Expression of homothorax and extradenticle mRNA in the legs of the crustacean Parhyale hawaiensis: evidence for a reversal of gene expression regulation in the pancrustacean lineage

Abstract

In Drosophila leg development, the extradenticle (exd) gene is expressed ubiquitously and its co-factor homothorax (hth) is restricted to the proximal leg portion. This condition is conserved in other insect species but is reversed in chelicerates and myriapods. As the region of co-expression does not differ in the two groups and transcripts from both are necessary for function, this difference in expression is likely to be functionally neutral. Here, we report the expression patterns of exd and hth in a crustacean, the amphipod shrimp Parhyale hawaiensis. The patterns in P. hawaiensis are similar to the insect patterns, supporting the close relationship between crustaceans and insects in the taxon Tetraconata. However, mRNA expression of exd in P. hawaiensis is weak in the distal leg parts, thus being intermediate between the complete lack of distal exd expression in chelicerates and myriapods and the strong distal exd expression in insects. Our data suggest that the reversal of the gene expression regulation of hth and exd occurred in the pancrustacean lineage.

Fig. 1

Phylogenetic analysis of selected arthropod Hth and Exd sequences. a Puzzle tree for homothorax sequences. b Puzzle tree for extradenticle sequences. All trees show the unrooted majority-rule consensus phylogram for 1,000 intermediate trees computed with the Quartet Puzzling method (Strimmer and von Haeseler 1996). Reliability values are indicated at the tree edges. Species abbreviations: Cs, C. salei; Dm, D. melanogaster; Gb, G. bimaculatus; Gm, G. marginata; Nv, N. vitripennis; Of, O. fasciatus; Ph, P. hawaiensis; Tc, T. castaneum

Fig. 2

Alignment of a conserved alpha helix in Hth proteins from various arthropods. The helix is involved in Hth–Exd interactions in D. melanogaster and is highly conserved in other arthropods. The motif M2 (boxed) and the valine behind it (arrow) have been shown to be essential for protein–protein interactions (PCM = Pbx co-operation motif). Dashes indicate identical amino acid residues. Species abbreviations see Fig. 1

Fig. 3

Expression of Ph-hth and Ph-exd mRNA in P. hawaiensis peraeopods. a Expression of Ph-hth in the peraeopods of an embryo at stage S19 (for P. hawaiensis embryonic development see Browne et al. (2005)). b Expression of Ph-exd in the peraeopods of an embryo at stage S19. In both cases, expression is restricted to proximal areas. c Expression of Ph-hth in a peraeopod at stage S24. Podomeres are indicated next to the appendage. Expression is restricted to the three proximal podomeres. d Expression of Ph-exd in a peraeopod at stage S24. Expression is ubiquitous but is weaker in the distal portion of the appendage. Abbreviations: cx, coxa; bs, basis; is, ischium; me, merus; ca, carpus; pp, propodus; da, dactylus

Fig. 4

Expression of Ph-hth and Ph-exd mRNA in P. hawaiensis pleopods and uropods. a, b Rear ends of stage 24 embryos, anterior to the left. Expression of Ph-hth is excluded from distal parts of the pleopods and uropods (a). Please note that the dark staining at the tip of some pleopods in the figure is an artificial staining caused by attraction of the antibody towards the embryonic cuticle that starts developing at these late stages. Although the antibody was pre-absorbed against fixed old embryos, artificial staining of cuticle could not be suppressed completely in all specimens. Expression of Ph-exd is ubiquitous at a low level in the pleopods and uropods (b). c–e Dissected trunk legs stained for Ph-hth mRNA. c Maxilliped. d Pleopod. e Uropod. The inset shows a generalised sketch of an amphipod shrimp with the tagmata denoted with colours: red, cephalothorax; orange, peraeon; yellow, pleosome; green, urosome. Abbreviations: en, endites; ep, endopod; ex, exopod; mxp, maxilliped; pe, peraeopods; pl, pleopods; up, uropods

Fig. 5

Hypothesis for the transition from exd–hth expression in chelicerates to exd–hth expression in insects. The cartoons represent the ancestral states (except for D which represents an apomorphic state) for the respective group and are deduced from the extant patterns. The red cartoon legs show the expression of exd (top) and hth (bottom). The green cartoon legs show the area of co-expression. Note that this area is identical in all cases and denotes the area of Exd–Hth function. A is hypothesized to be the ancestral condition in the arthropods and is retained in the extant Chelicerata and is also unchanged in the common ancestor of Myriapoda, Crustacea and Insecta (B) and the extant Myriapoda. C represents the ancestral state for the Crustacea and Insecta and is retained in the Crustacea, whereas in the insect lineage a new apomorphic state is evolved (D). A is based on Prpic et al. (2003) and Prpic and Damen (2004). B is based on Prpic and Tautz (2003). C is based on this paper. D is based on Angelini and Kaufman (2004), Inoue et al. (2002) and Prpic et al. (2003). The blue arrow denotes the origin of the gene expression regulation reversal by the release of exd proximal restriction and the proximal restriction of hth expression. In this view, the expression patterns in crustaceans mediate between the reversed conditions in chelicerates–myriapods and insects. For details, please see text