GENE REGULATORY MECHANISMS AND THE CONTROL OF EARLY EMBRYOGENESIS

多年来，我们的研究计划一直专注于早期海胆胚胎的基因调控机制，这将继续是其主要推动力。我们已经开发并成功地开发了一套优秀的技术，用于对胚胎顺式调控系统进行完整的结构/功能分析。这些技术包括：允许同时在多组数百个胚胎上表达的基因转移程序，以便能够以各种方式研究带有突变或由合成DNA的一部分组成的表达载体的空间和时间行为；在实验基因控制下抑制转录因子活性的新方法；亲和纯化后克隆转录因子的方法；大规模高密度阵列克隆文库的使用；用于数字记录荧光受体空间表达的先进成像技术；以及谱系追踪、细胞生物学、显微解剖和原位分析空间基因表达的完整程序。在下一阶段，我们打算扩展和深化我们对早期发育中基因调控的机制的理解，利用这些方法来从功能上表征位于早期胚胎遗传控制网络中关键位置的一些额外的顺式调控元件。我们将完成对复杂的Endo16顺式调控系统的分析，识别尚未确定特征的特定抑制子和激活子。通过高密度排列文库的差异筛选分离出的一些额外的差异表达的下游基因将受到类似的分析，以确定它们的顺式调控组织；并识别正向和负向空间调控相互作用&位于信号转导系统末端的元件。一个主要的努力将被指向控制编码转录因子的调节基因的早期合子表达的系统的顺式调节分析。在这些因素中，有一些已经被确定为我们已经研究过的末端基因的控制者。我们的总体目标是了解在卵裂和囊胚阶段，当控制从母体转录调控装置转移到合子转录调控装置时，启动的基因调控网络。我们将利用几种新的方法来分离基因组，即由同一组转录因子控制的一组下游基因。主要的努力将致力于试图了解母体转录因子的配置和复杂性，这些转录因子的功能是通过对其目标基因的顺式调控分析而知道的。在这里，我们有三个具体的目标：第一，通过传统的共聚焦免疫细胞学在早期胚胎中进行定位，并通过我们开发的基于微毛细管的移位程序来评估DNA靶点结合的区域活性；第二，通过质谱仪和其他方法分析转录因子变体；以及第三，在代表早期胚胎不同区域的大量分离的细胞部分中定位变体。我们还计划将我们的基因调控方法应用于囊胚壁中细胞接触的重要性。将测量β-连环蛋白的合成和周转率，以确定在卵裂囊胚期胚胎中对其可用性的控制是母体的还是合子的，如果是合子的，我们将研究其顺式调节系统。我们还将研究CyIIIa细胞骨架肌动蛋白基因在分解的外胚层细胞中下调的机制基础，以探索我们对CyIIIa顺式调控系统的完整理解。另一种在顺式调控系统中实现功能意义的方法是使用系间基因转移。因此，我们将确定海胆胚胎中控制关键转录因子在内胚层(GATA因子、一些同源结构域因子和叉头因子)和中胚层(如蜗牛和非蜗牛)早期表达的海鞘顺式调控系统的表达位点。同样，我们将提供相应的海胆顺式调控报告系统，用于海鞘胚胎的测试。由于转录因子可以直接从海胆提取物中分离出来，这种方法可能提供上游(通常是母体)调控因子在脊索状胚胎中产生早期合子调控基因表达的分子证据。

For many years our research program has been focused on the mechanisms of gene regulation in early sea urchin embryos, and this will continue to be its major thrust. We have developed and successfully exploited an excellent suite of technologies for complete structure/function analysis of embryonic cis-regulatory systems. Among these technologies are: a gene transfer procedure that permits expression on many groups of hundreds of embryos at a time, so that the spatial and temporal behavior of expression vectors bearing mutations or consisting in part of synthetic DNAs can be studied in great variety; a novel methodology for knocking our transcription factor activity under experimental genetic control; a methodology for cloning transcription factors after affinity purification; use of large-scale high density arrayed clone libraries; advanced imaging technology for digital recording of spatial expression of fluorescent receptors; and a complete armamentarium of lineage tracing, cell biological, microdissection and in situ procedures for assay of spatial gene expression. In the next period we intend to extend and deepen our mechanistic understanding of gene regulation in early development, by exploiting these methods to characterize functionally some additional cis-regulatory elements that lie in key positions in the genetic control network of the early embryo. We will complete our analysis of the complex Endo16 cis-regulatory system, identifying specific repressors and activators that are not yet characterized. Some additional differentially expressed, downstream genes isolated by differential screening of high density arrayed libraries will be subjected to similar analysis, to determined their cis-regulatory organization; and to identify positive and negative spatial regulatory interactions & elements that lie at the termini of signal transduction systems. A major effort will be directed to cis-regulatory analysis of the systems controlling early zygotic expression of regulatory genes encoding transcription factors. Among these will be factors already identified as controllers of the terminal genes we have already studied. Our overall objective is to understand the gene regulatory network that is set into operation as control is shifted from the maternal to the zygotic transcription regulatory apparatus in cleavage and blastula stages. We will utilize several new approaches to isolate gene batteries, i.e., sets of downstream genes that are controlled by the same set of transcription factors. Major efforts will be devoted to trying to understand the disposition and complexity of maternal transcription factors, the functions of which are known from cis-regulatory analysis of their target genes. Here we have three specific objectives; first, localization in the early embryo by conventional confocal immunocytology, and assessment of regional activity in respect to DNA target site binding by a microcapillary-based shift procedure that we developed; second, analysis of transcription factor variants by mass spectrometry and other methods; and third, localization of variants in mass-isolated cell fractions representing different territories of the early embryos. We are also planning to apply our gene regulatory approaches to the significance of cell contacts in the blastula wall. The synthesis and turnover rates of beta-catenin will be measured to determine whether control of its availability is maternal or zygotic in the cleavage-blastula-stage embryo, and if zygotic we will investigate its cis-regulatory system. We will also study the mechanistic basis on CyIIIa cytoskeletal actin gene down-regulation in disaggregated ectoderm cells, exploiting our complete understanding of the CyIIIa cis- regulatory system. An additional approach to functional significance in cis- regulatory systems is to use interphyletic gene transfer. Thus we will determine the locus of expression in sea urchin embryos of ascidian cis- regulatory systems that control early expression of key transcription factors in endoderm (GATA factors, some homeodomain factors, and forkhead factors); and in mesoderm (e.g., snail and not). Similarly we will provide the corresponding sea urchin cis-regulatory reporter systems for tests in ascidian embryos. Since transcription factors can be directly isolated from sea urchin extracts, this approach may provide molecular evidence of the upstream (often maternal) regulators that generate early zygotic regulatory gene expression in chordate embryos as well.