Deciphering T-box gene-dependent mesoderm development with synthetic probes

描述（由申请人提供）：脊椎动物中胚层向肌肉、软骨、骨、脊索、肾、性腺、血液和其它组织的转化是胚胎发育期间形态发生和细胞分化的经典实例。在过去的二十年中，诱变筛选和定位克隆方法已经揭示了控制这一过程的关键发育基因，包括形态发生素、细胞受体及其下游转录因子。特别是，对斑马鱼发育的研究已经证明，几种T-box（Tbx）转录因子协同工作以形成中胚层谱系，包括无尾（ntl）、spadelite（spt）和tbx 6。缺乏ntl功能的胚胎不能发育脊索和后中胚层，而spt突变体在躯干中胚层表现出严重的缺陷。虽然tbx 6突变体尚未产生，tbx 6的表达动力学和过表达表型表明，该T-box基因在中胚层模式中也具有重要作用。基于这些观察，已经假设ntl、spt和tbx 6联合作用以控制中胚层形态发生和分化。 虽然很明显，这些转录因子调节中胚层发育，但它们在空间和时间上如何作用以影响这种转化仍不清楚。在相应的斑马鱼突变体中ntl和/或spt功能的组成性和全局性丧失掩盖了这一过程的时空复杂性。此外，很少有转录靶点或下游效应的T-box基因已被确定。弥合我们知识中的这些差距将需要时空精确地控制ntl、spt和tbx 6功能的能力，并且申请人已经开发了一种新的化学技术，该技术将使这些遗传操作成为可能。该方法涉及用于光控基因沉默的笼状合成试剂，并建立在发育生物学社区广泛使用反义吗啉代进行靶向基因敲除的基础上。针对ntl基因的笼状吗啉代的初步研究已经证明了其对形态发生运动、脊索命运选择和脊索成熟的要求。还建立了一种基于吗啉代的笼式转录因子靶点发现策略。申请人现在提出应用这些技术来阐明ntl、spt和tbx 6在斑马鱼中胚层发育中的作用，重点是它们的活性及其转录靶标的时空方面。这三种转录因子将在不同的胚胎组织中单独和组合沉默，并将确定对细胞运动和命运选择的影响。直接靶基因和这些T盒因子的下游效应子也将通过结合笼状吗啉，荧光激活细胞分选和微阵列分析，以组织特异性的方式进行鉴定。使用这种跨学科的方法，申请人将破译中胚层图案化机制，这将是难以确定通过传统的遗传方法。 公共卫生相关性：在胎儿发育期间，组织形成和器官形成需要精确的时空控制细胞增殖、分化和运动。这项拟议中的研究调查了调节这一过程的分子机制，使用斑马鱼作为模式生物和一种称为笼式吗啉代的新化学技术。这些研究将揭示T-box转录因子no tail、spadetail和tbx 6如何在空间和时间上起作用，以在胚胎发生期间产生不同的中胚层组织。

DESCRIPTION (provided by applicant): The transformation of vertebrate mesoderm into muscle, cartilage, bone, notochord, kidneys, gonads, blood, and other tissues is a classic example of morphogenesis and cellular differentiation during embryonic development. During the past two decades, mutagenesis screens and positional cloning methods have revealed key developmental genes that control this process, including morphogens, cellular receptors, and their downstream transcription factors. In particular, studies of zebrafish development have demonstrated that several T-box (Tbx) transcription factors work in concert to pattern the mesoderm lineage, including no tail (ntl), spadetail (spt), and tbx6. Embryos lacking ntl function fail to develop a notochord and posterior mesoderm, and spt mutants exhibit severe deficits in trunk mesoderm. Although a tbx6 mutant has not yet been generated, tbx6 expression dynamics and overexpression phenotypes suggest that this T-box gene has an important role in mesoderm patterning as well. Based on these observations, it has been hypothesized that ntl, spt, and tbx6 act combinatorially to control the mesoderm morphogenesis and differentiation. While it is evident that these transcription factors regulate mesoderm development, precisely how they act in space and time to effect this transformation remains unclear. The constitutive and global loss of ntl and/or spt function in their corresponding zebrafish mutants masks the spatiotemporal complexity of this process. In addition, few transcription targets or downstream effectors of the T-box genes have been identified. Bridging these gaps in our knowledge will require an ability to control ntl, spt, and tbx6 function with spatiotemporal precision, and the applicant has developed a new chemical technology that will enable these genetic manipulations. This methodology involves caged synthetic reagents for light-controlled gene silencing and builds upon the extensive use of antisense morpholinos for targeted gene knockdowns by the developmental biology community. Preliminary studies with a caged morpholino targeting the ntl gene have demonstrated its requirement for morphogenetic movements, notochord fate choice, and notochord maturation. A caged morpholino-based strategy for transcription factor target discovery has also been established. The applicant now proposes to apply these technologies to elucidate the roles of ntl, spt, and tbx6 in zebrafish mesoderm development, focusing on spatiotemporal aspects of their activities and their transcriptional targets. The three transcription factors will be individually and combinatorially silenced in distinct embryonic tissues, and the resulting effects on cell movements and fate choice will be ascertained. Direct target genes and downstream effectors of these T-box factors will also be identified in a tissue-specific manner by combining caged morpholinos, fluorescence-activated cell sorting, and microarray analyses. Using this interdisciplinary approach, the applicant will decipher mesodermal patterning mechanisms that would be difficult to ascertain through conventional genetic methods. PUBLIC HEALTH RELEVANCE: During fetal development, tissue patterning and organogenesis require precise spatiotemporal control of cell proliferation, differentiation, and movement. The proposed research investigates the molecular mechanisms that regulate this process, using the zebrafish as a model organism and a new chemical technology called caged morpholinos. These studies will reveal how the T-box transcription factors no tail, spadetail, and tbx6 act in space and time to create distinct mesodermal tissues during embryogenesis.