Molecular mechanisms of cell fate specification

1)前神经外胚层Wnt信号的调控。（25%） （Ryan Range和Lynne Angerer）我们的目标是确定Wnt信号是如何控制神经元（神经外胚层）和非外胚层区域的发育的。Wnt被拮抗的前神经外胚层域和Wnt活跃的表皮外胚层形成。至少有三种不同的Wnt通路，Wnt/ -catenin， Wnt/PCP和Wnt/Ca+2参与了这两种类型外胚层的建立，至少有三种不同的Wnt信号调节因子，Dkk1， sFRP1/5和Dkk3在Wnt信号较低的前神经外胚层表达。我们已经发现了Wnt信号分支之间错综复杂的相互作用，消除了普遍存在的，母体驱动的前神经外胚层调节状态，除了胚胎的大部分前细胞。首先，通过Wnt/ β -连环蛋白的信号传导将其从后卵裂球中移除，并产生至少两个Wnt配体Wnt1和Wnt8，这些配体通过Wnt受体frizzle5 /8通过Wnt/JNK途径发出信号，以消除大多数前卵裂球的前神经外胚层命运。Wnt/ β -连环蛋白和Wnt/JNK通路均通过另一种Wnt受体frizzled1/2/7信号传导而减慢。fz5 /8依赖性的ANE调节状态的消除被Wnt拮抗剂Dkk1阻断。有趣的是，Dkk1的表达依赖于Fz5/8，然后负反馈抑制其活性。在大多数细胞中，Fz5/8的活性是其自身转录所必需的。在Dkk1存在的情况下，Fz5/8转录如何在前细胞中维持尚不清楚，但可能取决于Dkk3， Dkk3在前细胞中特异性表达，是Wnt信号传导的明显增强剂。这些研究揭示了早期模式中不同Wnt通路之间意想不到的和令人惊讶的复杂相互作用，以及Wnt/PCP和Wnt/Ca+2在调节早期外胚层细胞命运决定中的意想不到的作用。基于半足藻和头足藻的基因表达模式以及斑马鱼胚胎中分离的功能丧失研究，这种Wnt信号网络可能在后口胚胎中保守。手稿在修改中。

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.