Average area of endosomes analyzed in Garland cells from <i>wt, stam, hrs</i> and <i>hrs, stam</i> larvae.

<p>Average area of endosomes analyzed in Garland cells from <i>wt, stam, hrs</i> and <i>hrs, stam</i> larvae.</p

<i>stam</i> and <i>hrs</i> downregulate EGFR signalling activity during embryogenesis.

<p>Diphospho-ERK (dpMAPK) antibody staining in <i>wild type</i>, <i>hrs</i>, <i>stam</i> and <i>hrs, stam</i> maternal-zygotic loss-of-function embryos (A–H′). At stage 10, <i>hrs</i>, <i>stam</i> and <i>hrs, stam</i> mutants display enhancement of the dp-ERK staining (B–D), due to an expansion of their ventral fate (see black bar length) compared to a wild type embryo (A). Mutant embryos also display an expansion of the staining in tracheal placodes (F–H) compared to wild type (E). Close up views of the dp-ERK staining in the tracheal placodes (E′–H′).</p

<i>argos</i> expression is strongly reduced in <i>stam</i>, <i>hrs</i> and <i>hrs, stam</i> mutant cells in pupal wings.

<p><i>argos</i> expression was analysed in <i>wild type</i> (A), <i>stam^2L2896</i> (B) and <i>hrs, stam</i> (C) MARCM clones visualized with mCD8-GFP (green) in pupal wings (24–30 hrs APF) using a <i>argos</i>-<i>LacZ</i> enhancer trap line. White dotted squares (in A–C) indicate the position of close-up pictures. White doted lines (in A′–C′) indicated the position of the clones in the close-up pictures. The <i>argos</i> staining (red) is absent in stam and <i>hrs, stam</i> mutant cells.</p

<i>stam</i> is required to properly localise FGFR/Btl and fully activate FGFR signalling.

<p>A–C. Localisation of Btl in wild type and mutant tracheal cells. High magnification pictures of wild type (A), <i>stam</i> (B) and <i>hrs</i> (C) MARCM mutant cells in the ASP. Scale bar equals 15 µm. Mutant cells were visualised <i>via</i> the expression of <i>UAS-btl-GFP</i> (green). The tracheal cells were visualised with RFP-moesin (red). White arrow indicates the presence of Btl at the cell membrane in wild type cells while yellow arrows indicate Btl as dotted structures. The dotted structures are dramatically enlarged in <i>stam</i> and <i>hrs</i> mutant cells as compared to <i>wild type</i> cells. D–F. <i>pointed</i> expression in wild type and <i>stam</i> mutant tracheal cells. Scale bars: 15 µm. <i>pointed</i> expression is restricted to the distal part, the tip, of a wild type ASP (D–D″). In a <i>stam</i> mutant clone located at the proximal part of the ASP, <i>pointed</i> expression is unchanged (E–E″). When the <i>stam</i> clone is positioned close to or at the distal tip of the ASP: <i>pointed</i> expression is lost (F–F″). Dotted lines showed the position of the <i>stam</i> mutant cells in the ASP (E′, F′). Arrows indicate the distal tip of the ASP (E″, F″).</p

Two cellular models of vascular pruning.

<p>A pruning branch is initially a multicellular tube (A). The cellular rearrangements to follow depend on collapse or maintenance of lumen at this stage (pruning type I or II, respectively). If the lumen collapses before cell rearrangements (type I pruning, B’), cell rearrangements lead to formation of a unicellular connection (C’–D’). The last linking cell regresses (E’) and completely resolves the last connection (F’) to complete the pruning process (G). If the lumen is maintained, cell rearrangements lead most cells out of the branch (B‘‘, arrows) and force the remaining cell to undergo self-fusion and form a unicellular tube (C”, arrow). Transcellular lumen collapses in the unicellular tube, forming two separate luminal compartments (D”, arrows). The last cell reduces its contact to one of the major branches (E”) and eventually the last contact (F”) is resolved and pruning is complete (G). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002126#pbio.1002126.s007" target="_blank">S6 Fig</a>.</p

<i>stam</i> and <i>hrs</i> are required for tracheal cell migration in the air sac primordium.

<p>A. Schematic representation of the anterior part of a <i>Drosophila</i> third instar larva. The air sac primordium (ASP) (red) buds from the transverse connective branch (in grey) and is attached to the wing imaginal disc (orange). The tracheal system is drawn in grey and imaginal discs other than the wing disc are colored in yellow. B. Model for the formation of the air sac primordium during larval development. Tracheal cells divide and migrate during ASP formation. Migration occurs under the control of the FGFR signalling pathway. Tracheal cells at the distal tip of the primordium are extending filapodia in the direction of the FGF ligand source (blue). Double arrow indicates the position of ASP distal tip. C. Migration behaviour of <i>wild type</i>, <i>stam</i>, <i>hrs</i> and <i>stam hrs</i> mutant cells. Confocal micrographs of the ASP of a <i>Drosophila</i> third instar larva are shown. All tracheal cells are labelled in red (RFP-moesin) and MARCM clones are labelled in green (mCD8-GFP). The <i>FRT40A</i> chromosome was used as a wild-type control. MARCM clones were induced for <i>stam</i>, <i>hrs</i> and <i>stam</i>, <i>hrs</i>. Scale bar: 15 µm. White double arrows indicate the position of ASP distal tip. Percentages of distal clones are indicated for each genotype tested. Note the strong effect of mutations in <i>hrs</i> and <i>hrs, stam</i> on cell migration. For each genotype, more than 20 clones were scored.</p

<i>stam</i> and <i>hrs</i> are required for the efficient formation of fine cytoplasmic extensions in tracheal terminal cells and interact with FGFR signalling.

<p>Confocal micrographs of MARCM wild type and mutant dorsal terminal cells. Scale bar: 50 µm. The clones are visualised using UAS-mCD8-GFP. FRT40A line was used as a control (A). MARCM clones were induced for <i>stam</i> (B), <i>hrs</i> (C), <i>hrs, stam</i> (D). (E–F). The FRT40A and <i>stam</i> MARCM terminal cells with altered FGFR signalling. <i>bnl+/−</i> corresponds to a larvae heterozygous for <i>bnl</i>. Branch points (visualised in pink) were counted for mutant clonal cells for each genotype. The average number of branch points is given for each genotype.</p

Endothelial cells rearrange from a multicellular into a partially unicellular tube during blood vessel pruning.

<p>(A) Still images from a time-lapse movie of <i>Tg(kdrl:cytobow1.0)^mu125</i>; <i>Tg(HSP:Cre)^zdf13</i> embryos. Imaged area indicated with box in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075060#pone-0075060-g001" target="_blank">Figure 1D</a>. Bracket marks dorsal CrDI. At the 37.5 hpf time point, note unicellular connection between the dorsal CrDI and the NCA (single green cell, marked with red bracket). (B) Merged still images from time-lapse imaging of <i>Tg(UAS:RFP)</i>; <i>Tg(fliep:Gal4FF)^ubs4</i>; <i>Tg(UAS:VE-cadherin-deltaC-EGFP)^ubs12</i> between 36 and 45 hpf. Red bracket at 42.5 hpf indicates unicellular connection between dorsal CrDI and NCA. (C) Still images from <i>Tg(UAS:VE-cadherin-deltaC-EGFP)^ubs12</i> expression corresponding to images in B. (D) Schematic drawing of cell rearrangements shown in B, C. Brackets label dorsal CrDI, purple arrow highlights NCA.</p

Number of branching points of <i>hrs</i> and <i>stam</i> MARCM terminal cell clones in different FGFR signalling activity backgrounds.

<p>Number of branching points of <i>hrs</i> and <i>stam</i> MARCM terminal cell clones in different FGFR signalling activity backgrounds.</p

Dynamic of lumen collapse in a unicellular tube.

<p>(A) Stills from a time-lapse movie illustrating lumen collapse in a unicellular tube in a transgenic embryo Tg(<i>fliep</i>:GFF)^ubs3,(UAS:mRFP), (<i>5xUAS</i>:<i>cdh5-EGFP</i>)^ubs12. A single lumenized “last link” cell connects two major branches (1). The white/black arrow marks lumen length. The asterisk marks the nucleus; the red arrow points to the narrowest lumen part (next to the nucleus). The lumen splits first next to the nucleus (2, red arrow), forming two distinct luminal compartments within the cell (2, white arrows) that are separated by a nonlumenized part (grey dotted line). The nonlumenized part increases in length as the lower luminal compartment collapses (3). The cell body (nucleus, asterisk) moves towards the upper major branch. The last cell extension (gray line) contacts the lower major branch with a spot-like junction (4, arrow). (B) Stills from a time-lapse movie showing lumen collapse in a unicellular tube in a transgenic embryo TgBAC(<i>kdrl</i>:mKate-CAAX)^ubs16. Black arrows show continuous lumen, gray dotted lines show nonlumenized unicellular regions, the red arrow shows the point of lumen breakage, and asterisks mark the nucleus, where clearly distinguishable. Lumen breaks at the contact site to the lower major branch, next to the nucleus (1–3). The luminal compartment deflates and inflates again (4–5, arrows). After complete lumen collapse, the last connection is resolved (6–8). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002126#pbio.1002126.s021" target="_blank">S14 Movie</a>. (C) Stills from a time-lapse movie showing lumen collapse in higher time resolution. Inflated luminal compartments (1, arrows) are framed by apical membrane (marked by mKate2-CAAX) and separated by a thin bridge of cell body, most likely the nucleus (1, asterisk). The lumen expands and two apical membranes touch (2, arrow) and fuse (3), but the lumen does not completely inflate (4). The lumen breaks again (5–7) and reconnects in a similar fashion (8–11) within a short time. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002126#pbio.1002126.s022" target="_blank">S15 Movie</a>. (D) A schematic representing luminal instability, based on still pictures in C. The apical membrane is black, the cell body is dark gray, and the lumen is light gray.</p