Genetic regulation of progenitor cells in appendicular skeletal development

项目总结 先天性肢体畸形是由肢体发育异常引起的，每1000名活产儿中就有一名发生。 因此，了解肢体发育的机制具有重要的生物学和医学意义。四肢 发育始于将侧板中胚层的离散区域指定为肢体 祖细胞，这会产生肢芽。在过去的几十年里，这一研究领域备受关注。 在了解肢芽中的信号中心调节肢芽模式的机制后， 导致肢体骨骼的形成。然而，我们对肢体祖细胞的了解有限 在肢芽建立之前，它们的初始分化是由什么机制调节的？ 信号中枢；然而，这些过程对于正确的肢体发育是必不可少的。近几十年来的研究 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的缺失 在肢体发育开始前大约两天的因素，导致严重的缺陷，特别是在 后肢的缺失，而后期的缺失没有或微妙的影响。在我们的初步研究中，我们发现 同时失活的Sall4、IRX3和IRX5(IRX3/5)导致后肢缺失 后肢祖细胞特异性基因的表达，如Isl1。这一结果表明，这一组合功能 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 后肢祖细胞的分子机制规范。我们将确定SALL4和IRX3/5是否 通过其增强子对IsL1进行冗余的直接调控。我们将确定在下游起作用的基因 SALL4和IRX3/5通过基因组实验确定后肢祖细胞。我们将在中确定它们的功能 通过基因敲除方法确定后肢祖先。我们还获得了数据，强烈表明 Sall4基因敲除导致肢体祖细胞糖酵解增加，而内源性糖酵解是 从高活跃度到低活跃度。最近的研究提供了证据，除了提供能量外， 糖酵解介导成纤维细胞生长因子在尾芽中的信号传递，并调节身体的伸长。成纤维细胞 生长因子信号是调控肢体祖细胞分化的最早信号之一。在目标2中，我们将 检验一个耐人寻味的假设，即Sall4依赖的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突变胚胎，并确定它们的分化，以及增加胚胎的糖酵解。 突变。这项建议将产生关于规范和区分的重要基本信息 肢体祖细胞，这是肢体发育的基本初始过程。

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.