Molecular mechanisms of cell fate specification

细胞命运规范的分子机制

• 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
• 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

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）我们的目标是确定在神经源性外胚层中如何阻止神经发育的Wnt信号传导称为动物极域或APD。 在该区域中表达了至少两个可以拮抗Wnt（SFRP1/5和DKK1）的蛋白质的基因。 每个人的错误表达可以防止Wnt依赖性内胚层发育，并防止从侧外胚层中清除动物极域因子。 DKK1的损失允许Wnt信号消除整个APD。 出乎意料的是，SFRP1/5的丢失导致较大的APD。 因此，SFRP的角色尚不清楚，但可能会间接增加DKK1活性的域。 当APD在动物极处区分时，在动物半球中表达了几种Wnt受体。 其中之一是FZL1/2/7的APD开发所必需的，而第二个FZL5/8则对其进行了拮抗。 我们的结果支持一个模型，其中DKK1S的拮抗FZ5/8保护APD。手稿准备。 2）前肢中的从头神​​经发生。 （25％）（Zheng Wei，Lynne Angerer）我们做出了一个令人惊讶的发现，即海胆胚胎的咽神经元通过转录因子的活性，六，NKX3-2和BRN1/2/4，从内胚层从内胚层开始发展。 该结果是完全出乎意料的，因为发育生物学中的基本概念是神经来自外胚层。 我们排除了外胚层细胞迁移到咽部的迁移，通过使用光活化蛋白Kikgr跟踪所有假定的外胚层细胞。 我们观察到神经元出现在固醇外胚层之前的前肢中，并在前肢和exogastrulae中出现。 我们发现，NKX3-2与胃肠道的几个细胞中标记神经元的突触量B共表达，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，2010，Dev。Biol。340：188-199）表明，内胚层和神经基因调节网络在胃肠道之前在Foregut细胞谱系中起作用。 提交手稿。 3）内胚层分离的基础机制。 （25％）（Adi Sethi，Lynne Angerer）我们已经确定，在晚期间充质胚胎/早期胃阶段，内胚层和中胚层之间的主要区别是内胚层和中胚层核中的存在和不存在。 由于TCF是β-catenin的结合伙伴，因此它是典型Wnt信号的关键组成部分，这是内胚层开发所必需的。 在内胚层中，需要早期内胚层网络成分的转录因子在核内维持TCF，而在中胚层中，需要Notch信号来促进其从核中促进其出口。 虽然内胚层和中胚层中核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，但它并不是第一个规范步骤之一。 其中包括依赖于中胚层特异性基因表达程序的基因的Notch依赖性激活，并降低了在囊泡/早期间质阶段支持内胚层程序的因素的调节。 这些内胚层因素之一，尽管众所周知取决于规范的Wnt信号传导，但在中孔囊肿阶段支持它似乎也尤其重要。 另外，它支持其他内胚层调节蛋白在推定内胚层中出现后不久的表达，并且这些功能令人惊讶地取决于Notch信号传导。 正在进行进一步的研究，以确定内胚层细胞中的Notch信号如何影响规范Wnt信号，内胚层基因调节网络的运行以及TCF在内胚层核中的稳定。 手稿准备。 4）多巴胺能神经元调节胚胎对食物密度的反应（25％）（黛安·亚当斯，林恩·愤怒者）先前与多巴胺受体功能的药理抑制剂的先前作品表明，多巴胺信号传导参与了胚胎对食物密度的反应。 我们已经通过在多巴胺产生水平或消除多巴胺D2受体的水平上扰动该途径来证实这一假设。该反应的主要部分是将能量从母体脂质存储转移到加长臂以提高进食率。 我们已经开始对多巴胺能神经发生的研究，并定义了具有独特标记的新细胞类型，包括多巴胺生物合成酶Dopa dopa decarbosylase。 由于其中一些细胞位于骨骼生长的点附近，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颖的发展反应。 手稿准备