A Novel Expression Domain of extradenticle Underlies the Evolutionary Developmental Origin of the Chelicerate Patella

Abstract

Neofunctionalization of duplicated gene copies is thought to be an important process underlying the origin of evolutionary novelty and provides an elegant mechanism for the origin of new phenotypic traits. One putative case where a new gene copy has been linked to a novel morphological trait is the origin of the arachnid patella, a taxonomically restricted leg segment. In spiders, the origin of this segment has been linked to the origin of the paralog dachshund-2, suggesting that a new gene facilitated the expression of a new trait. However, various arachnid groups that possess patellae do not have a copy of dachshund-2, disfavoring the direct link between gene origin and trait origin. We investigated the developmental genetic basis for patellar patterning in the harvestman Phalangium opilio, which lacks dachshund-2. Here, we show that the harvestman patella is established by a novel expression domain of the transcription factor extradenticle. Leveraging this definition of patellar identity, we surveyed targeted groups across chelicerate phylogeny to assess when this trait evolved. We show that a patellar homolog is present in Pycnogonida (sea spiders) and various arachnid orders, suggesting a single origin of the patella in the ancestor of Chelicerata. A potential loss of the patella is observed in Ixodida. Our results suggest that the modification of an ancient gene, rather than the neofunctionalization of a new gene copy, underlies the origin of the patella. Broadly, this work underscores the value of comparative data and broad taxonomic sampling when testing hypotheses in evolutionary developmental biology.

Keywords: dachshund; Pycnogonida; daddy longlegs; segmentation; subfunctionalization.

Graphical abstract

Fig. 1.

The patella differentiates the appendages of most chelicerate orders from other arthropods. (a) Exemplars of leg architecture across major arthropod lineages. Appendage schematics from top to bottom: spider leg; centipede leg; amphipod cheliped; insect leg. (b) Simplified phylogeny of Chelicerata. Icons indicate condition of patella. Origin of dac-2 has been inferred to originate from whole-genome duplication in the arachnopulmonate ancestor. Abbreviations: cx, coxa; tr, trochanter; fe, femur; pa, patella; ti, tibia; mt, metatarsus; ta, tarsus; pfe, prefemur; ba, basis; is, ischium; mr, merus; ca, carpus; pr, propodus; pta, pretarsus. Chelicerate tree topology based on Ballesteros et al. (2022) with unstable nodes collapsed.

Fig. 2.

The patella is inconsistently defined across Chelicerata. (a–h) Scanning electron micrographs (SEMs) of legs for selected orders. (a) Leg four of sea spider Nymphon sp. (Pycnogonida). Inset: distal podomere morphology of Nymphon gracile leg. (b) Leg four of horseshoe crab Limulus polyphemus (Xiphosura). Inset: distal podomere morphology of the pusher leg (leg V). (c) Leg four of camel spider Mummucia sp. (Solifugae). (d) Leg four of tick Ixodes scapularis (Parasitiformes, Ixodida). (e) Leg one of velvet mite Trombidium sp. (Acariformes, Trombidiformes). (f) Leg three of harvestman Zalmoxis furcifer (Opiliones). (g) Leg one of pseudoscorpion Pselaphochernes scorpioides (Pseudoscorpiones). (h) Leg one of scorpion Scorpio palmatus (Scorpiones). Blue shading: putative patellar homologs. White and orange text indicate alternative nomenclature. Abbreviations: acl: auxiliary claw; cx, coxa; tr, trochanter; fe, femur; bfe, basifemur; tfe, telofemur; dfe, distifemur; pa, patella; gn, genu; ti, tibia; mt, metatarsus; ta, tarsus; bta, basitarsus; tta, telotarsus; pr, propodus; cl, claw; pta, pretarsus; ap, apotele.

Fig. 3.

A distal ring domain of Po-exd is established early in P. opilio embryonic development and localizes to the patella-tibia segmental boundary. (a–d) Leg mounts of L2 in selected stages with merged visualization of Hoechst counterstaining (white), Po-exd (magenta), and Po-dac (yellow). (a′–d′) Multiplexed expression of Po-exd and Po-dac. (a″–d″) Isolated Hoechst counterstaining. Abbreviations: cx, coxa; tr, trochanter; ti, tibia; mt, metatarsus. Scale bars: 250 µm.

Fig. 4.

RNAi against Po-exd incurs a fusion at the patella-tibia joint in P. opilio. Icons indicate morphology of early and late RNAi embryos. Dark grey corresponds to the germband. (a) Distribution of outcomes following early Po-exd RNAi or negative control injections. Embryos were scored as dead only if they exhibited no further development following RNAi microinjection. (b–e) Hoechst staining of late stage embryos for early knockdown. (b) Negative control embryo, lateral view. (c) Same embryo as in (b), ventral view. (d) Po-exd RNAi embryo, lateral view. (e) Po-exd RNAi embryo, ventral view. Note posterior truncation and proximal leg defects in RNAi embryos. (f) Distribution of outcomes following late Po-exd RNAi or negative control injections. (g–l′) Late knockdown of Po-exd. (g) Leg three of negative control hatchling. (g′) Same hatchling as in (g), showing magnification of patella-tibia joint. (h) Leg three of Po-exd RNAi hatchling exhibiting fusion of patella-tibia joint. Note the location of melanized cuticle at dorso-distal boundary of femur, patella, and tibia. (h′) Same hatchling as in (h), showing magnification of fused patella-tibia joint. Black arrowhead: wild type patella-tibia joint. White arrowhead: fused patella-tibia joint. (i) Negative control embryo with expression of Po-Serrate (blue) and Po-Notch (orange). (i′) Same embryo as in (i) with Hoechst nuclear counterstaining. (j) Late Po-exd RNAi embryo with expression of Po-Ser and Po-N. (j′) Same embryo as in (j) with nuclear counterstaining. (k) Leg four of negative control embryo with expression of Po-Ser and Po-N. Note strong expression of Po-Ser and Po-N in the distal and proximal compartments of each podomere, respectively. (k′) Same appendage as in (k) with nuclear counterstaining. (l) Leg four of late Po-exd RNAi embryo with expression of Po-Ser and Po-N. Note disrupted expression of Po-Ser and Po-N in the fused tibia and patella. (l′) Same appendage as in (l) with nuclear counterstaining. Orange arrowheads: ring domains of Po-N in the proximal territory of developing podomeres. Blue arrowheads: ring domains of Po-Ser in the distal territory of developing podomeres. Asterisks: disrupted Po-Ser and Po-N at the fused boundary of tibia and patella. Abbreviations: ch, chelicera; pp, pedipalp; L1, leg one; L2, leg two; L3, leg three; L4, leg four; sp, spiracle; O2, second opisthosomal segment; cx, coxa; tr, trochanter; fe, femur; pa, patella, ti, tibia; mt, metatarsus; ta, tarsus.

Fig. 5.

Knockdown of Po-N yields broad developmental defects and diminution of the Po-exd distal ring domain. (a) Stage 10 negative control embryo in ventral view with Hoechst counterstaining (cyan). (a′–a′″) Same embryo as in (a) with expression of Po-en (green, a′), Po-exd (magenta, a″), and Po-N (orange, a′″). (b) Stage 10 Po-N RNAi embryo with Hoechst counterstaining. (b′–b″′) Same embryo as in (b) with expression of Po-en (b′), Po-exd (b″), and Po-N (b′″). (c) Distribution of outcomes following Po-N RNAi or negative control injection. White arrowheads: distal ring domains of Po-exd in wild type embryo. Abbreviations: lb, labrum; ch, chelicera; pp, pedipalp; L1, leg one; L2, leg two; L3, leg three; L4, leg four.

Fig. 6.

Late RNAi against Po-dac results in segmental fusions of medial podomeres. Icons indicate early and late RNAi embryos. Dark grey corresponds to the germband. (a) Pedipalp of negative control hatchling. (b) Leg three of negative control hatchling. (c) Pedipalp of early knockdown hatchling. (d) Leg three of early knockdown hatchling. (e) Pedipalp of late knockdown hatchling. (f) Leg three of late knockdown hatchling. (g) Leg four of late knockdown hatchling. (h) Distribution of outcomes following late Po-dac RNAi or negative control injection. Black arrowheads: melanized cuticle patches at dorso-distal boundary of femur, patella, and tibia. Grey arrowheads: traces of melanized patches in knockdown phenotypes. Asterisks indicate segments with aberrant morphology.

Fig. 7.

Late RNAi against Po-dac does not diminish distal expression of Po-exd. (a) Negative control embryo in lateral view, with Hoechst counterstaining (cyan). (a′–a″) Same embryo as in (a) with single-channel expression of Po-dac (yellow, a′) and Po-exd (magenta, a″). (b–c″) Po-dac RNAi embryos in lateral view with Hoechst counterstaining (b, c), Po-dac expression (b′, c′), and Po-exd expression (b″, c″). Note the more severe medial appendage defects in (b) coincident with pronounced developmental delay. White arrowheads: distal ring domains of Po-exd expression in all embryos examined. Abbreviations: ch, chelicera; pp, pedipalp; L1, leg one; L2, leg two; L3, leg three; L4, leg four.

Fig. 8.

Pl-exd is expressed in a ring-like domain distal to Pl-dac expression in the developing legs of the sea spider P. litorale. All images apart from upper row in (c) show a ventral detail of the posterior body pole as indicated to the left of each row. Arrows: distal boundary of Pl-dac domain. Arrowheads: Pl-exd expression distal to the Pl-dac domain. Ovals and circles: limb bud primordia hidden under the cuticle. Asterisks indicate region of damaged tissue at the tip of the posterior body pole. (a) Detail of leg 1 bud and leg 2 primordium in early instars III. (b) Detail of leg 1 bud and leg 2 primordium in mid- to late-stage instars III. (c) Upper row: dissected leg one of mid-stage instars IV. Lower row: detail of leg 2 bud and leg 3 primordium in mid-stage instar IV. Abbreviations: cx, coxa; fe + ti1, femur-tibia 1 precursor; L, leg; mc, main claw; ta + pro, tarsus-propodus precursor; ti2, tibia 2.

Fig. 9.

Surveys of exd and dac expression across arachnid orders with disputed patellar homologs. (a) Embryo of the pseudoscorpion Pselaphochernes scorpioides visualized via cuticular autofluorescence (green). (a′) Same embryo as in (a) with multiplexed expression of Ps-exd-1 (magenta) and Ps-dac-1 (yellow). (b) Autofluorescent visualization of P. scorpioides embryo. (b′) Same embryo as in (b) with expression of Ps-exd-2 and Ps-dac-2. (c) Magnified view of Ps-exd-2 and Ps-dac-2 expression in the legs of an older P. scorpioides embryo. (d) Limb bud stage embryo of the acariform mite Archegozetes longisetosus with Hoechst nuclear counterstaining (cyan). (d′, d″) Same embryo as in (d) with multiplexed expression of Al-exd (magenta) and Al-dac (yellow) (d′), or single-channel expression of Al-exd (d″). (e–h) Limb mounts of the solifuge Titanopuga salinarum at leg elongation stage with nuclear counterstaining (cyan), and expression of Ts-exd (magenta) and Ts-dac (yellow). (e, e′) Chelicera. (f, f′) Pedipalp. (g, g) Leg one. (h, h′) Leg three. Note additional domain of overlapping dac and exd in the basifemur of leg three (unique to legs three and four; white arrow). White arrowheads indicate an exd boundary distal of dac. Abbreviations: ch, chelicera; pp, pedipalp; L1, leg one; L2, leg two; L3, leg three; L4, leg four. (i) Stage 9 embryo of the tick Ixodes scapularis with Hoechst nuclear counterstaining (cyan), Is-dacA expression (yellow), and Is-exd expression (magenta). (e′) Same embryo as in (e) without nuclear counterstaining. (j) Stage 10 embryo of I. scapularis with nuclear counterstaining. (j′) Same embryo as in (j) showing multiplexed expression of Is-dacA and Is-dacB (yellow), and Is-exd (magenta). Note autofluorescence of yolk and germ cells (gc) in magenta channel in (i′, j′).

Fig. 10.

A distal ring domain of exd, abutting a conserved medial domain of dac, during embryonic appendage formation is responsible for the origin of the chelicerate patella. Presence of the distal ring domain in Pycnogonida and many chelicerate orders supports its presence in the chelicerate common ancestor. Lineage-specific loss of distal exd expression in ixodid ticks suggests loss of a patellar homolog.