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在1000名活人出生中。 因此，了解肢体发育的机制与生物学和医学有关。肢 开发始于将侧板中胚层的离散区域的规范置于肢体 祖细胞，产生肢体芽。在过去的几十年中，研究领域非常集中 在理解信号中心在肢体芽中的机制调节肢体芽的模式， 导致肢体骨骼的形成。但是，我们对肢体祖细胞的了解有限 在建立肢体之前，指定和哪些机制调节其初始差异 信号传导中心；但是，这些过程对于正确的肢体开发至关重要。过去几十年的研究 表明在建立之前，不同的机制在侧板中胚层和肢体祖细胞上起作用 肢体芽信号中心。例如，我们发现SALL4的删除，编码锌指转录 在肢体发育开始前约两天，因素引起了严重的缺陷 后肢，而后来的删除却没有微妙的效果。在我们的初步研究中，我们发现 SALL4，IRX3和IRX5（IRX3/5）同时失活导致缺乏后肢 后肢祖细胞特异性基因的表达，例如ISL1。该结果表明 SALL4和IRX3/5将侧板中胚层指定为后肢祖细胞。在AIM 1中，我们的目标是阐明 后肢祖细胞规范的分子机制。我们将确定SALL4和IRX3/5是否 通过其增强子进行冗余而直接调节ISL1。我们将确定在下游的基因 SALL4和IRX3/5通过基因组实验指定后肢祖细胞。我们将确定它们的功能 通过遗传敲除方法指定后肢祖细胞。我们还获得了数据，强烈建议 当内源性糖酵解是 从高活动过渡到低活动。最近的研究提供了证据，表明除了提供能源外， 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身体伸长。成纤维细胞 生长因子信号传导是调节肢体祖细胞分化的最早信号传导之一。在AIM 2中，我们将 检验一个有趣的假设，即SALL4依赖性糖酵解调节肢体的分化 祖先。我们将构建野生型和SALL4突变体肢体祖细胞的代谢组 代谢状态和SALL4的变化。我们将通过减少糖酵解来测试糖酵解的作用 SALL4突变胚胎并确定其分化，并增加胚胎中的糖酵解 突变。该建议将生成有关规范和差异化的重要基本信息 肢体祖细胞，是肢体发育的基本初始过程。