5月week2 文獻閱讀： Combined Single-Cell Profiling of lncRNAs and Functional Screening Reveals that H19 Is Pivotal for Embryonic Hematopoietic Stem Cell Development

結(jié)合lncRNAs的單細胞分析和功能篩選顯示勘畔，H19對胚胎造血干細胞的發(fā)育至關(guān)重要.

SUMMARY

• The generation of hematopoietic stem cells (HSCs) from embryonic endothelial precursors and preHSCs is precisely regulated by signaling pathways and transcription factors.

  胚胎內(nèi)皮祖細胞和前體細胞造血干細胞的產(chǎn)生受信號通路和轉(zhuǎn)錄因子的調(diào)控北戏。

• Nevertheless, regulatory roles of non-coding RNAs remain unknown.

  然而，非編碼rna的調(diào)控作用仍然未知仗岸。

• Taking advantage of our ability to capture rare pre-HSCs and HSCs in vivo, we generated a single-cell landscape of long non-coding RNAs (lncRNAs) during HSC development.

  利用我們在體內(nèi)捕獲罕見的前造血干細胞和造血干細胞的能力勋乾，我們在HSC發(fā)育過程中生成了長非編碼rna (lncrna)的單細胞景觀徒欣。

• Combining bioinformatics and functional screening, we identified 6 lncRNAs influencing hematopoiesis in vitro.

  結(jié)合生物信息學(xué)和功能篩選，我們在體外鑒定了6種影響造血的lncrna仗哨。

• We further revealed that H19 lncRNA is pivotal for in vivo HSC emergence in aorta-gonads-mesonephros region.

  我們進一步發(fā)現(xiàn)H19 lncRNA在主動脈-性腺激素區(qū)體內(nèi)HSC的產(chǎn)生中起著關(guān)鍵作用澈侠。

• Early H19 lncRNA deficiency blocked endothelial-to-hematopoietic transition,which was independent of the H19-derived miR,miR-675.

  早期H19 lncRNA缺乏癥阻斷了內(nèi)皮細胞向造血細胞的轉(zhuǎn)化劫侧，這與H19來源的miR,miR-675無關(guān)。

• Moreover, H19-deficient pre-HSCs displayed promoter hypermethylation and concomitant downregulation of several master hematopoietic transcription factors, including Runx1 and Spi1.

  此外哨啃，h19缺失的前hscs表現(xiàn)為啟動子高甲基化烧栋，并伴有Runx1、Spi1等多種造血主轉(zhuǎn)錄因子的下調(diào)拳球。

• H19 deficiency increased the activity of S-adenosylhomocysteine hydrolase, a regulator of DNA methylation, which partially contributed to the observed hematopoietic defect.

  H19缺乏增加了s -腺苷基同型半胱氨酸水解酶的活性审姓，這是一種DNA甲基化的調(diào)節(jié)因子，這在一定程度上導(dǎo)致了觀察到的造血缺陷祝峻。

• Our findings provide a resource for further analysis of lncRNAs in embryonic HSC development.

  我們的發(fā)現(xiàn)為進一步分析胚胎HSC發(fā)育過程中的lncrna提供了資源魔吐。

INTRODUCTION

• Sitting at the apex of hematopoietic hierarchy, hematopoietic stem cells (HSCs) contribute to all mature blood lineages and continuously replenish lifetime hematopoiesis.

  造血干細胞(HSCs)位于造血系統(tǒng)的頂端，為所有成熟的血液系提供支持莱找，并不斷補充終生造血酬姆。

• In mouse embryos, the long-term adult-repopulating HSCs first appear in the aorta-gonad-mesonephros (AGM) region and also at other locations around embryonic day 11 (E11) (Dzierzak and Speck, 2008;Li et al., 2012;Samokhvalov et al., 2007).

  在小鼠胚胎中，長期成體再生的造血干細胞首先出現(xiàn)在主動脈-性腺-中腎(AGM)區(qū)域奥溺，也出現(xiàn)在胚胎的其他位置(第11天(E11)左右Dzierzak and Speck, 2008;李等轴踱，2012;Samokhvalov等，2007)谚赎。

• As recently recognized, several specialized vascular endothelial cells (ECs) in the embryonic major arteries show the capacity to produce hematopoietic stem and progenitor cells and are thus defined as hemogenic ECs (Boisset et al., 2010;Eilken et al., 2009;Zovein et al., 2008).

  最近發(fā)現(xiàn)，胚胎大動脈中有幾種特殊的血管內(nèi)皮細胞(ECs)具有產(chǎn)生he-造血干細胞和祖細胞的能力诱篷，因此被定義為血源性ECs (Boisset et al.壶唤， 2010;Eilken等，2009;Zovein等棕所，2008)闸盔。

• This dynamic process is termed the endothelial- to-hematopoietic transition.

  這一動態(tài)過程被稱為內(nèi)皮細胞向造血的轉(zhuǎn)變。

• More precisely, pre-HSCs are the key intermediates during stepwise HSC development, which includes two consecutive stages: CD45I- type 1 and CD45+ type 2 precursors (T1 and T2 pre-HSCs) (Rybtsov et al., 2011;Taoudi et al.Zhou et al., 2016).

  更準(zhǔn)確地說琳省，前造血干細胞是HSC逐步發(fā)展過程中的關(guān)鍵中間體迎吵，這包括兩個連續(xù)的階段:CD451型和CD45+ 2型前體(T1和T2 pre-HSCs) (Rybtsov等，2011;, 2008;陶迪等针贬，2008;周等击费，2016)。

• After E11.5, dozens of HSCs by maturation from pre-HSCs in the AGM enter the bloodstream and colonize the fetal liver (Medvinsky et al., 2011).

  E11.5后桦他，AGM中由前HSCs成熟而來的數(shù)十個HSCs進入血流并在胎兒肝臟中定植(Medvinsky etal .蔫巩， 2011)。

• Therefore, within a transient window, the dorsal aorta witnesses de novo the dramatic development of distinct HSC-competent populations, including the specification of hemogenic ECs as well as the formation and maturation of pre-HSCs, suggesting highly orchestrated intrinsic and extrinsic regulation.

  因此，在一個短暫的窗口內(nèi)圆仔，背主動脈見證了不同的造血干細胞群體的驚人的發(fā)展垃瞧，包括血源性ECs的明確表達以及前造血干細胞的形成和成熟，顯示出高度協(xié)調(diào)的內(nèi)在和外在調(diào)控坪郭。

（HSC形成的背景介紹）

• Accumulating evidence has documented various molecular mechanisms underlying this multi-step formation of HSCs in the AGM region.

  越來越多的證據(jù)已經(jīng)證明个从，在AGM區(qū)域這種多步驟HSCs形成的分子機制是多種多樣的。

• As one of the best investigated transcription factors, Runx1, is continuously expressed in the hemogenic ECs, pre-HSCs, and definitive HSCs and plays an indispensable role in the endothelial-to-hematopoietic transition, but not after the HSC fate decision (Chen et al., 2009;Yzaguirre et al., 2017).

  Runx1作為研究最好的轉(zhuǎn)錄因子之一歪沃，在造血干細胞嗦锐、前造血干細胞和最終造血干細胞中不斷表達，在內(nèi)皮細胞向造血細胞的轉(zhuǎn)化過程中發(fā)揮著不可或缺的作用绸罗，但在決定HSC的命運后卻沒有這種作用(Chen et al.意推， 2009;Yzaguirre等，2017)珊蟀。

• As the direct downstream target of Runx1, Gfi1 specif- ically marks hemogenic ECs and can accelerate hematopoietic commitment by silencing the endothelial program (Lancrin et al., 2012;Thambyrajah et al., 2016).

  Gfi1作為Runx1的直接下游靶點菊值，特異性地標(biāo)記了血源性ECs，并可通過沉默內(nèi)皮細胞程序加速造血形成(Lancrin et al.育灸， 2012;Thambyrajah等人腻窒，2016)。

• Likewise, another Runx1 target, Spi1, is upregulated and facilitates the endothelial-to-hematopoietic transition (Wilkinson et al., 2014).

  同樣磅崭，Runx1的另一個靶點Spi1上調(diào)儿子，促進了內(nèi)皮到血液的轉(zhuǎn)化(Wilkinson et al.2014)。

• In addition, hypomethylation of the Runx1 distal promoter is associated with its transcriptional activity and is a signature of definitive hematopoiesis (Webber et al., 2013).

  此外砸喻，Runx1遠端啟動子的低甲基化與其轉(zhuǎn)錄活性有關(guān)柔逼，是最終血液的標(biāo)志(Webber et al.， 2013)割岛。

• Nevertheless, the upstream mechanism modulating the DNA methylation of Runx1 promoters in embryos remains largely unknown.

  然而愉适，調(diào)控胚胎中Runx1 啟動子 DNA甲基化的上游機制仍不清楚。

• Most recently, using a newly defined surface-marker cocktail and concurrent massive single-cell RNA sequencing, the rare embryonic pre- HSCs were successfully purified and the transcriptomes were comprehensively surveyed at single-cell resolution (Zhou et al., 2016).

  最近癣漆，我們使用一種新定義的表面標(biāo)記cocktail维咸，并同時進行大規(guī)模單細胞RNA測序，成功純化了少量的胚胎前造血干細胞惠爽，并在單細胞分辨率下對轉(zhuǎn)錄組進行了全面調(diào)查(Zhou et al.癌蓖， 2016)。

• Relying on these valuable data, the unexpected critical role of mTOR signaling in the emergence of HSCs rather than hematopoietic progenitor cells was demonstrated, suggesting the research strategy enables in-depth characterization of key regulatory mechanisms.

  基于這些有價值的數(shù)據(jù)婚肆，意想不到的mTOR信號關(guān)鍵作用在造血干細胞而非造血祖細胞的出現(xiàn)中被證實了租副，這表明該研究策略能夠深入描述關(guān)鍵的調(diào)控機制。

（當(dāng)前研究和自己研究發(fā)現(xiàn)的對比性：Runx1作為研究最好的轉(zhuǎn)錄因子之一的現(xiàn)狀相光研究较性，mTOR信號關(guān)鍵的發(fā)現(xiàn)附井，單細胞數(shù)據(jù)揭示其調(diào)控機制的重要性讨越。）

• Nevertheless, the physiological function of non-coding genes in mammalian HSC emergence remains elusive.

  然而，非編碼基因的生理功能在哺乳動物中永毅，HSC的出現(xiàn)仍然是難以捉摸的把跨。

• Long non-cod- ing RNAs (lncRNAs), defined as transcripts longer than 200 nt with little protein-coding potential, constitute a large proportion of the transcriptome.

  長鏈非編碼 rna (Long non-coding RNAs, lncRNAs)在轉(zhuǎn)錄組中占很大比例，其定義為長度超過200 nt的轉(zhuǎn)錄本沼死，且?guī)缀鯖]有蛋白編碼潛能着逐。

• lncRNAs play pivotal roles in numerous bio- logical processes (Flynn and Chang, 2014;Kretz et al., 2013;Wang et al., 2014).

  lncrna在許多生物邏輯過程中發(fā)揮著關(guān)鍵作用(Flynn and Chang, 2014;Kretz等，2013;Wang等意蛀，2014)耸别。

• lncRNAs can regulate gene expression tran- scriptionally or post-transcriptionally, executing as signals, decoys, guides, or scaffolds (McHugh et al., 2015;Rinn et al., 2007;Wang and Chang, 2011).

  lncrna可以通過轉(zhuǎn)錄或轉(zhuǎn)錄后調(diào)控基因表達，作為信號县钥、誘導(dǎo)秀姐、指南或支架執(zhí)行(McHugh et al.， 2015;里恩等若贮，2007;Wang and Chang, 2011)省有。

• Previously, a deep-sequencing and comprehensive analysis study identified more than 150 un- annotated lncRNAs specifically enriched in adult HSCs.

  此前，一項深度測序和綜合分析研究發(fā)現(xiàn)谴麦，超過150種未加注釋的lncrna在成體造血干細胞中特異性富集蠢沿。

• Among them, two lncRNAs are functionally required in HSC self-renewal and differentiation (Luo et al., 2015).

  其中兩個lncrna在HSC自我更新和分化過程中具有功能上的需要(Luo et al.， 2015)匾效。

• However, the landscape and function of lncRNAs in the formation of embryonic HSCs are still unknown.

  然而舷蟀，lncrna在胚胎干細胞形成過程中的景觀和功能仍不清楚。

（IncRNA在哺乳動物種的重要重用面哼，IncRNA 在胚胎干細胞形成過程中的功能的模糊性）

• Here, using the robust and comprehensive single-cell transcriptome data of five continuous developmental HSC-related populations and adult HSCs (Zhou et al., 2016), we identified 約7,000 lncRNA genes and constructed the global lncRNA expression landscape during entire HSC ontogeny.

  在此野宜，我們利用5個持續(xù)發(fā)育的HSC相關(guān)人群和成年HSCs的健壯而全面的單細胞轉(zhuǎn)錄數(shù)據(jù)(Zhou et al.， 2016)魔策，鑒定了價值7000個的lncRNA基因匈子，并構(gòu)建了整個HSC個體發(fā)育過程中的全lncRNA表達景觀。

• Using bioinformatics and functional analyses, we screened for lncRNA candidates affecting embryonic hematopoiesis and further demon- strated the crucial role of H19 in the formation of HSCs in AGM region.

  通過生物信息學(xué)和功能分析代乃，我們篩選出了影響胚胎造血的lncRNA候選標(biāo)記 ，并進一步闡明了H19在AGM區(qū)造血干細胞形成中的重要作用仿粹。

• Ourfindings also provide a resource for the future elucidation of other lncRNAs pivotal for embryonic HSC development.

  我們的發(fā)現(xiàn)也為將來闡明其他對胚胎HSC發(fā)育至關(guān)重要的lncrna提供了資源搁吓。

（本文章研究的主要成果：影響胚胎造血的lncRNA候選標(biāo)記，H19在AGM區(qū)造血干細胞形成中的重要作用）

RESULTS

Global lncRNA Expression Dynamics during HSC Development

HSC發(fā)育過程中l(wèi)ncRNA的表達動態(tài)

• We have recently reported the single-cell transcriptome analysis of six cell populations related to HSC ontogeny, including ECs and two types of pre-HSCs from the E11 AGM region, HSCs from E12 and E14 fetal liver, and HSCs from adult bone marrow (Zhou et al., 2016).

  我們最近報道了6個與HSC個體發(fā)生相關(guān)的細胞群的單細胞轉(zhuǎn)錄組分析吭历，包括來自E11 AGM區(qū)域的ECs和兩種類型的前HSCs堕仔，來自E12和E14胎兒肝臟的HSCs，以及來自成人骨髓的HSCs (Zhou etal 晌区， 2016)摩骨。

• Here, to elucidate the physiological role of lncRNAs during HSC formation, we carried out systematic bioinformatics analyses and functional evaluations of candidate lncRNAs.

  為了闡明lncrna在HSC形成過程中的生理作用通贞，我們對候選lncrna進行了系統(tǒng)的生物信息學(xué)分析和功能評價。

• In total, 128 single-cell lncRNA profiles involving the above six distinct cell types were obtained from the ployA+ transcriptome dataset, in which non polyadenylated lncRNAs would not be captured (Table S1)(Zhou et al., 2016).

  從ployA+轉(zhuǎn)錄數(shù)據(jù)集中共獲得128個單細胞lncRNA譜恼五，涉及上述六種不同的細胞類型昌罩，其中不捕獲非聚腺苷酸lncRNA(表S1)(Zhou et al.， 2016)灾馒。

• By using unique whole-genome alignment (mouse genome [mm10]), 7,312 lncRNA genes, including 6,911 annotated and 401 unannotated lncRNAs, were detected as fragments per kilobase of transcript sequence per millions base pairs mapped (FPKM》=1 in at least one sample (Figure 1A;Table S2).

  通過使用獨特的全基因組比對(小鼠基因組[mm10])茎用，在至少一個樣本中檢測到7,312個lncRNA基因，其中包括6,911個注釋lncRNA和401個未注釋lncRNA睬罗，它們是每千位轉(zhuǎn)錄序列中每千位酶的片段(FPKM》=1每個樣本中（圖1A;表S2)轨功。

  
  
  fig1A

• These lncRNA genes were largely devoid of protein-coding potential according to bioinformatics analysis

  根據(jù)生物信息學(xué)分析，這些lncRNA基因在很大程度上缺乏蛋白編碼潛能(圖S1A)容达。

• On average, both the transcript length and the number of exons per transcript of lncRNAs were less than that of protein-coding genes (Figures S1B and S1C).

  平均而言古涧，lncrna的轉(zhuǎn)錄長度和每個轉(zhuǎn)錄的外顯子數(shù)均小于蛋白編碼基因(圖S1B和S1C)。

• The average number of mapped lncRNA genes in each cell was significantly lower than that of mRNA genes, with both of them declined gradually from embryonic ECs to adult HSCs (Figure 1B).

  每個細胞中l(wèi)ncRNA基因圖譜的平均數(shù)量明顯低于mRNA基因花盐，從胚胎ECs到成體HSCs均呈逐漸下降趨勢(圖1B)羡滑。

  
  
  fig1B

• On the other hand, the average number of both mRNA and lncRNA transcripts in each cell increased sharply from ECs to pre-HSCs and then decreased during HSC develop- ment;

  另一方面，每個細胞中mRNA和lncRNA轉(zhuǎn)錄本的平均數(shù)量從ECs到pre-HSCs急劇增加卒暂，然后在HSC發(fā)育過程中下降;

• mRNA transcripts peaked in T1 pre-HSCs, whereas lncRNAs showed the highest expression level in T2 pre-HSCs (Figure 1C).

  mRNA轉(zhuǎn)錄在T1 pre-HSCs中達到高峰啄栓，lncRNAs在T2 pre-HSCs中表達水平最高(圖1C)。

（測序數(shù)據(jù)樣本介紹也祠，A圖和B圖 IncRNA的圖譜介紹）

fig1C

• Interestingly, unannotated lncRNAs showed a higher average expression than annotated lncRNAs and an apparent upregula- tion from EC to T1 pre-HSC stage (Figures S1D and S1E).

  有趣的是昙楚，未加注釋的lncrna的平均表達高于加注釋的lncrna，并且從EC到T1 pre-HSC階段有明顯的上調(diào)(圖S1D和S1E)诈嘿。

• Specifically, two known HSC-specific unannotated lncRNAs, lncHSC-1 and lncHSC-2 (Luo et al., 2015), exhibited different dynamic expression patterns along with HSC development (Figure S1F).

  事實上堪旧，lncHSC-1和lncHSC-2 (Luo et al.， 2015)這兩種已知的HSC特異性無注釋lncrna隨著HSC的發(fā)展呈現(xiàn)出不同的動態(tài)表達模式(圖 S1F)奖亚。

• We found 2,865 (39.2%) of these lncRNAs overlapping with HSC-related lncRNAs recently reported in two studies (Luo et al., 2015;Qian et al., 2016)(Figure S1G).

  我們發(fā)現(xiàn)這些lncrna中有2865個(39.2%)與最近兩項研究中報道的與hsc相關(guān)的lncrna重疊(Luo et al.淳梦， 2015;Qian等，2016)(圖S1G)昔字。

• More precisely, there were 208 (14.4%) bone marrow HSC-expressed lncRNAs, 213 (14.7%) fetal liver-expressed lncRNAs, and 285 (11.6%) pre- HSC-expressed lncRNAs identified in our dataset shared with the bone marrow HSC-expressed lncRNAs reported previously (Luo et al., 2015)(Figure S1H).

  更準(zhǔn)確地說爆袍，我們的數(shù)據(jù)集中發(fā)現(xiàn)了208個(14.4%)骨髓hsc表達的lncrna, 213個(14.7%)胎兒肝臟表達的lncrna, 285個(11.6%)前hsc表達的lncrna與之前報道的骨髓hsc表達的lncrna共享(Luo etal .惩淳， 2015)(圖S1H)稚补。

• Among the 401 unannotated lncRNAs, 314 lncRNAs demonstrated FPKM >=1 in at least two single cells, and 84 out of the remaining 87 lncRNAs were detectable (FPKM >0) in at least two samples (Table S2).

  在401個未加注釋的lncrna中割捅，314個lncrna在至少兩個單細胞中顯示出FPKM>1茴晋，其余87個lncrna中有84個至少在兩個樣本中檢測到(FPKM >0)乘瓤。

• To further validate the authenticity of these unannotated lncRNAs identi- fied here, we selected 40 of them to conduct RT-PCR and Sanger sequencing.

  為了進一步驗證這些未加注釋的lncrna的真實性蝴蜓，我們選擇了其中的40個進行RT-PCR和Sanger測序运授。

• The results demonstrated that 25 out of 40 unannotated lncRNAs can be amplified as expected, and 17 out of 40 amplification products showed consistent sequence with assembled lncRNA, suggesting approximately half of the unannotated lncRNAs were real (Figures S1I and S1J;Table S3).

  結(jié)果表明荧关，40個未加注釋的lncRNA中有25個可以按預(yù)期擴增咏尝，40個擴增產(chǎn)物中有17個與組裝的lncRNA序列一致压语，說明約有一半未加注釋的lncRNA是真實的(圖S1I和S1J;表S3)啸罢。

  (挑選出未注釋的IncRNA,進一步利用RT-PCR和Sanger測序驗證,進一步篩選出真實可靠的IncRNA)

• As lncRNAs can be co-regulated or mutually regulated with their neighboring genes (Engreitz et al., 2016;Guttman et al., 2011), we then determined their genomic distribution.

  lncrna可以與鄰近基因共同調(diào)控或相互調(diào)控(Engreitz et al.， 2016;)然后胎食，我們確定了它們的基因組分布扰才。

• Among 6,911 annotated lncRNA genes, 60.11% were intergenic and 39.89% were intragenic (Figure 1D).

  在6911個注釋lncRNA基因中，60.11%為基因間型斥季，39.89%為基因內(nèi)型(圖1D)训桶。

  
  
  fig1DE

• Pairwise Pearson correlation analysis revealed that the lncRNAs showed a higher expres- sion correlation with the closest genes rather than distal ones (Figure 1E).

  兩兩配對的Pearson相關(guān)分析顯示，lncrna與最近基因的表達相關(guān)性高于與遠端基因的表達相關(guān)性(圖1E)酣倾。

• The co-expression pattern analysis was used to calculate trans correlations (the pairwise with a distance >5 kb) and cis correlations (the pairwise with a distance < 5 kb) be- tween the lncRNA and nearest gene.

  采用共表達模式分析計算lncRNA與最近基因之間的反相關(guān)(距離為> 5kb的成對)和順相關(guān)(距離為< 5kb的成對)舵揭。

• Notably, we found a higher proportion of positive correlations between cis pairs than trans pairs (Figures 1F and 1G).

  值得注意的是，我們發(fā)現(xiàn)順式對之間的正相關(guān)比反式對之間的正相關(guān)比例更高(圖1F和1G)躁锡。

  
  
  fig1FG

• The lncRNAs involved in cis pairs were then classified into six locus biotypes according to their genomic locations relative to the neighboring protein-coding genes,genes, including antisense lncRNA-mRNA pairs in the head-to- head (‘‘XH’’) or tail-to-tail position (‘‘XT’’), antisense lncRNAs located within (‘‘XI’’) or encompassing a protein-coding gene (‘‘XO’’), and sense lncRNAs downstream (‘‘SD’’) or upstream (‘‘SU’’) of protein-coding genes (Figure 1H).

  lncRNAs參與cis對被分為6個位點生物型根據(jù)他們的基因位置相對于鄰近的蛋白編碼基因,基因,包括反義lncRNA-mRNA雙頭-頭(“XH”)或tail-to-tail位置(XT)反義lncRNAs位于(XI)或包含蛋白質(zhì)編碼基因(XO)和lncRNAs下游(SD)或上游(“SU”)的蛋白編碼基因(圖1 h)午绳。
  

  fig1H

)

• For all six biotypes, XH (55.36%) was the dominant one (Figure 1I).

  在所有6個生物型中，XH(55.36%)為優(yōu)勢生物型(圖1I)映之。

  
  
  fig1I

• Consistent with previous work (Luo et al., 2016), the expression of lncRNA- mRNA pairs tended to be positively correlated for sense pairs (SD and SU) (Figures 1J, 1K, and S2;Table S4).

  與之前的工作一致(Luo et al.拦焚， 2016)， lncRNA- mRNA對的表達傾向于與sense pairs(SD和SU)呈正相關(guān)(圖1J, 1K杠输，和S2;表S4)赎败。

(分類分析Inc-RNA 與mRNA相關(guān)性,Inc-RNA--->Intr/Inter---->cis/Trans)

fig1J

fig1K

• We also analyzed the evolutionary state of these lncRNAs using the reported lncRNA repertoires of 11 tetrapod species (Nec- sulea et al., 2014).

  我們還利用已報道的11種四足動物的lncRNA序列分析了這些lncRNA的進化狀態(tài)(Nec- sulea et al.， 2014)蠢甲。

• Among 336 annotated lncRNAs transcribed in at least one species other than mouse, lncRNAs distal from their neighboring genes (>5 kb, trans) are expressed in more species than those that are closer (<5 kb, cis)(Figure 1L;Table S4).

  在336個帶注釋的lncrna轉(zhuǎn)錄中在小鼠以外至少一個物種表達僵刮，遠端與其鄰近基因(> 5kb, trans)的lncrna表達的物種數(shù)多于近端(% 5kb, cis)(圖1L;表S4)。

  (被注釋到的IncRNA遠端模式表達的物種數(shù)多于近端)
  

  fig1L

Distinct HSC-Competent Populations Are More Distinguished by lncRNAs than mRNAs

lncrna比mrna更能區(qū)分不同的hsc的群體

• To explore expression characteristics of lncRNAs during HSC formation, we first identified 849 differentially expressed lncRNAs (Figures 2Aand S3A;Table S5).

  為了探討lncrna在HSC形成過程中的表達特點鹦牛，我們首先鑒定了849個差異表達的lncrna(圖2a和S3A;表S5)搞糕。
  

  fig2A

• Next, principal-compo- nent analysis (PCA) clustered 128 single cells into six distinct populations, in line with the six sample populations from different stages (Figure 2B).

  接下來，主成分分析(PCA)將128個單細胞聚集成6個不同的群體曼追，與來自不同階段的6個樣本群體一致(圖2B)窍仰。

  
  
  fig2B

• Of note, pre-HSCs of different types and fetal liver HSCs of different stages were better distinguished by lncRNAs than by mRNAs, suggesting that lncRNAs exhibited higher stage specificity than mRNAs during HSC development (Figures 2B, 2C, and S3B;Table S5).

  值得注意的是，lncrna對不同類型的pre-HSCs和不同階段的胎兒肝臟HSCs的區(qū)分能力強于mRNAs礼殊，說明lncrna在HSC發(fā)育過程中驹吮，階段特異性高于mRNAs(圖2B, 2C, S3B;表S5)。

  
  
  fig2C

• Furthermore, qPCR was performed for the 186 most differentially expressed lncRNAs in the six distinct sample populations, and the expression pat- terns were generally consistent with that of single-cell RNA sequencing (RNA-seq) data, independently verifying the repeat- ability of the results (Figures 2D and S3C;Table S3).

  此外晶伦，對6個不同樣本群體中差異表達最多的186個lncrna進行qPCR, 表達模式與單細胞RNA測序(RNA-seq)數(shù)據(jù)基本一致碟狞，獨立驗證了結(jié)果的重復(fù)能力(圖2D和S3C;表S3)。
  

  fig2D

(差異IncRNA 表達熱圖,PCA主成分分析和PCR結(jié)果一致)

Function Prediction of lncRNAs by Protein-Coding Genes via Genomic Location and Expression Correlation Analyses

通過基因組定位和表達相關(guān)性分析坝辫，利用蛋白編碼基因預(yù)測lncrna的功能

• To further obtain signature lncRNAs associated with HSC potential, we compared pre-HSCs with closely related populations without pre-HSC potential as previously described (Zhou et al., 2016).

  為了進一步獲得與HSC潛能相關(guān)的簽名lncrna篷就，我們將前HSC干細胞與之前描述的無pre-HSC潛能的群體進行密切相關(guān)比較(Zhou et al.2016)射亏。

• 71 T1 pre-HSC signature lncRNAs were specifically expressed in T1 pre-HSCs (CD31+CD45-CD41lowKit+CD201high), but not in the CD31+CD45-CD41lowKit+CD201low/-population.

  在T1 pre-HSCs (CD31+CD45-CD41lowKit+CD201high)中特異性表達71 T1 pre-HSC簽名lncrna近忙，而在CD31+CD45?CD41lowKit+CD201low/-群體不表達竭业。

• Similarly, 56 T2 pre-HSC signature lncRNAs were highly enriched in the functional T2 pre-HSCs (CD31+CD45+CD41lowCD201+ and CD31+CD45+c-Kit+CD201high), but not in the CD31+CD45+ CD41lowCD201-population (Figure 2E;Table S5).

  同樣，功能T2pre-hscs (CD31+CD45+CD41lowCD201+和CD31+CD45+c-Kit+CD201high)中也富集了56個T2前hsc簽名lncrna及舍，而CD31+CD45+CD41lowCD201中群體中則沒有表達(圖2 e;表S5)未辆。

  
  
  fig2E

• 37 lncRNAs overlapped between T1 and T2 pre-HSCs and were designated as pre-HSC signature lncRNAs.

  在T1和T2 pre-HSCs之間重疊的37個lncrna被指定為pre-HSC特征lncrna。

• Among them, 12 lncRNA genes shared with adult bone marrow HSCs were designated as HSC signature lncRNAs.

  其中锯玛，與成體骨髓間充質(zhì)干細胞共享的12個lncRNA基因被指定為HSC特征lncRNA咐柜。

(通過對比無pre-HSC潛能的群體的群體,找出T1和T2 pre-HSCs之間重疊的37個lncrna,指定其為pre-HSC特征lncrna,與成體骨髓間充質(zhì)干細胞重疊的12個lncRNA基因被指定為HSC特征lncRNA)

• To determine the association between 37 pre-HSC signature lncRNAs and neighboring protein-coding genes, we built a non- coding to coding network using Circos (Figure 2F;Table S5).

  為了確定37個hsc前簽名lncrna與鄰近蛋白編碼基因之間的關(guān)系，我們利用Circos構(gòu)建了一個非編碼到編碼的網(wǎng)絡(luò)(圖2F;表S5)攘残。
  

  fig2F
  
  
  fig2F

• Of note, the neighboring coding genes of 5 signature lncRNAs (Gm20467,Gm16548,Gm13571, 4930538E20Rik, andGm28177) were also previously identified pre-HSC signature mRNA genes, namely Nkx2-3, Selp, Znf512b, Bcl11a,and Stat4, respectively (Zhou et al., 2016).

  值得注意的是拙友，5個特征lncrna (Gm20467,Gm16548,Gm13571, 4930538E20Rik, gm28177)的鄰近編碼基因也已被預(yù)先鑒定為pre-hsc特征mRNA基因，分別為Nkx2-3, Selp, Znf512b, Bcl11a, Stat4 (Zhou et al.歼郭， 2016)遗契。

• In addition, some pre-HSC signature lncRNAs were locatedadjacent to the critical hematopoietic genes, suchas Hoxb5, Meis1, Mecom,and Runx1 (Azcoitia et al., 2005;Chen et al., 2009;2016;Kataoka et al., 2011).

  此外，一些pre-hsc特征 lncrna與關(guān)鍵造血基因相鄰病曾，如Hoxb5牍蜂、Meis1、Mecom和Runx1 (Azcoitia et al.泰涂， 2005;陳等鲫竞，2009;2016;Kataoka等，2011)逼蒙。

• Based on the correlation analysis of genomic location, the 37 pre-HSC signature lncRNAs were mainly enriched in ‘‘regulation of hematopoiesis,’’ ‘‘regula- tion ofmulticellular organismal development,’’ and ‘‘positive reguation of cell communication.

  通過基因組定位的相關(guān)性分析从绘，37個pre-hsc特征lncrna主要富集在“造血調(diào)控”、“多細胞生物發(fā)育調(diào)控”其做、“細胞通訊調(diào)控”等方面顶考。

• ’’ The 12 HSC signature lncRNAs were mainly enriched in ‘‘regulation of gene expression’’ and ‘‘developmental process’’ (Figure S3D;Table S5).

  12個HSC特征lncrna主要富集于“基因表達調(diào)控”和“發(fā)育過程”(圖S3D;表S5)。

• These results suggested that the signature lncRNAs might play a role in HSC specification and function.

  這些結(jié)果表明妖泄，簽名lncrna可能在HSC規(guī)范和功能中發(fā)揮作用驹沿。

  (在特征IncRNA 中根據(jù)其與鄰近蛋白編碼基因的關(guān)系挑選出特征mRNA基因:Nkx2-3, Selp, Znf512b, Bcl11a, Stat4 ,并對37個pre-hsc特征lncrna和12個HSC特征lncrna進行功能富集分析)

• To further obtain the dynamic profiling of lncRNAs during HSC specification, self-organizing maps (SOMs) were used as an intuitional way to spatially visualize and investigate the heterogeneous expression pattern of single-cell transcriptome data.

  為了在HSC分化中進一步獲得lncrna的動態(tài)分析，我們使用自組織映射(SOMs)作為一種直觀的空間可視化方法蹈胡，研究單細胞轉(zhuǎn)錄組數(shù)據(jù)的異質(zhì)表達模式渊季。

• Each cell appeared as a two-dimensional heatmap, in which a set of genes with similar expression patterns were assembled as a unit;the units were clustered and located at a fixed location across all samples.

  每個細胞以二維熱圖的形式出現(xiàn)，其中一組表達模式相似的基因被組裝成一個單元;這些單元被聚集在所有樣本的一個固定位置罚渐。

• 10,241 lncRNA and mRNA genes expressed at FPKM >5 of the whole transcriptome were mapped onto an SOM (Figure S4A).

  10241個lncRNA和mRNA基因表達于整個轉(zhuǎn)錄組的FPKM >5上却汉，并映射到SOM上(圖S4A)。

• 28 hierarchical clusters with significantly dynamic expression were found during the six developmental stages, and the functions of lncRNAs could be predicted through the protein-coding genes in the same cluster (Figures S4B and S4C;Table S5).

  在6個發(fā)育階段共發(fā)現(xiàn)28個具有顯著動態(tài)表達的層次簇荷并，通過同一簇內(nèi)的蛋白編碼基因可以預(yù)測lncrna的功能(圖S4B和S4C;表S5)合砂。

  (在SOM圖中,10241個lncRNA和mRNA基因表達不變的固定在一個位置,有動態(tài)變化的基因可以被觀察到)

Functional Screening of lncRNAs Regulating Hematopoiesis In Vitro

體外調(diào)節(jié)造血的lncrna的功能篩選

• Next, lncRNAs meeting the following three criteria were selected for further functional evaluation: (1) upregulated from the EC to T1 pre-HSC stage, (2) belonging to the pre-HSC signature lncRNAs, and (3) highly conserved in more than three species (Figure 3A).

接下來，選取符合以下三個標(biāo)準(zhǔn)的lncrna進行進一步的功能評價:(1)從EC至T1 pre-HSC階段上調(diào)源织，(2)屬于pre-HSC特征lncrna翩伪，(3)在三個以上物種中高度保守(圖3A)微猖。

fig3A

• A total of 10 candidate lncRNAs were screened out, including AI662270, Gm28875, 4930538E20Rik, Gm28177, RP23-95l4.3, Gm15135, 4933439C10Rik, 1700113A116Rik, Gm17275, and H19 (Figure 3B).

  共篩選出10個候選lncrna，包括AI662270缘屹、Gm28875凛剥、4930538E20Rik、Gm28177轻姿、RP23-95l4.3犁珠、Gm15135、4933439C10Rik互亮、1700113A116Rik犁享、Gm17275、H19(圖3B)豹休。

  
  
  fig3B

• They belonged to seven SOM units.

  它們屬于七個SOM單位饼疙。

• Six of the 10 candidate IncRNAs were classified into SOM cluster 11, showing a similar expression pattern as that of transcription fac- tor Hoxb3 (Figures 3C and S4B).

  10個候選IncRNAs中有6個被歸為SOM cluster 11，表達模式與轉(zhuǎn)錄因子Hoxb3相似(圖3C和S4B)慕爬。

  
  
  fig3C

• Notably, H19 was associated with Runx1 and Gfi1b in cluster 8 (Figures 3C, S4B, and S4C).

  值得注意的是窑眯，H19與集群8中的Runx1和Gfi1b相關(guān)(圖3C、S4B和S4C對體外調(diào)節(jié)造血有影像的Inc-RNA)医窿。

  (通過找出的特征IncRNA,和動態(tài)觀察到的上調(diào)的IncRNA,以及在三個物種以上高度保守的Inc-RNA挑選出10個候選IncRNAs,并說明其在SOM中的分群)

• In order to determine the function of candidate lncRNAs, we generated lentivirus-expressing small hairpin RNAs (shRNAs) targeting each lncRNA to knock down their expression in E11 AGM-derived CD31+ cells, which contained nearly all of the pre-HSCs and hematopoietic stem and progenitor cells (HSPCs) in addition to ECs (Figure 3D).

  為了確定候選lncRNAs的功能,我們生成entivirus-expressingsmall hairpin(shRNAs)靶向每個lncRNA在E11 AGM-derived CD31 +細胞中調(diào)低他們表達,含有幾乎所有的pre-HSCs和造血干細胞和祖細胞(公司)除了ECs(圖3 d)磅甩。

  
  
  fig3D

• The infection efficiency of CD31+ cells was ~80%, and the expression of candidate lncRNAs was reduced by at least 50% (Figures S5A and S5B;Table S3).

  CD31+細胞的感染效率為~80%，候選lncrna的表達降低至少50%(圖S5A和S5B;表S3)姥卢。

• The knockdown of 6 out of 10 lncRNA candidates led to reduced hematopoietic colony formation compared to the vector controls, including H19, AI66270, 4933439C10Rik, Gm15135, Gm17275, and 1700113A16Rik.

  10個lncRNA候選基因中有6個被敲除卷要，包括H19、AI66270独榴、4933439C10Rik僧叉、Gm15135、Gm17275和1700113A16Rik,導(dǎo)致血液菌落形成減少棺榔，

• Among them, the effect of H19 knockdown was the most significant (Figure 3E), which promoted us to focus on the physiological role of H19 lncRNA in HSC development in vivo.

  其中H19敲除作用最為顯著(圖3E)瓶堕，這促使我們關(guān)注H19 lncRNA在體內(nèi)HSC發(fā)育中的生理作用。

  
  
  fig3E

(生成entivirus-expressingsmall hairpin(shRNAs)靶向每個lncRNA在E11 AGM-derived CD31 +細胞中調(diào)低他們表達,比較每個候選Inc-RNA對CFC-U形成的影響,找出H19的影響最大)

Essential Role of H19 in HSC Development in the AGM Region

H19在AGM地區(qū)HSC發(fā)展中的重要作用

(動脈-性腺-中腎(AGM)區(qū)域)

• The expression of H19 is controlled by the upstream differentially methylated region (DMR), an epigenetic regulatory element that directs H19 gene expression (Thorvaldsen et al., 1998;2006).

  H19的表達受上游差異甲基化區(qū)(DMR)控制症歇，DMR是作用H19基因表達的表觀遺傳調(diào)控元件(Thorvaldsen et al.郎笆， 1998;2006)。

• Conditional disruption of the H19-DMR with inducible Mx1-Cre in adult mice shows that H19 is pivotal for maintaining the balance of HSC quiescence and differentiation (Venkatraman et al., 2013).

  誘導(dǎo)Mx1-Cre對成年小鼠H19- dmr的條件破壞表明忘晤，H19在維持HSC沉默和分化的過程中起著關(guān)鍵作用(Venkatraman et al.宛蚓， 2013)。

• Using the same targeting strategy, we deleted the H19-DMR from the embryonic endothelial stage with Tie2-Cre transgenic mice to generate maternal allele-specific mutants (Tie2-Cre;mH19flDMR/+), in which the paternally inherited wildtype allele phenocopied the null allele due to exclusive expresion of the maternal allele.

  采用相同的靶向策略设塔，我們用Tie2-Cre轉(zhuǎn)基因小鼠從胚胎內(nèi)皮階段刪除H19-DMR凄吏，產(chǎn)生母體等位基因特異性突變體(Tie2-Cre;mH19flDMR/+)，其中父系遺傳的野生型等位基因由于母體等位基因的排他表達而抑制了空等位基因。

• No gross phenotype was observed in the E11 (41–43 somite pairs) mutant embryos, and the cellularity and cell viability in different hematopoietic tissues were comparable between mutants and littermate controls.

  E11(41-43對體細胞對)突變體胚胎未見明顯表型痕钢，不同造血組織中細胞的大小和細胞活力在突變體和胎鼠對照中具有可比性表谊。

  (對H19的調(diào)控引用文獻說明其調(diào)控作用,采用動物模型,做突變體,在E11中,分別在卵黃囊(CFU-C)和(動脈-性腺-中腎(AGM)區(qū)域,對比突變體和胎鼠對照中造血組織中細胞的大小和細胞活力)

• Mutant embryos showed a normal colony-forming unit in culture (CFU-C) number in the yolk sacs (Figure 4A).

  突變胚胎在卵黃囊培養(yǎng)(CFU-C)數(shù)目上顯示出正常的集落形成單元(圖4A)。

  
  
  fig4A

• Of note, the CFU-C number was significantly lower in the mutant AGM regions, suggesting that H19-DMR deletion might specifically in- fluence HSPC generation in the AGM region (Figure 4B).

  值得注意的是盖喷，突變的AGM區(qū)域的CFU-C數(shù)明顯較低，這表明H19-DMR的缺失可能在AGM區(qū)域的HSPC產(chǎn)生中具有特異性(圖4B)难咕。

  flDMR(對照組)

• As considerable hematopoietic progenitors in the mid-gestational AGM regions are derived from the yolk sac (McGrath and Palis, 2005), the defect in AGM hematopoiesis might be underesti- mated by the CFU-C assay.

  由于妊娠中期AGM區(qū)域的大量造血祖細胞來自卵黃囊(McGrath和Palis, 2005)课梳， CFU-C檢測可能沒有充分考慮AGM造血的缺陷。

• We further used another strategy to more precisely reflect the hemogenic capacity of in situ ECs in AGM regions.

  我們進一步使用另一種策略來更精確地反映AGM區(qū)域原位ECs的生血能力余佃。

• By conditionally deleting H19-DMR from the embryonic endothelial stage with a VEC-Cre transgene, we then sorted pure ECs (CD31+CD45?CD41?Ter119?) from AGM regions to perform hematopoietic induction by OP9 co-culture in vitro.

  我們利用VEC-Cre轉(zhuǎn)基因有條件地從胚胎內(nèi)皮細胞階段刪除H19-DMR暮刃，然后從AGM區(qū)域中篩選出純ECs (CD31+CD45-CD41-Ter119-)，通過體外OP9共培養(yǎng)進行造血誘導(dǎo)爆土。

• H19 mutant cells generated fewer hematopoietic cells than the controls (Figures 4C and 4D), together with remarkably decreased frequencies of CD19+ B lymphocytes and Gr1/Mac1+ myeloid cells (Figure 4E).

  與對照組相比椭懊，H19突變細胞產(chǎn)生的造血細胞更少(圖4C和4D)， CD19+ B淋巴細胞和Gr1/Mac1+髓細胞的頻率顯著降低(圖4E)步势。

  (找出差異后,進一步利用VEC-Cre轉(zhuǎn)基因有條件地從胚胎內(nèi)皮細胞階段刪除H19-DMR氧猬，然后從AGM區(qū)域中篩選出純ECs (CD31+CD45-CD41-Ter119-)通過體外OP9共培養(yǎng)進行造血誘導(dǎo),發(fā)現(xiàn)H19突變細胞產(chǎn)生的造血細胞更少(圖4C和4D)， CD19+ B淋巴細胞和Gr1/Mac1+髓細胞的頻率顯著降低)
  

  fig4CD

fig4E

• We next determined whether the ontogeny of T1 pre-HSCs was affected by H19-DMR deletion from the endothelial stage.

  接下來我們確定T1前造血干細胞的個體發(fā)育是否受到內(nèi)皮細胞階段H19-DMR缺失的影響坏瘩。

• We sorted CD31+CD45-Kit+ AGM cells for co-culture assay, which contained the functional CD45-T1 pre-HSCs.

  我們分類CD31 + CD45 -試劑盒+ AGM細胞共培養(yǎng)檢測盅抚，其中含有功能CD45-T1 pre-HSCs。

• The cells from mutant embryos generated remarkably fewer hematopoietic progenies than those from control embryos (Figures 4F and 4G).

  來自突變胚胎的細胞產(chǎn)生的造血后代明顯少于來自對照胚胎的細胞(圖4F和4G)倔矾。

  
  
  fig4FG

• Co-culture plus transplantation further demonstrated that no repopulation was detected in the recipients transplanted with the derivatives from mutant embryos (Figures 4H and S5C).

  共培養(yǎng)加移植進一步證明妄均，用突變胚胎的衍生物移植的受體中沒有檢測到再生群體(圖4H和S5C)。

  
  
  fi4H

fig4H

(確定實驗確定T1前造血干細胞的個體發(fā)育也受到內(nèi)皮細胞階段H19-DMR缺失的影響)

• To further examine the generation of mature HSCs, E11 AGM cells were directly transplanted into lethally irradiated adult recipients.

  為了進一步研究成熟HSCs的生成哪自，E11 AGM細胞被直接移植到致死輻照的成年受者體內(nèi)丰包。

• 8 of 9 recipients showed an average of 68.8% chimerism in the peripheral blood at 16 weeks post-transplantation using cells from control embryos.

  9例受者中有8例在使用對照胚胎細胞移植后16周外周血中平均出現(xiàn)68.8%的嵌合

• In sharp contrast, 0 of 8 recipients showed successful reconstitution when transplanted with cells from the H19 mutant embryos (Figures 4Iand S5D).

  與此形成鮮明對比的是，8例受體中有0例在移植H19突變胚胎的細胞后成功重組(圖4Iand S5D)壤巷。

fig4I

• We also analyzed the functional HSCs at later developmental stage.

  并對發(fā)育后期的功能干細胞進行了分析邑彪。

• The immunophenotypically defined HSC population was iso- lated from the E14.5 fetal liver, and a 30-HSC transplantation assay was performed (Benz et al., 2012).

  免疫表型定義的HSC人群是從E14.5胎肝中分離出來的，并進行了30-HSC移植試驗(Benz etal .胧华， 2012)锌蓄。

• Of note, the repopulat- ing capacity was significantly reduced by H19 deficiency, espe- cially at 16 weeks post-transplantation (Figure S5E).

  值得注意的是，H19缺乏癥顯著降低了移植后16周的再生能力撑柔，尤其是在移植后16周(圖S5E)瘸爽。

• Among several possibilities that might underlie the functional defects of fetal liver HSCs in the H19 mutants, the dramatically reduced generation of HSCs in the AGM region should be one of them.

  在可能導(dǎo)致H19突變體胎兒肝干細胞功能缺陷的幾種可能性中，AGM區(qū)肝干細胞的生成顯著減少應(yīng)該是其中之一铅忿。

• To exclude the defects in homing or survival capacities of HSPCs by H19 deficiency, we performed homing assay as reported previously (Zhao et al., 2015).

  為了排除H19缺陷對HSPCs歸巢或生存能力的影響剪决，我們采用了前人報道的歸巢評估(Zhao et al.， 2015)。

• No difference in homing and survival in the bone marrow of recipients was detected between the cells from H19 mutant embryos and littermate controls (Fig- ures S5F and S5G).

  來自H19突變胚胎的細胞和胎鼠對照細胞在歸巢和存活方面沒有差異(圖S5F和S5G)柑潦。

• Other possibilities might also make a contri- bution to the fetal liver HSC defects in the H19 mutants, such as abnormalities in HSC activation and quiescence caused by H19 deficiency as reported in adult HSCs (Venkatraman et al., 2013), which need further study.

  其他的可能性也可能對H19突變體中胎兒肝臟HSC缺陷產(chǎn)生抑制作用享言，如成人HSCs中報道的H19缺陷導(dǎo)致HSC活化異常和靜止(Venkatraman etal .， 2013)渗鬼，這需要進一步研究览露。

• Collectively, these in vivo functional data revealed that H19-DMR deletion dramatically impaired the generation of AGM HSCs, but not yolk sac hematopoietic progenitors.

  總體而言，這些體內(nèi)功能數(shù)據(jù)顯示譬胎，H19-DMR缺失顯著影響AGM HSCs的生成差牛，但不影響卵黃囊造血祖細胞的生成。

  (實驗和文獻結(jié)合論證H19-DMR缺失顯著影響AGM HSCs的生成堰乔，但不影響卵黃囊造血祖細胞的生成偏化。)

H19-DMR Deletion from the Endothelial Stage Disrupts Pre-HSC Development

內(nèi)皮細胞階段H19-DMR的缺失破壞了hsc前期的發(fā)育

• As pre-HSCs are the pivotal intermediates during the endothelial-to-HSC transition, we investigated the changes in molecular programs in T1 pre-HSCs of H19 mutant embryos.

  由于前造血干細胞是內(nèi)內(nèi)皮到 hsc轉(zhuǎn)化過程中的關(guān)鍵中間體，我們研究了H19突變胚T1前造血干細胞分子程序的變化镐侯。

• We sorted 10 and 12 immunophenotypically defined T1 pre-HSCs (CD31+ CD45-CD41lowKit+CD201+/high), which have been verified as highly enriched functional T1 pre-HSCs in wild-type embryos (Zhou et al., 2016), from the E11 AGM region of control and mutant embryos, respectively, and performed single-cell RNA- seq (Figure 5A;Table S1).

  我們分類10和12免疫表型定義T1 pre-HSCs (CD31 + CD45 - CD41lowKit + CD201 + /高),已被證實為高純度功能T1 pre-HSCs野生型胚胎(周et al ., 2016),從E11 AGM區(qū)控制和突變體胚胎,分別進行單細胞RNA - seq(圖5A;表S1)侦讨。

  
  
  fig5A

• 2,782 genes showed significantly dif- ferential expression between the two genotypes, including 1,484 downregulated genes and 1,298 upregulated genes in the mu- tants.

  兩種基因型間有2782個基因表達差異顯著，包括1,484個下調(diào)基因和1,298個上調(diào)基因苟翻。

• As expected, H19 was downregulated, accompanied by many hematopoietic transcription factors, including Gfi1, Nfe2, Runx1, Spi1, Etv6, Erg, and Lyl1.

  正如所料韵卤，H19表達下調(diào)，同時伴有多種造血轉(zhuǎn)錄因子崇猫，包括Gfi1怜俐、Nfe2、Runx1邓尤、Spi1拍鲤、Etv6、Erg汞扎、Lyl1季稳。

• In contrast, endothelial tran- scription factor Sox7 was among the upregulated genes in the cells from the mutant embryos (Figure 5B;Table S6).

  與此相反，內(nèi)皮轉(zhuǎn)運因子Sox7是突變胚胎細胞中上調(diào)的基因之一(圖5B;表S6)澈魄。

  (研究了H19突變胚T1前造血干細胞分子程序的變化,單細胞RNA-seq 分析,對比突變體和非突變體的差異表達基因)

• T-distributed stochastic neighboring embedding analysis using 98 pre-HSC signature genes (Zhou et al., 2016) demonstrated that the T1 pre-HSCs of the H19 mutants were distributed close to wildtype ECs and far away from those of control embryos, which clustered together with wild-type T1 pre-HSCs as expected (Figure 5C).

  利用98個pre-HSC特征基因進行隨機鄰近嵌入分析(Zhou et al.景鼠， 2016)，結(jié)果表明痹扇，H19突變體的T1 pre-HSCs分布在野生型ECs附近铛漓，遠離對照胚胎的T1 pre-HSCs，它與野生型T1 pre-HSCs聚集在一起(圖5C)鲫构。

  
  
  fi5C

• We further performed pseudotime analysis to reconstitute the developmental trajectory of embryonic HSCs by combining the sequencing data of H19 mutant and control T1 pre-HSCs with those of previously reported popula- tions from E11 ECs to E12 fetal liver HSCs (Zhou et al., 2016).

  我們結(jié)合H19突變體和對照T1 pre-HSCs的測序數(shù)據(jù)浓恶，結(jié)合之前報道的E11 ECs到E12胎肝HSCs的人群，進一步進行偽時間分析结笨，重建胚胎HSCs的發(fā)育軌跡(Zhou etal .包晰， 2016)湿镀。

• As expected, T1 pre-HSCs of control embryos were largely indistinguishable from wild-type T1 pre-HSCs.

  正如所料，對照胚胎T1前造血干細胞與野生型T1前造血干細胞在很大程度上難以區(qū)分伐憾。

• In contrast, those of mutant embryos were located near wild-type ECs, which is indicative of their retarded development along the path of endo- thelial-to-hematopoietic transition, which was also supported by the cell proportion analysis along the pseudotemporal axis (Fig- ures 5D and 5E).

  相比之下勉痴，突變胚胎位于野生型ECs附近，這表明它們在endo- thelial-to-hematopoietic transition過程中發(fā)育遲緩树肃，偽時間軸上的細胞比例分析也支持這一觀點(圖5D和5E)蒸矛。

  (單細胞的偽時間分析,發(fā)現(xiàn)H19突變體的T1 pre-HSCs的特點:發(fā)育遲緩)

  
  
  fig5D

fig5E

• In addition, we constructed a transcription factor network enriched in ECs and T1 pre-HSCs, respectively, on the basis of pairwise correlation of transcription factor expression across six stages of HSC development.

  此外，我們還構(gòu)建了一個富含ECs和T1 pre-HSCs的轉(zhuǎn)錄因子網(wǎng)絡(luò)胸嘴，基于轉(zhuǎn)錄因子在HSC發(fā)展的6個階段中表達的兩兩相關(guān)雏掠。

  
  
  fig5F

• Notably, most differentially ex- pressed transcription factors between T1 pre-HSCs from the H19 mutants and their littermate controls were those involved in the T1 pre-HSC network and obviously downregulated in the H19 mutants (Figure 5F).

  值得注意的是，來自H19突變體的T1 pre-HSCs和它們的同窩對照之間差異最大的轉(zhuǎn)錄因子是那些參與T1 pre-HSC網(wǎng)絡(luò)的轉(zhuǎn)錄因子筛谚，并且在H19突變體中明顯下調(diào)(圖5F)。

• These molecular features confirmed that H19 is pivotal for the transition from ECs to pre-HSCs.

  這些分子特征證實了H19是ECs向前hscs轉(zhuǎn)化的關(guān)鍵停忿。

  (富含ECs和T1 pre-HSCs的轉(zhuǎn)錄因子網(wǎng)絡(luò)分析:來自H19突變體的T1 pre-HSCs和它們的同窩對照之間差異最大的轉(zhuǎn)錄因子是那些參與T1 pre-HSC網(wǎng)絡(luò)的轉(zhuǎn)錄因子驾讲，并且在H19突變體中明顯下調(diào))

Elevated S-Adenosylhomocysteine Hydrolase Activity Partially Contributes to the Hematopoietic Defects Caused by H19 Deficienc

-腺苷基同型半胱氨酸水解酶活性升高是H19缺乏所致造血功能缺陷的部分原因

• Because H19 is the precursor for miR-675 (Dey et al., 2014;Ke- niry et al., 2012), we quantified H19 lncRNA and miR-675 in the E11 AGM cells.

  因為H19是miR-675的前體(Dey et al.， 2014;Ke- niry等席赂，2012)吮铭，我們定量了E11 AGM細胞中的H19 lncRNA和miR-675。

• The H19 lncRNA was the predominant product with the expression about 100-fold more abundant than miR-675 (Figure 6A).

  
  
  fig6A

H19 lncRNA是優(yōu)勢產(chǎn)物颅停，其表達量約為miR-675的100倍(圖6A)谓晌。

• H19-DMR deletion caused H19 lncRNA deficiency without influencing Igf2 (sharing the same gene locus with H19) and Igf1r (the major target of miR-675) expression (Figures 5B and S6A).

  H19- dmr缺失導(dǎo)致H19 lncRNA缺失，但不影響Igf2(與H19基因座相同)和Igf1r (miR-675的主要靶基因)表達(圖5B和S6A)癞揉。

• Moreover, the knockdown of H19 lncRNA in either E11 AGM cells or CD31+ cells had no effect on Igf1r expression at protein level (Figure S6B).

  此外纸肉，E11 AGM細胞或CD31+細胞中H19 lncRNA的下調(diào)對Igf1r蛋白表達無影響(圖S6B)。

• These data suggested that H19 lncRNA presumably functioned during embryonic HSC development independent of the miR-675 and Igf2-Igfr1 pathway.

  這些數(shù)據(jù)表明喊熟，H19 lncRNA可能在胚胎HSC發(fā)育過程中獨立于miR-675和Igf2-Igfr1通路發(fā)揮作用柏肪。

• Using either miR-675 inhibitors or mimics, we failed to detect any effect of miR-675 on the formation of hematopoietic progenitors from embryonic AGM CD31+ cells (Figures 6B, 6C, and S6C).

  使用miR-675抑制劑或模擬物，我們未能檢測到miR-675對胚胎AGM CD31+細胞造血祖細胞形成的任何影響(圖6B芥牌、6C和S6C)烦味。

  
  
  fig6B

fig6CD

• No significant change in Igfr1 expres- sion was observed with miR-675 inhibitor treatment (Figure S6C), which might be due to the low endogenous expression level of miR-675 in AGM cells (Figure 6A).

  miR-675 抑制處理后Igfr1表達無明顯變化(圖S6C)，這可能是由于miR-675在AGM細胞內(nèi)源性表達水平較低所致(圖6A)壁拉。

• More importantly, overexpres- sion of miR-675, accompanied by the obvious downregulation of its target, Igf1r, did not rescue the deficiency in hematopoietic progenitor formation by H19 lncRNA knockdown in AGM CD31+ cells (Figures 6B, 6D, and S6D).

  更重要的是谬俄，miR-675的過度表達，伴隨著其靶基因Igf1r的明顯下調(diào)弃理，并沒有挽救AGM CD31+細胞中H19 lncRNA敲低導(dǎo)致造血祖細胞形成的缺陷(圖6B溃论、6D和S6D)。

• Therefore, miR-675 was unlikely playing a role in embryonic hematopoiesis.

  因此痘昌，miR-675不太可能在胚胎造血中發(fā)揮作用蔬芥。

  (H19 lncRNA可能在胚胎HSC發(fā)育過程中獨立于miR-675和Igf2-Igfr1通路發(fā)揮作用)

• We next determined the subcellular localization of H19 lncRNAs, which might provide clues for its regulatory mechanism.

  接下來我們確定了H19 lncrna的亞細胞定位梆靖，這可能為其調(diào)控機制提供線索。

• RNA scope showed that H19 was predominantly localized in the cytoplasm of E11 AGM cells (Figures 6E and S6E).

  RNA顯示H19主要定位于E11 AGM細胞的細胞質(zhì)中(圖6E和S6E)笔诵。

  
  
  fig6EF

• Cyto-plasmicplasmic H19 has been previously shown to bind to and inhibit S-adenosylhomocysteine hydrolase (SAHH), which can hydro- lyze SAH, a strong inhibitor of DNA methyltransferases, thus leading to decreased DNMT3a/b-mediated methylation (Zhou et al., 2015).

  細胞質(zhì)H19與s -腺苷基同型半胱氨酸水解酶(SAHH)結(jié)合并抑制其活性返吻，SAH可水解DNA甲基轉(zhuǎn)移酶的強抑制劑SAH，從而降低DNMT3a/b介導(dǎo)的甲基化(Zhou et al.乎婿， 2015)测僵。

• We first detected the localization of H19 lncRNA and SAHH protein and observed clear co-localization between them in the cytoplasm of E11 AGM cells (Figures 6F and S6E).

  我們首先檢測了H19 lncRNA和SAHH蛋白的定位，并在E11 AGM細胞的細胞質(zhì)中觀察到它們之間明顯的共定位(圖6F和S6E)谢翎。

• Next, using the hydrolysate product homocysteine as a readout, we found that knockdown of H19 lncRNA in AGM cells could in- crease SAHH activity (Figure 6G).

  接下來捍靠，以水解產(chǎn)物同型半胱氨酸為讀數(shù)，我們發(fā)現(xiàn)AGM細胞中H19 lncRNA的下調(diào)可以增加SAHH活性(圖6G)森逮。

  
  
  fig6GHI

• Importantly, suppression of SAHH by knockdown partially rescued the deficiency in hemato- poietic colony formation induced by H19 lncRNA knockdown (Figures 6H, 6I, and S6F).

  重要的是榨婆，通過敲除抑制SAHH，部分挽救了H19 lncRNA敲除引起的造血集落形成缺陷(圖6H褒侧、6I和S6F)良风。

• Taken together, we speculated that the partial role of H19 in embryonic hematopoiesis was to bind to SAHH and inhibit its activity, thus mediating the demethylation of hematopoietic transcription factors.

  綜上所述，我們推測H19在胚胎造血中的部分作用是與SAHH結(jié)合并抑制其活性闷供，從而介導(dǎo)造血轉(zhuǎn)錄因子的去甲基化烟央。

  (實驗論證:H19在胚胎造血中的部分作用是與SAHH結(jié)合并抑制其活性，從而介導(dǎo)造血轉(zhuǎn)錄因子的去甲基化歪脏。)

Increased Promoter Methylation of Several Hematopoietic Transcription Factors by H19 Deficienc

H19缺陷增加了幾種造血轉(zhuǎn)錄因子的啟動子甲基化

• We hypothesized that H19 might regulate the DNA methylation of hematopoietic transcription factors during HSC deveopment.

  我們推測H19可能在造血干細胞發(fā)育過程中調(diào)控造血轉(zhuǎn)錄因子的DNA甲基化疑俭。

• To test this possibility, we performed genome-wide methylation sequencing of T1 pre-HSC-containing populations

  為了驗證這種可能性，我們對T1前含hsc的群體進行了全基因組甲基化測序

• (CD31+CD45?Kit+CD201+/high) from E11 H19 mutant and con- trol embryos, respectively.

  (CD31+CD45-Kit+CD201+/high)分別來自E11 H19突變體和對照胚胎婿失。

• The methylation level in the promoter region compared to other regions was the one most obviously affected by H19 deficiency (Figure 7A;Tables S1 and S7).

  與其他區(qū)域相比钞艇，啟動子區(qū)域的甲基化水平受H19缺乏癥影響最為明顯(圖7A;表S1和S7)。

  
  
  fig7A

• Of note, the genes with increased promoter methylation in the mutants were strongly enriched in hematopoiesis and chromatin modification (Figure 7B).

  值得注意的是豪硅，在突變中啟動子甲基化增加的基因在造血和染色質(zhì)修飾中被強烈富集(圖7B)香璃。

  
  
  fig7B

• Specifically, promoter methylation levels of several pivotal regulators of HSC development, including Runx1 and Spi1, were significantly higher in cells from the mutant embryos than in controls (Figures 7C, 7D,and S7), consistent with the decreased expression of these genes in the immunophenotypically defined T1 pre-HSCs of mutant embryos (Figure 5B).

  具體來說,啟動子甲基化水平的幾個關(guān)鍵HSC發(fā)展的調(diào)控因子,包括Runx1 Spi1,突變體胚胎細胞的顯著高于在控制組(圖7 c、7 d和S7),符合定義的這些基因的表達減少的突變體胚胎的T1 pre-HSCs免疫表型(圖5 b)舟误。

• In contrast, the promoter methyl- ation level of the endothelial transcription factor Sox7 was largely unchanged, suggestive of a divergent regulatory mech- anism (Figure 7C).

  
  
  fig7CD

相比之下葡秒，內(nèi)皮細胞轉(zhuǎn)錄因子Sox7的啟動子甲基化水平基本沒有變化，提示調(diào)控機制存在差異(圖7C)嵌溢。

• Taken together, these results suggested that the promoter hypermethylation of several crucial hemato- poietic transcription factors by H19 deficiency might underlie the decreased expression of these genes and thus lead to the defect in embryonic HSC formation (Figure 7E).

  綜上所述眯牧，H19缺乏可能導(dǎo)致幾個關(guān)鍵的造血轉(zhuǎn)錄因子啟動子高甲基化，從而導(dǎo)致這些基因表達下降赖草，從而導(dǎo)致胚胎HSC形成缺陷(圖7E)学少。

  
  
  fig7E

（推測H19可能在造血干細胞發(fā)育過程中調(diào)控造血轉(zhuǎn)錄因子的DNA甲基化，數(shù)據(jù)分析與實驗驗證秧骑，H19的作用機制）

Discussion

• In the present study, we combined systematic bioinformatics an- alyses of single-cell RNA-seq datasets and endothelial-specific knockout mouse models and revealed a previously unknown lncRNA functionally required for the formation of HSCs in early mouse embryos.

  在本研究中版确，我們將單細胞RNA-seq數(shù)據(jù)集的系統(tǒng)生物信息學(xué)分析和內(nèi)皮特異性敲除小鼠模型相結(jié)合扣囊，揭示了一種以前未知的lncRNA在小鼠早期胚胎中形成造血干細胞的功能需要。

• The H19 gene, which belongs to a highly conserved imprinted gene cluster, encodes a ~2.3 kb lncRNA (Brannan et al., 1990).

  H19基因?qū)儆诟叨缺Ｊ氐挠≯E基因簇绒疗，編碼一個~2.3 kb的lncRNA (Brannan et al.侵歇， 1990)。

• H19 is highly expressed during embryo- genesis and sharply downregulated after birth, except for high levels of expression in a subset of postnatal and adult tissues (Gabory et al., 2009).

  H19在胚胎發(fā)生期間高表達吓蘑，在出生后急劇下調(diào)惕虑，但在出生后和成年組織的一個子集中表達水平較高(Gabory et al.， 2009)磨镶。

• For example, H19 is preferentially expressed in long-term HSCs compared to short-term HSCs or multipotent progenitors in the adult blood system (Venkatraman et al., 2013).

  例如溃蔫，與成人血液系統(tǒng)中的短期造血干細胞或多能祖細胞相比，H19在主要在長期造血干細胞中表達(Venkatraman et al.琳猫， 2013)伟叛。

• Here, we showed that H19 lncRNAs played an important role in HSC formation from embryonic ECs.

  在此，我們證明了H19 lncrna在胚胎ECs形成HSC過程中發(fā)揮了重要作用脐嫂。

• By com- parison, H19 deficiency in adult HSCs, achieved by induced deletion of H19-DMR from adulthood but not from embryonic stage, reduces adult HSC quiescence (Venkatraman et al., 2013), suggesting functional diversity of H19 in regulating embryonic emergence versus adult homeostasis of HSCs.

  通過比較统刮，H19在成體HSCs中的缺失(H19- dmr在成體誘導(dǎo)缺失而非胚胎階段缺失)導(dǎo)致了成體HSC的沉默(Venkatraman et al.， 2013)雹锣，這表明H19在調(diào)節(jié)胚胎出現(xiàn)和成體HSCs穩(wěn)態(tài)方面的功能多樣性网沾。

• Interestingly, H19-DMR deletion had little effect on yolk sac hemato- poietic progenitors.

  H19-DMR的缺失對卵黃囊造血祖細胞影響不大癞蚕。

• Although both are derived from hemogenic ECs and belong to definitive hematopoiesis, the generation of erythroid-myeloid progenitors and HSCs is temporally and spatially distinct (Chen et al., 2011;Frame et al., 2016).

  雖然兩者都起源于血源性ECs蕊爵，屬于最終造血，但紅髓祖細胞和造血干細胞的生成在時間和空間上是不同的(Chen et al.桦山， 2011;Frame等攒射，2016)。

• Accumulating evidence suggests different regulatory mechanisms involved in the generation of these hematopoietic precursors.

  積累的證據(jù)表明恒水，這些造血前體的生成涉及不同的調(diào)節(jié)機制会放。

• For example, despite the absence of HSC formation, a nearly normal number of erythroid-myeloid progenitors are detected in the Notch1-deficient embryos (Hadland et al., 2004;Kumano et al., 2003).

  例如，盡管沒有HSC的形成钉凌，在notch1-缺陷的胚胎中檢測到幾乎正常數(shù)量的紅髓樣祖細胞(Hadland et al.咧最， 2004;Kumano等，2003)御雕。

• The underlying reason for the different effect on yolk sac and AGM hematopoiesis by H19-DMR deletion requires further investigation.

  H19-DMR缺失對卵黃囊和AGM造血作用不同的根本原因還有待進一步研究矢沿。

（當(dāng)前研究，和本研究的研究總結(jié)酸纲，依據(jù)還需研究的方向）

• Here, we proposed that H19 may promote the pre-HSC and HSC specification via demethylation of a series of master hematopoietic transcription factors such as Runx1 and Spi1.

  在此捣鲸，我們提出H19可能通過Runx1、Spi1等一系列造血主轉(zhuǎn)錄因子的去甲基化促進pre-HSC前闽坡、HSC特意分化栽惶。

• H19 plays important roles in embryonic development and growth control, at least in part by serving as a trans-regulator of the imprinted gene network (Gabory et al., 2009;Monnier et al., 2013).

  H19在胚胎發(fā)育和生長控制中發(fā)揮著重要的作用愁溜，至少在一定程度上是作為印跡基因網(wǎng)絡(luò)的跨調(diào)節(jié)因子(Gabory et al.， 2009;Monnier et al.外厂， 2013)冕象。

• However, we did not detect any apparent expression changes in these imprinted genes (data not shown), indicative of the distinct regulatory mechanisms of H19 regarding different cell types.

  然而，我們沒有檢測到這些印跡基因中任何明顯的表達變化(數(shù)據(jù)未顯示)酣衷，這表明H19對不同細胞類型具有不同的調(diào)控機制何荚。

• H19 maintains HSC quiescence in the adult bone marrow by serving as a source of miR-675 to restrict IGF2-IGF1R pathway activation (Venkatraman et al., 2013).

  H19作為miR-675的來源，抑制IGF2-IGF1R通路激活壳嚎，從而維持成體骨髓中HSC的沉默(Venkatraman et al.框舔， 2013)。

• Nevertheless, such a mechanism unlikely exists during embryonic hematopoiesis.

  然而啊片，這種機制不太可能存在于胚胎造血過程中只锻。

• The impaired AGMHSC generation by endothelial-specific deletion of maternal H19-DMR can be largely ascribed to the down- regulation of H19 lncRNA.

  母體H19- dmr內(nèi)皮特異性缺失導(dǎo)致AGMHSC生成受損，這在很大程度上可以歸因于H19 lncRNA的下調(diào)紫谷。

• It has been reported that H19 is highly transcribed but miR-675 is barely detectable in multiple embryo tissues except for placenta, as miR-675 processing is inhibited by the RNA-binding protein HuR during embryogenesis (Flynn and Chang, 2014).

  有報道稱H19具有高轉(zhuǎn)錄齐饮，但miR-675在除胎盤外的多個胚胎組織中幾乎檢測不到，因為miR-675在胚胎發(fā)生過程中受到rna結(jié)合蛋白HuR的抑制(Flynn and Chang, 2014)笤昨。

• We speculate that miR-675 processing might also be inhibited during HSC formation in the AGM region.

  我們推測祖驱，在AGM區(qū)域HSC的形成過程中，miR-675的處理也可能受到抑制瞒窒。

• Due to the rarity of embryonic HSC-related populations and technical limitations, an elegant delineation of how H19 functions in the developmental scenario remains extremely difficult.

  由于與胚胎期人類干細胞相關(guān)的種群數(shù)量稀少捺僻，加上技術(shù)上的限制，要精確描述H19在發(fā)育過程中的作用仍然極其困難崇裁。

• Cyto- plasmic H19 can control mRNA decay through binding with homology-type splicing-regulatory protein to promote its func- tion (Giovarelli et al., 2014).

  細胞質(zhì)H19可以通過與同源剪接調(diào)控蛋白結(jié)合來控制mRNA的衰減匕坯，促進其功能的發(fā)揮(Giovarelli et al.， 2014)拔稳。

• H19 also modulates let-7 activity by acting as a molecular sponge (Kallen et al., 2013).

  H19還通過充當(dāng)分子海綿調(diào)節(jié)let-7活性(Kallen et al.葛峻， 2013)。

• Further, H19 can regulate DNA methylation genome-wide by interacting with SAHH and inhibiting SAHH activity (Zhou et al., 2015).

  此外巴比，H19可以通過與SAHH相互作用术奖，抑制SAHH活性，在全基因組范圍內(nèi)調(diào)控DNA甲基化(Zhou et al.轻绞， 2015)采记。

• We speculated that HSC generation in the AGM region may engage a similar mechanism.

  我們推測AGM區(qū)域HSC的生成可能具有類似的機制。

• Overcoming technical limitations to sys- tematically identify the H19-interacting proteins and RNAs in very few cells would promisingly help to uncover the comprehen- sive regulatory mechanism of H19 during HSC generation.

  克服技術(shù)上的限制铲球，系統(tǒng)地鑒定極少數(shù)細胞中與H19相互作用的蛋白和rna挺庞，有望有助于揭示H19在HSC生成過程中的綜合調(diào)控機制。

  （本研究與當(dāng)前研究的尚存的一些待驗證的調(diào)控機制）

• In view of the ubiquitous regulation of lncRNAs in chromatin modifications and gene expression (Bo¨ hmdorfer and Wierzbicki, 2015;Fatica and Bozzoni, 2014), we constructed here the inte- grated transcriptome map of protein-coding and lncRNA genes during the early stage of HSC development.

  鑒于lncRNA在染色質(zhì)修飾和基因表達中普遍受調(diào)控(Bo¨hmdorfer and Wierzbicki, 2015;Fatica and Bozzoni, 2014)稼病，我們在這里構(gòu)建了HSC發(fā)育早期蛋白編碼和lncRNA基因的整合轉(zhuǎn)錄組圖譜选侨。

• Our following thorough analysis provides a searchable lncRNA resource for HSC formation at single-cell resolution.

  我們接下來通過單細胞分辨率下分析的為HSC的形成提供了一個可搜索的lncRNA資源掖鱼。

• Additionally, we identified 401 unannotated lncRNAs expressed in developmental HSC- related populations.

  此外，我們還鑒定了401個未加注釋的lncrna在發(fā)育中的HSC相關(guān)人群中表達援制。

• Among them, 74 lncRNAs are shared with previously described adult HSC-specific unannotated lncRNAs, implicative of their continuous roles along with HSC ontogeny (Luo et al., 2015).

  其中戏挡，74個lncrna與之前描述的成人HSC特異性無注釋lncrna被共享，暗示了它們在HSC個體發(fā)生過程中的持續(xù)作用(Luo et al.晨仑， 2015)褐墅。

• The expression pattern and functional require- ment of these newly identified lncRNAs during embryonic hema- topoiesis require further systematic investigations.

  這些新發(fā)現(xiàn)的lncrna在胚胎造血過程中的表達模式和功能需要進一步的系統(tǒng)研究。

• Particularly for those unannotated lncRNAs that cannot be verified by RT- PCR combined with Sanger sequencing, more sensitive strategies should be employed to precisely confirm their existence

  尤其對于那些無法通過RT- PCR結(jié)合Sanger測序驗證的未注釋lncrna洪己，應(yīng)采用更靈敏的反應(yīng)策略來準(zhǔn)確確認(rèn)其存在妥凳。

  （本研究新發(fā)現(xiàn)的IncRNA的局限性和待驗證性）