Toll Genes Have an Ancestral Role in Axis Elongation Matthew A. Benton, Matthias Pechmann, Nadine Frey, Dominik Stappert, Kai H. Conrads, Yen-Ta Chen, Evangelia Stamataki, Anastasios Pavlopoulos, Siegfried Roth  Current Biology  Volume 26, Issue 12, Pages 1609-1615 (June 2016) DOI: 10.1016/j.cub.2016.04.055 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Expression and Function of Tc-Tl7 and Tc-Tl10 in Tribolium (A and B) Whole-mount (A1, A 2, B1, and B2) and flat-mount (A3–A8 and B3–B8) embryos from the blastoderm to the extended germband stage showing Tc-Tl7 and Tc-Tl10 expression (magenta) and nuclei (blue). Primary (1°) and secondary (2°) domains are annotated in (A6) and (B6). (C) Flat-mounted embryos double stained for Tc-Tl7+Tc-Tl10 from the differentiated blastoderm stage (inset) to the extended germband stage. The first segmental domains of Tc-Tl7 and Tc-Tl10 are annotated with an arrowhead and an arrow, respectively. Nuclei are shown in the inset in blue. (D–G) Flat-mounted embryos double stained for Tc-eve+Tc-Tl7 (D), Tc-wg+Tc-Tl7 (E), Tc-eve+Tc-Tl10 (F), and Tc-wg+Tc-Tl10 (G; the anterior segmental stripe in this embryo appears to overlap the Tc-wg domain due to expression in the intercalary segment). (H) Stylized schematic showing expression of Tc-Tl7 and Tc-Tl10 with respect to marker genes and parasegment boundaries (PS; shown where possible; white lines). Note that segments along the anterior-posterior axis of Tribolium embryos exhibit different stages of maturity, with posterior segments being youngest and anterior segments being oldest. (I and J) Frames from time lapses of wild-type (I) or Tc-Tl7+Tc-Tl10 double RNAi (J) embryos transiently labeled with GAP43YFP annotated to show tracked cells. Timing is minutes post-cellularization (mpc). (K–N) Frames from time lapses of wild-type (K and M) or Tc-Tl7+Tc-Tl10 double RNAi (L and N) embryos transiently labeled with GAP43YFP as either ventral views 4.5 hr after the completion of cellularization (hpc; K and L) [6, 11] or lateral views 9 hpc (M and N). Germbands are outlined with dotted lines. (O and P) Flat-mounted embryos at the extended germband stage showing Tc-wg expression in wild-type (O) and Tc-Tl7+Tc-Tl10 double RNAi (P) embryos. Segment labels are mandible (Md); maxilla (Mx); labial (Lb); thoracic segments 1–3 (T1, T2, and T3); and abdominal segments 1, 5, and 10 (A1, A5, and A10). (A1), (A2), (B1), and (B2) are maximum-focus projections of one egg hemisphere, (I) and (J) are average-intensity projections spanning 6 μm, and (K)–(N) are average-intensity projections of one egg hemisphere. Anterior is to the left in all panels, and ventral is to the bottom in (A1), (A2), (B1), (B2), (I), (J), (M), and (N). Scale bars represent 25 μm in the inset of (C) and in (I) and (J) and 100 μm in all other panels. See also Figure S1 and Movies S1 and S2. Current Biology 2016 26, 1609-1615DOI: (10.1016/j.cub.2016.04.055) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Toll Genes Show Striped Expression in Diverse Arthropods Embryos from a wide range of arthropods showing Toll gene expression together with a cladogram to show evolutionary relationships. In Nasonia, Nv-Tl7 and Nv-Tl10 both show striped expression. In Oncopeltus and Gryllus, orthologs of Tl7, Tl8, and Tl10 all show striped expression. In Parhyale and Strigamia, only single Toll genes show striped expression. In Parasteatoda, Pt-LotoA is expressed in stripes and Pt-LotoB has broad expression in the posterior of the embryo where segments are specified. The Parhyale, Strigamia, and Parasteatoda, panels also show nuclei in blue. All embryos are mounted with the anterior to the left. The Nasonia embryos, Oncopeltus blastoderm embryos, and Strigamia embryo are shown as lateral views with ventral to the bottom; all others are flat-mount ventral views. Scale bars represent 100 μm for each animal. See also Figures S2 and S3. Current Biology 2016 26, 1609-1615DOI: (10.1016/j.cub.2016.04.055) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Pt-LotoA and Pt-LotoB Are Required for Normal Germband Morphogenesis (A–D) Flat-mounted wild-type (A and C) and Pt-LotoA+B double knockdown (B and D) embryos showing expression of the Parasteatoda ortholog of Orthodenticle-1 (Pt-Otd1) [34] to mark midline tissue (A and B) or the Parasteatoda ortholog of the segment polarity gene engrailed (Pt-en) [35] to mark segments (C and D). Expression is false-colored in red, nuclei are shown in blue. Embryos are ventral views with anterior to the left. The scale bar represents 200 μm. (E) Box-and-whisker graphs showing width of the first opisthosomal segment in wild-type and knockdown embryos staged to either 10 or 11 Pt-en stripes (ES). Numbers of embryos are shown for each treatment. ∗∗∗p < 0.001 (unpaired t test). The horizontal line is the median, boxes are first and third quartiles, and whiskers are minimum and maximum data points. See also Figure S3. Current Biology 2016 26, 1609-1615DOI: (10.1016/j.cub.2016.04.055) Copyright © 2016 Elsevier Ltd Terms and Conditions