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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要由肢体发育异常引起的先天性肢体畸形的发生率为千分之一。因此，了解肢体发育的机制与生物学和医学相关。肢发育始于将侧板中胚层的离散区域指定为肢体祖细胞，产生肢芽。在过去的几十年里，研究领域集中于了解肢芽信号中心调节肢芽模式的机制，从而形成四肢骨骼。然而，我们对肢体祖细胞的了解有限。指定的以及在肢芽建立之前调节其初始分化的机制信号中心；然而，这些过程对于正确的肢体发育至关重要。近几十年的研究研究表明，在建立之前，不同的机制作用于侧板中胚层和肢体祖细胞肢芽信号中心。例如，我们发现编码锌指转录的 Sall4 缺失大约在肢体发育开始前两天，该因素导致了严重的缺陷，特别是在后肢，而后期的删除没有或微妙的影响。在我们的初步研究中，我们发现 Sall4、Irx3 和 Irx5 (Irx3/5) 同时失活导致后肢缺失，并丧失后肢祖细胞特异性基因的表达，例如 Isl1。该结果表明，组合函数 Sall4 和 Irx3/5 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规范的分子机制。我们将确定是否SALL4和IRX3/5 通过其增强子冗余地直接调节 Isl1。我们将确定作用下游的基因 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基因敲除方法指定后肢祖细胞。我们还获得了数据，强烈表明当内源性糖酵解发生时，Sall4 敲除会导致肢体祖细胞糖酵解增加从高活性过渡到低活性。最近的研究提供的证据表明，除了提供能源之外，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。在目标 2 中，我们将测试一个有趣的假设，即 Sall4 依赖性糖酵解抑制调节肢体分化祖先。我们将构建野生型和 Sall4 突变肢体祖细胞的代谢组来了解代谢状态以及 Sall4 缺失引起的变化。我们将通过减少糖酵解来测试糖酵解的作用 Sall4 突变体胚胎并确定其分化，以及在没有突变的情况下增加胚胎中的糖酵解突变。该提案将产生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。