Fig. 4. The Pairberry pattern comprises both pby1 and pby2 in Schistocerca. Grasshopper embryos stained for pby1 mRNA (A,F,K) and pby2 mRNA (B,G,L), En protein (C,H,M), Pby protein (D,I,N), or both Pby (black) and En (brown) protein (E,J,O). Approximate stages are as indicated. Open arrowheads (<) mark T1 in Pby embryos. (A-E) 
18%: pby1 mRNA is found in seven stripes that correspond to the antennal, gnathal and thoracic segments, as well as a broad posterior domain and pre-antennal eyespots (brown arrows, A,D,E). (B,C) pby2, by contrast, is not expressed at this stage (B) while only the antennal stripes of En (C) have appeared. Anti-Pby staining reveals a pattern similar to pby1 mRNA as well as several, more anterior, pre-antennal domains of the head lobes (brown arrowheads, D,E). (F-J) 23%: additional pby1 stripes have appeared in the intercalary segment and A1-A6, along with a broad posterior domain (A7/A8). Note that the gnathal, thoracic, and A1 pby1 stripes are now restricted to the neuroectoderm (F). pby2 mRNA is found in stripes, coincident with pby1, that are entirely restricted to the neuroectoderm and are, in this embryo, only just appearing in A4 (G). pby2 mRNA appears about the same time as En, which in this embryo is found only as far posterior as A3 (H). The pby1 eyespots have refined to two lateral domains, which are still not present in the pby2 pattern (compare F with G). The two Pby embryos are slightly older, showing laterally restricted stripes in A3 and A4 as well individuated A7 and A8 stripes (I,J). The Pby/En embryo reveals that En stripes appear before the restriction of Pby1 protein (J). (K-O) 35%: ventrally restricted stripes of pby1, as well as pby2, are found in the antennal, intercalary as well as all gnathal, thoracic and abdominal segments (K,L). pby1 and pby2 stripes are also found in A11 (black arrows, K,L), which does not express En (the A11 stripe is present, but not visible in O). At this stage, pby1 is also expressed in at least one ring in the limb primordia, of which three are visible in the Pby embryos (red arrows, K,N,O). For the first time, pby2 is also expressed in pre-antennal domains coincident with pby1, as well as the gnathal appendages (red arrowheads, L,N,O). Anti-Pby staining recapitulates the combined pby1 and pby2 patterns, while also revealing non-PgIII domains found in the pre-antennal regions of the head lobes, the mandibles (in which staining appears before the appearance of pby2 mRNA in the mandible) (asterisk, I,J,N,O), as well as tips of gnathal palps, limbs, abdominal buds and the telson (where En is also expressed) (black arrowheads, M-O). Scale bar: 1.0 mm.
