Molecular mechanisms of cell fate specification

1)我们的目标是确定Wnt信号是如何在动物的前神经源性外胚层中被阻止的，它对抗神经发育，被称为动物极域或雪崩。至少有两个编码能拮抗Wnt的蛋白(SFRP1/5和Dkk1)的基因在该区域表达。每种基因的错误表达都可以阻止依赖Wnt的内胚层发育，并阻止动物极域因子从外侧外胚层中清除。Dkk1的丢失允许WNT信令消除整个apd。出乎意料的是，SFRP1/5的丢失导致了更大的雪崩。因此，sFRPs的作用尚不清楚，但它可能间接增加Dkk1的活性区域。当动物体顶端的动作电位发生分化时，几种Wnt受体在动物大脑半球表达。其中一个，Fzl1/2/7，是动作障碍发育所必需的，而第二个，Fzl5/8，则拮抗它。我们的结果支持Dkk1s拮抗Fz5/8保护动作电位的模型。手稿正在准备中。 2)前肠的新生神经发生。(25%)(郑伟，Lynne Angerer)我们有一个令人惊讶的发现，海胆胚胎的咽神经细胞通过转录因子Six3、Nkx3-2和Brn1/2/4的活性从前肠的内胚层重新发育。这一结果完全出人意料，因为发育生物学中的一个基本概念是神经来自外胚层。我们通过追踪所有具有光激活蛋白KikGR的推测外胚层细胞，排除了外胚层细胞向咽部迁移的可能性。我们观察到，在气孔外胚层与前肠连接之前，神经元出现在前肠和外肠中。我们发现Nkx3-2与标记分化神经元的突触素B在原肠前部的几个细胞中共表达，这与这些神经元对Nkx3-2的依赖一致。这些和其他结果(Peter等人，2010，Dev.比奥尔。340：188-199)表明，内胚层和神经基因调控网络在原肠形成之前在前肠细胞谱系中起作用。已提交手稿。 3)内胚层分离的机制。(25%)(Adi Sethi，Lynne Angerer)我们已经确定，在间叶胚泡晚期/原肠胚期早期，内胚层和中胚层之间的一个主要区别是内胚层和中胚层细胞核中分别存在和不存在TCF。由于Tcf是β-catenin的结合伙伴，因此它是内胚层发育所必需的规范Wnt信号的关键组成部分。在内胚层中，作为早期内胚层网络的组成部分的转录因子需要维持细胞核内的TCF，而在中胚层中，需要Notch信号来促进其从细胞核中输出。虽然对内胚层和中胚层核TCF水平的不同调节可能有助于在这些组织中建立稳定的调节状态，但这不是第一个规范步骤。这些包括依赖Notch激活中胚层特异性基因表达程序所需的基因，以及在胚泡/早期间充质阶段下调支持内胚层程序的因子。这些内胚层因子中的一种，虽然已知依赖于典型的Wnt信号，但在间充质中期囊胚期的支持中似乎也特别重要。此外，它还支持在推测的内胚层出现后不久表达其他内胚层调节蛋白，而这些功能出人意料地依赖于Notch信号。进一步的研究正在进行中，以确定内胚层细胞中的Notch信号如何影响规范的Wnt信号、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。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)(Diane Adams，Lynne Angerer)以前用药物抑制多巴胺受体功能的工作表明，多巴胺信号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的水平上干扰这一途径或通过消除多巴胺D2受体来证实这一假设。这种反应的一个主要部分是从母体脂肪储存中转移能量来延长手臂，以提高喂食率。我们已经开始了对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。由于这些细胞中的一些位于骨骼生长点附近，它们是调节骨骼生长反应的极佳候选者。这种机制是胚胎对环境的一种新的发育反应。正在筹备中的手稿

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