- Short report
- Open Access
Parasegmental appendage allocation in annelids and arthropods and the homology of parapodia and arthropodia
© Prpic; licensee BioMed Central Ltd. 2008
Received: 01 April 2008
Accepted: 20 October 2008
Published: 20 October 2008
The new animal phylogeny disrupts the traditional taxon Articulata (uniting arthropods and annelids) and thus calls into question the homology of the body segments and appendages in the two groups. Recent work in the annelid Platynereis dumerilii has shown that although the set of genes involved in body segmentation is similar in the two groups, the body units of annelids correspond to arthropod parasegments not segments. This challenges traditional ideas about the homology of "segmental" organs in annelids and arthropods, including their appendages. Here I use the expression of engrailed, wingless and Distal-less in the arthropod Artemia franciscana to identify the parasegment boundary and the appendage primordia. I show that the early body organization including the appendage primordia is parasegmental and thus identical to the annelid organization and by deriving the different adult appendages from a common ground plan I suggest that annelid and arthropod appendages are homologous structures despite their different positions in the adult animals. This also has implications for the new animal phylogeny, because it suggests that Urprotostomia was not only parasegmented but also had parasegmental appendages similar to extant annelids, and that limb-less forms in the Protostomia are derived from limb-bearing forms.
Arthropods and annelids have their body divided into a series of repeated units that bear pairs of appendages in most cases. These body units and their appendages have long been regarded as homologous structures and have been the basis for uniting annelids and arthropods as sister taxa in the taxon Articulata . The so-called "new animal phylogeny", however, does not support this close relationship between annelids and arthropods and rather places them in two different branches of protostome phylogeny termed Lophotrochozoa and Ecdysozoa, respectively . This suggests that body segments and appendages in annelids and arthropods might have originated separately and are therefore not homologous. On the other hand recent results suggest that at least segmentation might have an ancient origin that predates or coincides with the origin of the Bilateria .
In arthropods the body units are first specified in a parasegmental register  and later these parasegments are transformed into segments by re-segmentation during embryonic development . Intriguingly, recent work using the expression of the segmentation genes engrailed (en) and wingless (wg) has demonstrated that re-segmentation does not occur in annelids and the body units in annelids thus remain parasegments [6, 7]. This also makes annelid appendages (parapodia) parasegmental rather than segmental structures and this is further evidenced by the expression of the appendage marker Distal-less (Dll/Dlx) . This calls into question the homology between annelid parapodia and the appendages of arthropods (arthropodia). Here I show that in an arthropod species, the brine shrimp Artemia franciscana, the early body organisation including the limb primordia is identical to the annelid condition. This demonstrates that arthropodia like parapodia are initially parasegmental organs and suggests that both share a common evolutionary origin.
Hejnol and Scholtz  have studied limb primordium formation (as marked by Dll protein expression) in the crustaceans Orchestia cavimana and Porcellio scaber. Because in these species the cell lineage is known, these authors were able to demonstrate that the limb primordium starts as a single cell and expands by activation of Dll in adjacent cells. I propose that arthropods use this expansion mechanism to transform their initially parasegmental appendage primordia into segmental appendages after re-segmentation (Figure 1F, upper row). By contrast, annelids do not re-segment and the clonal boundaries of the parasegments are retained as the definite morphological borders between the body units. I suggest that because of this, the restrictive influence of the morphological borders between the parasegments cannot be overcome by the primordia of the parapodia, resulting in a lack of expansion of the primordia (Figure 1F, lower row). This model thus derives the adult arthropod condition from a common body organisation in immature arthropods and annelids by the arthropod-specific processes of re-segmentation and subsequent expansion of the appendage primordium across the parasegment boundary.
Artemia culture and fixation
Artemia franciscana cysts were purchased from Dohse Aquaristik (Grafschaft-Gelsdorf, Germany) and were activated in seawater (34 g seasalt per litre) with constant oxygen supply at 25°C. Larvae were harvested after 24, 48 or 72 hours. Fixation of nauplii proved to be difficult. Best results were achieved after washing the nauplii in DanKlorix cleaner (Colgate-Palmolive, Hamburg, Germany) for 5 minutes and subsequent fixation in PEMFAH (3 ml PEMS, 450 μl formaldehyde (37%), 5 ml heptane) at 4°C over night. After fixation the nauplii were transferred to methanol and stored at -20°C. Best possible tissue fixation was determined to be the case when after the methanol treatment the orange pigment of the nauplius eyes was preserved and the tissue within the appendages was white rather than clear.
In situ hybridisation
Artemia nauplii were rehydrated stepwise in PBST and then sonicated for 5 seconds with a tip sonifier (Branson Cell Disruptor B15). Sonication was optimal when more than 50% of all nauplii were destroyed. They were then treated according to the published protocol for Glomeris marginata  with the following modifications: acetylation lasted for 1 hour; anti-Dig antibodies were preabsorbed against fixed and sonicated nauplii for 24 hours at 4°C in PBST supplemented with 2% sheep serum.
For RNA extraction, cysts were activated in sterilized and filtered sea water and the nauplii were harvested 48 hours later. Total RNA was extracted using the Trizol reagent (Invitrogen). cDNA was synthesized from this total RNA using the SuperScript II system (Invitrogen). The Artemia engrailed gene has been reported previously  and a fragment was cloned with gene specific primers designed on the basis of the published sequence [GenBank:X70939]. A fragment of the homeobox of the Artemia Distal-less gene has been cloned using the previously published primers eDP fw, eDP bw, iDP fw and iDP bw . Within this sequence two nested gene specific primers were designed and were used in two subsequent PCR reactions together with the primer DP dlxm1  to amplify a larger portion of the gene that spans from the homeobox to the DLX-1 motif . A fragment of the Artemia wingless gene was cloned using the primers reported previously .
I thank Diethard Tautz and Wim Damen for support and comments on the manuscript. This work has been funded by the German Research Council (DFG grant PR1109/1-1).
- Westheide W: Articulata (Gliedertiere). Spezielle Zoologie, Teil 1, Einzeller und Wirbellose Tiere. Edited by: Westheide W, Rieger R. 1996, Stuttgart, Gustav Fischer Verlag, 350-352.Google Scholar
- Adoutte A, Balavoine G, Lartillot N, Lespinet O, Prud'homme B, de Rosa R: The new animal phylogeny: reliability and implications. Proc Natl Acad Sci USA. 2000, 97: 4453-4456. 10.1073/pnas.97.9.4453.PubMed CentralView ArticlePubMedGoogle Scholar
- Damen WGM: Evolutionary conservation and divergence of the segmentation process in arthropods. Dev Dyn. 2007, 236: 1379-1391. 10.1002/dvdy.21157.View ArticlePubMedGoogle Scholar
- Damen WGM: Parasegmental organization of the spider embryo implies that the parasegment is an evolutionary conserved entity in arthropod embryogenesis. Development. 2002, 129: 1239-1250.PubMedGoogle Scholar
- Piepenburg O, Vorbruggen G, Jaeckle H: Drosophila segment borders result from unilateral repression of Hedgehog activity by Wingless signaling. Mol Cell. 2000, 6: 203-209. 10.1016/S1097-2765(00)00022-8.View ArticlePubMedGoogle Scholar
- Prud'homme B, de Rosa R, Arendt D, Julien JF, Pajaziti R, Dorresteijn AW, Adoutte A, Wittbrodt J, Balavoine G: Arthropod-like expression patterns of engrailed and wingless in the annelid Platynereis dumerilii suggest a role in segment formation. Curr Biol. 2003, 13: 1876-1881. 10.1016/j.cub.2003.10.006.View ArticlePubMedGoogle Scholar
- Saudemont A, Dray N, Hudry B, Le Gouar M, Vervoort M, Balavoine G: Complementary striped expression patterns of NK homeobox genes during segment formation in the annelid Platynereis dumerilii. Dev Biol. 2008, doi: 10.1016/j.ydbio.2008.02.013.Google Scholar
- Benesch R: Zur Ontogenie und Morphologie von Artemia salina L. Zool Jb Anat. 1969, 86: 307-458.Google Scholar
- Hejnol A, Scholtz G: Clonal analysis of Distal-less and engrailed expression patterns during early morphogenesis of uniramous and biramous crustacean limbs. Dev Genes Evol. 2004, 214: 473-485.PubMedGoogle Scholar
- Philippe H, Lartillot N, Brinkmann H: Multigene analyses of bilaterian animals corroborate the monophyly of Ecdysozoa, Lophotrochozoa, and Protostomia. Mol Biol Evol. 2005, 22: 1246-1253. 10.1093/molbev/msi111.View ArticlePubMedGoogle Scholar
- Telford MJ, Bourlat SJ, Economou A, Papillon D, Rota-Stabelli O: The evolution of the Ecdysozoa. Phil Trans R Soc B. 2008, 363: 1529-1537. 10.1098/rstb.2007.2243.PubMed CentralView ArticlePubMedGoogle Scholar
- Eriksson BJ, Larson ET, Thörnqvist PO, Tait NN, Budd GE: Expression of engrailed in the developing brain and appendages of the onychophoran Euperipatoides kanangrensis (Reid). J exp Zool B (Mol Dev Evol). 2005, 304B: 220-228. 10.1002/jez.b.21043.View ArticleGoogle Scholar
- Prpic NM, Tautz D: The expression of the proximodistal axis patterning genes Distal-less and dachshund in the appendages of Glomeris marginata (Myriapoda: Diplopoda) suggests a special role of these genes in patterning the head appendages. Dev Biol. 2003, 260: 97-112. 10.1016/S0012-1606(03)00217-3.View ArticlePubMedGoogle Scholar
- Manzanares M, Marco R, Garesse R: Genomic organization and developmental pattern of expression of the engrailed gene from the brine shrimp Artemia. Development. 1993, 118: 1209-1219.PubMedGoogle Scholar
- Aspöck G, Bürglin TR: The Caenorhabditis elegans Distal-less ortholog ceh-43 is required for development of the anterior hypodermis. Dev Dyn. 2001, 222: 403-409. 10.1002/dvdy.1201.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.