Drosophila glypican Dally-like acts in FGF-receiving cells to modulate FGF signaling during tracheal morphogenesis

Abstract

Previous studies in Drosophila have shown that heparan sulfate proteoglycans (HSPGs) are involved in both breathless (btl)- and heartless (htl)-mediated FGF signaling during embryogenesis. However, the mechanism(s) by which HSPGs control Btl and Htl signaling is unknown. Here we show that dally-like (dlp, a Drosophila glypican) mutant embryos exhibit severe defects in tracheal morphogenesis and show a reduction in btl-mediated FGF signaling activity. However, htl-dependent mesodermal cell migration is not affected in dlp mutant embryos. Furthermore, expression of Dlp, but not other Drosophila HSPGs, can restore effectively the tracheal morphogenesis in dlp embryos. Rescue experiments in dlp embryos demonstrate that Dlp functions only in Bnl/FGF receiving cells in a cell-autonomous manner, but is not essential for Bnl/FGF expression cells. To further dissect the mechanism(s) of Dlp in Btl signaling, we analyzed the role of Dlp in Btl-mediated air sac tracheoblast formation in wing discs. Mosaic analysis experiments show that removal of HSPG activity in FGF-producing or other surrounding cells does not affect tracheoblasts migration, while HSPG mutant tracheoblast cells fail to receive FGF signaling. Together, our results argue strongly that HSPGs regulate Btl signaling exclusively in FGF-receiving cells as co-receptors, but are not essential for the secretion and distribution of the FGF ligand. This mechanism is distinct from HSPG functions in morphogen distribution, and is likely a general paradigm for HSPG functions in FGF signaling in Drosophila.

Figure 1. Dlp is required for bnl/btl-dependent tracheal cell migration during embryogenesis

All embryos are oriented anterior to the left. (A-D, H-I) Lateral views of stage15 embryos immunostained with 2A12 antibody. Insets show higher magnification of a single typical tracheal metamere. (A) Wild-type tracheal pattern. In maternal/zygotic null dlp embryo (B), virtually no tracheal branching occurs. This defect is completely paternally rescued (C). (D) bnl shows strong genetic interaction with dlp. One copy of bnl mutation can lead to strong tracheal defects combined with dlp maternal mutation. In wild-type embryos, overexpression of UAS-bnl by btl-Gal4 generates masses of fine branches (H). This phenotype is markedly suppressed in maternal/zygotic dlp mutant background (I). (E-F) Lateral views of stage 11 embryos stained with the diphospho-MAPK-specific antibody. The strong expression observed in wild-type (E) tracheal pits is markedly reduced in the corresponding positions in maternal/ zygotic null dlp embryos (F). (G) β-Gal antibody staining of stage 11 maternal/ zygotic dlp mutant embryos, which contain 1-eve-1. Tracheal pits form normally with respect to size and position in these mutant embryos. (J-M) Ventral views of stage 9 embryos immunostained for Twi expression. Unlike in htl mutant embryos (K), these Twi-positive mesodermal cells migrate normally in maternal/ zygotic null dlp (L) or dally-dlp (M) mutant embryos.

Figure 2. Rescue of dlp mutant embryos by ectopic expression of UAS-dlp in different domains or by other HSPG core proteins

(A-L) Lateral view of stage 15 embryos stained with the tracheal lumenal antibody 2A12. (A-D, F-G) dlp mutant embryos are rescued by ectopic expression of UAS-dlp in whole ectoderm cells (69B-Gal4) (A), FGF expression cells (bnl-Gal4) (B), tracheal cells (btl-Gal4) (C), both FGF expression and tracheal cells (D), ventral midline cells (sim-Gal4) (F), or ectoderm in every other segment (prd-Gal4) (G). Ectopic expression in whole ectoderm can almost completely rescue dlp embryos (A). Expressions in FGF expression cells or ventral midline cells fail to rescue (B, F). btl-Gal4 rescued embryos develop an extensive tracheal network which has an abnormal pattern (C). This is possibly due to segmentation defect associated with other signaling pathways. In fact, this phenotype is similar to that in Wg mutant embryos (E). Embryos rescued by both btl-Gal4 and bnl-Gal4 are similar to those rescued by btl-Gal4 alone (D). Ectopic expression in ectoderm of every other segment can rescue most of the tracheal defect with alternative truncations in dlp embryos (G). This phenotype is very similar to that in btl mutant embryos rescued by prd-Gal4/UAS-btl-GFP (I). (J-L) dlp mutant embryos ectopically expressing UAS-dally (J), UAS-syndecan (K) and UAS-perlecan (L) by prd-Gal4. None of these HSPG core proteins is able to rescue dlp embryos compared to Dlp expression (G). (M-N”’) Stage 11 (M-M”’) and 13 (N-N”’) dlp embryos rescued by prd-Gal4/UAS-dlp stained for trachealess-lacZ (M, N), Dlp (M’, N’) and diphospho-MAPK (M”’, N”’). The first two channels are merged in M” and N” to indicate overlapping region (arrows).

Figure 3. Both dlp and dally are expressed in the air sac tracheoblasts

(A) Schematic drawing of wing imaginal disc and associated air sac tracheoblast cells in the late third instar larvae. (B-B”) Air sac tracheoblasts are outlined by E-Cadherin staining (B). They migrate towards the gradient of Bnl expression, which is shown by β-Gal staining using a bnl-lacZ line (B’). Images from different focal planes are merged in (B”). (C-C”) Air sac tracheoblasts are outlined by btl-Gal4, UAS-CD8-GFP (C’), and are counterstained with E-Cadherin (C). (D-D”) Dlp immunostaining shows Dlp is expressed in columnar epithelial cells (D’). (D) shows β-Gal staining using a bnl-lacZ line. The first two channels are merged in (D”) to indicate relative position of Dlp and Bnl expression. (E-E””) Dlp immunostaining shows that Dlp protein is expressed in the air sac tracheoblasts (E’), which is marked by btl-Gal4, UAS-cytoplasmic GFP (E). In contrast, dlp is not expressed in mesodermal cells (E’), which are outlined by Twist staining (E”). (E) and (E’) are merged in (E”’); all three channels are merged in (E””). (F) Image of living tracheoblasts which express UAS-dlp-GFP by btl-Gal4. Dlp-GFP is localized in multiple filopodia extending from these cells (arrows). (G-G”) β-Gal staining using a dally-lacZ line demonstrates that dally is also expressed in the air sac tracheoblasts (F’). Dally expression is absent from mesodermal muscle precursor cells.

Figure 4. HSPGs are dispensable in FGF/Bnl producing cells and surrounding cells

All wing discs are oriented ventral up, anterior to the left. All mutant clones are generated in the columnar epithelial layer, which are marked by the absence of GFP (the second column). E-Cadherin staining (the first column) is used to outline migrating air sac tracheoblasts. bnl expression is demonstrated by β-Gal staining (the third column) utilizing a bnl-lacZ line. The E-Cadherin staining images are taken at the tracheoblast layer, while the GFP and β-Gal staining pictures are taken at the columnar epithelial layer. Different focal planes are merged in the fourth column. For dally-dlp or sfl, two representative mutant clones are shown. Big dally-dlp (A₁-A₂”’) or sfl (B₁-B₂”’) mutant clones in columnar epithelial cell layer do not interfere tracheoblast cell migration. bnl expression is also not affected in these mutant clones.

Figure 5. Both dally and dlp are essential in FGF/Bnl receiving cells

All mutant clones are generated in the air sac tracheoblast cells. The clones are positively marked by CD8-GFP; the entire air sac is outlined with E-Cadherin staining. (A₁-A₃) Three representative wild-type clones, which are located in the tip region of migrating tracheoblasts (the most distal part of air sac). (B₁-B₃) Three representative dlp mutant clones located in the tip region of migrating tracheoblasts. (C₁-C₃) Three representative dally mutant clones located in the tip region of migrating tracheoblasts. (D₁-D₃) Three representative dally-dlp mutant clones. These clones never reach the tip of air sac. (E₁-E₃) Three representative dally-dlp mutant clones rescued by UAS-dlp. They can localize in the tip region of migrating tracheoblasts. (F₁-F₃) Three representative sfl mutant clones. These clones never reach the tip of air sac. (G) Statistic data demonstrate ratio of clones that contribute to the tip of air sac. Among 45 Wild-type clones, 33% contribute to the tip region. Among 58 dlp mutant clones, 38% contribute to the tip region. Among 39 dally mutant clones, 25% contribute to the tip region. Among 45 dally-dlp or 30 sfl mutant clones, none of them reach the tip region. Among 38 dally-dlp mutant clones rescued by UAS-dlp, 24% contribute to the tip region.