Endothelial Cell-Selective Adhesion Molecule (ESAM) Is Required for the Ontogeny of Definitive Hematopoietic System in Mice
Endothelial Cell-Selective Adhesion Molecule (ESAM) Is Required for the Ontogeny of Definitive Hematopoietic System in Mice
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DOI：
10.1182/blood.v128.22.3874.3874
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发表时间：
2016-12
期刊：
影响因子：
20.3
通讯作者：
T. Ueda;T. Yokota;Y. Shingai;Y. Doi;Tomohiko Ishibashi;T. Sudo;Yasuhiro Nagate;Akira Tanimura
中科院分区：
文献类型：
--
作者：
T. Ueda;T. Yokota;Y. Shingai;Y. Doi;Tomohiko Ishibashi;T. Sudo;Yasuhiro Nagate;Akira Tanimura
我们先前报道，内皮细胞选择性黏附分子(ESAM)最初被确定为一种内皮细胞特异性抗原，在小鼠和人类中是一种有效的终生造血干细胞(HSC)标志物(Yokota Health 2009；Ishibashi Exp hematol 2016)。在第一个明确的HSC出现之前，ESAM已经在小鼠胚胎发育中的主动脉的血源性内膜上表达。我们还报道了ESAM在HSC上的表达对成人造血系统具有重要的功能，因为ESAM缺乏会导致5-FU(5-FU)治疗后危及生命的骨髓抑制，尤其是严重的贫血(SUDO J免疫学杂志2012，PLoS One 2016)。对新生的ESAM基因敲除(KO)小鼠进行基因分型的收集数据表明，根据孟德尔比率，纯合子(纯合子)ESAM KO小鼠的数量约为预期数量的一半。然而，ESAM在造血发育中的功能意义尚未确定。因此，在本研究中，我们分析了ESAM缺失对ESAM KO小鼠胚胎造血发育的影响。出乎意料的是，ESAM人KO胎儿的频率和大小在胚胎期(E)14.5天与野生型(WT)或杂合子KO产仔相当。然而，我们发现ESAM HOMO KO胎儿的肝脏含有明显较少的单核细胞。FACS分析显示，ESAM HOMO KO胎肝中所有受试造血细胞群，包括Lineage-Sca1+cKit High(LSK)和LSK CD150+CD48-HSCs、B220+B细胞、Gr1+髓系细胞和Ter119+红系细胞均显著减少。在ESAM HOMO KO胎儿中，红系分化被认为延迟，因为Ter119+成熟红系细胞显着减少，而CD71+Ter119-未成熟细胞显着增加。E14.5ESAM Homo KO小鼠的HSC富集型LSK细胞在MS5共培养中产生的血细胞数量少于其他来源的细胞，尤其是B系细胞，这表明在没有ESAM的情况下，HSC的生长和分化能力受到损害。虽然ESAM缺陷的胎儿在E14.5之前没有明显的器官发育障碍，但我们发现在E14.5之后的3天内发生了危及生命的事件。大约一半的人类KO胎儿在E15.5表现出严重的贫血，并在E17.5之前死亡。对E16.5Esam KO人胎肝进行的实时定量聚合酶链式反应分析显示，成年珠蛋白(主要是α和β)的信使核糖核酸水平显著降低。此外，在E16.5 Esam KO人胎儿的肝脏中，5-氨基乙酰丙酸合成酶2(ALAS2)的红系特异性同工酶(ALAS2)的mRNA水平也被发现降低。ALAS2是血红素生物合成途径中的第一个限速酶。为了更多地了解ESAM KO胎儿造血发育失败的分子机制，我们对E14.5WT和ESAM KO同系小鼠的LSK细胞进行了RNA测序(RNA-seq)。我们发现，虽然胚胎珠蛋白(ζ和Ey)的转录本大量保留，但成体珠蛋白(α，β主要和β次要)的转录本在ESAM-KO HSCs中显著下调。提示ESAM缺乏干扰了珠蛋白由胚胎型向成年型的转换。在Esam KO胎肝的LSK细胞中，ALAS2没有被充分诱导，这可能导致了血红素生物合成的缺陷。在胚胎发育过程中，红系细胞的快速和爆炸性生产对于支持胎儿的生长和存活是必不可少的。为了满足生理需要，最终的红细胞生成发生在发育中的肝脏，并取代原始的红细胞生成。我们的数据表明，ESAM的表达对于最终的红细胞生成的发展是不可或缺的。综上所述，我们揭示了ESAM在终止性造血的发展中起着关键作用。大约一半的Esam KO人胎儿在E15.5到E17.5之间死亡，至少部分原因是在没有ESAM的情况下成人血红蛋白合成延迟。披露：横田：Shionogi&Co.，Ltd.：研究资金。DOI：益力多本沙有限公司：音箱局。Shibayama：Novartis Pharma：荣誉、研究资助、发言人局；Celgene：荣誉、研究资助、发言人局；武田：发言人局；中外制药：发言人局；小野制药：发言人局。Kanakura：中外制药：研究资助；辉瑞：研究资助；Shionogi：研究资助；Kyowa Hakko Kirin：研究资助；Fujimotoseiyaku：研究资助；富山化学：研究资助；百时美施贵宝：研究资助；Alexion制药：研究资助；Nippon Shinyaku：研究资助；Astellas：研究资助；Eisai：研究资助。
We previously reported that endothelial cell-selective adhesion molecule (ESAM), which was initially identified as an endothelial cell-specific antigen, is an effective lifelong hematopoietic stem cell (HSC) marker in mice and humans (Yokota Blood 2009; Ishibashi Exp Hematol 2016). Prior to the advent of the first definitive HSC, ESAM was already expressed on hemogenic endotherium in the developing aorta of murine embryos. We also reported that ESAM expression on HSCs is functionally important for adult hematopoiesis because ESAM deficiency causes life-threatening myelo-suppression, especially severe anemia, after administration of 5-fluorouracil (5-FU) (Sudo J Immunol 2012, PLoS One 2016). Collective data obtained from the genotyping of newborn ESAM knockout (KO) mice suggested that the number of homozygous (homo) ESAM KO mice was about half of that expected as per the Mendelian ratio. The functional significance of ESAM in the development of hematopoiesis, however, has yet to be determined. Thus, in the present study we have analyzed how ESAM deletion affects hematopoietic development in fetuses of ESAM KO mice. Unexpectedly, the frequency and the size of ESAM homo KO fetuses were comparable to those of wildtype (WT) or heterozyqous KO littermates at embryonic day (E) 14.5. However, we found that the liver of ESAM homo KO fetuses contained significantly fewer mononuclear cells. FACS analyses revealed that all the tested hematopoietic cell populations, including lineage - Sca1 + cKit High (LSK) and LSK CD150 + CD48 - HSCs, B220 + B cells, Gr1 + myeloid cells, and Ter119 + erythroid cells, were significantly decreased in the ESAM homo KO fetal liver. Erythroid differentiation was thought to be delayed in ESAM homo KO fetuses because Ter119 + mature erythroid cells significantly decreased whereas CD71 + Ter119 - immature cells significantly increased. HSC-enriched LSK cells from E14.5 ESAM homo KO mice produced fewer numbers of blood cells in MS5 co-culture than those from the others, particularly B-lineage cells, suggesting that the growth and differentiation potential of HSCs is impaired in the absence of ESAM. Although ESAM-deficient fetuses grew without an apparent malfunction in developing organs until E14.5, we found that life-threatening events occurred in 3 days following E14.5. Approximately half of homo KO fetuses exhibited severe anemia at E15.5 and died before E17.5. Quantitative real-time PCR analyses from E16.5 ESAM KO homo fetal livers revealed a significant reduction in messenger RNA (mRNA) levels for adult globins (α and β major). In addition, the mRNA level for an erythroid-specific isoenzyme of 5-aminolevulinic acid synthase 2 (ALAS2), the first and rate-limiting enzyme in the heme biosynthesis pathway, was also found to be reduced in the liver of E16.5 ESAM KO homo fetuses. To learn more about molecular mechanisms involved in the developmental failure of hematopoiesis in ESAM KO fetuses, we performed RNA sequencing (RNA-seq) of LSK cells sorted from E14.5 WT and ESAM KO homo mice. We found that, while transcripts for embryonic globins (ζ and Ey) remained substantially, those for adult globins (α, β major, and β minor) were markedly down-regulated in ESAM-KO HSCs. The results suggested that ESAM deficiency disturbs the globin switch from embryonic to adult type. ALAS2 was insufficiently induced in LSK cells of ESAM KO fetal livers, which presumably results in defects in heme biosynthesis. During the embryonic development, rapid and explosive production of erythroid cells is imperative to support the growth and survival of fetuses. To meet the physiological requirement, definitive erythropoiesis occurs in the developing liver and replaces primitive erythropoiesis. Our data suggest that ESAM expression is indispensable for the development of definitive erythropoiesis. In conclusion, we have revealed that ESAM plays a critical role in the development of definitive hematopoiesis. Approximately half of ESAM KO homo fetuses died between E15.5 and E17.5, at least partly due to the delay of adult hemoglobin synthesis in the absence of ESAM. Disclosures Yokota: SHIONOGI & CO., LTD.: Research Funding. Doi: Yakult Honsha Co.,Ltd.: Speakers Bureau. Shibayama: Novartis Pharma: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding, Speakers Bureau; Takeda: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Ono Pharmaceutical: Speakers Bureau. Kanakura: Chugai Pharmaceutical: Research Funding; Pfizer: Research Funding; Shionogi: Research Funding; Kyowa Hakko Kirin: Research Funding; Fujimotoseiyaku: Research Funding; Toyama Chemical: Research Funding; Bristol Myers: Research Funding; Alexionpharma: Research Funding; Nippon Shinyaku: Research Funding; Astellas: Research Funding; Eisai: Research Funding.