Six3 demarcates the anterior-most developing brain region in bilaterian animals
- Patrick RH Steinmetz†1, 6,
- Rolf Urbach†2,
- Nico Posnien3, 7,
- Joakim Eriksson4, 8,
- Roman P Kostyuchenko5,
- Carlo Brena4,
- Keren Guy1,
- Michael Akam4Email author,
- Gregor Bucher3Email author and
- Detlev Arendt1Email author
© Steinmetz et al; licensee BioMed Central Ltd. 2010
Received: 24 March 2010
Accepted: 29 December 2010
Published: 29 December 2010
The heads of annelids (earthworms, polychaetes, and others) and arthropods (insects, myriapods, spiders, and others) and the arthropod-related onychophorans (velvet worms) show similar brain architecture and for this reason have long been considered homologous. However, this view is challenged by the 'new phylogeny' placing arthropods and annelids into distinct superphyla, Ecdysozoa and Lophotrochozoa, together with many other phyla lacking elaborate heads or brains. To compare the organisation of annelid and arthropod heads and brains at the molecular level, we investigated head regionalisation genes in various groups. Regionalisation genes subdivide developing animals into molecular regions and can be used to align head regions between remote animal phyla.
We find that in the marine annelid Platynereis dumerilii, expression of the homeobox gene six3 defines the apical region of the larval body, peripherally overlapping the equatorial otx+ expression. The six3+ and otx+ regions thus define the developing head in anterior-to-posterior sequence. In another annelid, the earthworm Pristina, as well as in the onychophoran Euperipatoides, the centipede Strigamia and the insects Tribolium and Drosophila, a six3/optix+ region likewise demarcates the tip of the developing animal, followed by a more posterior otx/otd+ region. Identification of six3+ head neuroectoderm in Drosophila reveals that this region gives rise to median neurosecretory brain parts, as is also the case in annelids. In insects, onychophorans and Platynereis, the otx+ region instead harbours the eye anlagen, which thus occupy a more posterior position.
These observations indicate that the annelid, onychophoran and arthropod head develops from a conserved anterior-posterior sequence of six3+ and otx+ regions. The six3+ anterior pole of the arthropod head and brain accordingly lies in an anterior-median embryonic region and, in consequence, the optic lobes do not represent the tip of the neuraxis. These results support the hypothesis that the last common ancestor of annelids and arthropods already possessed neurosecretory centres in the most anterior region of the brain. In light of its broad evolutionary conservation in protostomes and, as previously shown, in deuterostomes, the six3-otx head patterning system may be universal to bilaterian animals.
In arthropods, the cerebral ganglia are composed of the protocerebrum and two segmental neuromeres, the deuto- and tritocerebrum. The most anterior part, the protocerebrum, can be further subdivided into a more lateral region bearing, for example, the optic lobes (archicerebrum) and a median region that includes, for example, the pars intercerebralis (prosocerebrum). Most authors think that the archicerebrum represents the tip of the neuraxis [1, 5–8] but this has been disputed [9–11]. So far, it is unclear how the arthropod and annelid brain parts are related, if at all, and how they would align along the anterior-posterior axis [7, 8, 12, 13]. In order to molecularly reassess this long-standing question, we have compared the expression of the anterior regionalisation genes six3 and otx during the development of annelid, arthropod and onychophoran brains.
Results and discussion
To obtain independent evidence that six3 plays a conserved role in outlining the most anterior head region in annelids, we cloned and investigated the expression of otx and six3 orthologs (Additional file 1: Supplementary Figure 1) in the oligochaete annelid Pristina longiseta that asexually reproduces by fission into chains of individuals that each regenerate a full anterior-posterior axis . During early fission, both genes are expressed in stripes at the putative anterior part of the newly forming head in the middle of a segment (Figure 2g, h). At this stage, we were technically not able to resolve whether Plo-six3 lies anterior of Plo-otx. However, in later stages, using the developing antennae for spatial reference, we indeed observed a single patch of Plo-six3 expressing cells at the tip of a newly forming individual (Figure 2i), in front of otx expressing cells  (Figure 2k).
Our comparative expression data shows that the developing annelid, arthropod and onychophoran heads comprise an anterior-most six3+ region and a more posterior otx+ region. Both regions are overlapping to a variable degree but show a clear anterior-to-posterior sequence, allowing cross-phylum alignment of head regions. In arthropods, the six3+ and otx+ head regions give rise to the protocerebrum and to the eyes (Figure 1a). In annelids, the six3+ and otx+ regions cover the developing prostomium and the peristomium, from which the cerebral ganglia and eyes (and chemosensory appendages) develop (Figure 1b), but the six3/otx-based molecular subdivision does not fully match the morphological partition. While neuroectodermal six3 is restricted to the larval episphere and thus to the prostomium, the more posterior/equatorial otx expression covers the peristomium but also part of the prostomium where it overlaps with six3. Our data thus align annelid cerebral ganglia with arthropod protocerebrum (that is, the most anterior part of the arthropod cerebral ganglia, see "Background").
Many authors have argued that the most anterior structures in the arthropod brain are the anterior-lateral regions mainly consisting of the optic lobe [1, 5–8]. These ocular regions largely coincide with the otx+ region (Figure 1a). Yet, the clear anterior location of the six3+ region in the early embryos of diverse arthropods, together with the role of six3 in defining the most anterior structures in other phyla, strongly suggest that it is this median six3+ region, giving rise to the neurosecretory pars intercerebralis and pars lateralis that represents the most anterior extreme of the arthropod brain (arrow in Figure 1a) and corresponds to the neurosecretory brain parts in annelids. This has hitherto been a minority view [9–11]. As the terms "archicerebrum" and "prosocerebrum" are tightly connected with the Articulata theory - unsupported by almost all molecular phylogenies - and have been inconsistently used to include different brain regions, we suggest abandoning these terms. Instead, our comparative studies reveal the existence of a conserved, ancient neurosecretory brain part at the tip of the neuraxis (Figure 1). It is followed by a more posterior part of the head (and brain) anlage expressing otx that often exhibits an early ring or arc-like pattern [29, 37, 38], consistent with the radial head hypothesis , and includes the eye anlagen (Figure 1). In the animals investigated, the position of the mouth opening is not reliably connected to the six3 or otx region: while it comes to lie within the otx region of Platynereis and onychophorans, its origin in arthropods is unclear. The fact that the annelid and onychophoran antennae develop from the six3+ region, in contrast to the arthropod antennae that develop posterior to the otx+ protocerebrum, is consistent with the previous assumption of homology between annelid and onychophoran antennae, but not with arthropod antennae . The striking overall evolutionary conservation of a six3+ region in front of otx+ and hox+ regions in protostomes documented here (Figure 1), as well as in vertebrates and hemichordates, indicates that this anterior-posterior series may be universal to bilaterian animals.
Animal culture and collecting
Platynereis larvae obtained from an established breeding culture at EMBL, Heidelberg. Strigamia maritima eggs collected at Brora, Scotland (June 2006). Fly strains: Oregon R (wildtype). Female Euperipatoides kanangrensis Reid, 1996 were collected from decomposing logs of Eucalyptus trees in Kanangra Boyd National Park, NSW, Australia (33° 59'S 150° 08'E). Females were kept in containers with dampened sphagnum moss at 13°C and were fed crickets once every second week. Gravid females were relaxed and killed with ethyl acetate vapour from October to December in order to acquire embryos of correct stages. Embryos were dissected from the females in phosphate buffered saline (PBS) and, after removal of the egg membranes, fixed in 4% formaldehyde in PBS overnight at 4°C. Fixed embryos were dehydrated in a graded series of methanol (25, 50, 75% in PBS with 0.1% Tween-20 for 10 minutes each) and stored in 100% methanol at -20°C.
Cloning of six3, otx and tryptophane-2,3-dioxygenase genes
All primers, PCR programs and template DNA source are given in Additional file 2. Tc-six3 gene was identified by in silico analysis of the Tribolium genome and amplified from a mixed stages (0 to 24h) cDNA library. Full length Pdu-six3 was isolated by screening a 48 h λ-ZAP phage library (provided by C. Heimann, Mainz). Pdu-tryptophane-2,3-dioxygenase gene was identified during a sequencing screen of a 48 h Platynereis EST library. Gene orthology was confirmed by using NCBI Protein BLAST, MUSCLE  multiple sequence alignments and CLUSTALX v.2 neighbour-joining phylogenetic analysis  for complete proteins.
Database accession numbers
Eka-otx: EU347401, Eka-six3: EU347400, Plo-otx: EU330201; Plo-six3: EU330202; Tc-six3: AM922337; Stm-Six3: EU340980; Stm-otx: EU340979; Pdu-six3: FM210809; Pdu-tryptophane-2,3-dioxygenase: FN868644
Whole-mount in situ hybridisation and immunohistochemistry
Established protocols were used for single- and two-colour fluorescent whole-mount in situ hybridisations of Platynereis and Pristina , Euperipatoides , Strigamia , Drosophila , and Tribolium . A Drosophila six3/optix RNA probe was synthesized from EST clone LD05472 (Berkeley Drosophila Genome Project). Subsequent immunostainings were done using Vector Red (Vector Laboratories, Burlingame, CA, USA) or NBT/BCIP (Roche Diagnostics Penzberg, Germany)). Primary antibodies were: mouse anti-Crumbs (1:50; Developmental Studies Hybridoma Bank, DSHB), mouse anti-Fas2 (1:20; DSHB), rat anti-Orthodenticle  (1:1000, provided by T. Cook), guinea pig anti-Dchx1 antibody (1:1000; provided by T. Erclik), rabbit anti-Six3/Optix antibody (1:300; provided by F. Pignoni), alkaline phosphatase (AP)-coupled sheep anti-digoxygenin (1:1000, Roche). Secondary antibodies: AP-coupled donkey anti-rat, AP-coupled donkey anti-mouse, Cy5-coupled goat anti-rabbit (Dianova, Hamburg, Germany), Cy3-coupled goat anti-mouse (Dianova, , Hamburg, Germany). SYBRGreen (Invitrogen, San Diego, CA, USA) diluted 1:10.000.
Developmental Studies Hybridoma Bank EST: expressed sequence tags
Nitro-Blue Tetrazolium chloride
phosphate buffered saline
polymerase chain reaction
We thank Tiffany Cook (Cincinnati Children's Hospital Medical Center) for providing a Drosophila Orthodenticle-antibody. This work was funded by a fellowship from the Luxembourg Ministry of Culture, Higher Education and Research to P.R.H.S., by grants of the Deutsche Forschungsgemeinschaft (DFG) to U.R. (UR 163/1-3, 1-4), by a grant of the Russian Foundation for Basic Research (RFBR) to RPK (09-04-00866-a), through the DFG-Research Center for Molecular Physiology of the Brain and BU-1443/2-2 to G.B, by a BBSRC grant (BBS/B/07519) to C.B and by the Marie Curie RTN ZOONET (MRTN-CT-2004-005624) to M.A. and D.A.
- Siewing R: Lehrbuch der Zoologie. Systematik. 1985, Stuttgart, New York: Gustav Fischer VerlagGoogle Scholar
- Orrhage L, Müller MCM: Morphology of the nervous system of Polychaeta (Annelida). Hydrobiologia. 2005, 535/536: 79-111. 10.1007/s10750-004-4375-4.View ArticleGoogle Scholar
- Schroeder PC, Hermans CO: Annelida: Polychaeta. Reproduction of marine invertebrates. Edited by: Giese AC, Pearse JS. 1975, New York: Academic Press, 3: 1-213.View ArticleGoogle Scholar
- Ackermann C, Dorresteijn A, Fischer A: Clonal domains in postlarval Platynereis dumerilii (Annelida: Polychaeta). J Morphol. 2005, 266: 258-280. 10.1002/jmor.10375.View ArticlePubMedGoogle Scholar
- Jürgens G, Hartenstein V: The terminal regions of the body pattern. The development of Drosophila melanogaster. Edited by: Bate M, Martinez-Arias A. 1993, Cold Spring Harbor: CSHL Press, 1: 687-746.Google Scholar
- Haas MS, Brown SJ, Beeman RW: Pondering the procephalon: the segmental origin of the labrum. Dev Genes Evol. 2001, 211: 89-95. 10.1007/s004270000128.View ArticlePubMedGoogle Scholar
- Rempel JG: The evolution of the insect head: the endless dispute. Quaestiones Entomologicae. 1975, 11: 7-24.Google Scholar
- Siewing R: Zum Problem der Arthropodenkopfsegmentierung. Zoologischer Anzeiger. 1963, 170: 429-468.Google Scholar
- Urbach R, Technau GM: Early steps in building the insect brain: neuroblast formation and segmental patterning in the developing brain of different insect species. Arthropod Structure & Development. 2003, 32: 103-123. 10.1016/S1467-8039(03)00042-2.View ArticleGoogle Scholar
- Schmidt-Ott U, Gonzalez-Gaitan M, Technau GM: Analysis of neural elements in head-mutant Drosophila embryos suggests segmental origin of the optic lobes. Roux Arch dev Biol. 1995, 205: 31-44. 10.1007/BF00188841.View ArticleGoogle Scholar
- Melnikov OA, Rasnitsyn AP: Zur Metamerie des Arthropoden-Kopfes: Das Acron. Beitr Ent Berlin. 1984, 34: 3-90.Google Scholar
- Goodrich ES: On the relation of the arthopod head to the annelid prostomium. Quarterly Journal of Microscopical Science. 1897, 40: 247-268.Google Scholar
- Scholtz G, Edgecombe GD: The evolution of arthropod heads: reconciling morphological, developmental and palaeontological evidence. Dev Genes Evol. 2006, 216: 395-415. 10.1007/s00427-006-0085-4.View ArticlePubMedGoogle Scholar
- Tessmar-Raible K, Raible F, Christodoulou F, Guy K, Rembold M, Hausen H, Arendt D: Conserved sensory-neurosecretory cell types in annelid and fish forebrain: insights into hypothalamus evolution. Cell. 2007, 129: 1389-1400. 10.1016/j.cell.2007.04.041.View ArticlePubMedGoogle Scholar
- Steinmetz PR, Zelada-Gonzáles F, Burgtorf C, Wittbrodt J, Arendt D: Polychaete trunk neuroectoderm converges and extends by mediolateral cell intercalation. Proc Natl Acad Sci USA. 2007, 104: 2727-2732. 10.1073/pnas.0606589104.PubMed CentralView ArticlePubMedGoogle Scholar
- Arendt D, Technau U, Wittbrodt J: Evolution of the bilaterian larval foregut. Nature. 2001, 409: 81-85. 10.1038/35051075.View ArticlePubMedGoogle Scholar
- Kulakova M, Bakalenko N, Novikova E, Cook CE, Eliseeva E, Steinmetz PR, Kostyuchenko RP, Dondua A, Arendt D, Akam M, Andreeva T: Hox gene expression in larval development of the polychaetes Nereis virens and Platynereis dumerilii (Annelida, Lophotrochozoa). Dev Genes Evol. 2007, 217: 39-54. 10.1007/s00427-006-0119-y.View ArticlePubMedGoogle Scholar
- Oliver G, Mailhos A, Wehr R, Copeland NG, Jenkins NA, Gruss P: six3, a murine homologue of the sine oculis gene, demarcates the most anterior border of the developing neural plate and is expressed during eye development. Development. 1995, 121: 4045-4055.PubMedGoogle Scholar
- Carl M, Loosli F, Wittbrodt J: six3 inactivation reveals its essential role for the formation and patterning of the vertebrate eye. Development. 2002, 129: 4057-4063.PubMedGoogle Scholar
- Lowe CJ, Wu M, Salic A, Evans L, Lander E, Stange-Thomann N, Gruber CE, Gerhart J, Kirschner M: Anteroposterior patterning in hemichordates and the origins of the chordate nervous system. Cell. 2003, 113: 853-865. 10.1016/S0092-8674(03)00469-0.View ArticlePubMedGoogle Scholar
- Wei Z, Yaguchi J, Yaguchi S, Angerer RC, Angerer LM: The sea urchin animal pole domain is a six3-dependent neurogenic patterning center. Development. 2009, 136: 1179-1189. 10.1242/dev.032300.PubMed CentralView ArticlePubMedGoogle Scholar
- Tomer R, Denes A, Tessmar-Raible K, Arendt D: Profiling by image registration reveals common origin of annelid mushroom bodies and vertebrate pallium. Cell. 2010, 142: 800-809. 10.1016/j.cell.2010.07.043.View ArticlePubMedGoogle Scholar
- Arendt D, Tessmar K, de Campos-Baptista MI, Dorresteijn A, Wittbrodt J: Development of pigment-cup eyes in the polychaete Platynereis dumerilii and evolutionary conservation of larval eyes in Bilateria. Development. 2002, 129: 1143-1154.PubMedGoogle Scholar
- Friedrich M: Ancient mechanisms of visual sense organ development based on comparison of the gene networks controlling larval eye, ocellus, and compound eye specification in Drosophila. Arthropod Structure & Development. 2006, 35: 357-378. 10.1016/j.asd.2006.08.010.View ArticleGoogle Scholar
- Van Cleave CD: A study of the process of fission in the naid Pristina longiseta. Physiological Zool. 1937, 10: 299-314.Google Scholar
- Bely AE, Wray GA: Evolution of regeneration and fission in annelids: insights from engrailed- and orthodenticle-class gene expression. Development. 2001, 128: 2781-2791.PubMedGoogle Scholar
- Schröder R: The genes orthodenticle and hunchback substitute for bicoid in the beetle Tribolium. Nature. 2003, 422: 621-625. 10.1038/nature01536.View ArticlePubMedGoogle Scholar
- Posnien N, Bashasab F, Bucher G: The insect upper lip (labrum) is a nonsegmental appendage-like structure. Evol Dev. 2009, 11: 480-488. 10.1111/j.1525-142X.2009.00356.x.View ArticlePubMedGoogle Scholar
- Li Y, Brown SJ, Hausdorf B, Tautz D, Denell RE, Finkelstein R: Two orthodenticle-related genes in the short-germ beetle Tribolium castaneum. Dev Genes Evol. 1996, 206: 35-45. 10.1007/s004270050028.View ArticlePubMedGoogle Scholar
- Coiffier D, Charroux B, Kerridge S: Common functions of central and posterior Hox genes for the repression of head in the trunk of Drosophila. Development. 2008, 135: 291-300. 10.1242/dev.009662.View ArticlePubMedGoogle Scholar
- Manton SM: Studies on the Onychophora. VII. The early embryonic stages of Peripatopsis, and some general considerations concerning the morphology and phylogeny of the Arthropoda. Philos Trans R Soc Lond B Biol Sci. 1949, 233: 483-580. 10.1098/rstb.1949.0003.View ArticleGoogle Scholar
- Walker MH, Tait NN: Studies of embryonic development and the reproductive cycle in ovoviviparous Australian Onychophora (Peripatopsidae). Journal of Zoology. 2004, 264: 10.1017/S0952836904005837.Google Scholar
- Bovolenta P, Mallamaci A, Puelles L, Boncinelli E: Expression pattern of cSix3, a member of the six/sine oculis family of transcription factors. Mechanisms of Development. 1998, 70: 201-203. 10.1016/S0925-4773(97)00183-4.View ArticlePubMedGoogle Scholar
- Hartenstein V: The neuroendocrine system of invertebrates: a developmental and evolutionary perspective. J Endocrinol. 2006, 190: 555-570. 10.1677/joe.1.06964.View ArticlePubMedGoogle Scholar
- de Velasco B, Erclik T, Shy D, Sclafani J, Lipshitz H, McInnes R, Hartenstein V: Specification and development of the pars intercerebralis and pars lateralis, neuroendocrine command centers in the Drosophila brain. Dev Biol. 2007, 302: 309-323. 10.1016/j.ydbio.2006.09.035.View ArticlePubMedGoogle Scholar
- Urbach R, Technau GM: Molecular markers for identified neuroblasts in the developing brain of Drosophila. Development. 2003, 130: 3621-3637. 10.1242/dev.00533.View ArticlePubMedGoogle Scholar
- Simonnet F, Célérier M-L, Quéinnec E: Orthodenticle and empty spiracles genes are expressed in a segmental pattern in chelicerates. Dev Genes Evol. 2006, 216: 467-480. 10.1007/s00427-006-0093-4.View ArticlePubMedGoogle Scholar
- Browne WE, Schmid BG, Wimmer EA, Martindale MQ: Expression of otd orthologs in the amphipod crustacean, Parhyale hawaiensis. Dev Genes Evol. 2006, 216: 581-595. 10.1007/s00427-006-0074-7.View ArticlePubMedGoogle Scholar
- Bruce AE, Shankland M: Expression of the head gene Lox22-Otx in the leech Helobdella and the origin of the bilaterian body plan. Dev Biol. 1998, 201: 101-112. 10.1006/dbio.1998.8968.View ArticlePubMedGoogle Scholar
- Edgar RC: MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004, 32: 1792-1797. 10.1093/nar/gkh340.PubMed CentralView ArticlePubMedGoogle Scholar
- Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG: Clustal W and Clustal × version 2.0. Bioinformatics. 2007, 23: 2947-2948. 10.1093/bioinformatics/btm404.View ArticlePubMedGoogle Scholar
- Tessmar-Raible K, Steinmetz PRH, Snyman H, Hassel M, Arendt D: Fluorescent two color whole-mount in situ hybridization in Platynereis dumerilii (Polychaeta, Annelida), an emerging marine molecular model for evolution and development. BioTechniques. 2005, 39: 460-10.2144/000112023. 462, 464View ArticlePubMedGoogle Scholar
- Eriksson B, Tait N, Budd GE, Akam M: The involvement of engrailed and wingless during segmentation in the onychophoran Euperipatoides kanangrensis (Peripatopsidae: Onychophora) (Reid 1996). Dev Genes Evol. 2009, 219: 249-264. 10.1007/s00427-009-0287-7.View ArticlePubMedGoogle Scholar
- Chipman AD, Arthur W, Akam M: Early development and segment formation in the centipede, Strigamia maritima (Geophilomorpha). Evol Dev. 2004, 6: 78-89. 10.1111/j.1525-142X.2004.04016.x.View ArticlePubMedGoogle Scholar
- Plickert G, Gajewski M, Gehrke G, Gausepohl H, Schlossherr J, Ibrahim H: Automated in situ detection (AISD) of biomolecules. Dev Genes Evol. 1997, 207: 362-367. 10.1007/s004270050124.View ArticleGoogle Scholar
- Wohlfrom H, Schinko JB, Klingler M, Bucher G: Maintenance of segment and appendage primordia by the Tribolium gene knodel. Mech Dev. 2006, 123: 430-439. 10.1016/j.mod.2006.04.003.View ArticlePubMedGoogle Scholar
- Xie B, Charlton-Perkins M, McDonald E, Gebelein B, Cook T: senseless functions as a molecular switch for color photoreceptor differentiation in Drosophila. Development. 2007, 134: 4243-4253. 10.1242/dev.012781.View ArticlePubMedGoogle Scholar
- Urbach R: A procephalic territory in Drosophila exhibiting similarities and dissimilarities compared to the vertebrate midbrain/hindbrain boundary region. Neural Dev. 2007, 2: 23-10.1186/1749-8104-2-23.PubMed CentralView ArticlePubMedGoogle Scholar
- Telford MJ: Evidence for the derivation of the Drosophila fushi tarazu gene from a Hox gene orthologous to lophotrochozoan Lox5. Curr Biol. 2000, 10: 349-352. 10.1016/S0960-9822(00)00387-0.View ArticlePubMedGoogle Scholar
- Telford MJ, Thomas RH: Expression of homeobox genes shows chelicerate arthropods retain their deutocerebral segment. Proc Natl Acad Sci USA. 1998, 95: 10671-10675. 10.1073/pnas.95.18.10671.PubMed CentralView ArticlePubMedGoogle Scholar
- Hughes CL, Kaufman TC: Hox genes and the evolution of the arthropod body plan. Evol Dev. 2002, 4: 459-499. 10.1046/j.1525-142X.2002.02034.x.View ArticlePubMedGoogle Scholar
- Hirth F, Kammermeier L, Frei E, Walldorf U, Noll M, Reichert H: An urbilaterian origin of the tripartite brain: developmental genetic insights from Drosophila. Development. 2003, 130: 2365-2373. 10.1242/dev.00438.View ArticlePubMedGoogle Scholar
- Damen WG, Hausdorf M, Seyfarth EA, Tautz D: A conserved mode of head segmentation in arthropods revealed by the expression pattern of Hox genes in a spider. Proc Natl Acad Sci USA. 1998, 95: 10665-10670. 10.1073/pnas.95.18.10665.PubMed CentralView 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.