Fig. 5. Early Pairberry pattern in Shistocerca. (A-D) Anti-Pby immunostaining of early grasshopper embryos. (A) Dapi stain of embryo, 
9% of development. (B) Same embryo, showing weak posterior Pby domain also observed with pby1 mRNA (not shown). (C) 11% embryo showing gnathal arc (black arrowhead). The amniotic fold has been left intact (asterisk). (D) 15% embryo showing gnathal arc (black arrowhead) and T2/T3 broad domain with higher levels at the anterior edge where the T2 Pby stripe will form (open arrowhead). (E-G) Anti-Pby (black) and anti-Hb (brown) immunostaining of 16-17% embryos. (E) At 16% the Mn, La and T2 Pby stripes have formed, while T1 is just beginning to appear. The strong subdomain of Hb protein extends from the Mn Pby stripe to just posterior of the La Pby stripe, where the weak subdomain continues through the T1 Pby stripe. Low levels of Hb are also found throughout the more anterior head lobes (asterisk). (F) Slightly later, the Mx Pby stripe has formed while the Hb domain remains static. (G) At 18%, extension results in the concomitant separation of Pby stripes and increased length of the Hb domain, particularly in T1. (H) 17% embryo after the split of the gnathal arc and intercalation of the Mx and T1 Pby stripes, plus addition of antennal (red arrowhead) and T3 Pby stripes. Open arrowhead indicates position of T2 Pby stripe. Embryo also shows several pre-antennal domains, which include the eyespot present in the pby1 mRNA pattern (red arrow) and domains not seen in either the pby1 or pby2 pattern (black arrows). Scale bar: 300 µm for A-D,H; and 170 µm for E-G.
