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doi: 10.1242/10.1242/dev.00361

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Rac promotes epithelial cell rearrangement during tracheal tubulogenesis in Drosophila

Takahiro Chihara^1,2,*, Kagayaki Kato^1,3, Misako Taniguchi², Julian Ng⁴ and Shigeo Hayashi^1,2,3,

¹ Department of Genetics, Graduate University for Advanced Studies, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka-ken 411-8540 Japan
² Genetic Strain Research Center, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka-ken 411-8540 Japan
³ Morphogenetic Signaling Group, Riken Center for Developmental Biology, 2-2-3, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, 650-0047 Japan
⁴ Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA
^* Present address: Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA

View larger version (48K): [in a new window]   Fig. 1. Rac is required for apical localization of E-Cadherin and negatively regulates protein levels in the cadherin cell adhesion system. (A-B) Optical sections of the ventral epidermis of stage 15 embryos labeled for E-Cadherin (green) and Fasciclin 3 (purple). Grayscale images of each channel are shown below. In wild-type embryos, the apical concentration of E-Cadherin marks a distinct Fasciclin 3-free domain (A). In embryos zygotically null for Rac1 and Rac2 (Rac1, 2) this distinction is lost and many cells become flattened, with E-Cadherin localized over the entire cell surface (B). (C-D) Head epidermis of stage 6 embryos stained for E-Cadherin (green) and F-actin (purple). Cells in D have formed multiple cell layers and E-Cadherin is mislocalized. Grayscale images of each channel are shown below. (E) Western blot analysis of cadherins and their associated molecules in embryos with different levels of Rac activity. Arrows and arrowheads indicate mature and degradation products of each molecule, respectively. Arrowheads with an asterisk indicate degradation products of catenins, which were decreased in amount when Rac activity was reduced. Protein extracts of 15 embryos of each genotype were analyzed, and protein loading was monitored by Coomassie Brilliant Blue staining of the gel, which varied within a range of 10%. (F) Protein and RNA quantification. Protein was quantified by densitometric scanning of the films exposed to chemiluminescence. mRNA amounts were measured by quantitative RT-PCR. The amounts of cadherins and catenins were expressed as fold increase from the values for the control y w strain and were standardized by assuming that the amount of PP2AA remained constant. Asterisks on the bars for the amounts of ß-Catenin protein indicate that the measurement was underestimated because of overexposure of the chemiluminescence. Scale bars: in A, 10 µm for A-B; in C, 10 µm for C-D.

View larger version (124K): [in a new window]   Fig. 2. Mislocalization of E-Cadherin in tracheal cells that have reduced Rac activity. (A-B) Wild-type embryonic trachea at stage 12 (A) and stage 14 (B). Cell nuclei are marked with nuclear localized ß-galactosidase expressed by btl-Gal4 (green in A,B). The apical cell surface is labeled with anti-Crumbs (purple in A,B). Grayscale images of anti-Crumb are shown in A' and B'. Tracheal branches migrate toward the positions labeled with broken circles in which Bnl/FGF is expressed (Sutherland et al., 1996). While tracheal branches extend, cells of the dorsal branch (DB) and dorsal trunk (DT) rearrange their relative positions and form thinner tubules. (C,D) Wild-type (C) and btl-Gal4/+; btl-Gal4/UAS-DRac1N17 (D) embryos were stained with monoclonal antibody 2A12, which labels the tracheal lumen. In Rac1N17-expressing trachea, phenotypes of zigzagged and/or truncated DT were similar to those of Rac1, 2 mutants (compare D with G). (E-H,I) Wild-type (E,F), Rac1, 2 mutant (G,H) and btl-Gal4/+; btl-Gal4/UAS-DRac1N17 (I) embryos were stained with anti-E-Cadherin (purple), and tracheal cells were marked with GFP-moesin (green) to reveal F-actin. Grayscale images of anti-E-Cadherin are shown separately. F and H are high-magnification images of the broken frames shown in E and G. Arrows and arrowheads indicate basal and apical cell membranes of tracheal cells, respectively (F,H). E-Cadherin have accumulated at the apical side of the lateral membrane in wild-type trachea (E,F). In Rac1, 2 mutant and btl-Gal4/+; btl-Gal4/UAS-DRac1N17 embryos, E-Cadherin has abnormally accumulated at the basal membrane of tracheal cells (G-I). In I, E-Cadherin accumulation in cell protrusions is also evident (arrows). Scale bars: 20 µm in B,E,G,I; 5 µm in F,H; 40 µm in C.

View larger version (75K): [in a new window]   Fig. 3. Reduction in Rac activity inhibits tracheal cell rearrangement. Time-lapse observations of btl-Gal4, UAS-GFP-moesin/+ (A-E) and btl-Gal4, UAS-GFP-moesin/UAS-DRac1N17 (F-J) embryos. High-magnification views are shown for DB3 (D,E) and DB5 (I,J). White asterisks in F and G indicate absence of DB sprouts. Blue asterisks and broken red lines in D,E,I,J indicate DB cell nuclei and lumens, respectively. In an example shown in I and J, two cells at the tip of DB extend numerous filopodia and lead migration, but no additional cells follows, leaving only a thin stretch of the cytoplasm. Scale bars: in A, 40 µm for A-C; in F, 40 µm for F-H; in D, 20 µm for D,E; in I, 20 µm for I,J.

View larger version (96K): [in a new window]   Fig. 4. Elevated Rac activity disrupts tracheal cell adhesion. (A-D) Time-lapse observations of a btl-Gal4, UAS-GFP-moesin/UAS-DRac1V12 embryo. By stage 12, tracheal branching is already delayed (A), and in the next 4 hours many of the tracheal cells are expelled in clusters of one to five cells. (E-H) High-magnification images of a btl-Gal4, UAS-GFP-moesin/UAS-DRac1V12 embryo. Tracheal cells have become spherical and detached from the tracheal cell cluster. Arrows follow the same tracheal cell. The thin arrow in H indicates a thin stalk of cytoplasm. (I,J) Activated Rac1 transforms the tracheal epithelium into a mesenchymal state. gfp-moesin-labeled cells (green) form large cell aggregates in which expression of apical cell marker Crumbs (purple in I, grayscale in I') and E-Cadherin (purple in J, grayscale in J') were greatly reduced. In J, E-Cadherin is undetectable in the trachea, although its expression in the malpighian tubule appears to be normal (arrows). Scale bars: in A,I,J, 40 µm for A-D,I,J; in E, 5 µm for E-H.

View larger version (71K): [in a new window]   Fig. 5. Rac1V12 prevents newly synthesized E-Cadherin from accumulating at the apical cell membrane. (A-D) Embryos with the genotypes of btl-Gal4, UAS-E-Cadherin-GFP / + (control: A,C) and btl-Gal4, UAS-E-Cadherin-GFP / +; UAS-Rac1V12 / + (Rac1V12: B,D) at two different stages were labeled with anti-GFP (green) to reveal E-Cadherin-GFP and with DCAD2 (purple) to reveal both E-Cadherin-GFP and endogenous E-Cadherin. Note that in the control (A,C) and in a stage 11 Rac1V12 embryo (B), anti-GFP and DCAD2 signals overlap, but in D the signals are distributed differently.

View larger version (52K): [in a new window]   Fig. 6. Genetic interaction of Rac and FGF signaling. (A-C) Phenotypes of embryos deficient in Rac and FGF signaling classified into three classes. Rac1, 2/Rac1, 2 (A, mild), Rac1, 2/Rac1, 2 (B, intermediate) and Rac1, 2/bnlP1 laid by Rac1, 2/+ mother (C, severe) were stained with monoclonal antibody 2A12 to label the tracheal lumen. In Rac1, 2 mutants, the DT is disrupted (A,B) with a defect in germband retraction (arrow in B). In Rac1, 2/bnlP1 mutants, no tracheal cell migration has taken place (C). (D,E) Partial rescue of the btl mutant phenotype by the expression of the constitutively active form of Rac1. (D) A btlOh10 mutant embryo at stage 12. Tracheal cells are labeled with a nuclear ß-galactosidase marker. No sign of branching is apparent. (E) A btlOh10 mutant embryo at stage 12 expressing Rac1V12 by the btl enhancer. GFP-moesin and dp-MAPK are shown in green and purple, respectively. Tracheal cells were able to move. (F) Genetic interactions involving Rac. Maternal and zygotic genotypes of scored embryos are indicated. Tracheal phenotypes were classified into `normal' (white bar), `weak' (yellow bar) and `intermediate' (blue bar), according to the number of truncated DT of none, one to three, and four to nine, respectively, per one side of embryos. The `severe' class (red bar) corresponds to the phenotype of no tracheal migration at all. `n' is the number of embryos observed. Scale bars: in A, 40 µm for A-C; in D, 20 µm for D,E.