Distinct Signals Generate Repeating Striped Pattern in the Embryonic Parasegment  Victor Hatini, Stephen DiNardo  Molecular Cell  Volume 7, Issue 1, Pages 151-160 (January 2001) DOI: 10.1016/S1097-2765(01)00163-0

Figure 1 Mapping Sr Expression Each PS (brackets) Extends from the En Territory to the Next Posterior Wg Territory. (A) WT cuticle pattern; Sr protein expression (red stripes) is superimposed on cuticle pattern. Denticles rows (1°–6°) are colored in red due to non-specific staining. Sr row 1 and 2 (arrowheads, 1 and 2) are in epidermal cells that also secrete the second and fifth denticle rows, respectively; Sr row 3 (arrowhead, 3) is in one cell row in the middle of the expanse of smooth cuticle. B, C, and E are confocal sections of stage 13 embryos, ventral views; the first panel shows marker gene expression (green), the middle panels Anti-Sr (red), and the right-hand panel is a merged view. (B) Sr row 1 (arrowhead) is posteriorly adjacent to the En territory. Note that Sr row 1 is in anterior Ve-expressing cells (C, yellow in merged panel). (C) Sr row 2 is posteriorly adjacent to the Ve territory (arrowhead). Lower levels of Ve expression are detected in a few cells posterior to Sr row 3. However, no role for Ve at this region has been reported (O' Keefe et al. 1997; Szü ts et al. 1997). (D) Ser RNA (blue), Anti-Sr (brown) double label, stage 13 embryo. Sr row 2 (arrowhead, 2) is in anterior Ser expressing cells. (E) Sr row 3 (arrowhead) is anteriorly adjacent to the Wg territory. (F) Summary: the position of Sr expression across a PS relative to territories of gene expression. Bar = 20 μm in B, C, and E; 4 μm in D; 3 μm in A. Molecular Cell 2001 7, 151-160DOI: (10.1016/S1097-2765(01)00163-0)

Figure 2 A Distinct Ligand Induces Each Sr Row Confocal micrographs, Stage 13, ventral view; For A, C, and E: the first panel shows marker expression (green); the second panel, Anti-Sr (red); the third panel shows the merge. B, D, and F: show temperature-sensitive mutants used to inactivate the Hh, Egfr, and Wg pathways, respectively. Bracket marks one PS. (A) In wg mutants that maintain En expression, Sr (arrowhead) is expressed around the En territory. (B) Blocking Hh activity at 7 hr AEL selectively eliminates Sr row 1 (arrowhead shows loss of stripe). Sr row 2 and 3, identified by underlying tracheal expression, are unaffected in hh mutants. (C) In wg en double mutants, Sr (arrowhead) is induced around the Ve expressing territory. (D) Blocking Egfr function at 7 hr AEL specifically abolishes Sr row 2 (arrowhead shows loss of stripe). The normally slightly broad expression of Sr row 3 verifies that Sr row 2 was the affected row. (E) In hh mutants that maintain Wg expression, Sr (arrowhead) is expressed around the Wg domain. (F) Blocking Wg function at 7 hr AEL selectively eliminates Sr row 3 (arrowhead), as verified by marker Wg-lacZ expression (green). Bar = 20 μm in all panels. Molecular Cell 2001 7, 151-160DOI: (10.1016/S1097-2765(01)00163-0)

Figure 3 Altered Sr Expression Pattern in sr03999/+ and Ser−/− Embryos (A) Enhancer trap sr03999 insertion (sr03999 / +) faithfully expresses LacZ (green, left-hand panel) in Sr rows 2 and 3 but not Sr row 1 (merge panel, arrowhead points to endogenous Sr 1 in red, and absence of green). (B) WT shows the usual spacing between Sr row 1 and 2 (arrowhead). (C) In Ser mutants, the spacing between Sr row 1 and 2 is reduced (arrowhead). Bars = 20 μm in A, 50 μm in B–C. Molecular Cell 2001 7, 151-160DOI: (10.1016/S1097-2765(01)00163-0)

Figure 5 Generating a Repeating Striped Pattern across a Cellular Field (A,B) The primary organizing signals, Wg and Hh, establish four distinct territories across the PS. Early, Hh and Wg, secreted from the boundaries of each PS, form activity gradients across the PS (A). The activity landscape of each ligand is asymmetric, because Wg and Hh antagonize each other's function (Gritzan et al. 1999; Sanson et al. 1999). Wg and Hh define territories across the PS (B). (C,D) Ligands emanating from each of these territories, including Hh and Wg themselves, generate a repeating striped pattern. Local activity gradients of Hh, Spi (activated by Ve), and Wg form (C, this work; Alexandre et al. 1999; Gritzan et al. 1999). Then Sr is induced at positions of high signaling attained only adjacent to each ligand source (D). The directionality of the response is most likely dictated by the ability of each territory to induce Sr expression in response to each ligand (see Discussion). Molecular Cell 2001 7, 151-160DOI: (10.1016/S1097-2765(01)00163-0)

Figure 4 Signaling Levels Limit the Width of Sr Expression Zones A, C, E, and G: the first panel is marker expression (green); the second panel is Sr expression (red); the third panel is a merge, where boxed area is magnified in right-most panels, B, D, F, and H, respectively. (A–B) Expressing the secreted form of Hh (HhN) in the En territory leads to expansion of the Sr-expressing zone from one to six cell rows (arrowheads). (C–D) In such HhN embryos, all Sr expressing cells co-express Ve confirming that Sr row 1 is expanded in this condition. (E–F) Removing the inducible antagonist of Spi, Argos, leads to posterior expansion of Sr row 2, from one to two to three cell rows (arrowheads). (G–H) Reducing levels of the inducible antagonist of Wg signaling, Naked, leads to anterior expansion of Sr row 3 (arrowheads). Near the ventral midline, strong expression is seen in at least three cell rows further from the Wg source compared to one in wild type. Bar = 20 μm in A, C, and E; 6 μm in B, D, and F. Molecular Cell 2001 7, 151-160DOI: (10.1016/S1097-2765(01)00163-0)