Molecular mechanisms of cell fate specification

细胞命运规范的分子机制

基本信息

批准号：
8148640
负责人：
LYNNE M ANGERER
金额：
$ 112.5万
依托单位：
NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
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/8148640
关键词：
Molecular cell fate specification

项目摘要

1) Our objective was to determine how Wnt signaling, which antagonizes neural development, is prevented in the anterior neurogenic ectoderm, termed the animal pole domain or APD. At least two genes encoding proteins that can antagonize Wnt (sFRP1/5 and Dkk1) are expressed in this region. Mis-expression of each can prevent Wnt-dependent endomesoderm development and prevents clearance of the animal pole domain factors from the lateral ectoderm. Loss of Dkk1 allows Wnt signaling to eliminate the entire APD. Unexpectedly, loss of sFRP1/5 leads to a larger APD. Thus, sFRPs role is not yet clear, but it may indirectly increase the domain of Dkk1 activity. Several Wnt receptors are expressed in the animal hemisphere when the APD is differentiating at the animal pole. One of these, Fzl1/2/7, is required for APD development while the second, Fzl5/8, antagonizes it. Our results support a model in which Dkk1s antagonism of Fz5/8 protects the APD. Manuscript in preparation. 2) De novo neurogenesis in the foregut. (25%) (Zheng Wei, Lynne Angerer) We made the surprising discovery that pharyngeal neurons of sea urchin embryos develop de novo from the endoderm in the foregut through the activity of the transcription factors, Six3, Nkx3-2 and Brn1/2/4. This result is entirely unexpected because a fundamental concept in developmental biology is that nerves form from ectoderm. We ruled out migration of ectodermal cells to the pharynx by tracking all presumptive ectoderm cells with the photo-activatable protein, KikGR. We observed that neurons appear in the foregut before the stomodeal ectoderm joins the foregut and in exogastrulae. We found that Nkx3-2 is co-expressed with synaptotagmin B, which marks differentiating neurons, in several cells in the foregut of gastrulae, consistent with the dependence of these neurons on Nkx3-2. These and other results (Peter et al., 2010, Dev. Biol. 340: 188-199) suggest both endodermal and neural gene regulatory networks operate in foregut cell lineages before gastrulation. Manuscript submitted. 3) Mechanisms underlying endomesoderm segregation. (25%) (Adi Sethi, Lynne Angerer) We have determined that a major distinction between endoderm and mesoderm at late mesenchyme blastula/early gastrula stages is the presence and absence of TCF in endoderm and mesoderm nuclei, respectively. Since TCF is the binding partner of beta-catenin, it is a critical component of canonical Wnt signaling, which is required for endoderm development. In the endoderm, transcription factors that are constituents of the early endoderm network are required to maintain TCF within nuclei, whereas in the mesoderm, Notch signals are required to promote its export from nuclei. While differential regulation of nuclear TCF levels in endoderm and mesoderm is likely to help establish stable regulatory states in these tissues, it is not among the first specification steps. Those include Notch-dependent activation of genes required for mesoderm-specific gene expression programs and down regulation of factors supporting the endoderm program during blastula/early mesenchyme stages. One of these endoderm factors, although known to depend on canonical Wnt signaling, also appears to be especially important in supporting it during mid-mesenchyme blastula stages. In addition, it supports expression of other endoderm regulatory proteins shortly after its appearance in presumptive endoderm, and these functions surprisingly depend on Notch signaling. Further studies are underway to determine how Notch signals in endoderm cells affect canonical Wnt signaling, the operation of the endoderm gene regulatory network and the stabilization of TCF in endodermal nuclei. Manuscript in preparation. 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 or by eliminating a dopamine D2 receptor. A major part of this response is to divert energy from maternal lipid stores to lengthen arms in order to increase feeding rate. We have initiated studies on dopaminergic neurogenesis and defined a new cell type with unique markers, including the dopamine biosynthesis enzyme dopa decarboxylase. Because some of these cells are positioned near the points of skeletal growth, they are excellent candidates for mediating the skeletal growth response. This mechanism is a novel developmental response of the embryo to its environment. Manuscript in preparation

1) 我们的目标是确定如何在前神经源性外胚层（称为动物极结构域或 APD）中阻止拮抗神经发育的 Wnt 信号传导。至少有两个编码可拮抗 Wnt 的蛋白质的基因（sFRP1/5 和 Dkk1）在该区域表达。每种蛋白的错误表达都可以阻止 Wnt 依赖性内中胚层发育，并阻止动物极域因子从外侧外胚层清除。 Dkk1 的丢失使得 Wnt 信号传导消除整个 APD。出乎意料的是，sFRP1/5 的损失导致 APD 更大。因此，sFRPs的作用尚不清楚，但它可能间接增加Dkk1域的活性。当 APD 在动物极分化时，动物半球会表达多种 Wnt 受体。其中之一 Fzl1/2/7 是 APD 发育所必需的，而第二个 Fzl5/8 则与之拮抗。我们的结果支持 Dkk1s 对 Fz5/8 的拮抗作用保护 APD 的模型。手稿正在准备中。 2）前肠的从头神经发生。 (25%) (Zheng Wei, Lynne Angerer) 我们惊奇地发现，海胆胚胎的咽部神经元是通过转录因子 Six3、Nkx3-2 和 Brn1/2/ 的活性从前肠内胚层从头发育而来。 4. 这个结果完全出乎意料，因为发育生物学的一个基本概念是神经是由外胚层形成的。我们通过用光激活蛋白 KikGR 追踪所有假定的外胚层细胞，排除了外胚层细胞向咽部的迁移。我们观察到，在食道外胚层连接前肠之前和外原肠胚中，神经元出现在前肠中。我们发现，Nkx3-2 与突触结合蛋白 B 共表达，突触结合蛋白 B 在原肠胚前肠的几个细胞中标记分化神经元，这与这些神经元对 Nkx3-2 的依赖性一致。这些和其他结果（Peter 等人，2010，Dev. Biol. 340：188-199）表明内胚层和神经基因调控网络在原肠胚形成前在前肠细胞谱系中运作。稿件已提交。 3）内中胚层分离的机制。 (25%) (Adi Sethi, Lynne Angerer) 我们已经确定，内胚层和中胚层在囊胚晚期/原肠胚早期阶段的主要区别是内胚层和中胚层细胞核中是否存在 TCF。由于 TCF 是 β-连环蛋白的结合伴侣，因此它是内胚层发育所需的经典 Wnt 信号传导的关键组成部分。在内胚层中，转录因子是早期内胚层网络的组成部分，需要将 TCF 维持在细胞核内，而在中胚层中，需要 Notch 信号来促进其从细胞核中输出。虽然内胚层和中胚层核 TCF 水平的差异调节可能有助于在这些组织中建立稳定的调节状态，但这并不是第一个规范步骤。这些包括Notch依赖性激活中胚层特异性基因表达程序所需的基因，以及在囊胚/早期间充质阶段支持内胚层程序的因子的下调。尽管已知这些内胚层因子之一依赖于经典 Wnt 信号传导，但似乎在中期间充质囊胚阶段的支持中也特别重要。此外，它在假定的内胚层中出现后不久就支持其他内胚层调节蛋白的表达，并且这些功能令人惊讶地依赖于Notch信号传导。进一步的研究正在进行中，以确定内胚层细胞中的 Notch 信号如何影响经典 Wnt 信号传导、内胚层基因调控网络的运行以及内胚层细胞核中 TCF 的稳定性。手稿正在准备中。 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。这种反应的主要部分是将能量从母体脂质储存转移到延长手臂，以提高进食率。我们启动了多巴胺能神经发生的研究，并定义了一种具有独特标记的新细胞类型，包括多巴胺生物合成酶多巴脱羧酶。由于其中一些细胞位于骨骼生长点附近，因此它们是介导骨骼生长反应的极好候选者。这种机制是胚胎对其环境的一种新的发育反应。手稿正在准备中