Cavalieri V , Spinelli G .

	Figure 1. Expression of hbox12 and nodal genes during early embryogenesis of P. lividus.(A) Temporal expression profiles examined by qPCR. Values at the different stages are shown as a percentage of the maximum signal intensity. Absolute numbers of transcripts per embryo given at the 60-cell stage are averages of the results of two independent experiments using distinct batches of cDNA. Abbreviations of the examined developmental stages: 2, 2-cell; 4, 4-cell; 8, 8-cell; 16, 16-cell; 32, 32-cell; 60, 60-cell; Mor, morula; eB, early blastula; HB, hatching blastula. (B) Spatial restriction of the hbox12 and nodal transcripts observed following WMISH at the indicated stage.DOI: http://dx.doi.org/10.7554/eLife.04664.003
	Figure 2—figure supplement 1. Overexpression of the HD-En obligate repressor and effect on nodal and gsc gene expression.Changes in gene expression levels was assessed by qPCR in cDNA samples resulting from hd-En-injected embryos. Data are normalized and indicated as in Figure 2I. The gray region represents ΔΔCt values corresponding to less than threefold difference. See also Supplementary file 1.DOI: http://dx.doi.org/10.7554/eLife.04664.005
	Figure 2—figure supplement 2. Spatial distribution of ectoderm- and PMC-specific markers in control and hbox12 overexpressing embryos.Control strim1 out-of-frame transcript (A–D) and hbox12 mRNA injected (E–K) embryos were fixed at the mesenchyme blastula (A–C, E–G and I–K) or prism (D and H) stages and analysed by chromogenic WMISH with the indicated probes. The embryos shown in (D) and (H) are oriented in a oral/ventral and lateral view, respectively, while all the other embryos are in a vegetal view.DOI: http://dx.doi.org/10.7554/eLife.04664.006
	Figure 2. Disruption of embryonic DV polarity by ectopic expression of hbox12.(A–G) 0.1 pg of the full-length hbox12 mRNA (D–F) or the hd-En mRNA (G), as well as a control out-of-frame strim1 transcript (A–C), were injected into zygotes and embryos were observed at the indicated stages. Overexpression of either hbox12 or hd-En severely perturbed DV axis formation, inflicting morphological defects that appeared from the gastrula stage onward. (H) qPCR measurements of nodal transcript abundance in embryos injected with increasing amounts of the hbox12 mRNA. Values are shown as a percentage of the nodal mRNA level in control uninjected embryos. Further detail for the qPCR procedure is given in ‘Materials and methods’. (I) Changes in gene expression level of nodal and other territorial marker genes assessed by qPCR in hbox12-injected embryos. Data are indicated as normalized ΔCt (ΔΔCt, left ordinate), and as the corresponding fold difference in transcript abundance (right ordinate), with respect to control embryos, at the same stage of development, derived from zygotes injected with the strim1 out-of-frame transcript. The gray region represents ΔΔCt values corresponding to less than threefold difference. Although this is commonly considered the limit of significance for qPCR assays, the relevance of our measurements is reinforced by WMISH results (see text for details). Error bars are standard errors for the qPCR replicates. Oligonucleotide primer pairs used for qPCR reactions and amplicon lengths are indicated in Supplementary file 1. Abbreviations: lG, late gastrula; VEB, very early blastula.DOI: http://dx.doi.org/10.7554/eLife.04664.004
	Figure 3. Overexpression of hbox12 in chimeric embryos.(A) Schematic illustrating that, at the 16-cell stage, animal and vegetal halves of hbox12/TRCD-injected and control uninjected embryos were isolated and recombined. (B–C) Side views of representative examples of the resulting reciprocal chimeras examined at 48 hpf. The composition of the chimeras is shown in the diagrams in the left panels (Ba and Ca). Images for each embryo are shown under DIC optics with simultaneous plane polarized light illumination (Bb and Cb), epifluorescence (Bc and Cc), and aggregate merging (Bd and Cd).DOI: http://dx.doi.org/10.7554/eLife.04664.007
	Figure 4—figure supplement 1. Multiple comparison of the homeodomain of Hbox12 and Otp proteins from P. lividus.Identical residues are shaded by differently coloured boxes, while double and single dots indicate decreasing degree of conservation; divergent amino acids are indicated by blank spaces. The Glutamine at position 50 is marked by an asterisk. Accession numbers were: Hbox12, X83675.1, and Otp, AJ007501.1.DOI: http://dx.doi.org/10.7554/eLife.04664.009
	Figure 4—figure supplement 2. Overexpression of isolated homeodomains and effect on nodal and gsc gene transcription.Zygotes were injected with the synthetic mRNAs coding for the isolated homeodomain of either Hbox12 or Otp and observed at the early blastula stage (upper panels). Total RNA was extracted from embryos at the morula stage, and changes in gene expression level of nodal and gsc determined by qPCR measurements. Data are indicated as in Figure 2I. The gray region represents ΔΔCt values corresponding to non-significant variation (less than threefold difference). See also Supplementary file 1.DOI: http://dx.doi.org/10.7554/eLife.04664.010
	Figure 4. Impairing hbox12 function and effects on DV axis formation.(A) Zygotes were injected with the control out-of-frame strim1 RNA (Aa–Ab) or the hd mRNA (Ac–Ae), and the resulting embryos were observed from a vegetal view at the indicated stages. (B) Control- (Ba–Bd) and hd-injected (Be–Bh) embryos were fixed at the mesenchyme blastula stage and analysed by WMISH with the indicated probes. The embryo shown in (Ba) is oriented in a lateral view, while all the other embryos are in a vegetal view. (C) Changes in gene expression level of territorial marker genes assessed by qPCR during development of hd-injected embryos. Data are normalized and indicated as in Figure 2I. The gray region represents ΔΔCt values corresponding to non-significant variation (less than threefold difference). See also Supplementary file 1. Abbreviations: VEB, very early blastula; EB, early blastula; MB, mesenchyme blastula; lG, late gastrula.DOI: http://dx.doi.org/10.7554/eLife.04664.008
	Figure 5. Block of hbox12 function in the animal hemisphere of chimeric embryos, and effects on DV patterning.(A–B) Diagrams in (Aa) and (Ba) show the composition of the reciprocal chimeras resulting from animal and vegetal half recombination of hd/TRCD-injected and control uninjected embryos at the 16-cell stage. (Ab–Ae and Bb–Be) Side views of the resulting chimeras examined at 48 hpf. Images for each embryo are shown under DIC optics (Ab and Bb), plane polarized light illumination (Ac and Bc), epifluorescence (Ad and Bd), and aggregate merging (Ae and Be).DOI: http://dx.doi.org/10.7554/eLife.04664.011
	Figure 6—figure supplement 1. Expression of the HD-GFP fusion protein during early embryogenesis.(A–C) Zygotes were injected with either the phbox12-GFP (A) or phbox12-HD-GFP (B) DNA constructs or with the hd-GFP synthetic transcript (C), and embryos were observed at the indicated stages. (D) Control embryo injected with TRCD.DOI: http://dx.doi.org/10.7554/eLife.04664.013
	Figure 6. Block of hbox12 function in aboral ectoderm cells of transgenic embryos, and effects on DV patterning.(A–E) Zygotes were injected with the indicated transgenes, and the resulting embryos were observed from a vegetal view at the indicated stages. Bright field (Ca, Da, and Ea), DIC (Aa, Ba, Cb, Db, and Eb), Dark field (Ab, Bb, Cc, Dc, and Ec), epifluorescence (Ac, Bc, Cd, Dd, and Ed), and aggregate merging (Ad, Bd, Ce, De, and Ee) images are shown.DOI: http://dx.doi.org/10.7554/eLife.04664.012
	Figure 7. Rescue of DV polarity by clonal knock-down of nodal into hd-injected embryos.(A) At the 4-cell stage, one blastomere of hd-GFP mRNA injected embryos was co-injected with a morpholino oligonucleotide directed against nodal (Mo-nodal) together with the RLDX red fluorescent tracer. The resulting embryos were scored for simultaneous GFP/RLDX fluorescence at the early blastula stage, and eventually examined by microscopic observation at the pluteus stage. (B–G) Representative examples of embryos injected with either the hd-GFP mRNA (B and E) or the Mo-nodal alone (C and F), and of double-injected rescued embryos (D–G). Note that in both the rescued pluteus larvae shown in (D) and (G), the progeny of the blastomere that received Mo-nodal was embedded into the dorsal structures.DOI: http://dx.doi.org/10.7554/eLife.04664.014
	Figure 8. Functional correlation between hbox12 and p38 MAPK.(A–C) Embryos injected with the p38-GFP mRNA either alone (A) or in the presence of the hd mRNA (B) or the hbox12 mRNA (C), respectively, and analyzed by live confocal microscopy at the 60-cell stage. Images are internal projections of 10–15 sections from a confocal z-series. (D) At the 4-cell stage, one blastomere of p38-GFP mRNA injected embryos was co-injected with the hbox12 mRNA, and the resulting embryos were examined by live confocal microscopy at the early blastula stage. Representative examples of control p38-GFP mRNA injected (E) and double-injected (F) embryos are shown.DOI: http://dx.doi.org/10.7554/eLife.04664.015
	Figure 9. Model for establishment of the DV organizing centre in the sea urchin embryo.In the early embryo, hbox12 transcription is initiated by combinatorial positive inputs from Otx and probably Sox in the future dorsal ectoderm (Cavalieri et al., 2008). hbox12-dependent suppression of nodal gene expression in these cells is mediated by the transient inactivation of p38 and/or probably by direct repression. On the ventral side of the embryo, hbox12 expression is negatively regulated by unidentified repressors (Cavalieri et al., 2008). In these cells, active p38 stimulates nodal expression probably through Oct1/2 or other intermediate transcription factors (Range and Lepage, 2011), allowing the establishment of the DV organizer and patterning along the secondary axis.DOI: http://dx.doi.org/10.7554/eLife.04664.016

Agca, Respecification of ectoderm and altered Nodal expression in sea urchin embryos after cobalt and nickel treatment. 2009, Pubmed, Echinobase

Agca, Respecification of ectoderm and altered Nodal expression in sea urchin embryos after cobalt and nickel treatment. 2009, Pubmed , Echinobase
Angerer, A BMP pathway regulates cell fate allocation along the sea urchin animal-vegetal embryonic axis. 2000, Pubmed , Echinobase
Angerer, Patterning the sea urchin embryo: gene regulatory networks, signaling pathways, and cellular interactions. 2003, Pubmed , Echinobase
Ben-Tabou de-Leon, Gene regulatory control in the sea urchin aboral ectoderm: spatial initiation, signaling inputs, and cell fate lockdown. 2013, Pubmed , Echinobase
Bergeron, Oral-aboral patterning and gastrulation of sea urchin embryos depend on sulfated glycosaminoglycans. 2011, Pubmed , Echinobase
Bessodes, Reciprocal signaling between the ectoderm and a mesendodermal left-right organizer directs left-right determination in the sea urchin embryo. 2012, Pubmed , Echinobase
Bolouri, The gene regulatory network basis of the "community effect," and analysis of a sea urchin embryo example. 2010, Pubmed , Echinobase
Bradham, p38 MAPK is essential for secondary axis specification and patterning in sea urchin embryos. 2006, Pubmed , Echinobase
Bradham, Chordin is required for neural but not axial development in sea urchin embryos. 2009, Pubmed , Echinobase
Brandhorst, Molecular patterning along the sea urchin animal-vegetal axis. 2002, Pubmed , Echinobase
CHILD, Exogastrulation by sodium azide and other inhibiting conditions in Strongylocentrotus purpuratus. 1948, Pubmed , Echinobase
Cameron, The oral-aboral axis of a sea urchin embryo is specified by first cleavage. 1989, Pubmed , Echinobase
Cavalieri, Regulatory sequences driving expression of the sea urchin Otp homeobox gene in oral ectoderm cells. 2007, Pubmed , Echinobase
Cavalieri, cis-Regulatory sequences driving the expression of the Hbox12 homeobox-containing gene in the presumptive aboral ectoderm territory of the Paracentrotus lividus sea urchin embryo. 2008, Pubmed , Echinobase
Cavalieri, Functional studies of regulatory genes in the sea urchin embryo. 2009, Pubmed , Echinobase
Cavalieri, Promoter activity of the sea urchin (Paracentrotus lividus) nucleosomal H3 and H2A and linker H1 {alpha}-histone genes is modulated by enhancer and chromatin insulator. 2009, Pubmed , Echinobase
Cavalieri, Specific expression of a TRIM-containing factor in ectoderm cells affects the skeletal morphogenetic program of the sea urchin embryo. 2011, Pubmed , Echinobase
Cavalieri, The Compass-like locus, exclusive to the Ambulacrarians, encodes a chromatin insulator binding protein in the sea urchin embryo. 2013, Pubmed , Echinobase
Cavalieri, Impairing Otp homeodomain function in oral ectoderm cells affects skeletogenesis in sea urchin embryos. 2003, Pubmed , Echinobase
Chen, The dynamic gene expression patterns of transcription factors constituting the sea urchin aboral ectoderm gene regulatory network. 2011, Pubmed , Echinobase
Coffman, Oral-aboral axis specification in the sea urchin embryo. I. Axis entrainment by respiratory asymmetry. 2001, Pubmed , Echinobase
Coffman, Oral-aboral axis specification in the sea urchin embryo II. Mitochondrial distribution and redox state contribute to establishing polarity in Strongylocentrotus purpuratus. 2004, Pubmed , Echinobase
Coffman, Oral-aboral axis specification in the sea urchin embryo III. Role of mitochondrial redox signaling via H2O2. 2009, Pubmed , Echinobase
Coffman, Mitochondria and metazoan epigenesis. 2009, Pubmed , Echinobase
Czihak, [Not Available]. 1963, Pubmed
De Robertis, Spemann's organizer and the self-regulation of embryonic fields. 2009, Pubmed
Di Bernardo, Spatially restricted expression of PlOtp, a Paracentrotus lividus orthopedia-related homeobox gene, is correlated with oral ectodermal patterning and skeletal morphogenesis in late-cleavage sea urchin embryos. 1999, Pubmed , Echinobase
Di Bernardo, Homeobox-containing gene transiently expressed in a spatially restricted pattern in the early sea urchin embryo. 1995, Pubmed , Echinobase
Duboc, Nodal and BMP2/4 signaling organizes the oral-aboral axis of the sea urchin embryo. 2004, Pubmed , Echinobase
Duboc, Lefty acts as an essential modulator of Nodal activity during sea urchin oral-aboral axis formation. 2008, Pubmed , Echinobase
Duboc, Nodal and BMP2/4 pattern the mesoderm and endoderm during development of the sea urchin embryo. 2010, Pubmed , Echinobase
Flowers, Nodal/activin signaling establishes oral-aboral polarity in the early sea urchin embryo. 2004, Pubmed , Echinobase
Flytzanis, Persistence and integration of cloned DNA in postembryonic sea urchins. 1985, Pubmed , Echinobase
Franks, Direct introduction of cloned DNA into the sea urchin zygote nucleus, and fate of injected DNA. 1988, Pubmed , Echinobase
Hoey, Divergent homeo box proteins recognize similar DNA sequences in Drosophila. 1988, Pubmed
Houde, Intestinal epithelial cell differentiation involves activation of p38 mitogen-activated protein kinase that regulates the homeobox transcription factor CDX2. 2001, Pubmed
Hough-Evans, Mosaic incorporation and regulated expression of an exogenous gene in the sea urchin embryo. 1988, Pubmed , Echinobase
Hurley, Altered expression of spatially regulated embryonic genes in the progeny of separated sea urchin blastomeres. 1989, Pubmed , Echinobase
Hörstadius, [Not Available]. 1936, Pubmed
Kitamura, Transient activation of the micro1 homeobox gene family in the sea urchin ( Hemicentrotus pulcherrimus) micromere. 2002, Pubmed , Echinobase
Lapraz, Patterning of the dorsal-ventral axis in echinoderms: insights into the evolution of the BMP-chordin signaling network. 2009, Pubmed , Echinobase
Li, Direct and indirect control of oral ectoderm regulatory gene expression by Nodal signaling in the sea urchin embryo. 2012, Pubmed , Echinobase
Li, New regulatory circuit controlling spatial and temporal gene expression in the sea urchin embryo oral ectoderm GRN. 2013, Pubmed , Echinobase
Materna, High accuracy, high-resolution prevalence measurement for the majority of locally expressed regulatory genes in early sea urchin development. 2010, Pubmed , Echinobase
Materna, Diversification of oral and aboral mesodermal regulatory states in pregastrular sea urchin embryos. 2013, Pubmed , Echinobase
Molina, Nodal: master and commander of the dorsal-ventral and left-right axes in the sea urchin embryo. 2013, Pubmed , Echinobase
Nam, Cis-regulatory control of the nodal gene, initiator of the sea urchin oral ectoderm gene network. 2007, Pubmed , Echinobase
Oliveri, A regulatory gene network that directs micromere specification in the sea urchin embryo. 2002, Pubmed , Echinobase
Oliveri, Activation of pmar1 controls specification of micromeres in the sea urchin embryo. 2003, Pubmed , Echinobase
Oliveri, Development. Built to run, not fail. 2007, Pubmed
Range, Cis-regulatory analysis of nodal and maternal control of dorsal-ventral axis formation by Univin, a TGF-beta related to Vg1. 2007, Pubmed , Echinobase
Range, Maternal Oct1/2 is required for Nodal and Vg1/Univin expression during dorsal-ventral axis specification in the sea urchin embryo. 2011, Pubmed , Echinobase
Ransick, New early zygotic regulators expressed in endomesoderm of sea urchin embryos discovered by differential array hybridization. 2002, Pubmed , Echinobase
Revilla-i-Domingo, A missing link in the sea urchin embryo gene regulatory network: hesC and the double-negative specification of micromeres. 2007, Pubmed , Echinobase
Röttinger, Nemo-like kinase (NLK) acts downstream of Notch/Delta signalling to downregulate TCF during mesoderm induction in the sea urchin embryo. 2006, Pubmed , Echinobase
Saudemont, Ancestral regulatory circuits governing ectoderm patterning downstream of Nodal and BMP2/4 revealed by gene regulatory network analysis in an echinoderm. 2010, Pubmed , Echinobase
Su, Gene regulatory networks for ectoderm specification in sea urchin embryos. 2009, Pubmed , Echinobase
Svingen, Hox transcription factors and their elusive mammalian gene targets. 2006, Pubmed
Wang, Maternal and embryonic provenance of a sea urchin embryo transcription factor, SpZ12-1. 1995, Pubmed , Echinobase
Yaguchi, Sp-Smad2/3 mediates patterning of neurogenic ectoderm by nodal in the sea urchin embryo. 2007, Pubmed , Echinobase
Yaguchi, A Wnt-FoxQ2-nodal pathway links primary and secondary axis specification in sea urchin embryos. 2008, Pubmed , Echinobase
Yaguchi, TGFβ signaling positions the ciliary band and patterns neurons in the sea urchin embryo. 2010, Pubmed , Echinobase
Yamazaki, The micro1 gene is necessary and sufficient for micromere differentiation and mid/hindgut-inducing activity in the sea urchin embryo. 2005, Pubmed , Echinobase
Zhou, p38 MAP kinase interacts with and stabilizes pancreatic and duodenal homeobox-1. 2013, Pubmed