Six3 demarcates the anterior-most developing brain region in bilaterian animals
- Patrick RH Steinmetz1, 6Email author,
- Rolf Urbach2Email author,
- 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. 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.
Developmental Studies Hybridoma Bank
expressed sequence tags
Nitro-Blue Tetrazolium chloride
phosphate buffered saline
polymerase chain reaction
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.
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.
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