Jackson DJ , Meyer NP , Seaver E , Pang K , McDougall C , Moy VN , Gordon K , Degnan BM , Martindale MQ , Burke RD , Peterson KJ .

R01 GM093116 NIGMS NIH HHS

	Fig. 1. Phylogenetic reconstruction of COE evolutionary history. The topology shown is a 50% majority rule tree derived from a Bayesian analysis of unambiguously aligned positions (see supplementary material for alignment). Posterior probabilities following 1.8 million generations are indicated
	Fig. 2. Schematic representation of metazoan COE sequence architectures. The highly conserved nature of the DNA-binding domain (with the embedded zinc coordination motif), the IPT/TIG (immunoglobulin-like, plexins, transcription factors/transcription factor immunoglobulin) domain and the helix–loop–helix domains are indicated. Daburon et al. (2008) proposed that the second helix domain (H2d) is derived from the carboxyl-most helix domain (H2a), and we have followed this terminology here. Pairwise distances are indicated and were calculated using PAUP with human COE1 protein as the reference sequence. Only highly conserved alignable regions are shown, amino and carboxy regions are not included. All domain lengths are represented to scale
	Fig. 3. Developmental expression of MlCOE in the ctenophore, M. leidyi. a, c, e, g, h Lateral view with the asterisk denoting the blastopore. b, d, f Oral view. a, b At 3 hpf, MlCOE is expressed in the macromeres (endoderm). c, d At 4 hpf, the MlCOE+ macromeres have completely gastrulated and expression is present in the oral micromeres (mesoderm) which have entered the blastocoel. e, f At 6 hpf, MlCOE expression in the macromeres has decreased while expression remains in the descendents of the oral micromeres which line the aboral part of the blastocoel, as well as in oral ectoderm around the blastopore. g Expression is similar at 8 hpf in the mesoderm of the forming tentacle bulb and near the blastopore, which forms part of the pharynx. h At 9 hpf, when the comb plates have fully formed, MlCOE expression becomes more diffuse in the pharynx and tentacle bulb. In subsequent stages, we do not detect MlCOE expression
	Fig. 4. Developmental expression of CtCOE in the polychaete Capitella teleta. a, e, i, m, q Lateral; b, c, f, g, j, k, n, n', o, r, s ventral; and d, h, l, p, t anterior views. Anterior is to the left in all panels except d, h, l, p, t. In d, h, l, p, t, dorsal is up. a–d In St. 3 embryos, CtCOE is expressed in the trunk mesoderm and a small number of ectodermal cells in the head (arrows). e–h St. 4 larvae show expression in a subset of cells in the forming brain (e, f, h); ectodermal cells in the head (f, h); a mesodermal cell cluster (e, f) and cells in the presumptive subesophageal ganglion (g, arrows). i–l In St. 5 larvae, CtCOE is expressed in a subset of cells in the brain (br in i, j, l), in the forming ventral nerve cord (vnc in i, k) and subsurface anterior cells (j, arrow). m–p In St. 6 larvae, CtCOE is prominently expressed in the brain (m, p) and VNC (m, o). In addition, there are small CtCOE-expressing clusters associated with the foregut (m, n, arrowheads) and head (n, n' and p, arrows). n (more ventral) and n' (more dorsal) are different focal planes of the same specimen. q–t In late larval stages (St. 8, 9), CtCOE is expressed in a small subset of VNC cells (q, s, compare with the broad VNC expression during St. 6). Expression in the brain at this stage is largely undetectable (q, r) except in a small number of posterior cells (q, t, arrows). Abbreviations are as follows: br (brain), me (mesoderm), vnc (ventral nerve cord). The position of the mouth is demarcated with a black asterisk
	Fig. 5. Transient expression of CtCOE in lateral cell clusters and mesodermal cells. Anterior is to the left in all panels a, b ventral views, c lateral view. As development proceeds, CtCOE is sequentially expressed in one to two lateral subsurface clusters (arrowheads) in anterior (a, early St. 5), middle (b, mid-stage 5) and posterior (c, stage 6) segments. Abbreviations are as follows: br (brain), me (mesoderm), vnc (ventral nerve cord). The position of the mouth is demarcated with a black asterisk
	Fig. 6. Developmental expression of HasCOE and HasElav in the tropical abalone Haliotis asinina. Orientations are a–d lateral; e, f, p ventral; g and h dorsal; i–l apical/anterior; m–o posterior. a, e, iHasCOE expression in a hatched (10 hpf) trochophore larva, with expression detected in a group of posterior cells (pc), paraxial mesodermal bands (me), a pair of ventral ectodermal cells (ve) and within the apical tuft (at). Upper inset in (a) shows expression in the apical tuft (vertical arrow), lower inset shows an individual cell associated with the lateral ectoderm (black arrow, the white arrow in this inset indicates the COE+ paraxial mesodermal bands in a lower focal plane). b, f, j 13 hpf trochophore larva prior to torsion. A band of expression marking the division between the foot primordia (fp) and the expanding shell field (approximately indicated by the dashed line) has developed (arrow). The boxed region in F is expanded in the inset and highlights a triplet of COE+ cell within the vicinity of the stomodeum. c, g, k A 20-hpf larva (post-torsion) with HasCOE expression within the apical tuft and the presumed supraesophageal (spg) and subesophageal ganglia (sbg). Cells that will later form connectives (co) between the esophageal and pedal ganglia are also visible. The refractive operculum (op) can also be seen. d, h, l A 34-hpf veliger with a well-developed eyespot (e), operculum and digestive gland (dg). The supra- and subesophageal ganglia maintain expression of HasCOE and faint expression is also detected within the anterior of the foot (arrow). The fibres of the larval retractor muscle (lrm) are also visible. m–o Representative variation in the spatial expression of HasCOE+ posterior cells (labelled pc in a) viewed posteriorly between individual 10-h-old larvae. Expression ranges from (m) a distinct triplet of closely associated cells to (n) a group of cells with an additional lateral population of COE+ cells (arrows), through to (o) strong expression of HasCOE in the lateral and central groups. p A ventral view of a 10-hpf trochophore larva expressing HasElav in apical ectodermal cells associated with the apical tuft, a pair of lateral ectodermal cells (arrow) and a set of ectodermal posterior cells (pc)
	Fig. 7. Developmental expression of ChCOE in the polychaete Chaetopterusa–d lateral; e, f, h ventral; g dorsal; i–l anterior; m–o posterior views. Anterior is up in a–c and e–g and to the right in d and h. Dorsal is up in i–o. a, e, i, m In 11 hpf trochophore larvae, ChCOE transcripts are detected in two posterior groups of cells (e, m); two lateral groups of cells presumed to be mesoderm (me in a, e and i); and a dorsal, anterior ectodermal cell (de in a and i). The inset in a is a more superficial focal plane of the same animal showing the ChCOE+ mesodermal cells which vary in number (one to three) from left to right. The animal shown in the inset in e and in panel m is of a different animal for which the colour reaction was not incubated as long. The posterior group of cells also can be variable in number. At this stage, there are occasional ChCOE+ ectodermal cells in the region of the forming brain but not the apical tuft (position is marked with a red asterisk). ChCOE is also expressed in posterior, dorsolateral ectodermal patches (arrow in a). b, f, j, o 15 hpf trochophore larvae have similar clusters of ChCOE+ cells as the 11 hpf animals. By this stage, the dorsal ectodermal cluster (de) has expanded to three to five cells (j), and the ChCOE+ cells in the region of the forming brain are more visible but are still not localised around the apical tuft (b, f, j). The posterior region of ChCOE expression consists of a central group of cells and two ChCOE+ cells positioned just anterior and dorsal to the central posterior patch (o). This is similar to the posterior pattern of expression at 13 hpf (n), although the central, posterior ChCOE+ patch is just one cell at this stage. c, g, k At 19 hpf, ChCOE is expressed in clusters of ectodermal cells that appear to prefigure the central nervous system. ChCOE is expressed in the developing brain (c, g, k), in a dorsal ectodermal patch of cells (de in c) and in clusters of cells in the lateral (le) and posterior ectoderm (c, d). There are also two clusters of ChCOE+ cells on either side of the mouth, which may be neural or mesodermal. In addition, there are two small clusters of ChCOE+ cells in the ventral ectoderm (inset in g, anterior cluster of ChCOE+ cells). The animal in the inset in g is a different animal for which the colour reaction was carried out longer. The two posterior clusters of ChCOE+ cells correspond to the posterior ectodermal ChCOE+ cell clusters in g. d, h, l In L2–L3 animals (d, 46 hpf; h and l, 72 hpf), ChCOE is expressed in subsets of the forming central nervous system, including in the developing brain (br in d, right inset in h and l), the forming ventral nerve cord (vnc in d, h) and in clusters of posterior neural cells in the pygidium (left inset in h). There is a patch of ChCOE+ cells in the ventral ectoderm just posterior to the mouth (h) as well as single ChCOE+ cells around the posterior edge of the mouth (right inset in h), which spatially correspond to the subesophageal connective. br, brain; de, dorsal ectodermal cluster; le, lateral ectoderm; me, mesoderm; vnc, ventral nerve cord. The position of the mouth is demarcated with a black asterisk and the apical tuft with a red asterisk
	Fig. 8. Expression of SpCOE, SpNK2.1 and Sptektin3 in normal and MASO-injected embryos. a Expression of SpCOE in wild type (WT) embryo. Expression is clearly seen in three distinct loci at the apical end of the embryo (arrows). SpCOE expression is never detected in either the primary (pm) or secondary mesenchyme (sm) cells. b, c Injection of the SpCOE-MASO (b), unlike the SpNK2.1 MASO (c), has no affect on SpCOE expression, suggesting that SpCOE is downstream of SpNK2.1, but does not autoregulate. d Expression of SpNK2.1 in WT embryo in the apical domain (Takacs et al. 2004). e, fSpNK2.1 expression in embryo injected with SpCOE (e) and SpNK2.1 (f) MASO is unchanged suggesting that SpNK2.1 is not downstream of SpCOE, and does not autoregulate. g Expression of the cilia gene tektin3 in WT embryo in apical domain. h, i Injection of embryos with SpCOE-MASO does not alter tektin3 expression (h), whereas expression is abrogated upon introduction of the SpNK2.1 MASO (i) suggesting that SpCOE does not regulate apical tuft development. Abbreviations: a, archenteron; pm, primary mesenchyme; sm, secondary mesenchyme. The position of the blastopore is indicated with a black asterisk
	Fig. 9. Immunofluorescent images of morpholino injected larvae 72 h post-fertilisation. a Image of an uninjected larva combining the anti-serotonergic (green) and anti-synaptogamin (red) signals showing the location of the apical ganglion (arrow, ag). b Example of a control MASO-injected embryo that was used to quantify the number of serotonergic cells in the apical organs of 72-h larvae. c An example of an SpCOE-MASO-injected embryo in which there are supernumerary serotonergic neurons. dSpNK 2.1-MASO-injected embryos had more serotonergic cells than controls and projections with terminal growth cones (arrowheads) were not restricted to the neuropil of the apical ganglion. e Example of an apical organ in an uninjected control from the stage-matched set of embryos used to quantify cells in the apical organ. ag, apical organ; ax, axonal tracts of ciliary bands; cb, ciliary bands; m, mouth; st, stomach. Scale bar in a = 10 μM
	Fig. 10. Effect of SpCOE-MASO knockdown on the number of serotonergic neurons in the apical organ. Carefully staged embryos were prepared for immunofluorescence and the number of serotonergic neurons counted. Abrogation of SpCOE and SpNK2.1 translation results in a significantly higher number of serotonergic neurons in the apical organ compared to wild type and control larvae

Adamska, The evolutionary origin of hedgehog proteins. 2007, Pubmed

Adamska, The evolutionary origin of hedgehog proteins. 2007, Pubmed
Akerblad, Early B-cell factor (O/E-1) is a promoter of adipogenesis and involved in control of genes important for terminal adipocyte differentiation. 2002, Pubmed
Arendt, Genes and homology in nervous system evolution: comparing gene functions, expression patterns, and cell type molecular fingerprints. 2005, Pubmed
Arendt, The evolution of cell types in animals: emerging principles from molecular studies. 2008, Pubmed
Baumgardt, Specification of neuronal identities by feedforward combinatorial coding. 2007, Pubmed
Benito-Gutiérrez, Outlining the nascent nervous system of Branchiostoma floridae (amphioxus) by the pan-neural marker AmphiElav. 2005, Pubmed
Boyle, Expression of FoxA and GATA transcription factors correlates with regionalized gut development in two lophotrochozoan marine worms: Chaetopterus (Annelida) and Themiste lageniformis (Sipuncula). 2010, Pubmed
Chapman, The dynamic genome of Hydra. 2010, Pubmed
Corradi, Hypogonadotropic hypogonadism and peripheral neuropathy in Ebf2-null mice. 2003, Pubmed
Crozatier, Cellular immune response to parasitization in Drosophila requires the EBF orthologue collier. 2004, Pubmed
Crozatier, Head versus trunk patterning in the Drosophila embryo; collier requirement for formation of the intercalary segment. 1999, Pubmed
Crozatier, Collier, a novel regulator of Drosophila head development, is expressed in a single mitotic domain. 1996, Pubmed
Crozatier, Connecting Hh, Dpp and EGF signalling in patterning of the Drosophila wing; the pivotal role of collier/knot in the AP organiser. 2002, Pubmed
Crozatier, Requirement for the Drosophila COE transcription factor Collier in formation of an embryonic muscle: transcriptional response to notch signalling. 1999, Pubmed
Daburon, The metazoan history of the COE transcription factors. Selection of a variant HLH motif by mandatory inclusion of a duplicated exon in vertebrates. 2008, Pubmed
Dickinson, Development of the larval nervous system of the gastropod Ilyanassa obsoleta. 2003, Pubmed
Dickinson, Development of embryonic cells containing serotonin, catecholamines, and FMRFamide-related peptides in Aplysia californica. 2000, Pubmed
Dubois, XCoe2, a transcription factor of the Col/Olf-1/EBF family involved in the specification of primary neurons in Xenopus. 1998, Pubmed
Dubois, Collier transcription in a single Drosophila muscle lineage: the combinatorial control of muscle identity. 2007, Pubmed
Dunn, Molecular paleoecology: using gene regulatory analysis to address the origins of complex life cycles in the late Precambrian. 2007, Pubmed , Echinobase
Dunn, Broad phylogenomic sampling improves resolution of the animal tree of life. 2008, Pubmed
Edgar, MUSCLE: a multiple sequence alignment method with reduced time and space complexity. 2004, Pubmed
Garcia-Dominguez, Ebf gene function is required for coupling neuronal differentiation and cell cycle exit. 2003, Pubmed
Garel, Family of Ebf/Olf-1-related genes potentially involved in neuronal differentiation and regional specification in the central nervous system. 1997, Pubmed
Hadfield, The apical sensory organ of a gastropod veliger is a receptor for settlement cues. 2000, Pubmed
Hagman, Cloning and functional characterization of early B-cell factor, a regulator of lymphocyte-specific gene expression. 1993, Pubmed
Hartenstein, Blood cells and blood cell development in the animal kingdom. 2006, Pubmed
Hejnol, High-resolution fate map of the snail Crepidula fornicata: the origins of ciliary bands, nervous system, and muscular elements. 2007, Pubmed
Hinman, Expression of anterior Hox genes during larval development of the gastropod Haliotis asinina. 2003, Pubmed
Jackson, Ecological regulation of development: induction of marine invertebrate metamorphosis. 2002, Pubmed
Jackson, Dynamic expression of ancient and novel molluscan shell genes during ecological transitions. 2007, Pubmed
Krzemień, Control of blood cell homeostasis in Drosophila larvae by the posterior signalling centre. 2007, Pubmed
Larroux, Developmental expression of transcription factor genes in a demosponge: insights into the origin of metazoan multicellularity. 2006, Pubmed
Liberg, The EBF/Olf/Collier family of transcription factors: regulators of differentiation in cells originating from all three embryonal germ layers. 2002, Pubmed
Marlow, Anatomy and development of the nervous system of Nematostella vectensis, an anthozoan cnidarian. 2009, Pubmed
Martindale, A developmental perspective: changes in the position of the blastopore during bilaterian evolution. 2009, Pubmed
Martindale, The evolution of metazoan axial properties. 2005, Pubmed
Matus, Molecular evidence for deep evolutionary roots of bilaterality in animal development. 2006, Pubmed
Mella, Expression patterns of the coe/ebf transcription factor genes during chicken and mouse limb development. 2004, Pubmed
Nakajima, Divergent patterns of neural development in larval echinoids and asteroids. 2004, Pubmed , Echinobase
Nielsen, Patterns of gene expression: homology or homocracy? 2003, Pubmed
Pang, The ancestral role of COE genes may have been in chemoreception: evidence from the development of the sea anemone, Nematostella vectensis (Phylum Cnidaria; Class Anthozoa). 2004, Pubmed
Pang, Developmental expression of homeobox genes in the ctenophore Mnemiopsis leidyi. 2008, Pubmed
Perrone-Bizzozero, Role of HuD and other RNA-binding proteins in neural development and plasticity. 2002, Pubmed
Pick, Improved phylogenomic taxon sampling noticeably affects nonbilaterian relationships. 2010, Pubmed
Pozzoli, Xebf3 is a regulator of neuronal differentiation during primary neurogenesis in Xenopus. 2001, Pubmed
Prasad, unc-3, a gene required for axonal guidance in Caenorhabditis elegans, encodes a member of the O/E family of transcription factors. 1998, Pubmed
Putnam, Sea anemone genome reveals ancestral eumetazoan gene repertoire and genomic organization. 2007, Pubmed
Putnam, The amphioxus genome and the evolution of the chordate karyotype. 2008, Pubmed
Ryan, The cnidarian-bilaterian ancestor possessed at least 56 homeoboxes: evidence from the starlet sea anemone, Nematostella vectensis. 2006, Pubmed
Sodergren, The genome of the sea urchin Strongylocentrotus purpuratus. 2006, Pubmed , Echinobase
Srivastava, The Amphimedon queenslandica genome and the evolution of animal complexity. 2010, Pubmed
Stolfi, Early chordate origins of the vertebrate second heart field. 2010, Pubmed
Takacs, Expression of an NK2 homeodomain gene in the apical ectoderm defines a new territory in the early sea urchin embryo. 2004, Pubmed , Echinobase
Talavera, Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. 2007, Pubmed
Thamm, Notch signaling during larval and juvenile development in the polychaete annelid Capitella sp. I. 2008, Pubmed
Thavaradhara, Localization of nitric oxide synthase-like immunoreactivity in the developing nervous system of the snail Ilyanassa obsoleta. 2001, Pubmed
Travis, Purification of early-B-cell factor and characterization of its DNA-binding specificity. 1993, Pubmed
Wang, Molecular cloning of the olfactory neuronal transcription factor Olf-1 by genetic selection in yeast. 1993, Pubmed
Wheeler, The deep evolution of metazoan microRNAs. 2009, Pubmed