Tadao Usui

Flamingo, a Seven-Pass Transmembrane Cadherin, Regulates Planar Cell Polarity under the Control of Frizzled

We identified a seven-pass transmembrane receptor of the cadherin superfamily, designated Flamingo (Fmi), localized at cell-cell boundaries in the Drosophila wing. In the absence of Fmi, planar polarity was distorted. Before morphological polarization of wing cells along the proximal-distal (P-D) axis, Fmi was redistributed predominantly to proximal and distal cell edges. This biased localization of Fmi appears to be driven by an imbalance of the activity of Frizzled (Fz) across the proximal/distal cell boundary. These results, together with phenotypes caused by ectopic expression of fz and fmi, suggest that cells acquire the P-D polarity by way of the Fz-dependent boundary localization of Fmi.

Axon Patterning Requires D N-cadherin, a Novel Neuronal Adhesion Receptor, in the Drosophila Embryonic CNS

We identified DN-cadherin, a novel Drosophila cadherin that is expressed in axons and in the mesoderm. Although DN-cadherin has diverged from vertebrate classic cadherins in terms of its extracellular structure, it still can form a complex with catenins and induce cell aggregation, as do the vertebrate molecules. Loss-of-function mutations of the gene resulted in either embryonic lethality or uncoordinated locomotion of adults. In the central nervous system of null mutant embryos, subsets of ipsilateral axons displayed a variety of aberrant trajectories including failure of position shifts, defective bundling, and errors in directional migration of growth cones. These results suggest that processes of axon patterning critically depend on DN-cadherin-mediated axon-axon interactions.

Asymmetric colocalization of Flamingo, a seven-pass transmembrane cadherin, and Dishevelled in planar cell polarization

The Drosophila wing provides an appropriate model system for studying genetic programming of planar cell polarity (PCP) [1-4]. 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 [5]. 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) [6-8]. Here, we visualized endogenous Dsh in the developing wing. A portion of Dsh colocalized with Fmi, and the distributions of both proteins were interdependent. Furthermore, Fz controlled the association of Dsh with cell boundaries, which association was correlated with the presence of hyperphosphorylated forms of Dsh. Our results, together with a recent study on Fz distribution [9], support the possibility that Fz, Dsh, and Fmi constitute a signaling complex and that its restricted localization directs cytoskeletal reorganization only at the distal cell edge.

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 [1] [2]. 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 [3]. Instead, the Frizzled (Fz) protein mediates a planar polarity signal that controls the anteroposteriorly oriented first division (pl) of SOPs [4]. Here, we report that Flamingo (Fmi), a seven-transmembrane cadherin [5], controls the planar polarity of sensory bristles and the orientation of the SOP pl division. Both the loss of function and overexpression of fmi disrupted bristle polarity. During mitosis of the SOP, the axis of the pl division and the positioning of the Numb crescent were randomized in the absence of Fmi activity. Overexpression of Fmi and Fz caused similar effects. The dependence of proper Fmi localization on Fz activity suggests that Fmi functions downstream of Fz in controlling planar polarity. We also present evidence suggesting that Fz also functions in the Wingless pathway to pattern sensory organs.

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, we recovered mutations in flamingo (fmi). fmi encodes a seven-transmembrane cadherin, previously shown to function in planar cell polarity and in dendritic patterning. Here, we show that 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.

A mosaic genetic screen for genes necessary for Drosophila mushroom body neuronal morphogenesis.

Neurons undergo extensive morphogenesis during development. To systematically identify genes important for different aspects of neuronal morphogenesis, we performed a genetic screen using the MARCM system in the 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. We report mutations that affect these different aspects of morphogenesis and phenotypically characterize a subset. We found that 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. Our 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.

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, we performed extensive structure-function analysis of the Drosophila 7-TM cadherin Flamingo (Fmi) and investigated the activities of individual mutant forms 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 (ΔN) that lacks the entire cadherin repeat sequence, rescues flies - at least partially - from this phenotype. 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 overlapped. Overexpression of the ΔN form on the wild-type background phenocopied the overlap phenotype in the mutant, and analysis in heterologous systems supported the possibility that this effect might be because the Fmi-Fmi homophilic interaction is inhibited by ΔN. We propose 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 a sofar-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.

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. Here we show 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. We further show 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, we propose that the Fmi-Esn complex, together with the PCP regulators and the Tricornered (Trc) signaling pathway, executes the repulsive interaction between isoneuronal dendritic branches.

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. We found that Golden Goal (Gogo) and Flamingo (Fmi), two cell-surface proteins involved in photoreceptor targeting, functionally interact in R8 photoreceptor axons. Our 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. We propose 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.

Sensory-Neuron Subtype-Specific Transcriptional Programs Controlling Dendrite Morphogenesis: Genome-wide Analysis of Abrupt and Knot/Collier

The transcription factors Abrupt (Ab) and Knot (Kn) act as selectors of distinct dendritic arbor morphologies in two classes of Drosophila sensory neurons, termed class I and class IV, respectively. We performed binding-site mapping and transcriptional profiling of these isolated neurons. Their profiles were similarly enriched in cell-type-specific enhancers of genes implicated in neural development. We identified a total of 429 target genes, of which 56 were common to Ab and Kn; these targets included genes necessary to shape dendritic arbors in either or both of the two sensory subtypes. Furthermore, a common target gene, encoding the cell adhesion molecule Ten-m, was expressed more strongly in class I than class IV, and this differential was critical to the class-selective directional control of dendritic branch sprouting or extension. Our analyses illustrate how differentiating neurons employ distinct and shared repertoires of gene expression to produce class-selective morphological traits.