Molecular mechanisms of cell fate specification

细胞命运规范的分子机制

• 批准号: 8553341
• 负责人: LYNNE M ANGERER
• 金额: $ 118.57万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8553341
• 关键词: Affect; Anterior; Anus; Back; Biological Models; Blastopores; Cells; Commit; Communication; Complement; Complex; Development; Dopamine; Dopamine D2 Receptor; Dopamine Receptor; Dorsal; Ectoderm; Ectoderm Cell; Embryo; Endoderm; Endomesoderm; Environment; Equilibrium; Event; Evolution; Feeds; Food; Gene Expression Profile; Genetic Transcription; Goals; Growth; Head; Individual; Larva; Ligands; Lipids; MAPK8 gene; Manuscripts; Mediating; Mesoderm; Modeling; Molecular; Morphogenesis; Nature; Nervous system structure; Neuroectoderm; Neurons; Nodal; Nuclear Export; Oral; Oral cavity; Pathway interactions; Pattern; Positioning Attribute; Preparation; Process; Production; Prosencephalon; Regulator Genes; Research; Role; Science; Sea Urchins; Side; Signal Pathway; Signal Transduction; Staging; Structure; Testing; Tissues; Vertebrates; Work; Zebrafish; arm; base; beta catenin; cell fate specification; cephalochordate; comparative; density; developmental plasticity; dopaminergic neuron; embryo cell; embryo stage 2; environmental change; fascinate; feeding; gastrulation; genetic regulatory protein; hemichordate; inhibitor/antagonist; loss of function; neurodevelopment; notch protein; oral ectoderm; progenitor; programs; receptor; receptor function; response; segregation; skeletal; transcription factor

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.

1) 前神经外胚层中 Wnt 信号传导的控制。 (25%)（Ryan Range 和 Lynne Angerer）我们的目标是确定 Wnt 信号如何控制将产生神经元（神经外胚层）的区域与那些不产生神经元的区域的发育。 当 Wnt 被拮抗时，形成前神经外胚层结构域；当 Wnt 被激活时，形成表皮外胚层结构域。 至少三种不同的 Wnt 通路（Wnt/β-连环蛋白、Wnt/PCP 和 Wnt/Ca+2）参与建立这两种类型的外胚层，并且至少三种不同的 Wnt 信号调节因子（Dkk1、sFRP1/5 和 Dkk3）在 Wnt 信号传导较低的前神经外胚层中表达。 我们发现了 Wnt 信号分支之间复杂且相互关联的一组相互作用，这些相互作用消除了除胚胎最前部细胞之外的所有细胞中普遍存在的、母体驱动的前部神经外胚层调节状态。 首先，通过 Wnt/β-连环蛋白的信号传导将其从后卵裂球中去除，并产生至少两个 Wnt 配体 Wnt1 和 Wnt8，这些配体通过 Wnt 受体 Frizzled 5/8 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 Frizzled 1/2/7 的信号传导而减慢。 Fz5/8 依赖性 ANE 调节状态的消除被 Wnt 拮抗剂 Dkk1 阻断。 有趣的是，Dkk1 表达依赖于 Fz5/8，然后负反馈抑制其活性。 除了最前面的细胞外，在所有细胞中，Fz5/8 活性都是其自身转录所必需的。 在 Dkk1 存在的情况下，Fz5/8 转录如何在前部细胞中维持尚不清楚，但可能取决于 Dkk3，Dkk3 在前部细胞中特异性表达，并且是 Wnt 信号传导的明显增强剂。 这些研究揭示了早期模式形成中不同 Wnt 通路之间的一系列意想不到且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。 根据半索动物和头索动物的基因表达模式以及斑马鱼胚胎中孤立的功能丧失研究，这种 Wnt 信号网络很可能在后口动物胚胎中保守。手稿正在修订中。 2) 单个Wnt配体在外胚层模式形成中的作用。 (25%)（Zheng Wei、Ryan Range 和 Lynne Angerer）我们意外地发现，Wnt1 活性需要在相对较晚的阶段才能维持细胞命运沿胚胎背腹 (DV) 轴的正确方向。 在Wnt1缺失的情况下，nodal的表达异位延伸到胚胎的后腹角，并将这些细胞的命运转变为口腔外胚层。 因此，睫状带的位置从胚孔或肛门的腹侧转移到背侧，反映了沿 DV 轴的变化。 此外，由于胚胎 AP 轴的曲率过大，标志着胚胎后极的胚孔位置现在位于胚胎靠近嘴的腹侧，是一种前部结构。 因此，在形态发生过程中，需要 Wnt 和 Nodal 信号之间的持续相互作用来维持胚胎的身体规划。 Wei 等人，Development 139, 1662-1669 (2012)（封面照片） 3）内中胚层分离的机制。 (25%) (Adi Sethi, Lynne Angerer) 虽然在脊椎动物胚胎中，已知 cWnt 信号传导是内中胚层规范所必需的，并且 Notch 涉及控制内胚层和中胚层之间的平衡，但这些信号在从内中胚层祖细胞到稳定定型的内胚层和中胚层的转变中实际上如何发挥作用尚不清楚。 我们发现，在海胆胚胎中，内中胚层分离是对内中胚层基因调控网络中 Notch 和 Wnt/β-连环蛋白 (cWnt) 信号之间串扰的连续反应。 Notch 通过抑制转录因子 Hox11/13b 的表达来启动中胚层祖细胞的分离，该转录因子领导着关键的早期内胚层调节回路。 在内中胚层分离的第二步中，由于中胚层中的Notch电路失活，该电路随后仅在推定的内胚层中激活cWnt配体wnt1的转录。 由此产生的 Wnt1 依赖性 cWnt 电路维持内胚层状态，增强了内胚层和中胚层之间的区别。 第三步发生在原肠胚形成开始之前，其中Notch信号通过促进TCF（典型Wnt功能所需的转录因子）的核输出，将中胚层与Wnt活性和内胚层命运完全隔离。这三个步骤的发现定义了内胚层基因调控网络的运作机制，该机制产生内胚层与中胚层逐步分离所需的最佳信号环境。 鉴于脊椎动物和海胆胚胎的内中胚层发育中两种信号通路的参与，脊椎动物胚胎很可能也使用这种 cWnt/Notch 串扰模型的密切相关版本来控制内中胚层分离的基本过程。 Sethi 等人，科学 335, 590-593。 （《科学信号》中重点介绍） 4) 多巴胺能神经元调节胚胎对食物密度的反应 (25%) (Diane Adams, Lynne Angerer) 先前对多巴胺受体功能的药理学抑制剂的研究表明，多巴胺信号传导参与胚胎对食物密度的反应。 我们通过在多巴胺产生、多巴胺活性水平上干扰该通路或通过消除多巴胺 D2 受体证实了这一假设。这项工作令人惊讶的发现是，在没有食物的情况下发生的默认发育程序支持长臂的生长。 相反，当多巴胺信号传导受到刺激（在高食物密度下发生）时，发育程序就会受到抑制。 因此，普遍认为发育可塑性涉及幼体生长更长的臂以优化食物采集潜力的观点是不正确的。相反，可塑性需要多巴胺信号传导，而多巴胺信号传导会抑制手臂生长。 因此，对发育可塑性的选择并不是为了增强食物采集潜力；而是为了增强食物采集潜力。相反，它必须有利于保护母性储备。 与这一假设一致，我们发现长臂胚胎的脂质储备显着减少。 由于产生多巴胺的神经元位于骨骼生长点附近，因此它们是介导骨骼生长反应的绝佳候选者。 Adams 等人，《自然通讯》，DOI：10.1038/ncomms1603。 （专题文章） 我们还研究了整个棘皮动物中这种发育可塑性对食物的反应的演变，这些发育可塑性在过去 600 年的过程中彼此分化。 尽管目前的教条是对环境的敏感性是祖先的状态，但比较分析表明，对食物的反应在规则海胆中最近才进化出来，而在不规则海胆和铅笔海胆中并不存在。 （亚当斯等人，手稿正在准备中。