Molecular mechanisms of cell fate specification

我们关注的主要问题是：1)海胆胚胎的动物极域的上游调控成分是什么，它包含承载着长而不动的纤毛的神经和细胞？2)在早期的卵裂和囊胚阶段，内胚层的指定和适时的原肠形成所需的信号传递是什么？3)胚胎改变其形态以适应不断变化的食物浓度的分子基础是什么？在每种情况下，我们分析基因组中所有预测基因的表达，以获得对这些过程背后的基因调控网络的完整了解。 对于apd规范，Six3是必要的且充分的(50%)(郑伟、Ryan Range、Lynne Angerer) 在过去的几年里，利用生物信息学和分子筛选，我们鉴定了许多编码调控蛋白的基因，这些基因在海胆胚胎的初级神经源性区域中特异表达。其中最早的两个，FoxQ2和Six3，控制着外胚层图案的早期决定。2008年进行的实验表明，Six3对于所有已知的动作障碍发育特征是必要的，也是充分的。这包括在胚胎发育过程中形成的所有神经元，以及之前发现的绝大多数apd特异性调控蛋白，包括FoxQ2。我们确定了依赖于Six3的基因的完整调控谱，并发现该因子还可以抑制形成胚胎其余部分的规范Wnt和转化生长因子-β信号。我们的工作揭示了Six3在海胆胚胎apd中的功能与脊椎动物前脑中的Six3有一些共同的特征。我们的研究已经确定了许多额外的调控基因，这些基因是依赖于Six3的，并且在apd中表达。因此，确定这些基因的脊椎动物同源基因是否在脊椎动物前脑发育中发挥作用是有意义的。这项工作已由魏等人发表，《发展》136,1179-1189(2009)。 FoxQ2不仅可以抑制动作电位中的转化生长因子-β信号(Yaguchi等人，发育细胞14，97-107,2008)，而且它也是在这一领域控制Wnt信号所必需的。微阵列筛选显示，它是近500个基因的表达所必需的，包括编码早期apd调节蛋白的基因的子集，以及特定于apd的特殊纤毛。此外，FoxQ2是生产Wnt信号调节因子所必需的。当这些调控因子被消除或过度表达时，外胚层中的动作电位调节器边界的位置就会改变。这些结果进一步证实了在海胆胚胎早期外胚层中，APDGRN和WNT信号输出之间的相互拮抗作用调节着APD值的发育。 APDGRN的激活取决于母体的积极调节活动。其中之一是SoxB1。当SoxB1被消除时，在囊胚期胚胎中，apd内神经的分化被阻止，apd的上游关键成分GRN、Six3、FoxQ2和hBN的表达减少。SoxB1还在激活口腔和流产的外胚层发育的GRN以及抑制典型的Wnt信号方面发挥关键作用。确定SoxB1对外胚层模式的相对贡献是如何控制的，这对于理解决定外胚层内细胞命运的初始事件至关重要。 ActivinB/ALK4-5-7和Delta/Notch是诱导内胚层和内胚层规范的早期信号(25%)(Adi Sethi，Radhika Wikramanayake，Lynne Angerer) 先前的实验表明，异位和正常的内胚层诱导都需要ActivinB，并且这种信号因子执行先前未知的、但长期寻找的早期微米信号的所有内胚层诱导功能。这项工作(Sethi，AJ，Angerer，RC，Angerer，LM(PLOS Biology 7(2)：ee1000029,2009)首次将ActivinB信号与内胚层基因调控(GRN)网络的特定组件连接起来，该网络是所有胚胎中最大和最发达的，并为其运作带来了新的见解。阻断Delta/Notch信号通路的实验揭示了这一途径的新功能，即在内胚层区域内分离两个初级胚层--内胚层和中胚层。微米Delta信号不仅激活了上覆大粒子代的GCM，而且还抑制了早期内胚层指定所需的几个关键基因。最近的结果表明，在推测中胚层细胞的规范中，一个关键因素是Notch信号下调典型的Wnt信号通路的能力。 多巴胺能神经元调节胚胎对食物密度的反应(25%)(Diane Adams，Lynne Angerer) 一项新的研究旨在了解幼虫通过调节臂长来最大限度地摄取食物来感知和反应食物浓度的分子途径。这是一个连接幼虫发育生物学、幼虫生理学和幼虫扩散生态学的高风险项目。黛安发现，多巴胺D2受体的活性调节了口腔后骨骼杆长的变化，以回应食物密度的变化。她已经确定了口腔后手臂中表达多巴胺和酪氨酸羟基酶的细胞，那里发生了对食物密度的反应。目前正在进行实验，以确定哪种D2受体介导了这一反应。为了确定食物感知途径的其他组成部分，她正在使用微阵列来分析整个基因组对食物浓度以及多巴胺拮抗剂和激动剂的反应。

The major questions we focus on are: 1) What are the upstream regulatory components that specify the animal pole domain (APD) of the sea urchin embryo, which contains nerves and cells bearing long, immotile cilia? 2) What are the signals transmitted during early cleavage and blastula stages that are required for endomesoderm specification and timely gastrulation? 3) What is the molecular basis for the embryo's ability to change its morphology to adapt to changing food concentrations? In each case, we assay the expression of all predicted genes in the genome in order to gain a complete understanding of the gene regulatory networks that underlie these processes. Six3 is necessary and sufficient for APD specification (50%) (Zheng Wei, Ryan Range, Lynne Angerer) During the last several years, using bioinformatics and molecular screens, we identified many genes encoding regulatory proteins expressed specifically in the primary neurogenic domain of the sea urchin embryo. Two of the earliest, FoxQ2 and Six3, control early decisions in ectodermal patterning. Experiments conducted during 2008 showed that Six3 is necessary and sufficient for all known features of APD development. This includes all neurons formed during embryogenesis and the large majority of APD-specific regulatory proteins previously identified, including FoxQ2. We identified the full regulatory repertoire of genes that depend on Six3, and discovered that this factor can also suppress canonical Wnt and TGF-beta signals that pattern the rest of the embryo. Our work revealed that the function of Six3 in the sea urchin embryo APD shares some features with Six3 in the vertebrate forebrain. Our studies have identified many additional regulatory genes that are Six3-dependent and expressed in the APD. It is therefore of interest to determine whether the vertebrate orthologs of these genes function in vertebrate forebrain development. This work has been published by Wei et al., Development 136, 1179-1189 (2009). FoxQ2 not only can suppress TGF-beta signaling in the APD(Yaguchi et al., Developmental Cell 14, 97-107, 2008), but it also required for controlling Wnt signaling in this territory. Microarray screens show that it is required for the expression of nearly 500 genes including a subset of the genes encoding early APD regulatory proteins and for specialized cilia specifically in the APD. Further, FoxQ2 is required for production of regulators of Wnt signaling. When these regulators are either eliminated or over expressed, the position of the boundary of the APD within the ectoderm is altered. These results reinforce the model that mutual antagonism between the outputs of the APD GRN and Wnt signaling regulates APD development within the early ectoderm of the sea urchin embryo. Activation of the APD GRN depends upon maternal positive regulatory activities. One of these is SoxB1. When SoxB1 is eliminated, differentiation of nerves within the APD is blocked and expression of the key upstream components of the APD GRN, Six3, FoxQ2 and Hbn (homeobrain) are reduced in the blastula-stage embryo. SoxB1 also plays a key role in activating the oral and aboral ectodermal developmental GRNs as well as in suppressing canonical Wnt signaling. Determining how the relative contributions of SoxB1 to ectodermal patterning are controlled will be critical for understanding the initial events that specify cell fates within the ectoderm. ActivinB/ALK4-5-7 and Delta/Notch are early signals that induce specification of endomesoderm and endoderm (25%)(Adi Sethi, Radhika Wikramanayake, Lynne Angerer) Previous experiments showed that both ectopic and normal endomesoderm induction requires ActivinB and that this signaling factor executes all of the endomesoderm inducing functions of the previously unknown, but long-sought, early micromere signal. This work (Sethi, AJ, Angerer, RC, Angerer, LM (PLOS Biology 7(2): ee1000029, 2009) is the first to connect ActivinB signaling to specific components of an endomesoderm gene regulatory (GRN) network, the largest and best developed in any embryo, and it has led to new insights into its operation. Experiments in which Delta/Notch signaling is blocked reveal new functions for this pathway in separating two primary germ layers, the endoderm and mesoderm, within the endomesodermal field. Micromere Delta signals not only activate gcm in overlying macromere progeny, but also repress several critical genes required for early endoderm specification. Recent results suggest that a key element in the specification of presumptive mesodermal cells is the ability of Notch signals to down regulate the canonical Wnt signaling pathway. Dopaminergic neurons regulate the embryo's response to food density (25%) (Diane Adams, Lynne Angerer) A new line of investigation aims to understand the molecular pathways by which larvae sense and respond to food concentration by adjusting arm length to maximize food intake. It is a high-risk project that bridges larval developmental biology, larval physiology and the ecology of larval dispersal. Diane has found that dopamine D2receptor activity regulates changes in the post-oral skeletal rod length in response to food density. She has identified cells expressing dopamine and tyrosine hydroxylase in the post-oral arms where the response to food density occurs. Experiments are underway to identify which D2 receptor mediates this response. To determine other components of the food sensing pathway, she is using microarrays to assay the whole genome reponse to food concentration as well as to dopamine antagonists and agonists.