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

描述(申请人提供)：脊椎动物中胚层向肌肉、软骨、骨、脊索、肾脏、性腺、血液和其他组织的转变是胚胎发育过程中形态发生和细胞分化的经典例子。在过去的二十年里，突变筛选和定位克隆方法揭示了控制这一过程的关键发育基因，包括形态原、细胞受体及其下游转录因子。特别是，对斑马鱼发育的研究表明，几种T-box(Tbx)转录因子协同作用形成中胚层谱系，包括无尾(Ntl)、spaDetail(Spt)和tbx6。缺乏NTL功能的胚胎不能发育脊索和后中胚层，而SPT突变体在主干中胚层中表现出严重的缺陷。虽然还没有产生一个tbx6突变体，但tbx6的表达动态和过表达表型表明，这个T-box基因在中胚层模式形成中也起着重要的作用。基于这些观察结果，我们推测NTL、SPT和TBX6共同作用于中胚层的形态发生和分化。虽然这些转录因子显然调节中胚层的发育，但它们在空间和时间上如何作用于这种转化仍不清楚。在相应的斑马鱼突变体中，NTL和/或SPT功能的结构性和全局性丢失掩盖了这一过程的时空复杂性。此外，几乎没有发现T-box基因的转录靶点或下游效应因子。弥合我们知识中的这些差距将需要有能力以时空精度控制NTL、SPT和TBX6功能，申请人已经开发出一种新的化学技术，将使这些遗传操作成为可能。这种方法包括笼式合成试剂用于光控制的基因沉默，并建立在发育生物学社区广泛使用反义吗啉进行靶向基因敲除的基础上。针对NTL基因的笼养吗啡的初步研究表明，NTL基因对形态发生运动、脊索命运选择和脊索成熟是必需的。还建立了一种基于笼式吗啉的转录因子靶标发现策略。申请人现在建议应用这些技术来阐明NTL、SPT和TBX6在斑马鱼中胚层发育中的作用，重点关注它们活动的时空方面及其转录靶标。这三种转录因子将在不同的胚胎组织中单独和组合沉默，由此对细胞运动和命运选择的影响将被确定。这些T-box因子的直接靶基因和下游效应因子也将通过结合笼养吗啉、荧光激活细胞分选和微阵列分析以组织特异性的方式识别。使用这种跨学科的方法，申请人将破译通过传统遗传方法难以确定的中胚层模式形成机制。 公共卫生相关性：在胎儿发育过程中，组织构型和器官发生需要对细胞增殖、分化和运动进行精确的时空控制。这项拟议的研究使用斑马鱼作为模式生物，并使用一种名为笼养吗啉的新化学技术来研究调节这一过程的分子机制。这些研究将揭示T-box转录因子NOTail、SpaDetail和tbx6如何在胚胎发育过程中在空间和时间上作用于创建不同的中胚层组织。

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.