A conserved genetic mechanism specifies deutocerebral appendage identity in insects and arachnids

Abstract

The segmental architecture of the arthropod head is one of the most controversial topics in the evolutionary developmental biology of arthropods. The deutocerebral (second) segment of the head is putatively homologous across Arthropoda, as inferred from the segmental distribution of the tripartite brain and the absence of Hox gene expression of this anterior-most, appendage-bearing segment. While this homology statement implies a putative common mechanism for differentiation of deutocerebral appendages across arthropods, experimental data for deutocerebral appendage fate specification are limited to winged insects. Mandibulates (hexapods, crustaceans and myriapods) bear a characteristic pair of antennae on the deutocerebral segment, whereas chelicerates (e.g. spiders, scorpions, harvestmen) bear the eponymous chelicerae. In such hexapods as the fruit fly, Drosophila melanogaster, and the cricket, Gryllus bimaculatus, cephalic appendages are differentiated from the thoracic appendages (legs) by the activity of the appendage patterning gene homothorax (hth). Here we show that embryonic RNA interference against hth in the harvestman Phalangium opilio results in homeonotic chelicera-to-leg transformations, and also in some cases pedipalp-to-leg transformations. In more strongly affected embryos, adjacent appendages undergo fusion and/or truncation, and legs display proximal defects, suggesting conservation of additional functions of hth in patterning the antero-posterior and proximo-distal appendage axes. Expression signal of anterior Hox genes labial, proboscipedia and Deformed is diminished, but not absent, in hth RNAi embryos, consistent with results previously obtained with the insect G. bimaculatus. Our results substantiate a deep homology across arthropods of the mechanism whereby cephalic appendages are differentiated from locomotory appendages.

Keywords: Arthropoda; antenna; chelicera; deutocerebrum; opiliones; serial homology.

Figure 1.

Developmental dynamics of hth expression in deutocerebral and locomotory appendages. (a) Expression domains of Antp, hth, Dll and ss in the antenna and walking leg of D. melanogaster. In the antenna, hth knockdown or Antp overexpression results in antenna-to-leg transformation. In the leg, hth overexpression or Antp knockdown results in leg-to-antenna transformation. Gene interactions are shown to the right. (b) Comparative gene expression patterns of the Hox genes Antp and Ubx in an archetypal insect and arachnid. Note that chelicerate Antp is not expressed in the leg-bearing segments. (Online version in colour.)

Figure 2.

Reported expression boundaries of hth (green; upper bar) and Dll (red; lower bar) for deutocerebral and walking leg appendages across Arthropoda. Broken lines indicate uncertainty of expression boundary with respect to specific podomeres. Boxed orders indicate availability of functional data for hth orthologues (including from this study). References provided in the electronic supplementary material. (Online version in colour.)

Figure 3.

hth expression patterns in embryonic appendages of (a–c) the harvestman, P. opilio; (d–f) the scorpion, C. sculpturatus; and (g–i) the horseshoe crab, L. polyphemus. Arrowheads indicate segmental boundaries. Note absence of hth expression from both termini of all chelate appendages (arrows). Expression data for multiple spider species are closely comparable with harvestman counterparts and are not shown (figure 2). Scale bars, 100 µm. bt, basitarsus; da, distal article; fe, femur; mt, metatarsus; pa, patella; px, proximal segment; ta, tarsus; ti, tibia; tr, trochanter; tt, telotarsus. Expression data for Dll of C. sculpturatus are provided as the electronic supplementary material, figure S4. (Online version in colour.)

Figure 4.

Knockdown of hth results in homeotic transformations of gnathal appendages to legs in a chelicerate (Class II phenotype). (a) Control-injected hatchling of P. opilio, demonstrating wild-type morphology (ventral view). White arrowheads indicate pedipalpal spurs, which distinguish these appendages. (b,c) hth-dsRNA-injected hatchling of P. opilio in ventral view, exhibiting homeotic chelicera-to-leg transformation on one side (animal's left). (c) Same figure as in (b), with deutocerebral appendages outlined for clarity. (d–f) Appendage mounts of control-injected hatchlings. White arrowheads in (e) indicate pedipalpal spurs. (g–j) Appendage mounts of hth-dsRNA-injected hatchlings. Homeotic chelicera-to-leg transformation (g) and pedipalp-to-leg transformation (h) are accompanied by proximal leg defects (i). Note absence of pedipalpal spurs in (h). (j) Loss of mobile digit in a chelicera (black arrowhead). Scale bar for (a–c): 200 µm. Scale bar for (d–j): 50 µm. Abbreviations as in figure 3. (Online version in colour.)