Erythroid Development in the Mammalian Embryo

描述（由申请人提供）：EryP是哺乳动物胚胎中形成的第一种分化细胞类型，在氧气输送和产生正常血管发育所需的剪切力方面起着至关重要的作用。尽管它们具有丰富和不可或缺的功能，但EryP的发育和成熟仍然不明确。当卵黄囊和胚胎脉管系统之间的连接成熟时，卵黄囊的血岛在E7.5开始出现大的有核的EryP，并在E9.5左右开始循环。几天后，确定的红系（EryD）的小细胞开始在胎儿肝脏内分化并进入循环，因此两种谱系不容易区分。在之前的资助期间，我们开发了转基因小鼠系统，可以在整个妊娠期间对EryP及其细胞核进行标记和跟踪。从这项工作中得出的主要发现是，EryP通过以前未被识别的阶段导致其成熟，它们在整个妊娠期间都是一个稳定的群体，不会逐渐消失，并且它们在胎儿肝脏（FL）的红母细胞岛（EBIs）内短暂积累。伴随着EryP向FL的迁移，粘附分子的表达急剧增加，结合胎儿肝巨噬细胞（FLMs）的能力也显著增强。EryP与flm结合的能力受到发育调控，在胎儿肝脏内发现flm的时间窗口内达到最大，部分依赖于VCAM-1。在血液中检测到第一次去核的EryP时，在胎儿肝脏内发现大量挤压的EryP核。在共培养后的flm和体内的天然胎肝中，可以识别出EryP核，这表明它们被巨噬细胞清除和降解。去核后，循环中的EryP粘附巨噬细胞的能力丧失，巨噬细胞在FL中的数量下降。我们假设胎儿肝脏是原始红母细胞成熟的发育生态位，而EryP成熟的最终步骤，包括去核，发生在胎儿肝脏的ebi中，并涉及与巨噬细胞的粘附相互作用。胎儿肝脏正在发育，EryP开始循环，约为E9.5。因此，我们的观察结果提出了一个简单的解决方案，以解释为什么在出现后几天才检测到EryP的去核：终末成熟，包括核挤压，发生在胎儿肝脏，直到妊娠中期才形成。我们在之前的资助期间开发的工具将使我们能够以以前不可能的分辨率研究原始红细胞生成的生物学。我们建议(1)确定巨噬细胞是否为胎儿肝脏内的EryP成熟提供了微环境；(2)评价整合素及其受体在原始红母细胞成熟中的作用；(3)研究红体成熟最后阶段的分子事件。公共卫生相关性：祖细胞群的特征和胚胎与成年红细胞发育的共同特征以及区别特征的阐明，将是用于患者治疗目的的人类胚胎干细胞、造血干细胞或造血祖细胞的定向分化和用于输血的纯红细胞群的有效生产的先决条件。参与胚胎红系发育的途径可能在白血病和骨髓增生异常疾病中失调。因此，拟议的研究应具有广泛的生物医学意义。

DESCRIPTION (provided by applicant): EryP are the first differentiated cell type to form in the mammalian embryo and play a vital role in oxygen delivery and in generating shear forces necessary for normal vascular development. Despite their abundance and indispensable functions, the development and maturation of EryP remain poorly defined. Large, nucleated EryP arise within the blood islands of the yolk sac beginning ~E7.5 and begin to circulate around E9.5, when connections between the yolk sac and embryonic vasculature mature. Several days later, small cells of the definitive erythroid lineage (EryD) begin to differentiate within the fetal liver and enter the circulation, so that the two lineages are not easily distinguished. During the previous funding period, we developed transgenic mouse systems that allow the tagging and tracking of EryP and their nuclei throughout gestation. Major findings to emerge from this work were that EryP progress through previously unrecognized stages leading to their maturation, that they are a stable population present throughout gestation and do not gradually disappear, and that they accumulate transiently within the erythroblastic islands (EBIs) of the fetal liver (FL). Concomitant with EryP migration into the FL, a dramatic increase in adhesion molecule expression occurs along with significantly increased ability to bind fetal liver macrophages (FLMs). The ability of EryP to bind to FLMs is developmentally regulated, maximal during the window of time when they are found within the fetal liver, and partly dependent on VCAM-1. Large numbers of extruded EryP nuclei are found within the fetal liver at the time the first enucleated EryP are detected in the blood. EryP nuclei can be identified within FLMs after co- culture and in the native fetal liver, in vivo, suggesting that they are cleared and degraded by macrophages. After enucleation, the ability of circulating EryP to adhere to macrophages is lost and their numbers in the FL decline. We hypothesize that the fetal liver is a developmental niche for the maturation of primitive erythroblasts and that terminal steps in EryP maturation, including enucleation, occur in the EBIs of the fetal liver and involve adhesive interactions with macrophages. The fetal liver is just developing as EryP begin to circulate, around E9.5. Our observations therefore suggest a simple solution to the puzzling question of why enucleation of EryP is not detected until days after their appearance: terminal maturation, including nuclear extrusion, occurs in the fetal liver, which does not form until midgestation. The tools we have developed during the previous funding period will allow us to study the biology of primitive erythropoiesis at a resolution not previously possible. We propose to (1) determine whether macrophages provide a microenvironment for EryP maturation within the fetal liver; (2) evaluate the roles of integrins and their receptors in the maturation of primitive erythroblasts; and (3) investigate molecular events underlying the final stages of erythroid maturation. PUBLIC HEALTH RELEVANCE: Characterization of progenitor cell populations and elucidation of the common as well as the distinguishing features of embryonic versus adult erythroid development will be a prerequisite for the directed differentiation of human ES cells, HSCs or hematopoietic progenitors for therapeutic purposes in patients and for the efficient production of pure populations of red blood cells for transfusion. Pathways involved in erythroid development in the embryo may be dysregulated in leukemias and myelodysplastic disorders. The proposed studies should therefore be of broad biomedical significance.