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First published online 28 February 2006
doi: 10.1242/jcs.02832

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

¹ Department of Biophysics, Graduate School of Science, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8507, Japan
² Graduate School of Biostudies, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8507, Japan
³ Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Kawaguchi, Saitama, 332-0012, Japan

View larger version (18K): [in this window] [in a new window]   Fig. 1. Activities of mutant forms of Fmi under various developmental contexts. (Left) Schematic representation of the Fmi protein (3575 aa long) and various mutant forms designed by us. The extracellular region includes eight tandemly repeated cadherin repeats (Cadherin), five EGF-like domains (EGF-like), two laminin G domains (Laminin G), and a hormone receptor domain (HRM). (Right) Results corresponding to the mutant forms shown on the left. `Rescue from lethality' indicates whether the lethality of fmi-null mutants was prevented by expression of either form by using the pan-neuronal driver Gal4-1407 or not. Relevant genotypes were fmiE45 Gal4-1407/fmiE59; UAS-transgene/+. Relevant genotypes of `rescue from overgrowth phenotype', `overexpression in all da', `rescue of overlap phenotype' and `overexpression in ddaC' were as described in the legends of Figs 2, 3, 4, 5, 6, respectively. Data from the `cell aggregation assay' are shown in Fig. 8. `Effect of overexpression on PCP' indicates effects of overexpression on reorientation of wing hairs, as shown in Fig. 9A-D. +, rescue or formation of cell aggregates; - rescue did not occur or cells did not aggregate. Fmi and Fz, effect of overexpression of individual forms resembling that of Fmi and Fz overexpression, respectively (see details in Fig. 9A-D). N.D., not determined. Inactivity of HR::EYFP and CR:EYFP was reminiscent of results of in vivo structure-function analysis of DE-cadehrin (Oda and Tsukita, 1999), and we discussed this result and other constructs in Materials and Methods.

View larger version (83K): [in this window] [in a new window]   Fig. 2. Rescue of fmi mutant embryos from the dendritic overgrowth phenotype by mutant forms of Fmi. (A-I) Visualization by use of GFP of a subset of da neurons in the abdominal dorsal cluster in embryos at 20-22 hours AEL. In these and all subsequent panels of embryos and larvae, dorsal is at the top and anterior is to the left unless described otherwise. (A-C,F-I,L,M) Class I da neurons (ddaD and ddaE) and es neurons are labeled in the (A) wild type, (C) fmiE59/fmiE59 mutant and (F) a mutant expressing a transgene of either fmi, fmi::EYFP (G), C (H), or N::EYFP (I). Arrows indicate terminals of two da neurons, ddaD and ddaE [green cells in (B) tracing]; arrowheads indicate terminals of es neurons [magenta cell in (B) tracing]. In the tracing, one of the es neuron accessory cells (yellow) is also drawn. In contrast to the wild type (A,B), the fmi mutant extended the da dendrites more dorsally than es dendrites (C). Yellow bracket in C indicates lateral branches that showed undergrowth and/or misrouting. Neuronal expression of (F) Fmi and (G) Fmi::EYFP but not that of (H) Ctail, prevented both the dorsal overgrowth and malformation of lateral branches (bracket in F), whereas the rescue effect of N::EYFP was partial (I,J). Overextended dendrites of the contralateral counterpart are indicated by a magenta arrow (H). Detailed genotypes of individual panels are described in K. The Gal4 driver used in this rescue experiment was IH1. Although IH1-driven Fmi expression prevented the phenotype, it did not cure embryonic lethality of the fmiE59/fmiE59 mutant, probably because IH1 drove transgene expression only in a small subset of neurons of the CNS and PNS. (D,E) Dendritic morphology of a class IV ddaC in the (D) wild type or in (E) the fmi mutant. ddaC dendrites in the mutant overextended dorsally and a contralateral branch terminal is indicated by an arrow in E. Genotypes were (D) NP1015 Venus-pm and (E) NP7028 GFP[S65T] fmiE59/NP7028 GFP[S65T] fmiE59. (J,K) Quantitative analysis of the rescue experiments. Bars represent percentages of hemi-segments that showed the overgrowth in embryos of individual genotypes. Each of the full-length or mutant forms of Fmi was produced from two copies of each transgene in the mutant, except fmi; UAS-Fmi::EYFP (X1). *, statistically significant rescue (P<0.005), compared with the phenotypic penetrance of the mutant (Student's t-test). Table K summarizes genotypes and quantification. (L,M) Subcellular localizations of (L) Fmi::EYFP and (M) N::EYFP. Image of EYFP fluorescence of (L) IH1-GAL4/IH1-GAL4; UAS-fmi::EYFP/UAS-fmi::EYFP and of (M) IH1-GAL4/IH1-GAL4; N::EYFP/N::EYFP. Bar in M: 14.5 µm for A, 20 µm for C-I, 10 µm for L,M.

View larger version (86K): [in this window] [in a new window]   Fig. 3. Experiment showing partial rescue from the overgrowth phenotype by expression of N::EYFP. (A,B) Dorsal front views of embryos of 21-22 hours AEL in which a large subset of da neuron expressed GFP. Broken lines represent dorsal midlines. (A) In the wild-type embryo, dendritic terminals of ddaD and ddaE (D and E) did not reach the dorsal midline, and dendrite-free zones were observed (bracket). (B) In contrast, branch terminals in the fmi null mutant reached the midline before hatching. (C) N::EYFP expression partially prevented the dorsal overgrowth (bracket). Bar, 20 µm. (D) Distribution of the distance between the most dorsal tips of dendritic terminals of ddaD and ddaE and the dorsal midline in each hemi-segment. The label <0 on x-axis indicates that branch terminals extended beyond the midline and invaded contralateral hemi-segments. Relevant genotypes were Gal109(2)80 UAS-GFP[S65T]/IH1 UAS-GFP[S65T] (A, and Wild type in D), Gal109(2)80 UAS-GFP[S65T] fmi72/IH1 UAS-GFP[S65T] fmiE59 (B, and fmi mutant in D), Gal109(2)80 UAS-GFP[S65T] fmi72/IH1 UAS-GFP[S65T] fmiE59; UAS-fmi::EYFP/UAS-fmi::EYFP (Fmi::EYFP rescue in D), and Gal109(2)80 GFP[S65T] fmi72/IH1 GFP fmiE59; UAS-N::EYFP/UAS-N::EYFP (C, and N::EYFP rescue in D). Numbers of abdominal segments examined for individual genotypes are indicated.

View larger version (65K): [in this window] [in a new window]   Fig. 4. Overproduction of Fmi in the wild-type background caused underdevelopment of dendrites. (A) Dorsal clusters in a control embryo of 20-22 hours AEL where all da neurons expressed GFP. (B) Overproduction of the wild-type form of Fmi resulted in poor dendritic growth and a decrease in the number of branches. (C) Overproduction of N::EYFP did not cause an obvious morphological defect. Genotypes were IG1-2 Gal109(2)80 UAS-GFP[S65T]/IG1-2 Gal109(2)80 UAS-GFP[S65T] (A), IG1-2 Gal109(2)80 UAS-GFP[S65T]/IG1-2 Gal109(2)80 UAS-GFP[S65T]; UAS-fmi/UAS-fmi (B), and IG1-2 Gal109(2)80 UAS-GFP[S65T]/IG1-2 Gal109(2)80 UAS-GFP[S65T]; UAS-N::EYFP/UAS-N::EYFP (C). Bar, 30 µm. Embryos that were homozygous for IG1-2 Gal109(2)80 UAS-GFP[S65T] did not hatch, so we could not observe how Fmi overexpression in this genotype that had total four copies of Gal4 affected dendrite morphogenesis at larval stages.

View larger version (132K): [in this window] [in a new window]   Fig. 5. Fmi::EYFP prevented the dorsal-dendrite-overlap phenotype, whereas N::EYFP did not. (A-C and D-F) Dorsal front views of abdominal segments at 32-35 hours AEL and those at about 40 hours AEL, respectively. (A-C) A large subset of da neurons in dorsal clusters expressed GFP. (D-F) Essentially all da neurons except for ddaC were ablated in dorsal clusters of one hemi-segment and its contralateral counterpart by laser. Blue circles indicate cell bodies of the ddaC. Brackets indicate dendritic terminals that met midway between contralateral da neurons. Anterior is at the top left. (G-I) Tracing of dendritic branches that are shown in the respective panels D-F above. Dendrites that belonged to one of the adjacent hemi-segments are green or magenta, whereas intertwining branches that were difficult to track down are white. (A,D) Control larvae in which dendritic branches covered the whole body walls with minimum overlap. (B and E) In fmi mutant larvae, dendritic terminals that had extended from contralateral sides did not turn away, but rather overlapped or crossed with each other (bracket). Fmi::EYFP expression in da neurons in the fmi mutants rescued the larvae from the overlap phenotype (C), whereas N::EYFP expression did not confer an inhibitory interaction between terminals (F). Genotypes were Gal109(2)80 UAS-GFP[S65T]/IH1 UAS-GFP[S65T] (A and D), Gal109(2)80 UAS-GFP[S65T] fmi72/IH1 UAS-GFP[S65T] fmiE59 (B and E), and Gal109(2)80 UAS-GFP[S65T] fmi72/IH1 UAS-GFP[S65T] fmiE59; UAS-fmi/UAS-fmi (C), and Gal109(2)80 UAS-GFP[S65T] fmi72/IH1 UAS-GFP[S65T]fmiE59; UAS-N::EYFP/UAS-N::EYFP (F). Bars in I: 20 µm for A-C and 17 µm for D-I.

View larger version (119K): [in this window] [in a new window]   Fig. 6. N::EYFP overexpression on the wild-type background caused dendritic terminals to cross each other. Dorsal views of larvae at about 50 hours AEL in which dendrites of ddaC (class IV) were visualized. Anterior is at the bottom left. (A-C) Dendritic morphology of ddaC in a control larva (A) and a larva that expressed N::EYFP by using NP1161 in ddaC (B). The boxed area in B is magnified in C. (A'-C') Tracing of dendritic branches that are shown in each of the upper panels. The braches were colored like in Fig. 5G-5I. (D-F) Tracing of dendrites of other ddaC cells visualized by another driver, ppk-Gal4. (D) A control larva. (E and F) Larva that expressed N::EYFP. In the control larva, dendrites of ddaC did not cross over with those of contralateral counterparts (A, A' and D). By contrast, dendrites of N::EYFP-expressing ddaC failed to avoid each other and sometimes invaded contralateral hemi-segments (arrowhead in B', C,C',F). Genotypes were NP1161/NP1161; ppk-EGFP/ppk-EGFP (A and A'), NP1161/UAS-N::EYFP; ppk-EGFP/ppk-EGFP (B,C,B',C'), ppk-Gal4 UAS-mCD8-GFP/ppk-Gal4 UAS-mCD8-GFP (D), and ppk-Gal4 UAS-mCD8-GFP/ppk-Gal4 UAS-mCD8-GFP; UAS-N::EYFP/UAS-N::EYFP (E and F). Bars in F: 50 µm for A,B,A',B'; 25 µm for C,C'; 60 µm for D-F.

View larger version (144K): [in this window] [in a new window]   Fig. 7. N::EYFP expression decreased the level of Fmi at intercellular boundaries in wing epithelia. (A-I) N::EYFP was expressed by ptc-Gal4 in wing imaginal discs of 3rd instar larvae. Anterior is to the left. Imaginal discs were stained for endogenous Fmi (A, D, G; magenta in C,F,I), EYFP (B,E,H; green in C,F,I). (C,F,I) Merged images. (D-F) Higher-magnification images of AP boundaries. (A-F) When adjacent cells strongly expressed N::EYFP, the level of endogenous Fmi was reduced at cell-cell boundaries. (G-I) Boxed area in F shown higher-magnification image in I. Endogenous Fmi was localized at boundaries of cells that expressed N::EYFP at distinct levels (arrowheads in G,H). Bar in I: 11.5 µm for A-C; 5.5 µm for D-F; 2 µm for G-I.

View larger version (77K): [in this window] [in a new window]   Fig. 8. N::EYFP inhibited Fmi-dependent cell aggregation and might have interacted with Fmi in cultured cells. (A-F) S2 cells were transfected with a plasmid without insert (A) or with one of plasmids encoding various Fmi forms (B-F), together with an EGFP plasmid; and they were examined to see whether they formed aggregates or not. Cells that expressed Fmi (B), Fmi::EYFP (C), or Ctail (D) assembled, but those that expressed CR::EYFP (E) or N::EYFP (F) did not. (G,H) Coexpression of N::EYFP with Fmi inhibited Fmi-dependent cell aggregate formation. S2 cells were co-transfected with the EGFP plasmid and the Fmi expression plasmid, together with one without insert (G) or with the N::EYFP plasmid (H). (I) Fmi and HA-N::EYFP were expressed in HEK293T cells. The cell lysate (lane 1) and immunoprecipitates (IP) obtained with either anti-myc (negative control, lane 2), anti-HA (lane 3), or anti-Fmi (lane 4) were blotted with anti-Fmi antibodies. The arrowhead points to Fmi molecules. Fmi was coimmunoprecipitated with HA-N::EYFP (lane 3). (J,K) S2 cells were transfected with a plasmid expressing (J) membrane-bound Venus (Venus-pm) or with (K) N::EYFP plasmid, together with the Fmi expression plasmid. Cells were spread on ConA-coated dishes and stained for GFP (left panels; green in merged right panels) and Fmi (middle panels; magenta in merged right panels). We used 0.05% saponin to permeabilize plasma membranes in order to preserve intracellular vesicular structures. Over 20 cells were observed for each transfection experiment and all cells showed similar protein distributions to cells shown in J and K.

View larger version (92K): [in this window] [in a new window]   Fig. 9. Effects of Fmi or N::EYFP overexpression on planar cell polarity. (A-M) The effect of N::EYFP expression on planar polarity was addressed in (A-D,I,J) adult wings, (E-H) a pupal wing at 30 hours after puparium formation, and (K-M) an adult notum. Anterior is at the top (A-M), and proximal is to the left (A-J). (A) Control wing. Inset shows higher-magnification image of the boxed area. (B-D) Either (B) full-length Fmi, (C) N::EYFP or (D) Fz were expressed in a region between vein 3 and vein 4 by using ptc-Gal4, which drives short-range gradient expression along the anterior-posterior axis of the wing. Arrowhead in D indicates probable peak of the expression level in. Arrows indicate directions of reoriented wing. (E-H) Triple-staining of a N::EYFP-expressing pupal wing for (E) EYFP, (F) endogenous Fmi (see magenta in H) and (G) Dsh (see green in H). A small area that straddled the boundary of the expression domain is shown at high magnification. In contrast to the normal preferential P-D distribution (arrow in H), endogenous Fmi was located substantially at A-P boundaries (arrowhead in H) in the 2-3 cells-wide stripe adjacent to the N::EYFP-producing cells. See text for details. Bar in E: 5 µm for E-H. (I-M) N::EYFP-expressing clones were identified by X-Gal staining. Yellow-boxed areas in I and K are shown magnified in J and L; magenta-boxed area in K is shown enlarged in M. Notice that wing hairs (black box in J) and trichomes (L,M) are pointing away from the clone boundaries. The relevant genotype was hs-FLP; AyGal4 UAS-lacZ/UAS-N::EYFP.

View larger version (31K): [in this window] [in a new window]   Fig. 10. Models for dual Fmi functions in dendritic morphogenesis. Diagrams show models of putative Fmi function at two distinct phases of dendritic morphogenesis. (A) Branch outgrowth in the embryo. For simplicity, only ddaD and ddaE are illustrated. Fmi (indigo bars) that is expressed in neurons binds to an unknown ligand (red diamonds). Fmi-ligand binding (yellow star) elicits inhibitory signaling against branch extension. A dendritic branch that responded to this signal is indicated as a green bar (right panel). In this illustration, a source of this ligand is hypothesized to be located dorsally in non-neural cells. The secreted ligand is postulated to not propagate very far, but to stay close to its source. (B) Inhibitory communication at dendro-dendritic interfaces in larvae. Class IV da (ddaC) neurons extend dendritic terminals that encounter terminals of contralateral counterparts along the dorsal midline. Signaling occurs at the interfaces, triggerd by Fmi-Fmi homophilic interaction (magenta star). In turn, this triggers local signal transduction of heteroneuronal avoidance (green dendritic branches at the right).