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First published online May 23, 2006
doi: 10.1242/10.1242/dev.02404

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Drosophila talin and integrin genes are required for maintenance of tracheal terminal branches and luminal organization

Department of Biochemistry and Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305-5307, USA.

View larger version (126K): [in a new window]   Fig. 1. Tracheal luminal phenotype of tendrils mutations. (A,B) Dorsal and lateral views of third instar larval tracheal system (Rühle, 1932). Positions of lateral trunk (LT), dorsal trunk (DT), dorsal branch (DB), fat body branch (FB) and visceral branch (VB) are indicated. Anterior is leftwards. Dorsal is upwards in B. (C) Fluorescence micrograph montage of two DBs and their terminal cells (corresponding to boxed region in A) in mosaic third instar larva. Tracheal system is labeled with DsRed; DB terminal cell clone on the right is marked with GFP. Each terminal cell forms over a dozen terminal branches that attach tightly to the underlying muscles. s, DB stalk connecting DB to DT. f, fusion branch formed by individual cell connecting DB to contralateral DB. Arrowhead, DB terminal cell nucleus. Broken line indicates continuation of DB outside focal plane. (D) Schematic of terminal cell with multiple terminal branches (cellular projections), each with a membrane-bound lumen coursing through it. (E) Wild-type (tendrils+) DB terminal cell clone induced in 2- to 4-hour-old embryo marked with cytoplasmic GFP and examined in wandering third instar larvae. (F) Fluorescent (left; `cytoplasm'), bright-field (middle; `lumen') and merged (right) images of boxed region in E. There is a single lumen (GFP-excluded region in left panel, refractile region in middle panel) in the center of each branch. (G) Similar view of a homozygous tendrils6-66 DB terminal cell clone. Multiple convoluted lumens of different sizes are present in a single thick branch. (H-J) TEM analysis of tendrils+ DB terminal cell clone induced as above and marked with GFP and CD2-HRP. (H) Clone visualized by DAB staining prior to sectioning. Broken line indicates approximate sectioning plane. (I) Section through terminal branch of the clone. An electron dense layer of DAB staining is present at the plasma membrane. A single lumen (red dot) is present in cross section through branch. tc, terminal cell; m, muscle. (J) Higher magnification of the lumen in I. (K-M) Similar analysis of a tendrils- (rhea79) clone. More than 25 lumen cross-sections are visible in L, all in the same terminal branch (as shown by absence of basal plasma membrane between lumens in M). (N) Fluorescence (left) and bright-field (right) images of tendrils13-8 DB clones (marked with GFP) that include one of the two terminal cells shown (arrow) as well as neighboring fusion (f) and stalk (s) cells. Only the mutant terminal cell (arrow) is affected. Mutant stalk and fusion cells, and neighboring tendrils+ terminal cell (arrowhead) are indistinguishable from wild type. (O) tendrils13-8 fat body (FB) terminal cell clone as in N. Multiple convoluted lumens are present in single terminal branch, similar to DB terminal cell clones in G and N. Scale bars: 100 µm in C; 25 µm in E; in G, 25 µm for F,G; 25 µm in N,O; in I, 2 µm for I,L; in J, 0.5 µm for J,M.

View larger version (69K): [in a new window]   Fig. 2. Effect of tendrils mutations on terminal cell branching. (A) Fluorescent images of homozygous tendrils+ and tendrils- DB terminal cells clones in sixth tracheal metamere of wandering third instar larvae. White boxes, region enlarged in bright-field image below showing lumen. Scale bars: 20 µm in cytoplasm; 10 µm in lumen. (B) Quantification of terminal branching phenotype of tendrils alleles. Number of terminal branches in DB terminal cell clone was counted and compared with number in control contralateral tendrils+ terminal cell. Values shown are mean (±s.e.m.). n, number of terminal cells analyzed. (C) Quantification of lumen phenotype of tendrils alleles. The number of lumens in each clone was counted and divided by the number of terminal branches. Values above six lumens per branch are plotted together because of difficulty in obtaining accurate counts at high lumen density. The severity of terminal branch loss (B) parallels the severity of the lumen phenotype (C) in this series of alleles.

View larger version (92K): [in a new window]   Fig. 3. Onset and progression of tendrils phenotype. (A-C) tendrils13-8 DB terminal cell clones (yellow) and contralateral tendrils+ control (red) at larval stages indicated. Lumen tracings of corresponding bright-field images are shown at right. At early L2 tendrils13-8 mutant cells resemble tendrils+ cells, whereas tendrils13-8 clones examined later in development have progressively more severe phenotypes. (D,E) tendrils13-8 DB terminal cell clone imaged live in early L3 larva (D, GFP fluorescence; D', bright-field) and again 48 hours later (E,E'). Numbers indicate specific branches; dots indicate distal end of air-filled lumen. Terminal branches (1, 2, 3) are lost and their lumens compact into soma during a 48-hour period. Contralateral tendrils+ control cell (not shown) continued to develop normally. (F,G) Similar analysis of tendrils6-66 DB terminal cell clone. The convoluted lumens of branches 2 and 3 are displaced into the more proximal branch and branch 2 becomes extremely thin (broken line) during 48-hour period. The lumen of branch 4 is also displaced, as monitored by the position of a lumen spur (arrowhead) with respect to branch 3. (H-K) Effect of elimination of maternal tendrils+ function on tendrils phenotype. Fluorescent images (H-K) and bright-field close-ups (H'-K') of tendrils+ (H) and tendrils- (I,J) DB terminal cell clones generated in animals derived from tendrils+ (H-J) or tendrils- (K) eggs. tendrils phenotype (I,I',J,J') is not enhanced when maternal tendrils+ is eliminated (K,K'). Scale bars: 25 µm. Bar in C applies to A-C; bar in E' applies to D,E; bar in G' applies to F,G; bar in K applies to H-K; bar in K' applies to H'-K'.

View larger version (29K): [in a new window]   Fig. 4. Mapping and molecular characterization of tendrils mutations. (A) Meiotic and deficiency mapping of tendrils13-8. Initial mapping placed tendrils13-8 between roughoid (ru) and hairy (h) on chromosome 3 (top line). Markers used for mapping, FRT sites (triangles) and centromere (closed circle) are indicated. Second line shows ru-h interval with SNP markers used for mapping. Number of recombinant breakpoints in each SNP interval (out of 20 ru-h recombinants from first round of mapping) is shown below the line; arrows show direction of tendrils13-8 relative to SNP markers. Third line shows 3L075-h interval along with the combined results of first and second rounds of mapping (235 ru-h recombinants). The structure of three deficiency chromosomes and complementation results with tendrils13-8 are shown below the line. Gaps indicate minimal sizes of the deficiencies from published cytology. Fourth line shows genes (black boxes) in region between Df(3L)BSC13 proximal breakpoint and 3L078A. tendrils13-8 failed to complement rhea1 (gold box), which encodes talin. (B) Changes in talin-coding sequence in tendrils alleles. tendrils13-8 alters a 3' splice site in the fourth intron (see C) that results in mis-splicing, which changes codon D268 and beyond (see panel C). Putative head and rod regions, FERM- and F-actin-binding domain (hatched rectangle), and F-actin-binding domains (stippled rectangles) are indicated (Brown et al., 2002). (C) Sequences of fourth intron splice junctions of talin gene in wild-type (tendrils+) and tendrils13-8 mutant. tendrils13-8 has a G>A mutation (asterisk) in the 3' splice site that leads to use of a cryptic 3' splice site seven nucleotides downstream, as determined by RT-PCR analysis of tendrils13-8 RNA. (D) Diagram of talin dimer linking cell-surface integrin to actin cytoskeleton. Talin head binds to cytoplasmic domain of ß-integrin subunit; rod domain contains actin binding sites. Modified, with permission, from Calderwood and Ginsberg (Calderwood and Ginsberg, 2003). (E) Model of talin function in tracheal terminal cells. Talin associates with a ß-integrin at the basal surface of the terminal branches and stabilizes binding to the muscle. We speculate that talin also associates with the cytoskeleton and stabilizes lumen (Lu) position in the cell.

View larger version (145K): [in a new window]   Fig. 5. Tracheal terminal cell expression and mutant phenotype of myospheroid integrins. (A-B') Confocal fluorescent images of tendrils+ (A) and tendrils13-8 (B) terminal cell clones in larva fillets stained with mAb CF6G11 to show ßmys-integrin (red) and with anti-GFP (green) to show terminal cell cytoplasm. A' and B' show ßmys staining in gray scale. ßmys is found at higher levels in the periphery (arrowhead) of tendrils+ but not in tendrils13-8 terminal branches. Terminal branches adhere to muscles that also express ßmys. Bright ßmys staining in B (asterisk) is outside the tracheal system. (C-G) Fluorescent images and brightfield close ups (C'-G') of wild-type and homozygous DB terminal cell clones of integrin pathway mutations indicated. (C,C') Wild-type control. (D,D') rhea79, (E,E') mysXG43, amorphic ßmys-integrin allele. (F,F') mysG4, point mutation that disrupts ECM-binding by ßmys (Jannuzi et al., 2002). (G,G') mewM6, ifk27e double mutant. mys (E,F) and mew if (G) phenotypes are similar to tendrils phenotype (D). (H,I) mysXG43 DB terminal cell clone imaged in early L3 and again 48 hours later as in Fig. 3D-G. Terminal branches are lost and there is an increase in lumen density and complexity in remaining branches, as in tendrils mutants. Specific terminal branches are numbered; dots indicate distal extent of lumens. After 48 hours, branch 5 has been lost, branches 1-4 (broken lines) are barely detectable and their lumens (dots and arrowhead) have been displaced into the proximal branch, as in tendrils clones (Fig. 3F,G). Lumen displacement is also evident from changes in position of luminal bifurcation (arrowhead) with respect to branches 2 and 3. Panel I is a montage. (J-L) TEM analysis of mysXG43 DB terminal cell clone as in Fig. 1H-M. Terminal cell contains multiple lumens (red dots) without any intervening basal plasma membranes, as shown by close-up (L). tc, terminal cell; m, muscle; h, hemocyte. Scale bars: in A, 20 µm for A,B; in C, 25 µm for C-G; in C' 25 µm for C'-G'; in I, 25 µm for H,I; 2 µm in K; 0.5 µm in L.

View larger version (24K): [in a new window]   Fig. 6. Model of integrin-talin adhesion complexes in terminal branch maintenance. (Top) Branches sprout from wild-type terminal cells (budding and outgrowth) early in development and are stabilized on their targets (maintenance) by integrin-talin adhesion complexes (red dots). Stabilization may be a reversible process to allow redistribution of branches in response to physiological need (remodeling). (Bottom) Terminal branches sprout normally early in development from rhea-, mys- or mew- if- terminal cells that lack integrin adhesion complexes. However, branches are unstable and their lumens retract into proximal branches as the rest of the branch degenerates (degeneration).

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