Functional analysis of Scr during embryonic and post-embryonic development in the cockroach, Periplaneta americana

Abstract

The cockroach, Periplaneta americana represents a basal insect lineage that undergoes the ancestral hemimetabolous mode of development. Here, we examine the embryonic and post-embryonic functions of the hox gene Scr in Periplaneta as a way of better understanding the roles of this gene in the evolution of insect body plans. During embryogenesis, Scr function is strictly limited to the head with no role in the prothorax. This indicates that the ancestral embryonic function of Scr was likely restricted to the head, and that the posterior expansion of expression in the T1 legs may have preceded any apparent gain of function during evolution. In addition, Scr plays a pivotal role in the formation of the dorsal ridge, a structure that separates the head and thorax in all insects. This is evidenced by the presence of a supernumerary segment that occurs between the labial and T1 segments of RNAiScr first nymphs and is attributed to an alteration in engrailed (en) expression. The fact that similar Scr phenotypes are observed in Tribolium but not in Drosophila or Oncopeltus reveals the presence of lineage-specific variation in the genetic architecture that controls the formation of the dorsal ridge. In direct contrast to the embryonic roles, Scr has no function in the head region during post-embryogenesis in Periplaneta, and instead, strictly acts to provide identity to the T1 segment. Furthermore, the strongest Periplaneta RNAiScr phenotypes develop ectopic wing-like tissue that originates from the posterior region of the prothoracic segment. This finding provides a novel insight into the current debate on the morphological origin of insect wings.

Fig. 1

Embryonic expression patterns of Scr in Periplaneta americana. (A) At ≈10% development, Scr mRNA is broadly expressed in the mid-posterior region with two strong bands that correspond to the future Lb/T1 region. (B) At ≈20% development, strong Scr mRNA localizes to the labial segment. (C) Slightly later at ≈25%, Scr continues to be solely expressed in the labium with no signal in the Mx or T1 segments. (D) SCR protein accumulation in an embryo at ≈75% development. While strong expression remains in the labial palps, SCR has now expanded into the T1 segment with clear signal in the dorsal T1 region (orange arrowhead) and in two discrete clusters of cells in the T1 leg (white arrowheads). (E) RNAiScr embryo that has been stained for Scr mRNA accumulation. The lack of signal indicates the complete depletion of Scr. Abbreviations: Mn = Mandibles, Mx = Maxillary, Lb =Labial, T1 = First thoracic segment

Fig. 2

Embryonic RNAiScr phenotypes in the labial palps and T1 legs of Periplaneta. (A–C) Wild type and RNAiScr labial phenotypes. (A) Wild type labium of first nymph. The articulated labial palps are composed of three sub-segments: S1 (proximal), S2 (middle), and S3 (distal). (B) The labial palps of a moderate RNAiScr first nymph. The phenotypic effects are most noticeable in S2 and S3 where large thoracic leg-like ectopic bristles form (black arrowheads). The distal half of S3 develops numerous hairs (*) resembling those covering the antennae; compare to (C1). (C) Strong RNAiScr first nymph showing a transformation of the labial palp into a mixed leg-antennal identity. S2 shows a complete transformation, assuming the identity of the T1 femur. Note the row of bristles (black arrowheads) and the large spur at its distal end (black arrow; compare to 1D, black arrow). S3 transforms into a mixed leg (proximal) and antennal (distal) identity. (C1) Distal tip of a wild type antenna; compare with distal half of transformed S3 in B and C (*). (D–F) Wild type and RNAiScr T1 and T2 leg phenotypes. (D) Wild type T1 leg. (E) T1 leg of RNAiScr first nymph. The femur is characterized by a row of approximately seventeen small, closely organized bristles along the entire length of the ventral side. These features remain unaffected in Scr-depleted nymphs, compare to wild type T1 femur in D. (F) Wild type T2 leg showing similar morphology to T1 legs. The only observable difference is the row of bristles along the ventral margin, which are fewer in number (approximately 10) and more spaced out. (D1–F1) Magnified view of boxes in D–F showing that the T1 leg of RNAiScr first nymphs retains its identity.

Fig. 3

Dorsal ridge phenotypes of RNAiScr Periplaneta americana first nymphs. (A) Wild type first nymph showing the characteristically large pronotum that conceals most of the head. (A1) Ventral view of the head and thoracic boundary. (B) Lateral view of wild type first nymph. (B1) Magnified view of lateral head and T1 of wild type first nymph shown in (B). Black arrows in A, B, and B1 point to the dorsal ridge (C) Strong RNAiScr nymph phenotype showing the development of a supernumerary segment between the head and the prothorax. (C1) Extra segment viewed from the ventral side (open black arrowheads point to the ectopic segment). (D) Lateral view of RNAiScr first nymph. (D1) Close up of supernumerary segment shown in (D). Black brackets depict the length of the ectopic segment in (D–D1). Abbreviations: ant = antenna; fe = femur; gl = glossa; pg = paraglossa; lp = labial palp; S1 = sub-segment 1 of lp; S2 = sub-segment 2 of lp; S3 = sub-segment 3 of lp.

Fig. 4

Engrailed (en) mRNA accumulation in wild type and RNAiScr Periplaneta americana embryos. (A) Wild type embryo showing a combined Mx/Lb stripe of engrailed expression that circumvents the embryo. (A1) Close up of the embryo shown in (A). Green arrowhead points to the single Mx/Lb stripe of en expression. (B) Similarly staged RNAiScr embryo stained for en mRNA accumulation. The single band of Mx/Lb expression bifurcates (green arrowhead) and results in the formation of a de novo band of en expression anterior to the Lb stripe. Abbreviations: Mx = Maxillary, Lb = Labial

Fig. 5

Comparison of the labial appendages and the thoracic legs of wild type and RNAiScr Periplaneta americana adults. (A) Dissected head of a wild type adult. (B) Dissected head of an RNAiScr adult. Note that the labial appendages are unaffected and appear wild type. (C) Wild type adult T1 leg. (D) T1 leg of an RNAiScr adult. The legs remain unaltered and do not take on a T2 identity. (E) Wild type adult T2 leg.

Fig. 6

Adult RNAiScr phenotypes of the prothoracic (T1) segment in Periplaneta americana. (A) Dorsal view of the prothoracic (T1) and mesothoracic (T2) segments of a wild type adult. The posterior margin of T1 has a rounded, smooth morphology while T2 exhibits a thickening of the cuticle that forms a ridge-like structure (white arrowheads). (B) Dorsal view of the prothoracic (T1) and mesothoracic (T2) segments of a RNAiScr adult. The posterior margin of T1 now exhibits a thickening of the cuticle and starts to assume a T2-like identity. In addition, an ectopic groove appears along the midline of the T1 segment that is normally only found on T2 (green arrowheads). (C1) Close up of the posterior portion of the T1 segment of the wild type adult shown in (A). (C2) Close up of the posterior portion of the T1 segment of the RNAiScr adult shown in (B). Note that the cuticle exhibits a thickening and appears like the ridge-like structure present on T2 (compare to (C3)). (C3) Close up of the posterior portion of the T2 segment of the RNAiScr adult shown in (B). This segment is unaffected and appears wild type (compare to (A)). (D) Strong RNAiScr adult phenotype. Ectopic wing-like tissue develops from the posterior lateral portion of T1. (D1) Close up view of the left ectopic wing-like tissue of the strong RNAiScr adult shown in (D). (E) RT-PCR analysis of Scr mRNA in the prothoacic plates of seventh nymphs. RNAiScr nymphs show a complete depletion of Scr mRNA in T1 as compared to wild type.

Fig. 7

Morphology of Periplaneta wild type and RNAiScr seventh instars. (A–B2) Wild type. (C–D1) RNAiScr. (A) Wild type seventh nymph pronotum (T1) and mesonotum (T2). Note the large wing pads (arrows) on the lateral margins of T2 making this segment morphologically distinct from T1. (B) Dorsal image of wild type T1 and T2 illuminated by indirect light. The branching wing veins (trachea) along the lateral margins of T2 are quite evident, while trachea are not observed in the pronotum. (B1) Magnified image of lateral margin of T1 (upper box in B1). Note the absence of wing veins. (B2) Magnified image of lateral margin of T2 (lower box in B). Branched veins are clearly present in the developing wing pad. (C) T1 and T2 of an Scr-RNAi seventh nymph. Compared to wild type, the morphology of T1 is altered due to the development of ectopic wing pads at the lateral margins of this segment. (D) Close-up view of lateral margins of T1 and T2 illuminated with indirect light showing the development of trachea in the ectopic wing pads. (D1) The development of veins in T1 is unmistakable (compare to B1). Note, however, that the developing trachea are not identical to those in wild type T2, suggesting an incomplete transformation of T1 toward T2. Legend: T1 = prothorax; T2 = mesothorax; wp = wing pad.