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Linear signaling in the Toll-Dorsal pathway of Drosophila: activated Pelle kinase specifies all threshold outputs of gene expression while the bHLH protein Twist specifies a subset

Department of Molecular and Cell Biology, Division of Genetics and Development, 401 Barker Hall, University of California, Berkeley, CA 94720, USA

View larger version (104K): [in a new window]   Fig. 1. sna, vnd and sog expression in transgenic embryos with an ectopic, anterior-posterior Dorsal nuclear gradient. Cellularizing mutant embryos were collected from females homozygous for a null mutation in the gastrulation defective (gd) gene. Mutant embryos lack a dorsoventral Dorsal nuclear gradient. The embryos were hybridized with different digoxigenin-labeled antisense RNA probes, and visualized by histochemical staining. Each image is representative of the predominant pattern exhibited in the majority of embryos stained. In addition, at least three independent transgenic lines were analyzed for this and subsequent figures. Embryos are oriented with anterior to the left and dorsal up. (A-C) snail, vnd, and sog patterns, respectively, in mutant embryos. There is no staining above background levels. (D-F) snail, vnd, and sog patterns, respectively, in mutant embryos that contain the Toll10b transgene [P(bcdToll10bcd)]. snail expression is detected at the anterior pole, a band of vnd staining is detected in anterior regions (E), and a broad band of sog staining is detected in the anterior third of the embryo (F). The absence of vnd and sog expression at the anterior pole is probably due to repression by Snail (D).

View larger version (119K): [in a new window]   Fig. 2. Activated Pelle kinases generate multiple dorsoventral patterning thresholds. Mutant embryos at the cellular blastoderm stage were collected from females homozygous for a null mutation in gd. The embryos lack an endogenous dorsoventral Dorsal nuclear gradient. The embryos in C-E contain the Pelle-Tor4021 transgene, while the embryos in F-H contain a Pelle-Tor fusion gene. Both transgenes were expressed at the anterior pole of mutant embryos using the bcd 3' UTR. Embryos were stained with snail (C,F), vnd (D,G), and sog (E,H) hybridization probes. (A) Total protein extracts from wild-type and transgenic embryos were fractionated on a polyacrylamide gel, transferred to a membrane, and then incubated with an anti-Pelle antiserum. Lane 1 contains an extract from wild-type embryos (yw). The Pelle kinase has an apparent molecular weight of 75kDa. Lanes 2 and 3 contain extracts from transgenic embryos carrying either the Pelle-Tor4021 (lane 2) or the Pelle-Tor (lane 3) fusion protein. Both full-length proteins have an apparent molecular weight of 140kDa (the band at 95kDa is a putative degradation product). There are slightly higher levels of Pelle-Tor (lane 3) than Pelle-Tor4021 (lane 2). (B) The diagram depicts a precellular embryo that contains either the Pelle-Tor4021 or the Pelle-Tor transgene. Pelle-Tor4021 leads to the activation of snail at the anterior pole, whereas even high levels of the Pelle-Tor transgene fail to induce snail expression. (C) snail expression in mutant embryos that contain the Pelle-Tor4021 transgene. Staining is restricted to anterior regions containing peak levels of the transgene. (D) vnd expression pattern. Staining is detected in a stripe just posterior to the snail expression pattern. (E) sog expression pattern. Staining is detected in a broad band in the anterior third of the embryo. Both vnd and sog are repressed at the anterior pole, probably by Snail.(F) snail is not expressed in mutant embryos that contain the Pelle-Tor transgene. (G) vnd is activated only at the very anterior tip of embryos that express the Pelle-Tor transgene. (H) sog exhibits a broader pattern of expression in these embryos.

View larger version (99K): [in a new window]   Fig. 3. Twist is required for Dorsal gradient thresholds. Embryos were hybridized with a digoxigenin-labeled snail antisense RNA probe. They are oriented with anterior to the left. (A,B) Lateral (A) and ventral (B) views of wild-type embryos. snail staining is restricted to the ventral mesoderm. (C,D) Lateral (C) and ventral (D) views of twist–/twist– mutant embryos. The snail staining pattern is diminished and exhibits periodic repression along the anterior-posterior axis. (E,F) Wild-type (E) and mutant embryos (F) expressing a Toll10b transgene [P(hspToll10bbcd)], which produces a broad ectopic Dorsal nuclear gradient along the anterior-posterior axis. In a wild-type background the transgene produces uniformly intense activation of snail in the anterior third of the embryo, in addition to the endogenous pattern in the ventral mesoderm (E). There is a gap in staining where the ectopic and endogenous snail patterns intersect (arrow). There is a severe reduction in both the endogenous and ectopic snail expression patterns in twist–/twist– mutant embryos (F).

View larger version (76K): [in a new window]   Fig. 4. An anterior-posterior Twist gradient induces ectopic expression of sim. Embryos were hybridized with a digoxigenin-labeled sim (A-F) or Sex-lethal (G-I) antisense RNA probe and are oriented with anterior to the left. (A,D) Wild-type embryos that lack (A) or express (D) a twist-bcd transgene. sim is normally expressed in two lines that straddle the presumptive mesoderm (A). Staining does not extend to the anterior pole. In contrast, the twist-bcd transgene induces ectopic expression of sim at the anterior pole (D). This staining is first detected in precellular embryos (not shown), and persists during cellularization (D) and gastrulation (not shown). (B,C) snail–/snail– mutant embryos that lack the twist-bcd transgene. There is a delay in the onset of expression, and staining is not detected in advanced cellularizing embryos (B). Sporadic and weak expression is first detected at the onset of gastrulation in ventral regions (C). B and C display lateral and ventral views, respectively. (E,F) snail–/snail– mutant embryos that express the twist-bcd transgene. There is strong activation of sim in anterior regions of cellularizing embryos (E and F display lateral and ventral reviews, respectively). Strong ectopic expression persists during germband elongation (not shown). (G-I) Sex-lethal expression in wild-type embryos and those expressing the twist-bcd transgene. Sex-lethal is ubiquitously expressed in female embryos (H) and is not expressed in male embryos (G). In embryos from females containing the twist-bcd transgene, Sex-lethal is repressed at the anterior end of the embryo (I) in the domain that coincides with ectopic Twist expression. All Sex-lethal expressing embryos exhibit this repression, which accounts for the daughterless phenotype exhibited by females containing the twist-bcd transgene.

View larger version (107K): [in a new window]   Fig. 5. An anterior-posterior Twist gradient specifies two dorsoventral patterning thresholds in the absence of a Dorsal gradient. Embryos were hybridized with a digoxigenin-labeled snail antisense RNA probe, and are oriented with anterior to the left and dorsal up. (A) snail is not expressed in mutant embryos derived from Tollrm9/Tollrm10 transheterozygous females. sim is also silent in these mutants (data not shown). (B) snail staining pattern in a mutant embryo that contain the twist-bcd transgene. snail is broadly activated in anterior regions of cellularizing embryos (compare with A). (C) The snail staining pattern sharpens in cellularized mutant embryos that express the twist-bcd transgene. Staining is lost at the anterior pole. (D) sog is expressed throughout Tollrm9/Tollrm10 mutant, with the exception of the termini. (E) sog staining pattern in mutant embryos that contain the twist-bcd transgene. Staining is repressed in anterior regions (compare with D) where the Snail repressor is ectopically activated (see B). (F) sim staining pattern in mutant embryos that express the twist-bcd transgene. Expression is restricted to the anterior pole, in regions where the Snail repressor is lost (C).

View larger version (115K): [in a new window]   Fig. 6. Twist can activate vnd and sim without Dorsal. Embryos were stained with twist (A,B), sim (C), and vnd (D-F) RNA probes, and are oriented with anterior to the left and dorsal up. (A,B) Mutant embryos were obtained from gd–/gd– females and completely lack Dorsal nuclear protein. There is no twist expression in these mutants (A), but twist exhibits intense expression in anterior regions of mutant embryos that contain the twist-bcd transgene (B). (C,D) sim (C) and vnd (D) staining patterns in gd–/gd– mutant embryos that contain the twist-bcd transgene. Staining is detected in anterior regions where there are high levels of Twist. (E,F) The vnd expression pattern in precellular (E) and cellularized (F) wild-type embryos that contain the twist-bcd transgene. vnd expression is induced at the anterior pole in early embryos by the twist-bcd transgene (E), but this anterior expression is lost during the onset of the endogenous expression pattern (ventral stripes) after cellularization (F).

View larger version (24K): [in a new window]   Fig. 7. Dorsal-Twist patterning activities. (A) Previous studies identified a cluster of three low affinity Dorsal operator sites in a distal region of the snail 5' cis-regulatory region. The proximal regulatory region contains two Twist binding sites. The diagrams above the representation of the snail gene depict cross sections of embryos with Dorsal (left) and Twist (right) protein gradients (there are peak levels in ventral regions). The diagram to the far right summarizes a mutant embryo derived from Tollrm9/Tollrm10 females that contains a twist-bcd transgene. Normally, these embryos lack snail expression, but the twist-bcd transgene leads to the activation of snail in anterior regions. These results suggest that Twist plays a critical role in the on/off regulation of snail expression since the mutant embryos lack a Dorsal gradient. (B) Summary of Dorsal/Twist-regulated expression of snail and sim. We find that high amounts of Twist-bcd support ectopic sim expression (blue) that requires at least low levels of nuclear Dorsal and is repressed by Snail. High levels of ectopic nuclear Dorsal supported by the Toll10b-bcd transgene produces only very low levels of snail expression (red) and no sim expression (data not shown) in the absence of Twist. We conclude that Dorsal and Twist transcription factors function interdependently in order to generate multiple thresholds of gene expression. (sna-: no Snail protein; gd-: no nuclear Dorsal protein; twi–: no Twist protein; Tollrm9/rm10: low levels of nuclear Dorsal protein).