<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

<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

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

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

<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