The Drosophila Protein Dof Is Specifically Required for FGF Signaling Stéphane Vincent, Robert Wilson, Carmen Coelho, Markus Affolter, Maria Leptin  Molecular Cell  Volume 2, Issue 4, Pages 515-525 (October 1998) DOI: 10.1016/S1097-2765(00)80151-3

Figure 1 Defects in Mesoderm and Tracheal Morphogenesis in dof Mutants Stage 16 embryos stained with monoclonal antibody 2A12 to visualize the tracheal lumen. (A) Wild type; (B) btl mutant; (C) dof mutant. (D–G) Cross sections of gastrulating embryos stained with antibodies against Twist to visualize the invaginated mesoderm. In wild-type embryos ([D] and [E], stages 7 and 8), the invaginated mesodermal tube flattens against the ectoderm and then disperses into individual cells that migrate dorsally on the underlying ectoderm. In dof mutants (F and G), the mesodermal tube does not flatten. However, it does disperse into individual cells, and the cells that contact the ectoderm eventually migrate dorsally. This phenotype is identical to that of htl mutants (Gisselbrecht et al. 1996). (H). Rescue of the tracheal defects in a dof mutant embryo by expression of a dof transgene (see Experimental Procedures). (I and J) Dof protein expression in wild type ([I], dof1/+ or +/+) and dof1 (J) mutant embryos from the same staining reaction. The embryos were overstained to allow detection of even the lowest levels of Dof protein. Molecular Cell 1998 2, 515-525DOI: (10.1016/S1097-2765(00)80151-3)

Figure 2 Expression of dof in Developing Embryos (A) dof RNA is first detected at the syncytial blastoderm stage in the region of the mesoderm. (B) Dof remains detectable in the mesoderm until the extended germband but then disappears. (C and D) As dof expression fades in the mesoderm, dof is activated in the tracheal placodes and then the tracheal branches (both panels show a focal plane near the surface of the embryo; brackets, region enlarged in [F]). (E) Toward the end of embryogenesis, expression is seen in some cells in the CNS (possibly glia cells lying on top of the ventral nerve cord), parts of the visceral mesoderm, and a few other locations. (F) Higher magnification of the edge of the germ band of the embryo shown in (C), focused on the mesoderm. While dof RNA has disappeared in most of the mesoderm, it is maintained at low level in the dorsal edge of the mesoderm (white arrows), seen clearly in the regions between the tracheal invaginations, t. (G) Cross section through the embryo shown in (C), showing expression in the invaginated tracheae, t, and in a population of cells migrating along the mesoderm (arrow). (H) Higher magnification of the embryo shown in (D), focused on a deeper plane, showing the migrating cells (arrow). All embryos were hybridized with a probe prepared from a fragment common to both dof transcripts. Molecular Cell 1998 2, 515-525DOI: (10.1016/S1097-2765(00)80151-3)

Figure 3 dof Gene and Protein Structure (A) Diagram of the genomic region containing the dof gene. Restriction sites: S, SacI; H, HindIII; B, BamHI; N, NcoI. The insertion site of the P element in strain P837/10 is indicated by a triangle. The two different splice forms represented by our cDNAs are shown with the specific 5′ exons of transcript I in dark gray, and those of transcript II in light gray. The common 3′ exon is shown as a white box. We have not proven the absence of further upstream exons. The translation start site of the rescuing transcript II is marked by an arrow. (B) Protein sequence translated from the open reading frame of transcript II. The first amino acid shared by the two different splice products (D16) is indicated in bold type. Gray underlay, ankyrin repeats (details in [C]). Gray shaded box, coiled coil region. White box, positively charged cluster. Environments of tyrosines with similarities to known motifs surrounding phosphorylated tyrosines are bold and underlined. The proteins thought to interact with these motifs are indicated in (C). (C) Top, schematic representation of predicted motifs and structures in the Dof protein, showing the position of the ankyrin repeats (gray boxes), the coiled coil (hatched box), the positively charged cluster (white box with +), and six tyrosines, which, judged by their environment, might be binding sites for the proteins indicated above the diagram. The coiled coil structure prediction was according to the algorithm of A. Lupas (Lupas 1996). Bottom, alignment of Dof ankyrin repeats with known ankyrin repeats. Sequences (top to bottom): Dof, human ankyrin 3, human ankyrin R, human hypothetical protein KIAA0148, and mouse Rho-GEF. The numbers at the right are those of the last amino acid shown of each sequence. Amino acid identities to Dof are underlaid in dark gray, and conservative substitutions in light gray. Boxes above the sequence indicate the α helices found in ankyrin repeats; the zig-zag line denotes the sequence in Dof that is predicted to be able to form the β turn of an ankyrin repeat (Gorina and Pavletich 1996; Luh et al. 1997; Batchelor et al. 1998). Molecular Cell 1998 2, 515-525DOI: (10.1016/S1097-2765(00)80151-3)

Figure 4 Activation of ERK in the Mesoderm of Wild-Type and Mutant Embryos Cross sections through embryos during gastrulation, stained with antibodies against dp-ERK (A–C, E, and F) or Dof protein (D). (A–C) When the mesoderm has invaginated, ERK is activated in mesodermal cells adjacent to the ectoderm (arrows). Phosphorylated ERK is also seen in the overlying ectoderm. (D) The cells in the “stem” of the invaginated mesodermal tube, in which ERK is not phosphorylated, do not express Dof. (E and F) In embryos mutant for dof, no ERK activation is seen in the mesoderm, even at later stages, when some mesodermal cells do contact the ectoderm. Molecular Cell 1998 2, 515-525DOI: (10.1016/S1097-2765(00)80151-3)

Figure 5 Effects of Constitutively Active Signaling Molecules on Tracheal Defects in btl and dof Mutants Constitutively active forms of the FGFR Btl (Btl*), the Ras GTPase (Ras*), and the Raf kinase (Raf*) were expressed in the tracheal system of wild-type embryos and of btl and dof mutant embryos. The embryos were stained with antibody 2A12 (A–J) to visualize the tracheae, or (K–T) with an antibody against Drosophila SRF (DSRF), which is activated in the tracheal end cells in response to FGF receptor signaling. Note that expression of Btl* in wild-type embryos (B and L) leads to ectopic expression of DSRF in the tracheae. Arrows indicate examples of excessive terminal branching. Tracheae do not branch (C and G), and DSRF is not expressed (M and Q) in btl or dof mutants. Expression of Ras* or Raf* can induce DSRF expression (O, P, S, and T) and tracheal morphogenesis (E, F, I, and J) in both mutants, showing that activating the signaling cascade at these points circumvents the requirement for the FGFR and for the Dof protein. Btl*, in contrast, rescues these defects in btl mutants (D and N), but not in dof mutants (H and R), showing that even a constitutively active receptor still needs Dof for further transmission of the signal to the MAPK cascade and that Dof thus acts downstream of the FGFR. The phenotypic differences between (D), (E), (F), (I), and (J) are due to variabilty in the phenotypes rather than differences between genotypes. Btl* and Ras* were expressed under the control of the GAL4 driver line btl-GAL4. Raf* was expressed under heat shock control (two heat shocks of 20 min at an interval of 4 hr), and embryos were fixed 4 hr after the second heat treatment. Molecular Cell 1998 2, 515-525DOI: (10.1016/S1097-2765(00)80151-3)

Figure 6 Activation of DSRF by Activated Torso in dof Mutants Embryos stained with monoclonal antibody 2A12 recognizing the tracheal lumen (black arrows) and antibodies against DSRF (white arrows). Expression of a constitutively active Torso receptor (Tor*) in wild-type embryos (A) induces ectopic expression of DSRF in the nuclei of a number of tracheal cells (white arrow); compare to Figure 5K for wild-type control without activated receptor and to Figure 5L for wild-type with activated Btl (Btl*). (B) Expression of activated Torso in a dof mutant, focused on the level of DSRF-expressing cells. The activated Torso receptor is able to induce DSRF (white arrow) and some branching (black arrow) in dof mutants, whereas the activated FGFR Btl (C) does not. Molecular Cell 1998 2, 515-525DOI: (10.1016/S1097-2765(00)80151-3)

Figure 7 Dof Protein Expression and Migration of Gut Muscle Precursor Cells (A–C) Stage 10 embryo stained with antibodies against Dof. (A) Low power ventral view. (B and C) High magnification of individual cells (marked by arrowheads in [A]). The outlines of one cell in each panel are marked by white dots. (D–G) Dof in migrating visceral muscle precursors. Embryos stained with antibodies against Dof (brown) to distinguish wild-type (D and F) from dof mutants (E and G) and against β-galactosidase (blue) to mark a group of mesodermal cells at the posterior end of the germ band (expression of β-galactosidase under control of the croc-promoter). (F and G) The mesodermal cells migrate in an anterior direction and will eventually spread over more than half of the length of the embryo. Even in the absence of Dof (E and G), these cells are able to migrate as efficiently as in the wild type (D and F). White arrow in (D), expression of Dof in the anterior midgut primordium. (H) The migrating visceral mesodermal cells (marked by an arrow) also express the Htl protein. (I) Immunofluorescence labeling shows coexpression of β-galactosidase (red) and Dof (blue) in the migrating cells. Molecular Cell 1998 2, 515-525DOI: (10.1016/S1097-2765(00)80151-3)