Expression of Distal-Less, Dachshund, and Optomotor Blind in Neanthes Arenaceodentata

Dev Genes Evol (2010) 220:275–295 DOI 10.1007/s00427-010-0346-0 ORIGINAL ARTICLE Expression of Distal-less, dachshund, and optomotor blind in Neanthes arenaceodentata (Annelida, Nereididae) does not support homology of appendage-forming mechanisms across the Bilateria Christopher J. Winchell & Jonathan E. Valencia & David K. Jacobs Received: 26 July 2010 /Accepted: 9 November 2010 /Published online: 30 November 2010 # The Author(s) 2010. This article is published with open access at Springerlink.com Abstract The similarity in the genetic regulation of mesoderm. Domains of omb expression include the brain, arthropod and vertebrate appendage formation has been nerve cord ganglia, one pair of anterior cirri, presumed interpreted as the product of a plesiomorphic gene precursors of dorsal musculature, and the same pharyngeal network that was primitively involved in bilaterian ganglia and presumed interneurons that express dac. appendage development and co-opted to build appen- Contrary to their roles in outgrowing arthropod and dages (in modern phyla) that are not historically related vertebrate appendages, Dll, dac,andomb lack comparable as structures. Data from lophotrochozoans are needed to expression in Neanthes appendages, implying independent clarify the pervasiveness of plesiomorphic appendage- evolution of annelid appendage development. We infer forming mechanisms. We assayed the expression of three that parapodia and arthropodia are not structurally or arthropod and vertebrate limb gene orthologs, Distal-less mechanistically homologous (but their primordia might (Dll), dachshund (dac), and optomotor blind (omb), in be), that Dll’s ancestral bilaterian function was in sensory direct-developing juveniles of the polychaete Neanthes and central nervous system differentiation, and that arenaceodentata. Parapodial Dll expression marks pre- locomotory appendages possibly evolved from sensory morphogenetic notopodia and neuropodia, becoming re- outgrowths. stricted to the bases of notopodial cirri and to ventral portions of neuropodia. In outgrowing cephalic appen- Keywords Polychaete . Appendages . Nervous system . dages, Dll activity is primarily restricted to proximal Distal-less . dachshund . optomotor blind domains. Dll expression is also prominent in the brain. dac expression occurs in the brain, nerve cord ganglia, a pair of pharyngeal ganglia, presumed interneurons linking a Introduction pair of segmental nerves, and in newly differentiating As specialized body outgrowths, appendages facilitate numerous processes basic to animal life such as sensing, Communicated by D.A. Weisblat feeding, locomotion, mating, and defense. One central : C. J. Winchell D. K. Jacobs (*) question is which appendages among disparate animal taxa Department of Ecology and Evolutionary Biology, evolved from specific appendages of a common ancestor University of California, Los Angeles, (classical homology) and which appendages arose as 621 Charles E. Young Drive South, Los Angeles, CA 90095-1606, USA structural novelties (homoplasy)? Many appendage types e-mail: [email protected] are clearly apomorphic structures, so classical homology can be ruled out for certain comparisons. However, J. E. Valencia homology is an independent property of the various levels Division of Biology, California Institute of Technology, 1200 East California Boulevard; MC 156-29, of biological organization (genes, developmental mecha- Pasadena, CA 91125, USA nisms, cell types, organs, etc.) (e.g., Abouheif 1997; 276 Dev Genes Evol (2010) 220:275–295 Dickinson 1995; Minelli 1998; Striedter and Northcutt developmental stages in an individual. In the following 1991), thus evoking further relevant questions: can homol- paragraphs, we justify the selection of Distal-less, dachs- ogy be found by investigating mechanisms of appendage hund, and optomotor blind as genes of interest in the study formation? What evolutionary inferences can be drawn of appendage evo-devo. from such investigations? Distal-less (Dll) is well known for its expression in the Comparative evidence from model experimental ani- distal portions of developing annelid, arthropod, onychoph- mals provides the initial answers to these questions. oran, echinoderm, urochordate, and vertebrate appendages Drosophila appendages and vertebrate limbs share striking (Panganiban et al. 1997). Dll function has been examined in developmental–genetic similarities (reviews: Pueyo and mice and several arthropods. In general, loss-of-function Couso 2005; Shubin et al. 1997; Tabin et al. 1999). These mutations or elimination of endogenous Dll mRNA causes observations (among others) led to the concept of “deep distal truncation or severe distal malformations in appen- homology” (Shubin et al. 1997, 2009): historical continu- dages (Angelini and Kaufman 2004; Cohen et al. 1989; ity of developmental mechanisms in morphologically/ Robledo et al. 2002; Schoppmeier and Damen 2001). phylogenetically disparate structures. Given the lack of Appendicular Dll expression has been documented in two structural similarity between fly and vertebrate appen- polychaetes. In Chaetopterus variopedatus,itoccurs dages and because phylogenetically intervening groups mainly in antennae and throughout nascent parapodia (later (e.g., protochordates) evidently never possessed appen- in their distal tips) (Panganiban et al. 1997). In early dages comparable to wings or limbs, Shubin et al. (1997) trochophore larvae of the nereidid Platynereis dumerilii,it and Tabin et al. (1999) reasoned that fly and vertebrate marks the lateral appendage (parapodial) fields (Denes et al. appendages are not classical homologs. Instead they 2007; Saudemont et al. 2008), but expression has not been consider them “paralogs”, novel appendages originating observed in later stages undergoing appendage outgrowth. via the co-option of an ancient, conserved genetic Knowledge of Dll function combined with observations of network. Tabin et al. (1999) contended that this network its expression in diverse phyla has led to Dll’s reputation as evolved prior to the arthropod–tetrapod common ancestor the chief regulator of distal morphogenesis in appendages. and that it was used to build primitive appendages and has Assuming continuity of developmental processes, Dll since been used to build appendage paralogs in arthropods, expression in Neanthes is expected to occur in the distal vertebrates, and possibly other bilaterian phyla. Shubin et portions of developing parapodia. al. (1997), Panganiban et al. (1997), Arthur et al. (1999), dachshund (dac) shows a conserved pattern of expres- and Pueyo and Couso (2005) arrived at similar conclu- sion in the developing legs of broadly divergent sions. Minelli (2000) proposed an alternative scenario: arthropod taxa, for example: spider (Prpic et al. 2003), “axis paramorphism”. Based on comparative morphology millipede (Prpic and Tautz 2003), isopod (Abzhanov and and a different set of developmental–genetic criteria, he Kaufman 2000), branchiopod (Sewell et al. 2008), and posited an appendage-less ancestor and that appendages insect (Inoue et al. 2002). This occurs in an intermediate arose as homoplastic duplicates (paramorphs) of classical domain along the proximodistal leg axis, between the homologs, namely, the anteroposterior body axes of proximal domain of extradenticle and homothorax coex- bilaterians. pression and the distal domain of Dll expression. Together Flies and vertebrates belong to two separate and major these genes are referred to as “leg gap genes”;they bilaterian clades: Ecdysozoa and Deuterostomia, respec- function antagonistically in a regulatory network to divide tively. These clades have been the primary subjects of developing legs into three distinct units (Kojima 2004). appendage study to date. Comparatively little work has The recent demonstration of arthropod-like gap gene been done on the remaining major bilaterian clade, the expression in developing appendages of an onychophoran Lophotrochozoa, which together with Ecdysozoa comprise supports the homology of this network across panarthro- the protostomes. To examine whether the deep homology of pods and indicates that it evolved to fulfill a role in appendage-forming mechanisms is shared more broadly appendage development unrelated to limb segmentation among bilaterians, developmental studies focusing on (Janssen et al. 2010). dac function has been analyzed with appendage genes must be extended to the Lophotrochozoa. loss-of-function experiments in Drosophila (Mardon et al. In this study, three “appendage genes” (homologs of 1994) and mRNA depletion in the hemipteran Oncopeltus arthropod and vertebrate genes known to function in (Angelini and Kaufman 2004). Both studies revealed appendage morphogenesis) were isolated from the lopho- mutant phenotypes in which intermediate leg segments trochozoan Neanthes arenaceodentata, an errant polychaete were shortened and fused. Vertebrate dac homologs also with an array of appendages, including locomotor/sensory control limb differentiation along the proximodistal axis. segmental parapodia, which are added serially at a terminal Dach1, for example, maintains the apical ectodermal growth zone and can thus be observed at multiple ridge, an organizing center that drives limb outgrowth Dev Genes Evol (2010) 220:275–295 277 (Kida et al. 2004). This gene is also active in the broad Gene isolation mesodermal domains of the limb bud, and as development proceeds its expression becomes limited to the cartilage of Total RNA was extracted

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