Genetic and Transcriptional Control of Spleen Development

描述(申请人提供)：器官发生始于特定于器官的细胞类型的规范、定位和组装成器官原基(原基)。活跃的细胞增殖也会发生，以形成器官形态发生和扩张的临界质量。在这个提案中，我们将使用脊椎动物的脾作为模型来研究这些基本步骤。脾的复杂结构和功能源于不同细胞类型之间的密切相互作用：间充质细胞(“基本实质”)、侵袭内皮细胞和定植造血细胞。在人类中，脾在早期的造血、免疫和血液过滤中起着关键作用，它的缺失(如先天性脾功能不全，一种通常只有在尸检时才能认识到的被低估的疾病)会导致新生儿和儿童发生危及生命的细菌感染的高风险。我们的长期目标是确定控制脾发育的后续阶段的遗传途径：即造血细胞和内皮细胞的形态发生、扩张和流入，因为这些相互关联的器官发生过程对脾功能至关重要，但大多数情况下尚不清楚。使用遗传方法和脾小鼠品系，我们确定了控制早期脾发育的遗传途径中的关键步骤。我们报道了同源盒基因Pbx1是脾细胞命运所必需的，是NKX2.5和Hox11(这两个基因对脾的形成也是必不可少的)的分级共同调节因子。我们还发现，Pbx1在侧板中胚层(LPM)的表达早于NKX2.5和Hox11。此外，我们还发现Pbx1在发育中的脾血管内皮细胞中表达。鉴于这些发现，我们的假设是，LPM内Pbx1阳性的祖细胞亚群是脾实质特化、形态发生和扩张所必需的，内皮细胞中PBX的表达也有助于其在脾形态发生和扩张中的作用。此外，我们假设一个完整的间充质原始细胞和内皮细胞对于正常的脾造血定植和功能都是必不可少的。利用现有的基因靶向和转基因小鼠品系，我们将通过胚胎学、遗传学和分子方法来验证我们的假设。首先，我们将建立控制脾形态发生和扩张的遗传和分子途径。为此，我们将在一个条件失活的小鼠系中描述脾的形态发生和细胞增殖缺陷，该系小鼠的脾间充质中的Pbx1有条件失活，而内皮中没有。我们将进一步利用这些胚胎脾产生的永生化细胞培养来确定PBX在细胞周期调节中的作用。其次，我们将通过描述一个只有内皮细胞因Pbx1基因失活而改变的小鼠品系，来评估完整的内皮细胞是否对脾的形态形成和扩张是必不可少的。此外，通过Pbx1诱导失活，我们将在脾内皮细胞中建立PBX时间需求。第三，我们将分别从遗传学角度剖析间充质和内皮在脾造血定植、发育和功能中的作用。我们的研究将揭示新的遗传和分子网络，这些网络是脾发育的基础，而脾是个体发育中被忽视的器官。鉴于间充质脾细胞、侵袭内皮细胞和造血细胞之间的密切相互作用，这项工作产生的新知识将对脾功能的理解产生深远的影响。最后，我们的研究旨在为先天性脾功能不全的发病机制提供更好的理解，为先天性脾功能不全的产前分子诊断提供必要的基本遗传学背景。

DESCRIPTION (provided by applicant): Organogenesis begins with the specification, positioning and assembly of the cell types specific to an organ into the organ primordium (anlage). Active cell proliferation also takes place to build a critical mass for organ morphogenesis and expansion to occur. In this proposal, we will use the vertebrate spleen as a model to investigate these fundamental steps. The complex architecture and functions of the spleen result from intimate interactions among different cell types: mesenchymal cells ("basic parenchyma"), invading endothelial cells and colonizing hematopoietic cells. In humans, the spleen has critical roles in early hematopoiesis, immunity and blood filtering and its absence (as in congenital asplenia, an under-diagnosed disorder often recognized only at autopsy) results in a high risk for life-threatening bacterial infections in newborns and children. Our long-term objective is to identify genetic pathways that control the successive stages of spleen development: i.e. morphogenesis, expansion, and influx of hematopoietic and endothelial cells, since these interrelated organogenetic processes are of utmost importance to spleen function and yet mostly unknown. Using genetic approaches and asplenic mouse strains, we defined key steps in the genetic pathways that govern early spleen development. We reported that the homeobox gene Pbx1 is required for spleen cell fates and is a hierarchical co-regulator of Nkx2.5 and Hox11 (which are also essential for spleen formation). We also found that Pbx1 expression commences earlier than that of both Nkx2.5 and Hox11 in the Lateral Plate Mesoderm (LPM). Additionally, we uncovered that Pbx1 is expressed in the endothelium of the developing spleen anlage. In view of these findings, our hypothesis is that a distinct sub-population of Pbx1-positive progenitor cells within the LPM is required for spleen parenchyma specification, morphogenesis, and expansion and that Pbx expression in the endothelium also contributes to its function in spleen morphogenesis and expansion. In addition, we hypothesize that both an intact mesenchymal anlage and endothelium are essential for normal spleen hematopoietic colonization and function. Using available lines of gene-targeted and transgenic mice, we will test our hypothesis through embryologic, genetic, and molecular approaches. First, we will establish genetic and molecular pathways that control spleen morphogenesis and expansion. To this end, we will characterize the spleen morphogenesis and cellular proliferation defects in a mouse line with conditional inactivation of Pbx1 in the spleen mesenchymal parenchyma, but not in the endothelium. We will further utilize immortalized cell cultures generated from these embryonic spleens to determine the roles of Pbx in cell cycle regulation. Second, we will assess whether an intact endothelium is essential for spleen morphogenesis and expansion by characterizing a mouse line in which only the endothelium is altered by genetic inactivation of Pbx1. Also, by Pbx1 inducible inactivation, we will establish Pbx temporal requirements in the spleen endothelium. Third, we will genetically dissect the role of the mesenchyme and endothelium, respectively, in spleen hematopoietic colonization, development, and function. Our studies will shed light on novel genetic and molecular networks that underlie the development of the spleen, a neglected organ in regard to its ontogeny. In light of the intimate interactions among the mesenchymal spleen anlage, invading endothelial cells and hematopoietic cells, the new knowledge generated from this work will have a deep impact on the understanding of spleen function. Lastly, our studies aspire to provide a better comprehension of the pathogenesis of congenital asplenia, as we put forth the prerequisite basic genetic background towards prenatal molecular diagnosis of this condition.