We would like to thank Dr. Robert Angerer for his careful reading and editing of the manuscript. NIH R15HD088272-01 as well as the Office of Research and Development, and Department of Biological Sciences at Mississippi State University provided support for this project to RCR.

1. High Throughput Morpholino Design Strategy
<ol>
	<li>Identify a gene(s) of interest (e.g. candidate gene approach, cis-regulatory analysis, RNAseq and/or proteomic differential screens).</li>
	<li>Use genomic, transcriptomic, and gene expression data available on frequently updated websites (e.g. SpBase http://www.echinobase.org&#8203;20&#160;and S. purpuratus Genome Search http:///urchin.nidcr.nih.gov/blast/index.html) to determine that&#160;the spatiote...

<ol>
	<li>Erwin, D. H., Davidson, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+hierarchical+gene+regulatory+networks.">The evolution of hierarchical gene regulatory networks.</a> Nature reviews. Genetics. 10, 141-148 (2009).</li><li>Peter, I. S., Davidson, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+gene+regulatory+networks+controlling+body+plan+development.">Evolution of gene regulatory networks controlling body plan development.</a> Cell. 144, 970-985 (2011).</li><li>Borggrefe, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Notch+intracellular+domain+integrates+signals+from+Wnt,+Hedgehog,+TGFbeta/BMP+and+hypoxia+pathways.">The Notch intracellular domain integrates signals from Wnt, Hedgehog, TGFbeta/BMP and hypoxia pathways.</a> Biochimica et biophysica acta. 1863, 303-313 (2016).</li><li>Range, R. C., Angerer, R. C., Angerer, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+canonical+and+noncanonical+Wnt+signaling+pathways+patterns+the+neuroectoderm+along+the+anterior-posterior+axis+of+sea+urchin+embryos.">Integration of canonical and noncanonical Wnt signaling pathways patterns the neuroectoderm along the anterior-posterior axis of sea urchin embryos.</a> PLoS Biol. 11, e1001467 (2013).</li><li>Cleary, M. A., van Osch, G. J., Brama, P. A., Hellingman, C. A., Narcisi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF,+TGFbeta+and+Wnt+crosstalk:+embryonic+to+in+vitro+cartilage+development+from+mesenchymal+stem+cells.">FGF, TGFbeta and Wnt crosstalk: embryonic to in vitro cartilage development from mesenchymal stem cells.</a> Journal of tissue engineering and regenerative medicine. 9, 332-342 (2015).</li><li>Lapraz, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RTK+and+TGF-beta+signaling+pathways+genes+in+the+sea+urchin+genome.">RTK and TGF-beta signaling pathways genes in the sea urchin genome.</a> Dev Biol. 300, 132-152 (2006).</li><li>Pires-daSilva, A., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+signalling+pathways+in+animal+development.">The evolution of signalling pathways in animal development.</a> Nature reviews. Genetics. 4, 39-49 (2003).</li><li>Sethi, A. J., Wikramanayake, R. M., Angerer, R. C., Range, R. C., Angerer, L. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signaling+and+the+polarity+of+the+primary+body+axis.">Wnt signaling and the polarity of the primary body axis.</a> Cell. 139, 1056-1068 (2009).</li><li>Lapraz, F., Haillot, E., Lepage, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+deuterostome+origin+of+the+Spemann+organiser+suggested+by+Nodal+and+ADMPs+functions+in+Echinoderms.">A deuterostome origin of the Spemann organiser suggested by Nodal and ADMPs functions in Echinoderms.</a> Nature communications. 6, 8434 (2015).</li><li>Kikuchi, A., Yamamoto, H., Sato, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+activation+mechanisms+of+Wnt+signaling+pathways.">Selective activation mechanisms of Wnt signaling pathways.</a> Trends in cell biology. 19, 119-129 (2009).</li><li>Hogan, B. 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A., Samanta, M., Yuan, A., He, D., Davidson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SpBase:+the+sea+urchin+genome+database+and+web+site.">SpBase: the sea urchin genome database and web site.</a> Nucleic Acids Res. 37, D750-D754 (2009).</li><li>Stepicheva, N. A., Song, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+microinjections+of+sea+urchin+zygotes.">High throughput microinjections of sea urchin zygotes.</a> Journal of visualized experiments : JoVE. , e50841 (2014).</li><li>Cheers, M. S., Ettensohn, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+microinjection+of+fertilized+eggs.">Rapid microinjection of fertilized eggs.</a> Methods in cell biology. 74, 287-310 (2004).</li><li>Arenas-Mena, C., Cameron, A. R., Davidson, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+expression+of+Hox+cluster+genes+in+the+ontogeny+of+a+sea+urchin.">Spatial expression of Hox cluster genes in the ontogeny of a sea urchin.</a> Development. , 4631-4643 (2000).</li><li>Sethi, A. J., Angerer, R. C., Angerer, L. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Wnt-FoxQ2-nodal+pathway+links+primary+and+secondary+axis+specification+in+sea+urchin+embryos.">A Wnt-FoxQ2-nodal pathway links primary and secondary axis specification in sea urchin embryos.</a> Dev Cell. 14, 97-107 (2008).</li><li>Molina, M. D., de Croze, N., Haillot, E., Lepage, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodal:+master+and+commander+of+the+dorsal-ventral+and+left-right+axes+in+the+sea+urchin+embryo.">Nodal: master and commander of the dorsal-ventral and left-right axes in the sea urchin embryo.</a> Curr Opin Genet Dev. 23, 445-453 (2013).</li><li>Range, R. C., Glenn, T. D., Miranda, E., McClay, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LvNumb+works+synergistically+with+Notch+signaling+to+specify+non-skeletal+mesoderm+cells+in+the+sea+urchin+embryo.">LvNumb works synergistically with Notch signaling to specify non-skeletal mesoderm cells in the sea urchin embryo.</a> Development. 135, 2445-2454 (2008).</li><li>Range, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cis-regulatory+analysis+of+nodal+and+maternal+control+of+dorsal-ventral+axis+formation+by+Univin,+a+TGF-beta+related+to+Vg1.">Cis-regulatory analysis of nodal and maternal control of dorsal-ventral axis formation by Univin, a TGF-beta related to Vg1.</a> Development. 134, 3649-3664 (2007).</li><li>Warner, J. F., Miranda, E. L., McClay, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+hedgehog+signaling+to+the+establishment+of+left-right+asymmetry+in+the+sea+urchin.">Contribution of hedgehog signaling to the establishment of left-right asymmetry in the sea urchin.</a> Dev Biol. 411, 314-324 (2016).</li><li>Rottinger, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF+signals+guide+migration+of+mesenchymal+cells,+control+skeletal+morphogenesis+[corrected]+and+regulate+gastrulation+during+sea+urchin+development.">FGF signals guide migration of mesenchymal cells, control skeletal morphogenesis [corrected] and regulate gastrulation during sea urchin development.</a> Development. 135, 353-365 (2008).</li><li>Warner, J. F., McCarthy, A. M., Morris, R. L., McClay, D. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chordin+is+required+for+neural+but+not+axial+development+in+sea+urchin+embryos.">Chordin is required for neural but not axial development in sea urchin embryos.</a> Dev Biol. 328, 221-233 (2009).</li><li>Su, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulatory+networks+for+ectoderm+specification+in+sea+urchin+embryos.">Gene regulatory networks for ectoderm specification in sea urchin embryos.</a> Biochimica et biophysica acta. 1789, 261-267 (2009).</li><li>Lin, C. Y., Su, Y. 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The authors have nothing to disclose.

The methodology presented here is an example that illustrates the power of using embryos with less genomic and morphological complexity than vertebrates to understand the signaling transduction pathways and GRNs governing fundamental developmental mechanisms.. Many labs are using similar assays during early sea urchin development to dissect the signaling pathways involved in other cell fate specification events (e.g. Notch, Hedgehog, TGF- &#946;, and FGF signaling)27,<sup class...

Gene regulatory networks (GRNs) and signal transduction pathways establish the spatial and temporal expression of genes during embryonic development that are used to build the adult animal body plan. Cell-to-cell signal transduction pathways are essential components of these regulatory networks, providing the means by which cells communicate. These cellular interactions establish and refine the expression of regulatory and differentiation genes in and among the various territories during embryogenesis1,2. Interactions among secreted extracellular modulators (ligands, antagonists), receptors, and co-receptors control the activities of signal transduction pathways. An assortment of intracellular molecules transduces these inputs resulting in altered gene expression, division, and/or shape of a cell. While many of the key molecules used at the extracellular and intracellular levels in the major pathways are known, it is an incomplete knowledge due in large part to the complexity of individual signaling pathways. In addition, different signaling pathways often interact with one another either positively or negatively at the extracellular, intracellular, and transcriptional levels3,4,5,6. Importantly, the core components of signal transduction pathways are highly conserved in all metazoan species, and, remarkably, most of the major signaling pathways often perform similar developmental functions in many species when comparing organisms from closely related phyla in particular 7,8,9,10,11.
The study of signaling during development is a daunting task in any organism, and there are several significant challenges to studying signaling pathways in most deuterostome models (vertebrates, invertebrate chordates, hemichordates, and echinoderms): 1) In vertebrates there are large numbers of possible ligand and receptor/co-modulator interactions, intracellular transduction molecules, as well as potential interactions among different signaling pathways due to the complexity of the genome12,13,14; 2) The complex morphology and morphogenetic movements in vertebrates often make it more difficult to interpret functional interactions in and among signal transduction pathways; 3) Analyses in most non-echinoderm invertebrate deuterostome model species are limited by short windows of gravidity with the exception of some tunicate species15,16.
The sea urchin embryo has few of the above-mentioned limitations and offers many unique qualities for performing a detailed analysis of signal transduction pathways in vivo. These include the following: 1) The relative simplicity of the sea urchin genome significantly reduces the number of possible ligand, receptor/co-receptor and intracellular transduction molecule interactions17; 2) The GRNs controlling the specification and patterning of the germ layers and major embryonic axes are well established in sea urchin embryos, aiding in the understanding of the regulatory context of the cell/territory receiving the signals18,19; 3) Many signal transduction pathways can be studied between early cleavage and gastrula stages when embryos consist of a single layered epithelium whose morphology is easier to analyze; 4) The molecules involved in signaling pathways in the sea urchins are easily manipulated; 5) Many sea urchins are gravid for 10 to 11 months a year (e.g. Strongylocentrotus purpuratus and Lytechinus variegatus).
Here, we present a method to systematically and efficiently characterize components of the signaling pathways that specify and pattern territories in sea urchin embryos to illustrate the advantages that several invertebrate model systems offer in the study of complex molecular mechanisms.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Translational-blocking morpholino and/or splice-blocking morpholino</td>
			<td>Gene Tools LLC</td>
			<td>Customized</td>
			<td>More information at www.gene-tools.com</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Invitrogen</td>
			<td>15514-011</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC (dextran fluorescein isothiocyanate)</td>
			<td>Invitrogen, Life Technologies</td>
			<td>D1821</td>
			<td>Make 25mg/mL stock solution</td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16% solution EM Grade</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma Aldrich</td>
			<td>M1254-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma Aldrich</td>
			<td>23336-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Sigma Aldrich</td>
			<td>47671-1L-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast tRNA</td>
			<td>Invitrogen</td>
			<td>15401-029</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Sigma Aldrich</td>
			<td>G9023-10mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase-conjugated anti-digoxigenin antibody</td>
			<td>Roche</td>
			<td>11 093 274 910</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramisole hydrochloride (levamisole)</td>
			<td>Sigma Aldrich</td>
			<td>L9756-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base UltraPure</td>
			<td>Research Products Internationall Corp</td>
			<td>56-40-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma Aldrich</td>
			<td>7786-30-3</td>
			<td></td>
		</tr>
		<tr>
			<td>BCIP (5-Bromo-4-Chloro-3-indolyl-phosphate</td>
			<td>Roche</td>
			<td>11 383 221 001</td>
			<td></td>
		</tr>
		<tr>
			<td>4 Nitro blue tetrazolium chloride (NBT)</td>
			<td>Roche</td>
			<td>11 383 213 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Formamide</td>
			<td>Sigma Aldrich</td>
			<td>D4551-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>P9541-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S5761-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma Aldrich</td>
			<td>M7506-2KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>C1016-500G</td>
			<td></td>
		</tr>
	</tbody>
</table>

the power of simplicity: sea urchin embryos as in vivo developmental models for studying complex cell-to-cell signaling network interactions

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and tissues in the adult body form. Yet, each year more components of individual signaling pathways are discovered, and studies indicate that depending on the context there is significant cross-talk among most of these pathways. This complexity makes studying cell-to-cell signaling in any in vivo developmental model system a difficult task. In addition, efficient functional analyses are required to characterize molecules associated with signaling pathways identified from the large data sets generated by next generation differential screens. Here, we illustrate a straightforward method to efficiently identify components of signal transduction pathways governing cell fate and axis specification in sea urchin embryos. The genomic and morphological simplicity of embryos similar to those of the sea urchin make them powerful in vivo developmental models for understanding complex signaling interactions. The methodology described here can be used as a template for identifying novel signal transduction molecules in individual pathways as well as the interactions among the molecules in the various pathways in many other organisms.

In the sea urchin embryo we have shown that 3 different Wnt signaling branches (Wnt/&#946;-catenin, Wnt/JNK, and Wnt/PKC)4,25 interact to form a Wnt signaling network that governs anterior-posterior (AP) patterning. One of the most important consequences of these signaling events is that the initial broadly expressed anterior neuroectoderm (ANE) GRN becomes restricted to a small territory around the anterior pole by the beginning ...

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The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

This video article details a straightforward in vivo methodology that can be used to systematically and efficiently characterize components of complex signaling pathways and regulatory networks in many invertebrate embryos.

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and ...

The overall goal of these experiments is to illustrate the advantages that simple organisms offer in the study of complex interactions among gene regulatory networks that direct developmental processes.This methodology can help answer key questions in evolutionary and developmental biology, such as how signaling pathways govern cell fate specification along the major embryonic axes in metazoans.One of the main advantages of this methodology is it can be used to efficiently characterize the signaling pathways and the gene regulatory networks that they activate in an in vivo developmental context.Demonstrating this procedure will be Marina Martinez-Bartolom

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7.8K visualizações.  Mississippi State University. The overall goal of these experiments is to illustrate the advantages that simple organisms offer in the study of complex interactions among gene regulatory networks that direct developmental processes.This methodology can help answer key questions in evolutionary and developmental biology, such as how signaling pathways govern cell fate specification along the major embryonic axes in metazoans.One of the main advantages of this methodology is it can be used to efficiently characterize the si...