Genetic Regulation of Progenitor Cells in Appendicular Skeletal Development

描述(由申请人提供)：附肢骨骼系统代表了具有独特形状和位置的骨骼元素的发展范例。此外，该系统还提供了一个研究同一位置的骨/软骨如何在前肢和后肢两种类型的肢体中发育成不同形状的平台。阐明这些骨骼元素是如何由祖细胞产生的，对于理解附件骨骼系统的发育至关重要。在胚胎发育过程中，“后生长区”，即位于身体后部的结构，生长并向后延伸。这个过程被称为身体延伸，逐渐产生各种组织的祖细胞，包括侧板中胚层(LPM)。LPM中的细胞产生肢芽间充质，该间充质扩张并经历区域特有的生长和分化，导致骨骼元素从最近端(顶足类)到中部(斑足类)和远端(自足类)的渐进性形态发生。我们获得的初步数据支持这样一种观点，即早于肢芽形成的遗传系统调节着附件骨骼随后发育的祖细胞的生成和扩张。在肢体生长之前，编码含锌指蛋白的Sall4有条件地失活，导致仅在后肢近侧-前部骨骼元素的丢失，如股骨、胫骨和前指。我们还获得了初步的数据，表明在后肢形成区表达Isl1的细胞，编码LIM同源结构域蛋白，有助于后肢后-远端骨骼元素的形成，如腓骨和后趾。这些结果表明，Isl1和Sall4的遗传系统在LPM中工作，并调节后肢祖细胞发育成特定的骨骼元素。此外，我们的初步数据表明，在早期的上游事件中，Sall4及其同源物SALL1的组合功能调节身体伸展以产生后肢祖细胞。该提案的目标是确定在祖细胞中工作的Sall4、Isl1和SALL1的遗传和分子机制。我们将为这一目标设定三个具体目标。目的1验证Sall4通过调控细胞周期蛋白依赖性激酶抑制因子基因调控后肢祖细胞增殖和存活的假说。目的2验证Sall4和Isl1对ss-catenin的不同调控控制hindlmb祖细胞增殖和/或存活的假设。目的3验证SALL1和Sall4的联合功能通过调节身体伸展来控制LPM后肢祖细胞的形成的假设。该项目的完成将揭示一个新的概念，即遗传系统调节LPM中祖细胞的扩张，以促进附件骨骼系统的后期发育。阐明这种调节附件骨骼发育的新机制将极大地促进我们目前对附件骨骼发生的理解

DESCRIPTION (provided by applicant): The appendicular skeletal system represents a paradigm for development of skeletal elements with unique shapes and locations. Moreover, this system provides a platform to study how bone/cartilage in the same position develops into distinct shapes in two types of limbs, namely the forelimbs and hindlimbs. Elucidating how these skeletal elements are generated from progenitor cells is crucial for understanding the development of the appendicular skeletal system. During embryonic development, "the posterior growth zone", the structure located at the posterior of the body, grows and posteriorly extends the body. This process, called the body extension, progressively generates progenitor cells for various tissues, including the lateral plate mesoderm (LPM). Cells in the LPM give rise to the limb bud mesenchyme, which expand and undergo region-specific growth and differentiation, leading to progressive morphogenesis of skeletal elements from the most proximal (stylopod) to the middle (zeugopod) and distal (autopod) region. We obtained preliminary data supporting the idea that genetic systems earlier than limb bud formation regulate generation and expansion of progenitor cells for subsequent development of the appendicular skeletons. Conditional inactivation of Sall4, which encodes a zinc finger containing protein, prior to limb outgrowth, resulted in the loss of proximal-anterior skeletal elements only in the hindlimb, such as the femur, tibia and anterior digits. We also obtained preliminary data, suggesting that cells expressing Isl1, encoding a LIM homeodomain protein, in the hindlimb-forming region contribute to the formation of posterior-distal skeletal elements in hindlimbs, such as fibula and posterior digits. These results suggest that genetic systems by Isl1 and Sall4 operate in the LPM and regulate hindlimb progenitor cells to develop into specific skeletal elements. Furthermore, our preliminary data suggest that, in an earlier, upstream event, combined function of Sall4 and its homolog, Sall1, regulates body extension to generate hindlimb progenitors. The goal of the proposal is to determine the genetic and molecular mechanisms of Sall4, Isl1 and Sall1 operating in progenitor cells. We will have three specific aims for this goal. Aim 1 will test the hypothesis that Sall4 regulates proliferation and survival of hindlimb progenitors through regulation of the cyclin-dependent kinase inhibitor genes. Aim 2 will test the hypothesis that distinct regulation of ss-catenin by Sall4 and Isl1 controls proliferation and/or survival of hindlmb progenitor cells. Aim 3 will test the hypothesis that the combined function of Sall1 and Sall4 controls the formation of the hindlimb progenitors in the LPM through regulation of body extension. Completion of this project will reveal a novel concept that genetic systems regulate the expansion of progenitors in the LPM for development of the appendicular skeletal system in later stages. Elucidating this novel mechanism that regulates appendicular skeletal development will significantly advance our current understanding of appendicular skeletogenesis