1) Control of Wnt signaling in the anterior neuroectoderm. (25%) (Ryan Range and Lynne Angerer) Our objective was to determine how Wnt signaling controls the development of regions that will give to neurons (neuroectoderm) versus those that do not. The anterior neuroectoderm domain forms where Wnt is antagonized and epidermal ectoderm where Wnt is active. At least three different Wnt pathways, Wnt/β-catenin, Wnt/PCP and Wnt/Ca+2, are involved in setting up these two types of ectoderm and at least three different regulators of Wnt signaling, Dkk1, sFRP1/5 and Dkk3, are expressed in the anterior neuroectoderm where Wnt signaling is low. We have uncovered an intricate, interconnected set of interactions among the Wnt signaling branches that eliminates the ubiquitous, maternally driven anterior neuroectoderm regulatory state from all but the anterior-most cells of the embryo. First, signaling through Wnt/beta-catenin removes it from posterior blastomeres and produces at least two Wnt ligands, Wnt1 and Wnt8, that signal through the Wnt/JNK pathway via the Wnt receptor, Frizzled 5/8, to eliminate anterior neuroectoderm fate from most of the anterior blastomeres. Both Wnt/beta-catenin and Wnt/JNK pathways are slowed by signaling through another Wnt receptor, Frizzled 1/2/7. Fz5/8-dependent elimination of the ANE regulatory state is blocked by the Wnt antagonist, Dkk1. Interestingly, Dkk1 expression depends on Fz5/8 and then negatively feeds back to inhibit its activity. In all but the anterior-most cells Fz5/8 activity is required for its own transcription. How Fz5/8 transcription is maintained in anterior cells in the presence of Dkk1 is not yet understood, but may depend on Dkk3, which is expressed specifically in anterior cells and is an apparent potentiator of Wnt signaling. These studies have uncovered a set of unexpected and surprisingly complex interactions among different Wnt pathways in early patterning as well as unexpected roles for Wnt/PCP and Wnt/Ca+2 in regulating early ectodermal cell fate decisions. This network of Wnt signaling is likely conserved among deuterostome embryos, based on gene expression patterns in hemichordates and cephalochordates and isolated loss-of-function studies in zebrafish embryos. manuscript in revision. 2) Role of individual Wnt ligands in ectoderm patterning. (25%) (Zheng Wei, Ryan Range and Lynne Angerer) We made the unexpected discovery that Wnt1 activity was required at a relatively late stage to maintain the correct orientation of the cell fates along the dorsal ventral (DV) axis of the embryo. In the absence of Wnt1, the expression of nodal extends ectopically into the posterior ventral corner of the embryo and converts the fates of these cells to oral ectoderm. As a consequence the position of the ciliary band shifts from the ventral to the dorsal side of the blastopore or anus, reflecting a change along the DV axis. Furthermore, the position of the blastopore, which marks the posterior pole of the embryo, is now on the ventral side of the embryo near the mouth, an anterior structure, as a result of the exaggerated curvature of the AP axis of the embryo. Thus, during morphogenesis continued interactions between Wnt and Nodal signaling are required to maintain the body plan of the embryo. Wei et al., Development 139, 1662-1669 (2012) (cover photo) 3) Mechanisms underlying endomesoderm segregation. (25%) (Adi Sethi, Lynne Angerer) Although, in vertebrate embryos, cWnt signaling is known to be required for endomesoderm specification and Notch is implicated in controlling the balance between endoderm and mesoderm, how these actually work in the transition from endomesoderm progenitor to stably committed endoderm and mesoderm is not understood. We showed that, in sea urchin embryos, endomesoderm segregation is a sequential response to crosstalk between Notch and Wnt/β-catenin (cWnt) signaling within the endomesoderm gene regulatory network. Notch initiates segregation in mesoderm progenitors by inhibiting expression of the transcription factor, Hox11/13b, which heads a key early endoderm regulatory circuit. In the second step of endomesoderm segregation, this circuit subsequently activates transcription of the cWnt ligand, wnt1, only in the presumptive endoderm as a result of circuit inactivation by Notch in the mesoderm. The resulting Wnt1-dependent cWnt circuit maintains the endoderm state, reinforcing the distinction between endoderm and mesoderm. A third step occurs just before gastrulation commences in which Notch signals completely insulate the mesoderm from Wnt activity and an endoderm fate by promoting the nuclear export of TCF, a transcription factor required for canonical Wnt function. The discovery of these three steps has defined the mechanism operating in the endoderm gene regulatory network that generates optimal signaling environments required for the progressive separation of endoderm from mesoderm. Given the involvement of both signaling pathways in endomesoderm development in both vertebrates and sea urchin embryos, it is likely that vertebrate embryos also use a closely related version of this cWnt/Notch crosstalk model to control the fundamental process of endomesoderm segregation. Sethi et al., Science 335, 590-593. (Highlighted in Science Signaling) 4) Dopaminergic neurons regulate the embryos response to food density (25%) (Diane Adams, Lynne Angerer) Previous work with pharmacological inhibitors of dopamine receptor function suggested that dopamine signaling was involved in the embryos response to food density. We have confirmed this hypothesis by perturbing this pathway at the level of dopamine production, dopamine activity or by eliminating a dopamine D2 receptor. The surprising finding from this work is that the default developmental program, which occurs in the absence of food, supports the growth of long arms. In contrast, when dopamine signaling is stimulated, which occurs at high food densities, the developmental program is suppressed. Thus, the commonly held view that the developmental plasticity involves growth longer larval arms to optimize food gathering potential is incorrect;instead plasticity requires dopamine signaling, which inhibits arm growth. Thus, selection for developmental plasticity is not to enhance food gathering potential;instead it must favor conservation of maternal reserves. Consistent with this hypothesis, we found that embryos with long arms have a significant loss of lipid reserves. Because neurons producing dopamine are positioned near the points of skeletal growth, they are excellent candidates for mediating the skeletal growth response. Adams et al., Nature Communications, DOI:10.1038/ncomms1603. (Featured article) We have also examined the evolution of this developmental plasticity in response to food throughout echinoderms that diverged from each other over the course of the last 600 my. Although the current dogma is that sensitivity to the environment is the ancestral state, comparative analysis suggests that the response to food evolved more recently in the regular urchins and is not present in the irregular and pencil urchins. (Adams et al., manuscript in preparation.

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Wei, Zheng; Angerer, Lynne M; Angerer, Robert C (2016) Neurogenic gene regulatory pathways in the sea urchin embryo. Development 143:298-305
Sethi, Aditya J; Angerer, Robert C; Angerer, Lynne M (2014) Multicolor labeling in developmental gene regulatory network analysis. Methods Mol Biol 1128:249-62
Range, Ryan C; Angerer, Robert C; Angerer, Lynne M (2013) Integration of canonical and noncanonical Wnt signaling pathways patterns the neuroectoderm along the anterior-posterior axis of sea urchin embryos. PLoS Biol 11:e1001467
Sethi, Aditya J; Wikramanayake, Radhika M; Angerer, Robert C et al. (2012) Sequential signaling crosstalk regulates endomesoderm segregation in sea urchin embryos. Science 335:590-3
Yaguchi, Junko; Angerer, Lynne M; Inaba, Kazuo et al. (2012) Zinc finger homeobox is required for the differentiation of serotonergic neurons in the sea urchin embryo. Dev Biol 363:74-83
Wei, Zheng; Range, Ryan; Angerer, Robert et al. (2012) Axial patterning interactions in the sea urchin embryo: suppression of nodal by Wnt1 signaling. Development 139:1662-9
Wei, Zheng; Angerer, Robert C; Angerer, Lynne M (2011) Direct development of neurons within foregut endoderm of sea urchin embryos. Proc Natl Acad Sci U S A 108:9143-7
Yaguchi, Shunsuke; Yaguchi, Junko; Wei, Zheng et al. (2011) Fez function is required to maintain the size of the animal plate in the sea urchin embryo. Development 138:4233-43
Adams, Diane K; Sewell, Mary A; Angerer, Robert C et al. (2011) Rapid adaptation to food availability by a dopamine-mediated morphogenetic response. Nat Commun 2:592
Angerer, Lynne M; Yaguchi, Shunsuke; Angerer, Robert C et al. (2011) The evolution of nervous system patterning: insights from sea urchin development. Development 138:3613-23

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