Two ligands signal through the Drosophila PDGF/VEGF receptor to ensure proper salivary gland positioning

Abstract

The Drosophila embryonic salivary gland is a migrating tissue that undergoes a stereotypic pattern of migration into the embryo. We demonstrate that the migratory path of the salivary gland requires the PDGF/VEGF pathway. The PDGF/VEGF receptor, Pvr, is strongly expressed in the salivary glands, and Pvr mutations cause abnormal ventral curving of the glands, suggesting that Pvr is involved in gland migration. Although the Pvr ligands, Pvf1 and Pvf2, have distinct expression patterns in the Drosophila embryo, mutations for either one of the ligands result in salivary gland migration defects similar to those seen in embryos that lack Pvr. Rescue experiments indicate that the PDGF/VEGF pathway functions autonomously in the salivary gland. The results of this study demonstrate that the Drosophila PDGF/VEGF pathway is essential for proper positioning of the salivary glands.

Figure 1. Morphogenesis of the salivary glands

(A–H) Salivary gland cells (arrows) are stained for FKH in green and the circular visceral mesoderm is stained for FASIII in red. (A, C, E G) Lateral and (B, D, F, H) corresponding ventral views of embryos stages 11 through 14. (A, B) Salivary glands begin as a pair of single-layered epithelial disks, the salivary placodes that invaginate by apical constriction to form slender tubes. (C, D) As they leave the surface these tubes extend dorsally and posteriorly at a 45 degree angle until they reach the visceral mesoderm. (E, F) Then they change paths and migrate actively along the mesoderm until they lie horizontally within the embryo (G, H) By stage 15 the glands reach their final position within the embryo.

Figure 2. Pvr and Pvf1 are expressed in the developing salivary glands

(A) w¹¹¹⁸ embryos were hybridized in situ with Fkh and Pvr probes. Lateral view of a stage 11 embryo. (A-A″) The Pvr receptor is expressed in the salivary placode. (B–C) Lateral view of wild-type embryos stained for FKH and PVR proteins. (B-B″) PVR is first detected as the salivary gland invaginates into the embryo (stage 12) and is enriched at the apical membrane. (C-C″) PVR expression reaches peak levels by stage 15. (D–E) Lateral view of wild-type embryos stained for FKH and PVF1 proteins. (D-D″) PVF1 expression begins in the salivary glands at stage 12. (E-E″) PVF1 expression increases during salivary gland development and reaches peak levels by stage 15.

Figure 3. Pvr and the Pvf ligands are required for proper salivary gland positioning

(A–E) Lateral views of stage 15 embryos stained with FKH. (B) In Pvr null embryos the salivary glands curve ventrally. (C–D) Embryos lacking Pvf1 or Pvf2 have salivary glands that bend ventrally, similar to Pvr mutants. Two Pvf1 alleles (Pvf1^G0146, Pvf1^EP1624) and two Pvf2 alleles (Pvf2^C06947, Pvf2^d02444) were tested. All caused similar phenotypes at 40–60% penetrance. (E) Pvf3 mutant embryos have an impenetrant phenotype that resembles Pvr mutants. (F) Graphical representation of phenotypic penetrance in embryos scored for salivary gland migration defects at stages 14–16. At least 30 embryos were scored for each genotype represented on the graph.

Figure 4. Pvr function is autonomous to the salivary gland

(A–F) Lateral views of stage 15 embryos stained with FKH. (B) In Pvr mutant embryos the salivary glands curve ventrally. (C) Expression of a dominant negative transgene for Pvr in the gland phenocopies the Pvr mutant phenotype (compare B and C). (D,G) Expression of dominant negative Pvr in the hemocytes has little effect on salivary gland positioning. (E,G) The Pvr mutant phenotype can be rescued by UAS-Pvr using a salivary gland specific GAL4 driver, fkh-GAL4. (F,G) A constitutively active Pvr construct is also able to rescue the Pvr mutant phenotype when driven in the salivary gland. (G) Graphical representation of phenotypic penetrance in embryos scored for salivary gland migration defects at stages 14–16. At least 30 embryos were scored for each genotype represented on the graph.

Figure 5. Ectopic expression of the Pvf ligands results in salivary gland positioning defects

(A–D) Lateral views of stage 15 embryos stained with FKH. (B) Overexpression of Pvf1 using the salivary gland specific GAL4 driver, fkh-GAL4, results in ventrally curved salivary glands. (C–D) Misexpression of either Pvf2 (C) or Pvf3 (D) in the salivary gland also results in ventrally curved glands. (E) Similar results, though at a higher frequency, are seen when the constitutively active receptor, λPvr, is expressed in the salivary glands. (F) Graphical representation of phenotypic penetrance in embryos scored for salivary gland migration defects at stages 14–16. At least 30 embryos were scored for each genotype represented on the graph.