Gautam S , Fenner JL , Wang B , Range RC .

R15 HD088272 NICHD NIH HHS

	Figure 1. Spatiotemporal expression patterns of sp5 during early germ layer formation(A) Model summarizing the progressive posterior-to-anterior patterning of early sea urchin germ layer GRNs by the AP Wnt signaling network involving canonical (Wnt/β-catenin) and non-canonical (Wnt1/Wnt8-Fzd5/8-JNK and Fzd1/2/7-PKC) signaling pathways.12,18,19,22 Posterior Wnt/β-catenin and anterior Fzd5/8-JNK signaling pathways are active in distinct territories while Fzd1/2/7-PKC signaling is active throughout the early embryo during this process. See text for details.(B) Three-color whole mount HCR-FISH for sp5 (magenta), six3 (blue), and foxa (orange). Anterior is top, and posterior is bottom. (Ba, e, i, m) 120-cell stage embryo showing sp5 expression in all germ layers. (Bd, f, j, n) Hatched blastula (Bc, g, k, o) late blastula, and (Bd, h, l, p) mesenchyme blastula stage embryos showing that sp5 expression is downregulated from the ANE where six3 is expressed and restricted to the equatorial ectoderm and the endomesoderm regions overlapping with foxa expression. (Bq-t) Schematic diagrams of the merged expression patterns observed in the HCR RNA-FISH images in Bm-p. six3 (light blue) marks the ANE and later the endomesoderm territory and foxa (orange) marks the endomesoderm. The solid lines in the diagrams demarcate the ANE, equatorial ectoderm (EE) and endomesoderm (EM) GRN territories during early AP patterning. Scale bars: 20 μm.
	Figure 2. Wnt/β-catenin signaling activates Sp5 expression in the endomesoderm(A) Posterior/vegetal views of three-color whole mount HCR-FISH embryos showing sp5 (magenta) expression in relation to foxa (orange) and gata4/5/6 (blue) during blastula stages. sp5, foxa and gata4/5/6 expression overlap in hatched blastula (Aa, d, g, j, m) and late blastula embryos (Ab, e, h, k, n). Mesenchyme blastula embryos showing co-expression of sp5 and foxa in anterior/veg1 and downregulation in posterior/veg2 endoderm cells marked by gata4/5/6 (Ac, f, i, j, o). (Am-o) Schematic diagrams of the merged expression patterns observed in Aj-l. foxa (orange) marks anterior/veg1endoderm, and gata4/5/6 (orange) marks posterior/veg2 endoderm cells by mesenchyme blastula stage (Ao).(B) Data show colorimetric WMISH images of mesenchyme blastula stage embryos from the same mating pairs. Embryos injected with axin mRNA show downregulation of sp5 transcripts (Ba, b) and broad expression of foxq2 (Bd, e). Ectopic activation of the Wnt/β-catenin pathway by LiCl treatment shows ectopic sp5 (Ba, c) and downregulation of foxq2 (Bd, f). VV, vegetal views. Scale bars: 20 μm.
	Figure 3. Sp5 is a component of the posterior endomesoderm gene regulatory network(A) blimp1b, gata4/5/6, eve and foxa expression in 120-cell stage control (Aa-e) and Sp5 morpholino (Af-j) injected embryos (Af-j).(B) Sp5 morphants at mesenchyme blastula stage showing blimp1b, gata4/5/6, foxa, eve, and otx expression (Bf,g,h,i).(C) Data show mesenchyme blastula stage embryos injected with Eve morpholinos. (Ca, b) Colorimetric WMISH showing sp5 expression in (Ca) control and (Cb) Eve morpholino injected embryos. (Cc-e) HCR-FISH using probes for sp5 and foxa in the same embryo. (Cc) Control embryo showing co-expression of foxa and sp5. (Cd, e) Separate images of the same Sp5 morphant showing the foxa (Cd) and sp5 (Ce) expression.(D) Diagram of our new model for the endoderm kernel gene regulatory network. The solid lines indicate previously assessed direct regulation among the transcription factors.45,46,47,48,49,50 Dotted lines show interactions based on the functional evidence in this study. Scale bars: 20 μm. MO, Morpholino. LiCl, Lithium Chloride.
	Figure 4. Sp5 signaling is necessary for Wnt1/Wnt8-Fzd5/8-JNK mediated ANE restriction mechanism in anterior blastomeres(A) Wnt1, Wnt8, Fzd5/8, and JNK knockdown embryos show downregulated sp5 expression (compare Aa to Ab-e).(B) Embryos treated with wnt1 mRNA (Bb), Wnt8 mRNA (Bd), Fzd1/2/7 MO (Bd), and Dkk1 MO (Be) show ectopic sp5 expression compared control (Ba).(C) Injection of Sp5 morpholino 1 causes posterior expansion of foxq2 expression (compare Ca, b, c), and ectopic sp5 mRNA expression downregulates foxq2 expression (compare Ca, d). The angle α shown in Ca-c was measured with ImageJ. Volume = 0.5(1-cos α/2) was used to calculate the percentage of the surface area (±sem) occupied by foxq2 expression in control and Sp5 knockdowns.(D) Sp5 MO2 rescue experiment. (Db) Sp5 MO2 morphant showing foxq2 expansion. (Dc) A representative sp5 mRNA-injected embryo with downregulated foxq2 expression. (Da, d, e) foxq2 expression rescued by double injection of Sp5MO2 and sp5 mRNA in a majority of embryos (Ed, e).(E) Model based on the data from this figure for the Wnt8-Fzd5/8-JNK-Sp5 ANE restriction mechanism in equatorial ectoderm cells from late cleavage to mesenchyme blastula. All embryos in this figure are mesenchyme blastulae. MO, Morpholino. Δ, Dominant-negative. Scale bars: 20 μm.
	Figure 5. Regulatory feedback loops among Wnt8, Fzd5/8, and Sp5 in the equatorial ectoderm(A) HCR RNA-FISH in situ images of sp5 (magenta), fzd5/8 (orange), wnt1 and wnt8 (both turquoise) transcripts during in mesenchyme blastula stage embryos. sp5 (Aa and e) is co-expressed with wnt1 (Ab) and fzd5/8 (Ac, g) in the endoderm and with wnt8 (Af) in the equatorial ectoderm.(B) wnt8 expression is downregulated in ΔFzd5/8 injected embryos (compare Ba to Bb). Sp5 morphants show downregulated wnt8 expression (compare Ba with Bc) and embryos injected with sp5 mRNA show ectopic wnt8 expression (compare Ba to Bd).(C) Sp5 morphants express sp5 transcripts ectopically at the mesenchyme blastula stage.(D) Model for the Wnt8-Fzd5/8-JNK-Sp5 ANE restriction mechanism in equatorial ectoderm cells based on the data represented in this figure. MO, Morpholino. Δ, Dominant-negative. Scale bars: 20 μm.
	Figure 6. The role of Sp5 in early germ layer GRNs and schematic diagrams depicting similarities among the Wnt-mediated ANE GRN restriction mechanisms in deuterostome embryos(A) A model of endoderm and equatorial ectoderm GRNs incorporating the data from this study. The solid lines indicate previously assessed direct regulation among the transcription factors.45,46,47,48,49,50,76 Dotted lines show interactions based on the functional evidence in this study.(B) Embryos are shown at the approximate termination of ANE GRN restriction to territories around the anterior pole during gastrulation. The diagrams for each embryo depict the germ layer territories/sub-territories generated by the end of this developmental process. Dark blue boxes above each embryo indicate the early ANE GRN components conserved among each species. In each of the animals, a posterior-to-anterior Wnt-signaling dependent mechanism restricts initially broadly expressed ANE GRN components to a territory around the anterior pole (see text for details).2 Conserved epistatic relationships among the known Wnt signaling components involved in posterior-to-anterior ANE GRN restriction are indicated. Factors shown in light gray with question marks indicate that the function of the gene has not been assessed. The colored bars next to each embryo indicate the spatial expression domains along the anterior-posterior axis for sp5, wnt8, and fzd5/8 orthologs. In hemichordate embryos, the expression of sp5 has not been characterized (shaded gray bar). Expression and functional data were taken from.2,23,25,26,77,78,79,80,81

Angerer, The evolution of nervous system patterning: insights from sea urchin development. 2011, Pubmed, Echinobase

Angerer, The evolution of nervous system patterning: insights from sea urchin development. 2011, Pubmed , Echinobase
Aronson, Role of GATA factors in development, differentiation, and homeostasis of the small intestinal epithelium. 2014, Pubmed
Azambuja, A regulatory sub-circuit downstream of Wnt signaling controls developmental transitions in neural crest formation. 2021, Pubmed
Bikoff, An expanding job description for Blimp-1/PRDM1. 2009, Pubmed
Cary, Systematic comparison of sea urchin and sea star developmental gene regulatory networks explains how novelty is incorporated in early development. 2020, Pubmed , Echinobase
Castro-Mondragon, JASPAR 2022: the 9th release of the open-access database of transcription factor binding profiles. 2022, Pubmed
Chan, Developmental gene regulatory networks in the zebrafish embryo. 2009, Pubmed
Choi, Mapping a multiplexed zoo of mRNA expression. 2016, Pubmed , Echinobase
Cirillo, Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4. 2002, Pubmed
Croce, Frizzled5/8 is required in secondary mesenchyme cells to initiate archenteron invagination during sea urchin development. 2006, Pubmed , Echinobase
Cui, Specific functions of the Wnt signaling system in gene regulatory networks throughout the early sea urchin embryo. 2014, Pubmed , Echinobase
Dailey, Amphioxus Sp5 is a member of a conserved Specificity Protein complement and is modulated by Wnt/β-catenin signalling. 2017, Pubmed
Darras, Anteroposterior axis patterning by early canonical Wnt signaling during hemichordate development. 2018, Pubmed
Davidson, A provisional regulatory gene network for specification of endomesoderm in the sea urchin embryo. 2002, Pubmed , Echinobase
Davidson, A genomic regulatory network for development. 2002, Pubmed , Echinobase
de-Leon, Information processing at the foxa node of the sea urchin endomesoderm specification network. 2010, Pubmed , Echinobase
Duffy, Wnt signaling promotes oral but suppresses aboral structures in Hydractinia metamorphosis and regeneration. 2010, Pubmed
Dunty, Transcriptional profiling of Wnt3a mutants identifies Sp transcription factors as essential effectors of the Wnt/β-catenin pathway in neuromesodermal stem cells. 2014, Pubmed
Emily-Fenouil, GSK3beta/shaggy mediates patterning along the animal-vegetal axis of the sea urchin embryo. 1998, Pubmed , Echinobase
Erkenbrack, Conserved regulatory state expression controlled by divergent developmental gene regulatory networks in echinoids. 2018, Pubmed , Echinobase
Erkenbrack, Whole mount in situ hybridization techniques for analysis of the spatial distribution of mRNAs in sea urchin embryos and early larvae. 2019, Pubmed , Echinobase
Erkenbrack, Evolutionary rewiring of gene regulatory network linkages at divergence of the echinoid subclasses. 2015, Pubmed , Echinobase
Feuda, Homologous gene regulatory networks control development of apical organs and brains in Bilateria. 2022, Pubmed , Echinobase
Garriock, A dorsal-ventral gradient of Wnt3a/β-catenin signals controls mouse hindgut extension and colon formation. 2020, Pubmed
Gray, Saccoglossus kowalevskii: Evo-devo insights from the mud. 2022, Pubmed
Harrison, Sp5, a new member of the Sp1 family, is dynamically expressed during development and genetically interacts with Brachyury. 2000, Pubmed
Hinman, Caught in the evolutionary act: precise cis-regulatory basis of difference in the organization of gene networks of sea stars and sea urchins. 2007, Pubmed , Echinobase
Hinman, Developmental gene regulatory network architecture across 500 million years of echinoderm evolution. 2003, Pubmed , Echinobase
Hohenauer, The Prdm family: expanding roles in stem cells and development. 2012, Pubmed
Holstein, The role of cnidarian developmental biology in unraveling axis formation and Wnt signaling. 2022, Pubmed
Huggins, The WNT target SP5 negatively regulates WNT transcriptional programs in human pluripotent stem cells. 2017, Pubmed
Ka, Receptor Tyrosine Kinases ror1/2 and ryk Are Co-expressed with Multiple Wnt Signaling Components During Early Development of Sea Urchin Embryos. 2021, Pubmed , Echinobase
Kennedy, Sp5 and Sp8 recruit β-catenin and Tcf1-Lef1 to select enhancers to activate Wnt target gene transcription. 2016, Pubmed
Khadka, A novel gene's role in an ancient mechanism: secreted Frizzled-related protein 1 is a critical component in the anterior-posterior Wnt signaling network that governs the establishment of the anterior neuroectoderm in sea urchin embryos. 2018, Pubmed , Echinobase
Kiecker, A morphogen gradient of Wnt/beta-catenin signalling regulates anteroposterior neural patterning in Xenopus. 2001, Pubmed
Kim, Specification of an anterior neuroectoderm patterning by Frizzled8a-mediated Wnt8b signalling during late gastrulation in zebrafish. 2002, Pubmed
Kim, Characterization of two frizzled8 homologues expressed in the embryonic shield and prechordal plate of zebrafish embryos. 1998, Pubmed
Lagutin, Six3 repression of Wnt signaling in the anterior neuroectoderm is essential for vertebrate forebrain development. 2003, Pubmed
Lebedeva, Cnidarian-bilaterian comparison reveals the ancestral regulatory logic of the β-catenin dependent axial patterning. 2021, Pubmed , Echinobase
Leclère, Development of the aboral domain in Nematostella requires β-catenin and the opposing activities of Six3/6 and Frizzled5/8. 2016, Pubmed
Lee, Exclusive developmental functions of gatae cis-regulatory modules in the Strongylocentrorus purpuratus embryo. 2007, Pubmed , Echinobase
Lekven, Zebrafish wnt8 encodes two wnt8 proteins on a bicistronic transcript and is required for mesoderm and neurectoderm patterning. 2001, Pubmed
Leonard, Analysis of dishevelled localization and function in the early sea urchin embryo. 2007, Pubmed , Echinobase
Levine, Gene regulatory networks for development. 2005, Pubmed , Echinobase
Livi, Expression and function of blimp1/krox, an alternatively transcribed regulatory gene of the sea urchin endomesoderm network. 2006, Pubmed , Echinobase
Logan, Nuclear beta-catenin is required to specify vegetal cell fates in the sea urchin embryo. 1999, Pubmed , Echinobase
Martik, Developmental gene regulatory networks in sea urchins and what we can learn from them. 2016, Pubmed , Echinobase
Martínez-Bartolomé, A biphasic role of non-canonical Wnt16 signaling during early anterior-posterior patterning and morphogenesis of the sea urchin embryo. 2019, 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
McClay, Conditional specification of endomesoderm. 2021, Pubmed , Echinobase
McClay, Evolutionary crossroads in developmental biology: sea urchins. 2011, Pubmed , Echinobase
McKnight, Foxh1 and Foxa2 are not required for formation of the midgut and hindgut definitive endoderm. 2010, Pubmed
Miao, Opposite Roles of Wnt7a and Sfrp1 in Modulating Proper Development of Neural Progenitors in the Mouse Cerebral Cortex. 2018, Pubmed
Molina, MAPK and GSK3/ß-TRCP-mediated degradation of the maternal Ets domain transcriptional repressor Yan/Tel controls the spatial expression of nodal in the sea urchin embryo. 2018, Pubmed , Echinobase
Morley, A gene regulatory network directed by zebrafish No tail accounts for its roles in mesoderm formation. 2009, Pubmed
Niehrs, On growth and form: a Cartesian coordinate system of Wnt and BMP signaling specifies bilaterian body axes. 2010, Pubmed
Nikaido, A systematic survey of expression and function of zebrafish frizzled genes. 2013, Pubmed
Oliveri, Repression of mesodermal fate by foxa, a key endoderm regulator of the sea urchin embryo. 2006, Pubmed , Echinobase
Onai, Essential role of Dkk3 for head formation by inhibiting Wnt/β-catenin and Nodal/Vg1 signaling pathways in the basal chordate amphioxus. 2012, Pubmed
Peng, Differential regulation of disheveled in a novel vegetal cortical domain in sea urchin eggs and embryos: implications for the localized activation of canonical Wnt signaling. 2013, Pubmed , Echinobase
Peter, Methods for the experimental and computational analysis of gene regulatory networks in sea urchins. 2019, Pubmed , Echinobase
Peter, The endoderm gene regulatory network in sea urchin embryos up to mid-blastula stage. 2010, Pubmed , Echinobase
Peter, A gene regulatory network controlling the embryonic specification of endoderm. 2011, Pubmed , Echinobase
Petersen, Wnt signaling and the polarity of the primary body axis. 2009, Pubmed
Poustka, A global view of gene expression in lithium and zinc treated sea urchin embryos: new components of gene regulatory networks. 2007, Pubmed , Echinobase
Range, LvGroucho and nuclear beta-catenin functionally compete for Tcf binding to influence activation of the endomesoderm gene regulatory network in the sea urchin embryo. 2005, Pubmed , Echinobase
Range, Canonical and non-canonical Wnt signaling pathways define the expression domains of Frizzled 5/8 and Frizzled 1/2/7 along the early anterior-posterior axis in sea urchin embryos. 2018, Pubmed , Echinobase
Range, Specification and positioning of the anterior neuroectoderm in deuterostome embryos. 2014, Pubmed , Echinobase
Range, An anterior signaling center patterns and sizes the anterior neuroectoderm of the sea urchin embryo. 2016, Pubmed , Echinobase
Range, Integration of canonical and noncanonical Wnt signaling pathways patterns the neuroectoderm along the anterior-posterior axis of sea urchin embryos. 2013, Pubmed , Echinobase
Robert, A comprehensive survey of wnt and frizzled expression in the sea urchin Paracentrotus lividus. 2014, Pubmed , Echinobase
Rojas, An endoderm-specific transcriptional enhancer from the mouse Gata4 gene requires GATA and homeodomain protein-binding sites for function in vivo. 2009, Pubmed
Schaeper, A clustered set of three Sp-family genes is ancestral in the Metazoa: evidence from sequence analysis, protein domain structure, developmental expression patterns and chromosomal location. 2010, Pubmed
Schindelin, Fiji: an open-source platform for biological-image analysis. 2012, Pubmed
Sethi, Gene regulatory network interactions in sea urchin endomesoderm induction. 2009, Pubmed , Echinobase
Sethi, Sequential signaling crosstalk regulates endomesoderm segregation in sea urchin embryos. 2012, Pubmed , Echinobase
Shi, Wnt/planar cell polarity signaling controls morphogenetic movements of gastrulation and neural tube closure. 2022, Pubmed
Shinya, Zebrafish Dkk1, induced by the pre-MBT Wnt signaling, is secreted from the prechordal plate and patterns the anterior neural plate. 2000, Pubmed
Smith, A spatially dynamic cohort of regulatory genes in the endomesodermal gene network of the sea urchin embryo. 2008, Pubmed , Echinobase
Smith, Gene regulatory network subcircuit controlling a dynamic spatial pattern of signaling in the sea urchin embryo. 2008, Pubmed , Echinobase
Soudais, Targeted mutagenesis of the transcription factor GATA-4 gene in mouse embryonic stem cells disrupts visceral endoderm differentiation in vitro. 1995, Pubmed
Steinmetz, Six3 demarcates the anterior-most developing brain region in bilaterian animals. 2010, Pubmed
Sumanas, Zebrafish frizzled-2 morphant displays defects in body axis elongation. 2001, Pubmed
Sun, An early global role for Axin is required for correct patterning of the anterior-posterior axis in the sea urchin embryo. 2021, Pubmed , Echinobase
Technau, Evolutionary crossroads in developmental biology: Cnidaria. 2011, Pubmed
Tewari, A small set of conserved genes, including sp5 and Hox, are activated by Wnt signaling in the posterior of planarians and acoels. 2019, Pubmed
Thorpe, Wnt/beta-catenin regulation of the Sp1-related transcription factor sp5l promotes tail development in zebrafish. 2005, Pubmed
Tseng, An evolutionarily conserved kernel of gata5, gata6, otx2 and prdm1a operates in the formation of endoderm in zebrafish. 2011, Pubmed
Varga, Correct anteroposterior patterning of the zebrafish neurectoderm in the absence of the early dorsal organizer. 2011, Pubmed
Vogg, An evolutionarily-conserved Wnt3/β-catenin/Sp5 feedback loop restricts head organizer activity in Hydra. 2019, Pubmed
Vonica, TCF is the nuclear effector of the beta-catenin signal that patterns the sea urchin animal-vegetal axis. 2000, Pubmed , Echinobase
Wang, SpZ12-1, a negative regulator required for spatial control of the territory-specific CyIIIa gene in the sea urchin embryo. 1995, Pubmed , Echinobase
Weber, A role for GATA5 in Xenopus endoderm specification. 2000, Pubmed
Wei, The sea urchin animal pole domain is a Six3-dependent neurogenic patterning center. 2009, Pubmed , Echinobase
Weidinger, The Sp1-related transcription factors sp5 and sp5-like act downstream of Wnt/beta-catenin signaling in mesoderm and neuroectoderm patterning. 2005, Pubmed
Weitzel, Differential stability of beta-catenin along the animal-vegetal axis of the sea urchin embryo mediated by dishevelled. 2004, Pubmed , Echinobase
Wikramanayake, Nuclear beta-catenin-dependent Wnt8 signaling in vegetal cells of the early sea urchin embryo regulates gastrulation and differentiation of endoderm and mesodermal cell lineages. 2004, Pubmed , Echinobase
Wilm, Essential roles of a zebrafish prdm1/blimp1 homolog in embryo patterning and organogenesis. 2005, Pubmed
Yaguchi, A Wnt-FoxQ2-nodal pathway links primary and secondary axis specification in sea urchin embryos. 2008, Pubmed , Echinobase
Ye, Wnt/β-catenin and LIF-Stat3 signaling pathways converge on Sp5 to promote mouse embryonic stem cell self-renewal. 2016, Pubmed
Yu, Axial patterning in cephalochordates and the evolution of the organizer. 2007, Pubmed
Yuh, An otx cis-regulatory module: a key node in the sea urchin endomesoderm gene regulatory network. 2004, Pubmed , Echinobase
Zhao, An SP1-like transcription factor Spr2 acts downstream of Fgf signaling to mediate mesoderm induction. 2003, Pubmed
Zhao, Sp1-like transcription factors are regulators of embryonic development in vertebrates. 2005, Pubmed