Lymphopoietic development in the extra-embryonic yolk sac

描述（由申请人提供）：目前工作的总体目标是确定允许胚外卵黄囊和主动脉帕拉内脏胸膜（P-Sp）中出现B淋巴细胞的机制，并促进这些细胞归巢到腹膜腔中。尽管胚外卵黄囊是原始红细胞、巨核细胞和巨噬细胞产生的第一个部位，但它是否是B淋巴样细胞出现的部位仍有争议。一个胚胎内位点，即来源于P-Sp的性腺-中肾（AGM）区域，目前被认为是造血干细胞产生的第一个位点，该造血干细胞能够重新填充致死性照射的成年小鼠的造血区室，并且据报道具有在体外与某种基质细胞系共培养时产生B淋巴样细胞的潜力。然而，在体循环开始后，B细胞祖细胞是否从卵黄囊进入P-Sp或反之亦然仍然不清楚和未经证实。事实上，当试图确定发育中的小鼠胚胎中B淋巴细胞生成的开始时，遇到的最难克服的障碍是心脏收缩的开始和循环的建立。我们已经开发了一种新的方法，允许确定造血细胞出现在卵黄囊和P-Sp的确切位置和时间。Ncx 1编码一种钠钙交换蛋白，该蛋白仅在胚胎（E）11.0天的心肌细胞中表达。缺乏Ncx 1的胚胎无法启动心脏收缩，从而阻止体循环的发生。缺乏血流最初是耐受的，没有发育不良的证据，然而胚胎在E10.0时表现出生长迟缓，并在E11.0时死亡。使用Ncx 1突变和野生型（WT）小鼠，我们最近报道，所有造血永久祖细胞种子的E 10.0胎肝是卵黄囊来源。我们还报道了在野生型和Ncx 1缺失小鼠的E 9.5卵黄囊和P-Sp中存在形成B淋巴细胞的潜力;证实了B淋巴细胞在卵黄囊和P-Sp中自主产生。现在出现了一系列问题，我们在以下目标中解决：目标1：描述卵黄囊和P-Sp中B淋巴细胞的时空出现，并从这些部位的出现细胞中定义B细胞向B1和/或B2淋巴谱系的特化。目的2：明确卵黄囊和P-Sp B淋巴细胞生成所需的生长因子信号通路。目的3：明确卵黄囊和P-Sp来源的B细胞归巢至腹腔的分子途径。这些研究将确定B淋巴细胞出现的机制，确定B细胞特化的差异是否是内在的和受限制的，并可能澄清分泌天然抗体的B1淋巴细胞的起源; B1淋巴细胞是在人类自身免疫性疾病和白血病发展中重要的细胞群体。公共卫生相关性：本项目旨在确定允许B淋巴细胞在小鼠卵黄囊和胚胎内出现所需的因素，并确定卵黄囊和胚胎内B淋巴细胞向腹膜腔归巢所需的分子。

DESCRIPTION (provided by applicant): The overall goal of the present work is to determine the mechanisms that permit the emergence of B lymphoid cells in the extraembryonic yolk sac and the para aortic splanchnopleura (P-Sp) and facilitate the homing of these cells into the peritoneal cavity. Though the extraembryonic yolk sac is the first site of primitive red blood cell, megakaryocyte, and macrophage production, it is controversial whether it is a site of B lymphoid emergence. An intraembryonic site, the aorta-gonad-mesonephros (AGM) region derived from the P-Sp, is currently recognized as the first site of production of HSCs capable of repopulating the hematopoietic compartment of lethally irradiated adult mice and is reported to possess the potential to give rise to B lymphoid cells upon in vitro co-culture with a certain stromal cell line. However, after the systemic circulation has commenced, it remains unclear and unproven whether the B cell progenitors are moving from the yolk sac into the P-Sp or vice versa. Indeed, the most formidable obstacle encountered when attempting to define the onset of B lymphopoiesis in the developing murine embryo, is the onset of cardiac contractions and establishment of the circulation. We have developed a novel approach that permits determination of exactly where and when hematopoietic cells emerge in the yolk sac and P-Sp. Ncx1 encodes a sodium-calcium exchange protein that is expressed only in cardiomyocytes through embryonic day (E) 11.0. Embryos lacking Ncx1 are unable to initiate cardiac contractions preventing the onset of the systemic circulation. Lack of blood flow is initially tolerated without evidence of maldevelopment, however embryos demonstrate growth retardation by E10.0 and die by E11.0. Using Ncx1 mutant and wild type (WT) mice, we have recently reported that all hematopoietic definitive progenitor cells that seed the E 10.0 fetal liver are yolk sac-derived. We have also reported that the potential for forming B lymphoid cells is present in the E 9.5 yolk sac and P-Sp of wild type and Ncx1 null mice; confirming that B lymphoid cells arise autonomously in the yolk sac and P-Sp. A host of questions now emerge that we address in the following aims: Aim 1: To delineate the temporal and spatial emergence of B lymphoid cells in the yolk sac and P-Sp and to define B cell specification to B1 and/or B2 lymphoid lineages from the emergent cells in these sites. Aim 2: To define the growth factor signaling pathways required for yolk sac and P-Sp B lymphopoiesis. Aim 3: Define the molecular pathway for yolk sac and P-Sp derived B cell homing to the peritoneal cavity. These studies will define the mechanism of B lymphocyte emergence, determine whether differences in B cell specification are intrinsic and restricted, and may clarify the origin of the B1 lymphocytes that secrete natural antibodies; a population of cells important in human development of autoimmune diseases and leukemia. PUBLIC HEALTH RELEVANCE: This project aims to determine the factors required to permit the emergence of B lymphoid cells in the murine yolk sac and within the embryo and to define the molecules that are necessary for yolk sac and intraembryonic B lymphoid cells to home to the peritoneal cavity.