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First published online 20 August 2003
doi: 10.1242/dev.00691

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The Na⁺/K⁺ ATPase is required for septate junction function and epithelial tube-size control in the Drosophila tracheal system

Sarah M. Paul^1,*, Melissa Ternet^1,*, Paul M. Salvaterra² and Greg J. Beitel^1,

¹ Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA
² Division of Neurosciences, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA

View larger version (77K): [in a new window]   Fig. 1. nrv2 and ATP mutations cause tracheal length and diameter defects. When compared with wild-type (WT) animals (A,C-E), nrv2 (B,F-I) and ATP (M-O) mutant trachea have increased length, diameter expansions and missing lumen segments. The tracheal defects in nrv2 homozygotes can be completely rescued by expressing the nrv2.2 isoform using the e22c-Gal4 driver (J,K) or partially rescued using the btl-Gal4 driver (L). Similarly, expression of the nrv2.1 isoform by the e22C-Gal4 driver could also completely rescue all tracheal defects in nrv2 mutants (Fig. 6H), whereas the btl-Gal4 driver produced only the same partial rescue seen with nrv2.2 (data not shown). (A-C,F,J,L,M,O) Lateral views of the dorsal trunk (DT) and transverse connective (TC). (D,G,K,N) Ventral views of the ganglionic branches (GB). (E,H,I) Dorsal views of the dorsal trunk. The animal in H lacks both zygotic and maternal nrv2. All images are of stage 16 embryos, except E,H,I, which show stage 15 embryos. Examples of lumenal gap regions are indicated with brackets. UAS-nrv2.1 or UAS-nrv2.2 expressed under the control of the e22c driver rescued the nrv2 tracheal phenotype to wild type in 8/9 and 19/21 animals respectively. Genotypes: (A,C-E) Oregon R; (B,F,G,I) nrv2nwu3; (H) nrv2nwu3/nrv2-23B from nrv2nwu3 germline clone (glc); (J,K) nrv2k13315e22C-Gal4/nrv2l(2)k04223 UAS nrv2.2; (L) nrv2l(2)k04223 btl-Gal4/nrv2l(2)k04223 UAS nrv2.2; (M,N) ATP04694; (O) ATPDTS1R1. Scale bar: in B, 10 µm for A,B; in O, 5 µm for C,F,J,L,M,O; in N, 5 µm for D,G,K,N; in E, 10 µm for E,H,I.

View larger version (94K): [in a new window]   Fig. 2. Tracheal tube size defects do not result from trans-epithelial diffusion barrier defects. The integrity of the septate junction diffusion barrier in tracheal and septate junction mutants was assessed by a dye permeability assay. For each mutant, DIC images of the trachea are shown in black and white (A-H) and the matched fluorescence image taken in the same focal plane is shown in color (a-h). Wild-type trachea exclude fluorescently labeled 10 kDa dextran dye injected into the body cavity (a; dotted lines outline tracheal tubes), while the trachea of Na+/K+ ATPase and other mutants are permeable to the dye which enters and fills their lumens (b-g). Permeability defects do not cause the observed tube-size defects as cor14* mutants have defective diffusion barriers (e) but have normal tracheal morphology (E). Furthermore, convoluted (conv) mutants have the identical tube-size defects as nrv2 (compare H with B) but do not have permeability defects (h). All mutants that failed to exclude dye from the trachea also failed to exclude it from the salivary gland. Genotypes: nrv2nwu3, ATPDTS1R2, cor14* is coracle14 plus additional unidentified genetic background, cystick13717b, megatracheaEA97, convolutedk6507b. In addition, coracle5, varicose3953b, neurexin IV14, neuroglian17, gliotactinJ29-41bl, nrv2l(2)k04223 btl-Gal4/nrv2l(2)k04223 UAS-nrv2.1 and nrv2l(2)k04223 btlGal4/nrv2l(2)k04223 UAS nrv2.2 animals have tracheal diffusion barrier defects, while coracle15, hindsight1142, nrv2k13315 e22C-Gal4/nrv2l(2)k04223 UAS-nrv2.2, nrv2k13315 e22C-Gal4/nrv2l(2)k04223 UAS nrv2.1 and coracle14(backcrossed) do not (data not shown). Scale bar: 10 µm.

View larger version (72K): [in a new window]   Fig. 3. Septate junction components are mislocalized or dramatically reduced in Na+/K+ ATPase mutants. In nrv2 mutants (B,D,F,H), Coracle (red, B,H), Discs Large (green, D) and FasIII (red, F) appear to be reduced and/or no longer have the septate junction localization seen in wild-type animals (A,C,E,G). Neurexin staining closely resembled Coracle staining and was mislocalized in nrv2 mutants (data not shown). Localization of Discs Lost (green, E,F), E-cadherin (green, G,H) and Armadillo (data not shown) was unaffected by nrv2 mutations (F,H). ATP null mutations caused the same mislocalization and defects in protein levels as do nrv2 mutations (data not shown). Basal surfaces are outlined in white, apical/lumenal surfaces in blue. For each marker examined, the wild-type and mutant images are matched pairs (e.g. A and B, and C and D), where the wild type is a heterozygous embryo imaged with the same settings, on the same slide, and in the same session as the mutant embryo to provide an internal reference for protein levels. Scale bar: in H'', 5 µm for A-D,A''-D'',E-H''; in D', 5 µm for A'-D'.

View larger version (36K): [in a new window]   Fig. 4. Na+/K+ ATPase localization to the septate junction depends on Nrv2 but not Coracle. In wild-type (A,B,C) and coracle mutant animals (E), the Na+/K+ ATPase (red A,C,E) localizes to the septate junction marked by Coracle in wild-type animals (green A,B). However, in nrv2 mutants (D) Na+/K+ ATPase staining is reduced and/or not localized to the septate junction. Na+/K+ ATPase staining (5 anti- subunit monoclonal) is undetectable in ATP mutants (data not shown). Basal surfaces are outlined in white, apical/lumenal surfaces in blue. Scale bar: in E'', 5 µm for A-E,A''-E''; in E', 5 µm for A'-E'.

View larger version (92K): [in a new window]   Fig. 5. Septate junctions control tracheal tube size through multiple pathways. Null mutations in the known septate junction genes coracle and neurexin cause the identical tracheal tube size phenotypes as null nrv2 mutations (C-E). Double null mutant combinations of nrv2 and coracle have the same phenotype as either single mutant (compare F with D and E), suggesting nrv2 and coracle act in the same pathway. Similarly, double null mutant combinations of nrv2 and gli are not more severe than the nrv2-null mutants (compare H with D). By contrast, varicose, convoluted and cystic mutations exacerbate the tracheal phenotypes of null nrv2 mutations (compare J with D,F,H,I; L with D,F,H,K; N with D,F,H,M), suggesting that varicose, convoluted and cystic do not function only in a single linear pathway with nrv2. Dotted lines indicate tracheal lumens. Genotypes: (A) Oregon R; (B) nrg17; (C) nrx4865; (D) nrv223B; (E) cor5; (F) nrv2nwu3cor5; (G) gliAE2-45; (H) nrv2nwu3gliJ29-7b; (I) convk6507b; (J) nrv2nwu3convk6507b; (K) vari3953b; (L) nrv2k04223vari3953b; (M) cysk13717b; (N) nrv2nwu3cysk13717b. Scale bar: 5 µm.

View larger version (78K): [in a new window]   Fig. 6. Both nrv2.1 and nrv2.2, but not nrv1 and nrv3, are required for and possess tube-size control activity. (A) Organization of the nrv2 locus. Exons common to both nrv2 isoforms are shown as red boxes, while exons specific for the nrv2.1 and nrv2.2 isoforms are shown in blue and green, respectively. (B-G) Tracheal tube diameter and length defects are caused by the injection of dsRNA corresponding to coracle (C), nrv2 common exons (D), nrv2.1 and nrv2.2 specific exons (E,F) into otherwise wild-type animals with tracheal GFP expression. Injection of these dsRNAs also causes septate junction barrier defects (data not shown). Injection of buffer (B) or nrv3 dsRNA (G) did not cause tracheal tube-size defects. (H,I) Expression of nrv2.1 in nrv2 homozygotes using the e22C-Gal4 driver rescued all tracheal morphology defects (H), but expression of nrv1 (I) or nrv3 (J) did not detectably rescue the nrv2 defects. Rescue by nrv2.2 is shown in Fig. 1H. Genotypes: (B-G) btl-Gal4 UAS-GFP; (H) nrv2k13315e22C-Gal4/nrv2k04223 UAS-nrv2.1; (I) nrv2k13315e22C-Gal4/nrv2k04223UAS-nrv1; (J) nrv2k13315e22C-Gal4/nrv223BUAS-nrv3. Scale bar: 5 µm.

View larger version (21K): [in a new window]   Fig. 7. Models for the role of the Na+/K+ ATPase in tube-size control via the septate junction. See text for detailed discussions. (A) One possible formulation of the genetic pathways controlling tracheal tube size and septate junction barrier function. This model is based on the barrier phenotypes of the shown mutants and on genetic interactions between these mutants and nrv2-null mutants. Comparison of these phenotypes and interactions divides the mutants into three classes. nrv2 appears to act in a linear genetic pathway with gliotactin and coracle (shown in blue) but in a parallel, partially redundant, or branching pathway to varicose and cystic (shown in red). convoluted may act either downstream of a varicose/cystic pathway (indicated in red) and/or in parallel pathways (indicated in green). (B) A molecular representation of the pathways shown in A to illustrate how nrv2/coracle (blue) and varicose (red) could both be required for septate junction barrier function, but act in genetically distinguishable pathways for tube-size control. Question marks indicate uncertainty in identity or subcellular localization. Only a subset of known septate junction proteins are shown.

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