Genetic regulation of progenitor cells in appendicular skeletal development

祖细胞在阑尾骨骼发育中的遗传调控

• 批准号: 10251117
• 负责人: Yasuhiko Kawakami
• 金额: $ 32.86万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10251117
• 关键词: Affect; Apical Ectodermal Ridge; Biochemical; Biology; Birth; ChIP-seq; Characteristics; Congenital Limb Deformities; Data; Defect; Development; Developmental Process; Embryo; Energy Supply; Enhancers; Failure; Fibroblast Growth Factor; Forelimb; Foundations; Funding; Genes; Genetic; Genetic Transcription; Genomics; Glucose; Glycolysis; Goals; Hindlimb; Homeobox Genes; Human; Knock-out; Knowledge; Lateral; Limb Bud; Limb Development; Limb structure; Mediating; Medicine; Mesoderm; Metabolic; Methods; Modeling; Molecular; Morphogenesis; Mutation; Organ; Paraxial Mesoderm; Pattern; Play; Process; Regenerative Medicine; Regulation; Repression; Research; Role; Signal Transduction; Skeletal Development; Skeleton; Somites; Specific qualifier value; Specificity; Tail; Testing; Tissues; To specify; Work; Zinc Fingers; conditional knockout; design; enzyme activity; experimental study; gastrulation; genetic analysis; insight; metabolome; mutant; progenitor; skeletal; skeletal disorder; skeletal stem cell; stem cells; transcription factor; transcriptome sequencing

PROJECT SUMMARY Congenital limb malformations, caused by abnormal limb development, occur in one in 1,000 live human births. Therefore, understanding the mechanisms of limb development is relevant to biology and medicine. Limb development starts with the specification of a discrete region of the lateral plate mesoderm into limb progenitors, which gives rise to the limb bud. In the last several decades, the research field intensely focused on understanding the mechanisms by which signaling centers in limb buds regulate patterning of limb buds, leading to formation of limb skeletons. However, we have limited knowledge about how limb progenitors are specified and what mechanisms regulate their initial differentiation before the establishment of limb bud signaling centers; yet, these processes are essential for correct limb development. Studies in the last decades showed that distinct mechanisms operate on lateral plate mesoderm and limb progenitors prior to establishing limb bud signaling centers. For example, we found that deletion of Sall4, encoding a zinc finger transcription factor, approximately two days before the onset of limb development, resulted in severe defects specifically in hindlimbs, while deletion at later stages had no or subtle effect. In our preliminary studies, we found that simultaneous inactivation of Sall4, Irx3 and Irx5 (Irx3/5) caused the absence of hindlimbs with the loss of expression of hindlimb progenitor-specific genes, such as Isl1. This result indicates that combined function of Sall4 and Irx3/5 specifies lateral plate mesoderm into hindlimb progenitors. In Aim 1, our goal is to elucidate the molecular mechanisms of hindlimb progenitor specification. We will determine whether SALL4 and IRX3/5 redundantly and directly regulate Isl1 through its enhancer. We will determine genes that act downstream of Sall4 and Irx3/5 to specify hindlimb progenitors by genomic experiments. We will determine their functions in specifying hindlimb progenitors by genetic knockout approaches. We also obtained data, strongly suggesting that Sall4 knockout causes increased glycolysis in limb progenitors, when endogenous glycolysis is transitioning from high to low activity. Recent studies provided evidence that, beyond supplying energy, glycolysis mediates fibroblast growth factor signaling in the tail bud and regulates body elongation. Fibroblast growth factor signaling is one of earliest signaling that regulates limb progenitor differentiation. In Aim 2, we will test an intriguing hypothesis that Sall4-dependent repression of glycolysis regulates differentiation of limb progenitors. We will construct metabolomes of wild type and Sall4 mutant limb progenitors to understand metabolic status and the changes by loss of Sall4. We will test the role of glycolysis by reducing glycolysis in Sall4 mutant embryos and determine their differentiation, as well as increasing glycolysis in embryos without mutations. This proposal will generate important basic information on the specification and differentiation of limb progenitors, which are fundamental initial processes of limb development.

项目摘要 由肢体发育异常引起的先天性肢体畸形发生率为千分之一。 因此，了解肢体发育的机制与生物学和医学有关。肢体 发育开始于侧板中胚层的一个离散区域分化为肢 祖细胞，这产生了肢芽。在过去的几十年里，研究领域集中在 在理解肢芽中的信号中心调节肢芽模式的机制时， 导致肢体骨骼的形成。然而，我们对肢体祖细胞是如何 在肢芽形成之前，它们的分化是由什么样的机制调控的 信号中心;然而，这些过程对于正确的肢体发育至关重要。过去几十年的研究 表明，不同的机制运作侧板中胚层和肢体祖细胞建立之前， 肢芽信号中心例如，我们发现缺失编码锌指转录的Sall 4， 因素，大约两天前开始的肢体发育，导致严重的缺陷，特别是在 后肢，而在后期阶段删除没有或微妙的影响。在我们的初步研究中，我们发现， Sall 4、Irx 3和Irx 5（Irx 3/5）的同时失活导致后肢缺失， 表达后肢祖细胞特异性基因，如Isl 1。这一结果表明， Sall 4和Irx 3/5将侧板中胚层指定为后肢祖细胞。在目标1中，我们的目标是阐明 后肢祖细胞特化的分子机制。我们将确定SALL 4和IRX 3/5是否 通过其增强子冗余地直接调节Isl 1。我们将确定作用于下游的基因 Sall 4和Irx 3/5通过基因组实验指定后肢祖细胞。我们将确定它们的功能， 通过基因敲除方法指定后肢祖细胞。我们还获得了数据，有力地表明 Sall 4基因敲除导致肢体祖细胞糖酵解增加，当内源性糖酵解被激活时， 从高活性转变为低活性。最近的研究表明，除了提供能源， 糖酵解介导尾芽中的成纤维细胞生长因子信号传导并调节体伸长。成纤维 生长因子信号传导是调控肢体祖细胞分化的最早信号传导之一。在目标2中，我们将 测试一个有趣的假设，即糖酵解的Sall 4依赖性抑制调节肢体的分化 祖先我们将构建野生型和Sall 4突变体肢体祖细胞的代谢组，以了解 代谢状态和由Sall 4损失引起的变化。我们将通过减少糖酵解来测试糖酵解的作用， Sall 4突变胚胎并确定它们的分化，以及在没有突变的胚胎中增加糖酵解。 突变。该提案将产生关于以下各项的规格和区分的重要基本信息： 肢祖细胞，这是肢发育的基本初始过程。