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Specificity of FGF signaling in cell migration in Drosophila

Caroline Dossenbach^*, Salome Röck^* and Markus Affolter

Abteilung Zellbiologie, Biozentrum der Universität Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland
^* These authors contributed equally to this study

View larger version (38K): [in a new window]   Fig. 1. Receptor constructs tested in vivo. (A) Representation of a tracheal segment indicating the six different primary tracheal branches: DB, dorsal branch; DT, dorsal trunk; VB, visceral branch; LTa and LTp, lateral trunk anterior and posterior; GB, ganglionic branch. (B,C) Wild-type Btl and Htl constructs, and the chimeric receptor constructs. The extracellular domain of Btl is composed from left to right of a N-terminal signal peptide, three Ig domains, an acidic region, a CAM binding domain, two Ig domains and a transmembrane domain. The intracellular domain consists of the juxtamembrane domain followed by the split tyrosine kinase catalytic domain and a short C-terminal domain. Htl contains only two Ig extracellular domains; all the other domains are homologous to Btl. (B) The extracellular and transmenbrane domain of Btl were fused to the intracellular domain of the following RTKs: Htl, Tor and EGFR. (C) Additionally, the extracellular domain and the transmembrane domain of Htl were fused to the intracellular domain of Btl, Tor and EGFR.

View larger version (79K): [in a new window]   Fig. 2. Rescue of tracheal cell migration and patterning defects by hybrid receptors. The lumen of the tracheal system of stage 15 embryos was visualized with the 2A12 antibody (green, A,B,D,E,G,H,J,K,M,N; red, C,F,I,L,O) and terminal cells were visualized with the anti-DSRF antibody (green, C,F,I,L,O). Embryos obtained from the crosses as described in the Materials and Methods were collected at 18° C and 25°C. (A) Tracheal system of a wild-type embryo. (B) In homozygous btl mutant embryos, tracheal cells failed to migrate completely. (C) Tracheal system of a wild-type embryo outlining the DSRF/bs-expressing terminal cells. (D,E) All aspects of tracheal cell migration were fully rescued in btl mutant embryos expressing a btl transgene under the control of btl-Gal4. (F) The correct number of terminal cells differentiated at the proper positions as in wild-type embryos. (G,H) The Btl-Htl chimeric receptor construct was able to fully substitute for Btl and rescued tracheal cell migration. (I) The Btl-Htl chimeric receptor construct was able to induce proper terminal cell fates. In F,I, DSRF/bs expression in visceral branches is out of the focal plane. (J,K) The Btl-Tor fusion protein was able to rescue tracheal cell migration in btl mutant embryos. Some dorsal branches were missing (arrowheads) and the lateral trunk did not fuse in each segment (arrow). Apart from these defects, dorsal trunk fusion, visceral branch and ganglionic branch formation was not affected and the general pattern of the tracheal system was restored. Note the felted phenotype of the tracheal system, owing to the fine ectopic terminal branches (see K). (L) The Btl-Tor construct led to the ectopic formation of terminal cells (arrow). (M,N) The Btl-EGFR fusion protein was able to rescue tracheal cell migration in btl mutant embryos but less efficiently than Btl-Tor. Some dorsal branches were missing (dorsal arrowheads), the lateral trunk did not fuse (ventral arrow, N), ganglionic branches often failed to migrate (ventral arrowhead, M) and in some embryos, the visceral branches were misguided (arrow, M). (O) DSRF/bs expression and terminal cell formation was strongly reduced (arrow) when compared with wild type (C).

View larger version (88K): [in a new window]   Fig. 3. Rescue of tracheal cell migration in the absence of dof. Whole-mount antibody staining for the tracheal lumen of stage 15 embryos using the 2A12 antibody (green, A,B,D,E,G,H,J,K; red, C,F,I,L) and for the terminal cells using the anti-DSRF antibody (green, C,F,I,L). Embryos from crosses described in the Materials and Methods were collected at 18°C and 25°C. (A) Tracheal system of a homozygous dof mutant embryo upon the expression of a dof transgene driven by btl-Gal4. Apart from defects that are due to the malformation of the mesoderm in dof mutant embryos (for example, truncation of visceral branches), the tracheal system was fully rescued. (B) The Btl wild-type receptor construct was not able to rescue tracheal cell migration in btl,dof double mutant embryos. (C) DSRF/bs expression was not induced in any tracheal cell in btl,dof mutant embryos expressing the btl transgene. (D,E) The Btl-Htl chimeric receptor construct was not able to rescue tracheal cell migration in btl,dof mutant embryos. (F) DSRF/bs expression was not induced in the tracheal cells of btl,dof mutant embryos expressing Btl-Htl. (G,H) In contrast to Btl and Htl, the Btl-Tor fusion protein was able to rescue tracheal cell migration in btl,dof mutants. The general pattern of the tracheal system was rescued. Dorsal trunk fusion occurred efficiently but some dorsal branches failed to form (dorsal arrowheads). The lateral trunk did not fuse (arrows) and depending on the embryo, one or two ganglionic branches failed to form (ventral arrowhead). (I) Expression of the Btl-Tor construct led to ectopic activation of DSRF/bs and terminal cell formation in btl,dof mutant embryos (arrow). (J,K) The Btl-EGFR construct was able to rescue tracheal cell migration in btl,dof mutant embryos to a certain extent. General tracheal patterning was rescued, although the tracheal tree was more affected. In some embryos the dorsal trunk was disrupted, some dorsal branches were missing (dorsal arrowheads) or misguided (dorsal arrow) and the lateral trunk failed to fuse (ventral arrows). In contrast to the rescue by Btl-Tor, some of the ganglionic branches failed to form and did not migrate in proper direction (ventral arrowhead). (L) DSRF/bs expression was strongly reduced in btl,dof mutant embryos expressing Btl-EGFR (arrow).

View larger version (54K): [in a new window]   Fig. 4. Rescues of mesodermal cell migration defects by hybrid receptors. The embryos were generated in the crosses described in the Materials and Methods and collected at 25°C. The tracheal system was visualized with the 2A12 antibody. Mesoderm migration and pericardial cell formation was analyzed with an anti-Eve antibody (see also Materials and Methods). Evenskipped is specifically expressed in the pericardial cells and was used as a marker for these mesodermal cells. Arrows point to the pericardial cells. (A) Pericardial cells arranged in two rows at the dorsal side of a wild-type embryo. (B) Mesodermal cells were not able to migrate properly in htl mutant embryos. Consequently, pericardial cell fate determination did not occur. (C) Mesodermal cells did not migrate properly in dof mutant embryos and pericardial cells failed to form. (D) Mesodermal cell migration and pericardial cell differentiation were rescued in dof mutant embryos expressing the dof transgene. The arrangement of the cells and cell number were identical to wild-type embryos. (E) Pericardial cell formation was fully rescued by the Htl wild-type construct expressed in htl mutant embryos. (F) By contrast, the Htl wild-type construct was not able to rescue mesodermal cell migration and pericardial cell formation in the absence of Dof. (G) The chimeric receptor construct Htl-Btl was not able to fully replace the endogenous Htl receptor when expressed in htl mutant embryos. Only 12-16 pericardial cells were formed on each side of the embryo (instead of 20). (H) In dof mutant embryos, Htl-Btl failed to rescue pericardial cell formation. (I) Expression of the Htl-Tor fusion protein in htl mutant embryos led to excess cardiac cell formation. Nineteen to 30 scattered cells were observed along the midline in the dorsal part of the embryo. (J) The rescue of pericardial cells in dof mutant embryos expressing Htl-Tor was almost complete. 18-20 pericardial cells were arranged in a slightly disordered manner at the dorsal side of the embryo. (K) 17-20 pericardial cells were formed in htl mutant embryos expressing the Htl-EGFR receptor construct. (L) In dof mutant embryos, the Htl-EGFR construct was able to rescue 12-18 pericardial cells on each side of the embryo but the cells failed to arrange in an anterior-posterior row.