Genetic Regulation of Progenitor Cells in Appendicular Skeletal Development

描述（由申请人提供）：无骨骨骼系统代表了具有独特形状和位置的骨骼元件发育的范例。此外，该系统提供了一个平台，以研究如何在同一位置的骨/软骨发展成不同的形状在两种类型的肢体，即前肢和后肢。阐明这些骨骼元素是如何从祖细胞产生的是至关重要的理解的approximular骨骼系统的发展。在胚胎发育期间，“后部生长区”，即位于身体后部的结构，生长并向后延伸身体。这个过程，称为身体延伸，逐步产生祖细胞的各种组织，包括侧板中胚层（LPM）。LPM中的细胞产生肢芽间充质，其扩展并经历区域特异性生长和分化，导致骨骼元件从最近端（茎足）到中间（zeugopod）和远端（autopod）区域的渐进形态发生。我们获得了初步的数据支持的想法，即遗传系统早于肢芽形成调节祖细胞的产生和扩展，为随后的发展approximular骨骼。在肢体长出之前，编码含锌指蛋白质的Sall 4的条件性失活导致仅在后肢中的近端-前部骨骼元件的损失，例如股骨、胫骨和前趾。我们还获得了初步的数据，表明表达Isl 1的细胞，编码LIM同源结构域蛋白，在后肢形成区域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，遗传系统的Isl 1和Sall 4在LPM和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，在一个较早的上游事件，Sall 4和它的同源物，Sall 1的组合功能，调节身体的延伸，产生后肢祖细胞。该提案的目标是确定Sall 4，Isl 1和Sall 1在祖细胞中运作的遗传和分子机制。为此，我们将有三个具体目标。目的1验证Sall 4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将检验Sall 4和Isl 1对β-连环蛋白的不同调节控制hindlmb祖细胞的增殖和/或存活的假设。目的3将检验Sall 1和Sall 4的组合功能通过调节身体伸展来控制LPM中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中祖细胞的扩增，以促进后期无骨系统的发育。阐明这一调节无骨骨骼发育的新机制将大大推进我们目前对无骨骨骼发生的理解

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