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

结合lncRNAs的单细胞分析和功能筛选显示，H19对胚胎造血干细胞的发育至关重要.

SUMMARY

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

  胚胎内皮祖细胞和前体细胞造血干细胞的产生受信号通路和转录因子的调控。

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

  然而，非编码rna的调控作用仍然未知。

• 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.

  利用我们在体内捕获罕见的前造血干细胞和造血干细胞的能力，我们在HSC发育过程中生成了长非编码rna (lncrna)的单细胞景观。

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

  结合生物信息学和功能筛选，我们在体外鉴定了6种影响造血的lncrna。

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

  我们进一步发现H19 lncRNA在主动脉-性腺激素区体内HSC的产生中起着关键作用。

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

  早期H19 lncRNA缺乏症阻断了内皮细胞向造血细胞的转化，这与H19来源的miR,miR-675无关。

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

  此外，h19缺失的前hscs表现为启动子高甲基化，并伴有Runx1、Spi1等多种造血主转录因子的下调。

• 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，这是一种DNA甲基化的调节因子，这在一定程度上导致了观察到的造血缺陷。

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

  我们的发现为进一步分析胚胎HSC发育过程中的lncrna提供了资源。

INTRODUCTION

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

  造血干细胞(HSCs)位于造血系统的顶端，为所有成熟的血液系提供支持，并不断补充终生造血。

• 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，长期成体再生的造血干细胞首先出现在主动脉-性腺-中肾(AGM)区域，也出现在胚胎的其他位置(第11天(E11)左右Dzierzak and Speck, 2008;李等，2012;Samokhvalov等，2007)。

• 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).

  最近发现，胚胎大动脉中有几种特殊的血管内皮细胞(ECs)具有产生he-造血干细胞和祖细胞的能力，因此被定义为血源性ECs (Boisset et al.， 2010;Eilken等，2009;Zovein等，2008)。

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

  这一动态过程被称为内皮细胞向造血的转变。

• 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).

  更准确地说，前造血干细胞是HSC逐步发展过程中的关键中间体，这包括两个连续的阶段:CD451型和CD45+ 2型前体(T1和T2 pre-HSCs) (Rybtsov等，2011;, 2008;陶迪等，2008;周等，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，AGM中由前HSCs成熟而来的数十个HSCs进入血流并在胎儿肝脏中定植(Medvinsky etal .， 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，在一个短暂的窗口内，背主动脉见证了不同的造血干细胞群体的惊人的发展，包括血源性ECs的明确表达以及前造血干细胞的形成和成熟，显示出高度协调的内在和外在调控。

（HSC形成的背景介绍）

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

  越来越多的证据已经证明，在AGM区域这种多步骤HSCs形成的分子机制是多种多样的。

• 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作为研究最好的转录因子之一，在造血干细胞、前造血干细胞和最终造血干细胞中不断表达，在内皮细胞向造血细胞的转化过程中发挥着不可或缺的作用，但在决定HSC的命运后却没有这种作用(Chen et al.， 2009;Yzaguirre等，2017)。

• 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作为Runx1的直接下游靶点，特异性地标记了血源性ECs，并可通过沉默内皮细胞程序加速造血形成(Lancrin et al.， 2012;Thambyrajah等人，2016)。

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

  同样，Runx1的另一个靶点Spi1上调，促进了内皮到血液的转化(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，Runx1远端启动子的低甲基化与其转录活性有关，是最终血液的标志(Webber et al.， 2013)。

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

  然而，调控胚胎中Runx1 启动子 DNA甲基化的上游机制仍不清楚。

• 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，我们使用一种新定义的表面标记cocktail，并同时进行大规模单细胞RNA测序，成功纯化了少量的胚胎前造血干细胞，并在单细胞分辨率下对转录组进行了全面调查(Zhou et al.， 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.

  基于这些有价值的数据，意想不到的mTOR信号关键作用在造血干细胞而非造血祖细胞的出现中被证实了，这表明该研究策略能够深入描述关键的调控机制。

（当前研究和自己研究发现的对比性：Runx1作为研究最好的转录因子之一的现状相光研究，mTOR信号关键的发现，单细胞数据揭示其调控机制的重要性。）

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

  然而，非编码基因的生理功能在哺乳动物中，HSC的出现仍然是难以捉摸的。

• 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.

  长链非编码 rna (Long non-coding RNAs, lncRNAs)在转录组中占很大比例，其定义为长度超过200 nt的转录本，且几乎没有蛋白编码潜能。

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

  lncrna在许多生物逻辑过程中发挥着关键作用(Flynn and Chang, 2014;Kretz等，2013;Wang等，2014)。

• 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可以通过转录或转录后调控基因表达，作为信号、诱导、指南或支架执行(McHugh et al.， 2015;里恩等，2007;Wang and Chang, 2011)。

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

  此前，一项深度测序和综合分析研究发现，超过150种未加注释的lncrna在成体造血干细胞中特异性富集。

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

  其中两个lncrna在HSC自我更新和分化过程中具有功能上的需要(Luo et al.， 2015)。

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

  然而，lncrna在胚胎干细胞形成过程中的景观和功能仍不清楚。

（IncRNA在哺乳动物种的重要重用，IncRNA 在胚胎干细胞形成过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 约7,000 lncRNA genes and constructed the global lncRNA expression landscape during entire HSC ontogeny.

  在此，我们利用5个持续发育的HSC相关人群和成年HSCs的健壮而全面的单细胞转录数据(Zhou et al.， 2016)，鉴定了价值7000个的lncRNA基因，并构建了整个HSC个体发育过程中的全lncRNA表达景观。

• 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.

  通过生物信息学和功能分析，我们筛选出了影响胚胎造血的lncRNA候选标记 ，并进一步阐明了H19在AGM区造血干细胞形成中的重要作用。

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

  我们的发现也为将来阐明其他对胚胎HSC发育至关重要的lncrna提供了资源。

（本文章研究的主要成果：影响胚胎造血的lncRNA候选标记，H19在AGM区造血干细胞形成中的重要作用）

RESULTS

Global lncRNA Expression Dynamics during HSC Development

HSC发育过程中lncRNA的表达动态

• 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).

  我们最近报道了6个与HSC个体发生相关的细胞群的单细胞转录组分析，包括来自E11 AGM区域的ECs和两种类型的前HSCs，来自E12和E14胎儿肝脏的HSCs，以及来自成人骨髓的HSCs (Zhou etal ， 2016)。

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

  为了阐明lncrna在HSC形成过程中的生理作用，我们对候选lncrna进行了系统的生物信息学分析和功能评价。

• 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).

  从ployA+转录数据集中共获得128个单细胞lncRNA谱，涉及上述六种不同的细胞类型，其中不捕获非聚腺苷酸lncRNA(表S1)(Zhou et al.， 2016)。

• 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).

  通过使用独特的全基因组比对(小鼠基因组[mm10])，在至少一个样本中检测到7,312个lncRNA基因，其中包括6,911个注释lncRNA和401个未注释lncRNA，它们是每千位转录序列中每千位酶的片段(FPKM》=1每个样本中（图1A;表S2)。

  
  fig1A

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

  根据生物信息学分析，这些lncRNA基因在很大程度上缺乏蛋白编码潜能(图S1A)。

• 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，lncrna的转录长度和每个转录的外显子数均小于蛋白编码基因(图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).

  每个细胞中lncRNA基因图谱的平均数量明显低于mRNA基因，从胚胎ECs到成体HSCs均呈逐渐下降趋势(图1B)。

  
  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，每个细胞中mRNA和lncRNA转录本的平均数量从ECs到pre-HSCs急剧增加，然后在HSC发育过程中下降;

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

  mRNA转录在T1 pre-HSCs中达到高峰，lncRNAs在T2 pre-HSCs中表达水平最高(图1C)。

（测序数据样本介绍，A图和B图 IncRNA的图谱介绍）

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，未加注释的lncrna的平均表达高于加注释的lncrna，并且从EC到T1 pre-HSC阶段有明显的上调(图S1D和S1E)。

• 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).

  事实上，lncHSC-1和lncHSC-2 (Luo et al.， 2015)这两种已知的HSC特异性无注释lncrna随着HSC的发展呈现出不同的动态表达模式(图 S1F)。

• 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).

  我们发现这些lncrna中有2865个(39.2%)与最近两项研究中报道的与hsc相关的lncrna重叠(Luo et al.， 2015;Qian等，2016)(图S1G)。

• 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).

  更准确地说，我们的数据集中发现了208个(14.4%)骨髓hsc表达的lncrna, 213个(14.7%)胎儿肝脏表达的lncrna, 285个(11.6%)前hsc表达的lncrna与之前报道的骨髓hsc表达的lncrna共享(Luo etal .， 2015)(图S1H)。

• 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个未加注释的lncrna中，314个lncrna在至少两个单细胞中显示出FPKM>1，其余87个lncrna中有84个至少在两个样本中检测到(FPKM >0)。

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

  为了进一步验证这些未加注释的lncrna的真实性，我们选择了其中的40个进行RT-PCR和Sanger测序。

• 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).

  结果表明，40个未加注释的lncRNA中有25个可以按预期扩增，40个扩增产物中有17个与组装的lncRNA序列一致，说明约有一半未加注释的lncRNA是真实的(图S1I和S1J;表S3)。

  (挑选出未注释的IncRNA,进一步利用RT-PCR和Sanger测序验证,进一步筛选出真实可靠的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可以与邻近基因共同调控或相互调控(Engreitz et al.， 2016;)然后，我们确定了它们的基因组分布。

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

  在6911个注释lncRNA基因中，60.11%为基因间型，39.89%为基因内型(图1D)。

  
  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).

  两两配对的Pearson相关分析显示，lncrna与最近基因的表达相关性高于与远端基因的表达相关性(图1E)。

• 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.

  采用共表达模式分析计算lncRNA与最近基因之间的反相关(距离为> 5kb的成对)和顺相关(距离为< 5kb的成对)。

• 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，我们发现顺式对之间的正相关比反式对之间的正相关比例更高(图1F和1G)。

  
  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参与cis对被分为6个位点生物型根据他们的基因位置相对于邻近的蛋白编码基因,基因,包括反义lncRNA-mRNA双头-头(“XH”)或tail-to-tail位置(XT)反义lncRNAs位于(XI)或包含蛋白质编码基因(XO)和lncRNAs下游(SD)或上游(“SU”)的蛋白编码基因(图1 h)。
  

  fig1H

)

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

  在所有6个生物型中，XH(55.36%)为优势生物型(图1I)。

  
  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).

  与之前的工作一致(Luo et al.， 2016)， lncRNA- mRNA对的表达倾向于与sense pairs(SD和SU)呈正相关(图1J, 1K，和S2;表S4)。

(分类分析Inc-RNA 与mRNA相关性,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).

  我们还利用已报道的11种四足动物的lncRNA序列分析了这些lncRNA的进化状态(Nec- sulea et al.， 2014)。

• 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个带注释的lncrna转录中在小鼠以外至少一个物种表达，远端与其邻近基因(> 5kb, trans)的lncrna表达的物种数多于近端(% 5kb, cis)(图1L;表S4)。

  (被注释到的IncRNA远端模式表达的物种数多于近端)
  

  fig1L

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

lncrna比mrna更能区分不同的hsc的群体

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

  为了探讨lncrna在HSC形成过程中的表达特点，我们首先鉴定了849个差异表达的lncrna(图2a和S3A;表S5)。
  

  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).

  接下来，主成分分析(PCA)将128个单细胞聚集成6个不同的群体，与来自不同阶段的6个样本群体一致(图2B)。

  
  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对不同类型的pre-HSCs和不同阶段的胎儿肝脏HSCs的区分能力强于mRNAs，说明lncrna在HSC发育过程中，阶段特异性高于mRNAs(图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，对6个不同样本群体中差异表达最多的186个lncrna进行qPCR, 表达模式与单细胞RNA测序(RNA-seq)数据基本一致，独立验证了结果的重复能力(图2D和S3C;表S3)。
  

  fig2D

(差异IncRNA 表达热图,PCA主成分分析和PCR结果一致)

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

通过基因组定位和表达相关性分析，利用蛋白编码基因预测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).

  为了进一步获得与HSC潜能相关的签名lncrna，我们将前HSC干细胞与之前描述的无pre-HSC潜能的群体进行密切相关比较(Zhou et al.2016)。

• 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)中特异性表达71 T1 pre-HSC签名lncrna，而在CD31+CD45?CD41lowKit+CD201low/-群体不表达。

• 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).

  同样，功能T2pre-hscs (CD31+CD45+CD41lowCD201+和CD31+CD45+c-Kit+CD201high)中也富集了56个T2前hsc签名lncrna，而CD31+CD45+CD41lowCD201中群体中则没有表达(图2 e;表S5)。

  
  fig2E

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

  在T1和T2 pre-HSCs之间重叠的37个lncrna被指定为pre-HSC特征lncrna。

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

  其中，与成体骨髓间充质干细胞共享的12个lncRNA基因被指定为HSC特征lncRNA。

(通过对比无pre-HSC潜能的群体的群体,找出T1和T2 pre-HSCs之间重叠的37个lncrna,指定其为pre-HSC特征lncrna,与成体骨髓间充质干细胞重叠的12个lncRNA基因被指定为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).

  为了确定37个hsc前签名lncrna与邻近蛋白编码基因之间的关系，我们利用Circos构建了一个非编码到编码的网络(图2F;表S5)。
  

  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，5个特征lncrna (Gm20467,Gm16548,Gm13571, 4930538E20Rik, gm28177)的邻近编码基因也已被预先鉴定为pre-hsc特征mRNA基因，分别为Nkx2-3, Selp, Znf512b, Bcl11a, Stat4 (Zhou et al.， 2016)。

• 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与关键造血基因相邻，如Hoxb5、Meis1、Mecom和Runx1 (Azcoitia et al.， 2005;陈等，2009;2016;Kataoka等，2011)。

• 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.

  通过基因组定位的相关性分析，37个pre-hsc特征lncrna主要富集在“造血调控”、“多细胞生物发育调控”、“细胞通讯调控”等方面。

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

  12个HSC特征lncrna主要富集于“基因表达调控”和“发育过程”(图S3D;表S5)。

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

  这些结果表明，签名lncrna可能在HSC规范和功能中发挥作用。

  (在特征IncRNA 中根据其与邻近蛋白编码基因的关系挑选出特征mRNA基因:Nkx2-3, Selp, Znf512b, Bcl11a, Stat4 ,并对37个pre-hsc特征lncrna和12个HSC特征lncrna进行功能富集分析)

• 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.

  为了在HSC分化中进一步获得lncrna的动态分析，我们使用自组织映射(SOMs)作为一种直观的空间可视化方法，研究单细胞转录组数据的异质表达模式。

• 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.

  每个细胞以二维热图的形式出现，其中一组表达模式相似的基因被组装成一个单元;这些单元被聚集在所有样本的一个固定位置。

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

  10241个lncRNA和mRNA基因表达于整个转录组的FPKM >5上，并映射到SOM上(图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个发育阶段共发现28个具有显著动态表达的层次簇，通过同一簇内的蛋白编码基因可以预测lncrna的功能(图S4B和S4C;表S5)。

  (在SOM图中,10241个lncRNA和mRNA基因表达不变的固定在一个位置,有动态变化的基因可以被观察到)

Functional Screening of lncRNAs Regulating Hematopoiesis In Vitro

体外调节造血的lncrna的功能筛选

• 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).

接下来，选取符合以下三个标准的lncrna进行进一步的功能评价:(1)从EC至T1 pre-HSC阶段上调，(2)属于pre-HSC特征lncrna，(3)在三个以上物种中高度保守(图3A)。

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).

  共筛选出10个候选lncrna，包括AI662270、Gm28875、4930538E20Rik、Gm28177、RP23-95l4.3、Gm15135、4933439C10Rik、1700113A116Rik、Gm17275、H19(图3B)。

  
  fig3B

• They belonged to seven SOM units.

  它们属于七个SOM单位。

• 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个候选IncRNAs中有6个被归为SOM cluster 11，表达模式与转录因子Hoxb3相似(图3C和S4B)。

  
  fig3C

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

  值得注意的是，H19与集群8中的Runx1和Gfi1b相关(图3C、S4B和S4C对体外调节造血有影像的Inc-RNA)。

  (通过找出的特征IncRNA,和动态观察到的上调的IncRNA,以及在三个物种以上高度保守的Inc-RNA挑选出10个候选IncRNAs,并说明其在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).

  为了确定候选lncRNAs的功能,我们生成entivirus-expressingsmall hairpin(shRNAs)靶向每个lncRNA在E11 AGM-derived CD31 +细胞中调低他们表达,含有几乎所有的pre-HSCs和造血干细胞和祖细胞(公司)除了ECs(图3 d)。

  
  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+细胞的感染效率为~80%，候选lncrna的表达降低至少50%(图S5A和S5B;表S3)。

• 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个lncRNA候选基因中有6个被敲除，包括H19、AI66270、4933439C10Rik、Gm15135、Gm17275和1700113A16Rik,导致血液菌落形成减少，

• 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敲除作用最为显著(图3E)，这促使我们关注H19 lncRNA在体内HSC发育中的生理作用。

  
  fig3E

(生成entivirus-expressingsmall hairpin(shRNAs)靶向每个lncRNA在E11 AGM-derived CD31 +细胞中调低他们表达,比较每个候选Inc-RNA对CFC-U形成的影响,找出H19的影响最大)

Essential Role of H19 in HSC Development in the AGM Region

H19在AGM地区HSC发展中的重要作用

(动脉-性腺-中肾(AGM)区域)

• 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的表达受上游差异甲基化区(DMR)控制，DMR是作用H19基因表达的表观遗传调控元件(Thorvaldsen et al.， 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).

  诱导Mx1-Cre对成年小鼠H19- dmr的条件破坏表明，H19在维持HSC沉默和分化的过程中起着关键作用(Venkatraman et al.， 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，我们用Tie2-Cre转基因小鼠从胚胎内皮阶段删除H19-DMR，产生母体等位基因特异性突变体(Tie2-Cre;mH19flDMR/+)，其中父系遗传的野生型等位基因由于母体等位基因的排他表达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对体细胞对)突变体胚胎未见明显表型，不同造血组织中细胞的大小和细胞活力在突变体和胎鼠对照中具有可比性。

  (对H19的调控引用文献说明其调控作用,采用动物模型,做突变体,在E11中,分别在卵黄囊(CFU-C)和(动脉-性腺-中肾(AGM)区域,对比突变体和胎鼠对照中造血组织中细胞的大小和细胞活力)

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

  突变胚胎在卵黄囊培养(CFU-C)数目上显示出正常的集落形成单元(图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，突变的AGM区域的CFU-C数明显较低，这表明H19-DMR的缺失可能在AGM区域的HSPC产生中具有特异性(图4B)。

  flDMR(对照组)

• 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区域的大量造血祖细胞来自卵黄囊(McGrath和Palis, 2005)， CFU-C检测可能没有充分考虑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.

  我们进一步使用另一种策略来更精确地反映AGM区域原位ECs的生血能力。

• 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.

  我们利用VEC-Cre转基因有条件地从胚胎内皮细胞阶段删除H19-DMR，然后从AGM区域中筛选出纯ECs (CD31+CD45-CD41-Ter119-)，通过体外OP9共培养进行造血诱导。

• 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).

  与对照组相比，H19突变细胞产生的造血细胞更少(图4C和4D)， CD19+ B淋巴细胞和Gr1/Mac1+髓细胞的频率显著降低(图4E)。

  (找出差异后,进一步利用VEC-Cre转基因有条件地从胚胎内皮细胞阶段删除H19-DMR，然后从AGM区域中筛选出纯ECs (CD31+CD45-CD41-Ter119-)通过体外OP9共培养进行造血诱导,发现H19突变细胞产生的造血细胞更少(图4C和4D)， CD19+ B淋巴细胞和Gr1/Mac1+髓细胞的频率显著降低)
  

  fig4CD

fig4E

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

  接下来我们确定T1前造血干细胞的个体发育是否受到内皮细胞阶段H19-DMR缺失的影响。

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

  我们分类CD31 + CD45 -试剂盒+ AGM细胞共培养检测，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).

  来自突变胚胎的细胞产生的造血后代明显少于来自对照胚胎的细胞(图4F和4G)。

  
  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).

  共培养加移植进一步证明，用突变胚胎的衍生物移植的受体中没有检测到再生群体(图4H和S5C)。

  
  fi4H

fig4H

(确定实验确定T1前造血干细胞的个体发育也受到内皮细胞阶段H19-DMR缺失的影响)

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

  为了进一步研究成熟HSCs的生成，E11 AGM细胞被直接移植到致死辐照的成年受者体内。

• 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例在使用对照胚胎细胞移植后16周外周血中平均出现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).

  与此形成鲜明对比的是，8例受体中有0例在移植H19突变胚胎的细胞后成功重组(图4Iand S5D)。

fig4I

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

  并对发育后期的功能干细胞进行了分析。

• 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).

  免疫表型定义的HSC人群是从E14.5胎肝中分离出来的，并进行了30-HSC移植试验(Benz etal .， 2012)。

• 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缺乏症显著降低了移植后16周的再生能力，尤其是在移植后16周(图S5E)。

• 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.

  在可能导致H19突变体胎儿肝干细胞功能缺陷的几种可能性中，AGM区肝干细胞的生成显著减少应该是其中之一。

• 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).

  为了排除H19缺陷对HSPCs归巢或生存能力的影响，我们采用了前人报道的归巢评估(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).

  来自H19突变胚胎的细胞和胎鼠对照细胞在归巢和存活方面没有差异(图S5F和S5G)。

• 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对H19突变体中胎儿肝脏HSC缺陷产生抑制作用，如成人HSCs中报道的H19缺陷导致HSC活化异常和静止(Venkatraman etal .， 2013)，这需要进一步研究。

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

  总体而言，这些体内功能数据显示，H19-DMR缺失显著影响AGM HSCs的生成，但不影响卵黄囊造血祖细胞的生成。

  (实验和文献结合论证H19-DMR缺失显著影响AGM HSCs的生成，但不影响卵黄囊造血祖细胞的生成。)

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

内皮细胞阶段H19-DMR的缺失破坏了hsc前期的发育

• 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.

  由于前造血干细胞是内内皮到 hsc转化过程中的关键中间体，我们研究了H19突变胚T1前造血干细胞分子程序的变化。

• 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).

  我们分类10和12免疫表型定义T1 pre-HSCs (CD31 + CD45 - CD41lowKit + CD201 + /高),已被证实为高纯度功能T1 pre-HSCs野生型胚胎(周et al ., 2016),从E11 AGM区控制和突变体胚胎,分别进行单细胞RNA - seq(图5A;表S1)。

  
  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.

  两种基因型间有2782个基因表达差异显著，包括1,484个下调基因和1,298个上调基因。

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

  正如所料，H19表达下调，同时伴有多种造血转录因子，包括Gfi1、Nfe2、Runx1、Spi1、Etv6、Erg、Lyl1。

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

  与此相反，内皮转运因子Sox7是突变胚胎细胞中上调的基因之一(图5B;表S6)。

  (研究了H19突变胚T1前造血干细胞分子程序的变化,单细胞RNA-seq 分析,对比突变体和非突变体的差异表达基因)

• 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个pre-HSC特征基因进行随机邻近嵌入分析(Zhou et al.， 2016)，结果表明，H19突变体的T1 pre-HSCs分布在野生型ECs附近，远离对照胚胎的T1 pre-HSCs，它与野生型T1 pre-HSCs聚集在一起(图5C)。

  
  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).

  我们结合H19突变体和对照T1 pre-HSCs的测序数据，结合之前报道的E11 ECs到E12胎肝HSCs的人群，进一步进行伪时间分析，重建胚胎HSCs的发育轨迹(Zhou etal .， 2016)。

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

  正如所料，对照胚胎T1前造血干细胞与野生型T1前造血干细胞在很大程度上难以区分。

• 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，突变胚胎位于野生型ECs附近，这表明它们在endo- thelial-to-hematopoietic transition过程中发育迟缓，伪时间轴上的细胞比例分析也支持这一观点(图5D和5E)。

  (单细胞的伪时间分析,发现H19突变体的T1 pre-HSCs的特点:发育迟缓)

  
  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，我们还构建了一个富含ECs和T1 pre-HSCs的转录因子网络，基于转录因子在HSC发展的6个阶段中表达的两两相关。

  
  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，来自H19突变体的T1 pre-HSCs和它们的同窝对照之间差异最大的转录因子是那些参与T1 pre-HSC网络的转录因子，并且在H19突变体中明显下调(图5F)。

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

  这些分子特征证实了H19是ECs向前hscs转化的关键。

  (富含ECs和T1 pre-HSCs的转录因子网络分析:来自H19突变体的T1 pre-HSCs和它们的同窝对照之间差异最大的转录因子是那些参与T1 pre-HSC网络的转录因子，并且在H19突变体中明显下调)

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.

  因为H19是miR-675的前体(Dey et al.， 2014;Ke- niry等，2012)，我们定量了E11 AGM细胞中的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是优势产物，其表达量约为miR-675的100倍(图6A)。

• 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缺失导致H19 lncRNA缺失，但不影响Igf2(与H19基因座相同)和Igf1r (miR-675的主要靶基因)表达(图5B和S6A)。

• 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，E11 AGM细胞或CD31+细胞中H19 lncRNA的下调对Igf1r蛋白表达无影响(图S6B)。

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

  这些数据表明，H19 lncRNA可能在胚胎HSC发育过程中独立于miR-675和Igf2-Igfr1通路发挥作用。

• 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抑制剂或模拟物，我们未能检测到miR-675对胚胎AGM CD31+细胞造血祖细胞形成的任何影响(图6B、6C和S6C)。

  
  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 抑制处理后Igfr1表达无明显变化(图S6C)，这可能是由于miR-675在AGM细胞内源性表达水平较低所致(图6A)。

• 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，miR-675的过度表达，伴随着其靶基因Igf1r的明显下调，并没有挽救AGM CD31+细胞中H19 lncRNA敲低导致造血祖细胞形成的缺陷(图6B、6D和S6D)。

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

  因此，miR-675不太可能在胚胎造血中发挥作用。

  (H19 lncRNA可能在胚胎HSC发育过程中独立于miR-675和Igf2-Igfr1通路发挥作用)

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

  接下来我们确定了H19 lncrna的亚细胞定位，这可能为其调控机制提供线索。

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

  RNA显示H19主要定位于E11 AGM细胞的细胞质中(图6E和S6E)。

  
  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).

  细胞质H19与s -腺苷基同型半胱氨酸水解酶(SAHH)结合并抑制其活性，SAH可水解DNA甲基转移酶的强抑制剂SAH，从而降低DNMT3a/b介导的甲基化(Zhou et al.， 2015)。

• 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).

  我们首先检测了H19 lncRNA和SAHH蛋白的定位，并在E11 AGM细胞的细胞质中观察到它们之间明显的共定位(图6F和S6E)。

• 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).

  接下来，以水解产物同型半胱氨酸为读数，我们发现AGM细胞中H19 lncRNA的下调可以增加SAHH活性(图6G)。

  
  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，通过敲除抑制SAHH，部分挽救了H19 lncRNA敲除引起的造血集落形成缺陷(图6H、6I和S6F)。

• 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.

  综上所述，我们推测H19在胚胎造血中的部分作用是与SAHH结合并抑制其活性，从而介导造血转录因子的去甲基化。

  (实验论证:H19在胚胎造血中的部分作用是与SAHH结合并抑制其活性，从而介导造血转录因子的去甲基化。)

Increased Promoter Methylation of Several Hematopoietic Transcription Factors by H19 Deficienc

H19缺陷增加了几种造血转录因子的启动子甲基化

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

  我们推测H19可能在造血干细胞发育过程中调控造血转录因子的DNA甲基化。

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

  为了验证这种可能性，我们对T1前含hsc的群体进行了全基因组甲基化测序

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

  (CD31+CD45-Kit+CD201+/high)分别来自E11 H19突变体和对照胚胎。

• 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).

  与其他区域相比，启动子区域的甲基化水平受H19缺乏症影响最为明显(图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，在突变中启动子甲基化增加的基因在造血和染色质修饰中被强烈富集(图7B)。

  
  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).

  具体来说,启动子甲基化水平的几个关键HSC发展的调控因子,包括Runx1 Spi1,突变体胚胎细胞的显著高于在控制组(图7 c、7 d和S7),符合定义的这些基因的表达减少的突变体胚胎的T1 pre-HSCs免疫表型(图5 b)。

• 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，内皮细胞转录因子Sox7的启动子甲基化水平基本没有变化，提示调控机制存在差异(图7C)。

• 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).

  综上所述，H19缺乏可能导致几个关键的造血转录因子启动子高甲基化，从而导致这些基因表达下降，从而导致胚胎HSC形成缺陷(图7E)。

  
  fig7E

（推测H19可能在造血干细胞发育过程中调控造血转录因子的DNA甲基化，数据分析与实验验证，H19的作用机制）

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，我们将单细胞RNA-seq数据集的系统生物信息学分析和内皮特异性敲除小鼠模型相结合，揭示了一种以前未知的lncRNA在小鼠早期胚胎中形成造血干细胞的功能需要。

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

  H19基因属于高度保守的印迹基因簇，编码一个~2.3 kb的lncRNA (Brannan et al.， 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在胚胎发生期间高表达，在出生后急剧下调，但在出生后和成年组织的一个子集中表达水平较高(Gabory et al.， 2009)。

• 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，与成人血液系统中的短期造血干细胞或多能祖细胞相比，H19在主要在长期造血干细胞中表达(Venkatraman et al.， 2013)。

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

  在此，我们证明了H19 lncrna在胚胎ECs形成HSC过程中发挥了重要作用。

• 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.

  通过比较，H19在成体HSCs中的缺失(H19- dmr在成体诱导缺失而非胚胎阶段缺失)导致了成体HSC的沉默(Venkatraman et al.， 2013)，这表明H19在调节胚胎出现和成体HSCs稳态方面的功能多样性。

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

  H19-DMR的缺失对卵黄囊造血祖细胞影响不大。

• 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).

  虽然两者都起源于血源性ECs，属于最终造血，但红髓祖细胞和造血干细胞的生成在时间和空间上是不同的(Chen et al.， 2011;Frame等，2016)。

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

  积累的证据表明，这些造血前体的生成涉及不同的调节机制。

• 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，尽管没有HSC的形成，在notch1-缺陷的胚胎中检测到几乎正常数量的红髓样祖细胞(Hadland et al.， 2004;Kumano等，2003)。

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

  H19-DMR缺失对卵黄囊和AGM造血作用不同的根本原因还有待进一步研究。

（当前研究，和本研究的研究总结，依据还需研究的方向）

• 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，我们提出H19可能通过Runx1、Spi1等一系列造血主转录因子的去甲基化促进pre-HSC前、HSC特意分化。

• 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在胚胎发育和生长控制中发挥着重要的作用，至少在一定程度上是作为印迹基因网络的跨调节因子(Gabory et al.， 2009;Monnier et al.， 2013)。

• 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，我们没有检测到这些印迹基因中任何明显的表达变化(数据未显示)，这表明H19对不同细胞类型具有不同的调控机制。

• 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作为miR-675的来源，抑制IGF2-IGF1R通路激活，从而维持成体骨髓中HSC的沉默(Venkatraman et al.， 2013)。

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

  然而，这种机制不太可能存在于胚胎造血过程中。

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

  母体H19- dmr内皮特异性缺失导致AGMHSC生成受损，这在很大程度上可以归因于H19 lncRNA的下调。

• 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).

  有报道称H19具有高转录，但miR-675在除胎盘外的多个胚胎组织中几乎检测不到，因为miR-675在胚胎发生过程中受到rna结合蛋白HuR的抑制(Flynn and Chang, 2014)。

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

  我们推测，在AGM区域HSC的形成过程中，miR-675的处理也可能受到抑制。

• 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.

  由于与胚胎期人类干细胞相关的种群数量稀少，加上技术上的限制，要精确描述H19在发育过程中的作用仍然极其困难。

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

  细胞质H19可以通过与同源剪接调控蛋白结合来控制mRNA的衰减，促进其功能的发挥(Giovarelli et al.， 2014)。

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

  H19还通过充当分子海绵调节let-7活性(Kallen et al.， 2013)。

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

  此外，H19可以通过与SAHH相互作用，抑制SAHH活性，在全基因组范围内调控DNA甲基化(Zhou et al.， 2015)。

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

  我们推测AGM区域HSC的生成可能具有类似的机制。

• 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.

  克服技术上的限制，系统地鉴定极少数细胞中与H19相互作用的蛋白和rna，有望有助于揭示H19在HSC生成过程中的综合调控机制。

  （本研究与当前研究的尚存的一些待验证的调控机制）

• 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.

  鉴于lncRNA在染色质修饰和基因表达中普遍受调控(Bo¨hmdorfer and Wierzbicki, 2015;Fatica and Bozzoni, 2014)，我们在这里构建了HSC发育早期蛋白编码和lncRNA基因的整合转录组图谱。

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

  我们接下来通过单细胞分辨率下分析的为HSC的形成提供了一个可搜索的lncRNA资源。

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

  此外，我们还鉴定了401个未加注释的lncrna在发育中的HSC相关人群中表达。

• 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，74个lncrna与之前描述的成人HSC特异性无注释lncrna被共享，暗示了它们在HSC个体发生过程中的持续作用(Luo et al.， 2015)。

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

  这些新发现的lncrna在胚胎造血过程中的表达模式和功能需要进一步的系统研究。

• 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

  尤其对于那些无法通过RT- PCR结合Sanger测序验证的未注释lncrna，应采用更灵敏的反应策略来准确确认其存在。

  （本研究新发现的IncRNA的局限性和待验证性）