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First published online May 16, 2007
doi: 10.1242/10.1242/dev.001164

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Different Wnt signals act through the Frizzled and RYK receptors during Drosophila salivary gland migration

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.

View larger version (97K): [in this window] [in a new window]   Fig. 1. Morphogenesis of the Drosophila salivary glands. (A-H) Salivary gland cells (arrow) are stained for FKH in green, the CNS is visualized in red by ELAV staining and the CVM is stained for FASIII, also 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° angle on either side of the CNS 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, dorsal and lateral to the CNS. (G,H) By stage 14, the glands encounter the longitudinal visceral mesoderm (not shown) and separate from the circular visceral mesoderm. (I-K) Lateral views of embryos stage 13-15 stained with the salivary gland marker SG2 in green, the mesodermal marker Titin in red and the CVM marker FASIII also in green. In these panels the CVM appears yellow due to FASIII-Titin co-staining; LVM (arrows) is red due to Titin, but not FASIII, staining. (I) At the end of stage 13, cells of the LVM migrate anteriorly and displace the distal end of the salivary gland from the CVM. (J,K) From stage 14, the tip of the salivary gland remains in contact with the LVM.

View larger version (57K): [in this window] [in a new window]   Fig. 2. drl and Wnt5 are required for proper positioning of the tip of the salivary gland. (A-A'') Wild-type Drosophila embryos were hybridized in situ with fkh and drl probes. Ventral view of a stage 11 embryo. The drl receptor is expressed in the dorsoposterior of the salivary placode (arrow), which later corresponds to the salivary gland cells that lead invagination into the embryo. (B-B'') Lateral view of stage 15 embryo with an enhancer trap insertion at the drl locus. ß-galactosidase expression at the salivary gland is confined to the distal tip. Arrows denote the location of drl expression in the salivary gland. (C-H) Lateral view of stage 15-16 embryos stained for FKH and the apical cell marker CRB. (I) Graphical representation of phenotypic penetrance in embryos scored for salivary gland migration defects at stages 14-16. (C) Wild-type control. (D,I) In drl-null embryos, the salivary glands curve ventromedially. (E,I) The drlR343 mutant phenotype can be rescued by UAS-drl using a salivary-gland-specific GAL4 driver, fkh-GAL4. (F,I) The intracellular domain of drl is important for drl function, as UAS-drli does not rescue the salivary gland migration phenotype in drlR343 mutant embryos. (G,I) In embryos lacking Wnt5 the tip of the salivary gland is bent toward the CNS. (H) Lateral view of a stage 15 embryo stained with the salivary gland marker SG2 in green, the mesodermal marker Titin in red and the CVM marker FASIII also in green. The salivary glands of drlR343 mutant embryos do not maintain contact with LVM (compare with Fig. 1K).

View larger version (79K): [in this window] [in a new window]   Fig. 3. Expression of drl in the salivary gland is dependent on Scr and fkh. (A-C) In situ hybridizations for drl were performed on stage 11 wild-type (A) Screk6 (B) and fkhXT6 (C) mutant Drosophila embryos. drl expression in the salivary placode is absent in both Scr and fkh mutants, indicating that drl is a transcriptional target of FKH. Arrow in A denotes the location of drl in wild-type salivary placodes.

View larger version (50K): [in this window] [in a new window]   Fig. 4. Salivary gland positioning is disrupted in dnt and Drl-2 mutant embryos. (A-D) Lateral views of stage 15-16 Drosophila embryos stained with FKH and CRB. (E) Graphical representation of phenotypic penetrance in embryos scored for salivary gland migration defects at stages 14-16. (A) Wild-type control. (B) dnte00722 mutant embryos have salivary glands that curve ventrally at the tip of the salivary gland. (C) Similarly, Drl-2-null embryos also show a derailed-like phenotype. (D,E) Salivary gland curving is also seen in drlR343 Drl-2E124 double mutants, but the penetrance of the phenotype is not enhanced compared to either of the single mutants.

View larger version (63K): [in this window] [in a new window]   Fig. 5. Two independent Wnt pathways regulate salivary gland positioning. (A-I) Lateral views of stage 15-16 Drosophila embryos stained with FKH and CRB. (J) Graphical representation of phenotypic penetrance in embryos scored for salivary gland migration defects at stages 14-16. (A) Wild-type control. (B) Wnt4-null embryos have a ventral curving defect that affects a large portion of the salivary gland. (C,J) Embryos mutant for both Wnt4EMS23 and Wnt5D7 show an enhanced penetrance (68%) of the salivary gland guidance phenotype compared with the single mutants. (D) fz1 mutant embryos show a phenotype very similar to that of Wnt4EMS23. (E,F) Using the GAL4/UAS system, dominant-negative transgenes of either fz or fz2 were ectopically expressed in the salivary gland using a salivary-gland-specific driver. The loss of fz signaling in the gland resulted in guidance defects similar to those seen in Wnt4 mutants. (G,J) Furthermore, embryos mutant for both drlR343 and fz1 show a higher penetrance of salivary gland guidance defects than drlR343 or fz1 alone. (H,I) Expression of dominant-negative transgenes for Tcf or dsh in the salivary glands, using a salivary-gland-specific GAL4 driver, results in ventral curving of a large portion of the salivary gland.

View larger version (46K): [in this window] [in a new window]   Fig. 6. Ectopic expression of either Wnt5 or Wnt4 is sufficient to repel the salivary glands. (A-C) Lateral views of stage 16 Drosophila embryos stained for FKH and CRB. (B) WNT5 was ectopically expressed in the visceral mesoderm, dorsal to the gland (located above the gland in these panels). The misexpression of WNT5 is sufficient to redirect the tip of the salivary gland ventrally. (C) When Wnt4 is ectopically expressed in the visceral mesoderm, it is sufficient to repel the entire salivary gland away from the visceral mesoderm. These data support the hypothesis that both Wnt proteins act as repellents.

View larger version (69K): [in this window] [in a new window]   Fig. 7. drl genetically interacts with Src64 and Src42 in the salivary glands. (A-F) Lateral views of stage 15-16 Drosophila embryos stained with FKH and CRB. (A) Wild-type control. (B) Src64P1 embryos closely resemble drlR343 embryos. (C) drlR343 and Src64P1 doubly heterozygous embryos exhibit ventral curving of the salivary gland tips. (D,E) The salivary glands in Src42E1 show a variety of defects, including ventral curving at the tip of the glands (D) and curving that affects a large portion of the length of the gland (E). Embryos heterozygous for both drlR343 and Src42E1 resemble drlR343 embryos. (G) Graphical representation of phenotypic penetrance in embryos scored for salivary gland migration defects at stages 14-16.

View larger version (31K): [in this window] [in a new window]   Fig. 8. Model of salivary gland migration in Drosophila. (A) As the salivary glands invaginate into the embryo during stage 12 they are attracted to the CVM by Netrin. The salivary glands also encounter both attractive (Netrin) and repulsive (WNT4 and Slit) signals from the CNS, which guide the salivary glands during their posterior migration. The receptors for these early signals are present throughout the entire salivary gland and appear to work, for the most part, independently of each other. (B) During stage 15, the salivary glands meet the LVM and rely on the WNT5 repulsive signal from the CNS to keep the distal tip of the salivary gland positioned so it can adhere to the longitudinal visceral mesoderm. This is accomplished through Derailed, which is present specifically in the cells at the tip of the salivary glands.