite of the apparent symmetrical distribution (Usui, 1999).

Polarization of the Stan distribution can be explained by two mechanisms that are not mutually exclusive. One is the sorting of Stan preferentially to P/D cell boundaries, and the other is involved in selective retention and degradation of Stan at P/D and A/P boundaries, respectively. Fz is able to recruit a signal transducer, Dsh, from cytoplasmic vesicles to cell-cell interfaces in a heterologous system (Axelrod, 1998). This finding may be suggestive of the intracellular sorting event. This Fz-dependent translocation of Dsh and aberrant localization of Stan in the dsh mutant imply a possibility that the sorting of Stan could be mediated by either direct or indirect interaction between Stan and Dsh. Whichever mechanism works, the observations made here suggest that this operation is initiated after 18 hr APF. Consistently, temperature-shift experiments using a cold-sensitive fz mutation suggest that fz function is required between approximately 15 hr APF at 25°C and the start of prehair morphogenesis. One obvious question that has not been rigorously answered is where Fz is localized within the cell. Staining for endogenous Fz proteins is challenging because of their low abundance; it is sometimes possible to detect Fz signals at both the apical-free surfaces and apical portions of cell boundaries, irrespective of the stage of prehair development. A more difficult task is visualizing the sites where Fz is activated; one reason for this difficulty is that the ligand for Fz, which is considered to be the polarizing signal, has not been identified (Usui, 1999).

Apart from Stan, the cell fate determinant Numb has been the only protein reported that displays biased distribution along a planar axis; its localization is also under the control of Fz signaling (Gho, 1998). Numb is a membrane-associated intracellular protein and forms a crescent that overlies one of the two spindle poles of cells that undergo asymmetric divisions. A search for proteins interacting with Numb led to the identification of Pon, which colocalizes with Numb during mitosis and directs Numb-asymmetric localization (Lu, 1998). Although polarization and depolarization of the Stan distribution occurs in postmitotic cells, a hunt for binding partners may help to disclose the molecular machinery that regulates Stan location (Usui, 1999).

Why do cells employ a system that is so complicated and requires spatially distinct activation of the two receptor species instead of Fz activation alone? What is Stan's role in breaking cellular symmetry along the P/D axis? It is speculated that the initial bias of the Fz activity is too subtle to drive cytoskeletal reorganization and that Stan is responsible for making this bias stronger. If active Stan molecules at the proximal edge downregulate activity of Fz molecules in the same domain of the plasma membrane, this inhibitory effect of Stan against Fz could enhance the difference in Fz activity across the P/D boundary. The antagonistic interplay between the two receptors could make the imbalance of Fz activity exceed a certain threshold to initiate prehair formation at the distal cell vertex. Planar polarity is also reflected in the arrangement of photoreceptor cells in the Drosophila eye, and it has been proposed that Fz sets up an initial small bias, which is amplified by the Delta-Notch pathway. Stan is also required for polarity formation in the eye; therefore, it is intriguing to investigate where Stan is localized in ommatidia and whether Stan is involved in the connection between Fz and Notch signaling pathways (Usui, 1999).

To test the function of Stan in regulating spindle orientation and Numb protein localization during the SOP pI division, the effects were analyzed of both the loss of function and the overexpression of stan on the pupal notum. In stan (fmiE59/fmi71) transheterozygous mutant pupae, the Numb crescent is randomly positioned within the epithelial plane during the SOP pI division. The adapter protein Partner of Numb (Pon), which controls Numb localization during SOP division (Lu, 1998) is also mispositioned, but the two proteins remain colocalized. Staining for tubulin reveals that spindle orientation and Numb crescent positioning are still tightly coupled. During the SOP pI division in apterous-Gal4;UAS-fmi pupae, the Numb crescent is also mispositioned and the mitotic spindle misoriented within the epithelial plane, but they remain aligned with each other. Therefore, loss of function and overexpression of stan both disrupts the cellular process that regulates mitotic spindle orientation and protein localization during the SOP pI division (Lu, 1999).

To gain a better insight into the mechanism of Stan function, its subcellular localization was examined in SOP cells using an antibody against the ectodomain of Stan. Consistent with Stan being a seven-transmembrane cell-adhesion molecule, Stan is localized to cell-cell boundaries in both the SOPs and their surrounding epithelial cells. There was also a low level of punctate cytoplasmic staining, suggesting that cytoplasmic Stan may be associated with intracellular vesicles. The Stan staining in SOP cells is stronger than that in the surrounding epithelial cells, indicating elevated expression or stability of Stan in the SOPs. No apparent polarized distribution of Stan is observed in mitotic SOPs. In nota of apterous-Gal4;UAS-fz flies, the localization of Stan in the SOPs is similar to that in wild-type flies. In nota of fzr54/fzKD4a mutant flies, Stan staining at the cell-cell boundary is reduced, whereas cytoplasmic Stan staining is increased. A similar effect is observed in the notum of dsh1 mutant flies. In the wing epithelia, localization of Stan at the cell-cell boundary is also affected in fz and dsh mutant backgrounds. Therefore, the proper recruitment of Stan from the cytoplasm to the cell-cell boundary depends on Fz signaling. It has been shown that Dsh can be selectively recruited to the membrane by Fz but not by Dfz2. Whether a similar mechanism is involved in recruiting Stan and Dsh to the membrane remains to be determined (Lu, 1999).

These data indicate that Stan functions largely in a cell-autonomous manner to control the planar polarity of sensory bristles on the Drosophila notum. The similar polarity phenotypes caused by overexpression and loss of function of stan or fz, together with the dependence of proper Stan localization on Fz activity, suggest that Stan functions downstream of Fz in the planar polarity pathway. It should be noted that, although loss of stan function affects both bristle polarity and the positioning of Numb crescents, the correlation between these two phenotypes is not strict. Although the orientation of the Numb crescent is largely random in the stan mutant, this is not the case for bristle polarity. It is possible that there exist some other mechanical constraints in the developing imaginal epithelia that influence the orientation of bristles or that Fz-independent cues can direct bristle orientation. The tight coupling of misoriented mitotic spindle and mislocalized Numb crescent during SOP pI divisions suggests that a downstream activity which coordinates these two processes is still intact when stan or fz are overexpressed or inactive. During neuroblast division, Inscuteable acts downstream of Bazooka to coordinate spindle orientation and Numb localization. It is unlikely, however, that Inscuteable is the activity required to couple these two processes during the SOP pI division. It is possible that, in the SOP lineage, Stan/Fz functions like Bazooka to regulate an Inscuteable-like activity, which in turn couples spindle orientation and protein localization. Despite the difference between the planar polarity pathway and the Bazooka/Inscuteable pathway for the orthogonal apical-basal polarity, both pathways appear to regulate the localization of Numb through Pon. Further studies will help clarify how the two pathways impinge on Pon to control Numb localization (Lu, 1999).

Golden Goal collaborates with Flamingo in conferring synaptic-layer specificity in the visual system

Neuronal connections are often organized in layers that contain synapses between neurons that have similar functions. In Drosophila, R7 and R8 photoreceptors, which detect different wavelengths, form synapses in distinct medulla layers. The mechanisms underlying the specificity of synaptic-layer selection remain unclear. This study found that Golden Goal (Gogo) and Flamingo (Fmi), two cell-surface proteins involved in photoreceptor targeting, functionally interact in R8 photoreceptor axons. The results indicate that Gogo promotes R8 photoreceptor axon adhesion to the temporary layer M1, whereas Gogo and Fmi collaborate to mediate axon targeting to the final layer M3. Structure-function analysis suggested that Gogo and Fmi interact with intracellular components through the Gogo cytoplasmic domain. Moreover, Fmi was also required in target cells for R8 photoreceptor axon targeting. It is proposed that Gogo acts as a functional partner of Fmi for R8 photoreceptor axon targeting and that the dynamic regulation of their interaction specifies synaptic-layer selection of photoreceptors (Hakeda-Suzuki, 2011).

The results suggest that the transmembrane receptor Gogo physically interacts (directly or indirectly) with the atypical cadherin Fmi in cis to cooperatively guide R8 photoreceptor axons to their correct target. However, a robust direct interaction between Gogo and Fmi could not be demonstrated by co-immunoprecipitation, bimolecular fluorescent complementation (BiFC) or proximity ligation assay. The failure in co-immunoprecipitation was probably a result of technical difficulties in solubilizing the seven-pass transmembrane Fmi and maintaining a huge complex during the procedure (Fmi is about 400 kDa). Nevertheless, a close interaction of these proteins is supported by three lines of evidence. First, ectopic expression of Gogo in wing epithelial cells was able to relocate Fmi in cis. Second, Fmi and Gogo colocalized at cell-cell contacts in cultured cells via their ectodomains. Finally, Gogo accumulation at the growth cone was strongly reduced in fmi mutant photoreceptor axons, suggesting that Fmi is at least partially required to localize and/or stabilize Gogo at the growth cone through a close association (Hakeda-Suzuki, 2011).

It has been suggested that Fmi binds homophilically in cis. This study also found that Gogo formed oligomers in cultured cells. These observations suggest that, even if Gogo and Fmi physically interact with each other, they may multimerize and form a protein cluster. Alternatively, Gogo-Gogo, Gogo-Fmi and Fmi-Fmi interactions may happen separately at distinct locations and have different functions (Hakeda-Suzuki, 2011).

Fmi controls the nervous system development broadly. It regulates axon guidance, but also synaptic target selection and dendritic field development. The phenotypic similarities and the genetic interactions of gogo and fmi in diverse aspects of neuronal development in Drosophila suggest that the collaboration of Gogo and Fmi is a general molecular mechanism (Hakeda-Suzuki, 2011).

Notably, however, in the dendrites of multi-dendritic neurons, it has been reported that the ectodomain deletion of Fmi (FmiΔN) is able to partially rescue the fmi dorsal-overgrowth phenotype in dendrites, but the cytoplasmic deletion of Fmi (FmiΔC) cannot. On the contrary, FmiΔN was not able to rescue the phenotype in R8 photoreceptor axon, but FmiΔIntra could. These observations indicate that the underlying molecular mechanisms may be different between axons and dendrites. It will be interesting to investigate the molecular mechanisms of Gogo in dendrite formation to decipher the general principles versus unique, diversified mechanisms mediated by the Gogo-Fmi interaction (Hakeda-Suzuki, 2011).

What is the function of Gogo when interacting with Fmi? Three scenarios are envisioned that are not mutually exclusive. First, Fmi homophilic adhesion properties change when it is associated with Gogo. Second, Gogo mediates intracellular signaling to transduce axon pathfinding information in the growth cone. Third, Gogo adds a specificity code to the Fmi-Fmi homotypic asymmetric interaction. To test the first scenario, a cell aggregation assay was used, mixing Fmi-expressing cells with cells co-expressing Gogo and Fmi. As the two populations of cells were equally distributed in the aggregate, Gogo seems to not have an effect on Fmi homophilic adhesion in S2 cells. The second scenario is supported by the fact that the Gogo cytoplasmic domain mediated the R7 photoreceptor co-overexpression phenotype and that the Fmi cytoplasmic domain was dispensable for R8 photoreceptor axon pathfinding. In addition, the interaction between Gogo and Fmi seems to add molecular specificity to R8 photoreceptor axons, allowing them to recognize the proper layer M3, suggesting the third scenario. Fmi seems to be the cue on the target layers, as elimination of Fmi from a population of brain cells, but not from photoreceptors, resulted in targeting defects in R8 photoreceptor axons. This suggests that Fmi-Fmi homotypic interactions take place between R8 photoreceptor axons and the target cells. However, the interaction seems to be asymmetric, as Gogo is not required in the brain for photoreceptor axon pathfinding (Hakeda-Suzuki, 2011).

Overall, it is proposed that Gogo alone promotes adherence between R8 photoreceptor axons and the M1 layer, that, at mid-pupal stages, Fmi acts antagonistically with Gogo at the M1 layer and Gogo and Fmi collaborate to mediate R8 targeting to the M3 layer, and that Fmi on the target cells mediates homophilic interaction with Fmi on R8 photoreceptor axons at the M3 layer. Fmi is detected on R8 photoreceptor axons when R8 photoreceptor axons extend their tip to the M3 layer, if Fmi protein level is reduced from surrounding neuropils, consistent with the idea that Gogo and Fmi act together to guide the M3 targeting growth cones. The above model is supported by five lines of evidence. First, overexpression of both Fmi and Gogo retargets R7 photoreceptor axons to the M3 layer. Second, removing Fmi from presumptive target cells induces R8 photoreceptor axon stopping at the M1 layer. Third, a combination of Gogo overexpression and fmi hypomorphic background induces more R8 photoreceptor axon stopping at M1 layer than is observed in each of these genotypes individually. Fourth, the Gogo overexpression phenotype is suppressed by mild fmi overexpression. Taking into account that gogo overexpression in a fmi hypomorph does not enhance the axon bundling that is typical in fmi mutant axons, it is unlikely that gogo overexpression merely has a dominant-negative effect on Fmi function. Fifth, in fmi mutants, R8 photoreceptors commonly stall at the M1 layer, whereas in gogo mutants or in the double mutants, R8 photoreceptors have a tendency to stray at the M1 layer. This difference is thought to be a result of a reduced adhesion of R8 photoreceptors to M1 layer in gogo mutants; adhesion is not impaired in fmi mutants (Hakeda-Suzuki, 2011).

The cell identity of the M3 layer that is recognized by Gogo and Fmi in R8 photoreceptor axons is not clear. fmi was knocked out almost completely from the lamina neurons. Although the R8 photoreceptor axon stopping phenotype was not completely penetrant, substantial R8 photoreceptor axon stopping was observed at the M1 layer, indicating that the lamina neurons might be the target cells in which Fmi functions as a ligand. Lamina neurons innervate into medulla layers during early pupal stages. Their processes take over R8 photoreceptor axons when R8 photoreceptor axons rest at the temporary layer, and they arborize between developing R7 and R8 photoreceptor termini. Notably, L3 lamina neurons spread their terminal processes at the M3 layer. The functional importance of L3 neurons in this context should be addressed in the future. In any case, it seems that mutual interactions between lamina neuron processes and photoreceptor axons account for the two-step targeting mechanism of R8 photoreceptor axons (Hakeda-Suzuki, 2011).


Transcriptional regulation

The Drosophila wing is covered by an array of distally pointing hairs. This tissue planar polarity is regulated by the frizzled pathway. The function of the grainy head transcription factor is essential for the function of the frizzled pathway. grainy head mutant cells fail to localize planar polarity proteins at either the proximal or distal sides of wing cells and produce multiple hairs of abnormal polarity. Levels of the Starry night protein are strongly reduced in grainy head mutants in both larval wing discs and pupal wings, which is sufficient to account for much of the polarity phenotype. In addition, grh has frizzled pathway independent functions during the development of the adult cuticle (Lee, 2004).

grh function is required for several different processes during the differentiation of the adult Drosophila epidermis. These include the function of the fz dependent tissue polarity pathway, pigmentation, the timing of differentiation, epidermal hair morphogenesis and wing vein/blade specification. The Grh protein was originally isolated by virtue of its ability to bind to DNA in a sequence specific manner and to regulate the expression of target genes. These and later experiments led to the conclusion that grh functions as a transcription factor for development specific gene regulation. Experiments on vertebrate homologs of grh also suggest a similar cellular function. It is likely that it serves a similar function in the development of the adult epidermis (Lee, 2004).

The analysis of grh function in regulating gene expression appears complex. The first studies on grh argued that it acted as a positive regulator of Ddc and Ubx expression. Curiously, although Grh was isolated by virtue of its ability to bind to a sequence essential for the neuronal activation of Ddc, grh mutations alter the epidermal and not neuronal expression of Ddc. More recently it was found that grh positively regulates tll expression and negatively regulates ventral dpp expression (Lee, 2004).

The function of the grh transcription factor is shown in this study to be required for the function of the fz pathway in the wing. In the absence of grh function the Fz, Dsh and Vang proteins fail to accumulate apically and the levels of the Stan protein are dramatically decreased. Furthermore, Stan levels are increased in cells with two versus one copy of grh. Thus, stan expression is directly related to grh dose suggesting that stan might be a direct target of Grh. The direct relationship between stan expression and grh dose is seen in both pupal wing cells where Stan is localized assymetrically and in third instar wing disc cells where it is evenly distributed. Thus, it is concluded that the decreased levels of Stan protein in grh cells is not due to a failure of assymetric localization. Grh does not affect Stan stability; stan expression from the endogenous stan gene is altered, consistent with Grh having an important role in promoting stan transcription. It is suspected that this could be due to a direct interaction of Grh protein with stan genomic DNA. stan does not appear to be highly enriched in putative Grh binding sites but this may be a reflection of the variability in identified Grh binding sites not providing an ideal consensus site. It is also concluded that the decreased level of Stan protein is neither the cause or effect of the the delay in hair morphogenesis in grh cells. Thus far, all of the proteins that localize assymetrically are co-required for the asymmetric localization of the others, however only Stan is required for the apical accumulation of all of the other proteins. The alterations in tissue polarity protein localization seen in grh mutant cells could be explained entirely by the effect of grh on Stan expression. It remains possible however, that grh could be important for the expression of several or all members of the tissue polarity group. These experiments did not allow the assessment of possible changes in Fz or Vang levels due to altered expression of these genes, since the localization was examined of proteins produced from transgenes that did not utilize the normal promoters. Decreased levels of Dsh were not seen by antibody staining, however the staining background was relatively high in these experiments which could have hidden a modest effect on Dsh levels. The finding that Arm cortical localization is not altered in grh clone cells indicates that apical-basal polarity is not altered and suggests that gross cellular physiology is not altered in grh clones (Lee, 2004).

While it is possible that the grh mutant planar polarity phenotype could be due solely to a lack of stan expression in grh mutant cells, this may not be the case since there are a number of differences between the phenotypes of grh and stan clones. For example, the multiple hair phenotype of grh is much stronger than stan. There is also a difference in the non-autonomy of grh and stan clones. For mutations in both of these genes the domineering nonautonomy of clones is much weaker than that of fz or Vang. However, the weak domineering nonautonomy is seen much more frequently with grh than stan clones, suggesting that grh mutations alter the expression of additional tissue polarity genes or other cellular genes that interact with the planar polarity system. Genetic screens for enhancers or suppressors of the dominant negative grhFK2131 allele could be useful in identifying such genes (Lee, 2004).

grh has both fz pathway dependent and independent functions during wing development. Epistasis experiments showed that the ectopic wing vein, cuticle pigmentation, disturbed marginal bristle row and extreme multiple hair cell phenotypes of grh mutations are not altered in a null fz, in or mwh mutants. Thus, it is quite likely that some of the target genes whose transcription is altered by grh mutations are not part of the fz pathway (Lee, 2004).

grh cells are often dramatically delayed in hair morphogenesis. This is not seen in cells mutant for fz or stan and hence is unlikely to be an indirect consequence of a failure of stan expression or in the inactivation of the frizzled pathway. The time course of pupal development is controlled by ecdysone and it is possible that grh functions as part of the ecdysone cascade. The delay in hair morphogenesis could be due to a failure to induce the expression of genes such as kojak, where a loss of function results in a similar delay (Lee, 2004).

The grh multiple hair cell phenotype differs from that of downstream members of the fz pathway such as inturned, in not showing the typical fz/in abnormal polarity pattern and in the hairs being much more erect. The identity of the targets responsible for this phenotype are unkown. The grh hair phenotype is somewhat reminiscent of that seen with mutations in genes such as Rho kinase or crinkled (myosin VII) suggesting these or related genes as possible targets (Lee, 2004 and references therein).

The transcription of the Ddc gene has previously been shown to be regulated by grh and Ddc activity is required for melanization. Is ddc likely to be the target gene whose altered expression leads to the lowered pigmentation of grh clone cells? This is certainly possible but it seems unlikely to be the entire story. Ddcts2 flies raised at the restrictive condition have more profound pigmentation defects than grh clones. However, clones of ddc null alleles typically have a less severe pigmentation phenotype than grh clones due to partial rescue of the pigmentation phenotype by neighboring cells (i.e. ddc displays submissive cell non-autonomy). Based on these observations it is argued that grh must have other targets that contribute to the decreased pigmentation (Lee, 2004).

The data reported in this paper argue that grh has multiple functions during the development of the adult epidermis. In this context it is not clear to what extent grh functions in a permissive fashion to promote the expression of developmentally important genes and/or to promote changes in gene expression that are associated with the differentiation of the adult cuticle. The data are consistent with grh functioning in both ways. The requirement for grh for the expression of stan was seen at multiple stages consistent with grh having a permissive role. The effects on the timing of hair morphogenesis are consistent with, but do not demand an instructive role (Lee, 2004).

Membrane protein location-dependent regulation of Flamingo by PI3K (III) and rabenosyn-5 in Drosophila wing cells

The class III phosphatidylinositol-3 kinase [PI3K (III)] regulates intracellular vesicular transport at multiple steps through the production of phosphatidylinositol-3-phosphate [PI(3)P]. While the localization of proteins at distinct membrane domains are likely regulated in different ways, the roles of PI3K (III) and its effectors have not been extensively investigated in a polarized cell during tissue development. This study, in vivo functions of PI3K (III) and its effector candidate Rabenosyn-5 (Rbsn-5) were examined in Drosophila wing primordial cells, which are polarized along the apical-basal axis. Knockdown of the PI3K (III) subunit Vps15 resulted in an accumulation of the apical junctional proteins DE-cadherin and Flamingo and also the basal membrane protein beta-integrin in intracellular vesicles. By contrast, knockdown of PI3K (III) increased lateral membrane-localized Fasciclin III (Fas III). Importantly, loss-of-function mutation of Rbsn-5 recapitulated the aberrant localization phenotypes of beta-integrin and Fas III, but not those of DE-cadherin and Flamingo. These results suggest that PI3K (III) differentially regulates localization of proteins at distinct membrane domains and that Rbsn-5 mediates only a part of the PI3K (III)-dependent processes (Abe, 2009).

Cell polarity along the apical-basal axis is essential for the function of epithelial cells. This polarity is formed and maintained by distinct localization of membrane spanning and associated proteins, to apical, lateral or basal membrane domains. Membrane proteins localized to the apical or basolateral plasma membrane are endocytosed into early and apical or basolateral endosomes. For example, horseradish peroxidase (HRP) administered to the apical cell surface is incorporated into the apical early endosome. By contrast, HRP or dimeric IgA administered to the basolateral cell surface or transferring receptor (TfR) in the basolateral domain are internalized into the basolateral early endosome, which remain distinct. Sorting of proteins for transcytosis, recycling and degradation takes place in these early endosomes. The proteins, incorporated into apical and basolateral early endosomes, meet in common endosomes, a process that can be observed within 15 min after the onset of internalization in MDCK cells. The significance of keeping the apical and basolateral early endosomes distinct is thought to ensure that proteins from the apical and basolateral plasma membrane remain apart before the sorting processes proceeds. Although it is plausible that the trafficking of proteins in distinct membrane domains is regulated differently, the factors involved in such a differential regulation remain elusive (Abe, 2009).

One of the key molecules regulating membrane trafficking is PI3K (III), a heterodimer of Vps34p and Vps15p/p150, which produces phosphatidylinositol-3-phosphate (PI(3)P). PI(3)P is found to localize with early endosome and internal vesicles of multivesicular bodies (MVBs) in mammalian cells in culture. Genetic and pharmacological analysis, using yeast and mammalian cells in culture, suggests that PI3K (III) is required for five distinct processes. These are: (1) the fusion of clathrin-coated vesicles and early endosomes as well as the fusion between early endosomes; (2) the recycling from early endosomes back to the Golgi complex or other destinations; (3) the entry of proteins into the lysosomal degradation pathway; (4) the formation of internal vesicles of MVBs and (5) autophagy. Moreover, inactivation of PI3K (III) by Vps34 mutation leads to an expansion of the outer nuclear membrane and an abnormal reduction of the LDL receptor at the apical membrane in C. elegans. In Drosophila, dVps34 mutation results in defective endocytosis of the apical membrane protein Notch and a defective onset of autophagy. It has been suggested that PI3K (III) utilizes different effectors at apical and basolateral endosomes. However, the role of PI3K (III) in the regulation of protein localization at different membrane domains has remained unclear (Abe, 2009 and references therein).

To understand the various functions of PI3K (III), it is crucial to clarify which downstream effectors are involved in each of the processes it regulates. PI3K (III) is thought to exert its function through the recruitment of proteins that contain PI(3)P-binding motifs such as FYVE or PX domains. Among such proteins, Rabenosyn-5 (Rbsn-5) has been shown to contribute to endosome fusion and recycling processes in mammalian cells. Genetic studies on C. elegans and Drosophila also show that Rbsn-5 is essential for receptor-mediated endocytosis and endosome fusion, although it is not clear whether or not Rbsn-5 is involved in other PI3K (III)-related phenomena (Abe, 2009).

To determine how the proteins in distinct membrane domains are regulated by PI3K (III) and its effector Rbsn-5 this study analyzed Drosophila wing development. This provides a good model since wing primordial cells have a clear polarity along the apical-basal axis. In addition a number of membrane proteins are known to be transported in an organized manner along the apical-basal axis. For example DE-cadherin, a cell adhesion protein and Fmi, a planar cell polarity (PCP) core protein, are localized in the apical junctions or zonula adherens (ZA), whereas the cell adhesion molecules FasIII and β-integrin are localized in lateral and basal membranes, respectively. This study found that inactivation of PI3K (III) in the wing primordial cells by knockdown of dVps15 affects the localization of these membrane proteins differently. In particular, it was found that dVps15 knockdown results in the accumulation of FasIII at the lateral membrane, whereas it results in intracellular accumulation of DE-cadherin, Fmi and β-integrin. Importantly, inactivation of Rbsn-5 shows accumulation of FasIII and β-integrin at the lateral membrane and intracellular vesicles, respectively, but no effects of DE-cadherin and Fmi localization (see in contrast Mottola, 2010). These results provide evidence for a differential regulation of protein localization by PI3K (III) and Rbsn-5 at distinct membrane domains (Abe, 2009).

This study demonstrated that PI3K (III) differentially regulates the localization of proteins at distinct membrane domains. The intracellular accumulation of Fmi, DE-cadherin and β-integrin induced by the dVps15 knockdown might be due to defects in the degradation pathway, since the maturation of MVBs and the lysosomal trafficking were defective in these cells. However, unlike these proteins, Fas III did not accumulate in the intracellular compartments, but rather accumulated at the surface of the lateral plasma membrane. It is possible that PI3K (III) regulates proteins at the lateral membrane differently from those localized at other membrane domains. It is also possible that PI3K (III) regulates Fas III in a different way, irrespective of the membrane domain to which it is localized. Whichever is the case it will be important to elucidate the mechanism underlying this difference in a future study (Abe, 2009).

Rbsn-5, a FYVE domain-containing protein, shares a part of the functions of PI3K (III), in that it is necessary for the regulation of Fas III and β-integrin localization, but not that of DE-cadherin and Fmi localization. Although the Rbsn-5C241 null mutant clones may not completely lack Rbsn-5 activity, the requirement of Rbsn-5, or at least the requirement of an appropriate amount, differs between these proteins with respect to normal trafficking. It appears that Rbsn-5 preferentially controls the events at the basolateral regions, given that Rbsn-5 is necessary for the formation of large endosomes at the basal region, whereas it is indispensable for the formation of actin bundles at the apical surface (Abe, 2009).

PI3K (III) has been implicated in the differential regulation of vesicle trafficking at apical and basolateral regions. For instance, a reduction of PI(3)P dissociates EEA1, a FYVE-domain containing protein essential for early endosome fusion, selectively from basolateral endosomes. However, which proteins, including EEA1, regulate the different trafficking pathways downstream of PI3K (III) has remained unknown. Rbsn-5 has been proposed to be a PI3K (III) effector, since Rbsn-5 harbors a FYVE domain. The current results provide further evidence supporting a possible functional interaction between these two molecules, based on their genetic interaction on the wing morphogenesis and the PI3K (III)-dependent Rbsn-5 immunostaining. Importantly, the different requirement of Rbsn-5 for trafficking at apical junction and basolateral membrane domains suggests that Rbsn-5 may a selective regulator under the control of PI3K (III) (Abe, 2009).

Anterograde Jelly belly and Alk receptor tyrosine kinase signaling mediates retinal axon targeting in Drosophila

Anaplastic lymphoma kinase (Alk) has been proposed to regulate neuronal development based on its expression pattern in vertebrates and invertebrates; however, its function in vivo is unknown. This study demonstrated that Alk and its ligand Jelly belly (Jeb) play a central role as an anterograde signaling pathway mediating neuronal circuit assembly in the Drosophila visual system. Alk is expressed and required in target neurons in the optic lobe, whereas Jeb is primarily generated by photoreceptor axons and functions in the eye to control target selection of R1-R6 axons in the lamina and R8 axons in the medulla. Impaired Jeb/Alk function affects layer-specific expression of three cell-adhesion molecules, Dumbfounded/Kirre, Roughest/IrreC, and Flamingo, in the medulla. Moreover, loss of flamingo in target neurons causes some R8-axon targeting errors observed in Jeb and Alk mosaic animals. Together, these findings suggest that Jeb/Alk signaling helps R-cell axons to shape their environment for target recognition (Bazigou, 2007).

These genetic studies in Drosophila provide functional evidence in vivo that Alk plays a crucial role in the developing central nervous system. This study shows that Alk and its cognate ligand Jeb form an anterograde signaling pathway in the fly visual system, which is required for target selection by R cell axons within the lamina and medulla. It is proposed that R cell axons release Jeb to activate Alk signaling in target neurons and, through direct or indirect regulation of downstream guidance molecules, contribute to creating the appropriate environment for target recognition (Bazigou, 2007).

In the visual system, R cell axons provide two known anterograde signals to the optic lobe to promote neuronal proliferation and differentiation of target neurons during the third instar larval stage. R cell-derived Hh induces mitotic divisions of lamina precursor cells (LPCs), as well as expression of the early neuronal marker Dachshund in both LPCs and postmitotic lamina neurons. Dachshund in turn is required to control the expression of the EGF receptor in lamina neurons, thus making them competent for the second anterograde R cell-derived signal Spitz that induces the next step of lamina neuron differentiation. A third so far unidentified signal controls glial cell development and migration in the optic lobe. The current findings show that R cell axons provide an unexpected fourth anterograde signal -- Jeb -- that is required to mediate target selection of R cell axons during pupal development. Unlike the Hh and Spitz signals, Jeb represents an anterograde signal delivered by R cell axons not only to the lamina but also to the medulla (Bazigou, 2007).

That Jeb and Alk form an anterograde signaling pathway in the visual system that is supported by three lines of evidence: first, Jeb and Alk are expressed in a largely complementary pattern from the third instar larval to midpupal stages. The ligand Jeb is produced in R cells, whereas the receptor Alk is specifically expressed by target neurons. Since the Jeb protein has been shown to be secreted in vitro, it is highly likely released from R cell growth cones. Second, jeb is genetically required in R cells, whereas Alk functions in target neurons. Third, in the converse experiment, removal of jeb function in the target or Alk in the eye does not produce any conspicuous targeting phenotypes (Bazigou, 2007).

It is proposed that Jeb/Alk signaling plays a role in regulating late events of target-neuron maturation to control R1-R6 axons in the lamina and R8 axons in the medulla. Consistent with this model, the data indicate that loss of Jeb/Alk signaling affects the expression of three guidance molecules, Duf/Kirre, Rst/IrreC, and Fmi, in the R8 recipient layer of the medulla, while Caps, LAR, PTP69D, and CadN appear normal at this level of resolution. Interestingly, animals lacking Jeb/Alk signaling display similar R8 projection defects as fmi and caps eye mosaics. It was further shown that loss of fmi in target neurons causes R8-targeting defects, which qualitatively resemble those observed in Jeb/Alk mosaics. As Jeb/Alk signaling acts upstream of multiple cell-adhesion molecules, loss of one factor likely results in milder targeting defects. In support of this notion, it was observed that phenotypes in jeb or Alk mosaics were more frequent in comparison to fmi knockdown or fmi ELF mosaics. Moreover, loss of fmi in the target appeared to cause one prevalent targeting defect, i.e., the fasciculation of R8 axons with processes in adjacent medulla columns. Notably, loss of sec15 in R cells, which encodes an exocyst component regulating the localization of cell-adhesion molecules to axon terminals, also causes distinct targeting errors. This is consistent with the model that regulating the precise expression of guidance molecules by Jeb/Alk signaling is indeed important for axon targeting in the visual system (Bazigou, 2007).

R cell-targeting defects occurred in both null and kinase domain mutant alleles of Alk, showing that tyrosine kinase activity is essential. Furthermore, studies of vertebrate Alk in vitro, as well as Drosophila Alk in vivo, demonstrate that this RTK drives an ERK/MAPK-mediated signaling pathway, suggesting that Alk may also act through this pathway in the visual system. There are three possible mechanisms as to how Jeb/Alk signaling could regulate downstream guidance molecules: (1) Jeb and Alk may directly regulate the expression of guidance molecules, (2) they could indirectly regulate the expression pattern of guidance molecules via the activation of transcriptional programs determining target neuron identities, or (3) they could separately control both the expression of guidance molecules and transcription factors. Such mechanisms would be analogous to what has been observed in the developing visceral mesoderm, where Jeb/Alk signaling induces the expression of both Duf/Kirre and Org-1, a transcription factor and mammalian Tbx1 homolog, to drive muscle fusion. At present, it cannot be excluded that Alk additionally modulates the activity of downstream targets (Bazigou, 2007).

Anterograde Jeb/Alk signaling would make it possible to coordinate the timing of R cell growth-cone extension with local expression of guidance factors in the target. These in turn could directly regulate afferent axon targeting. Alternatively, guidance factors may be required to shape dendritic and axonal arbors of target neurons and to mediate R cell-targeting decisions. Fmi could indeed take part in both processes, as it can control dendrite development, as well as axon guidance by afferent/afferent and afferent/target interactions. Similar to CadN or LAR eye mosaics, some R1-R6 axons lacking jeb function failed to extend from their original bundle. Extension and cartridge assembly phenotypes were also detected in jeb eye or Alk target mosaics, which qualitatively resembled those described for fmi eye mosaics. Future studies will require the identification and validation of (other) downstream guidance molecules, as well as the isolation of transcriptional regulators controlling target neuron subtype specificity in both the lamina and medulla to provide further insights into the mechanisms underlying Jeb/Alk function (Bazigou, 2007).

It was observed that ectopic expression of Jeb in the visual system strongly reduces the number of activated Caspase 3-positive cells in the medulla at 24 hr APF, when many postmitotic medulla neurons normally undergo apoptosis in wild-type. Thus, Jeb/Alk signaling may also mediate cell survival in parallel to neuronal maturation. The mechanism could be similar to the pleiotropic function of EGF-receptor signaling, which, depending on low or high level of activation regulates cell-cycle withdrawal, mitosis, cell survival, and differentiation in the developing eye imaginal disc of Drosophila (Bazigou, 2007).

Although Jeb shares some sequence similarity with proteins such as the secreted bovine glycoprotein Sco-Spondin , no Jeb homolog has been isolated so far in vertebrates. However, the growth factors Pleiotrophin and Midkine have been reported to act as ligands for Alk in vertebrates, and both have been linked to neuronal development and neurodegenerative diseases. Therefore, Alk may work with different ligands in the vertebrate nervous system. The C. elegans homolog of Alk is localized presynaptically at the neuromuscular junction and has been proposed to mediate synapse stabilization. Also, the vertebrate homologs of Alk are strongly expressed in the developing and adult nervous systems. This includes motor-neuron columns in the spinal cord and, intriguingly, also the superior colliculus, a higher-order processing center for visual information in the brain. That Alk may play a role in neuronal development in vertebrates is further supported by in vitro studies indicating that activated Alk can promote neuronal differentiation and neurite outgrowth in specific cell line. These observations suggest that the function of Alk in regulating specific aspects of neuronal development may be conserved (Bazigou, 2007).

Protein Interactions

To address whether Stan has an intercellular adhesion activity, stan was expressed in Drosophila S2 cells that exhibit a very weak self-aggregating property. Cell aggregates are formed by transfection of the S2 cells with a cDNA construct that encodes the full-length Stan protein (Usui, 1999).

During planar polarization of the Drosophila wing epithelium, the homophilic adhesion molecule Flamingo localizes to proximal/distal cell boundaries in response to Frizzled signaling; perturbing Frizzled signaling alters Flamingo/Starry night distribution, many cell diameters distant, by a mechanism that is not well understood. diego, a tissue polarity gene, encodes a protein comprised of six ankyrin repeats that colocalizes with Flamingo at proximal/distal boundaries. Diego is specifically required for polarized accumulation of Flamingo and drives ectopic clustering of Flamingo when overexpressed. It is suggested that Frizzled acts through Diego to promote local clustering of Flamingo, and that the clustering of Diego and Flamingo in one cell nonautonomously propagates to others. Localized Frizzled signaling would modify the properties of the Diego protein at proximal-distal boundaries, increasing Diego's ability to promote clustering of itself and Flamingo. A bias in Diego localization might then be enhanced via homophilic interactions between Flamingo molecules on adjacent cells. This model is consistent with the reciprocal effects that Flamingo and Diego have on each other's localization (Feiguin, 2001).

Does cortical localization of Diego depend on Flamingo? The distribution of Diego was examined in clones of Flamingo mutant cells. Diego is undetectable at the cortex of flamingo mutant cells and is also absent from both proximal and distal interfaces between wild-type and flamingo mutant tissue. These data suggest that Diego is localized to the cortex via Flamingo, either directly or indirectly. Like Diego, Flamingo also fails to accumulate at either the proximal or distal interface between wild-type and Flamingo mutant clones. This has been interpreted to mean that Flamingo is normally enriched both proximally and distally and that homophilic interactions are required for its polarized accumulation. Similarly, it can be argued that Diego is normally enriched on both proximal and distal sides of the cell and that stable cortical Diego localization occurs only where homophilic Flamingo interactions are possible (Feiguin, 2001).

Clearly, Flamingo is required for cortical accumulation of Diego. To ask whether it was also sufficient, that is, whether Diego simply accumulates wherever Flamingo is present, the distribution of Diego was examined in cells that overexpressed Flamingo. Although some Flamingo can be detected at cell boundaries when the protein is overexpressed, Flamingo accumulates most obviously in bright spots inside the cell. Since Flamingo is a transmembrane protein, these spots might either represent protein that is trapped in the secretory pathway or that has accumulated in the endocytic pathway. In Flamingo-overexpressing cells, Diego protein appears less abundant on the cortex overall, and what Diego remains is no longer restricted to proximal-distal cell boundaries. Diego does not colocalize with Flamingo inside the cell where Flamingo accumulates to the highest level. This indicates that other proteins besides Flamingo must promote the cortical localization of Diego. Colocalization of Diego and Flamingo upon transfection of S2 cells could not be seen, indicating that other proteins may regulate whether Diego and Flamingo are present in the same complexes. Nevertheless, at least at the cortex, dominant mislocalization of Flamingo by overexpression can relocalize Diego (Feiguin, 2001).

In wild-type cells adjacent to Flamingo-overexpressing cells, Diego disappears from the proximal-distal boundaries and appears to accumulate to a higher level at the boundary with the Flamingo-overexpression domain, perpendicular to its normal proximal-distal pattern. Furthermore, overaccumulation of Diego is also seen on the cell boundary opposite that which is in contact with the Flamingo-overexpressing cells. In some wings, reorientation of both Diego and Flamingo occurs up to three cell diameters away from the overexpression stripe. Thus, despite the fact that loss of Flamingo activity in clones has an essentially autonomous effect, depleting Diego and Flamingo only from the directly adjacent cell boundary, flamingo overexpression causes nonautonomous relocalization of both itself and Diego over several cell diameters (Feiguin, 2001).

Is Diego required for Flamingo localization? The distribution of Flamingo in wild-type versus homozygous diego mutant wings was compared. In all of the wild-type wings examined, the Flamingo protein is enriched on proximal-distal boundaries by 27 hr after puparium formation. In contrast, Flamingo in dgo380 wings localizes uniformly around the junctional region at this time, similar to its distribution in frizzled mutant wings. The ratio of Flamingo staining intensity on proximal-distal versus anterior-posterior cell boundaries was quantified in images from four wild-type wings and four dgo380 mutant wings. In wild-type wings, Flamingo is enriched between 2.5- and 3.2-fold on proximal-distal boundaries at 27 hr after puparium formation. In contrast, the Flamingo in dgo380 mutant wings is not significantly enriched (ratios ranged between 0.8 and 1.3). These data suggest that Diego promotes Flamingo polarization (Feiguin, 2001).

Flamingo sometimes polarizes its distribution in parts of dgo380 mutant wings at later times (29-30 hr after puparium formation), but the axis of polarity is often abnormal, presaging the whorls of misoriented hairs observed in dgo380 adult wings. In all cases, the effect of diego is specific to Flamingo and does not affect the distribution of cortical actin. Clonal analysis shows that diego loss of function does not affect Flamingo localization outside of diego clones. These data suggest that Diego increases the efficiency of Flamingo polarization and promotes its correspondence with the proximal-distal axis determined by Frizzled. The fact that Flamingo is localized similarly in frizzled and diego mutant cells may indicate that Frizzled acts through Diego to mediate Flamingo polarization (Feiguin, 2001).

To assess the effects of excess Diego protein on polarity, the effect of Diego overexpression on Flamingo localization was examined. When Diego protein is overexpressed with the ptc:GAL4 driver, it still localizes exclusively to the junctional region, but is present in large aggregates with no proximal-distal polarity. Flamingo protein accumulates in these cortical clusters, and its distribution is no longer polarized along the proximal-distal axis. These data suggest that Diego specifically promotes clustering of Flamingo. It is suggested that under wild-type conditions, Diego-mediated Flamingo clustering is regulated in response to Frizzled signaling and occurs only at proximal-distal boundaries (Feiguin, 2001).

Interestingly, Diego overexpression has nonautonomous effects on Flamingo localization in adjacent cells. In the first row of wild-type cells adjacent to Diego overexpressers, Flamingo is missing from proximal-distal boundaries and presumably relocalizes to maximize homophilic interactions with the clustered Flamingo on the Diego-overexpressing cells. Interestingly, Flamingo protein levels are also modestly elevated on the cell boundary opposite that of the Diego-overexpressing cells. As a result, the Flamingo in the second row of cells seems less unambiguously polarized to proximal-distal boundaries than in the cells at a greater distance from the overexpression stripe (Feiguin, 2001).

The ability of adjacent pupal wing cells to compare the activity of different tissue polarity genes and orient hairs accordingly is intrinsic to the mechanism of planar polarization. When artificial expression gradients are produced, Frizzled and Flamingo cause opposite effects on hair polarity; hairs point away from cells with higher Frizzled expression but toward cells with higher Flamingo expression. To ask how cells with differing levels of Diego behaved, the adult wings from UAS:dgo * ptc:GAL4 crosses were examined. Wing hairs were oriented from high- to low-expressing cells in ptc:GAL4;UAS:dgo wings -- similar to the effects caused by Frizzled and the opposite of those caused by Flamingo. These data suggest that Diego responds positively to Frizzled signaling and that the subsequent Diego-mediated clustering of Flamingo has negative effects on Frizzled signaling activity (Feiguin, 2001).

The mechanism by which Frizzled proteins mediates cell polarization events is not well understood. In wing epithelial cells, one consequence of Frizzled signaling is the polarization of Flamingo. Diego protein has been shown in this study to be required for normal polarization of Flamingo. Diego is itself localized at proximal-distal boundaries in response to Frizzled signaling. In the wing, proximal-distal localization of Diego and Flamingo proteins is interdependent. Perturbing the distribution of either protein disrupts that of the other. In the absence of Flamingo, Diego fails to localize to the cortex at all. However, in diego mutant cells, Flamingo is cortically localized but fails to polarize efficiently or accurately along the proximal-distal axis. The localization of each of these proteins can also be perturbed by overexpression of the other. When Diego is overexpressed, it accumulates in large nonpolarized junctional clusters that recruit high levels of Flamingo protein as well. In contrast, overexpression of Flamingo has a dispersive rather than a clustering effect on Diego in the same cell, causing it to delocalize from proximal-distal boundaries and assume a more uniform cortical distribution. Furthermore, in cells adjacent to Flamingo overexpressers, Diego is dominantly relocalized with Flamingo to face the overexpression domain. Clearly, Flamingo and Diego each affect the localization of the other (Feiguin, 2001).

It is suggested that Diego associates with the cortical membrane and helps to polarize the cell in response to Frizzled signaling. Diego's cortical localization and ability to cluster make this model more likely than one in which Diego promotes polarized delivery of the Flamingo protein from the Golgi apparatus. How might Diego and Flamingo polarize in response to Frizzled? Although one possibility is that Diego mediates polarization by directly interacting with components of the activated Frizzled pathway, the interdependence of Flamingo and Diego localization speaks against such a linear model. A more reasonable model is that localized Frizzled signaling might modify the properties of the Diego protein at proximal-distal boundaries, increasing its ability to promote clustering of itself and Flamingo. This initial bias in localization caused by clustering might then be potentiated via homophilic interactions between Flamingo molecules on adjacent cells. This model is consistent with the reciprocal effects that Flamingo and Diego have on each other's localization (Feiguin, 2001).

The ability of polarization signals to propagate from cell to cell is one of the most intriguing aspects of tissue polarity and is one of the least understood. It has been known for some time that locally perturbing Frizzled signaling has long-range effects on the polarity of the surrounding tissue. This repolarization is reflected, at the molecular level, by the altered distribution of Flamingo and Diego. Interestingly, in the absence of any direct disruption of Frizzled, Flamingo overexpression causes nonautonomous repolarization of Diego and Flamingo that propagates over several cell diameters. Both Flamingo and Diego accumulate ectopically to a high level at the interface between frizzled mutant and wild-type tissue. The data suggest that the accumulation of Diego and Flamingo at frizzled clonal boundaries might play an important role in propagating repolarization of these proteins into the surrounding tissue (Feiguin, 2001).

How might this propagation occur? Although it is easy to understand how Flamingo-mediated homophilic interactions could recruit Flamingo and Diego from a directly adjacent cell to the boundary with the overexpression domain, it is less clear how this would result in a corresponding accumulation on the opposite side of the cell. One possibility is that when Diego and Flamingo are polarized via Flamingo-mediated homophilic interactions, a locally acting intracellular signal is generated that prevents similar interactions nearby. Flamingo and Diego might then tend to accumulate on the opposite side of the cell, where such interactions were not discouraged. Increased levels of Diego and Flamingo on the opposite side of the cell might, in turn, recruit Diego and Flamingo from the next cell in line. Such a mechanism might be sufficient to propagate polarization of these proteins over many cell diameters (Feiguin, 2001).

Asymmetric homotypic interactions of the atypical cadherin Flamingo mediate intercellular polarity signaling

Acquisition of planar cell polarity (PCP) in epithelia involves intercellular communication, during which cells align their polarity with that of their neighbors. The transmembrane proteins Frizzled (Fz) and Van Gogh (Vang) are essential components of the intercellular communication mechanism, as loss of either strongly perturbs the polarity of neighboring cells. How Fz and Vang communicate polarity information between neighboring cells is poorly understood. The atypical cadherin, Flamingo (Fmi), is implicated in this process, yet whether Fmi acts permissively as a scaffold or instructively as a signal is unclear. This study provides evidence that Fmi functions instructively to mediate Fz-Vang intercellular signal relay, recruiting Fz and Vang to opposite sides of cell boundaries. It is proposed that two functional forms of Fmi, one of which is induced by and physically interacts with Fz, bind each other to create cadherin homodimers that signal bidirectionally and asymmetrically, instructing unequal responses in adjacent cell membranes to establish molecular asymmetry (Chen, 2008).

Nonclassical cadherins generally exhibit weak homophilic binding in vitro, raising the possibility that they regulate signaling rather than adhesion. Moreover, after cell-cell recognition, cadherins are thought to function either homophilically and symmetrically or heterophilically and asymmetrically between cells. This study shows that the atypical cadherin Fmi acts homophilically to communicate PCP signals between neighboring cells, yet its action is asymmetric, serving to link the accumulation of Fz on one cell boundary with Vang on the adjacent cell boundary, and vice versa. These data lead to the proposal of a model in which Fmi exists in two functional forms on opposite sides of intercellular borders, one of which selectively and cell-autonomously interacts with Fz (F-Fmi), and the other with Vang (V-Fmi). The native form of Fmi is V-Fmi, but upon interaction with Fz, V-Fmi is converted to F-Fmi. It is inferred that Fmi homodimers consist preferentially of opposite forms, thereby producing asymmetric function of the complex. By virtue of this mechanism, Fmi-mediated intercellular signaling communicates information about PCP protein asymmetry between neighboring cells (Chen, 2008).

How might Fmi achieve its homophilic yet asymmetric function? Although two splice forms exist, a single form can fulfill both V- and F-Fmi functions. A second possibility is that different stoichiometries of Fmi interact on opposite sides of the boundary -- for example, cis-dimers of Fmi might behave as V-Fmi whereas monomers function as F-Fmi. However, a reproducible proximal-distal difference in levels of tagged Fmi have not been detected when expressed in a mosaic pattern. A third possibility is that posttranslational regulation results in two distinct forms of Fmi that are selectively recruited or retained, directly or indirectly, by Fz or Vang. A fourth model is that V-Fmi and F-Fmi are alternate conformers or modified forms of Fmi where conversion of V-Fmi to F-Fmi depends on interaction with Fz. Although it is not possible to distinguish between the latter three possibilities, the finding that Fz and Fmi directly interact favors models in which Fz physically alters the properties of V-Fmi, thereby inducing the F-Fmi form. Extensive evidence shows that interacting proteins can modify the activity of cadherins. Of note, Xenopus Fz7, which mediates convergent extension during gastrulation, has been reported to directly bind a protocadherin through its extracellular domain. Detailed molecular and structural studies will be required to determine the precise nature of V-Fmi and F-Fmi and how they interact with Fz and Vang (Chen, 2008).

During PCP signaling, cells each receive a signal that orients polarization. Cells then consolidate this information by amplifying the asymmetry in a process that involves communicating and aligning polarity with surrounding cells. By signaling to a neighbor that a given cell boundary is enriched for either Fz or Vang, asymmetric Fmi homodimers transmit this information bidirectionally between cells. In the wild-type, amplification through feedback control is required to produce sharp differences between Fz and Vang levels on adjacent cell surfaces. Pk, Dgo, and Dsh are required for this amplification, though they are not required for intercellular signaling per se. The mechanism by which this amplification occurs is unknown, but the result is a mutual exclusion of Fz and Vang from a given region of the cell surface. Fmi therefore serves to link the action of feedback loops in neighboring cells, assuring a coordinated polarization (Chen, 2008).

It has been proposed that asymmetric placement of core PCP proteins is itself the signal that controls morphological polarization. However, an alternative model has been proposed in which the absolute, scalar value of Fz activity within each cell varies in a gradient across the tissue in response to an unidentified ligand. Scalar Fz levels are proposed to be refined by an averaging process between neighboring cells. According to this view, asymmetric PCP protein localization is only an epiphenomenon and is not required for function. It is suggested that several observations are inconsistent with this model. (1) The model predicts that the extent of domineering nonautonomy near fz mutant clones should vary according to position in the Fz activity gradient. However, its extent was reported to be equal throughout the abdomen, and no proximal-distal difference is evident in the fly wing. (2) FzΔCRD rescues polarity in fz null mutant flies despite deletion of the CRD, leaving little protein to which an extracellular ligand might bind (Chen, 2008).

In essence, the scalar Fz model argues for a Fz gradient across the tissue, whereas the asymmetric protein localization model invokes gradients of core PCP protein localization or activity within each cell but not across the tissue. As an additional test to distinguish between these two models, small wild-type islands of ~20 cells in size surrounded by fmi mutant cells were exsmained. These wild-type cells are prevented from communicating with and receive no repolarizing signal from the surrounding fmi mutant cells. The scalar Fz model predicts that these small wild-type islands should still be directly responsive to the proposed morphogen gradient and should therefore generate a normal Fz activity slope, resulting in normal polarity. In contrast, because the asymmetric PCP protein model posits only subcellular gradients of PCP protein activity but no tissue level gradient of Fz or other core PCP protein activity, each cell's tendency to align polarity with its neighbors could lead to other patterns of local alignment. Consistent with this latter possibility, prehairs in many of these islands exhibited PCP defects and formed swirling patterns. Because there is no discontinuity as one follows the polarity of cells in these islands, the scalar Fz activity model cannot accommodate this result without invoking an Escher's staircase of infinitely rising Fz levels. In contrast, the asymmetric protein model easily explains this result by organizing proximal and distal PCP protein domains in a spoke-like pattern between the cells of the swirl, as is indeed observed. In light of the evidence presented in this study that Fmi homodimers can instructively generate asymmetric Fz and Vang localization and locally align the polarity of neighboring cells, a model is favored in which instructive protein localization mediated by Fmi homodimers is itself the signal that transmits PCP information between cells. It is thought that this is the only known example of a cadherin homodimer providing dissimilar signals across intercellular boundaries (Chen, 2008).

The Frizzled extracellular domain is a ligand for Van Gogh/Stbm during nonautonomous planar cell polarity signaling

The Frizzled (Fz) receptor is required cell autonomously in Wnt/β-catenin and planar cell polarity (PCP) signaling. In addition to these requirements, Fz acts nonautonomously during PCP establishment: wild-type cells surrounding fz- patches reorient toward the fz- cells. The molecular mechanism(s) of nonautonomous Fz signaling are unknown. Un vivo studies identify the extracellular domain (ECD) of Fz, in particular its CRD (cysteine rich domain), as critical for nonautonomous Fz-PCP activity. Importantly, biochemical and physical interactions have been demonstrated between the FzECD and the transmembrane protein Van Gogh/Strabismus (Vang/Stbm). This function precedes cell-autonomous interactions and visible asymmetric PCP factor localization. The data suggest that Vang/Stbm can act as a FzECD receptor, allowing cells to sense Fz activity/levels of their neighbors. Thus, direct Fz-Vang/Stbm interactions represent an intriguing mechanism that may account for the global orientation of cells within the plane of their epithelial field (Wu, 2008).

The data suggest that the Fz ECD, including the CRD, acts as a Vang/Stbm ligand in nonautonomous signaling. How do these data and interpretations fit with other existing results and models? The Fz CRD is clearly dispensable for canonical Wg signaling in vivo. Previous studies have shown that it is essential for PCP signaling; however, the specifics of when and where have been controversial. A recent paper suggests that the CRD is not strictly required for PCP establishment, as FzΔCRD can partially rescue fz mutant phenotypes in the wing. However, some PCP defects remain. It is also worth noting that in experiments where the multiple wing hair phenotype of fz wings were assayed, this serves as a marker for late stage cell-autonomous Fz functions and does not address whether FzΔCRD is functional in intercellular nonautonomous communication. The experiments indicate that FzΔCRD does not fully rescue fz PCP phenotypes and does not affect domineering nonautonomy of fz mutant clones. Thus, it is concluded that the CRD of Fz is necessary for cells to send polarizing signals to neighboring cells (Wu, 2008).

Genetic and physical interaction data suggest that at the early PCP signaling stage (14-24 hr APF), Vang/Stbm functions as a receptor for FzCRD. As such, it would appear that Vang/Stbm senses how much Fz (activity) is present on adjacent cells and relays this information, causing a cell to orient toward the neighboring cell with lower Fz level/activity. The conclusion that Fz 'signals' and Vang 'receives the signal' is consistent with previous models, in that a fz cell at the clone boundary will orient toward the center of the mutant area as it compares levels of its two neighboring cells (one of which is the wild-type cell adjacent to the clone). Similarly, it has been shown that Vang is not needed in the 'sending' cell, which is consistent with the result that fz Vang double mutant clones behave like fz clones. How do these observations fit with the nonautonomous behavior of Vang clones? In Vang mutant cells, all Fz protein accumulates at the membranes abutting wild-type cells; wild-type cells at the clonal border would therefore presumably detect more Fz in Vang cells. The model would predict that this relocalization of Fz causes these cells to orient away from the Vang neighbors. This interpretation is also consistent with the Vang phenotype being suppressed in fz Vang double mutant clones, suggesting that the nonautonomous effect is mediated largely through Fz (Wu, 2008).

Fz-Vang/Stbm interactions are dependent on the presence of the Fmi (also known as Stan) protein but are independent of the core PCP factor Dsh. Similarly, they are independent of Pk, which mediates the cell-autonomous requirement for Vang/Stbm. It is important to note that Vang/stbm mutants affect fz nonautonomy differently from pk mutants: Vang/stbm backgrounds suppress the domineering nonautonomy of fz clones (consistent with the model), whereas pk mutants enhance the nonautonomous effects. These data suggest that during early nonautonomous PCP signaling, Fz-Vang/Stbm effects are independent of Pk. It is thus likely that there are two distinguishable phases of Fz-Vang/Stbm interactions: the nonautonomous phase addressed here (14-24 hr APF and a later autonomous phase involving Dsh and Pk (Wu, 2008).

The simplest interpretation of the data suggests signaling from Fz to Vang/Stbm during nonautonomous signaling. It cannot excluded, however, that the interaction is bidirectional and that Fz activity is also influenced by binding to Vang/Stbm (in a Dsh-independent manner). Nevertheless, comparing the gain-of-function data of Fz and Vang/Stbm, the effects of Fz in repolarizing neighboring cells are always robust, whereas those with Vang/Stbm are milder and more cell autonomous. Despite this observation, bidirectional signaling is possible, either through the Fz-Vang/Stbm interaction or through their links to the atypical cadherin Fmi as suggested in several models. The data do not exclude an instructive role for a Fmi-Fmi interaction as proposed earlier. Indeed, this latter idea is supported by the observation that nonautonomous fz clonal phenotypes are not completely suppressed in a Vang mutant background, as some nonautonomy is still observed (~25% of fz clones still display weak nonautonomy in a Vang background) (Wu, 2008).

Fmi has recently been shown to associate with Fz, and thus the homophilic cell adhesion behavior of Fmi could also contribute to an instructive directional signal. The observations that Fz can associate extracellularly with Vang/Stbm (this work) and within the membrane with Fmi suggest a complex scenario. A cross-cell interaction mediated by the homophilic Fmi interaction could display asymmetric properties, as Fmi-Fz and/or Fmi-Vang complexes could have different qualities and signal in either direction. However, fmi null clones show little nonautonomous behavior (a 1 cell wide effect), while the fz and fz Vang clones with widespread nonautonomy are nevertheless striking. Fmi causes significant nonautonomy when overexpressed, and this effect seems not to depend on the presence of Fz or Vang in the overexpressing clone. Multiple parallel mechanisms are thus likely to exist that contribute to cell-cell communication in transmitting the polarity signal (Wu, 2008).

In conclusion, this study provides molecular evidence for a mechanism of nonautonomous Fz signaling through direct interactions with Vang/Stbm on neighboring cells. It remains unclear how the levels of the initial Fz-Vang/Stbm interaction are established in wild-type. Both Fz and Vang/Stbm are expressed evenly and their initial subcellular localization is not polarized. Thus, in wild-type, the generation of a polarized Fz-Vang/Stbm interaction (across cells) must be mediated by other factors that modify Fz, Vang/Stbm, or their interaction in a graded manner (Wu, 2008).

Differential stability of flamingo protein complexes underlies the establishment of planar polarity

The planar polarization of developing tissues is controlled by a conserved set of core planar polarity proteins. In the Drosophila pupal wing, these proteins adopt distinct proximal and distal localizations in apicolateral junctions that act as subcellular polarity cues to control morphological events. The core polarity protein Flamingo (Fmi) localizes to both proximal and distal cell boundaries and is known to have asymmetric activity, but the molecular basis of this asymmetric activity is unknown. This study examined the role of Fmi in controlling asymmetric localization of polarity proteins in pupal wing cells. Fmi was found to interact preferentially with distal-complex components, rather than with proximal components, and evidence is presented that there are different domain requirements for Fmi to associate with distal and proximal components. Distally and proximally localized proteins cooperate to allow stable accumulation of Fmi at apicolateral junctions, and evidence is presented that the rates of endocytic trafficking of Fmi are increased when Fmi is not in a stable asymmetric complex. Finally, evidence is provided that Fmi is trafficked from junctions via both Dishevelled-dependent and Dishevelled-independent mechanisms. A model is presented in which the primary function of Fmi is to participate in the formation of inherently stable asymmetric junctional complexes: Removal from junctions of Fmi that is not in stable complexes, combined with directional trafficking of Frizzled and Fmi to the distal cell edge, drives the establishment of cellular asymmetry (Strutt, 2009).

The differing ability of overexpressed Fmi to modulate Fz:Dsh and Stbm:Pk levels at junctions could be explained by a number of mechanisms. One likely hypothesis is that Fmi may require a cofactor for a robust interaction with Stbm, and that this cofactor is limiting when Fmi is overexpressed. Alternatively, Fmi may require posttranslational modification or a conformational change to interact with Stbm, and a factor needed for this modification is limiting. The cytoplasmic C-terminal tail of Fmi is a likely region to mediate an interaction with Fz:Dsh or Stbm:Pk; therefore, a truncated form of Fmi was constructed, in which this region is either absent or replaced with GFP (Strutt, 2009).

When overexpressed in pupal wing cells, FmideltaIntra is much more efficient at recruiting Fz and Dsh to junctions than full-length Fmi, an effect similar to that caused by removal of stbm or pk. Stbm is still reduced at junctions, although less than when full-length Fmi is overexpressed. This suggests that the C-terminal intracellular domain of Fmi is dispensible for the interaction of Fmi with Fz:Dsh and, importantly, that Fz:Dsh no longer have to compete with Stbm:Pk for access to Fmi (Strutt, 2009).

Interestingly, two isoforms of Fmi have been identified, one of which contains a PDZ binding motif (PBM) at its C terminus. It is possible that loss of the PBM alone could account for the failure of overexpressed Fmi or FmideltaIntra to associate with Stbm:Pk. However, this is unlikely, because Fmi that lacks the PBM can rescue the planar polarity phenotype of fmi mutants (Strutt, 2009).

Endogenous Fmi is thought to be localized on both proximal and distal cell boundaries. This was confirmed by expressing CFP-tagged Fmi at physiological levels in clones in pupal wings, and it was observed that levels of staining appear similar at each end of the cell, consistent with the homophilic-interaction model. Notably, expression of a GFP-tagged form of FmideltaIntra results in its preferential localization to distal cell edges, where Fz and Dsh also localize (Strutt, 2009).

Interestingly, junctional localization of FmideltaIntra-EGFP is not dependent on endogenous, full-length Fmi, suggesting that this molecule is still able to participate in homophilic interactions. Hence, the ability of FmideltaIntra-EGFP to functionally rescue the polarity phenotype of fmi null mutant clones was investigated. If FmideltaIntra-EGFP interacts preferentially with the distal Fz:Dsh complex, then Stbm recruitment to junctions inside clones would be compromised. Consequently, FmideltaIntra-EGFP:Fz complexes inside the clone would preferentially interact with Fmi:Stbm outside the clone, leading to a reversal in polarity on proximal clone edges. Importantly, this prediction is upheld, and fmi clones rescued with FmideltaIntra-EGFP exhibit weak proximal polarity inversions, such that trichomes point away from the clone, and polarity proteins are recruited to the clone boundary (Strutt, 2009).

Nevertheless, Stbm localizes asymmetrically inside the clone, although not always at the correct site, whereas in a fmi null mutant it lacks any asymmetric localization. Thus, FmideltaIntra-EGFP must retain some ability to interact with Stbm. To confirm this, the ability of full-length Fmi or FmideltaIntra-EGFP to interact with Fz and Stbm in Drosophila S2 cells was analyzed. In this assay, Fmi and FmideltaIntra-EGFP are recruited to sites of cell contact, as a result of homophilic interactions between their extracellular domains. Cotransfection of Fz or Stbm with either full-length Fmi or FmideltaIntra-EGFP in Drosophila S2 cells results in the recruitment of both to sites of cell contact (Strutt, 2009).

Interestingly, if S2 cells were transfected with either Fz or Stbm and then mixed, weak recruitment is also observed to sites of cell contact, arguing that their extracellular domains can interact independently of Fmi. Nevertheless, recruitment was weaker and less frequent than when Fmi was cotransfected, suggesting that Fmi:Fmi interactions are more important than Fz:Stbm interactions in stabilizing complexes between adjacent cells (Strutt, 2009).

The data suggest that Fz:Dsh and Stbm:Pk complexes differ in their ability to associate with Fmi. Whereas endogenous levels of Fmi result in the formation of asymmetric complexes with Fz:Dsh on one side of the boundary and Stbm:Pk on the other, overexpressing Fmi favors Fz:Dsh recruitment. Furthermore, a C-terminally deleted form of Fmi preferentially localizes distally with Fz, and overexpression of this form has an even greater preference for Fz:Dsh recruitment. Thus, the C terminus of Fmi is important in promoting the interaction with Stbm:Pk. The Fmi truncation data could be explained simply by the possibility that the C terminus of Fmi contains a direct binding site for Stbm; however, this fails to explain why overexpressed full-length Fmi prefers to recruit Fz:Dsh. It is therefore proposed that the association of Fmi with Stbm:Pk requires a limiting factor that is saturated by Fmi overexpression. The most plausible hypothesis is a requirement for a cofactor for Stbm:Pk binding, but other possibilities include saturation of the machinery for a posttranslational modification or a conformational change in Fmi (Strutt, 2009).

The data also suggest that Fmi itself needs to associate with both proximal and distal components in order to be stably localized to apicolateral junctions. Although it can form homophilic dimers between adjacent cell membranes in tissue culture, in pupal wings Fmi does not localize strongly to apical junctions and presumably fails to form stable homodimers in trans. Fz on one side of the junction and Stbm:Pk on the opposite side stabilize Fmi at junctions, most likely by promoting homophilic interactions or preventing internalization. However, Fmi appears to be capable of forming complexes with either distal or proximal components alone, but these complexes (particularly the proximal complex) are apparently less stable at junctions. Taken together with overexpression experiments, this would suggest that the most stable configuration is Fz:Fmi on one side of the boundary and Fmi*:Stbm:Pk on the other (where Fmi* denotes the modified form able to preferentially associate with Stbm:Pk) (Strutt, 2009).

In order for an asymmetric complex to be stabilized across junctions, the extracellular domains must somehow 'look' different. One possibility is that the Fz and Stbm extracellular loops interact - a view supported by S2 cell data. Alternatively, the Fmi extracellular domain, when associated with either Fz or Stbm:Pk, could undergo a conformational change that promotes homophilic Fmi interactions (Strutt, 2009).

An intriguing question is why clones of cells that overexpress Fmi behave like fz loss-of-function clones. It is suggested that within the clones, excess Fmi associates with the entire available pools of both Fz and Stbm. However, there is still a pool of uncomplexed Fmi that can associate with Fmi:Fz in adjacent wild-type cells, forming the relatively stable Fmi-Fmi:Fz configuration, thus causing polarity to be reversed on distal clone boundaries. In support of this model, an identical nonautonomous effect is seen when FmideltaIntra is overexpressed, which itself interacts only poorly with Stbm but presumably can interact with Fmi:Fz in adjacent cells outside the clone (Strutt, 2009).

Interestingly, Fmi accumulates in excess at junctions in a dsh, stbm double mutant, whereas Fz does not. Thus, although Fz acts to stabilize Fmi at junctions, Fmi does not always need to associate with Fz in a stoichiometric fashion in order to be stabilized. Perhaps as long as there is some Fz associated with Fmi, this may permit local stabilization of other Fmi molecules in cis. Alternatively, this excess accumulation of Fmi might simply represent 'unstable' Fmi homodimers that are no longer being removed from junctions by the actions of Dsh and Stbm (Strutt, 2009).

The composition of the complex with which Fmi is associated appears to be critical for determining the frequency and manner by which Fmi is turned over from the plasma membrane. Most compellingly, Fmi accumulates more strongly in an enlarged endosomal compartment in Rab7TN mutant tissue when stbm and fz are absent than when they are present. Thus, it is suggested that more Fmi is resident in the endocytic pathway when it is unable to form stable asymmetric complexes. Fmi:Fz puncta have been observed that are selectively trafficked to distal cell edges. In the current experiments, these puncta colocalize with YFP-Rab4, suggesting that Fmi and Fz are recycled back to the plasma membrane by a Rab4-dependent mechanism. Furthermore, the increased intracellular and junctional levels of Fz and Fmi in dor mutant clones suggests that in addition to being recycled to the plasma membrane, a significant fraction of internalized Fmi and Fz is also sent for degradation. It is formally possible that the intracellular accumulation of Fmi and Fz seen when lysosomal trafficking is blocked by loss of Rab7 or in dor clones is due to their being sent for degradation immediately after synthesis (e.g., if damaged or misfolded); however this is unlikely because newly synthesized Fmi-ECFP appears first at junctions before been seen in puncta (Strutt, 2009).

Stbm has not been observed in large intracellular puncta, but it seems likely that it is also internalized and recycled, possibly together with Fmi, although it must do so by alternative pathways involving smaller or more rapidly recycling particles that are not visible by confocal microscopy. Indeed, the data suggest a potential role for Dsh and Stbm in regulating junctional levels of Fmi. A stbm mutant alone results in a loss of Fmi from junctions, consistent with a need for Stbm in stabilizing Fmi in asymmetric complexes. In contrast, loss of Dsh and Stbm together increases Fmi levels at junctions, suggesting a role for Stbm in internalization. It is suggested that the outcome of any interaction of Stbm with Fmi is dependent upon whether Fmi is able to form stable homodimers with Fz on the opposite cell membrane. In a wild-type situation, one could envisage that Fmi forms stable homodimers in a Fz:Fmi-Fmi*:Stbm configuration, and that both Dsh and Stbm promote internalization of any Fmi that is not in this configuration, the majority of which is subsequently recycled back to the plasma membrane. In dsh mutants, there is reduced internalization, but the effect on Fmi levels is subtle; Fz and Stbm are still present to promote Fmi homodimer formation, and Stbm still promotes internalization of any unstable Fmi. In contrast, in stbm mutants, the number of less stable Fmi complexes (associating only with Fz) is greatly increased, favoring internalization by Dsh. Finally in dsh, stbm double mutants, Fmi is again less stable (associating only with Fz), but there is no Dsh- or Stbm-mediated internalization, leading to an overall increase of Fmi at junctions (Strutt, 2009).

How do Dsh and Stbm regulate Fmi levels at junctions? Stbm contains potential interaction motifs for the endocytic adaptor AP2, but their role has not been functionally tested. In addition, in vertebrate Wnt signaling, there is evidence that Dsh interacts with the endocytic adaptor protein β-arrestin and mu2 subunit of AP2 to mediate Wnt/Fz endocytosis and downregulation of Wnt signaling. Interestingly, in planar polarity this is no evidence that Dsh directly mediates internalization of Fz, but the data rather point to Dsh promoting Fmi internalization when it is not associated with Fz. Instead, the trafficking of Fmi together with Fz into the lysosomal pathway is Dsh independent (Strutt, 2009).

In summary, it is proposed that a number of mechanisms exist by which Fmi contributes to the generation of asymmetry at the molecular level. First, the characterization of the previously inferred asymmetry in Fmi activity indicates that Fmi normally prefers to bind to Fz and requires a limiting factor for association with Stbm:Pk. Second, Fmi stability at junctions is dependent on both Fz and Stbm:Pk, with the most stable form being Fz:Fmi bound to Fmi*:Stbm. Finally, it is proposed that entry of Fmi into the endocytic trafficking pathway is decreased if it is in a stable complex, and this is regulated either by Dsh and Stbm or independently of Dsh and Stbm, depending on whether it is associated with Fz (Strutt, 2009).

An outstanding question is how these mechanisms translate into cellular asymmetry, such that in any particular cell, heterophilic polarity complexes preferentially form with Fz:Dsh at the distal junctions, rather than having heterophilic complexes in both orientations. It is thought that the acquisition of cellular asymmetry is likely to be driven by directional trafficking of Fmi:Fz, although other models, such as a mechanism for preferential stabilization of Fmi:Fz interactions at the distal cell edge, are also possible. In addition, it seems likely that an amplification mechanism would be required, although the molecular mechanisms remain to be elucidated (Strutt, 2009).

While this manuscript was in preparation, another manuscript was published, in which Fmi was proposed to mediate an asymmetric and instructive signal between proximal and distal complexes to generate asymmetry, and thus does not act merely as a scaffold for Fz:Stbm interactions across membranes. It is argued that the current data do not provide evidence for a specific signaling function of Fmi. Instead, the hypothesis is favored that the composition of the proximal and distal complexes is distinct, and that heterophilic complexes are inherently more stable than homophilic complexes. Together, removal of unstable nonasymmetric complexes through increased endocytic turnover, in concert with directional trafficking and an unknown amplification mechanism, may be sufficient to generate asymmetry without the need to invoke a specific signaling function for any components of the complexes (Strutt, 2009).

Echinoid regulates Flamingo endocytosis to control ommatidial rotation in the Drosophila eye

Planar cell polarity (PCP) refers to a second polarity axis orthogonal to the apicobasal axis in the plane of the epithelium. The molecular link between apicobasal polarity and PCP is largely unknown. During Drosophila eye development, differentiated photoreceptors form clusters that rotate independently of the surrounding interommatidial cells (ICs). This study demonstrates that both Echinoid (Ed), an adherens junction-associated cell adhesion molecule, and Flamingo (Fmi), a PCP determinant, are endocytosed via a clathrin-mediated pathway in ICs. Interestingly, it was found that Ed binds AP-2, an adaptor that acts between cargo receptors and clathrin-coated vesicles during endocytosis, and is required for the internalization of Fmi into ICs. Loss of ed led to increased amounts of Fmi on the cell membrane of non-rotating ICs and also to the misrotation of photoreceptor clusters. Importantly, overexpression of fmi in ICs alone was sufficient to cause misrotation of the adjacent photoreceptor clusters. Together, it is proposed that Ed, when internalized by AP-2, undergoes co-endocytosis with, and thereby decreases, Fmi levels on non-rotating ICs to permit correct rotation of ommatidial clusters. Thus, co-endocytosis of Ed and Fmi provides a link between apicobasal polarity and PCP (Ho, 2010).

This study found that Ed interacts with AP-2 and promotes Fmi internalization via a clathrin-dependent pathway in ICs. Moreover, loss of ed leads to accumulation of Fmi (and of several receptors/CAMs of AJs) on the membrane of these non-rotating ICs. This, together with the observation that overexpression of fmi in the non-rotating ICs is sufficient to cause photoreceptor misrotation, led to a proposal of an Ed-mediated co-endocytosis model to explain the rotation defects associated with the ed mutant tissue. Thus, the homophilic CAMs, Ed and Fmi, play crucial roles in ICs to allow coordinated rotation of ommatidial clusters (Ho, 2010).

Ed specifically regulates Fmi endocytosis in ICs. Although Fmi was previously shown to be endocytosed in the photoreceptor cluster, it is argued that Fmi levels in the rotating photoreceptor clusters are regulated by an Ed-independent mechanism. First, Fmi levels in the photoreceptor clusters are not affected in the ed mutant clones. Second, the Fmi distribution pattern in R3/R4 is largely unchanged even when Ed-GFP is overexpressed in photoreceptors by Elav-Gal4 to mimic the ed-expressing ICs. It was also demonstrated that Ed levels in ICs, but not in photoreceptor clusters, are regulated via an AP-2-dependent endocytic pathway. It remains unknown how Ed is downregulated in the photoreceptor cluster. Egfr signaling has been proposed to regulate the morphological and adhesive changes of cells within the photoreceptor cluster, and it is possible that Egfr signaling directly or indirectly downregulates the Ed levels in R8/R2/R5 and later in R3/R4 (Ho, 2010).

Interestingly, ed affects the levels of Fmi, DE-cad and Egfr, but not of Dlg. Therefore, Ed seems to only affect receptors/CAMs at AJs. One intriguing possibility is that Ed, via its interaction with AP-2, triggers the co-endocytosis of most, if not all, of the receptor/CAM at AJs. Although Ed has been shown to associate with Egfr, there is currently no evidence that Ed interacts directly with Fmi. Thus, Ed might undergo co-endocytosis either directly or indirectly. Although Ed contains five putative protein-sorting motifs, it is not the only molecule with a protein-sorting motif: Egfr, for example, also contains the YXXphi signal (phi is a bulky hydrophobic amino acid which could potentially interact with the AP-50 subunit of the AP-2 complex). It is possible that different receptors/CAMs might cooperate, via their interaction with AP-2 or other adaptors, to promote the co-endocytosis of other receptors/CAMs (Ho, 2010).

Because Ed facilitates the endocytosis of many receptors/CAMs of AJs, although to different extents, multiple functions of ed in the eye disc would be expected. Indeed, ed plays crucial roles in PCP (this study) and in Egfr signaling during eye development. It was previously shown that loss of ed leads to sustained MAPK (Rolled - FlyBase) activation only in cells of the proneural clusters and over several rows. This is consistent with the observation that Egfr is upregulated in an enlarged group of arc cells that contains two R8 photoreceptors as well as in cells of developing ommatidia up to two rows posterior to the MF. Thus, it is plausible that Ed was co-endocytosed with Egfr in the proneural clusters to downregulate Egfr activity within these cells and thus ensure that only one R8 is selected from the two to three R8 cell-equivalent group. When ed is absent, Egfr cannot be internalized efficiently and therefore persists on the membrane to cause sustained MAPK activation and multiple R8 selection. Although the level of Egfr is also upregulated in cells more posterior to the MF, these levels might not be high enough to cause sustained MAPK activation. In the wing disc, Ed also facilitates Notch signaling to promote mesothorax bristle patterning. In fact, Ed colocalizes with Notch/Delta in Hrs-containing early endosomes. However, it remains unclear whether the endocytosis of Ed plays any role in facilitating Notch signaling in the wing discs (Ho, 2010).

It has been shown that each photoreceptor cluster, as a group, moves independently of the adjacent ICs. Most rotation-specific genes identified thus far have been proposed to function mainly in the rotating clusters to modulate rotation. This study provides evidence that Ed plays crucial roles in the ICs to modulate ommatidial rotation. It is proposed that Ed, via co-endocytosis, reduces the level of Fmi on the non-rotating ICs to prevent homotypic interactions with the enriched Fmi on the rotating cluster. This allows free and coordinated rotation of photoreceptor clusters, a process regulated by effectors such as Zipper and Nemo. It is reasoned that in the absence of ed, as seen in ed mutant clones, the upregulated Fmi on the non-rotating ICs might affect the free rotation of ommatidial clusters not only within the ed clone, but also in the adjacent wild-type clusters abutting the ed clones. This might contribute, at least in part, to the non-autonomous effect of ed on ommatidial rotation. The dynamic and differential expression of Ed (and of its paralog Friend of Echinoid) in the rotating clusters and non-rotating ICs has also been proposed to modulate ommatidial rotation (Fetting, 2009). Thus, differential expression of Ed, Fmi, DE-cad and Friend of Echinoid in the rotating clusters and non-rotating ICs prevents the homotypic interaction of these four CAMs to allow free rotation of photoreceptor clusters. The largely complementary expression pattern between Ed and DE-cad/Arm in a photoreceptor cluster is similar to that observed during the generation of ed mutant clones in the wing discs, where Ed-non-expressing cells accumulate high levels of DE-cad/Arm and sort out from the surrounding Ed-expressing cells. Thus, cell sorting-like behavior of a photoreceptor cluster, mediated by differential expression of Ed and DE-cad/Arm, might help photoreceptors in the cluster to rotate as a group (Ho, 2010).

Fetting (2009) recently showed that ed genetically interacts with Egfr pathway members, and proposed that ed, via inhibiting Egfr signaling in the photoreceptors, regulates ommatidial rotation. However, this study demonstrated that, after row 2, Ed facilitates the endocytosis of Egfr only in the non-rotating ICs, but not in the photoreceptor clusters. Thus, if Ed indeed inhibits Egfr signaling in the photoreceptors as suggested, it probably employs mechanisms other than to reduce the levels of Egfr on the photoreceptors. It is currently unknown whether the effect of Ed on Egfr levels in ICs plays any role in the modulation of ommatidial rotation. Moreover, this study found that in the absence of ed, not only Fmi, Fz and Dsh (the R3-specific PCP proteins), but also Strabismus (Van Gogh) and its associated Prickle (the R4-specific PCP proteins) were all upregulated in ICs, but their enrichment at R3/R4 borders in the photoreceptor cluster was largely unaffected. As ed affects the levels of all the core PCP proteins tested in ICs, it remains unclear how ed only interacts genetically with the R3-specific PCP genes to modulate the degree of ommatidial rotation (Fetting, 2009). Finally, ed also weakly affects the initial R3/R4 specification, as a small proportion of ed mutant cells also show randomized chirality and symmetrical ommatidia. It is possible that ed might exert this effect through mechanisms other than the promotion of Fmi endocytosis. Alternatively, as overexpression of Fmi in the non-rotating ICs (generated by fmi-overexpressing clones) can affect both ommatidial rotation and reversal of R3/R4 cell fate of the adjacent clusters, it is possible that the Fmi upregulation in the ICs (generated by ed mutant clones) might also affect, to some extent, the asymmetric distribution of Fmi in R3/R4 and, thereby, the R3/R4 specification of the adjacent clusters (Ho, 2010).

The seven-pass transmembrane cadherin Flamingo controls dendritic self-avoidance via its binding to a LIM domain protein, Espinas, in Drosophila sensory neurons

Members of the Flamingo cadherin family are required in a number of different in vivo contexts of neural development. Even so, molecular identities downstream from the family have been poorly understood. This study shows that a LIM domain protein, Espinas (Esn), binds to an intracellular juxtamembrane domain of Flamingo (Fmi), and that this Fmi-Esn interplay elicits repulsion between dendritic branches of Drosophila sensory neurons. In wild-type larvae, branches of the same class IV dendritic arborization neuron achieve efficient coverage of its two-dimensional receptive field with minimum overlap with each other. However, this self-avoidance was disrupted in a fmi hypomorphic mutant, in an esn knockout homozygote, and in the fmi/esn trans-heterozygote. A functional fusion protein, Fmi:3eGFP, was localized at most of the branch tips, and in a heterologous system, assembly of Esn at cell contact sites required its LIM domain and Fmi. It was further shown that genes controlling epithelial planar cell polarity (PCP), such as Van Gogh (Vang) and RhoA, are also necessary for the self-avoidance, and that fmi genetically interacts with these loci. On the basis of these and other results, it is proposed that the Fmi-Esn complex, together with the PCP regulators and the Tricornered (Trc) signaling pathway, executes the repulsive interaction between isoneuronal dendritic branches (Matsubara, 2011).

A yeast two-hybrid screen highlighted members of the Drosophila PET-LIM domain family: Prickle (Pk), Espinas (Esn), and Testin. Pk has been well studied as one of the core group members of PCP, whereas any physiological roles of Esn were unknown. Because of prominent expression of esn in the nervous system, Esn was considered to be a strong candidate for a Fmi binder in neurons, and this protein was analyzed further. The JM domain of Fmi interacted with the LIM domain of Esn or Pk in a targeted yeast two-hybrid assay and also in a coimmunoprecipitation (co-IP) experiment using Drosophila S2 cells. JM was dissected and the N-terminal subdomain (JM-A) was shown to bound LIM much more strongly than the C-terminal JM-B. Although the detection of the JM-LIM interaction was straightforward, no Fmi-Esn interaction was shown by co-IP when both full-length proteins were expressed in S2 cells. A technical difficulty with the demonstration of the Fmi-Esn interaction by co-IP is anticipated, considering that Fmi is a multipass transmembrane protein of 3579 residues. Nonetheless, attempts were made to demonstrate the physical interaction between the endogenous proteins by using a larval CNS lysate as a source material. In two out of two co-IP experiments, endogenous Esn was detected in the anti-Fmi immunoprecipitates, but not in the immunoprecipitates with negative control antibodies, although the efficiency of Esn co-IP with Fmi was low. These results imply that the association of Esn to Fmi might be transient and/or dependent on the cellular milieu (Matsubara, 2011).

The Fmi-Esn interaction was further investigated in a cell adhesion assay. Fmi-expressing S2 cells adhere to each other due to Fmi-Fmi homophilic interactions, and Fmi often accumulates at sites of cell-cell contacts. This assay was applied to test whether Esn was colocalized with Fmi at the cell contact sites; however, no clear conclusions could be drawn. As an alternative approach to assess the possibility of Fmi-Esn colocalization in cells, pupal wing epithelia was used where endogenous Fmi is enriched at cell boundaries. When Esn was ectopically expressed in the wing, it was colocalized with Fmi at cell boundaries. In contrast, an Esn mutant form that lacked the LIM domain (Esn?LIM) was diffusely distributed in the cells and did not exhibit obvious concentration at the boundaries where Fmi was accumulated. Furthermore, the full-length Esn was no longer localized at the apical cell contact sites when fmi was knocked down. These results show that Esn can be localized at cell contact sites in a LIM domain- and Fmi-dependent manner (Matsubara, 2011).

This study shows both physical and genetic interactions between Fmi and Esn, and has proposed a role of the Fmi-Esn interplay in contact-dependent repulsive signaling between isoneuronal dendritic branches of class IV da neurons. The LIM domain is shared by a number of proteins and is recognized as a modular protein-binding interface. Esn belongs to the Drosophila PET-LIM domain family, where two other members are Pk and Testin. The the co-IP results raise the possibility that the binding of Esn to Fmi in da neurons might be under the control of a conformational change and/or post-translational modifications of Esn. In fact, it has been shown for human Testin (hTes) that its LIM domains mediate the intramolecular interaction (Zhong, 2009). More specifically, the C-terminal LIM interacts directly with N-terminal hTes sequences, and this intramolecular association seems to preclude the docking of partners such as α-actinin. Furthermore, there might be a regulation at the level of tethering Esn to the plasma membrane. The CAAX motif at the C terminus of Esn could be the target of such a regulation. Potential candidates that may be involved in such a hypothetical regulation of Esn would be PCP regulators such as Vang (Matsubara, 2011).

A group of key molecules play pivotal roles in dendrite self-avoidance. That said, the underlying mechanisms involving these molecules (the Hpo-Trc kinase cascade, Dscam1, Tutl, and the Fmi-Esn interplay) seem to be complex, as suggested by the fact that phenotypic analyses of these genes show similarities as well as differences in terms of class specificity and self-avoidance versus tiling. Moreover, understanding of the molecular mechanisms is currently limited. For example, unknown are the identities of interbranch signals and their sensors that are connected to the Trc pathway. Genetic interactions have been sought between trc and Dscam1 or tutl, but positive results have not been obtained. This study found an interaction between fmi and trc; between fmi and fry, which encodes an activator of Trc; and between fmi and hpo, an upstream kinase. These genetic interactions by themselves do not necessarily support the idea that Fmi acts in close proximity to Trc, Fry, or Hpo; analysis of the Fmi-Esn and Trc-Fry complexes, and verification of their cross-talk, are required at the molecular level (Matsubara, 2011).

The search for genetic interactions was targeted toward PCP genes, and several positive results were found. It should be emphasized that PCP regulators play important roles not only in epithelial sheets, but also in rearranging cell populations. In connection with the avoidance behavior of the dendrites, intriguing analogies are found in contact inhibition of locomotion of neural crest cells in Xenopus and in stereotyping a trajectory of migrating facial motor neurons in zebrafish. Vertebrate homologs of Pk, Vang, and Dsh are required for neural crest cells to initiate sustained directional migration away from the contact with another neural crest cell, although roles of Fmi homologs were not examined in this system. Importantly, the PCP regulators are localized at the site of cell contact, leading to activation of RhoA. In the context of migration of the motor neurons, homologs of Fmi and Fz act in the surrounding neuroepithelium to prevent the integration of the neurons into the neuroepithelium, thus restricting them to the correct path (see Wada, 2009). Although the mechanisms are unknown, homologs of Fmi and Fz are also proposed to act together in axonal navigation and tract formation in mouse forebrain and C. elegans nerve cord on the basis of similar mutant phenotypes (Matsubara, 2011).

It is considered likely that Fmi, Esn, Vang, and Fz are localized closely within branches and activate RhoA locally upon contact with other isoneuronal branches, redirecting those branches away from one another. However, in contrast to prominent puncta of Fmi:eGFP at most of the tips, mCherry:Esn was not necessarily enriched there and distributed more diffusely inside branches. These differential distributions do not necessarily exclude the hypothesis. As for the Trc kinase and its activator, Fry, both physical and genetic interactions are substantiated, and yet, those two molecules show distinct features of subcellular localization in da neurons (Fang, 2010). In mammals, localization of Pk1, a mammalian member of the PET-LIM domain family, is regulated by degradation that depends on the interaction of E3 ubiquitin ligases with one of the other PCP regulators, Dvl2 (Narimatsu, 2009). It could be that the contact between isoneuronal branches may trigger the Fmi-Esn interaction locally, with subsequent stabilization of the bound Esn, which is otherwise degraded. Another, mutually nonexclusive possibility would be that the association/dissociation of Esn and Fmi may be coupled to a switch of Esn signaling activity. There could be a third intriguing possibility: Esn may play a role in the trafficking of Fmi, such as promotion of endocytic recycling of Fmi. It has been proposed that Fmi is subject to high rates of endocytic turnover in the pupal wing (Strutt. 2008; Strutt, 2011), and a cytoplasmic motif of a mouse homolog of Fmi, Celsr1, governs its internalization (Devenport, 2011; Matsubara, 2011 and references therein).

If the PCP proteins are localized at contact sites at least transiently, then how do they assemble on both sides of the interface? The assembly of the PCP proteins at cell boundaries has been best studied in the Drosophila wing epithelia, where the arrangement of the proteins is asymmetrical across proximodistal (P/D) cell boundaries: Vang and Pk at the proximal cell cortex, Fz and Dsh at the distal cortex, and Fmi at both. In the dendritic arbor of a single da neuron, would it be possible that some terminal branches are Vang-rich 'proximal' types, whereas others are Fz-rich 'distal' ones? Alternatively, a single terminal branch or even a single tip could be a mosaic of Vang-rich and Fz-rich membrane domains. Coexpression of Fz and Vang with distinct tags in the same neuron and tracking pairs of terminal branches that are approaching each other at high-enough resolution may answer this question (Matsubara, 2011).


Northern blot analysis shows that the large STAN mRNA is present in a number of developmental stages. It is most abundant in 6-9 hour embryos and more abundant in pupae than larvae. In situ hybridization was used to examine the expression of stan in pupal wings. STAN mRNA is present at relatively even levels in all regions of the pupal wing. This is consistent with the genetic experiments showing that stan mutations have a phenotype over most if not all of the wing (Chae, 1999).

Hairs in adult wings are derived from prehairs that emerge 30-36 hr after puparium formation (hr APF) at 25°C. To gain insight into where Stan functions, researchers stained wing epithelia for Stan at various pupal stages and found dynamic transitions in Stan's subcellular distribution. In 18 hr APF wings, immunofluorescence signals of Stan are present almost entirely at cell-to-cell boundaries. However, at 24 hr APF, Stan becomes redistributed; its localization appears to be biased toward the proximal/distal (P/D) cell boundaries in many cells. At 30 hr APF, the polarized pattern becomes most prominent; signals run zigzag, orthogonal to the P/D axis, indicating that Stan molecules are predominantly localized at the P/D boundaries rather than at the anterior/posterior boundaries (A/P boundaries). In every typical hexagonal cell aligned parallel to the P/D axis, the emergence of a prehair at its distal cell vertex, where two Stan-rich distal boundaries meet, could easily be confirmed. The zigzag pattern is seen throughout the dorsal and ventral surfaces of the wing. This biased localization of Stan provides a striking contrast to the honeycomb distribution of an epithelial classic-type cadherin, Shotgun. Along the apicobasal cell axis at the cell-cell junction, Stan is present apically and its distribution is at least partially overlapped with that of Shotgun concentrated at the adherens junction. Once prehairs have emerged and initiated outgrowth, the distribution starts to be depolarized, although a temporal coordination between the extent of prehair length and that of depolarization of Stan is not strictly fixed in this transition phase (30-36 hr APF). The staining pattern becomes almost nonpolar by 36 hr APF, when prehairs are shifting toward cell centers (Usui, 1999).

Strong Stan signals at P/D boundaries may be explained by a concentration of Stan at either the proximal or distal edge of each cell; alternatively, the protein could be abundant at both edges. To distinguish between these possibilities, mutant clones were generated using an almost protein-null allele, fmiE59, and an examination was made of whether Stan is localized at interfaces between stan+ cells (fmiE59/+ or +/+) and mutant cells (fmiE59/fmiE59) along clone borders. Along the borders of more than 50 clones observed, Stan was missing at all interfaces between stan+ and stan- cells, no matter where the interface was positioned. Assuming that Stan molecules bind to each other in a homophilic fashion in vivo as in vitro, the most plausible interpretation of this mosaic analysis would be that both of the two apposed cells need to produce Stan to localize this receptor at the interface and that retention of Stan at P/D boundaries is achieved through homophilic interaction between its ectodomains. If this interpretation is correct, it follows that Stan is present at both proximal and distal edges in normal epithelial cells (Usui, 1999).

The Drosophila wing provides an appropriate model system for studying genetic programming of planar cell polarity (PCP). Each wing cell respects the proximodistal (PD) axis; i.e., it localizes an assembly of actin bundles to its distalmost vertex and produces a single prehair. This PD polarization requires the redistribution of Flamingo (Fmi), a seven-pass transmembrane cadherin, to proximal/distal cell boundaries; otherwise, the cell mislocalizes the prehair. Achievement of the biased Fmi pattern depends on two upstream components in the PCP signaling pathway: Frizzled (Fz), a receptor for a hypothetical polarity signal, and an intracellular protein, Dishevelled (Dsh). In this study, endogenous Dsh was visualized in the developing wing. A portion of Dsh colocalizes with Fmi, and the distributions of both proteins are interdependent. Furthermore, Fz controls the association of Dsh with cell boundaries: this association is correlated with the presence of hyperphosphorylated forms of Dsh. These results, together with studies on Fz distribution, support the possibility that Fz, Dsh, and Fmi constitute a signaling complex and that the restricted localization of this complex directs cytoskeletal reorganization only at the distal cell edge (Shimada, 2001).

Dishevelled was visualized in the developing wing by using specific antibodies. Some of the Dsh molecules are present at cell-to-cell boundaries in third-instar wing discs and in wings 2 hr after puparium formation (hr APF). By 18 hr APF, a larger fraction of Dsh molecules appear to be associated with cell boundaries, and then they are redistributed preferentially at proximal/distal (P/D), but not anterior/posterior (A/P), boundaries. This asymmetrical pattern is detectable at 24 hr APF and is most prominent at 30 hr APF, just prior to the onset of prehair outgrowth. This conversion produces a zigzag pattern on the epidermal plane: this pattern is highly reminiscent of the distribution of Flamingo (Fmi), a seven-pass transmembrane cadherin. In fact, Dsh and Fmi appeared to colocalize until 30 hr APF, and in terms of the time course, generation of the asymmetrical pattern of Dsh is indistinguishable from that of Fmi. Dsh, like Fmi, was present apically along the apicobasal axis at cell boundaries; curiously, Dsh distribution appears to be more restricted than that of Fmi. Besides Dsh molecules at cell boundaries, diffuse or punctate signals are also seen in the cytoplasm. Once prehairs emerge and initiate outgrowth at around 32–34 hr APF, the Fmi pattern starts to become depolarized and becomes almost nonpolar by 36 hr APF. In contrast, the asymmetrical Dsh distribution appears to persist, and patchy signals are found at distal cell vertexes and in outgrowing prehairs (Shimada, 2001).

Fmi localization has been studied under various genetic conditions of fz that alter polarity. Dsh was examined under the same genetic conditions and it was found that Dsh, like Fmi, is redistributed to cell boundaries where there is an imbalance of Fz activity. One example was seen along borders of clones of cells homozygous for fzR52, a strong fz allele. Another example was shown in an experiment of graded fz expression. A fz gradient expression along the anterior-posterior axis of the wing reorients hairs from high to low levels of fz expression. In these wings, ectopic Dsh and Fmi accumulation at A/P cell boundaries, instead of P/D ones, prefigures prehair outgrowth in the anterior or posterior direction. A tight coupling of Dsh and Fmi mislocalization with altered polarity is also seen in areas distal to fzR52 clones. All of these results are consistent with the ideas that (1) an imbalance of Fz activity at boundaries is necessary and sufficient to localize Dsh and Fmi there, and (2) in the wild type, distributions of the two proteins at the P/D boundaries direct the cell to choose the distal edge for prehair development. Nevertheless, to rigorously demonstrate that the distributions of Dsh and Fmi play instructive roles in polarizing cells, one would need to design each molecule to mislocalize at A/P boundaries and then investigate how cells are reoriented (Shimada, 2001).

To investigate how colocalization of Dsh and Fmi at P/D boundaries is controlled, the subcellular distribution of one of the two was studied in the absence of the other. Fmi distribution is nonpolar in dsh1, and the requirement of Dsh for making the Fmi pattern asymmetric was confirmed by staining dsh1 or dshV26 clones for Fmi. Fmi is uniformly present at boundaries within these clones. Curiously, the intensity of Fmi signals seems to increase in the dsh mutant cells, and this increase implies the possibility that Dsh may be involved in the destabilization of Fmi. Inside the clone and at the clone border, Fmi remains at apical cell boundaries whether Dsh is colocalized or not. This is perhaps due to the intrinsic nature of Fmi as a transmembrane molecule. In contrast, the boundary localization of Dsh is dependent on Fmi, as demonstrated by the staining of fmi mutant clones for Dsh. Fmi is missing at cell boundaries inside the clones and at clone borders, and Dsh is hardly detected at those boundaries. At boundaries outside the clones, Dsh always coexists with Fmi. These results support a reciprocal dependence between Dsh and Fmi for subcellular localization until prehair formation and may imply a complex formation. Under the experimental immunoprecipitation conditions, however, evidence could not be provided for a physical association between Dsh and Fmi (Shimada, 2001).

Although it has been shown that Dsh is phosphorylated in response to Wingless in a cell culture system and in the embryo, posttranslational modification of Dsh has not been studied in the context of PCP signaling. Western blot analysis shows that a fraction of Dsh molecules in pupae is hyperphosphorylated and that those forms are hardly detectable in fz or fmi mutants. Thus, the absence of the hyperphosphorylated forms correlates with the loss of Dsh at cell boundaries and suggests that the hyperphosphorylation is either required for, or is a downstream readout of, the cell boundary localization. Besides reduction in the level of the hyperphosphorylated forms, the overall amount of Dsh also appears to decrease in fz or fmi mutants; an exception may be the most quickly migrating band, which most likely represents an unphosphorylated or poorly phosphorylated form (Shimada, 2001).

Where is Fz localized within the cell? It has been shown recently that the ubiquitous expression of Fz-GFP rescues a fz polarity defect and that Fz-GFP colocalizes with Fmi: these findings strongly suggest that endogenous Fz assembles with Dsh and Fmi at the P/D boundary. Furthermore, Fz-GFP distribution is regulated by Dsh and Fmi. Therefore, in the sense of subcellular localization, there seems to exist an interdependence between any two of Fz, Dsh, and Fmi. This triangular relationship can be summarized as follows: (1) in the absence of Fz or Fmi function, the intracellular protein Dsh cannot be attached to cell boundaries; (2) without Dsh function, boundary distributions of Fz and Fmi no longer become asymmetric along the P/D axis; and (3) Fz localization at cell boundaries is abolished by a loss of Fmi (Shimada, 2001).

Other data strongly suggest bilateral distribution of Fmi at the P/D boundary, and it is pointed out that this bilateral pattern per se does not explain how the distal cell vertex, not the proximal one, is selected for prehair formation. Importantly, Fz-GFP localization is unipolar; i.e., it is present only at the distal boundary. A distal concentration of Dsh molecules could be likely because Dsh positively relays Fz signaling, although the possibility of the bilateral localization still exists. It had been expected that one could answer this question by tracing Dsh signals in wild-type cells contacting proximal and distal borders of dsh mutant clones; what underlies this approach is the fact that dsh controls PCP in a cell-autonomous fashion. Given that Dsh is localized only at the distal cell edge and that dsh mutant cells do not affect Dsh localization in wild-type neighbors, Dsh signals could have been detected at interfaces between wild-type and mutant cells only along proximal borders of the clones. Unexpectedly, Dsh was not always localized at those cell boundaries along proximal borders; furthermore, 50% of wild-type cells in direct contact with proximal borders mispositioned Dsh at anterior/posterior boundaries. Therefore, dsh mutations appear to exert a one-cell nonautonomous effect on Dsh distribution, and this did not allow a conclusion about unipolar versus bilateral localization of Dsh. This one-cell nonautonomy could be due to misplaced Fz and Fmi molecules in dsh mutant cells, which might send an illegitimate message to wild-type neighbors. In any case, the local assembly of a tripartite signaling complex of Fz, Dsh, and Fmi in the cell most likely amplifies Fz signaling only at the distal cell vertex and induces cytoskeletal reorganization (Shimada, 2001).

It should be noted that in larval wing discs and early pupal wings, Dsh distribution is not asymmetric along the presumptive PD axis; nonetheless, it is associated with cell boundaries, and this association is dependent on Fz. Functional relevance of the cell boundary localization of Dsh has been suggested in vertebrate embryos, in which Dsh controls cell polarization in convergent extension movements. A fusion protein of a Xenopus Dsh homolog and GFP (Xdsh-GFP) is associated with boundaries in cells undergoing morphogenetic movement, but this protein remains in the cytoplasm of cells that are not undergoing such movement. It would be interesting to examine whether the boundary association of Xdsh-GFP or endogenous Xdsh, as well as distributions of Xenopus homologs of Fz and Fmi, are biased toward the direction of the cell movement (Shimada, 2001).

Drosophila epithelia acquire a planar cell polarity (PCP) orthogonal to their apical-basal axes. Frizzled (Fz) is the receptor for the PCP signal, and Dishevelled (Dsh) transduces the signal. Unipolar relocalization of Dsh to the membrane is required to mediate PCP, but not Wingless (Wg) signaling. Dsh membrane localization reflects the activation of Fz/PCP signaling, revealing that the initially symmetric signal evolves to one that displays unipolar asymmetry, specifying the cells' ultimate polarity. This transition from symmetric to asymmetric Dsh localization requires Dsh function, and reflects an amplification process that generates a steep intracellular activity gradient necessary to determine PCP (Axelrod, 2001).

To investigate a possible role for Dsh membrane association during Fz/PCP signaling in vivo, Dsh subcellular localization during PCP signaling was examined in the developing wing. Transgenes were produced that express a Dsh::green fluorescent protein (GFP) C-terminal fusion, driven by native dsh regulatory sequences. One or two copies of these transgenes rescue dshv26 null mutants to viability and produce wild-type PCP, indicating that they fully replace the function of endogenous Dsh in both Wg and PCP signaling (Axelrod, 2001).

The pattern of Dsh localization observed in pupal wings is reminiscent of that for Fmi, a seven-pass transmembrane cadherin required for PCP signaling. By 30 h apf, both are seen at the proximal-distal boundaries, though Dsh is strictly distal, whereas Fmi was proposed to be at both proximal and distal edges. Double labeling for Fmi and Dsh reveals significant colocalization in 30-h apf pupal wings, each demonstrating a zigzag pattern. However, at later times, the Dsh asymmetry persists, whereas the Fmi asymmetry decays. Transverse sections taken in wing-edge cells indicate that both are located at the most apical region of cell-cell contact, and that low levels of Dsh are also seen throughout the cytoplasm. Fmi localization depends on both Fz and Dsh, but not on Multiple wing hairs (Mwh), suggesting that Fmi functions downstream of Dsh. To study this relationship further, Dsh localization was examined in fmi mutant wings. At 30 h apf, little Dsh is associated with the membrane in fmi mutant wings. This reveals a reciprocal dependence between Dsh and Fmi for persistent membrane association, and suggests that Fmi does not simply function downstream of Dsh (Axelrod, 2001).

Fz and Fmi colocalize at proximal-distal boundaries at 30 h apf. Furthermore, the asymmetric pattern of Fz localization depends on Fmi, whereas the asymmetric pattern of Fmi localization depends on Fz. Taken together, these data are consistent with the possibility that Fz, Dsh, and Fmi function together, perhaps in a complex, during PCP signaling, with both Fz and Dsh localizing to the distal edge, and Fmi apparently localizing to both the proximal and distal edges of the cell. A mutual dependence for asymmetric localization exists between these three proteins (Axelrod, 2001).

Asymmetric localization of Frizzled and the determination of Notch-dependent cell fate in the Drosophila eye

During patterning of the Drosophila eye, the Notch-mediated cell fate decision is a critical step that determines the identities of the R3/R4 photoreceptor pair in each ommatidium. Depending on the decision taken, the ommatidium adopts either the dorsal or ventral chiral form. This decision is directed by the activity of the planar polarity genes, and, in particular, higher activity of the receptor Frizzled confers R3 fate. Evidence is presented that Frizzled does not modulate Notch activity via Rho GTPases and a JNK cascade as previously proposed. The planar polarity proteins Frizzled, Dishevelled, Flamingo, and Strabismus adopt asymmetric protein localizations in the developing photoreceptors. These protein localizations correlate with the bias of Notch activity between R3/R4, suggesting that they are necessary to modulate Notch activity between these cells. Additional data support a mechanism for regulation of Notch activity that could involve direct interactions between Dishevelled and Notch at the cell cortex. In the light of these findings, it is concluded that Rho GTPases/JNK cascades are not major effectors of planar polarity in the Drosophila eye. A new model is proposed for the control of R3/R4 photoreceptor fate by Frizzled, whereby asymmetric protein localization is likely to be a critical step in modulation of Notch activity. This modulation may occur via direct interactions between Notch and Dishevelled (Strutt, 2002).

The polarity gene stbm is required for R4 fate: whether Stbm protein also shows an asymmetrical localization in R3/R4 was investigated using a Stbm-YFP transgene. Stbm-YFP is apically localized in cells posterior to the furrow, and, subsequently, its distribution is similar but distinct from that exhibited by Fz-GFP. In row 4, a symmetric pattern is observed, with Stbm-YFP around R3/R4, except where these cells contact R2/R5, and enriched on the posterior face of R8. This symmetric pattern is maintained until the ommatidia are already rotated in row 6 and more posteriorly. Staining then fades around R3, except where R3 contacts R4. Mosaic analysis revealed that, in contrast to Fz-GFP, Stbm-YFP is enriched on the R4 side of the R3/R4 boundary from row 4 onward, i.e., Stbm is on the opposite side of the boundary with Fz (Strutt, 2002).

Therefore Fz, Dsh, Fmi, and Stbm localize to the apical region of the R3/R4 cell boundary, where they become asymmetrically distributed prior to or concomitant with R3/R4 fate determination. Normally, Fz/Dsh are enriched on one particular side of the cell boundary, in the presumptive R3 cell. However, in mosaic ommatidia where one or other cell is mutant for polarity genes, the assembly of the asymmetrical complexes can be reversed. In all conditions examined, the polarity of Notch signaling between R3/R4 is consistent with the polarity of the asymmetric complexes, with Notch activity being lowest in the cell where Fz/Dsh accumulate. Finally, evidence is provided that the domain of N, which is known to interact directly with Dsh, is required for efficient R3/R4 fate decisions (Strutt, 2002).

The adoption of the asymmetric pattern occurs in two phases. The first involves symmetric apicolateral localization of Fz, Dsh, Fmi, and Stbm in R3/R4 (and in all other cells except R2/R5); this is evident in ommatidial row 4. As in the wing, the initial apical recruitment of Fz is dependent on Fmi, and the recruitment of Dsh is in turn dependent on Fz. Subsequently, the distribution evolves rapidly into an asymmetric pattern. Adoption of asymmetry requires the function of dsh, stbm, and the LIM domain protein Prickle-Spiny-legs (Pk-Sple), and if any of these are missing, Fz distribution remains symmetric in ommatidial rows 5/6, and ommatidial rotation is delayed. It is likely that the asymmetry evolves through the same mechanisms as in the wing, where it has been proposed that an extrinsic signal leads to a small bias in Fz/Dsh signaling on either side of the cell boundary, which subsequently becomes amplified by feedback loops that lead to Fz/Dsh becoming concentrated on one side of the interface and Pk-Sple/Stbm on the other (Strutt, 2002).

One notable difference between the eye and the wing is that asymmetric Fz/Dsh distribution is eventually observed in stbm and pk-sple eye discs, but in both cases it occurs with a random bias and is delayed by about one to two ommatidial rows. This correlates well with the fact that the adult phenotypes of stbm and pk-sple exhibit a low incidence of achiral ommatidia. Conversely, in fmi, fz and dsh, negligible asymmetric protein localization occurs, and there is a relatively high proportion of 'achiral' ommatidia in the adult eye, suggesting that achirality is a result of poor asymmetric complex formation. In general, the aquisition of asymmetry also correlates with mDelta0.5 activity, particularly in pk-sple and sple mutations where its expression usually resolves into a single cell by row 10 (Strutt, 2002).

In the pupal wing, asymmetric localization of Fz/Dsh/Pk-Sple is proposed to involve a signaling feedback loop that amplifies an initially small bias in Fz/Dsh activity across the axis of each cell. In the eye, the N/Dl feedback loop was proposed to perform a similar function, amplifying an initially small difference in Fz/Dsh activity between R3/R4. With the observation that Fz/Dsh are also distributed in asymmetric complexes in the eye, it appears that both mechanisms are operating in R3/R4, although it is not clear why both would be required, since either alone should be sufficient to amplify small biases in signaling activity (Strutt, 2002).

One possible explanation is that use of both mechanisms increases the speed and robustness of the R3/R4 fate decision. A fast fate decision may be necessary because of the dynamic nature of eye patterning, in which the R3/R4 decision is only part of a complex series of events involving cell recruitment and movement to generate the final polarized ommatidium. It is also possible that a rapid decision is required because the extrinsic polarity cue is transient in nature. It is noted that the rapidity of the decision would be further enhanced if N/Dl signaling also influenced Fz/Dsh localization. While there is no direct evidence for this, it could explain the eventual, randomly polarized, asymmetric protein localization seen in stbm and pk-sple backgrounds in the eye. In this case, the inability of Fz/Dsh to efficiently localize asymmetrically in the absence of Stbm/Pk-Sple might lead to N/Dl making a stochastic decision that then leads to Fz/Dsh asymmetry. Conversely, in the pupal wing, where N/Dl are not active in planar polarity decisions, Stbm/Pk-sple activity would be absolutely required, since their absence would not be compensated for by the N/Dl feedback loop (Strutt, 2002).

Egfr signaling regulates ommatidial rotation and cell motility in the Drosophila eye via MAPK/Pnt signaling and the Ras effector Canoe/AF6

Egfr signaling is evolutionarily conserved and controls a variety of different cellular processes. In Drosophila these include proliferation, patterning, cell-fate determination, migration and survival. Evidence is provided for a new role of Egfr signaling in controlling ommatidial rotation during planar cell polarity (PCP) establishment in the Drosophila eye. Although the signaling pathways involved in PCP establishment and photoreceptor cell-type specification are beginning to be unraveled, very little is known about the associated 90° rotation process. One of the few rotation-specific mutations known is roulette (rlt) in which ommatidia rotate to a random degree, often more than 90°. rlt is shown to be a rotation-specific allele of the inhibitory Egfr ligand Argos; modulation of Egfr activity shows defects in ommatidial rotation. The data indicate that, beside the Raf/MAPK cascade, the Ras effector Canoe/AF6 acts downstream of Egfr/Ras and provides a link from Egfr to cytoskeletal elements in this developmentally regulated cell motility process. Evidence is provided for an involvement of cadherins and non-muscle myosin II as downstream components controlling rotation. In particular, the involvement of the cadherin Flamingo, a PCP gene, downstream of Egfr signaling provides the first link between PCP establishment and the Egfr pathway (Gaengel, 2003).

To specifically test the involvement of cytoskeletal elements and adhesion as well as junctional components, candidate genes were tested for dominant interaction of the mild Star rotation phenotype. These genetic data argue for an involvement of E-Cadherin/shotgun, the atypical cadherin Flamingo (Fmi), the adherens junction protein canoe, non-muscle myosin II (zipper), the septin peanut, and capulet, a protein with actin and adenylate cyclase-binding ability (Gaengel, 2003).

Next, the expression of Fmi and Shotgun in ommatidial preclusters was examined during rotation. Strong LOF alleles of Egfr and its signaling components also affect cell proliferation, fate specification and survival, making the analysis of cell adhesion and junctional components in the context of rotation rather difficult. Thus localization of the cadherins and Arm/ß-catenin was examined in imaginal discs of the rotation-specific aosrlt allele (Gaengel, 2003).

Although the overall expression and localization of Shotgun and Arm/ß-catenin are largely unaffected, the localization of Fmi is changed in aosrlt discs. In WT, Fmi is initially present apically in all cells of the morphogenetic furrow and subsequently becomes asymmetrically enriched in the R3/R4 precursor pair. In and posterior to column 6, Fmi is expressed at the membrane of R4, and largely depleted from R3 membranes that do not touch R4, forming a horseshoe-like R4-specific pattern. In contrast, in aosrlt discs, Fmi restriction to the R4 precursor is generally delayed, and often not established even in columns 8-12, where high levels of Fmi are still seen around the apical membrane cortex of R3 and R4. Since Fmi is thought to act as a homophyllic cell-adhesion molecule, its increased presence on R3 membranes should have a direct effect on Fmi localization in neighboring cells and thus possibly the adhesive properties of the precluster. It is worth noting that although Fmi is required during PCP establishment and R3/R4 cell-fate specification, the delay in Fmi restriction to R4 has no significant effect on the R3/R4 cell-fate decision. Although Fmi interacts with Fz and Notch in this context, the R4-specific mDelta-lacZ marker does not differ significantly from WT and adult aosrlt eyes also display no defects in R3/R4 specification. Thus, it appears that the delay in Fmi localization specifically affects ommatidial rotation, probably through adhesion, and possibly explains the broad range of rotation angles in aosrlt and other Egfr pathway mutants (Gaengel, 2003).

Thus Egfr/Ras signaling plays a general role in the regulation of ommatidial rotation. Canoe has been identified as an effector of Ras in this context. Although much is known about how ommatidial chirality and the associated R3/R4 cell-fate decision are regulated (Fz/PCP-Notch signaling), no clear link between the mechanistic aspects of ommatidial rotation and Fz/PCP signaling previously existed. This is the first link to be demonstrated between Egfr signaling and PCP genes, namely Fmi. A further connection between Egfr signaling and PCP establishment is provided by Zipper, which acts downstream of Fz/Dsh and Rok in wing PCP and modifies the Star rotation phenotype. The identification of the Egfr pathway and its regulation of Fmi/cadherin-mediated cell adhesion will serve as an important entry point to further such studies (Gaengel, 2003).

Cell interactions and planar polarity in the abdominal epidermis of Drosophila

The integument of the Drosophila adult abdomen bears oriented hairs and bristles that indicate the planar polarity of the epidermal cells. Four polarity genes, frizzled (fz), prickle (pk), Van gogh/strabismus (Vang/stbm) and starry night/flamingo (stan/fmi) were examined in this study, and what happens when these genes are either removed or overexpressed in clones of cells was examined. The edges of the clones are interfaces between cells that carry different amounts of gene products, interfaces that can cause reversals of planar polarity in the clone and wild-type cells outside them. To explain, a model is presented that builds on an earlier picture of a gradient of X, the vector of which specifies planar polarity and depends on two cadherin proteins, Dachsous and Fat. It is conjectured that the X gradient is read out, cell by cell, as a scalar value of Fz activity, and that Pk acts in this process, possibly to determine the sign of the Fz activity gradient (Lawrence, 2004).

Evidence is discussed that cells compare their scalar readout of the level of X with that of their neighbors and set their own readout toward an average of these. This averaging, when it occurs near the edges of clones, changes the scalar response of cells inside and outside the clones, leading to new vectors that change polarity. The results argue that Stan must be present in both cells being compared and acts as a conduit between them for the transfer of information, and that Vang assists in the receipt of this information. The comparison between neighbors is crucial, because it gives the vector that orients hairs: these hairs point toward the neighbor cell that has the lowest level of Fz activity (Lawrence, 2004).

Recently, it has been shown that, for a limited period shortly before hair outgrowth in the wing, the four proteins studied, as well as others, become asymmetrically localised in the cell membrane, and this process is thought to be instrumental in the acquisition of cell polarity. However, some results do not fit with this view -- it is suggested that these localisations may be more a consequence than a cause of planar polarity (Lawrence, 2004).

There are a number of simple systems in which isolated cells orient to a polarising signal. These include the localized outgrowth, or 'schmooing' of yeast in response to mating pheromone and directed migration of Dictyostelium cells up a gradient of cyclic AMP. Small differences (as little as 1%-5%) in receptor activation across single cells are sufficient to polarise them, a response that, in yeast and elsewhere probably depends on localised exocytosis. It is not known whether the polarisation of single, isolated cells is a model for planar polarity of cells in an epithelium, but it is likely that they share at least some of the mechanisms (Lawrence, 2004).

It has been proposed that, in the abdomen of Drosophila, morphogen gradients (Hh in the A compartment and Wg in the P compartment) organise a secondary gradient ('X'); the vector of X specifying the polarity of each cell. Although the composition of X is unknown, at least three proteins, Fj, Ds and Ft, are implicated. All three may be expressed, or be active, in bell-shaped distributions that peak near the A/P (Ds) or P/A (Fj, Ft) boundaries. Ds and Ft are transmembrane proteins in the cadherin superfamily; Fj probably acts in the Golgi. Ds and Ft are integrated into the membrane, suggesting that the X gradient itself may not be diffusible but instead might depend on information transfer from cell to cell (Lawrence, 2004).

How does Hh set up the X gradient? Although changing the real or perceived level of Hh does affect polarity, many clones (for example clones that lack Smo, an essential component of Hh reception) show there is no simple correlation between Hh concentration and polarity. For instance, large smo- clones in the center of the A compartment are polarised normally, even though they are blind to Hh. Also, while smo- clones in some regions of the A compartment do affect polarity, both mutant and wild-type cells are repolarised. Both these observations argue for some transfer of information about polarity between cells, a process that would be at least partly Hh independent. This paper explores this process and is concerned with four genes (stan, fz, Vang and pk) that probably act downstream of ds, ft and fj (Lawrence, 2004).

Perhaps normal cells could transfer information from one to another (this might be particularly important for nascent cells following mitosis) to help keep the readout of X as a smooth gradient? To do this they might make a comparison of their neighbors and modify this readout of X toward an average of those neighbors. X might be read by a receptor molecule and the results point to Fz being the most likely candidate. The results indicate that the comparison itself requires the cadherin Stan. Thus, a cell would need to read and compare (using Stan) the levels of X (recorded in the activity of Fz) in neighboring cells. Then, in a way analogous to how a Dictyostelium amoeba reads the vector of a cAMP gradient, a cell would determine its polarity from the vector of Fz activity. The results suggest that Vang also acts in this step, helping cells to sense the level of Fz activity in neighboring cells (Lawrence, 2004).

Some of the results are discussed in terms of the model (Lawrence, 2004). Clones that lack, or overexpress Fz cause local and consistent repolarisations of cells that extend from within the clone and affect normal wild-type cells outside it. Because simply removing the fz gene from all cells randomizes polarity in the ventral pleura, it is self-evident that these organised polarity reversals must result from an interaction between the clone and the surrounding cells. It has been argued that Stan and Fz act in this process, but how? Note that stan and fz are the only mutants that have randomised hairs in the pleura, and the results indicate that neither Stan nor Fz can function properly without the other. Averaging might depend on the capacity of Stan to form homophilic dimers as bridges between neighboring cells, with such Stan:Stan dimers serving as a conduit for information about the relative level of Fz activity in each cell. However, with respect to non-autonomy, the results with the two genes differ:

  1. stan- cells cannot be repolarised by, and cannot repolarise, neighboring cells. This shows that Stan is essential in both neighboring cells for the transfer of information between them. Without Stan, the cells cannot compare and cannot therefore determine any vector. However, a stan+ cell, even if it is adjacent to a stan- cell, can be polarised normally; having Stan it should be able to read the levels of all neighboring cells except the stan- one and, having Fz, it should be able to set its own level (Lawrence, 2004).

  2. But, fz- cells, unlike stan- cells, can repolarise neighboring wild-type cells. Also, a fz- cell, again unlike a stan- cell, can itself be repolarised. The results also show that to be repolarised, or to polarise a neighbor, a fz- cell must be adjacent to a stan+ fz+ cell. Such a fz- cell will accumulate Stan in that membrane which abuts the stan+ neighbor so it should be able to read the level of the neighboring wild-type cell and be polarised accordingly. Consider a fz- cell at the outer edge of a fz- clone: lacking Fz, its activity level would be zero, but this level would be communicated by Stan to the neighboring cells. Wild-type cells outside would obviously have a higher level (than zero). The result would be that the two cells abutting the interface, the fz- cell inside, and a fz+ cell outside, would both make hairs that point into the center of the clone. The nextmost interior cell would not be polarised, since all its neighbors would be cells with level zero. In contrast, the next most exterior cell would be repolarised, since its scalar level would be brought down by the averaging process (Lawrence, 2004).

How far does the non-autonomy spread into wild-type cells? This process can be stimulated. According to the model this range would depend on the value of a single adjustable parameter, a that relates to how much a cell's scalar is read from X. At one extreme for this parameter (a=0), when the scalar of a cell depends only on X, a wild-type cell just posterior to a clone of fz- cells would reset its scalar as it was before; there could be no averaging and only that cell and its fz- neighbor will be repolarised. Thus the non-autonomy would be limited to one cell. At the other extreme (a=1), any local disturbance produced by a clone would decay rapidly because of averaging, and the repolarisation will tend to be lost altogether. In between these extremes, the non-autonomy spreads more than one cell, but over diverse values for this parameter, the range is near the amount usually observed (2-4 cell diameters) (Lawrence, 2004).

It has been observed that fz- clones have effects over longer range in backgrounds such as ds- where the X gradient might be flatter than normal. Similarly, cells are normally polarised in large smo- clones in the middle of the A compartment, where, because there can be no input from Hh, the X gradient could also be flat. Both these results are consistent with the model, because the range affected by averaging will increase (Lawrence, 2004).

Many of the proteins required for normal cell polarity, including Fz, Dsh, Dgo, Pk, Vang and Stan are found to be asymmetrically localised in the proximodistal axis of wing cells. This localisation is restricted to a brief period of just a few hours shortly before the wing hairs grow out, but, nevertheless it is assumed to be mechanistically important to planar polarity. For example, non-autonomy could be explained if localised proteins were components of one or more molecular complexes that propagate polarity from cell to cell. In support of this, note that loss of any of these proteins, including the removal of both Pk and Sple, prevents the asymmetric localisation of the others (Lawrence, 2004).

But the results do not seem to fit with such a mechanism, mainly because they provide evidence that polarity can propagate into cells that lack, or fail to localise all of these proteins. In particular, pk- cells are normally polarised throughout the P compartment and can be repolarised in both compartments by sharp discontinuities in Fz activity even in the pleura (where polarity is randomized in fz- and stan- animals). At a minimum, these findings challenge the hypothesis that Pk itself is an essential component of a feedback amplification mechanism responsible for polarising cells. Furthermore, if it is assumed that the observed failure of Fz, Dsh, Vang and Stan to localize in pk- wing cells reflects a general property, these results also challenge the idea that Fz, Dsh, Pk, Vang, Diego and Stan must be able to accumulate asymmetrically in order for cells to detect, and be polarised by, the X gradient, or by disparities in Fz activity. Indeed, Adler (2002) has already hinted that there is no convincing evidence that the asymmetric localisation of these proteins actually functions in planar polarity: 'the preferential accumulation [of proteins] along the...edges of wing cells is a process that intuitively seems likely to be part of a core system...but perhaps it is not and if not...this would leave rather little in the core' (Lawrence, 2004).

Are wing cells polarised only briefly just prior to the hair outgrowth? The reason for raising this possibility is that the proteins are apparently only asymmetrically localised at that time. If this localisation were not causal, as it is now suggested, it could be that the cells are polarised for all or most of development -- again arguing that the ephemeral localisation of the proteins is more a consequence than a cause of polarisation (Lawrence, 2004).

spalt genes are required for proper localization of Flamingo in the equatorial side of R3 and R4

The establishment of planar cell polarity in the Drosophila eye requires correct specification of the R3/R4 pair of photoreceptor cells. In response to a polarizing factor, Frizzled signaling specifies R3 and induces Delta, which activates Notch in the neighboring cell, specifying it as R4. The spalt zinc-finger transcription factors (spalt major and spalt-related) are part of the molecular mechanisms regulating R3/R4 specification and planar cell polarity establishment. In mosaic analysis, spalt genes have been shown to be specifically required in R3 for the establishment of correct ommatidial polarity. In addition, spalt genes are required for proper localization of Flamingo in the equatorial side of R3 and R4, and for the upregulation of Delta in R3. These requirements are very similar to those of frizzled during R3/R4 specification. spalt genes are required cell-autonomously for the expression of seven-up in R3 and R4, and seven-up is downstream of spalt genes in the genetic hierarchy of R3/R4 specification. Thus, spalt and seven-up are necessary for the correct interpretation of the Frizzled-mediated polarity signal in R3. Finally, it has been shown that, posterior to row seven, seven-up represses spalt in R3/R4 in order to maintain the R3/R4 identity and to inhibit the transformation of these cells to the R7 cell fate (Domingos, 2004).

Therefore, the results suggest that sal is required upstream or in parallel to the Fz/PCP pathway for R3/R4 specification. Also, in support of this model, sal expression is not affected in R3/R4, either in gain- or loss-of-function experiments with members of the Fz/PCP and Notch signaling pathways. sal is required cell-autonomously in R4 for normal levels of mdelta0.5-lacZ expression. This requirement of sal in R4 could be due to a defect in the activation of Notch signaling [e.g. sal may be required for the expression of Notch or Su(H)]. Alternatively, sal may be required for transcriptional activation of E(spl)mdelta, in parallel to Notch signaling. The latter possibility is favored, since the expression of a transgenic line, where lacZ is under the regulation of 12Suppressor of Hairless multimerized-binding sites [12Su(H)-lacZ], is not affected when R4 is sal. The 12Su(H)-lacZ transgenic line is a reporter for Su(H)-dependent Notch signaling, and thus, sal is not required for the expression or activation of Notch, Su(H) or other components required for signaling. In addition, exogenous expression of a constitutively activated Notch (sev-Nact) can rescue mdelta0.5-lacZ expression in sal clones. Altogether, these results suggest that sal acts in parallel to Notch signaling for the transcriptional activation of E(spl)mdelta. Finally, although there is a reduction of E(spl)mdelta expression when R4 is sal, this does not correspond to chirality defects in mature ommatidia. This suggests that other genes may be redundant to sal in R4 for PCP establishment (Domingos, 2004).

In conclusion, these results demonstrate that sal is required in R3 to allow normal Fz/PCP signaling to specify the R3 and R4 cell fates. Ommatidia mutant for sal show defects that are very similar to those observed in fz and dsh mutants, as judged by the loss of asymmetric Fmi localization at the equatorial side of the R3/R4 precursors, and by the lack of Dl and E(spl)mdelta upregulation within the R3/R4 pair. In addition, sal is required upstream of svp for normal R3/R4 specification. Finally, these results show that, posterior to row seven, svp represses sal in R3/R4 in order to maintain R3/R4 identity and to inhibit transformation of these cells to the R7 cell fate (Domingos, 2004).

Polarized transport of Frizzled along the planar microtubule arrays in Drosophila wing epithelium

Cells in a variety of developmental contexts sense extracellular cues that are given locally on their surfaces, and subsequently amplify the initial signal to achieve cell polarization. Drosophila wing cells acquire planar polarity along the proximal-distal (P-D) axis, in which the amplification of the presumptive cue involves assembly of a multiprotein complex that spans distal and proximal boundaries of adjacent cells. This study pursues the mechanisms that place one of the components, Frizzled (Fz), at the distal side. Intracellular particles of GFP-tagged Fz move preferentially toward distal boundaries before Fz::GFP and other components are tightly localized at the P/D cortex. Arrays of microtubules (MTs) are approximately oriented along the P-D axis and these MTs contribute to the formation of the cortical complex. Furthermore, there appears to be a bias in the P-D MTs, with slightly more plus ends oriented distally. The hypothesis of polarized vesicular trafficking of Fz is discussed (Shimada, 2006).

It is proposed that Flamingo- and Fz::GFP-containing vesicles are transported preferentially toward the distal cell cortex along P-D-oriented MTs. It has been considered that a cassette of transmembrane proteins (Four-jointed, Dachsous, and Fat) functions upstream of Fz and confers an initial small asymmetry of Fz activity across the cell. This small imbalance is amplified through formation of the multiprotein complex across the P/D boundary, reinforces Fz signaling, and eventually leads to specification of prehair formation at the distal cell end. The polarized transport of Fz that was observed may reflect one of the outputs of the upstream cassette to set an initial bias of Fz activity and/or an intermediate step of the Fz signaling feedback amplification (Shimada, 2006).

Immuno-EM studies revealed intracellular vesicles that were associated with Fz::GFP or Flamingo (Fmi) on the AJ plane. The number of these vesicles per cell was an order of magnitude higher than that of Fz::GFP particles visualized by confocal microscopy. Only a subpopulation of the vesicles, one containing large numbers of Fz::GFP molecules, may have been detected by confocal microscopic observations. Although both the multiprotein complex of the cortical PCP signaling components and the DE-cadherin-catenin adhesion complex are located at AJ in an almost overlapping manner, this study supports the possibility that components of these two complexes were separated into distinct vesicles at their exit from the trans-Golgi network (TGN). Sorting of Fz::GFP, Fmi, and possibly Dsh as well from DE-cadherin should also take place when molecules on the plasma membrane are incorporated into recycling endosomes. Experiments using mammalian epithelial cell lines have elucidated biosynthetic and recycling pathways for sorting apical and basolateral plasma membrane proteins. Wing epidermal cells appear to have a machinery to subdivide molecules that are targeted to AJ (Shimada, 2006).

The results of double staining for Fz::GFP and Fmi indicated that the majority of Fmi-containing vesicles are transported distally, not to the proximal boundary. Then how are Fmi, Stbm/Vang, and Pk distributed to the proximal cortex (Shimada, 2006)?

Fmi, Stbm/Vang, and Pk have already been distributed rather uniformly at the cell cortex in imaginal and early pupal epithelia before the polarized transport starts, and these molecules may be allowed to diffuse laterally within the plasma membrane. Fmi, which is transported to the distal boundary together with Fz and Dsh, can lock a counterpart on the proximal membrane of the adjacent cell through its homophilic binding property. Formation of this Fmi-Fmi bridge across the P/D boundary would anchor the Fz-Dsh complex at the distal cortex, increasing local activity and the copy number of Fz-Dsh. This slight input may initiate recruitment of Stbm/Vang-Pk on the opposing proximal cortex by means of mutual exclusion between Dsh and Pk, and by means of hypothetical ectodomain interaction between Fz and Stbm/Vang. Then the nascent asymmetric complex amplifies the imbalance of Fz activity by unknown mechanisms. This amplification could be involved in either facilitating loading Fz-Dsh and Fmi into vesicles at their exit from the TGN, accelerating the transport of the vesicles to the distal cortex, and/or restricting diffusion of Fz-Dsh and Stbm/Vang-Pk out of the distal and proximal membrane domains, respectively (Shimada, 2006).

In setting up the initial bias, the polarized transport may not necessarily require Stbm and Pk, whereas the subsequent amplification presumably depends on the formation of the cortical complex. This hypothesis is consistent with the observation that bright Fz::GFP particles were hardly seen in the dsh, fmi, or pk mutant backgrounds. It also explains why FzP278L::YFP particles did not move distally, because FzP278L is postulated to lose its interaction with Dsh and most FzP278L::YFP particles did not contain Fmi (Shimada, 2006).

Besides noncentrosomal MTs along the apicobasal axis, P-D-oriented MTs were unequivocally visualized in the AJ plane, and the important role of this MT array in localizing the cortical PCP proteins and the distal-oriented movement of Fz::GFP particles was shown. Assuming the importance of the P-D MT alignment, how is the Fz::GFP particle transported or recycled back along the MT track in terms of vesicular trafficking? The particles showed staggered trajectories when followed at 1 or 10 s intervals, and this back-and-forth motion of the Fz::GFP particle can be interpreted as follows: (1) individual Fz::GFP particles bind multiple motors of different classes, and (2) activities of the motors of opposing directionality are coordinated. This bidirectional transport has been shown for organelle trafficking; despite such back-and-forth motion, cargos can still achieve net transport on longer time scales. Another, not mutually exclusive, possibility could be that the particle/cargo motor complex may repeat cycles of running, falling off the MT track, and reengaging. Whether specific members of MT motors contribute to the Fz::GFP transport or not awaits further genetic as well as biochemical characterization of the particle (Shimada, 2006).

Given that either of the above possibilities of motor-driven motions is the case, why did the Fz::GFP particle move predominantly toward distal cell boundaries when followed at 1 min intervals? Attempts were made to trace the origin of this asymmetry to the polarity of individual P-D MTs and it was found that the wing cell had slightly more + end-distal MTs than + end-proximal ones. Nevertheless, it is an open question as to whether such a small difference can be causally related to the distally oriented transport and whether the subtle difference in MT polarity can be one of the critical parameters in operating the cortical feedback loop (Shimada, 2006).

How are noncentrosomal MTs aligned approximately along the P-D axis at the level of adherens junction? P-D-oriented MTs were still observed in the absence of Fz function, and this strongly suggests that generation of the P-D MT array is controlled by a mechanism upstream of, or separate from, the cortical complex. One clue for this mechanism involving MTs may be Widerborst (Wdb), a B' regulatory subunit of PP2A. It would be necessary to investigate how exactly MTs are disorganized when Wdb function is abrogated. Another clue may be a recent report that the presence of apical MTs is related to Dpp signaling activity, although a cytoskeletal function for Dpp remains to be shown (Shimada, 2006).

A large gap remains in cell biological understanding of the global cue that is considered to be provided by the cassette of transmembrane proteins (Fj, Ds, and Ft). It should be pointed out that each of Fj, Ds, and Ft controls the ratio of proximal-distal to anterior-posterior growth of appendages and that Ds and Ft have been recently shown to control the shape of the growing organs by regulating orientations of cell divisions in imaginal discs. This supports the hypothesis that Ds and Ft can orient the mitotic spindle, and hence MT organization, during wing morphogenesis. It would be intriguing to investigate whether the MT orientation and polarity observed in this study are also under the control of the upstream cassette, and to pursue a molecular connection between tissue shape and polarity (Shimada, 2006).

Dynamics of core planar polarity protein turnover and stable assembly into discrete membrane subdomains

The core planar polarity proteins localize asymmetrically to the adherens junctions of epithelial cells, where they have been hypothesized to assemble into intercellular complexes. This study shows that the core proteins are preferentially distributed to discrete membrane subdomains ('puncta'), where they form asymmetric contacts between neighboring cells. Using an antibody internalization assay and fluorescence recovery after photobleaching in prepupal and pupal wings, the turnover of two key core proteins, Flamingo and Frizzled, was investigated, and it was found that the localization of both within puncta is highly stable. Furthermore, the transmembrane core proteins, Flamingo, Frizzled, and Strabismus, are necessary for stable localization of core proteins to junctions, whereas the cytoplasmic core proteins are required for their concentration into puncta. Thus, this study defines the distinct roles of specific core proteins in the formation of asymmetric contacts between cells, which is a key event in the generation of coordinated cellular asymmetry (Strutt, 2011).

Since the first report over a decade ago of the asymmetric subcellular localization of Fmi in the Drosophila pupal wing, the mechanisms underlying the distribution of the core polarity proteins have been extensively investigated. A growing number of models have been presented to describe how the core proteins might achieve asymmetric localization, with a common feature being the general assumption that the core proteins assemble together into a stable asymmetric intercellular complex. However, the existence of such a complex is largely inferred from the distributions of the proteins, and the actual roles of individual proteins in the formation, stabilization, and subcellular distribution of such complexes are poorly understood (Strutt, 2011).

This study shows that a fundamental organizing principle for core protein asymmetry is their distribution into discrete plasma membrane subdomains in the apicolateral junctions, which are referred to as 'puncta.' Using the independent methodologies of antibody internalization and FRAP, this study demonstrates that the populations of Fmi and Fz in puncta are highly persistent, supporting the view that the core polarity proteins do indeed form stable asymmetric complexes, and that these complexes are preferentially clustered together in puncta (Strutt, 2011).

The data lead to make several inferences about the formation of such asymmetric complexes. It was previously observed that in the absence of Stbm, an asymmetric Fz-Fmi:Fmi complex was preferentially formed between neighboring cells. The results suggest that this Fz-Fmi:Fmi complex is the primary building block for the core protein complex. In the absence of Fmi, Fz does not localize to junctions, and in the absence of Fz, Fmi is also poorly localized to junctions and subject to endocytic turnover. Importantly, loss of other core proteins (Stbm, Pk, Dsh) has less or no effect on Fmi endocytosis, and similarly does not eliminate the stable fraction of Fz, indicating that Fz and Fmi stably localize to junctions in the absence of these factors. Nevertheless, although Stbm does not preferentially form an asymmetric complex with Fmi in the absence of Fz, its ability to further stabilize Fmi at junctions in the presence of Fz indicates an important secondary role in formation of the asymmetric complex (Strutt, 2011).

Although the cytoplasmic core proteins do not appear to play any role in the formation of stable complexes, they do promote the 'clustering' of such complexes into puncta. This is consistent with previous data suggesting that the cytoplasmic factors are not required for intercellular communication but that they have an intracellular function in generating asymmetry. This absence of a requirement for the cytoplasmic factors in polarized intercellular communication, and the ability of Fz and Fmi to form asymmetric complexes in the absence of Stbm both suggest that protein complexes are already asymmetric in the absence of clustering (Strutt, 2011).

Several lines of data suggest that puncta are functionally important for generation of cellular asymmetry. First, they are the major sites of asymmetric localization of the core proteins. Second, their size, and the degree to which they contain a stable faction of Fz, varies over time and correlates with the degree of cellular asymmetry observed. Third, core polarity gene mutations that affect cellular asymmetry to different extents have a corresponding effect on the size of the stable fraction of Fz in puncta (Strutt, 2011).

The mechanism by which asymmetric complexes are clustered into puncta is unknown. The simplest model is that cytoplasmic factors act as 'glue' to hold complexes of the same orientation together and reduce their rates of lateral diffusion in the membrane. The alternative hypothesis that the cytoplasmic factors promote clustering by reducing rates of endocytic turnover is inconsistent with the observation that the overall stable fraction is not altered in the absence of cytoplasmic core protein function. The preference for clustering complexes of the same polarity may also be promoted by inhibitory interactions between proximal and distal complex components (Strutt, 2011).

A key question is how such clustering might lead to the establishment of cellular asymmetry. One possibility is a process of self-organization involving local self-enhancement and longer-range inhibition. If planar polarity represents such a self-organizing process, clustering of asymmetric complexes into puncta is likely to provide local enhancement, whereas formation of intrinsically asymmetric complexes between cells may effectively provide longer-range subcellular inhibition that prevents all the clusters within a cell having the same orientation. In support of such self-organization in the pupal wing, it is noted that induction of Fz, Fmi, or Stbm expression as late as 24 hr APF can lead to locally organized cellular polarity within a few hours that is not oriented on the PD axis and as such is unlikely to be specified by long-range patterning cues (Strutt, 2011).

Overall, a model is proposed in which molecular asymmetry is initially established by formation of Fz-Fmi:Fmi complexes that are intrinsically stable and in which Fmi endocytosis is attenuated. Entry of Stbm into the complex further promotes Fmi localization to junctions. The cytoplasmic components Dsh, Pk, and Dgo can then be recruited into the complex but do not increase its stability. Instead, they are required for clustering of asymmetric complexes of common polarity into junctional puncta, which are sites of local asymmetry. Through a self-organization process, which would normally be globally biased by an upstream patterning cue, locally organized puncta adopt an asymmetric distribution within the cell, linking the polarity of neighboring cells (Strutt, 2011).

Time-lapse experiments indicate that individual puncta are stable for several hours. Nevertheless, in the Drosophila wing, morphogenetic changes such as wing eversion, hinge contraction, and junctional remodeling necessitate some rearrangement of junctions, and this appears to be accompanied by reduced puncta size and loss of cellular asymmetry. Interestingly, although during junctional remodeling (at 20 hr APF), brighter regions are still visible in the junctions, FRAP experiments reveal that these regions are no longer enriched for the stable fraction of Fz. This suggests that the membrane subdomains in which puncta form may be persistent, but the mechanisms that promote accumulation of asymmetric complexes in puncta are not active. This may allow the remodeling of planar polarity, following morphogenetic changes (Strutt, 2011).

Transient asymmetric localization of polarity proteins is also seen in more dynamic systems, for example in vertebrate gastrulation, where their distribution is also highly punctate. It is possible that in cells that are undergoing movement and changing their contacts, local organization of polarity proteins into puncta allows more rapid reestablishment of polarized interactions between neighboring cells (Strutt, 2011).

A novel function for the Rab5 effector Rabenosyn-5 in planar cell polarity

In addition to apicobasal polarization, some epithelia also display polarity within the plane of the epithelium. To what extent polarized endocytosis plays a role in the establishment and maintenance of planar cell polarity (PCP) is at present unclear. This study investigated the role of Rabenosyn-5 (Rbsn-5), an evolutionarily conserved effector of the small GTPase Rab5, in the development of Drosophila wing epithelium. It was found that Rbsn-5 regulates endocytosis at the apical side of the wing epithelium and, surprisingly, a novel function was discovered of this protein in PCP. At early stages of pupal wing development, the PCP protein Flamingo (Fmi) redistributes between the cortex and Rab5- and Rbsn-5-positive early endosomes. During planar polarization, Rbsn-5 is recruited at the apical cell boundaries and redistributes along the proximodistal axis in an Fmi-dependent manner. At pre-hair formation, Rbsn-5 accumulates at the bottom of emerging hairs. Loss of Rbsn-5 causes intracellular accumulation of Fmi and typical PCP alterations such as defects in cell packing, in the polarized distribution of PCP proteins, and in hair orientation and formation. These results suggest that establishment of planar polarity requires the activity of Rbsn-5 in regulating both the endocytic trafficking of Fmi at the apical cell boundaries and hair morphology (Mottola, 2010).

This study uncovered a novel role of Rbsn-5 in the establishment of PCP during pupal wing development, and it was further demonstrated that the PCP protein Fmi undergoes endocytic trafficking in a process that is dependent on Rbsn-5 and required for the establishment of PCP (Mottola, 2010).

Rbsn-5 shares with its mammalian orthologue Rabenosyn-5 several structural features, as well as the function of molecular coordinator of endocytosis and recycling. First, the inhibition of fluid-phase endocytosis observed in rbsn34 cells is consistent with the impairment of early endocytic transport described for both Rabenosyn-5 and Vps45 in mammalian cells, C. elegans and Drosophila. Second, although Rbsn-5 does not bind Rab4, it interacts with EHD/RME1, a protein that is required for recycling cargo from endosomes to the surface. Third, the formation of expanded Rab5-positive endosomes in Rbsn-5 mutant cells phenocopies the endosomal enlargement observed upon inhibition of recycling. Moreover, the accumulation of Fmi in late endocytic compartments, which is also consistent with the requirement of Rbsn-5 (and the yeast orthologue Vac1p) for protein sorting to the degradative pathway, resembles the phenotype previously described for sec5E13 clones in Drosophila oocytes (Mottola, 2010).

The function of Rbsn-5 in endocytic transport is required for the re-distribution of Fmi between endosomes and the apical cell boundaries during the establishment of PCP in the Drosophila wing. Before PD asymmetry is established in the whole tissue, endogenous Fmi is detected on Rbsn-5- and Rab5-positive early endosomes. At later stages, Fmi must recycle back to the plasma membrane because it subsequently localizes to the apical cell boundaries concomitantly with Rbsn-5. Recycling from endosomes to the cell surface is also consistent with the dependence on Fmi for the recruitment of the exocyst subunit Sec5 at the apical cell boundaries. Loss of Rbsn-5 causes intracellular accumulation of Fmi, which correlates with defects in PD polarity and hair orientation. Consistently, Rab5 overexpression, which influences Rbsn-5 redistribution, also alters Fmi trafficking and causes PCP defects. These data therefore indicate that Rbsn-5-dependent trafficking of Fmi is relevant for the establishment of PCP. Clearly, the data do not exclude the possibility that other (e.g. biosynthetic) trafficking events of Fmi might contribute to this process (Mottola, 2010).

Why is Fmi endocytosed and recycled during establishment of PCP in the pupal wing? It has been recently proposed that a combination of polarized secretion, Fmi endocytosis and stabilization of Fmi and Fz to the distal apical cell boundaries might underlie the establishment of cellular asymmetry. In line with this proposal, Rbsn-5-dependent trafficking might be required to remove unstable Fmi (associating only to Fz) from the apical cell boundaries and relocate it in regions of the plasma membrane where it can be stabilized in proximal PCP complexes. Therefore, the weak proximal non-autonomy in trichome orientation observed for rbsn34 clones, which resembles the phenotype of fmi clones rescued with a GFP-tagged Fmi mutant lacking the cytoplasmic domain (Fmiδintra-EGFP), might be explained with the blockade of Fmi at the plasma membrane preferentially bound to Fz:Dsh complexes (Mottola, 2010).

Some defects observed for rbsn34 clones, such as Fmi redistribution as swirls and proximal perturbation of trichome polarity inside mutant clones, together with weak proximal non-autonomy are also reminiscent of defects in Fat and Ds mutant clones. Interestingly, it was observed that big rbsn34 clones could be found only on the distal side of the pupal wing. This might reflect a less important requirement for Rbsn-5 on this side compared with the proximal one. However, whether Rbsn-5 is also involved in the global propagation of PCP signalling via the upstream module Ds-Fat-Fj remains to be determined (Mottola, 2010).

Additionally, Rbsn-5 mutant clones show defects in hair formation and elongation. As endocytosis and actin cytoskeleton remodelling are functionally connected, these defects might be indirect consequences of endocytosis impairment. However, the specific accumulation of Rbsn-5 at the bottom of emerging hairs would be consistent with the idea that Rbsn-5 mediated endocytic/recycling trafficking might actively contribute to outgrowth of wing hairs, possibly by regulating specific membrane delivery (Mottola, 2010).

While preparing this manuscript, a study on the regulation of membrane protein localization by PI3K (III) and Rabenosyn-5 in Drosophila wing cells reported (Abe, 2009) that loss-of-function mutation of Rbsn-5 does not affect Fmi localization and hair formation and orientation. The discrepancy with the current data could be explained by the fact that, in that study, the analysis was conducted at 25°C instead of 18°C. Indeed, it was noticed that rbsn34 clones are less healthy and tend to be smaller when grown at higher temperatures. Under these conditions, the intracellular accumulation of Fmi might well be less noticeable and the rate of lysosomal degradation may be higher (Mottola, 2010).

In conclusion, the characterization of Rbsn-5 during Drosophila wing development allowed discovery of a novel function for this Rab5 effector in vivo in a developmental context and provided evidence in favor of a role of the apical endocytic trafficking of Fmi in the establishment of PCP. Future studies will hopefully provide additional molecular links and mechanistic insights into the functional interplay between the endocytic and the PCP machineries (Mottola, 2010).

Establishment of global patterns of planar polarity during growth of the Drosophila wing epithelium

Epithelial tissues develop planar polarity that is reflected in the global alignment of hairs and cilia with respect to the tissue axes. The planar cell polarity (PCP) proteins form asymmetric and polarized domains across epithelial junctions that are aligned locally between cells and orient these external structures. Although feedback mechanisms can polarize PCP proteins intracellularly and locally align polarity between cells, how global PCP patterns are specified is not understood. It has been proposed that the graded distribution of a biasing factor could guide long-range PCP. However, epithelial morphogenesis has been identified as a mechanism that can reorganize global PCP patterns; in the Drosophila pupal wing, oriented cell divisions and rearrangements reorient PCP from a margin-oriented pattern to one that points distally. This study used quantitative image analysis to study how PCP patterns first emerge in the wing. PCP appears during larval growth and is spatially oriented through the activities of three organizer regions that control disc growth and patterning. Flattening morphogen gradients emanating from these regions does not reduce intracellular polarity but distorts growth and alters specific features of the PCP pattern. Thus, PCP may be guided by morphogenesis rather than morphogen gradients (Sagner, 2012).

To study the emergence of polarity in the wing disc, the subcellular distribution of the PCP proteins Flamingo (Fmi) and Prickle (Pk) were quantified. Planar cell polarity (PCP) nematics were calculated based on Fmi staining and PCP vectors based on the perimeter intensity of EGFP::Pk clones. At 72 hr after egg laying (hAEL), the wing pouch has just been specified and is small. EGFP::Pk localizes to punctate structures at the cell cortex that are asymmetrically distributed in some cells, but PCP vectors exhibit no long-range alignment. By 96 hAEL, PCP vector magnitude increases and a global pattern emerges. Later, PCP vector magnitude increases further and the same global polarity pattern is clearly apparent. It is oriented with respect to three signaling centers: the dorsal-ventral (DV) boundary (where Wingless [Wg] and Notch signaling occur), the anterior-posterior (AP) compartment boundary (where Hedgehog [Hh] and Decapentaplegic [Dpp] signaling occur), and with respect to the hinge fold (where levels of the atypical Cadherin Dachsous [Ds] change sharply) (Sagner, 2012).

PCP vectors in the wing pouch near the hinge fold point away from it toward the center of the pouch. Within the Wg expression domain at the DV boundary, PCP vectors parallel the DV boundary and point toward the AP boundary. Just outside this domain, PCP nematics and vectors turn sharply to point toward the DV boundary in central regions of the wing pouch. However, where the DV boundary intersects the hinge-pouch interface, they remain parallel to the DV boundary over larger distances such that PCP vectors orient away from the hinge around the entire perimeter of the wing pouch (Sagner, 2012).

The AP boundary is associated with sharp reorientations of PCP. First, PCP vectors that parallel the DV boundary point toward the AP boundary in both anterior and posterior compartments. Second, although PCP vectors in the central wing pouch are generally orthogonal to the DV boundary, they deflect toward the AP boundary where Hh signaling is most active (as defined by upregulation of the Hh receptor Patched [Ptc]). On either side of this region, PCP vectors turn sharply to realign parallel to the AP boundary. Third, PCP vectors in the hinge point away from the AP boundary and align parallel to the hinge fold (Sagner, 2012).

The atypical Cadherins Fat (Ft) and Ds limit disc growth and orient growth perpendicular to the hinge. Their loss perturbs the PCP pattern in pupal wings and alters hair polarity. To investigate whether they influence the larval pattern, PCP was was quantified in ft and ds mutant discs. The PCP pattern is similar to wild-type (WT) in the central wing pouch but altered in proximal regions close to the hinge fold. Polarity vectors deviate from their normal orientation (away from the hinge fold) in many regions of the proximal wing pouch. This is especially clear near the intersection of the DV boundary with the hinge - here, PCP vectors orient toward the DV boundary rather than away from the hinge. Furthermore, near the AP boundary, vectors form a reproducible point defect, with vectors pointing away from the defect center (Sagner, 2012).

After pupariation, morphogenesis reshapes the wing disc, apposing its dorsal and ventral surfaces such that the DV boundary defines the margin of the wing blade. During reshaping the PCP pattern evolves, but specific local features are retained through pupal development. Consistent with this, hair polarity in ds adult wings proximal wing near the anterior wing margin orient toward the margin rather than away from the hinge. Near the AP boundary, hairs form swirling patterns. Thus, Ft and Ds are required during larval growth to ensure that PCP vectors in the proximal wing orient away from the hinge (Sagner, 2012).

Notch and Wg signaling at the DV boundary organize growth and patterning in the developing wing. These pathways maintain each other via a positive feedback loop; Notch induces transcription of Wg at the DV interface, and Wg signaling upregulates expression of the Notch ligands Delta (Dl) and Serrate (Ser) adjacent to the Wg expression domain, further activating Notch signaling at the DV boundary. To study how the DV boundary organizer affects PCP, Ser was ectopically expressed along the AP boundary with ptc-Gal4 (ptc > Ser). In the ventral compartment, Ser induces two adjacent stripes of Wg expression, which then upregulate Dl expression in flanking regions (dorsally, Fringe prevents Notch activation by Ser. The posterior Wg and Dl stripes are distinct, but the anterior stripes are broader due to the graded activity of ptc-Gal4. In these discs, the ventral compartment overgrows along the AP boundary, parallel to the ectopic 'organizers'. PCP nematics and vectors near the posterior Wg/Dl stripes are organized similarly to those flanking the normal DV boundary, running parallel to the stripe and turning sharply outside this region to orient toward the ectopic organizer). PCP nematics anterior to the ectopic Ser stripe run parallel to it over larger distances before turning sharply, consistent with the broader Wg/Dl expression in this region. In resulting adult wings, hairs orient toward the ectopic wing margin that forms along the AP boundary. Ectopically expressing Wg along the AP boundary also generates an ectopic organizer that reorients growth and PCP (Sagner, 2012).

To ask how loss of the DV boundary organizer affected PCP, a temperature-sensitive allele of wg was used that blocks Wg secretion (wgTS), or wings were populated with wg null mutant clones. Loss of Wg signaling severs the feedback loop with Notch such that both decay. PCP nematics were quantified in wgTS discs shifted to the restrictive temperature shortly after the second to third-instar transition (earlier, Wg is required to specify the wing pouch). wgTS discs have smaller wing pouches than WT and are missing a large fraction of the central region of the pouch where polarity orients perpendicular to the DV boundary. Polarity still orients away from the hinge, thus the PCP pattern in wgTS discs appears more radial (i.e., oriented toward the center of the wing pouch). Analogously, adult wings populated by wg null clones are missing those regions of the distal wing blade where hairs normally point perpendicular to the wing margin. The remaining proximal tissue is normally polarized except at its distal edges. Here, polarity deflects from the proximal-distal axis to parallel the edge of the wing. Normally, hair polarity in the wing blade parallels the margin only in proximal regions, where Ft/Ds influences polarity. Thus, the DV organizer is needed to orient PCP in distal regions perpendicular to the margin. Ft/Ds is required for a complementary subset of the PCP pattern in the proximal wing. Their influences largely reinforce each other (i.e., away from the hinge and toward the DV boundary or wing margin) except where the hinge and wing margin intersect. Here, loss of one signaling system expands the influence of the other. Wg is distributed in a graded fashion and is a ligand for Frizzled (Fz). Thus, it could bias the PCP pattern directly, e.g., by asymmetrically inhibiting interactions between Fz, Strabismus (Stbm), and Fmi or causing Fz internalization. If so, uniform Wg overexpression should prevent intracellular polarization or reduce cortical localization of PCP proteins. To investigate this, Wg was overexpressed uniformly (C765 > wg::HA). Uniform Wg expression elongates the wing pouch parallel to the AP boundary. It broadens the pattern of Dl expression, such that sharp Dl stripes at the DV boundary are lost, but Dl expression remains excluded from the Hh signaling domain anterior to the AP boundary. Fmi and EGFP::Pk polarize robustly in these discs; thus, the Wg gradient does not act directly on PCP proteins to induce or orient polarity. However, the pattern of PCP vectors and nematics is altered. PCP points away from the hinge (rather than perpendicular to the DV boundary) over larger distances compared to WT and then turns sharply to face theDV boundary in the middle of the wing pouch. Because specific alterations in the PCP pattern are induced by uniform Wg overexpression, Wg protein distribution does not directly specify the new PCP pattern (Sagner, 2012).

To identify signals that influence the PCP pattern near the AP boundary, the effects of uniform high-level expression of Dpp and Hh, two morphogens that form graded distributions near the AP boundary, were examined. Uniform Dpp expression does not influence the magnitude of PCP or the range over which PCP deflects toward the AP boundary. Interestingly, uniform Hh expression dramatically increases the range over which PCP deflects toward the AP boundary, suggesting that Hh is important for this aspect of the pattern. However it clearly indicates that PCP vectors are not oriented directly by the graded distribution of Hh or by the graded activity of Hh signaling, because both are uniformly high in the anterior compartment of Hh overexpressing discs. Whether the apposition of cells with very different levels of Hh signaling might produce sharp bends in the PCP pattern was therefore considered. In WT discs, Hh signaling levels change at two interfaces: one along the AP boundary and one along a parallel line outside the region of highest Hh signaling where Ptc is upregulated. PCP vectors orient parallel to the AP boundary in the cells posterior to it, deflect toward the boundary anteriorly, and then reorient sharply outside of this region to align parallel to the AP boundary. Discs uniformly overexpressing Hh have only one signaling discontinuity (at the AP boundary), because Hh signaling is high throughout the anterior compartment. This could explain why PCP in these discs remains deflected toward the AP boundary over longer distances (Sagner, 2012).

To test this, clones mutant for the Hh receptor Ptc, which constitutively activate signaling in the absence of ligand, were generated. Quantifying PCP nematics in these discs reveals reproducible patterns of polarity reorientation at interfaces between WT and ptc- tissue. In WT tissue adjacent to ptc- clones, PCP aligns parallel to the clone interface. Due to the typical clone shape, this orientation is often consistent with the normal PCP pattern. However, PCP also aligns parallel to ptc- clones in regions where this is not so. Thus, ptc- clones exert a dominant effect on adjacent WT tissue. In contrast, on the mutant side of the clone interface, polarity tends to orient perpendicular to the interface. Thus, apposition of high and low levels of Hh signaling causes a sharp bend in the PCP pattern. Corresponding polarity reorientation by ptc- clones is also seen in adult wing. Thus, Hh signaling has two effects in WT discs: within the Hh signaling domain, it deflects PCP toward the AP boundary, and just outside the Hh signaling domain, it orients PCP parallel to the AP boundary. In this region, the tendency for polarity to align parallel to Hh signaling interfaces is consistent with the orientation of polarity toward the DV boundary and away from the hinge. Thus, these three polarity cues reinforce each other throughout much of the wing pouch, making the global PCP pattern robust (Sagner, 2012).

Simulations have highlighted the difficulty of establishing long-range polarity alignment in a large field of cells from an initially disordered arrangement. The pattern typically becomes trapped in local energy minima, forming swirling defects. Introducing a small bias in each cell removes such defects - this has been an attractive argument for the involvement of large - scale gradients in orienting PCP. The graded distribution of Ds along the proximal-distal axis (orthogonal to the hinge-pouch interface) suggested a plausible candidate for such a signal. Strikingly, the Ds expression gradient gives rise to intracellular polarization of both Ft and Ds, and the recruitment of the atypical myosin Dachs to the distal side of each cell. Nevertheless, most of the PCP defects in ft mutants can be rescued by uniform overexpression of a truncated Ft version that cannot interact with Ds, and PCP defects in ds mutants can be rescued by uniform overexpression of Ds. Moreover, blocking overgrowth through removal of dachs also suppresses PCP phenotypes in both mutants. The remaining disturbances in PCP in each of these backgrounds are restricted to very proximal regions, both in adult wings and the wing disc. Thus, the graded distribution of Ds does not provide a direct cue to orient PCP over long distances; rather, it appears to be important only locally near the hinge. Furthermore, this study shows that the two other key signaling pathways that contribute to the global PCP pattern in the disc do not act directly through long-range gradients. How do these signals specify the PCP pattern, if not through gradients (Sagner, 2012)?

Simulations in the vertex model have suggested that long-range polarity can be established in the absence of global biasing cues if PCP is allowed to develop during growth. PCP easily aligns in a small system, and globally aligned polarity can then be maintained as the system grows. Such a model obviates the necessity of long-range biasing cues like gradients, at least to maintain long-range alignment of PCP domains. The finding that a global PCP pattern develops early during growth of the wing makes this idea plausible. It may be that a combination of local signals at the different organizer regions specifies the vector orientation of PCP when the disc is still small, and that the pattern is maintained during growth. This may explain why loss-of-function studies have failed to identify the signaling pathways at the AP and DV boundaries as important organizers of the PCP pattern (Sagner, 2012).

In addition to local signals, the orientation of growth may provide additional cues that help shape the PCP pattern. Simulating the interplay between PCP and growth in the vertex model showed that oriented cell divisions and cell rearrangements orient PCP either parallel or perpendicular to the axis of tissue elongation, depending on parameters. Interestingly, each of the signaling pathways that influence PCP in the disc also influences the disc growth pattern. Wg/Notch signaling at the DV boundary drives growth parallel to the DV boundary, consistent with the pattern of clone elongation at the DV boundary. Growth near the AP boundary, where Hh signaling is most active, is oriented parallel to the AP boundary. This behavior probably reflects oriented cell rearrangements rather than oriented cell divisions. Finally, Ft and Ds orient growth away from the hinge. Suppressing overgrowth in ft or ds mutant wings by altering downstream components of the Hippo pathway rescues normal PCP except in the most proximal regions of the wing. Thus, altered growth orientation may contribute to the PCP defects seen in ft and ds mutants (Sagner, 2012).

Growth orientation reflects the orientation of both cell divisions and neighbor exchanges, and these can each exert different effects on the axis of PCP. Understanding the influence of local growth patterns on PCP will require a quantitative description of the patterns of cell divisions and rearrangements in the disc. More refined simulations incorporating local differences in the orientation of cell divisions and rearrangements will allow exploration of how planar polarity patterns can be guided by different growth patterns (Sagner, 2012).

Dissecting the molecular bridges that mediate the function of Frizzled in planar cell polarity

Many epithelia have a common planar cell polarity (PCP), as exemplified by the consistent orientation of hairs on mammalian skin and insect cuticle. One conserved system of PCP depends on Starry night (Stan, also called Flamingo), an atypical cadherin that forms homodimeric bridges between adjacent cells. Stan acts together with other transmembrane proteins, most notably Frizzled (Fz) and Van Gogh (Vang, also called Strabismus). In this study, using an in vivo assay for function, it was shown that the quintessential core of the Stan system is an asymmetric intercellular bridge between Stan in one cell and Stan acting together with Fz in its neighbour: such bridges are necessary and sufficient to polarise hairs in both cells, even in the absence of Vang. By contrast, Vang cannot polarise cells in the absence of Fz; instead, it appears to help Stan in each cell form effective bridges with Stan plus Fz in its neighbours. Finally, it was shown that cells containing Stan but lacking both Fz and Vang can be polarised to make hairs that point away from abutting cells that express Fz. It is deduced that each cell has a mechanism to estimate and compare the numbers of asymmetric bridges, made between Stan and Stan plus Fz, that link it with its neighbouring cells. It is proposed that cells normally use this mechanism to read the local slope of tissue-wide gradients of Fz activity, so that all cells come to point in the same direction (Struhl, 2012).

In Drosophila and other animals, including vertebrates, there appear to be at least two conserved genetic systems responsible for planar cell polarity (PCP); this study is concerned with the Stan system. In Drosophila, epithelial cells become polarised by a multicellular gradient of Fz activity. To read this gradient, the Stan system builds intercellular bridges of Stan-Stan homodimers that allow neighbouring cells to compare their levels of Fz activity. Under this hypothesis, Fz and Stan are essential components, as without Fz there is nothing to compare and without Stan there is no means to make comparisons. The Stan system also depends on a third protein, Vang, which appears to act in a complementary way to Fz. This study has dissected the function of these proteins by confronting adjacent cells of different fz, Vang and stan genotypes and assaying the effects on PCP. The main finding is that, even in the absence of Vang, Fz can function to polarise cells if it is present in at least one of the two abutting cells. By contrast, Vang has no detectable function when Fz is absent. Based on these and on other results, it follows that, at the core of the Stan system, intercellular bridges form between Stan on its own and Stan complexed with Fz (StanFz), and these act to polarise cells on both sides. It is concluded that Vang acts as an auxiliary component, helping Stan bridge with StanFz. Furthermore, it is posited that the numbers and disposition of asymmetric Stan<<StanFz bridges linking each cell with its neighbours are the consequence of the Fz activity gradient and serve to polarise the cell (Struhl, 2012).

A model has been built for how bridges between Stan and StanFz might determine the polarity of a cell (see The Stan system in PCP - a model). In the absence of Vang, expression of Fz in a sending cell can bias the polarity of a receiving cell that lacks Fz. Previous results indicate that within the receiving cell, Stan should accumulate only on the surface that faces the sending cell - because it is the only interface where it can form bridges with StanFz - and it is now proposed that it is this localised accumulation of Stan that biases the Vang- fz- receiving cell to make hairs on the other side, pointing away from the sending cell. A parsimonious hypothesis is that the apical membrane of each cell would have an unpolarised propensity to form hairs, and that an excess of Stan on one side locally inhibits this propensity, directing the production of hairs to the opposite side where there is least Stan. The response by a Vang- fz- cell eloquently suggests that the local accumulation of Stan bridged to StanFz in neighbouring cells is the main, and possibly the only, intracellular transducer of Stan system PCP (Struhl, 2012).

Next consider the finding that Vang functions in receiving cells to help Stan interact productively with StanFz in sending cells. In the key experiment Vang is added to just the Vang- fz- receiving cell: this cell is now more strongly polarised by StanFz signal coming from the sending cell. Thus, Vang can act in the same cell as Stan to help it receive incoming StanFz signal. The model also explains why the polarising effect of the Fz-expressing cell propagates only one cell into the fz- surround, even when Vang activity is restored to the receiving cells - as Stan-Stan bridges do not form, and/or do not function, between neighbouring cells that lack Fz (Struhl, 2012).

Last, consider the finding that cells lacking Fz can polarise cells devoid of Vang. In this case, only Stan<<StanFz and StanV<<StanFz bridges can form between the two cells, and as a consequence, only the StanFz form of Stan will accumulate on the surface of the Vang- receiving cell where it abuts the fz- sending cell. It is conjectured that Fz, when in a complex with Stan, acts to inhibit the normal action of Stan to block hair outgrowth. Therefore, the only place within the Vang- receiving cell where Stan can accumulate and block hair formation is on the far side, where it can form intercellular bridges with StanFz in the next Vang- cell. Accordingly, the receiving cell would be directed to make a hair on the near side, where it abuts the fz- sending cell. This reasoning also explains why the polarising effect of fz- sending cells on Vang- receiving cells appears to be limited mostly to the adjacent Vang- cell; because Stan<<StanFz and StanFz>>Stan bridges should form and/or function poorly between this cell and the next Vang- cell. Nevertheless, some imbalance between these two kinds of bridges probably does spread further than one cell; indeed fz- sending cells can polarise receiving cells up to two rows away in Vang- pupal wings (Struhl, 2012).

All the many other experiments fit with the simple model, in which Stan accumulates at the cell surface only where it can form intercellular bridges with StanFz, and each cell is polarised by differences in the amounts of Stan that accumulate along each of its interfaces with adjacent cells. Vang is not essential for these bridges, but by acting on Stan it helps them form and/or makes them more effective (Struhl, 2012).

How do wild-type cells acquire different numbers and dispositions of asymmetric bridges on opposite sides of the cell? In the Drosophila abdomen, in the anterior compartment of each segment, it has been argued that the Hh morphogen gradient drives a gradient of Fz activity. The slope of the vector of the Fz gradient would then be read by each cell via a comparison of the amount of Stan in its membranes. Within each cell, most Stan will accumulate on the cell surface that abuts the neighbour with most Fz activity, whereas most StanFz will accumulate on the opposite surface, where it confronts the neighbour with least Fz activity. This differential would then be amplified by feedback interactions both between and within cells. The result in each cell is a steep asymmetry in Stan activity that represses hair formation on one side, while allowing it at the other, directing all cells to make hairs that point 'down' the Fz gradient. The model differs in various and simplifying ways from the several and overlapping hypotheses published before. It makes Stan, rather than Fz, the main mediator of PCP, with differences in Fz activity between cells serving to regulate the local accumulation and transducing activity of Stan within cells (Struhl, 2012).

A central premise of this model is that morphogen gradients do not act directly on each cell to polarise Fz activity, but rather indirectly, by first specifying stepwise differences in Fz activity between adjacent cells. Such an indirect mechanism is favored for two reasons. First, PCP in much of the abdominal epidermis is organised by Hh, which is transduced primarily by its effects on the transcription factor Cubitus interruptus (Ci). It is difficult to understand how graded extracellular Hh could act directly - without cell interactions and only through the regulation of transcription - to polarise Fz activity within each cell. In addition, previous studies used temperature to drive tissue-wide gradients of transcription of a fz transgene under the control of a heat shock promoter; these studies nicely establish that cell-by-cell differences in Fz activity generated by transcriptional regulation are sufficient to polarise cells. Second, it has been previously shown that the polarising action of Hh depends on the Stan system. Specifically, cells in which the Hh transduction pathway is autonomously activated by the removal of the negative regulator Patched require Stan to polarise neighbouring cells. That result adds to evidence that graded Hh creates differences in Fz activity between cells - presumably via transcriptional regulation - that lead to asymmetries in Fz and Stan activities within cells. The target gene could be either fz itself or any other gene whose activity might bias the formation of Stan<<StanFz versus StanFz>>Stan bridges (Struhl, 2012).

Two previously developed staining experiments provide further support for this model with respect to Stan<<StanFz bridges. First, when Vang- clones are made in fz- flies (generating patches of Vang- fz- cells within fz- territory), a situation in which no Stan<<StanFz bridges can form, there is no accumulation of Stan near or at the border between the clone and the surround - and indeed this study now finds no polarisation of the fz- cells across the clone border. Second, and by contrast, when fz- clones are made in Vang- flies (generating patches of Vang- fz- cells within Vang- territory) Stan accumulates strongly along cell interfaces at the clone borders. Moreover, it is depleted from the cytoplasm of those cells of a clone that abut that border, indicating that Stan in Vang- fz- cells is accumulating at the apicolateral cell membrane where it can form stable intercellular Stan<<StanFz bridges. Previously, there was no evidence that this localisation of Stan within such Vang- fz- cells would polarise them. However, this study now shows that the Vang- fz- cells are polarised by their Fz-expressing neighbours and, also that the effect is reciprocal, their Fz-expressing neighbours are polarised in the same direction (Struhl, 2012).

The molecular mechanisms by which Fz and Vang control the formation and activity of Stan bridges remain unknown. Consistent with a direct action of Fz on Stan, both in vivo and in vitro studies suggest a physical interaction between the two proteins. Thus, Fz might act in a StanFz complex to regulate both the bridging and transducing activities of Stan. There is no comparable evidence in Drosophila for direct interactions between Vang and Stan. However, their mammalian counterparts have been shown to interact with each other (Devenport, 2008). But Drosophila Vang does interact directly with Pk, while a different Pk-related protein, Espinas, appears to interact directly with Stan during Drosophila neuronal development. Hence, Vang and Pk might form a cis-complex with Stan in epidermal cells, allowing Vang to act directly on Stan and help it form intercellular bridges with StanFz. Intriguingly, there is some evidence that Vang in one cell can interact directly with Fz in adjacent cells. Such an interaction might enhance the capacity of Stan to bridge with StanFz by providing an additional binding surface between the two forms of Stan. Alternatively, Vang might affect the formation or stability of Stan<<StanFz bridges indirectly, consistent with evidence implicating it in the trafficking of proteins and lipids to the cell surface. For example, evidence has been presented that any Stan or StanFz on the cell surface that is not engaged in Stan<<StanFz bridges is rapidly endocytosed and recycled to other sites on the cell surface. Vang activity could bias this process in favour of Stan, thereby enhancing its capacity to form bridges with StanFz (Struhl, 2012).

The results point to parallels between the Stan and Ds/Ft systems of PCP. First, both systems depend on the formation of asymmetric intercellular bridges between two distinct protocadherin-like molecules. For the Ds/Ft system, these are the Ds and Ft proteins themselves; for the Stan system, it is argued that these are two forms of Stan, either alone or in complex with Fz (StanFz). Second, morphogens may organise both systems by driving the graded transcription of target genes to create opposing gradients of bridging molecules. For the Ds/Ft system, at least two such target genes have been identified: ds itself and four-jointed (fj), a modulator of Ds/Ft interactions. For the Stan system, the existence of at least one such target gene induced by Hh is inferred. Third, for both systems, the two kinds of asymmetric bridges become distributed unequally on opposite faces of each cell, providing the information necessary to point all cells in the same direction. Thus for the Ds/Ft system, it is proposed that different amounts of Ds-Ft heterodimers would be distributed asymmetrically in the cell and this has been recently observed. Similarly, for the Stan system, there is plenty of evidence showing that Stan, Fz and Vang are unequally distributed within each cell. Finally, both systems have self-propagating properties: sharp disparities in Stan, Vang or Fz activity repolarise neighbouring cells over several cell diameters, even in the absence of the Ds/Ft syste, and the same is true of sharp disparities in Ds or Ft activity in the absence of the Stan system. Thus, the Stan and Ds/Ft systems may share a common logic that links morphogen gradients via the oriented assembly of asymmetric molecular bridges and feedback amplification, to cell polarisation (Struhl, 2012).


Viable stan mutants (particularly stan3/stan3 and stan3/Df-stan) were used to examine the stan wing phenotype. As is the case for other tissue polarity mutants, stan mutants do not show a complete loss or randomization of hair polarity across the wing. Rather, they show a stereotypic abnormal polarity pattern. The polarity patterns that result from mutations in many tissue polarity genes are quite similar, albeit not identical. This pattern has been called the fz/inturned (in) pattern after two of the best-studied genes in fz pathway. The stan mutant wings also have this general pattern. A second criterion that has been used to characterize tissue polarity mutants is the frequency of wing cells that form more than the normal one hair. Wings homozygous for stan3 have relatively few multiple hair cells (an average of 1.03 hairs/cell in a typical test region). This is similar to wings mutant for fz (1.02 hairs/cell), dsh (1.01 hairs/cell) and pk (1.02 hairs/cell). In contrast, it is much lower than that seen in wings mutant for inturned (in) (1.82 hairs/cell), fuzzy (fy) (1.92 hairs/cell) and multiple wing hair (mwh) (3.94 hairs/cell). The process of hair morphogenesis in stan was examined pupal wings. In wild-type wings, the prehairs that develop into the adult cuticular hairs are formed in the vicinity of the distal-most vertex of the cell and extend away from the cell in a distal direction. In stan mutants, many cells form prehairs at a relatively central location on the apical cell surface. Other cells form prehairs at abnormal locations along the cell periphery. This is similar to what has been found for mutations in the fz-like genes. Thus, based on several phenotypic criteria stan can be placed in the fz-like group of genes (Chae, 1999).

Flamingo controls the planar polarity of sensory bristles and asymmetric division of sensory organ precursors in Drosophila

In both embryos and imaginal tissues, stan is broadly expressed in epithelia and the nervous system. Mutations of the stan gene were isolated and base substitutions have been identified in the protein coding sequences of two genetically null alleles, fmiE45 and fmiE59, neither of which give a signal in immunostained epithelia. The stan null mutations are embryonic lethal, and the mutant embryos show local disconnection of longitudinal axon fascicles in the central nervous system. This lethality and the axonal defect can be rescued by stan cDNA expression in the nervous system of the mutant, fmiE45/fmiE59, indicating that the role of Stan in axon outgrowth is essential for viability. In the rescued animals, epithelia and imaginal photoreceptor cells are devoid of stan expression, and those adults exhibit defects in planar cell polarity in three classes of structures: ommatidia, sensory bristles, and wing hairs. This finding classifies stan into the core group of tissue polarity genes that includes frizzled and dishevelled. In region D of the wing of the fmiE45/fmiE59 escaper, hairs are deflected from the P/D axis and oriented toward the posterior wing margin. This pattern alteration is similar to that reported for the same region of fz mutant wings. As reported in null mutants in all the known tissue polarity genes, most hairs of the stan mutant do not point in random directions but possess an abnormal, nondistal polarity, similar to that of their neighbors. This 'stream' pattern suggests that a stan-independent system is working to align adjacent cells (Usui, 1999).

Polarity disruption of the above structures is reproduced in homozygous stan (fmiE59) clones, and prehairs emerge near the cell center or at wrong locations along the cell periphery, which is again similar to a phenotype of fz mutations. In the core group, several mutations in fz and strabismus/Van Gogh (stbm/Vang) show non-cell autonomy in specifying hair polarity, and those nonautonomous effects range over ten cells when clones are large. In contrast, the stan null mutation behaves essentially in a cell-autonomous way (Usui, 1999).

It is known that most fz mutations, including fzR52, show non-cell autonomy in orienting wing hairs with perfect penetrance. In areas distal to but not proximal to fz mutant clones, hairs of fz+ cells swirl toward the clone; this is due to initiation of prehair formation at abnormal subcellular sites, for example, at posterior and proximal cell vertexes. The mis-selection of prehair sites appears to be prepatterned by mislocalization of Stan. The Stan zigzag pattern is distorted in areas distal to the clone; many zigzags run obliquely rather than orthogonally to the P/D axis, and at a subcellular level, Stan is present at A/P cell boundaries in some fz+ cells. Deformation of the Stan pattern is minimal in areas proximal to the clone (Usui, 1999).

Whether prehair morphogenesis always occurs in the vicinities of such Stan-rich A/P boundaries is difficult to determine because Stan localization is being depolarized once prehairs start outgrowth. Nevertheless, the directional nonautonomous effect of the fz mutation on Stan localization was observed in 20 out of 20 clones examined that were well isolated from one another, suggesting a tight coupling of Stan mislocalization with incorrect placement of prehair sites. This observation is consistent with the hypothesis that assembled Stan molecules play an important role in initiating prehair morphogenesis (Usui, 1999).

Flamingo controls the planar polarity of sensory bristles and asymmetric division of sensory organ precursors in Drosophila

The sensory bristles of the fruit fly Drosophila are organized in a polarized fashion such that bristles on the thorax point posteriorly. These bristles are derived from asymmetric division of sensory organ precursors (SOPs). The Numb protein, which is localized asymmetrically in a cortical crescent in each SOP, segregates into only one of the two daughter cells during cell division, thereby conferring distinct fates to the daughter cells. In neuroblasts, establishment of apical-basal polarity by the protein Inscuteable is crucial for orienting asymmetric division, but this is not the case for division of SOPs. Instead, the Frizzled (Fz) protein mediates a planar polarity signal that controls the anteroposteriorly oriented first division (pl) of SOPs. Flamingo/Stan, controls the planar polarity of sensory bristles and the orientation of the SOP pl division. Both the loss of function and overexpression of stan disrupted bristle polarity. During mitosis of the SOP, the axis of the pl division and the positioning of the Numb crescent are randomized in the absence of Stan activity. Overexpression of Stan and Frizzled cause similar effects. The dependence of proper Stan localization on Fz activity suggests that Fmi functions downstream of Fz in controlling planar polarity (Lu, 1999).

Loss of starry night/flamingo function disrupts the planar polarity of non-innervated hairs on the Drosophila wing. To investigate the role of stan in regulating the planar polarity of sensory bristles, the bristles on the adult notum (dorsal thorax) were studied in stan mutants. Null mutants of stan are embryonic lethal. A transheterozygous combination between fmiE59, a putative null allele carrying a nonsense mutation in the segment of the gene encoding the ectodomain of Stan, and fmi71, for which the molecular lesion has not yet been determined, gives viable adult flies that display abnormal polarity in both types of sensory bristles (macrochaetes and microchaetes). Instead of pointing posteriorly within the epithelial plane as in wild-type flies, these bristles point in different directions: posteriorly, laterally and, less frequently, anteriorly. Moreover, some bristles also point upwards. The macrochaetes tend to be less affected than the microchaetes, and bristles located at different positions on the notum are affected to different degrees. Although only a small percentage of microchaetes located at the anterior (10%) and dorsocentral (13%) positions exhibit abnormal polarity, a higher percentage at the posterior (57%) and lateral (61%) positions are affected (Lu, 1999).

A similar planar polarity phenotype is observed in fmiE59 mutant clones. As in fmiE59/fmi71 transheterozygotes, fmiE59 mutant clones generated at different positions on the notum exhibit position-dependent penetrance of bristle polarity phenotypes. Marking the mutant clones with the yellow marker allows for an examination of whether Fmi activity is cell autonomous. In most cases, bristles that display abnormal polarity are confined within stan mutant clones. Infrequently, a wild-type bristle immediately adjacent to a mutant clone exhibits abnormal polarity. Thus, unlike fz, which has a long-range distal non-cell-autonomous effect on the wing, stan acts largely cell autonomously on the notum to control the planar polarity of sensory bristles. A similar cell-autonomous function of Stan is observed for the polarity of non-innervated hairs on the wing (Lu, 1999).

Not only does loss of stan function disrupt bristle planar polarity, overexpression of stan causes similar effects. stan was overexpressed on the adult notum using the apterous gene to drive expression of the Gal4-encoded transcription factor and a stan transgene driven by the upstream activator sequence (UAS) to which Gal4 binds. The apterous gene is expressed throughout the parts of the wing and haltere discs that give rise to the dorsal surface of wing blades and haltere, and in the regions that form the notum, scutellum and wing hinge. The microchaetes in apterous-Gal4 driven UAS-stan flies are affected to similar degrees as in fmiE59/fmi71 transheterozygotes; the macrochaetes are more affected than those in fmiE59/fmi71 flies. On the wing, overexpression of stan with a patched-Gal4 driver alters the planar polarity of non-innervated wing hairs. The disruption of bristle planar polarity by both overexpression and loss of function of stan suggests that the planar polarity signaling pathway is sensitive to the level of Stan activity (Lu, 1999).

The planar polarity pathway is also sensitive to the level of Fz activity. Overexpression of fz using UAS-fz and apterous-Gal4 also disrupted bristle planar polarity, consistent with earlier experiments using fz constructs driven by the heat shock gene (hs) promoter. Besides the planar polarity phenotype, overexpression of fz also leads to the formation of ectopic macrochaetes. The apterous-Gal4 driven UAS-fz flies exhibit frequent (~80%) ectopic induction of scutellar bristles, a phenotype not observed in apterous-Gal4 driven UAS-stan flies. Further overexpression studies with Drosophila Frizzled 2 (Dfz2) and dominant-negative forms of Fz and Dfz2 have suggested that, in addition to its role in controlling planar polarity, Fz also functions together with Dfz2 in the Wingless pathway to pattern the sensory bristles (Lu, 1999).

The orientation of the SOP pI division axis and the positioning of Numb protein crescent on the cortex both respond to polarity cues that are regulated by Fz signaling. In fz or dishevelled (dsh) loss-of-function backgrounds, the SOP pI division axis and the positioning of the Numb crescent are randomized. These two processes were examined in the pupal notum in apterous-Gal4;UAS-fz flies, and it was found that both the spindle orientation and positioning of Numb crescent no longer follows the anteroposterior axis and becomes randomized. Nevertheless, the Numb crescent still overlays one pole of the misoriented spindles and the randomization of spindle orientation is restricted two-dimensionally within the epithelial plane, as is observed in fz or dsh loss-of-function mutants (Lu, 1999).

The domineering non-autonomy of frizzled and Van Gogh clones in the Drosophila wing is a consequence of a disruption in local signaling

The frizzled gene is required for the development of distally pointing hairs on the Drosophila wing. It has been suggested that fz is needed for the propagation of a signal along the proximal distal axis of the wing. The directional domineering non-autonomy of fz clones could be a consequence of a failure in the propagation of this signal. This hypothesis was tested in two ways. In one set of experiments the domineering non-autonomy of fz and Vang Gogh (Vang) clones was used to assess the direction of planar polarity signaling in the wing. prickle (pk) mutations alter wing hair polarity in a cell autonomous way, so pk cannot be altering a global polarity signal. However, pk mutations alter the direction of the domineering non-autonomy of fz and Vang clones, arguing that this domineering non-autonomy is not due to an alteration in a global signal. In a second series of experiments, cells in the pupal wing were ablated. A lack of cells that could be propagating a long-range signal does not alter hair polarity. It is suggested that fz and Vang clones result in altered levels of a locally acting signal and the domineering non-autonomy results from wild-type cells responding to this abnormal signal (Adler, 2000).

The complementary nature of the domineering nonautonomy of fz and Vang clones is striking. It is true for the anatomical direction of the non-autonomy (i.e. distal vs. proximal); the relationship of the domineering non-autonomy to the clone (i.e. affected wild-type hairs pointing toward or away form the clone), and for the interactions with pk mutations. It is suggested that the domineering non-autonomy of fz clones is a consequence of a failure of the clone cells to send a locally acting polarity signal. The domineering non-autonomy of Vang clones could be due to the Vang clone sending excess signal (models that reverse this arrangement are also possible) (Adler, 2000).

A model for tissue polarity signaling in the wing is presented. Early models to explain planar polarity in the insect epidermis suggested it could be a reflection of the vector of a concentration gradient and this idea has remained popular. It is suggested that a distal/proximal gradient of fz activity is produced in the early prepupal wing (or wing disc). One way this could be achieved is by a gradient of a Wnt (or other type of ligand) resulting in a gradient of ligand bound Fz. Later in development cells would produce a locally acting second signal in amounts proportional to Fz activity. This hypothetical signal is referred to as Z and it is suggested that ligand bound Fz activates more Z production than unbound Fz. In this way a gradient of Fz activity would be translated into a gradient of Z. Cells would respond by initiating prehair morphogenesis on the side of the cell where Z level was lowest. This would result in hair polarity being oriented in the same direction as the vector of the Z concentration gradient. This is consistent with previous results showing that a directed gradient of fz expression results in cells with higher Fz levels producing hairs that point toward cells of lower levels. The absence of fz activity in clone cells would result in no Z being produced by the clone and a local decrease in Z levels that would cause surrounding cells to produce hairs that point toward the clone as is observed. Such a model can effectively incorporate the affects of pk and ds mutations on the direction of fz domineering non-autonomy. Mutations in these genes could alter the relationship between the ligand bound state of fz and Z production. For example, in a new antimorphic dominant pk allele, pkD wing unbound Fz receptor could act as a super-activator of Z production. This would lead to a reversed gradient of Z and to the reversal of both polarity and the direction of fz domineering non-autonomy. This model can also explain the observation that cells inside of a pk clone display the same polarity as do cells in a similar position in an entirely pk wing, since the alternative polarity caused by pk mutations would be due to abnormal amounts of Z. Such a model can also explain some of the results seen with Vang. The domineering non-autonomy of Vang could be due to Vang cells being constitutive for the production of high levels of Z. This would lead to locally elevated Z levels and cells surrounding Vang clones producing hairs that point away from the clone, as is observed. The model can also explain the ability of pkD to enhance the extent of fz domineering non-autonomy and suppress the extent of Vang domineering non-autonomy. In the model the level of Z will be higher in all regions of a pkD wing since now both bound and unbound Fz receptor will be strongly activating the production of Z. Thus, when a clone of cells lacking functional Fz protein is produced, the difference between the amount of Z produced by the clone cells and their neighbors will be increased over that seen in an otherwise wild-type wing. The ability of pkD to inhibit the extent of domineering non-autonomy of Vang clones can be explained by the reduced difference in the level of Z produced by the clone and neighboring cells (Adler, 2000).

At first glance the model cannot explain the suppression of Vang domineering non-autonomy in a fz mutant background, because the clone should produce high levels of Z in a background where there is little or no Z produced. One possibility is that in the absence of functional Fz no Z can be produced. A second possibility is that fz has multiple functions in wing tissue polarity and that an additional function is what suppresses the domineering non-autonomy of Vang. The model can also explain the relatively weak and poorly penetrant domineering non-autonomy of pk clones. The cells in such clones would produce aberrant amounts of Z, however the difference between the normal and mutant levels would be less than is seen in a fz mutant clone (that produces no Z) or in a Vang mutant clone (that produces constitutive high levels of Z). Thus, it is reasonable that pk (and ds) clones would show weak domineering non-autonomy (Adler, 2000).

The results of the cell ablation experiments are also consistent with the model. After surgery wound healing took place in the pupal wing. To form a permanent hole it was necessary for the healing to juxtapose neighboring dorsal and ventral wing cells. In those cases the juxtaposed cells were of similar position along the proximal/distal axis and therefore would be expected to be producing similar levels of Z. Thus polarity disruptions, equivalent to a clone of fz cells that juxtapose cells that produce normal levels of Z with cells that produce none, would not be seen. The model also predicts that the domineering non-autonomy of fz clones should be greater in proximal regions and weaker in the most distal regions of the wing. Some evidence for this sort of variation was found, although over a large middle region of the wing no significant difference in the strength of domineering non-autonomy is seen. Perhaps the hypothesized gradient of Z is shallow in this region of the wing or the assay is not sensitive enough. The model also predicts that the domineering non-autonomy of Vang clones should be greater in distal and weaker in proximal regions, but this was not seen. The nature of the hypothesized factor Z and its receptor are unknown. The function of the genes that encode these factors should be required for cells to sense differences in fz activity. An attractive candidate for the receptor is fz itself, since previous experiments have indicated that fz has both cell non-autonomous and cell-autonomous functions in the development of wing tissue polarity. Two roles for fz are also suggested by experiments that found both Vang and starry night/flamingo are required for some, but not all fz functions. This could be explained by fz functioning both upstream and downstream of Vang and starry night. Alternative candidates for factor Z and its receptor are Delta and Notch. These genes have been shown to be downstream of fz in the eye and to interact with dishevelled (dsh) during wing development. An interesting feature of the model is that the fz-dependent production of factor Z should be dsh independent because dsh is acting cell autonomously. It is worth noting that models suggested to explain the development of ommatidial polarity also rely on two sets of gradients along the polar/equator axis. Indeed, there are substantial similarities between the models. One difference is that in contrast to this model for the wing, in the eye fz is suggested to be essential only in the read out of the secondary gradient (Adler, 2000).

The core planar cell polarity gene prickle interacts with flamingo to promote sensory axon advance in the Drosophila embryo

The atypical cadherin Drosophila protein Flamingo and its vertebrate homologues play widespread roles in the regulation of both dendrite and axon growth. However, little is understood about the molecular mechanisms that underpin these functions. Whereas flamingo interacts with a well-defined group of genes in regulating planar cell polarity, previous studies have uncovered little evidence that the other core planar cell polarity genes are involved in regulation of neurite growth. This study presents data showing that the planar cell polarity gene prickle interacts with flamingo in regulating sensory axon advance at a key choice point - the transition between the peripheral nervous system and the central nervous system. The cytoplasmic tail of the Flamingo protein is not required for this interaction. Overexpression of another core planar cell polarity gene dishevelled produces a similar phenotype to prickle mutants, suggesting that this gene may also play a role in regulation of sensory axon advance (Mrkusich, 2011).

The dorsal cluster sensory axon stalling defect reported in this study in the pk mutant is the first demonstration in Drosophila of a role for a core PCP gene other than flamingo in regulation of neurite growth. This finding raises the question as to whether prickle and flamingo interact in this context. The similarity in the phenotypes of fmi RNAi and pk mutant embryos supports this proposition and further weight is provided by the finding that embryos lacking a single copy of both fmi and pk show the same axon stalling phenotype as pk homozygotes. A pk-fmi interaction is not required for axon advance in all sensory neurons, since lateral cluster axons grow into the CNS normally in pk mutant embryos (Mrkusich, 2011).

One possible molecular mechanism for a Prickle-Flamingo interaction -- direct binding between Prickle protein and the intracellular tail of Flamingo -- is not supported by the current study. A mutant Flamingo protein lacking the C-terminal tail is at least partially effective in rescuing the fmi mutant phenotype. Whether Prickle interacts directly with Flamingo in some other way, or whether an intermediary protein is involved, remains to be established (Mrkusich, 2011).

Do other core PCP genes cooperate with flamingo and prickle in regulating sensory axon growth? Whereas Frizzled has been shown to be necessary for the formation of several axon tracts in the mouse brain, there is no apparent requirement for the Drosophila homologue to promote sensory axon advance. It was previously found that sensory axon growth is normal in frizzled mutant embryos and, in addition, no signs of sensory axon stalling was seen in frizzled RNAi embryos (Mrkusich, 2011).

However, an axon stalling phenotype similar to pk mutants was observed following overexpression of Dishevelled in all sensory neurons. This finding mirrors findings in mammalian cell culture systems: overexpression of murine Prickle1 or Prickle2 induces neurite outgrowth from neuroblastoma cells and this effect is repressed by over-expression of Dishevelled1. It is concluded that in Drosophila, Prickle promotes axon extension by inhibiting Dishevelled-mediated repression of axon growth, a hypothesis that could be tested by jointly manipulating levels of Prickle and Dishevelled expression in embryonic sensory neurons. Dishevelled has been implicated in regulation of neurite growth in other systems, but as part of a canonical Wnt signalling pathway that diverges downstream of glycogen synthase kinase (Gsk3β), leading to direct remodelling of microtubules in the growth cone. It will be interesting to see whether the same molecular components are employed downstream of Dishevelled in growing sensory axons in Drosophila (Mrkusich, 2011).

The site of sensory axon stalling in pk mutants -- the border between the CNS and the PNS -- has been previously recognised as a key choice point for growing axons in both insects and vertebrates. The entry point to the CNS is the most common stall point for dorsal cluster sensory axons in neuroglian mutants. In Drosophila, peripheral glial cells play an important role in enabling both efferent motor axons and afferent sensory to advance across this transition zone. Future studies should examine whether prickle mediates an interaction between axons and glial cells to promote their continued growth at this site (Mrkusich, 2011).

In conclustion, similarities in LOF mutant phenotypes and the presence of axonal growth defects in transheterozygous embryos provide evidence that flamingo and prickle interact in regulating sensory axon advance in the Drosophila embryo. The C-terminal cytoplasmic tail of Flamingo appears not to be necessary for this interaction. Overexpression experiments suggest that another core PCP gene, dishevelled, acts downstream of flamingo and prickle to regulate axon advance. Prickle may promote axon extension by inhibiting Dishevelled-mediated repression of axon growth. In summary, it appears that regulation of sensory axon advance in the Drosophila embryo is achieved by using some of the components of the PCP pathway in an altered molecular setting (Mrkusich, 2011).

Asymmetric localization of Frizzled and the establishment of cell polarity in the Drosophila wing

The frizzled gene of Drosophila encodes a transmembrane receptor molecule required for cell polarity decisions in the adult cuticle. In the wing, a single trichome is produced by each cell, which normally points distally. In the absence of frizzled function, the trichomes no longer point uniformly distalward. During cell polarization, the Frizzled receptor (visualized using Frizzled-Green fluorescent protein) is localized to the distal cell edge, probably resulting in asymmetric Frizzled activity across the axis of the cell. Furthermore, Frizzled localization correlates with subsequent trichome polarity, suggesting that it may be an instructive cue in the determination of cell polarity. This differential receptor distribution may represent a novel mechanism for amplifying small differences in signaling activity across the axis of a cell (Strutt, 2001).

To understand the asymmetric distribution of Fz-GFP, the distribution was studied in flies mutant for other genes involved in trichome polarity establishment. In clones of cells lacking starry night (stan) function, both the apical and PD localization of Fz-GFP is completely abolished. However, in cells lacking dsh function, in which Fz signal transduction is compromised, Fz-GFP apical localization is preserved, but there is no proximodistal (PD) localization, with a splotchy irregular distribution being seen instead. The same phenotype is observed for mutations in the prickle-spiny-legs (pkpk-sple) and Van Gogh (Vang) genes. This would be consistent with the trichome polarity phenotypes of these mutations being due to a failure of Fz localization (Strutt, 2001).

Genetic data indicate that the polarity genes in, fy, and mwh act downstream of Fz/Dsh, inhibiting trichome formation where Fz is not active. In agreement with this, mutations in these loci do not alter Fz-GFP distribution despite trichome polarity being disrupted (Strutt, 2001).

The localization of Stan has also been reported to be disrupted in cells lacking dsh function but not in those lacking mwh. Therefore, whether Fz-GFP and Stan remain colocalized in different mutant backgrounds was tested. In clones of cells lacking dsh function, it was found that both Fz-GFP and Stan remain predominantly apical, and although the distribution of both appears diffuse, they nevertheless show broad colocalization. Similarly, in an in background, both Fz-GFP and Stan remain apical and colocalized to the PD cell boundaries (Strutt, 2001).

It is concluded that one important function of Stan is to localize Fz apically in the cell during polarity establishment. Stan may also play a role in localizing and/or anchoring Fz at the distal cell edge. However, in the absence of fz autonomous polarity signaling activity, neither Fz-GFP nor Stan is localized to PD boundaries. This leads to the speculation that the Fz receptor is responsible for receiving an extracellular polarity signal, and that the interpretation of this signal drives the localization of Fz to the distal cell edge and Stan to the distal and proximal cell edges, Stan thus acting downstream of Fz. Nevertheless, the data would equally well support Fz acting downstream of Stan; the codependence of the localization of both proteins would support this possibility, as would the observation that Stan has homology to G protein-coupled receptors. Thus far, no ligand has been identified for the Fz receptor in polarity signaling, and it is conceivable that in fact Fz might be activated by association with another transmembrane receptor, a role for which Stan is clearly a candidate (Strutt, 2001).

Both Fz-GFP and Stan localization are also downstream of fz nonautonomous signaling activity. One of the functions of Stan is to measure differences in fz activity between adjacent cells, as Stan accumulates on the boundary between fz+ and fz- cells. These findings have been extended to show that Stan in fact accumulates on the boundaries between cells with different levels of fz nonautonomous signaling activity. This observation is consistent with there being a long-range gradient of fz nonautonomous signaling activity across the pupal wing, with each cell having different levels of fz nonautonomous activity relative to its neighbors (Strutt, 2001).

An intriguing observation is that in the cells bordering a clone deficient in only fz autonomous signaling, Stan appears to show a preference for localizing on the cell boundaries lying perpendicular to the clone boundary. Thus, in cells bordering the proximal and distal clone edges, Stan is sometimes seen lying on the lateral cell boundaries rather than the PD cell boundaries. Similarly, the same is true for Fz-GFP localization on the boundary of stan clones. This phenomenon seems to suggest that if Stan or Fz-GFP cannot localize on one of the PD boundaries of a cell, they show a preference for not localizing on the opposite PD boundary. This, in turn, opens up the possibility that there is an intracellular communication mechanism that couples Stan/Fz-GFP accumulation on one cell boundary to that on the opposite cell boundary. Interestingly, such a mechanism would enable the propagation of a wave of Stan/Fz-GFP polarization across the wing, starting from a single row of polarized cells at one edge. Obviously, such a mechanism for propagation of polarity would obviate the need for an external ligand gradient and would also argue against the existence of a long-range gradient of fz nonautonomous activity. However, long-range gradient models and cell-cell communication models for the propagation of polarity need not be mutually exclusive, and both may operate side by side (Strutt, 2001).

Taking these observations together, the following model is put forward for Fz function in the polarization of single cells in the developing wing. Initially, unlocalized Fz is required for the long-range propagation of a polarity signal. Fz is then recruited apically in a Stan-dependent manner and becomes stably localized at the distal cell edge in a process requiring Fz signaling and the activities of Stan, Dsh, Pkpk-sple, and Vang. This Fz localization then restricts the site of trichome initiation to the distal cell vertex. It is possible that Fz signaling activates Stan molecules to bind both to Fz (in the same cell) and to Stan molecules in the adjacent cell, and so anchors Fz at the distal edge of the cell. Localization of Fz may lead to further increased Fz signaling (possibly through the effects of receptor clustering), which could, in turn, recruit more Stan and Fz. Over time, increased activity of clustered Fz receptors at the distal cell edge would lead to the majority of the Fz in the cell being recruited to this location. In heterologous systems, Fz activity leads to recruitment of Dsh to the cell membrane, so it is likely that Dsh is also present at the distal cell boundary. A precedent for Fz-dependent localization of a cytoplasmic protein during planar polarity establishment is provided by the observation that the Numb protein requires Fz activity for correct asymmetric subcellular localization during sense organ precursor cell divisions (Strutt, 2001).

The stable PD localization of Fz also requires Pk-Sple and Vang activity, with their loss having a similar effect on Fz-GFP localization as loss of Dsh activity. It is possible that, like Dsh, they are required for the transduction of the Fz signal, or they may be involved in the function of Dsh itself. Interestingly, Vang activity on only one side of the PD boundary is sufficient for Fz-GFP localization to occur. Further investigations of the biochemical activities of these proteins will be required to fully elucidate their roles in planar polarity establishment (Strutt, 2001).

The function of the frizzled pathway in the Drosophila wing is dependent on inturned and fuzzy

The Drosophila epidermis is characterized by a dramatic planar or tissue polarity. The frizzled pathway has been shown to be a key regulator of planar polarity for hairs on the wing, ommatidia in the eye, and sensory bristles on the notum. The genetic relationships between putative frizzled pathway downstream genes inturned, fuzzy, and multiple wing hairs (inturned-like genes) and upstream genes such as frizzled, prickle, and starry night (frizzled-like genes) were investigated. Previous data has shown that the inturned-like genes are epistatic to (function downstream of) the frizzled-like genes when the entire wing is mutant. Consistent with this are observations showing that the asymmetric accumulation of Fz, Dsh, and Fmi is not altered in in, fy, or mwh mutants. Those experiments were extended and the behavior of frizzled clones in mutant wings was examined. The domineering nonautonomy of frizzled clones is not altered when the clone cells are simultaneously mutant for inturned, multiple wing hairs, or dishevelled but it is blocked when the entire wing is mutant for inturned, fuzzy, multiple wing hairs, or dishevelled. Thus, for the domineering nonautonomy phenotype of frizzled, both inturned and multiple wing hairs are needed in the responding cells but not in the clone itself. Expressing a number of frizzled pathway genes in a gradient across part of the wing repolarizes wing cells in that region. inturned, fuzzy, and multiple wing hairs are required for a gradient of frizzled, starry night, prickle, or spiny-legs expression to repolarize wing cells. These data argue that inturned, fuzzy, and multiple wing hairs are downstream components of the frizzled pathway. To further probe the relationship between the frizzled-like and inturned-like genes the consequences of altering the activity of frizzled-like genes was determined in wings that carried weak alleles of inturned or fuzzy. Interestingly, both increasing and decreasing the activity of frizzled and other upstream genes enhances the phenotypes of hypomorphic inturned and fuzzy mutants. Results from several different experimental paradigms support the hypotheses that in and fy function downstream of fz, dsh, stan, pk, and sple in the wing and that they are required for the transduction of the fz signal to regulate hair morphogenesis and hence the actin and microtubule cytoskeletons (Lee, 2002).

To determine the relative positions of in, fy, and mwh with respect to several other fz-like tissue polarity genes, their ability to block the gain-of-function phenotypes that result from the directed expression of stan/fmi, pk, and sple were examined. ptc-GAL4 was used to overexpress pk. This results in a band of expression in the C region of the wing, with a decreasing gradient of expression away from the center of the ptc expression domain. This results in hairs pointing toward the midline in the C region of the wing. This phenotype is blocked by mutations in in, fy, and mwh. The resulting wings (e.g., ptc-GAL4/UAS-pk; in/in) show no effects from the directed expression of pk. Two different GAL4 drivers (ms1096-GAL4 and act-GAL4) were used to direct the expression of UAS-sple. Both of these drivers result in relatively even expression across the wing (although in ms1096 the level in the dorsal cell layer is higher than that in the ventral cell layer). This results in a reversal of hair polarity over much of the wing that resembles that seen in pkD. Once again it was found that mutations in in, fy, and mwh are able to block this gain-of-function phenotype. The omb-GAL4 driver was used to drive expression of UAS-stan. In the wing disc omb-GAL4 drives expression in a band located centrally along the anterior/posterior axis of the wing. However, in the pupal wing the expression pattern is more complicated during the time for tissue polarity development. During this time in the distal region of the wing, a series of alternating bands of expression and no expression is seen. Driving expression of stan using omb-GAL4 leads to a series of bands of polarity reversals; polarity is oriented from cells of low toward cells of high stan expression. Mutations in in, fy, and mwh are able to block the consequences of overexpressing stan. These data argue that in, fy, and mwh functioned downstream of fz, pk, sple, and stan in tissue polarity (Lee, 2002).

Driving expression of dsh using the ptc-GAL4 driver results in the formation of multiple hair cells in the proximal part of the ptc expression domain. In addition, ectopic bristles are also often found, presumably due to the role of dsh in canonical wg signaling. When UAS-dsh +/+ ptc-GAL4; in/in flies were examined, an additive multiple hair cell phenotype was found (i.e., stronger than either single phenotype) in the proximal part of the ptc domain and the presence of ectopic bristles. Equivalent results were found in UAS-dsh fy; fy ptc-GAL4 wings. The formation of the ectopic bristles was not surprising, since this is likely an effect of dsh on canonical wg signaling and in and fy are not thought to play a role in wg signaling. The additive multiple hair cell phenotype was unexpected and argues either that in and fy are not downstream of dsh in the fz pathway or that when overexpressed, Dsh can bypass the requirement for in and fy function (Lee, 2002).

The overexpression of fz just prior to hair initiation results in a multiple hair cell phenotype, which resembles that of in and fy (this is sometimes referred to as the late fz gain of function). When fz was overexpressed just prior to hair initiation in pupae that were mutant for a null allele of either in or fy an increased number of multiple hair cells was found. Thus, for the late fz gain of function, as for the overexpression of dsh, either in and fy are not required for fz signal transduction or when fz is overexpressed late the requirement for in and fy can be bypassed (Lee, 2002).

To test whether the frizzled-like genes could be regulating the inturned-like genes negatively or positively, the effects of both decreased and increased fz-like gene activity on hypomorphic alleles of in and fy were examined. In these experiments advantage was taken of the much higher number of multiple hair cells found in in and fy mutants compared to fz-like mutants. In all cases examined, mutations (null or hypomorphic) in fz, pk, Vang, stan, and dsh acted as strong enhancers of a weak in or fy phenotype as assayed by the frequency of multiple hair cells. These data are consistent with the fz-like genes acting as positive regulators of in and fy (Lee, 2002).

If there were a simple positive relationship between the fz-like genes and in and fy then it would be expected that the overexpression of fz-like genes should suppress hypomorphic alleles of in and fy. To test this fz was overexpressed from a hs-fz transgene and it was found that this enhances the multiple hair cell phenotypes of weak inII53 and fyJN12 alleles. These results argued that fz antagonizes the activity of in and fy. The genetic relationship between pk, sple, and fmi and weak alleles of in and fy was similarly tested. Overexpression of sple and stan from ms1096-GAL4 (or act-GAL4); UAS-sple and omb-GAL4; UAS-stan, respectively, in hypomorphic mutant backgrounds of in and fy results in an increase in the multiple hair cell phenotypes in the region of the wing, where expression is driven by the GAL4 enhancer trap. A similar enhancement was not seen for the overexpression of pk from ptc-GAL4 UAS-pk. These results suggest that sple and stan antagonize the activity of the in and fy. Notably, the gain-of-function polarity phenotypes of overexpressed pk, sple, and stan are blocked even with weak alleles of in and fy, confirming that these genes are required for the function of the fz pathway. It is clear that the fz-like genes do not act as simple positive or negative regulators of in and fy. The interaction is reminiscent of the observations that similar phenotypes result from either overexpression or a lack of function of fz-like genes (Lee, 2002).

The disruption of the microtubule cytoskeleton by treatment with vinblastine or colchicine results in many wing cells forming more than one hair. At the level of the individual cell these multiple hair cells resemble those found in in and fy mutants since the hairs are formed at the cell periphery and appear to be the result of independent initiation events. The altered polarity seen in in and fy mutant cells is not induced by vinblastine or colchicine. Immunostaining has not revealed any defects in microtubule organization in in or fy mutants. Thus, it seems unlikely that the in and fy phenotypes are due to disrupting the microtubule cytoskeleton and in this way indirectly affecting hair morphogenesis. However, it seemed possible that the disruption of the microtubule cytoskeleton could be inducing multiple hairs by interfering with in and/or fy function. It was reasoned that if that is the case then treatment of in or fy null mutant wing cells with vinblastine or colchicine would not produce a stronger phenotype than the in or fy null cells by themselves. Vinblastine was injected into in or fy mutant pupae and an additive response was found. That is, injected in or fy mutant cells produces a stronger multiple hair cell phenotype than does untreated mutant cells or cells from injected wild-type pupae. This result shows that the microtubule disruption phenotype cannot be due to a simple interference with in/fy function and argues that the microtubule cytoskeleton has a function that is independent of the fz pathway. The additive response in this experiment stands in sharp contrast with the lack of additivity in double mutants of in, fy, frtz, and mwh (Lee, 2002).

The hypothesis that in, fy, and mwh function downstream of fz and dsh originally came from the observation that mutations in in, fy, and mwh are epistatic to mutations in fz and dsh. Consistent with this hypothesis, the distal accumulation of Fz, Dsh, Fmi, and Dgo is not altered in in, fy, or mwh mutants. in, fy, and mwh are required for cells to either detect or respond to the lack of fz activity in a clone of neighboring mutant cells. The function of in and fy was required for cells to respond to a directed gradient of fz, stan, pk, or sple expression. Thus, using both of these experimental approaches it was found that in and fy are required for the function of the fz pathway. The simplest interpretation of this is that in and fy are essential downstream components of the fz pathway in the wing. An alternative explanation is that in and fy function in a parallel pathway and that the function of the fz pathway is dependent on this putative in pathway. There were a couple of surprising exceptional results. The overexpression of dsh or the late overexpression of fz induces the formation of multiple hair cells and it was found that these phenotypes are additive with an in or fy mutation. This could indicate that there are multiple pathways downstream of fz and dsh in the wing and that one of them does not require in or fy function. It is thought more likely that this is an example of overexpression bypassing a normal requirement for downstream proteins. It is suggested that In and Fy normally function as adapters to allow distally accumulated Fz and Dsh to stimulate the cytoskeleton and induce hair initiation in the correct part of the cell. When Fz or Dsh is overexpressed just prior to hair initiation the high cellular concentration might allow these proteins to bypass the need for In and Fy and to interact with and hyperactivate the hair initiation machinery (Lee, 2002).

In vertebrate embryos, a fz-based planar polarity pathway regulates convergent extension. Thus far there has been no evidence for a fz-based planar pathway having an analogous role in Drosophila. The eversion defect seen in fz mutant wings may be such an example. Some fz flies have a deformed wing that is short, fat, and often kinked proximally. This is seen in all strong and null fz alleles examined. This is due to a defect in wing disc eversion. In a normal pupa the wing everts so that it extends posteriorly and ventrally. In a fz mutant many pupae contain wings that extend anteriorly and sometimes dorsally. The wing eversion phenotype is another phenotype where in and fy appear to be epistatic to fz. The observations are somewhat puzzling as the defect in fz appears to be rescued by a mutation in in or fy. Thus, it is not clear that a simple inactivation of the fz pathway is responsible for the eversion phenotype. Perhaps for this phenotype there are multiple inputs into the fz pathway upstream of in and fy and the eversion defect is due to an imbalance in the input to in and fy. Blocking the pathway at in or fy would eliminate the function of the pathway completely and suppress miseversion (Lee, 2002).

The cellular mechanisms involved in the eversion of the wing are only poorly understood, but it is possible that convergent extension plays a role. It is also possible that the joint defects seen in fz mutants have a similar basis. In vivo observations on everting wings and joint morphogenesis would make an important contribution toward determining if convergent extension plays a role in these morphogenetic events. The development of in vivo imaging approaches for Drosophila pupae makes such experiments possible (Lee, 2002).

Genetic manipulation of single neurons in vivo reveals specific roles of Flamingo in neuronal morphogenesis

To study the roles of intracellular factors in neuronal morphogenesis, the mosaic analysis with a repressible cell marker (MARCM) technique was used to visualize identifiable single multiple dendritic (MD) neurons in living Drosophila larvae. Individual neurons in the peripheral nervous system (PNS) develop clear morphological polarity and diverse dendritic branching patterns in larval stages. Each MD neuron in the same dorsal cluster develops a unique dendritic field, suggesting that they have specific physiological functions. Single-neuron analysis reveals that Flamingo does not affect the general dendritic branching patterns in postmitotic neurons. Instead, Flamingo limits the extension of one or more dorsal dendrites without grossly affecting lateral branches. The dendritic overextension phenotype is partially conferred by the precocious initiation of dorsal dendrites in flamingo mutant embryos. In addition, Flamingo is required cell autonomously to promote axonal growth and to prevent premature axonal branching of PNS neurons. Molecular analysis also indicates that the amino acid sequence near the first EGF motif is important for the proper localization and function of Flamingo. These results demonstrate that Flamingo plays a role in early neuronal differentiation and exerts specific effects on dendrites and axons (Sweeney, 2002).

In studies of neuronal morphogenesis, it is important to differentiate the direct and indirect effects of the gene of interest. Similar to other important regulators, Flamingo functions in different cell types and at different developmental stages. Evidence is provided that Flamingo has a direct role in controlling dorsal dendritic growth in postmitotic neurons. Although individual MD neurons in the dorsal cluster differ greatly in their dendritic fields, the defects caused by flamingo mutations appear to be similar: mostly one process of the mutant neurons overextends toward the dorsal midline. Surprisingly, the general dendritic architecture of these MD neurons is not affected dramatically. In addition, these findings suggest that Flamingo is not a cell-type-specific regulator of dendritic morphology, nor does it affect dendritic branching patterns in a global way. It seems that Flamingo functions cell autonomously in controlling dendritic fields of different MD neurons by limiting the overextension of their dorsal dendrites. On the contrary, other mutants identified, such as tumbleweed, appear to affect both dorsal and lateral dendrites in a more general way. The studies also demonstrate that Flamingo function in neuronal morphogenesis is independent of its function in precursor cells (Sweeney, 2002).

How does Flamingo mainly control dorsal dendrite extension in postmitotic neurons? Neuronal morphogenesis of dorsal cluster MD neurons can be separated into relatively discrete developmental phases. These neurons always extend their axons first toward the ventral nerve cord. The extension of dorsal dendrites toward the dorsal midline ceases at 16-17 h AEL, before lateral dendrites extend toward the adjacent segment boundaries. If different development phases are controlled by different mechanisms, Flamingo may function mainly during dorsal dendrite extension. Indeed, Flamingo also prevents precocious initiation of dorsal dendrites: this contributes to the longer dorsal dendrites before 16 h AEL in flamingo mutant embryos. The failure to stop after 17 AEL also contributes to the longer dorsal dendrites in flamingo mutant embryos. Accordingly, the level of flamingo mRNA expression decreases during late embryogenesis, and Flamingo expression also decreases in the first instar larvae. The results presented here indicate that Flamingo has a function during early neuronal differentiation to control the initiation and extension of dorsal dendrites (Sweeney, 2002).

How does Flamingo function at the molecular level? Flamingo contains seven transmembrane segments homologous to a subset of G protein-coupled receptors, however, whether Flamingo functions through G protein signaling pathway is still unknown. The finding that the extracellular domain of Flamingo is still present on MD neuron membranes in flamingo72 mutant embryos indicates that the transmembrane segments are important for Flamingo function. In addition, mutational analysis suggests that the amino acid sequence near the first EGF motif is required for the proper subcellular localization and function of Flamingo. Whether the first EGF motif is directly involved in protein-protein interactions or whether the mutation affects the folding and/or trafficking of Flamingo remains to be determined. It would be useful to identify the proteins that interact with the first EGF motif and other domains of Flamingo (Sweeney, 2002).

GFP labeling of single mutant neurons provides an opportunity to study whether genes that control dendrite development also affect other aspects of neuronal morphogenesis, such as axon growth. Flamingo is required cell autonomously for promoting axonal growth. Since axons extend several hours earlier than dendrites, it is possible that the perdurance of Flamingo prevents the appearance of axonal phenotypes in some mutant neurons. Indeed, more flamingo mutant neurons exhibited dendritic defects than axonal defects (Sweeney, 2002).

Different molecules downstream of Flamingo might mediate its differential functions in dendrites and axons; alternatively, the same downstream molecules could transduce different signals in the two compartments (e.g., through the cyclic nucleotide pathway). Further dissection of the Flamingo pathway will help to elucidate how dendritic initiation and axonal growth are coordinated during development. Since Flamingo is highly conserved from flies to humans, it is likely that Flamingo also plays an essential role in controlling neuronal morphogenesis in mammals (Sweeney, 2002).

Starry night/Flamingo, dendritic fields and tiling in the peripheral nervous system

Signaling between neurons requires highly specialized subcellular structures, including dendrites and axons. Dendrites exhibit diverse morphologies yet little is known about the mechanisms controlling dendrite formation in vivo. Methods have been developed to visualize the stereotyped dendritic morphogenesis in living Drosophila embryos. Dendrite development is altered in prospero mutants and in transgenic embryos expressing a constitutively active form of the small GTPase cdc42. From a genetic screen, several genes have been identified that control different aspects of dendrite development including dendritic outgrowth, branching, and routing. These genes include kakapo, a large cytoskeletal protein related to plectin and dystrophin; flamingo/starry night, a seven-transmembrane protein containing cadherin-like repeats; enabled, a substrate of the tyrosine kinase Abl; and nine potentially novel loci. These findings begin to reveal the molecular mechanisms controlling dendritic morphogenesis (Gao, 1999).

Neurons elaborate dendrites with stereotypic branching patterns, thereby defining their receptive fields. These branching patterns may arise from properties intrinsic to the neurons or competition between neighboring neurons. Genetic and laser ablation studies reported here reveal that different multiple dendritic neurons in the same dorsal cluster in the Drosophila embryonic PNS do not compete with one another for dendritic fields. In contrast, when dendrites from homologous neurons in the two hemisegments meet at the dorsal midline in larval stages, they appear to repel each other. The formation of normal dendritic fields and the competition between dendrites of homologous neurons require the proper expression level of Flamingo, a G protein-coupled receptor-like protein, in embryonic neurons. Whereas Flamingo functions downstream of Frizzled in specifying planar polarity, Flamingo-dependent dendritic outgrowth is independent of Frizzled (Gao, 2000).

To study the mechanisms controlling dendritic morphogenesis, the Drosophila embryonic peripheral nervous system (PNS) was used as a model system. Neurons in the Drosophila PNS can be individually identified based on their location and dendritic morphology. There are 44 peripheral neurons in each abdominal hemisegment. The formation of their precursors depends on the proneural genes that encode bHLH transcriptional factors. The cell lineages that give rise to these neurons and the mechanisms for determination of their cell fates have been extensively studied. Using the UAS-GAL4 system to express green fluorescent protein (GFP), dorsal sensory neuron dendrites can be visualized in living Drosophila embryos and their growth, branching, and remodeling can be followed in real time. Dendritic morphogenesis during development is highly dynamic; however, the dendritic branching pattern for specific multiple dendritic (md) neurons in each segment is fairly invariant from embryo to embryo. This suggests that a genetic program underlies the process of dendritic morphogenesis (Gao, 2000).

The goal of the present study was to investigate how the dendritic field of a particular md neuron is determined during development. Laser ablation experiments demonstrate that the dendritic fields of embryonic md neurons are not affected by the absence of other md neurons in the same cluster. This result suggests that competition between different neurons in the same neighborhood is not essential for patterning dendritic fields during embryogenesis. Further support for this notion was obtained by examining embryos that fail to form one or more md neurons due to the loss of function of the proneural genes achaete, scute, or atonal. At larval stages, a subset of dorsal cluster md neurons in the two contralateral hemisegments extend their dendrites dorsally and meet at the dorsal midline, but these dendrites appear to repel each other and have minimal overlap. In the absence of these dendrites in one hemisegment, dorsal dendrites from the other hemisegment extend across the dorsal midline. Thus, competition between dendrites of these homologous neurons does play a role in defining their dendritic fields during larval development. The extension of dorsal dendrites and the apparent repulsion between dendrites of homologous neurons require the proper function of Flamingo in neurons. Both loss of function and gain of function of Flamingo result in the overextension of dorsal dendrites. Thus, the proper level of Flamingo activity in neurons is important for the process. In addition, neither loss-of-function mutations in Frizzled nor overexpression of Frizzled or Dishevelled affect dendritic development. Flamingo, therefore, affects dendritic outgrowth in a pathway different from the pathway that involves Frizzled and Flamingo in planar polarity determination (Gao, 2000).

Tiling, a complete but nonredundant coverage of the receptive field by dendrites of neurons with the same physiological function, may be achieved via competition among these neurons during the formation of their dendritic fields. In vertebrates, different retinal cell types are distributed in regular mosaics in the retina. The dendritic trees of different subtypes of ganglion cells overlap extensively. However, within each subtype the ganglion cells have nonoverlapping dendritic fields that cover every part of the retina. This kind of spatial arrangement of ganglion cell dendrites ensures that each subtype of ganglion cells in the retina receives complete but not redundant information from the visual field. It has been suggested that adjacent ganglion cell dendrites compete for their afferents and that the competition contributes to their mature dendritic morphology. The competitive interaction occurs only among neurons of the same subtype, thereby allowing access of each subtype of ganglion cells with distinct physiological functions to the entire visual field. A competition mechanism not only operates in defining the dendritic fields of homologous neurons, but also is involved in the establishment of axonal arborization. Previous studies in leech suggest that the sibling axonal branches of a single mechanosensory neuron do not overlap with each other, a process known as 'neuronal self-avoidance'. Whether dendritic 'tiling' and axonal 'self-avoidance' share some common molecular mechanism is unknown at present (Gao, 2000).

In Drosophila, the competition between homologous md neurons, but not neighboring neurons of likely distinct biochemical and physiological characteristics, is reminiscent of tiling. If further studies substantiate this scenario, the molecular mechanisms that prevent homologous neurons from having overlapping dendrites in Drosophila may be of relevance in understanding how tiling is achieved in general (Gao, 2000).

To begin to understand the molecular basis that determines dendritic fields, a genetic screen was carried and Flamingo was identified as one of the important players. In flamingo loss-of-function mutants, the extension of dendrites fail to stop short of the dorsal midline during embryogenesis. An important question is whether the function of flamingo in shaping the dendritic fields is cell autonomous. Previously, it has been shown that Flamingo functions cell autonomously in planar polarity determination. Several lines of evidence strongly suggest that Flamingo indeed has a cell autonomous role in shaping dendritic fields of md neurons. (1) Flamingo protein is localized to the cell bodies, dendrites, and axons of dorsal sensory neurons, including md neurons -- an expression pattern consistent with a cell-autonomous role for Flamingo in md neurons. (2) Overexpression of Flamingo in the md neurons by using neuronal-specific Gal-4 drivers leads to a dorsal dendritic overextension phenotype similar to but much weaker than the flamingo loss-of-function phenotype. Thus, the proper level of Flamingo activity in md neurons is important for dendritic outgrowth. Flamingo contains a G protein-coupled receptor-like domain with seven transmembrane segments. Recent studies of G protein-coupled receptors have revealed that they may function as dimers. Thus, the overexpressed Flamingo molecules could have a dominant-negative function. (3) The dendritic phenotype in fmi72 mutant embryos can be partially rescued by expressing wild-type Flamingo protein specifically in neurons, again supporting the notion that Flamingo has a cell-autonomous function in dendrite development. The incomplete rescue of dendritic phenotype by neuronal expression of Flamingo in fmi72 mutant embryos could be due to an experimental inability to mimic the precise level and timing of the Flamingo expression in wild-type neurons. Taken together, these observations indicate that specification of dendritic fields requires proper function of Flamingo in neurons (Gao, 2000).

Flamingo belongs to a family of protocadherins also found in mammals and C. elegans. The presence of nine cadherin repeats in these molecules suggests that they may function in cell-cell adhesion. Indeed, Flamingo proteins can mediate homophilic interactions in transfected S2 cells. Since the dendrites of md neurons are in contact with the basal side of epithelial cells, the question arises as to whether Flamingo expressed in epithelial cells has a role in shaping dendritic fields. Whereas overexpression of Flamingo only in neurons alters the dendritic morphology, overexpression of Flamingo only in epithelial cells does not give rise to dendritic overextension phenotype. Furthermore, with the available anti-Flamingo antibody, it was found that although Flamingo is highly localized to the adherens junctions between embryonic epithelial cells, Flamingo proteins could not be detected at the basolateral side of epithelial cells where they come into contact with dendrites. These results suggest that Flamingo proteins on epithelial cells may not be involved in md neuron dendrite development during embryogenesis. However, the possibility that a very low level of Flamingo expression on the basolateral side of epithelial cells may play a role in such a process cannot be ruled out (Gao, 2000).

In larval stages, competition between dendrites of homologous neurons plays a role in defining their dendritic fields. The extensive overlap of dendrites from homologous neurons in fmi mutant larvae suggests that loss of Flamingo function can also affect the competition between these dendrites at later developmental stages. Whether this competition involves direct Flamingo action remains to be elucidated (Gao, 2000).

Since Flamingo contains a G protein-coupled receptor-like domain with seven transmembrane segments, the hypothesis is favored that Flamingo functions as a receptor or coreceptor for as yet unidentified ligand(s) to shape the dendritic fields of md neurons. The recent finding that RhoA is involved in controlling dendritic extension of mushroom body neurons in Drosophila raises an intriguing possibility that the Flamingo signaling pathway may involve or interact with the RhoA pathway. It would be useful in the future to identify the G proteins with which Flamingo may interact and to characterize the downstream effectors of Flamingo involved in controlling dendrite development (Gao, 2000).

Insect dendritic arborization (da) neurons (one of three classes of multiple dendritic neurons) provide an opportunity to examine how diverse dendrite morphologies and dendritic territories are established during development. The 15 da neurons in hemisegments A2-A6 are arranged in four clusters (ventral, ventral', lateral and dorsal). The nomenclature for the da neurons identifies their position within one of these four clusters with the prefix v, v', l or d, their status as a da neuron and an alphabetic suffix that orders the cells from ventral to dorsal within each cluster. Three neurons (vpda, v'ada and v'pda) do not conform to this naming scheme. In this study, each neuron has been named according to its typical position within a cluster; however, the primary criterion for identifying each cell is its peripheral dendritic morphology. Four neurons were identified grouped together in a ventral cluster (vdaA-D), one lone ventral neuron (vpda), the previously identified v'ada and v'pda neurons, the two lateral neurons ldaA and ldaB and six dorsal da neurons (ddaA-F) (Grueber, 2002).

The morphologies of Drosophila da neurons have been examined by using the MARCM (mosaic analysis with a repressible cell marker) system. Each of the 15 neurons per abdominal hemisegment spread dendrites to characteristic regions of the epidermis. These neurons were placed into four distinct morphological classes (termed class I, II, III and IV neurons) distinguished primarily by their dendrite branching complexities. Some class assignments correlate with known proneural gene requirements as well as with central axonal projections. The data indicate that cells within two morphological classes partition the body wall into distinct, non-overlapping territorial domains and thus are organized as separate tiled sensory systems (for a more complete treatment of the meaning of the word tiling, see The Tilings Around Us). In contrast, the dendritic domains of cells in different classes can overlap extensively. The cell-autonomous roles in tiling of sequoia (seq) and starry night (stan, also known as flamingo (fmi), were examined. Neurons with these genes mutated generally terminate their dendritic fields at normal locations at the lateral margin and segment border, where they meet or approach the like dendrites of adjacent neurons. However, stan mutant neurons occasionally send sparsely branched processes beyond these territories that could potentially mix with adjacent like dendrites. Together, these data suggest that widespread tiling of the larval body wall involves interactions between growing dendritic processes and as yet unidentified signals that allow avoidance by like dendrites (Grueber, 2002).

Morphological characterization of Drosophila da neurons indicates that they are similar to the da neurons of the moth Manduca sexta, of which there are at least three distinct morphological classes. In Manduca, the alpha, beta and gamma da neurons show morphological similarities to the class I/II, IV and III da neurons of Drosophila, respectively. Manduca alpha neurons appear to function as proprioceptors, whereas gamma neurons probably function as touch receptors. Whether the Drosophila da neurons are functionally similar to Manduca da neurons remains to be determined. At least two lines of evidence, however, suggest that the morphological classes that have been identified in Drosophila represent functionally distinct types of neurons. (1) pickpocket (ppk), a degenerin/epithelial sodium channel subunit, appears to be expressed only in the class IV neurons ddaC, v'ada and vdaB. Since ppk may have a physiological role in mechanotransduction, its expression in class IV neurons could underlie a functional specialization of these cells. (2) Drosophila da neurons have dichotomous axonal projections, which probably reflect their functional distinctions. Most da neurons project into the ventral neuropil, a characteristic of tactile projections. vpda and an unidentified neuron in the dorsal cluster, by contrast, have more dorsal projections, which is similar to proprioceptive neurons. The ventral-projecting neurons appear to correspond to the class II, III and IV neurons, whereas the dorsal projections belong to at least a subset of the class I neurons (Grueber, 2002).

An important issue arising from this characterization of the Drosophila da system is how the morphological properties of each neuron relate to their genetic specification. Previous studies have shown that the Drosophila da system consists of genetically distinct subgroups of neurons. Most da neurons require proneural genes in the achaete-scute complex (ASC) arise as components of external bristle lineages, and express the Cut protein. The only da neurons that do not share these characteristics are vpda and two dorsal neurons. vpda remains in ASC-mutant embryos but is lost in animals mutant for the proneural gene, atonal. One dorsal da neuron requires a third proneural gene, amos. Finally, vpda and two dorsal da neurons fail to express Cut (Grueber, 2002 and references therein).

Reconciling these accumulated data with a cell-by-cell characterization of the da system, it appears that the class II, III and IV neurons are those that require ASC genes and express Cut. In contrast, Class I neurons do not express detectable levels of Cut and could correspond to the da neurons that require atonal and/or amos (although only one ASC-independent neuron has been described in the dorsal cluster). Consistent with these assignments, during mosaic analysis class III and class IV neurons were often observed co-labeled with es neurons, suggesting that these cells could have arisen from a common precursor. The class II neurons are likely also es related because all four neurons in the ventral cluster are lineally related to es organs and two class II neurons reside here. Understanding how the genes required for early da specification are linked to the activation of distinct programs of dendritic morphogenesis is an important goal for future studies (Grueber, 2002).

Tiling is a principle of dendrite organization in which functionally similar neurons completely fill available receptive territories with little or no redundancy. Two independent tilings of the Drosophila epidermis by da neurons bearing similar morphologies have been identifed. A similar type of tiling has recently been identified in Manduca (Grueber, 2001), suggesting that this is an evolutionarily conserved plan for organizing the da sensory system. In Drosophila, tiling occurs between class III da neurons and between class IV da neurons, which each partitions the body wall into a collection of non-overlapping territories. Furthermore, class III and IV neurons each provide a nearly complete segmental coverage. Dendrites with distinct morphologies, by contrast, can cross extensively (Grueber, 2002).

The tiling of the Drosophila epidermis by class III and IV neurons appears analogous to the tiling among physiologically alike vertebrate retinal ganglion cells. In this system, ON-center and OFF-center, four ON-OFF direction-selective classes and several other cell types, including amacrine cells, provide independent tilings of the retina. Such an arrangement ensures that each region of the visual field is 'viewed' by each physiological type of ganglion cell. Additionally, as visual information is distributed to the appropriate centers of the brain, the location of its origin is unambiguous, thereby maintaining a coherent representation of sensory space. The same rules of organization applied to the insect da system would likewise be advantageous. Mechanical and thermal stimuli often necessitate rapid and finely directed behavioral responses, particularly when they could lead to damage to the cuticle. If future studies show that the da neurons comprising each tiling class are united by their physiology, as is suspected, then these modalities would have the capacity to provide accurate spatial resolution of stimuli landing anywhere on the body wall (Grueber, 2002).

Although the cellular mechanisms that control dendritic tiling are not yet understood, several developmental scenarios can be envisioned. Individual dendrites could repel like dendrites where they meet. Alternatively, neurons could be endowed with a limited capacity for dendritic growth (depending, for example, on cell size) and form their territories without influence from neighboring neurons. Finally, in the case of the da neurons, interactions with the epidermis or surrounding tissue, such as muscle, could provide permissive or restrictive growth signals. These mechanisms are not mutually exclusive and could conceivably act in concert. In the da system, however, dendritic boundaries could not always be correlated with physical boundaries, such as muscle insertion sites, and terminal dendrites typically turned abruptly where they met like dendrites. Thus, these data do not provide strong support for the latter two mechanisms (limited growth capacity and physical boundaries) but do suggest that branch interactions could contribute to tiling. Experimental studies of the effects of adding neurons to, and removing neurons from, the da system will provide essential tests of the importance of these mechanisms. Additionally, because the data are taken from mature da neurons, a crucial question still to be addressed is how dendrites of like neurons behave during development as their territories are established. Dendrites could show exclusion throughout their development or, alternatively, refine their boundaries as a maturational step (Grueber, 2002).

Regulation of tiling by dendritic branch interactions is a likely scenario in the vertebrate retina, where contact-mediated avoidance signals appear to operate in a cell-type-specific manner. Furthermore, morphological data from mammalian retinal neurons show that dendro-dendritic contacts are made between like neurons but not between unlike neurons. Such contacts could provide an opportunity for these neurons to signal to each other by their activity or cell surface composition. Similarly, typically single apparent dendritic contacts are observed between tiling class IV neurons. Whether these contacts are important for exclusion among the remaining branches remains to be determined (Grueber, 2002).

The molecular mechanisms of dendritic tiling in the vertebrate retina have not been established. Mutant screens of the second chromosome in Drosophila might be informative in this regard, since several candidate loci have been identified that cause early overextension of dendrites. Alleles of two of these genes, stan and seq, have been tested for possible roles in tiling. In seq22 and stan72 mutant embryos, dendrites show an overextension phenotype and exhibit abnormal crossing of the dorsal midline. MARCM analysis using the seq22 and stanE59 alleles suggests that such overextension might not reflect a widespread defect in dendritic exclusion, because a majority of the dendrites terminate or turn where contact with an adjacent like neuron occurs or would be expected. Importantly, however, one or two sparsely branched processes were seen extending beyond the normal boundary of the cell in 18% of the class IV stan-mutant neurons (Grueber, 2002).

If branch recognition and exclusion are required for tiling, which appears to be the case for the class IV neurons, one interpretation of the stan phenotype is that the dendrite is overextending because it does not receive or transduce a repulsive signal that requires Stan function. Alternatively, because exclusion occurs among terminal dendritic branches in wild-type neurons, a lack of exclusion in stan-mutant neurons could arise if the overextended processes are equivalent to primary trunks and thus lack the machinery for tiling. However, without information about the fields of surrounding like neurons, the possibility remains that exclusion is intact in stan-mutant neurons. By extending earlier, or more rapidly, than the rest of the dendritic field, these single processes could have successfully invaded an uninnervated region of the body wall. This latter scenario seems to provide a reasonable explanation for why a dorsal branch from ddaC is observed overextending along the dorsal midline (one of the last regions of the body wall to become innervated). Whether a similar 'invasion' scenario could account for the overextended processes from vdaB might ultimately depend on the timing and pattern of outgrowth of its class IV neighbors (i.e. how far can a dendrite of a stan- vdaB neuron extend before encountering like dendrites?). Because MARCM experiments suggest that stan acts cell autonomously in the dendritic arborization neurons, future studies might be conducted using cell-type specific markers of the class IV neurons in a stan (and seq) mutant background. Such markers would allow the visualization of all neurons together and, as a result, provide a better indication of the relationship between early dendritic overextension phenotypes and tiling (Grueber, 2002).

In addition to the dendritic exclusion that occurs between like neurons, exclusion between dendrites that belong to the same neuron is frequently observed. Such 'self-avoidance' has been identified in Manduca sensory neurons (Grueber, 2001) and characterized experimentally in leech sensory axons; however the underlying mechanisms are not understood. In theory, self-avoidance and tiling might not require distinct signals or signaling pathways (among like neurons) because isoneuronal dendrites could be developmentally identical to 'like' heteroneuronal dendrites. It will therefore be of special interest to compare the mechanisms of exclusion by isoneuronal and heteroneuronal branches during development. Ultimately, an understanding of the distinction between these two processes will require the elucidation of their molecular underpinnings (Grueber, 2002).

Starry night/Flamingo and planar cell polarity in the eye

Planar cell polarity is established in the Drosophila eye through distinct fate specification of photoreceptors R3 and R4 by a two-tiered mechanism employing Fz and Notch signaling: Fz signaling specifies R3 and induces Dl to activate Notch in R4. The atypical cadherin Flamingo (Fmi) plays critical, but distinct, roles in both R3 and R4. Fmi is first enriched at equatorial cell borders of R3/R4, positively interacting with Fz/Dsh. Subsequently, Fmi is upregulated in R4 by Notch and functions to downregulate Dl expression by antagonizing Fz signaling. This in turn amplifies and enforces the initial Fz-signaling bias in the R3/R4 pair. These results reveal differences in the planar cell polarity genetic circuitry between the eye and the wing (Das, 2002).

To investigate the role of flamingo in eye development, fmi mutant clones were induced with the eye specific ey-FLP/FRT system. Analysis of fmi- tissue in adult eyes shows typical PCP defects with randomized chirality, resulting in loss of mirror image symmetry. Reminiscent of fz, dsh, and stbm null alleles, fmi- clones display defects in ommatidial chirality establishment (random chirality and symmetrical clusters) and rotation. In addition, fmi- clones contain ~20% ommatidia lacking photoreceptors (Das, 2002).

PCP aspects of the fmi phenotype are apparent from the earliest stage in the five-cell precluster. All markers reflecting the arrangement and rotation of ommatidial preclusters (Spalt: R3/R4; Bar: R1/6; svp-lacZ: R3/R4 and R1/R6) show typical polarity defects in fmi- tissue, with a random selection of the direction of rotation and abnormal rotation degrees. Thus, PCP defects are the primary phenotypic features of fmi clones, confirming its critical role in ommatidial polarity establishment (Das, 2002).

During eye disc patterning, Fmi protein is localized apically in all cells anterior to the morphogenetic furrow (MF), within the MF, and in a few rows of developing ommatidia posterior to the MF. Subsequently, Fmi is detected in differentiating photoreceptor cells in perinuclear areas and growing axons, possibly reflecting a late function of fmi in photoreceptor differentiation (Das, 2002).

During the time of polarity signaling, Fmi is strictly apical. The protein is largely detected uniformly around the apical cortex of the cells. Strikingly, however, in the developing R3 and R4 cells of the precluster, Fmi is enriched asymmetrically and is largely localized to the equatorial border of these cells. This asymmetry is visible in the third ommatidial row and maintained for about two rows. Subsequently, Fmi localization changes, and, as the clusters initiate rotation, it becomes enriched predominantly in R4, where it is maintained for two to three rows. This dynamic expression pattern is in contrast to Fmi expression during PCP establishment in pupal wing cells, where Fmi is found at very similar levels on both proximodistal ends of each cell (Das, 2002).

The initial asymmetrical enrichment of Fmi in both R3 and R4, and the subsequent enrichment in R4 only, raised the question of in which cell(s) of the precluster is fmi required for PCP establishment. Interestingly, the analysis of mosaic clusters revealed a requirement for fmi in both R3 and R4. An ommatidium always adopts the correct orientation when both R3 and R4 are fmi+. When either R3 or R4 (or both) are fmi-, the ommatidium selects chirality randomly or stays symmetrical. Significantly, all ommatidia with wrong or no chirality had fmi- R3 and/or R4 cells. Loss of fmi function in any other R cells in any combination has no effect on ommatidial polarity. These data indicate that fmi is necessary and sufficient in both the R3 and R4 photoreceptor precursors for normal polarity establishment (Das, 2002).

The genetic requirement of fmi in both R3 and R4 is unique, since other PCP genes are required only in either cell (fz and dsh in R3 and stbm and N in R4), and raised the question of how Fmi relates to these genes in function and expression. Thus, the expression patterns of other PCP proteins were examined in the eye (Das, 2002).

Although Dsh is cytoplasmic, it colocalizes with Fmi at the equatorial membranes of R3 and R4. Subsequently, Dsh is found apically at membranes in R4, in a U-shaped pattern, again colocalizing with Fmi. Also, Fz, which colocalizes with Dsh in the wing, shows a similar asymmetric equatorial-polar expression pattern like that in Fmi and Dsh early in R3/R4. The later upregulation in R4 is, however, not apparent for Fz (Das, 2002).

The membrane association of Dsh is Fmi dependent because, in fmi- cells, Dsh fails to be membrane enriched. When Fmi is overexpressed in R3/R4 (using the sev enhancer), more Dsh is localized at the membrane within these and other sev-expressing cells, indicating that excess Fmi leads to greater Dsh membrane recruitment. These data suggest that Fmi is both necessary and sufficient to induce Dsh membrane recruitment (Das, 2002).

Next it was asked how Fmi localization is regulated in the R3/R4 pair. In the wing, Fmi, Fz, Dsh, and Dgo are codependent for their localization. This interdependence is only partially observed in the eye. Within fz- or dsh- eye tissue, Fmi is not polarized within the early R3/R4 pair and subsequently not found enriched in R4. Strikingly, borders of fz- clones display three specific features: (1) Fmi gets enriched at cell boundaries between fz+ and fz- cells; (2) this is only observed within the region of the five-cell precluster, where Fz signaling takes place, and (3) only when the fz-/fz+ boundaries are perpendicular to the equatorial-polar axis. This enrichment is reminiscent of the Fmi asymmetry seen in R3 and R4 and suggests that differences in Fz-signaling levels (but not absolute protein amounts) between neighboring cells lead to Fmi accumulation at the respective membranes (Das, 2002).

Thus, Fmi localizes to the membranes between cells that have different Fz-signaling levels in the axis of signaling. This also provides additional evidence to support the hypothesis that there is a significant difference in Fz-signaling levels between the R3 and R4 precursors (Das, 2002).

This study shows that Fmi is also important for the R3/R4 fate decisions and polarity establishment. Unlike other PCP genes fmi is required in both cells of the R3/R4 pair. Fmi protein is dynamically expressed within the R3/R4 pair, being first enriched at equatorial cell borders of R3 and R4 and subsequently detected in R4. The data suggest that Fmi has multiple roles in polarity establishment. The early R3/R4 equatorial enrichment is Fz dependent and results from Fz activity differences in this axis. The later R4-specific function is to downregulate Dl in R4, by antagonizing Fz signaling directly or indirectly (Das, 2002).

The requirement of fmi in both cells of the R3/R4 pair is unique, as compared to those of fz, dsh, and Dl (required in R3) or N and stbm (required in R4). It was shown in cell culture assays that Fmi can act as a homophilic cell adhesion molecule. Thus, a simple interpretation would be to propose a cell adhesion function for fmi. However, Fmi localization and genetic requirements do not support this scenario. Instead, the data in the eye suggest that Fmi plays both positive and negative roles in a complex signaling network, and the interdependence of Fmi, Fz, and Dsh localization suggests that they might form a signaling complex required in R3. At the initial time of PCP generation, prior to expression of markers distinguishing R3 from R4, Fmi is enriched at the equatorial cell boundaries of R3 and R4. Importantly, this novel expression pattern is similar to Fz and regulated by Fz-signaling differences. Moreover, Dsh colocalizes with Fmi at the equatorial cell boundaries, its membrane localization depends on fmi, and Fmi overexpression (sev --> Fmi) leads to extensive Dsh membrane recruitment. Thus, Fmi is necessary and sufficient to localize Dsh to the membrane. The asymmetry in Fmi localization depends not only on fz but also on dsh: Fmi shows a nonpolarized apical distribution in R3/R4 in fz and dsh mutants (Das, 2002).

Although the fmi phenotype is reminiscent of fz and dsh, the role of fmi is more complex: fmi is first required in an Fz-dependent manner in the R3/R4 pair and later is specifically upregulated in R4 (Das, 2002).

An indication for a late role of fmi in R4 and a link to Fz-Notch signaling comes from the sev --> Fmi experiment and its endogenous late expression in R4 (Das, 2002).

The sev-Fmi mosaics give insight into the Fmi function at this stage. In mosaic sev-Fmi R3/R4 pairs, the cell with higher Fmi levels adopts the R4 fate. However, when both cells have equally high levels of Fmi, the cluster has a high tendency to develop as an R3/R3 symmetrical ommatidium. How could this be explained? The sev --> Fmi genotype is both sensitive to Dl dosage and causes a downregulation of Dl transcription in R3/R4. As Dl is nonautonomously required for R4 induction, it serves as a link between the mosaic requirement and phenotypic features of sev --> Fmi: when both cells contain sev --> Fmi, Dl is downregulated in both cells, and Notch activation often fails in both cells of the pair. This leads to a lack of R4 induction, generating R3/R3 symmetrical ommatidia. However, when only one cell of the pair has high Fmi levels, Dl is kept lower in this cell (even if it was originally the R3 precursor). It then adopts an R4 fate because it receives higher Dl levels from its neighbor. Thus, the endogenous role of Fmi in R4 could be to inhibit Dl expression and enhance the differences in Dl levels between R3 and R4 (Das, 2002).

How does Fmi inhibit Dl expression in R4? This is likely mediated through Fz signaling, as it was speculated in PCP establishment in the wing that Fmi can antagonize Fz . Thus, Fmi-mediated inhibition of Fz/PCP signaling should be a general mechanism. The lack of genetic interactions between fz and fmi (in either direction) suggests that Fmi does not directly inhibit Fz, and the mechanistic dissection of this inhibition will require biochemical studies (Das, 2002).

Notch activation and Fmi upregulation are coincident in R4, and, thus, Notch signaling itself is a good candidate for Fmi upregulation. The sev-N* (expression of activated Notch using a sevenless promoter) data indicate that Notch activation indeed leads to Fmi upregulation in a cell autonomous manner: all cells that express the sev enhancer, including the nonneuronal cone cells normally not expressing Fmi significantly, show an upregulation. Since nuclear Notch (N-intra) shows the same effect, it is likely a direct transcriptional event (Das, 2002).

These data indicate that the initial two-tiered mechanism of Fz-Notch activation establishing the R3/R4 fates and polarity in the eye can be extended further to include Notch-mediated upregulation of Fmi in R4. This in turn inhibits Fz/PCP-mediated Dl induction in R4 and amplifies the signaling differences between R3 and R4, leading to a solid binary cell fate decision (Das, 2002).

Since Fmi is initially expressed in both cells of the pair, its inhibitory role on Dl can only be allowed in R4 and thus needs to be regulated. How is this achieved? Diego (Dgo) is a good candidate for this role. The cytoplasmic Dgo protein depends on Fmi for membrane association and generally colocalizes with Fmi at all membranes in the eye disc. The genetic interactions with sev --> Fmi identify dgo as a strong enhancer, suggesting that it is suppressing Fmi function in this context. Mosaic analysis of dgo shows that it is required in R3 and thus might keep the inhibitory function of Fmi off in R3. Since Fmi is necessary, but not sufficient, for Dgo membrane recruitment, other factor(s) are also required. Since Dgo and Fmi colocalize also in R4, a factor is needed there to antagonize Dgo function. Strabismus (Stbm), since it is required in R4, is a candidate. Since fmi mutants are enhancers of an Stbm overexpression phenotype, Stbm could serve this function in R4 (Das, 2002).

Several pieces of evidence argue for a positive requirement of Fmi in R3. Fmi is asymmetrically distributed in response to Fz/Dsh signaling, and it is in turn required to maintain Dsh membrane localization in R3 early. Both Fz and Dsh are required in R3, and Dsh needs to be associated with the membrane for its function in R3. Since this is disturbed in fmi- cells, Fz signaling might not function normally there in the absence of Fmi. This interdependence of Fz, Dsh, and Fmi is also supported by observations in the wing, where each component requires the presence of the other for normal localization. Thus, it is speculated that, initially, during the activation of Fz/PCP signaling, Fmi is required positively for Fz/Dsh function, prior to its inhibitory role on Fz/Dsh signaling in R4 (Das, 2002).

How could this be achieved? (1) There are the distinct requirements for dgo in R3 and stbm in R4; (2) the differences in Fmi levels in early R3/R4 versus late R4 could account for its individual functions. High levels of Fmi in R4 could lead to a formation of a different complex than that formed in R3. For example, an Fz/Dsh/Fmi/Dgo complex would promote Fz signaling, whereas, in R4, since there is significantly more Fmi, a different Fmi complex would inhibit Fz signaling by possibly sequestering Dsh from the Fz complex (Das, 2002).

Although the 'core polarity' genes control PCP in multiple tissues, there are differences in their function in different contexts. In wing cells, Fz, Dsh, Fmi, and Dgo are localized to membranes within the proximodistal axis, and all four depend on each other's function. While Fmi and Dgo localize on proximal and distal sides of the cells, Fz and Dsh do so only distally. In the eye, Fmi is initially enriched at the equatorial side of R3 and R4 in an fz/dsh-dependent manner, colocalizing with them. Subsequently, Fmi gets specifically upregulated in R4. In contrast to the wing, where dgo is required for Fmi localization, Fmi is normally localized in dgo- eye tissue, indicating that the circuitry between the 'core polarity' genes is different between the eye and wing (Das, 2002).

In the notum, where these genes are required to orient the mitotic spindle of the sensory organ precursor (SOP) cell, the scenario is again different. In the SOP cell no asymmetric distribution of Fmi is detected (Fz/Dsh have not been analyzed), with Fmi covering the entire apical circumference. In this context, Dgo is not required, since dgo- flies show normal polarity of the bristles. Thus, different tissues utilize the 'core polarity genes' in different ways (Das, 2002).
Despite these differences, many features are conserved and thus likely to serve as a general mechanism in PCP establishment: (1) Fmi is required to maintain asymmetric Fz/Dsh localization and thus allow Fz/PCP signaling. (2) Fmi subsequently inhibits Fz signaling. (3) Dgo antagonizes Fmi. (4) Stbm might provide the initial bias and/or antagonize Dgo's role on Fmi. In the context of setting up the initial bias and asymmetry, Stbm/Vang is particularly interesting. Fz signaling and associated asymmetric Fmi localization are not affected in stbm- tissue but are rather randomized. Thus, Stbm could act upstream of Fz and Fmi localization, making it a good candidate to set up an initial asymmetry (Das, 2002).

In the context of the related pathway in vertebrates, regulating cellular polarization during convergent extension, there is growing evidence that this mechanism involves not only Fz/PCP signaling but also other 'core planar polarity' genes. Stbm homologs are required in convergent extension signaling. It is thus likely that the function of core PCP genes and their interplay with Fz/PCP signaling is evolutionarily conserved (Das, 2002).

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).

The predominance of Fmi-containing vesicles at the junction of photoreceptors R8, R2, R5, R3 and R4 suggests that this internalization may be the means by which Fmi is removed from these cells, or at least from a subset of these cells. In an effort to identify the process underlying this internalization, the efficiency of this process was tested in two mutants that interfere with the endocytic pathway: shibire (shi) and hook (hk). It was found that the vesicularization of Fmi is altered in these mutants, indicating that Fmi is internalized via endocytosis (Rawls, 2003).

shi, which encodes the Drosophila dynamin, is required early in the endocytic pathway for the budding of clathrin-coated pits from the membrane upstream of the fusion of these structures with endosomes. Temperature-sensitive shi2ts larvae were heat shocked for 1 hour at the restrictive temperature, sacrificed immediately and immunostained with an antibody against Fmi. In shi2ts larvae, the multivesicular body-like, Fmi-containing vesicles normally found in wild type are abolished; Fmi is instead found in small puncta on cell membranes. The large Fmi-containing vesicles reappear in larvae allowed to recover for 1-6 hours at room temperature (Rawls, 2003).

hk, which encodes a novel component of the endocytic pathway, acts downstream of shi in this pathway and is required for the formation and/or maintenance of multivesicular bodies (MVBs). In hk11 mutants, the large, MVB-like vesicles are absent; instead, Fmi is localized to smaller cellular puncta at the junction where the large vesicles normally localize (R8/R2/R5/R3/R4). The failure of Fmi to accumulate in vesicles in shi and hk mutants suggests that the Fmi-containing vesicles in wild-type eyes result from the endocytosis of Fmi. While the internalization of Fmi into vesicles is dependent on endocytosis, earlier changes in distribution of the protein (for example, its removal from R8, R2, R5 and subsequent accumulation in R4) are not (Rawls, 2003).

The functional significance of Fmi endocytosis in the eye is not known. Clearly, this internalization is taking place too late to initiate or mediate rotation. Perhaps it is necessary for rotation to stop. It could also be important for other aspects of development given that the endocytosis of membrane-associated receptors is required for signaling in key developmental pathways [for example, Notch, Dpp, and Wg] (Rawls, 2003).

The regulation of Fmi localization in the larval eye disc shows a dependency on fz and Notch (N), genes implicated in R3 and R4 cell fate determination, respectively. The dependency of Fmi localization on fz has also been described for Fmi localization in the wing (Rawls, 2003).

The early pattern of Fmi localization is unaffected in the absence of Fz -- it is still localized to all cell membranes anterior to the furrow and in nascent photoreceptor clusters. Furthermore, slightly later in development, Fmi is still abundant in photoreceptors R3 and R4. However, whereas Fmi would ordinarily be removed from photoreceptors R8, R2 and R5 at this stage in wild type, it is only partially removed from these cells in the fzKD4A mutant. The most notable change in Fmi localization is that it no longer accumulates asymmetrically in R4. The size, number and location of Fmi-containing vesicles are also disrupted in fzKD4A larvae: there are more vesicles, they are smaller and they accumulate approximately four rows earlier in development. Similar defects in Fmi localization are observed in Nts1 larvae heat-shocked for 6 hours. Additionally, Fmi localization is also perturbed when N-mediated signaling is knocked down via overexpression of the sev-Su(H)-EnR transgene. While these data do suggest a role for N in the asymmetric localization of Fmi, one cannot yet be assigned, given the abundance of roles for N throughout development (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).

Flamingo as a negative regulator of neuronal process extension in mushroom bodies

Neurons undergo extensive morphogenesis during development. To systematically identify genes important for different aspects of neuronal morphogenesis, a genetic screen using the MARCM system was performed in mushroom body (MB) neurons of the Drosophila brain. Mutations on the right arm of chromosome 2 (which contains ~20% of the Drosophila genome) were made homozygous in a small subset of uniquely labeled MB neurons. Independently mutagenized chromosomes (4600) were screened, yielding defects in neuroblast proliferation, cell size, membrane trafficking, and axon and dendrite morphogenesis. Mutations that affect these different aspects of morphogenesis are reported; a subset have been phenotypically characterized. roadblock, which encodes a dynein light chain, exhibits reduced cell number in neuroblast clones, reduced dendritic complexity and defective axonal transport. These phenotypes are nearly identical to mutations in dynein heavy chain Dhc64 and in Lis1, the Drosophila homolog of human lissencephaly 1, reinforcing the role of the dynein complex in cell proliferation, dendritic morphogenesis and axonal transport. Phenotypic analysis of short stop/kakapo, which encodes a large cytoskeletal linker protein, reveals a novel function in regulating microtubule polarity in neurons. MB neurons mutant for flamingo, which encodes a seven transmembrane cadherin, extend processes beyond their wild-type dendritic territories. Overexpression of Flamingo results in axon retraction. These results suggest that most genes involved in neuronal morphogenesis play multiple roles in different aspects of neural development, rather than performing a dedicated function limited to a specific process (Reuter, 2002).

A mutant, 39B17, was isolated in which many processes extend beyond the typical MB dendritic field, often as far as the axon lobes. The number of short and long (defined as less or more than one calyx diameter, respectively) overextended processes were quantified in 39B17 mutants and for wild type. Neuroblast clones homozygous for 39B17 have a marked increase of long over-extended processes. Overextended processes from the calyx are also evident in adult, projecting along similar tracks as the inner antennal cerebral tract as in the case of shot mutant neurons. Mutant neuroblast clones also have fewer cells than wild type, which becomes more obvious in adult clones. These clones contain neither alpha nor alpha' dorsal lobes, indicating an arrest of neuroblast proliferation before the generation of alpha' neurons (Reuter, 2002).

Deficiency mapping uncovered a lethal mutation in the 47A1-47D2 region for 39B17. This mutation was tested for complementation against a mutant allele of flamingo (fmi), also known as starry night, which encodes a seven transmembrane cadherin. Loss-of-function mutations of fmi exhibit defects in planar polarity and excessive dendritic outgrowth and misguidance in embryonic sensory neurons. 39B17 fails to complement fmiE59, which has a stop codon early in the extracellular domain and is believed to be a null allele. Two additional lines of evidence demonstrate that the overextension phenotype in 39B17 is due to a mutation in fmi: (1) MARCM clones of fmiE59 also exhibited phenotypes of process overextension and reduction of neuroblast clone size similar to that of 39B17; (2) using MARCM, MB clones homozygous mutant for fmi39B17 or fmiE59 were created in which a full-length fmi cDNA was also expressed under the control of UAS; this UAS-fmi expression was able to rescue the process overextension and cell number reduction phenotypes in third instar larvae and adults. Thus, the 39B17 mutation is an allele of flamingo and this allele was named fmiMB (Reuter, 2002).

To determine the nature of the overextended processes, flies were constructed carrying fmiMB/E59 and UAS-Nod-ßgal, and clones were made using the MARCM system. Nod-ßgal staining is observed in a subset of overextended processes, suggesting that a portion of the overextended processes are dendrites. The remaining overextensions are either misguided axons in the dendritic field or dendrites in which Nod-ßgal transport was inefficient. Fmi regulates dendritic extension in embryonic and larval sensory neurons. fmi mutant sensory neurons extend their dorsal dendrites beyond their normal territory. Although dorsal dendrites from homologous neurons appear to repel each other at the dorsal midline in wild type, they do not do so in fmi mutants. These data extend previous findings into dendrites of CNS neurons and suggest a general function for fmi in regulating dendritic extension (Reuter, 2002).

While neuroblast clones expressing wild-type Fmi do not exhibit any phenotypes and can indeed rescue the fmi mutant phenotypes, it was found that whole MB overexpression of Fmi using GAL4-OK107 results in loss of the dorsal branches of axons when examined in the adult. FasII staining, which allows the three classes of MB neurons to be distinguished, suggests that ß, ß' and gamma lobes are present when Fmi is expressed in all MB neurons. Coupled with the lack of cell loss, it is suspected that either the alpha and alpha' axons fail to extend dorsally, or they extend and retract, as is the case for MB neurons expressing double-stranded RNA corresponding to Drosophila p190 RhoGAP (Reuter, 2002).

To distinguish between these two possibilities, a developmental study was performed; a progressive worsening of the phenotype was found. High level expression of Fmi in all MB neurons does not result in any detectable phenotypes in wandering third instar larvae. At 18 hours after puparium formation, wild-type MB gamma neurons undergo pruning whereas alpha'/ß' neurons retain their larval branches including the dorsal alpha' lobe. All MBs overexpressing Fmi retain at least a portion of the dorsal lobes, with 63% more than half the length of the normal dorsal lobe and 25% full length, indicating that at least 25% and perhaps all alpha'/ß' axons extend normally. Over the next 12-24 hours, dorsal lobes become progressively shorter until they are not detectable at 48 hours after puparium formation. Although failure of dorsal lobe extension could in theory also contribute to the phenotypes, these developmental studies indicate that dorsal lobe phenotypes mainly result from axon retraction (Reuter, 2002).

These phenotypes are qualitatively similar to (albeit stronger than) inhibition of p190 RhoGAP. The RhoGAP phenotypes are caused by activation of RhoA, Drok and phosphorylation of myosin regulatory light chain encoded by spaghetti squash (sqh). Whether Fmi may signal through the RhoA/Drok/Sqh pathway was tested. Despite considerable efforts, however, biochemical studies and genetic interaction experiments have failed to provide such a link. The opposite phenotypes (overextension versus retraction) observed in fmi mutant and Fmi overexpression neurons suggest a general role for Fmi as a negative regulator of neuronal process extension (Reuter, 2002).

Flamingo regulates R8 axon-axon and axon-target interactions in the Drosophila visual system

Photoreceptors (R cells) in the Drosophila retina connect to targets in three distinct layers of the optic lobe of the brain: R1-R6 connect to the lamina, and R7 and R8 connect to distinct layers in the medulla. In each of these layers, R axon termini are arranged in evenly spaced topographic arrays. In a genetic screen for mutants with abnormal R cell connectivity, mutations were uncovered in flamingo (fmi). fmi encodes a seven-transmembrane cadherin, previously shown to function in planar cell polarity and in dendritic patterning. fmi has two specific functions in R8 axon targeting: it facilitates competitive interactions between adjacent R8 axons to ensure their correct spacing, and it promotes the formation of stable connections between R8 axons and their target cells in the medulla. The former suggests a general role for Fmi in establishing nonoverlapping dendritic and axonal target fields. The latter, together with the finding that N-Cadherin has an analogous role in R7 axon-target interactions, points to a cadherin-based system for target layer specificity in the Drosophila visual system (Senti, 2003).

Nine alleles of fmi were recovered in a genetic screen for mutations disrupting photoreceptor (R cell) connectivity in the Drosophila visual system. In this screen, photoreceptor axon projections were examined in whole-eye mosaics generated with eyFLP. In wild-type animals and control mosaics, photoreceptor axons terminate in smooth topographic arrays in three distinct layers of the optic lobe: R1-R6 terminate in the lamina, R7 terminates in the M6 layer of the medulla, and R8 terminates in the more superficial M3 layer. In contrast, R axons in fmi mosaics terminate in a highly disorganized pattern, particularly within the medulla. These defects were rescued by restoring fmi function specifically in the eye by using a GMR-fmi transgene. This confirms that fmi function is required autonomously in the eye for correct R axon targeting. Importantly, this GMR-fmi transgene does not produce any dominant guidance or targeting defects, indicating that fmi's function does not require its restricted expression in just a subset of photoreceptor cells (Senti, 2003).

Since fmi is also required for correct ommatidial polarity in the eye, it was of interest to enquire whether these axon targeting defects might be secondary to polarity defects. To test this, R axon projections were examined in animals mutant for frizzled, dishevelled, strabismus/Van Gogh, and prickle-spiny legs, genes that act together with fmi in the establishment of ommatidial polarity. For all of these mutants, R axon projection patterns appeared normal, despite the defects in ommatidial polarity. It was concluded that ommatidial polarity defects do not necessarily cause strong axon targeting defects, and that the function of fmi in axon targeting is mediated by a pathway distinct from that used in establishing ommatidial polarity (Senti, 2003).

For a more detailed analysis of photoreceptor axon targeting in fmi mosaics, markers specific for each subclass of photoreceptors were used. These markers revealed a highly specific R8 targeting defect. In contrast, R1-R6 axons correctly target the lamina, and R7 axons generally appear to select their correct target layer in the medulla, although their termini are slightly disorganized (Senti, 2003).

Since the R7 axon from each ommatidium extends into the medulla some 12 hr after the R8 axon, it was of interest to find out if the mild R7 targeting defects might merely be secondary to the severe defects in R8 targeting. To test for a specific role of fmi in R7 targeting, GMR-FLP and the MARCM system were used to generate and label single mutant R7 cells in an otherwise heterozygous animal. The axons of these fmi mutant R7 cells always targeted the correct layer in the medulla (Senti, 2003).

From this analysis, it was concluded that fmi is required in the eye for R8 axons to select targets in the correct layer of the medulla, but not for the target layer specificity of R1-R6 or R7. This precludes neither an additional nonautonomous requirement for fmi within the target region nor a role for fmi in any R cell for the selection of the appropriate synaptic partners within the target layer (Senti, 2003).

R8 axons first extend from the eye imaginal disc into the optic lobe during the third instar larval stage. The Rh6 marker is not expressed at this stage, and so to follow the initial projections of the R8 axons, an early R8 marker ato-τmyc was generated . With this marker, it appears that most if not all R8 axons do initially reach their correct target layer in the medulla. Since many R8 axons terminate in more superficial layers in the adult, it is inferred that these R8 axons fail to form stable contacts in their target region and subsequently retract to more superficial layers (Senti, 2003).

In wild-type animals, R8 axons form evenly spaced topographic arrays in the medulla, with 'inverted-Y-shaped' growth cones. In fmi mosaics, the R8 growth cones are irregularly spaced and have a more 'club-like' morphology, but they have many elaborate fine processes. The processes of individual R8 growth cones often overlap extensively, something only rarely observed in control animals (Senti, 2003).

Despite this irregular spacing, the entire target field appears to be filled, and there does not appear to be any dramatic misrouting of axons within the optic lobe. This suggests that the overall topographic order is largely preserved in fmi mosaics. This was confirmed for the dorsoventral axis by using markers specific for polar (i.e., dorsal- and ventral-most) axons. In fmi mosaics, as in control mosaics, these axons maintain their correct topographic positions as they extend within the eye disc, through the optic stalk, and into the optic lobe. Analogous markers to assess topographic mapping along the anterior-posterior axis are unavailable, but the ordered posterior-to-anterior filling of the medulla target field in all of the preparations examined is strong evidence that, along this axis too, topographic order is preserved (Senti, 2003).

Since Fmi is a homophilic cell adhesion molecule, it was of interest to see whether it might also contribute to the bundling of photoreceptor axons into their discrete ommatidial fascicles. This possibility was investigated using electron microscopy to examine the composition and structure of ommatidial fascicles within the optic stalk. The only difference noted in fmi compared to control mosaics was a slight (5.0%) increase in the number of fascicles comprising more than eight R axons. This difference can be attributed to the low frequency of ommatidia containing extra R cells. Otherwise, fmi mosaics were indistinguishable from the controls, indicating that Fmi does not function in the formation of ommatidial fascicles (Senti, 2003).

Anti-Fmi mAb 74 was used to assess the distribution of Fmi protein in the developing visual system. At the third instar larval stage, Fmi protein is strongly expressed within the lamina plexus, where R1-R6 axons terminate, and in the R7/R8 termination region in the medulla. In photoreceptor axons, Fmi is highly localized to the growth cone; only very low levels of staining are seen along the axon shaft. Strong staining was also observed within the medulla and lobula. This staining appears to localize to the processes and termini, respectively, of medulla cortical neurons. No Fmi protein was detected in glia in the retina, lamina, or medulla. In whole-eye fmi mosaics, most Fmi staining is lost in the lamina plexus, while staining in the medulla is reduced but not eliminated. This confirms that Fmi protein in the lamina is largely confined to R1-R6 growth cones, while, in the medulla, some but not all Fmi protein is localized to R7 and/or R8 growth cones (Senti, 2003).

Fmi immunoreactivity persists in the lamina and medulla throughout early- and mid-pupal development, with increased staining of lamina and medulla cortical neurons. By the mid-pupal stage, the R7 and R8 growth cones have become more widely separated (by 10 μm), in part due to the intercalation of growth cones and processes from lamina and other neurons. Intense Fmi staining is seen in the medulla neuropil in the region between the R8 and R7 termini, with only low levels in the layers immediately above the R8 termini or below the R7 termini. Fmi also localizes to a single broad band deeper in the medulla and to the lobula (Senti, 2003).

These results define two distinct and specific functions for Fmi in R axon targeting: (1) Fmi facilitates competitive or inhibitory interactions between adjacent R8 growth cones; (2) it promotes R8 axon-target interactions. These inhibitory interactions between R8 growth cones may be mechanistically related to those previously demonstrated for R7 axons. Competitive interactions between retinal axons also contribute to the formation of an evenly spaced topographic map in the mammalian visual system. Mammalian Fmi proteins are also widely expressed in the developing nervous system and thus are strong candidates to mediate similar competitive axon-axon interactions. This function of Fmi in R axons may also be analogous to its role in the dendritic tiling of the embryonic PNS, where competitive interactions involving Fmi prevent overlap between the dendritic fields of homologous sensory neurons (Senti, 2003).

In addition to this negative role in R8 axon-axon interactions, Fmi also appears to act positively in R8 axon-target interactions. Here, parallels can be drawn with the function of the classical cadherin, N-Cadherin, in R7 targeting. In both cases, mutant axons initially contact their correct medulla target layer, but then a specific subclass retracts: R7 retracts in the case of N-Cadherin and R8 retracts in the case of fmi. Distinct cadherins thus regulate distinct targeting decisions in the medulla and possibly act in this case as homophilic cell adhesion molecules. However, since both N-Cadherin and Fmi are expressed on all photoreceptor axons and in multiple layers in the optic lobe, these two cadherins alone cannot account for the distinct target layer selections of R7 and R8. Additional determinants must exist. One of these is the receptor tyrosine phosphatase LAR, which is specifically required for R7 target layer selection and may act by modulating N-Cadherin-mediated adhesion. Other factors are likely to emerge from ongoing genetic screens for layer-specific axon targeting in the Drosophila visual system (Senti, 2003).

The protocadherin Flamingo is required for axon target selection in the Drosophila visual system

Photoreceptor neurons (R cells) in the Drosophila visual system elaborate a precise map of visual space in the brain. The eye contains some 750 identical modules called ommatidia, each containing eight photoreceptor cells (R1-R8). Cells R1-R6 synapse in the lamina; R7 and R8 extend through the lamina and terminate in the underlying medulla. In a screen for visual behavior mutants, alleles of flamingo (fmi) were identified that disrupt the precise maps elaborated by these neurons. These mutant R1-R6 neurons select spatially inappropriate targets in the lamina. During target selection, Flamingo protein is dynamically expressed in R1-R6 growth cones. Loss of fmi function in R cells also disrupts the local pattern of synaptic terminals in the medulla, and Flamingo is transiently expressed in R8 axons as they enter the target region. It is proposed that Flamingo-mediated interactions between R-cell growth cones within the target field regulate target selection (Lee, 2003).

To identify proteins that regulate R1-R6 targeting, a screen was conducted for mutations affecting the optomotor response, a behavior requiring correct R1-R6 function. In this screen, genetically mosaic animals were tested in which the eye was homozygous mutant and the target was not. Two alleles of fmi were isolated. To assess the functional requirement for fmi in photoreceptors, genetic mosaic animals were generated using the FLP/FRT system in which the FLP recombinase is expressed under the control of the eyeless promoter (ey-FLP). In these animals, virtually all R cells are made homozygous mutant. As in wild type, fmi mutant R1-R6 axons in such genetically mosaic animals terminate in the lamina. Unlike wild type, however, their growth cones selected as synaptic targets lamina neurons that were in inappropriate locations, as assessed by DiI labeling experiments in developing pupae. The penetrance of the phenotype was 100%, and the expressivity ranged in severity from complete loss to moderate disruption of the normal pattern of projections. There is no overlap between this phenotype and the R1-R6 targeting phenotypes previously described for N-cadherin and Lar mutants; Flamingo thus acts at a different step in target selection. Although small clones of mutant cells are found within lamina target neurons in mosaics generated using ey-FLP, the complete penetrance of the phenotype indicates that it must be the loss of fmi function in R cells, rather than in their targets, that disrupts targeting (Lee, 2003).

Flamingo, like other cadherins, mediates homotypic cell adhesion. To assess whether Flamingo mediates interactions between R1-R6 growth cones or between these growth cones and their targets, the pattern of Flamingo expression during target selection was characterized. Flamingo protein is transiently and dynamically expressed on R1-R6 growth cones during this period. At 24% of pupal development, Flamingo is expressed strongly on R1-R6 axons. Weak expression on lamina neuron cell bodies was also observed. By 30% of pupal development, Flamingo is expressed unequally in R1-R6 growth cones within the lamina plexus, as compared with uniform growth cone expression of the pan photoreceptor-specific marker, GMR-GFP. One likely explanation for this pattern is that specific subsets of R1-R6 growth cones express relatively high levels of Flamingo protein, while other subsets express little, if any. At the same stage, lamina neurons are almost entirely devoid of Flamingo expression, although in some preparations, a few scattered lamina cell bodies were labeled. Since all fmi mutant R cells showed targeting phenotypes, this very limited expression of Flamingo within the target field is unlikely to be of functional significance. By 46% of pupal development, Flamingo is no longer detected in photoreceptor axons and their growth cones. In contrast, N-cadherin is strongly expressed on both R1-R6 cell axons and on their lamina target neurons throughout development. On the basis of these and other observations, it has been proposed that N-cadherin mediates interactions between photoreceptor growth cones and their targets. The dynamic and highly restricted expression of Flamingo, a homophilic cell adhesion molecule, on R1-R6 growth cones supports the view that Flamingo is directly involved in mediating interactions between R1-R6 growth cones that are required for target selection (Lee, 2003).

Although the simplest interpretation of the data is that Flamingo mediates interactions between R1-R6 growth cones within the target region, the possibility that Flamingo could affect these interactions only indirectly was considered. Defects in projections could, for example, reflect disruptions in the polarity of the ommatidium, or an earlier role in R8, or defects in fascicle ordering before reaching the lamina target. A series of studies argues against these possibilities. (1) The projection defects seen in fmi mutants are strikingly different from those seen in frizzled and spiny legs, two mutants that show planar cell polarity defects in the eye similar to fmi. Whereas the orientation of the projection pattern in frizzled and spiny legs largely reflects the orientation of the cells in the eye (and is frequently reversed along the dorsoventral axis by inversion of the ommatidium), the relative pattern of targets chosen by R1-R6 from a single ommatidium in these mutants is indistinguishable from wild type. (2) The R1-R6 defects in fmi mutants are not a secondary consequence of defects in R8, because targeted expression of an fmi cDNA using the Gal4-UAS system rescues the R8 targeting phenotype but does not rescue defects in R1-R6 target selection. Finally, electron micrographs showed that the composition of individual R1-R8 axon bundles above the lamina are indistinguishable from those in wild type, indicating that all defects in target selection must arise from defective sorting at the lamina surface rather than as a secondary consequence of aberrant R1-R8 fasciculation into bundles of abnormal composition (Lee, 2003).

Does Flamingo mediate interactions between other growth cones, or is its function restricted to sorting mechanisms required for R1-R6 connections? In the initial characterization of the fmi phenotype, it was observed that fmi is also required to form precise retinotopic projections of R7 and R8 growth cones in the medulla. In developing third-instar eye-brain complexes or in adults labeled with monoclonal antibody 24B10, a jumbled arrangement of R7 and R8 growth cones was observed in the medulla. Genetic mosaic analyses reveal that targeting defects in R7 are non-cell autonomous (that is, Flamingo protein is not required in the R7 cell for target specificity), supporting the idea that defects in R7 projections are a consequence of an earlier role in R8 axons (Lee, 2003).

To assess how Flamingo contributes to R8 connection specificity, a detailed study of map formation was carried out. In wild-type larvae, axons exited the optic stalk and dispersed at regular intervals along the anterior edge of the lamina. In contrast to wild-type axons, fmi mutant axons dispersed in an irregular fashion. This disruption in local retinotopic assembly was quantified. In wild type, R8 axon terminals from the anterior-most two columns of ommatidia in the retina penetrated the anterior edge of the lamina, and their number establishes a 1:1 correspondence between R-cell terminals and ommatidia. In fmi mutants, R8 growth cones do not disperse appropriately, reducing this ratio. In wild type, an average R-cell axon terminal:ommatidia ratio of 53:51 was observed, whereas in fmi mutants, the ratio was 25:38. Targeted expression of Flamingo in R8 rescued the R8 mutant phenotype (mutant + transgene ratio, 43:40). Consistent with a role for flamingo in mediating interactions between R8 axons, Flamingo protein shows a transient early burst of expression on R-cell axons in the optic stalk and as they enter the optic lobe. Strong labeling was observed in the youngest axons, with decreased expression in more mature axon bundles. The earliest defect detected in the R8 pattern was seen at a stage in development when Flamingo is expressed in R8 axons but not on surrounding cells. These findings support a role for Flamingo in permitting R8 growth cones to separate from each other in an orderly fashion after exiting the optic stalk and entering their target field in the medulla (Lee, 2003).

Results indicate that the protocadherin Flamingo is crucially involved in regulating target selection in two distinct classes of photoreceptors. In particular, R1-R6 cells lacking flamingo function terminate in the correct ganglion, the lamina, but fail to select the appropriate pattern of post-synaptic targets. Within this layer, Flamingo protein expression is largely confined to afferent axons and is highly dynamic during the critical developmental stages when R1-R6 axons are extending to their targets. R8 cells lacking fmi function have defects in the local topographic mapping of their terminals. In this context, fmi seems to permit R8 growth cones to separate from one another in an orderly fashion as they exit the optic stalk and enter the target field, a phenomenon similar to its role in promoting the appropriate spatial separation of R1-R6 growth cones during target selection in the lamina. Given that Flamingo, like other cadherins, has been shown to mediate homotypic interactions in cell culture, it is proposed that Flamingo influences R-cell target selection by mediating specific interactions among afferent growth cones (Lee, 2003).

How does Flamingo contribute to the remarkable precision of R1-R6 target specificity? It is proposed that R1-R6 target selection within the lamina requires three sequential steps, two of which require Flamingo function. (1) The orientation of the projection pattern along the dorsoventral axis is set by the orientation of the ommatidium; this is reinforced by a weak dorsoventral signal in the target. Flamingo acts at this early step in the retina to promote the interactions between photoreceptor cell bodies underlying polarity. (2) At a later stage in development, Flamingo-mediated interactions between R1-R6 growth cones act prior to axon divergence to define the precise orientation, and hence the extension trajectory, of each growth cone. (3) In a third step, N-cadherin and Lar promote extension toward and/or recognition of specific lamina targets (Lee, 2003).

These results leave open several possibilities regarding the precise role of Flamingo in controlling R-cell growth cone trajectory. For example, Flamingo could mediate interactions between R1-R6 growth cones from the same ommatidium, between growth cones of neighboring ommatidia or both. In addition, the observation that Flamingo is strongly expressed by only a subset of R-cell axons at a critical developmental stage raises the possibility that Flamingo might mediate homotypic interactions between specific R-cell growth cones. Indeed, specific interactions between their growth cones are required for R cells to select their correct targets. On the other hand, it remains possible that Flamingo mediates heterotypic interactions with an as-yet unknown ligand, either amongst R-cell growth cones or between these growth cones and their targets (Lee, 2003).

It is important to note that Flamingo is unlikely to mediate a general or a non-specific adhesive interaction among R-cell axons that simply maintains fascicle structure. Indeed, no changes in the ultrastructural organization of R-cell axon fascicles were observed in animals in which all R-cell axons lack flamingo function. Since the cues that control the last steps in choosing a synaptic partner are likely to be delivered via cell-cell contact by short-range signals that function by changing growth cone shape and/or position, they are essentially adhesive in nature. Rather than being nonspecific, however, such interactions are likely to be highly regulated with respect to space, time and cell type. Viewed in this context, the precise and dynamic regulation of Flamingo expression in R-cell growth cones is found to be particularly striking (Lee, 2003).

The orientation process by which R-cell growth cones become polarized prior to extension is broadly similar to the orientation process that underlies the establishment of planar cell polarity in epithelial sheets. In both cases, for example, an underlying rearrangement of the cytoskeleton is likely to be critical. It is posited that the role of Flamingo protein in R-cell target selection may be molecularly related to its role in controlling the polarity of ommatidia and, more generally, of epithelial cells. It is important to note, however, that whereas Flamingo is required for this step, other planar cell polarity genes are not, indicating that Flamingo must act with a different, though perhaps overlapping, set of proteins in R-cell growth cones (Lee, 2003).

Using specific interactions with other R-cells to determine the precise orientation of their growth cones, R-cell axons encounter relatively few potential targets during subsequent extension to their appropriate targets. It is suggested that this may reflect a general strategy in which reducing the number of potential targets encountered by an afferent growth cone reduces the requirement for molecular complexity in the cellular recognition events regulating the formation of precise patterns of synaptic connections (Lee, 2003).

Hexagonal packing of Drosophila wing epithelial cells by the planar cell polarity pathway

The mechanisms that order cellular packing geometry are critical for the functioning of many tissues, but they are poorly understood. This problem was investigated in the developing wing of Drosophila. The surface of the wing is decorated by hexagonally packed hairs that are uniformly oriented by the planar cell polarity pathway. They are constructed by a hexagonal array of wing epithelial cells. Wing epithelial cells are irregularly arranged throughout most of development, but they become hexagonally packed shortly before hair formation. During the process, individual cell boundaries grow and shrink, resulting in local neighbor exchanges, and Cadherin is actively endocytosed and recycled through Rab11 endosomes. Hexagonal packing depends on the activity of the planar cell polarity proteins. It is proposed that these proteins polarize trafficking of Cadherin-containing exocyst vesicles during junction remodeling. This may be a common mechanism for the action of planar cell polarity proteins in diverse systems (Classen, 2005).

A link between the PCP pathway and epithelial repacking is suspected, because repacking occurs at the time that these proteins are thought to polarize. Therefore neighbor number and junction length variability was quantified at the time of hair outgrowth in different PCP mutants. For prickle (pk-sple13/26), neighbor number was quantitated over time (Classen, 2005).

pk-sple13/26 wings begin repacking at the same time as wild-type; however, the process is less successful. Whereas wild-type wings reduce the percentage of pentagonal cells from 34% to 13% by the time that hairs begin to emerge, pk-sple13/26 wings retain 21%. Thus, about 40% of the pentagonal cells that normally assemble boundaries with new neighbors (and become hexagonal) fail to do so in pk-sple mutants. Consistent with this, pk-sple wing epithelia contain abnormally high numbers of four-way vertices between cells. pk1 mutant wings are even more irregularly packed than pk-sple13/26 wings. A total of 62% of the pentagonal cells that would normally become hexagonal fail to assemble boundaries with new neighbors in pk1 wings. Even four-sided cells accumulate significantly in pk1 mutant wings. Individual cell contact lengths are also much more variable; while pk-sple13/26 boundary lengths were 9% more variable than wild-type, those of pk1 were 42% more variable. These data are consistent with the earlier observation that adult pk wings frequently contain pentagonal cells. These data suggest that the assembly of new cell boundaries and regularization of junction length do not occur efficiently in the absence of products of the Pk-Sple locus (Classen, 2005).

Packing defects of the hypomorphic Flamingo (fmi) allele, fmi(stan)3, are mild but significant. The null allele fmiE59 produces much stronger defects. The variability of individual junctional lengths in these cells is more than twice that of wild-type, and only 69% of fmiE59 mutant cells become hexagonal, compared with 78% in wild-type. Pentagonal cells persisted in fmiE59 mutants (27% compared with 13% in wild-type). This suggests that the majority of pentagonal cells fail to assemble boundaries with new neighbors when Fmi is missing (Classen, 2005).

The packing geometry was examined of two different frizzled (fz) alleles, fzR52 and fzP21. fzP21 mutant wings fall into two classes. While the majority of wild-type and PCP mutant wings initiate hair formation by 42 hr after puparium formation (APF) (at 22°C), a subset of fzP21 mutant wings does not. Since these wings were not apoptotic (as indicated by Caspase staining), they were included in the analysis and quantified separately. Even at 50 hr APF, their packing is much more irregular than that of wild-type . Defects in fzP21 mutant wings that do initiate hair formation by 42 hr APF are milder but still significant. fzR52 homozygotes do not produce viable pupae in these experiments, and homozygous mutant clones are small. These clones have even stronger packing defects than those of fzP21, suggesting that little repacking occurs in fzR52 homozygous tissue. Thus, Fz is needed to develop regular hexagonal packing (Classen, 2005).

stbm6 and dgo380 mutant wings have milder, but significant, alterations in the ratio of pentagons, hexagons, and heptagons and of four-way vertices. Both mutants, however, affect junction length variability more strongly than pk-sple13/26. Taken together, these data indicate that PCP mutant cells fail to efficiently assemble boundaries with new neighbors and cannot regularize their packing geometry (Classen, 2005).

To ask whether interfering with PCP polarity could alter the geometry of packing in wild-type cells, cells were examined surrounding PCP mutant clones with either autonomous (fmiE59) or nonautonomous (fzR52) effects on polarity. The frequency of pentagons, hexagons, and heptagons was examined in fzR52 and fmiE59 mutant clones, and in the areas of disturbed and normal Fmi polarity surrounding both. The mutant cells within both fzR52 and fmiE59 clones are abnormally packed. However, whereas the packing defects caused by Fmi clones are predominantly restricted to the clone and directly adjacent cells, Fz clones alter packing over long distances in wild-type tissue in the same regions where Fmi polarity is disturbed. The abnormal packing of wild-type cells surrounding fzR52 clones is unlikely to be a consequence of altered cell packing within the mutant clone, because fmiE59 mutant clones pack just as abnormally, but do not perturb packing in the surrounding tissue. This suggests that dominant reorientation of Fmi polarity by frizzled mutant clones disturbs the repacking of wild-type cells (Classen, 2005).

To investigate how the PCP proteins were localized during repacking, pupal wings were imaged for Fmi before, during, and after hexagonal packing. Since it is thought that PCP proteins do not polarize until shortly before hair formation, it was surprised to find that the subcellular distribution of Fmi is polarized in many areas of the wing before junction remodeling is initiated, even in late third instar wing discs and prepupal wings. Fz-GFP is distributed similarly. This polarity may have been missed because it exhibits less long-range coherence in imaginal discs and prepupal wings than it does later (Classen, 2005).

In prepupal wings, Fmi polarity is roughly proximal-distal in the region surrounding L3. Coherent Fmi polarity is lost at the beginning of the pupal period: this is exactly the time at which junction remodeling initiates. Although polarity is not coherent, Fmi is not uniformly distributed along cell boundaries. This can be clearly seen when Fmi localization is compared to that of E-Cadherin (Classen, 2005).

At pupal time TP1, Fmi polarization begins in vein cells as they contract their apical cross-section. Intervein regions contain only small groups of cells with coherent polarity, and the axes of these groups are not always proximal-distal. By TP2, Fmi polarity is coherent between larger groups of cells, although the axis of polarity is still mixed. Fmi polarity is aligned in large coherent domains along the proximal-distal axis by TP4, when hexagonal packing is completed, and it remains unchanged at TP5 when hairs emerge. In summary, PCP proteins polarize during larval and prepupal stages, alignment of polarity between cells is disturbed when junction remodeling begins, and long-range polarity is reestablished as hexagonal packing is completed. Early polarization of PCP proteins is consistent with the genetic requirement for fz and ds activity at this time to determine the axis of polarity, and it suggests that the feedback loop that organizes coupled proximal and distal domains probably acts during these early stages (Classen, 2005).

It was asked whether PCP proteins might affect packing by influencing recycling of junctional components. Therefore, it was asked whether PCP mutants enhance the hole formation caused by shi loss of function. Double mutant pupae were shifted to a subrestrictive temperature that never causes holes to form in shi mutants or in PCP mutants. When shi is combined with dgo380, stbm6, stbm153, stbmD, stan3, pk-spl1, or pk1, hole formation occurs even under these mild conditions. This raises the possibility that PCP proteins may worsen Cadherin recycling defects in dynamin mutant cells. Consistent with this, gaps in Cadherin arise more frequently in double shi;pk1 or shi;dgo380 mutant wings than in wings mutant for shi alone. This suggests that Cadherin is recycled less efficiently in the absence of PCP proteins (Classen, 2005).

Despite this enhancement, no striking abnormalities in Cadherin distribution were seen in most PCP mutants. fzP21 mutant cells sometimes show gaps in E-Cadherin that are similar to, but much less frequent than, those of shi mutants. In fmiE59 mutant cells, E-Cadherin levels are elevated, but no gaps in localization are observed. These observations suggest that PCP proteins are not required for delivery of Cadherin to cell contacts during remodeling. Nevertheless, the PCP mutants enhance Cadherin recycling defects caused by loss of Dynamin. One model consistent with this shows that PCP proteins bias Cadherin recycling to specific places on the cortex. Reducing both the rate of recycling and its elevation at a particular site could exacerbate the failure of Cadherin delivery to growing cell boundaries (Classen, 2005).

To test whether exocyst components were polarized by PCP proteins, Sec5 localization was examined during repacking of the wing epithelium. At this time, cell shapes are irregular, and Fmi polarity is not coherent between cells. Nevertheless, Fmi accumulates preferentially on specific regions of the cortex. Although Sec5 vesicles are seen throughout the cell, they are particularly enriched near Fmi-positive cell boundaries. Enrichment persists as Fmi polarity becomes aligned (Classen, 2005).

To test whether Fmi plays an active role in recruiting Sec5, Fmi was overexpressed and Sec5 localization was examined. Overexpressed Fmi is present uniformly around the cortex and in large punctate structures within the cell. Sec5 dramatically accumulates in cells overexpressing Fmi and is recruited to sites of Fmi localization. Large internal structures positive for Fmi and Sec5 also contain Cadherin. These observations indicate that Fmi can recruit Sec5-positive vesicles containing E-Cadherin, and they suggest that PCP proteins may promote hexagonal packing by polarizing membrane trafficking (Classen, 2005).

The conserved cassette of PCP proteins controls a variety of seemingly different developmental processes, and no common cell biological mechanism has ever been proposed for their action. Polarizing membrane trafficking by recruiting Sec5 is a basic function that could be utilized in many different contexts, and it may help explain the requirement of PCP proteins in a divergent set of processes. Both rotation of photoreceptor clusters and convergent extension movements depend on the ability of cells to make and break intercellular contacts, as they do during hexagonal packing in the wing. Consistent with this, Silberblick (Wnt-11) acts through the PCP pathway and appears to affect endocytic trafficking of Cadherin during zebrafish gastrulation. Recruitment of exocyst components might also be a plausible mechanism to explain the ability of PCP proteins to bias Notch Delta signaling between R3 and R4 photoreceptors, since Delta delivery is dependent on the exocyst. In the future, identifying the chain of events that leads from PCP protein localization to exocyst recruitment may increase the understanding of these important processes (Classen, 2005).

Potential dual molecular interaction of the Drosophila 7-pass transmembrane cadherin Flamingo in dendritic morphogenesis

Seven-pass transmembrane cadherins (7-TM cadherins) play pleiotropic roles in epithelial planar cell polarity, shaping dendritic arbors and in axonal outgrowth. In contrast to their role in planar polarity, how 7-TM cadherins control dendritic and axonal outgrowth at the molecular level is largely unknown. Therefore, extensive structure-function analysis was performed of the Drosophila 7-TM cadherin Flamingo (Fmi), and the activities of individual mutant forms were investigated mostly in dendritogenesis of dendritic arborization (da) neurons. One of the fmi-mutant phenotypes was overgrowth of branches in the early stage of dendrite development. In da neurons but not in their adjacent non-neuronal cells, expression of a truncated form (DeltaN) that lacks the entire cadherin repeat sequence, rescues flies, at least partially, from this phenotype. The truncated form still retains HRM (hormone-receptor domain), a motif of about 60 amino acids long conserved in the subfamily of G-protein coupled receptors (GPCRs); conserved cysteine residues in HRM have been implicated in transmitting signals of ligand binding to intracellular components. The HRM of the truncated protein might still be able to interact with a yet-to-be-identified ligand and therefore be able to limit branch elongation (Kimura, 2006).

Another phenotype is observed at a later stage, when dendritic terminals outgrowing from the contralateral sides meet and then avoid each other. In the fmi mutant, by contrast, those branches overlap. Overexpression of the DeltaN form on the wild-type background phenocopies the overlap phenotype in the mutant, and analysis in heterologous systems supports the possibility that this effect might be because the Fmi-Fmi homophilic interaction is inhibited by DeltaN. It is proposed that a dual molecular function of Fmi play pivotal roles in dendrite morphogenesis. In the initial growing phase, Fmi might function as a receptor for an as yet unidentified ligand and this hypothetical heterophilic interaction would be responsible for limiting branch elongation. At a later stage, homophilic Fmi-binding at dendro-dendritic interfaces would elicit avoidance between dendritic terminals (Kimura, 2006).

On the basis of extensive structure-function analysis, a hypothesis for the molecular functions of Fmi is proposed. To control dendritic morphogenesis in the embryo, one likely molecular function of Fmi is that of a receptor for a yet-to-be-identified ligand and that this hypothetical Fmi-ligand interaction is responsible for appropriate pausing of branch elongation. This hypothesis also explains an axon-retraction effect by Fmi overexpression in the mushroom body. The partial rescue activity of DeltaN::EYFP (the truncated form of Fmi attached to Enhanced yellow fluorescent protein) could be due to weak binding to such a hypothetical ligand. DeltaN::EYFP retains its HRM domain and, consistently, structure-function analysis of the mammalian 7-pass transmembrane cadherin Celsr2 also implies a functional role for an extracellular subregion that includes the HRM domain (Shima, 2004). These results suggest that the role of this domain to control dendritic growth is conserved among species (Kimura, 2006).

In contrast to rescue activity of DeltaN::EYFP towards the embryonic overextension phenotype, DeltaN::EYFP is a loss-of-function and dominant-negative form in the inhibitory interaction at dendro-dendritic interfaces in the larval stage. The molecular nature of DeltaN::EYFP was investigated in imaginal discs and in cultured cells. Fmi molecules in DeltaN::EYFP-expressing cells in the disc are not held back in the ER or Golgi on their way to cell membranes. In addition, coexpression experiments in cultured cells showed that two treatments, DeltaN::EYFP bound to Fmi and DeltaN::EYFP expression, did not dramatically alter the distribution of Fmi at the plasma-membrane. These results suggest the possibility that DeltaN::EYFP-Fmi complexes stay on the cell surface but out of contact sites, where abutting cells express DeltaN::EYFP. The simplest explanation of the effect of DeltaN::EYFP expression at dendro-dendritic interfaces is that Fmi-Fmi interaction plays a role in the mutual avoidance during dynamic cycles of terminal extension and retraction, and this interaction is supported by homophilic interaction of cadherin domains. In addition to the likely role of this trans Fmi-Fmi homophilic interaction, other possibilities are not excluded. For example, cis or lateral interaction of Fmi might recruit other cell surface receptors and ligands responsible for the bi-directional signaling for avoidance, such as Eph and ephrin (Kimura, 2006).

Different results of DeltaN::EYFP expression in the two distinct rescue experiments is interpreted such that Fmi exerts two types of molecular interactions. Although both the full-length form (Fmi::EYFP) and the short form (DeltaN::EYFP) are produced at similar levels in da neurons of transgenic flies, it is difficult to totally rule out the possibility that different processes of dendritogenesis (elongation vs interneuronal avoidance) require different threshold levels of protein activity. The hypothesis needs to be further tested by investigating functional interactions between Fmi and other molecules that operate in dendritogenesis, and by pursuing other approaches to identify binding partners of Fmi (Kimura, 2006).


Mcelsr1 encodes a protein of 3034 amino acids predicted to contain seven membrane spanning domains having homology to a group of peptide hormone binding G-protein coupled receptors. Its extracellular domain comprises epidermal growth factor-like repeats, laminin A G-domains and cadherin repeats. Homologous genes have been identified in C. elegans and D. melanogaster suggesting that the Celsr gene family is ancient. mCelsr1 mRNA expression precedes gastrulation, is subsequently restricted primarily to ectodermal derivatives and is tightly regulated in the developing central nervous system (CNS). Segmentally-restricted gene expression in the developing hindbrain and in the spinal cord dynamic dorso-ventrally restricted 'stripes' of expression is observed (Hadjantonakis, 1998).

To identify large proteins with an EGF-like-motif in a systematic manner, a computer-assisted method called motif-trap screening has been developed. The method exploits 5'-end single-pass sequence data obtained from a pool of cDNAs whose sizes exceed 5 kb. Using this screening procedure, five known and nine new genes for proteins with multiple EGF-like-motifs were identified from 8000 redundant human brain cDNA clones. These new genes were found to encode a novel mammalian homolog of Drosophila Fat protein; two seven-transmembrane proteins containing multiple cadherin and EGF-like motifs; two mammalian homologs of Drosophila Slit protein; an unidentified LDL receptor-like protein, and three totally uncharacterized proteins. The organization of the domains in the proteins, together with their expression profiles and fine chromosomal locations, has indicated their biological significance, demonstrating that motif-trap screening is a powerful tool for the discovery of new genes that have been difficult to identify by conventional methods (Nakayama, 1998).

cDNAs have been isolated for three members of a family of seven-pass transmembrane cadherins in mouse (Celsr1, 2 and 3). These three genes represent vertebrate homologs of flamingo/starry night, recently identified as an essential component of the Drosophila planar cell polarity pathway and for the correct formation of dendritic fields within the Drosophila peripheral nervous system. Each member of the mouse Celsr family exhibits distinct patterns of expression within a range of different tissues within the developing embryo. Celsr1 and Celsr2 expression is observed during gastrulation and within the developing nervous system. Celsr3 transcripts, however, are found only at sites of active neurogenesis (Formstone, 2001).

Celsr1 is expressed in the vicinity of the primitive streak during gastrulation. Further analysis within the late gastrulating embryo reveals that the Celsr1 expression domain lies predominately within the primitive streak. In contrast, Celsr2 expression is within anterior neural ectoderm. The contrasting expression patterns exhibited by Celsr1 and Celsr2 persist within the developing embryo at later stages. At the 7 somite stage, Celsr2 transcripts are up-regulated within the anterior ventral midline and restricted to rhombomere (r) 3 within the developing hindbrain. By 9.5 dpc, Celsr2 expression is uniformly expressed within the caudal neural tube and is absent from the isthmus region. Celsr2 transcripts are, however, up-regulated within r1 and are restricted to rhombomere boundaries. Celsr1 expression is also observed within rhombomere boundaries at this stage. At 9.5 dpc, Celsr3 exhibits punctate dorsal neural tube expression anterior to the level of the developing forelimb bud. Celsr3 expression is additionally observed within the ventral neural tube with expression restricted to r2 and r4 within the early developing hindbrain. Celsr3 is also expressed within the early peripheral nervous system. Further striking patterns of Celsr expression are revealed during spinal cord development. Within this tissue, the Celsr family appear to define different neuroepithelial cell populations. A complementary pattern of expression is clearly evident between Celsr2 and Celsr3 at 12 dpc. The sequential expression of the family within the spinal cord may reflect a progression in the maturation of neuronal precursors. Additionally, RNA transcripts for all three members of the Celsr family are observed within the dorsal root ganglia (Formstone, 2001).

Drosophila Flamingo is a 7-pass transmembrane cadherin that is necessary for dendritic patterning and axon guidance. How it works at the molecular level and whether homologs of Flamingo play similar roles in mammalian neurons or not have been unanswered questions. Loss-of-function analysis using an RNAi system and organotypic brain slice cultures have been performed to address the role of a mammalian Flamingo homolog, Celsr2. Knocking down Celsr2 results in prominent simplification of dendritic arbors of cortical pyramidal neurons and Purkinje neurons, and this phenotype seemed to be due to branch retraction. Cadherin domain-mediated homophilic interaction appears to be required for the maintenance of dendritic branches. Furthermore, expression of various Celsr2 forms elicits distinct responses that are dependent on an extracellular subregion outside the cadherin domains and on a portion within the carboxyl intracellular tail (Shima, 2004).

To gain mechanistic insight into the molecular function of Celsr2, whether or not the siRNA-induced phenotype could be rescued by coexpressing each of several deletion forms of Celsr2A was addressed. Expression constructs that contained the target recognition sequence of the siRNA were made from Celsr2AA2088T cDNA, which had a silent point mutation within the target sequence of siRNA2078. The extracellular region of Celsr2 was dissected into two subregions: a string of tandemly repeated cadherin domains (CR) and the more membrane-proximal subregion that contains motifs such as EGF-like domains, laminin G domains, and a hormone receptor domain (HRM). This membrane-proximal subregion was designated as the EGF-HRM region. Expression of deltaEGFHRM-A, an A form without the EGF-HRM region, rescued the knockdown phenotypes of pyramidal neurons and Purkinje neurons, whereas deltaCR-A and deltaEX-A, in which the cadherin domains were totally deleted, did not (Shima, 2004).

Two forms were studied that had modified intracellular or transmembrane domains. One was Celsr2B, which lacked part of the carboxyl intracellular tail that includes residues conserved among the Fmi/Celsr family, and the other was Ex-1TM, in which the entire 7-pass transmembrane domain and the carboxyl tail were substituted with the single-pass domain of N-cadherin. Neither Celsr2B nor Ex-1TM recovered the effect of the siRNA. The results of this structure-function analysis show that the cadherin repeats as well as the carboxyl intracellular portion are required for rescuing the siRNA-induced dendritic malformation. In all of these attempts to rescue the knockdown phenotypes described in this study, dendritic morphology as observed at 3 days in vitro (DIV). In parallel with the coexpression of each Celsr2 form with the siRNA, each form alone was also expressed; expression of any form by itself did not give rise to statistically significant morphological effects when compared with the control protein expression at 3 DIV (Shima, 2004).

Frizzled3a and Celsr2 function in the neuroepithelium to regulate migration of facial motor neurons in the developing zebrafish hindbrain

Migration of neurons from their birthplace to their final target area is a crucial step in brain development. This study shows that expression of the off-limits/frizzled3a (olt/fz3a) and off-road/celsr2 (ord/celsr2) genes in neuroepithelial cells maintains the facial (nVII) motor neurons near the pial surface during their caudal migration in the zebrafish hindbrain. Celsr2 (for cadherin, EGF-like, LAG-like and seven-pass receptor), is a vertebrate homolog of Drosophila Flamingo. In the absence of olt/fz3a expression in the neuroepithelium, nVII motor neurons extended aberrant radial processes towards the ventricular surface and mismigrated radially to the dorsomedial part of the hindbrain. These findings reveal a novel role for these genes, distinctive from their already known functions, in the regulation of the planar cell polarity (i.e. preventing integration of differentiated neurons into the neuroepithelial layer). This contrasts markedly with their reported role in reintegration of neuroepithelial daughter cells into the neuroepithelial layer after cell division (Wada, 2006).

The present finding that neuroepithelial cells are involved in positioning specific neurons near the pial surface suggests a fundamental role for the neuroepithelium in brain development. In the mammalian cortex, neurons are generated in ventricular germinal zones and migrate radially towards the pial surface to form architectural layered structures. In mouse embryos, Reelin signaling regulates the positioning of neurons during layer formation of the cerebrum, and is essential for radial migration of the nVII motor neurons. These data suggest that similar mechanisms regulate the proper positioning of both the hindbrain motor neurons and the cortical layer neurons (Wada, 2006).

In the mouse cerebral cortex, many wnt and frizzled family genes are expressed in gene-specific regional and lamina patterns. Such patterned expression suggests the possibility that these genes are involved in other aspects of brain development. Recent studies have shown that functional fzd3 and celsr3 genes are required for the development of the anterior commissure, and the cortico-subcortical, thalamocortical and corticospinal tracts. It is possible that the mouse fzd3 and celsr3 genes regulate neuroepithelial cells to guide these axonal tracts to the proper region in a similar manner to that by which the zebrafish fz3a and celsr genes act in neuroepithelial cells to restrict the migrating nVII motor neurons near the pial surface of the hindbrain. The demonstration of a role for neuroepithelial cells in preventing integration of differentiated neurons into the neuroepithelial layer may provide new insights into the general mechanisms underlying the formation of layered structures in the mammalian brain, such as in the cerebral cortex (Wada, 2006).

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).

Flamingo regulates epiboly and convergence/extension movements through cell cohesive and signalling functions during zebrafish gastrulation

During vertebrate gastrulation, the body axis is established by coordinated and directional movements of cells that include epiboly, involution, and convergence and extension (C&E). Recent work implicates a non-canonical Wnt/planar cell polarity (PCP) pathway in the regulation of C&E. The Drosophila atypical cadherin Flamingo (Fmi) and its vertebrate homologue Celsr, a 7-pass transmembrane protein with extracellular cadherin repeats, regulate several biological processes, including C&E, cochlear cell orientation, axonal pathfinding and neuronal migration. Fmi/Celsr can function together with molecules involved in PCP, such as Frizzled (Fz) and Dishevelled (Dsh), but there is also some evidence that it may act as a cell adhesion molecule in a PCP-pathway-independent manner. This study shows that abrogation of Celsr activity in zebrafish embryos results in epiboly defects that appear to be independent of the requirement for Celsr in PCP signalling during C&E. Using a C-terminal truncated form of Celsr that inhibits membrane presentation of wild-type Celsr through its putative pro-region, a hanging drop assay reveals that cells from embryos with compromised Celsr activity have different cohesive properties from wild-type cells. It is disruption of this ability of Celsr to affect cell cohesion that primarily leads to the in vivo epiboly defects. In addition, Lyn-Celsr, in which the intracellular domain of Celsr is fused to a membrane localisation signal (Lyn), inhibits Fz-Dsh complex formation during Wnt/PCP signalling without affecting epiboly. Fmi/Celsr therefore has a dual role in mediating two separate morphogenetic movements through its roles in mediating cell cohesion and Wnt/PCP signalling during zebrafish gastrulation (Carreira-Barbosa, 2009).

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).

Celsr3 is required in motor neurons to steer their axons in the hindlimb

The cadherin Celsr3, homolog of Drosophila Flamingo, regulates the directional growth and targeting of axons in the CNS, but whether it acts in collaboration with or in parallel to other guidance cues is unknown. Furthermore, the function of Celsr3 in the peripheral nervous system is still largely unexplored. This study shows that Celsr3 mediates pathfinding of motor axons innervating the hindlimb. In mice, Celsr3-deficient axons of the peroneal nerve segregate from those of the tibial nerve but fail to extend dorsally, and they stall near the branch point. Mutant axons respond to repulsive ephrinA-EphA forward signaling and glial cell-derived neurotrophic factor (GDNF). However, they are insensitive to attractive EphA-ephrinA reverse signaling. In transfected cells, Celsr3 immunoprecipitates with ephrinA2, ephrinA5, Ret, GDNF family receptor alpha1 (GFRalpha1) and Frizzled3 (Fzd3). The function of Celsr3 is Fzd3 dependent but Vangl2 independent. These results provide evidence that the Celsr3-Fzd3 pathway interacts with EphA-ephrinA reverse signaling to guide motor axons in the hindlimb (Chai, 2014).

Transient tissue-scale deformation coordinates alignment of planar cell polarity junctions in the mammalian skin

Planar cell polarity (PCP) refers to the collective alignment of polarity along the tissue plane. In skin, the largest mammalian organ, PCP aligns over extremely long distances, but the global cues that orient tissue polarity are unknown. This study shows that Celsr1 (homolog of Drosophila Starry night) asymmetry arises concomitant with a gradient of tissue deformation oriented along the medial-lateral axis. This uniaxial tissue tension, whose origin remains unknown, transiently transforms basal epithelial cells from initially isotropic and disordered states into highly elongated and aligned morphologies. Reorienting tissue deformation is sufficient to shift the global axis of polarity, suggesting that uniaxial tissue strain can act as a long-range polarizing cue. Observations both in vivo and in vitro suggest that the effect of tissue anisotropy on Celsr1 polarity is not a direct consequence of cell shape but rather reflects the restructuring of cell-cell interfaces during oriented cell divisions and cell rearrangements that serve to relax tissue strain. Cell intercalations remodel intercellular junctions predominantly between the mediolateral interfaces of neighboring cells. This restructuring of the cell surface polarizes Celsr1, which is slow to accumulate at nascent junctions yet stably associates with persistent junctions. It is proposed that tissue anisotropy globally aligns Celsr1 polarity by creating a directional bias in the formation of new cell interfaces while simultaneously aligning the persistent interfaces at which Celsr1 prefers to accumulate (Aw, 2016).

A non-autonomous function of the core PCP protein VANGL2 directs peripheral axon turning in the developing cochlea

The cochlea is innervated by neurons that relay sound information from hair cells to central auditory targets. A subset of these are the type II spiral ganglion neurons, which have nociceptive features and contribute to feedback circuits providing neuroprotection in extreme noise. Type II neurons make a distinctive 90 degrees turn towards the cochlear base to synapse with 10-15 outer hair cells. This study demonstrates that this axon turning event requires planar cell polarity (PCP) signaling and is disrupted in Vangl2 and Celsr1 knockout mice, and that VANGL2 acts non-autonomously from the cochlea to direct turning. Moreover, VANGL2 is asymmetrically distributed at intercellular junctions between cochlear supporting cells, and in a pattern that could allow it to act directly as an axon guidance cue. Together, these data reveal a non-autonomous function for PCP signaling during axon guidance occurring in the tissue that is innervated, rather than the navigating growth cone (Ghimire, 2018).


Search PubMed for articles about Drosophila starry night

Abe, M., et al. (2009). Membrane protein location-dependent regulation by PI3K (III) and rabenosyn-5 in Drosophila wing cells. PLoS ONE 4: e7306. PubMed Citation: 19798413

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Aw, W. Y., Heck, B. W., Joyce, B. and Devenport, D. (2016). Transient tissue-scale deformation coordinates alignment of planar cell polarity junctions in the mammalian skin. Curr Biol [Epub ahead of print]. PubMed ID: 27451904

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Carreira-Barbosa, F., et al. (2009). Flamingo regulates epiboly and convergence/extension movements through cell cohesive and signalling functions during zebrafish gastrulation. Development 136(3): 383-92. PubMed Citation: 19091770

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Chai, G., Zhou, L., Manto, M., Helmbacher, F., Clotman, F., Goffinet, A. M. and Tissir, F. (2014). Celsr3 is required in motor neurons to steer their axons in the hindlimb. Nat Neurosci 17: 1171-1179. PubMed ID: 25108913

Chen, W.-S., et al. (2008). Asymmetric homotypic interactions of the atypical cadherin Flamingo mediate intercellular polarity signaling. Cell 133: 1093-1105. PubMed Citation: 18555784

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Das, G., Reynolds-Kenneally, J. and Mlodzik, M. (2002). The atypical cadherin Flamingo links Frizzled and Notch signaling in planar polarity establishment in the Drosophila eye. Dev. Cell 2: 655-666. 12015972

Devenport, D. and Fuchs, E. (2008). Planar polarization in embryonic epidermis orchestrates global asymmetric morphogenesis of hair follicles. Nat Cell Biol 10: 1257-1268. PubMed ID: 18849982

Devenport, D., Oristian, D., Heller, E. and Fuchs, E. (2011). Mitotic internalization of planar cell polarity proteins preserves tissue polarity. Nat. Cell Biol. 13: 893-902. PubMed Citation: 21743464

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Gaengel, K. and Mlodzik, M. (2003). Egfr signaling regulates ommatidial rotation and cell motility in the Drosophila eye via MAPK/Pnt signaling and the Ras effector Canoe/AF6. Development 130: 5413-5423. 14507782

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Biological Overview

date revised: 29 September 2022

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