starry night: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | 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 links: Precomputed BLAST | Entrez Gene
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
Summary:
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.

BIOLOGICAL OVERVIEW

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).


PROTEIN STRUCTURE

Amino Acids - 3589

Structural Domains

The predicted translational product of stan has 3575 amino acids. The Stan ectodomain has nine cadherin repeats, three cysteine-rich domains (Cys-rich), and two laminin A globular domains (LmA-G). A combination of Cys-rich and LmA-G domains is a common feature of many invertebrate members and some vertebrate molecules of the cadherin superfamily. In contrast to those of classic-type cadherins, the carboxy-terminal intracellular tail of Stan does not possess catenin-binding sequences, nor does it bind to catenins. Thus, Stan can be classified into the nonclassic-type subfamily. Compared with functionally characterized members of this superfamily, Stan is structurally unusual, as it is predicted to be a seven-pass transmembrane (7TM) protein. Sequences of the 7TM region show similarity to those of one particular family of G protein-coupled receptors: the first such protein to be isolated is a receptor for a peptide hormone, secretin. Many proteins of this secretin receptor family have been shown to increase the intracellular levels of cAMP and/or inositol phosphates upon ligand binding. Whether Stan is coupled to G proteins remains to be demonstrated. The stan gene is conserved across species: a Caenorhabditis elegans counterpart gene is present in cosmid clones F15B9 and W07G4 (GenBank), and two paralogs of mammalian receptors have been reported, that is, mouse Celsr1 (Hadjantonakis, 1998) and rat MEGF2 (Nakayama, 1998). In addition, mouse cDNA clones have been isolated that encode the entire protein of a third paralog, mouse Flamingo1 (Usui, 1999).


EVOLUTIONARY HOMOLOGS

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).


Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 6 May 2000

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