Single-cell transcriptomic reconstruction reveals cell cycle and multi-lineage differentiation defects in Bcl11a-deficient hematopoietic stem cells

Background Hematopoietic stem cells (HSCs) are a rare cell type with the ability of long-term self-renewal and multipotency to reconstitute all blood lineages. HSCs are typically purified from the bone marrow using cell surface markers. Recent studies have identified significant cellular heterogeneities in the HSC compartment with subsets of HSCs displaying lineage bias. We previously discovered that the transcription factor Bcl11a has critical functions in the lymphoid development of the HSC compartment. Results In this report, we employ single-cell transcriptomic analysis to dissect the molecular heterogeneities in HSCs. We profile the transcriptomes of 180 highly purified HSCs (Bcl11a+/+ and Bcl11a−/−). Detailed analysis of the RNA-seq data identifies cell cycle activity as the major source of transcriptomic variation in the HSC compartment, which allows reconstruction of HSC cell cycle progression in silico. Single-cell RNA-seq profiling of Bcl11a−/− HSCs reveals abnormal proliferative phenotypes. Analysis of lineage gene expression suggests that the Bcl11a−/− HSCs are constituted of two distinct myeloerythroid-restricted subpopulations. Remarkably, similar myeloid-restricted cells could also be detected in the wild-type HSC compartment, suggesting selective elimination of lymphoid-competent HSCs after Bcl11a deletion. These defects are experimentally validated in serial transplantation experiments where Bcl11a−/− HSCs are myeloerythroid-restricted and defective in self-renewal. Conclusions Our study demonstrates the power of single-cell transcriptomics in dissecting cellular process and lineage heterogeneities in stem cell compartments, and further reveals the molecular and cellular defects in the Bcl11a-deficient HSC compartment. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0739-5) contains supplementary material, which is available to authorized users.

Conclusions: Our study demonstrates the power of single-cell transcriptomics in dissecting cellular process and lineage heterogeneities in stem cell compartments, and further reveals the molecular and cellular defects in the Bcl11a-deficient HSC compartment.

Background
Continuous and responsive hematopoiesis is essential for hematopoietic homeostasis throughout the life of a mammalian individual. The classical model of hematopoiesis described this process in a developmental hierarchy, where multipotent hematopoietic stem cells (HSCs) situate at the apex [1]. Purified HSCs are able to provide long-term reconstitution to all blood lineages in transplantation experiments [2][3][4][5]. In adult mice, HSCs reside in the bone marrow and remain predominantly mitotically quiescent. Only a small portion of HSCs are cycling to maintain hematopoietic homeostasis or in response to stress and injury [6]. Lymphoid and myeloid blood cells are derived from successive differentiation of respective lineage progenitors with the loss of reconstituting potential upon commitment. HSCs generate multipotent progenitors, which in turn give rise to lineage progenitors, i.e., the common myeloid progenitors (CMPs) and common lymphoid progenitors (CLP). CLPs subsequently produce B/T lymphocytes, whereas CMPs further differentiate to megakaryocyte-eryhroid progenitors (MEPs) and granulocyte-macrophage progenitors (GMPs) to reconstitute erythrocytes, platelets and myeloid cells. Transcription regulators play critical roles in the HSC compartment and direct stem cell differentiation [7,8].
We have previously reported that the C2H2 zinc finger transcription factor B cell CLL/lymphoma 11A (Bcl11a) is essential for both fetal and adult lymphoid development [9,10]. Bcl11a is also highly expressed in the adult HSC compartment, and Bcl11a-deficient HSCs fail to contribute to CLP development in vivo [9]. Increasing evidence suggests that the phenotypically defined HSC compartment is heterogeneous and a portion of HSCs display lineage bias or restriction [8,[11][12][13][14][15]. It is possible that the lymphoid defect in Bcl11a-deficient HSCs is dependent on its functions in the regulation of lineage-priming heterogeneities in the HSC compartment. However, unbiased transcriptomic dissection of the HSC compartment at the single-cell level was previously hampered by the rarity of HSCs. This technical obstacle has recently been overcome by the advance of RNA-seq technology [16,17], which allows genomewide transcriptome analysis to be conducted at the single-cell level.
In this report, we applied a semi-automated microfluidic system to dissect the transcriptomic heterogeneities and reconstruct the cell cycle progression in the Lin − Sca1 + Kit + (LSK) CD150 + 48 − and Lin − Sca1 + Kit + (LSK) CD150 + 48 − 34 − 135 − HSC compartments at the singlecell level. These exercises further revealed the molecular and cellular defects in the Bcl11a-deficient HSC compartment, which only contains myeloerythroidrestricted progenitor-like cells with substantial selfrenewal defects.

Results
Single-cell transcriptome profiling of adult HSCs using a microfluidic system We employed the Fluidigm microfluidic system coupled with multiplex barcoding to streamline the workflow in cell sorting and cDNA generation. The use of the microfluidic system allowed us to visually confirm the presence of single cells in individual capture sites under the microscope and to filter out debris that may otherwise be mistaken as single cells in flow cytometry. A maximum of 96 cells can be captured and cDNA reverse transcription could be generated in situ in each integrated fluidic circuit. We analyzed the LSK CD150 + 48 − HSC compartment (Bcl11a +/+ ) from eight Bcl11a-GFP reporter conditional knockout mice that we previously described [9] (Fig. 1a). An IRES-eGFP cassette was targeted at the 3' untranslated region of the Bcl11a locus, which enabled tracking of Bcl11a expression by green fluorescent protein (GFP). The reporter mice have normal HSC development and functions [9]. Additionally, we also purified HSCs (sBcl11a +/+ ) with a more stringent sorting scheme (LSK CD150 + 48 − 34 − 135 − ) from ten wild-type C57BL/6 mice (Fig. 1a). Notably, the majority (81 %) of the LSK CD150 + 48 − compartment is also LSK CD150 + 48 − 34 − 135 − , in contrast to the progenitor cell compartment (14 % of LSK CD150 − 48 + ), indicating significant overlap between the sorting schemes. The single-cell capture rate by the microfluidic integrated fluidic circuit was 86.5 % (83/96) and 54.2 % (52/96) for Bcl11a +/+ and sBcl11a +/+ HSCs, respectively (Fig. 1b). To investigate the role of Bcl11a in the HSC compartment, we used Bcl11a flox/flox ; ROSA26-Cre-ERT2 mice to obtain LSK CD150 + 48 − Bcl11a −/− HSCs (Bcl11a −/− ) [9]. Bcl11a deletion was induced by treating the mice with tamoxifen [9]. One week after tamoxifen treatment, HSCs were purified using fluorescence-activated cell sorting (FACS) from five mice and the single-cell capture rate was 85.4 % (82/96). Cell lysis, cDNA reverse transcription and pre-amplification by PCR were performed and harvested by the C1 Single-Cell Auto Prep system. The sequencing libraries of individual cells of each experiment group (Bcl11a +/+ , sBcl11a +/+ or Bcl11a −/− ) were then pooled separately and sequenced. After quality control, cells with low numbers of reads (<500,000 in annotated genes), low numbers of detectable genes (<3000 annotated genes), high fractions of mitochondrially encoded transcripts (>10 %) or anomalies seen under microscope were filtered out. We also removed cells showing incomplete Bcl11a exon 4 deletion in the Bcl11a −/− dataset (Additional file 1). In total, 76 Bcl11a +/+ HSCs, 44 sBcl11a +/+ HSCs and 61 Bcl11a −/− HSCs were further analyzed. The average number of unique counts of genes was 3.16 million (range 1.43-4.52 million) per cell (Bcl11a +/+ ), 0.89 million (range 0.55-1.71 million) per cell (sBcl11a +/+ ) and 3.67 million (range 0.54-12.5 million) per cell (Bcl11a −/− ). Standardized External RNA Controls Consortium (ERCC) RNA spike-ins were added to the sequencing library to account for the technical variability of the protocol [18]. One significant outlier cell from the Bcl11a +/+ dataset was removed from downstream analysis after principal component analysis (PCA; Fig. 1c). The lower count number from the sBcl11a +/+ dataset is not unexpected due to the lower sequencing depth ("Methods and materials"), but the numbers of genes detected (normalized count >1) between two wild-type datasets after size factor normalization are comparable (Wilcox rank sum test p = 0.362) (Fig. 1d).
Cell cycle activity represents the dominant source of transcriptional heterogeneity in the HSC compartment Single-cell transcriptomic analysis allows the detection of gene expression variability between individual cells and identification of cellular subpopulations. Expression variability of a particular gene could come from technical sources (e.g., stochasticity of reverse transcription reaction and library preparation) or genuine biological sources (e.g., differences in cellular states, distinct biological subpopulations and transcription kinetics). It is crucial, therefore, to properly account for the technical HSCs passed initial computational quality control. One significant outlier from the Bcl11a +/+ dataset was identified (marked by red arrow). It was removed from subsequent analysis. d Boxplot comparing the number of genes detected (normalized count >1) in the sBcl11a +/+ and Bcl11a +/+ datasets. The two datasets were comparable, despite low sequencing depth of the sBcl11a +/+ dataset variability in single-cell transcriptomic data interpretation. Brennecke et al. [18] recently described a statistical approach to address this problem by the addition of standardized external RNA spike-ins to the sequencing library. The null hypothesis is that the expression variability detected in a particular gene is not different from the technical variability measured by the external RNA spike-ins; thus, genes that display higher than expected variability imply genuine biological fluctuation from possible cellular subgroups. We identified 6,597, 7,716 and 5503 highly variable genes in the Bcl11a +/+ , sBcl11a +/+ and Bcl11a −/− datasets, respectively ( Fig. 2a; Additional file 2). Remarkably, gene ontology (GO) term enrichment analysis showed that terms related to cell cycle were significantly overrepresented in all three datasets (p < 0.0001; Fig. 2b; Additional file 2). This result suggested that cell cycle activity is the dominant source of transcriptomic heterogeneity among HSCs.

Transcriptional reconstruction of cell cycle progression in the HSC compartment
To resolve the cell cycle heterogeneities in the HSC compartment, we focused on the expression pattern of cell cycle-associated genes in the merged Bcl11a +/+ HSC dataset (Bcl11a +/+ and sBcl11a +/+ ). We compiled a list of 2212 cell cycle-associated genes based on the annotations in the GO term "cell cycle", the Cyclebase database and a recent study on G0/G1 transition by Oki et al. [19] (Additional file 3). PCA was performed to transform the high dimensional expression data into individual linearly uncorrelated principal components. Interestingly, we observed that HSCs displayed a characteristic distribution pattern in the first two principal components (PC1/PC2; Fig. 3a). To aid visualization, we grouped HSCs into five smaller clusters (C1-5) (Fig. 3a) based on their proximity in the PCA plot (Fig. 3b). The grouping is largely consistent with hierarchical clustering (Fig. 3c). To understand if the distribution of the HSCs depicted a cell cycle progression trajectory, we inspected the expression pattern of stage-specific genes based on their annotations in the Cyclebase database and from literature [20]. It appeared that genes situated at different spatial domains of the plot showed clustered expression of stage-specific genes. For instance, the C1 clusters showed high expression of the quiescence regulator of Cdkn1c (p57) and G1 arrest factor Txnip [21,22] and lack of the methylation and G1/S entry regulator Uhrf1 [23] (Fig. 3d). C3 was dominated by S phase-specific genes such as Rrm2 [24] and DNA replication-licensing factors (e.g., Mcm2, Mcm5, Mcm7), while the C4 cluster expressed high levels of genes involved in cell division (e.g., Prc1, Mki67 (Ki-67)) ( Fig. 3d). Correspondence of individual clusters to specific cell cycle stages is further supported by the distinct pattern of cyclin and cyclindependent kinase expression in specific clusters (Fig. 3d). Cyclins are known to oscillate at specific cell cycle stages when they form complexes with partnering cyclin-dependent kinases to drive cell cycle progression [25]. Cyclin E (Ccne2) regulates G1/S transition with CDK2 (Cdk2), cyclin A (Ccna2) is specifically active in S phase, and cyclin B (Ccnb2) functions at G2/M phase transition with CDK1 (Cdk1). Cyclin F expression closely followed that of cyclins A and B [26]. Cyclin D (Ccnd2) is first expressed in G1 phase and involved in G1/S transition, and its expression is sustained in proliferative cells. The expression levels of specific cyclin genes therefore provided a good molecular marker to define the cell cycle stages of different cell clusters. Concordant to the clustering pattern in PCA, the cyclin expression patterns across clusters C1-4 closely recapitulated the known cyclin oscillation during cell cycle progression (Fig. 3d). This correlation combined with the expression patterns of Cdkn1c and Txnip allowed us to reconstruct a transcriptomically defined cell cycle and assign the cell cycle stage of C1 (72/119 cells) as G0/early G1 phase, C2 (14/119) as late G1 phase, C3 (21/119) as S phase and C4 (10/119) as G2/M phase (Fig. 3b, d). The status of the C5 clusters is undetermined due to the low number of cells. Interestingly, the fraction of HSCs classified as G0/early G1 phase in both the Bcl11a +/+ (28 %, 54/75) and sBcl11a +/+ (27.3 %, 32/44) datasets are similar, consistent with our finding that the sorting schemes of LSK CD150 + 48 − and LSK CD150 + 48 − 34 − 135 − are mostly overlapping (Fig. 1a). We then applied this classification to explore the molecular differences between the "G0/early G1 phase" C1 and "S phase" C3 clusters. We performed gene set enrichment analysis (GSEA) on gene sets curated in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Gene sets related to "cell cycle" and "DNA replication" are significantly enriched as expected (Fig. 3e). In addition, we also detected significant enrichment of gene sets involved in DNA damage repair such as "mismatch repair" and "nucleotide/base excision repair". This finding is in accordance with recent reports that quiescent adult HSCs accumulate DNA damage and this damage is repaired upon entry into the cell cycle [27][28][29]. Similarly, enrichment of "p53 signaling pathway" is also detected in the proliferative C3 cluster, consistent with its role in the orchestration of DNA damage repair [30]. Furthermore, gene set enrichment of "oxidative phosphorylation" and "citrate cycle" in the C3 cluster is supported by the observation that quiescent HSCs undergo a glycolytic to aerobic metabolic transition upon activation [31]. Interestingly, gene sets associated with "ubiquitin-mediated proteolysis" is also significantly enriched, affirming the role of the ubiquitin proteasome system in the regulation of HSC cell cycle regulation [32]. These results suggest that Acute Bcl11a deletion in the HSC compartment alters cell cycle progression We next examined the HSC compartment when Bcl11a was deleted. Notably, the distribution of Bcl11a −/− HSCs in PCA closely mimicked that of merged Bcl11a +/+ HSCs in terms of cell cycle-related genes (Fig. 4a). Nevertheless, there were more Bcl11a −/− cells (59.0 %, 36/61 cells) in the space occupied by the proliferative C3 and C4 clusters of the control Bcl11a +/+ HSCs (26.1 %, 31/119) (Fig. 4a), suggesting that Bcl11a deficiency is associated with increased proliferation in the HSC compartment. Moreover, we observed a significant increase of transcription activity in the proliferating Bcl11a −/− HSCs compared with controls, estimated by the total number of ERCC-normalized counts per cell (Fig. 4b). This is consistent with a previous report that transcription rate periodically increases from G1 to S/G2/M phase [33] and suggests that Bcl11a −/− HSCs are more transcriptionally active. There was also evident general up-regulation of cyclin genes and down-regulation of the quiescence regulator Cdkn1c (p57) and G2/M markers such as Prc1, Plk1 and Mki67 (Ki-67) [20] (Fig. 4c). Notably, genes encoding HSC activation and progenitor markers such as Cd34 and Cd244 were also elevated [34,35]. Moreover, examination of the gene expression correlation of transcription factors revealed that there were two anti-correlative gene clusters (Fig. 4d, cluster I in red and II in blue) in the HSC compartment. Cluster I genes were enriched for GO cell cycle terms like "interphase", suggesting their actions in cell cycle regulation (Fig. 4e). Meanwhile, the expression of Bcl11a in cluster II correlated with multiple known HSC quiescence regulators such as Egr1 [36], Fos [37], Mecom [38], Meis1 [39], Hlf [40], and Nr4a1 [41] (Fig. 4d). Signaling mediators important for HSC quiescence, such as Notch signaling (Hes1) [42], Wnt signaling (Tcf7l2) [42] and TGFβ signaling (Id1 and Id3) [43] can also be found in cluster II. These suggest that Bcl11a may cooperate with these regulators in maintaining HSC quiescence. To confirm the prediction that Bcl11a −/− HSCs have reduced quiescence, we performed in vivo 5-bromodeoxyuridine (BrdU) staining for cell cycle profiling. As predicted, significantly higher numbers of BrdU + cells were found in either the Bcl11a +/− or Bcl11a −/− HSC compartment compared with the control (Fig. 4f, g). Furthermore, Bcl11a −/− HSC compartment had significantly more proliferative cells compared with haploinsufficient Bcl11a +/− HSCs, indicating a dose-dependent requirement for Bcl11a. These transcriptomic and experimental results demonstrate a cell cycle defect in the Bcl11a −/− HSC compartment.

Bcl11a-deficient HSCs have defects in long-term selfrenewal potential
The cell cycle defect in Bcl11a-deficient HSCs prompted us to examine their self-renewal potential. We first examined the expression of the self-renewal gene signature by GSEA. We retrieved the signature gene set described by Krivtsov et al. [44] as associated with HSC and leukemic stem cell self-renewal (Additional file 3). We detected a highly significant reduction of expression in Bcl11a −/− HSCs (Fig. 5a). To experimentally test this observation, we transplanted purified Bcl11a −/− or Bcl11a +/− LSKs (CD45.1 − ) to sublethally irradiated CD45.1 + recipient mice. Donor contribution in the peripheral blood was examined at different time points after the adoptive transfer. Bcl11a −/− LSKs had substantially lower capacity to generate hematopoietic progenies, and contributed much less efficiently in the recipients than the Bcl11a +/− LSKs (Fig. 5b). Furthermore, the secondary recipient mice had drastically decreased Bcl11a −/− HSCs, both percentage-wise and total cell numbers (Fig. 5c, d), which was further confirmed by using an alternative HSC sorting scheme (LSK CD34 − 135 − ) (Fig. 5d).
Bcl11a deletion eliminated lymphoid-competent HSCs and resulted in expansion of two myeloerythroid-restricted subpopulations in the HSC compartment We have previously demonstrated experimentally that Bcl11a is essential in the generation of lymphoid progenitors, including lymphoid-primed multipotent progenitors and CLPs, but its effects on HSCs at the single-cell level were unclear [9]. We thus attempted to interrogate the effect of Bcl11a deficiency on the lineage multipotency of HSCs with our single-cell transcriptomic dataset. We compiled a list of genes from the annotation "hematopoiesis and lymphoid organ development" in the GO database and the literature (Additional file 3) and studied their expression in HSCs with PCA. Annotated hematopoietic genes also involved in cell cycle activity were removed from the compilation to reduce their effect on heterogeneity dissection. Notably, significant overlap of Bcl11a +/+ and sBcl11a +/+ HSCs can be observed in the PCA (Fig. 6a, upper panel). The Bcl11a −/− HSCs are segregated into two uneven subpopulations, with the larger subpopulation partially overlapping the merged Bcl11a +/+ (Bcl11a +/+ and sBcl11a +/+ ) HSCs. The smaller and more scattered Bcl11a −/− HSC subpopulation clusters with few sBcl11a +/+ HSCs at the right lower quadrant of the loading plot of the first two principal components (Fig. 6a,  upper panel).
To aid visualization and downstream comparison, we again grouped the HSCs shown in Fig. 6a into four subgroups (H1-4) based on their locations on the loading plot (Fig. 6a, middle panel). Intriguingly, the expression pattern of hematopoietic genes pointed to a clustered expression of markers associated with specific lineages (Fig. 6b). For instance, expression of mega-erythroid genes such as Epor, Selp, Gp9, and Gfi1b [45,46] were clustered in subgroups H2 and H3, while myelopoietic genes such as Mpo, Cebpa [47], Gfi1 [48], and Csf1r were concentrated at the H4 subgroup. Interestingly, the known antagonistic regulation between Gfi1 and Gfi1b is recapitulated by the reciprocal exclusion of the two regulators in the H3 and H4 subgroups [49,50]. Compared with the H1 subgroup, the H3 and H4 subgroups showed reduced expression of lymphoid and HSC activation regulators such as Egr1, Bcl11a, and Jun, but prevalent expression of progenitor markers like Cd34 and Cd48 (Fig. 6b).
Overlaying the cell cycle staging information on the PCA plot of hematopoietic lineage genes (Fig. 6a, lower panel), we found that most of the cells in the H1 subgroup belong to the C1 (G0/early G1 phase) and C2 (late G1 phase) clusters. Wild-type HSCs which clustered with Bcl11a −/− HSCs in the H4 subgroup belong to either C3 (S phase) or C4 (G2/M phase) (Fig. 6a, lower panel).
We then performed GSEA to further compare lineage progenitor signature enrichment between the different subgroups (Fig. 6c, d). The signature gene sets were retrieved from Sanjuan-Pla et al. [13] (Additional file 3) and comparison between Bcl11a +/+ and Bcl11a −/− HSCs showed an expected depletion of the lymphoid signature, consistent with our previous report [9] (Fig. 6c). In addition, the H1 subgroup is enriched with the lymphoid and self-renewal signature, while the H3 and H4 subgroups are enriched with the erythroid and granulocytemacrophage signatures, respectively (Fig. 6d). Further HSC transplantation experiments also confirmed that Bcl11a −/− HSCs, which dominated the H3 and H4 subgroups, are myeloid-restricted, and that the myeloid progenitor compartments (CMPs, GMPs and MEPs) were not affected in both acute Bcl11a deletion mice and primary recipients after Bcl11a deletion (Fig. 6e-h).
Collectively, a trajectory of gradual loss of lymphoid and self-renewal potential can be discerned as wild-type HSCs become activated and proliferative when transiting from the lymphoid-competent H1 subgroup to the megaerythroid-restricted H3 subgroup or the granulocytemacrophage-restricted progenitor-like H4 subgroup. More importantly, the existence of sBcl11a +/+ HSCs in the H4 subgroup suggest that myeloid-restricted cells similar to Bcl11a −/− HSCs have already existed in the wild-type HSC compartment. The Bcl11a −/− HSCs likely come from expansion or activation of existing myeloid-restricted HSCs in the wild-type compartment after the elimination of lymphoid-competent HSCs by Bcl11a deletion.

Discussion
The hematopoietic system has been an area of intense research because of its paramount clinical potential. The discovery of specific surface markers for purification of HSCs in the adult mouse model allowed us to study the key biological properties of self-renewal and lineage commitment. Recently, increasing evidence from clonal transplantation assays supports the phenotypically defined HSC compartment as being functionally heterogeneous, with subpopulations of platelet-primed or myeloidrestricted HSCs [13,14]. In this study, we have profiled the transcriptomes of 180 HSCs in the mouse adult HSC compartment and studied the transcriptome structure and the role of Bcl11a in the HSC compartment. We show that single-cell transcriptomics could be used to reconstruct biological processes, dissect (See figure on previous page.) Fig. 3 Transcriptomic reconstruction of cell cycle progression by single-cell RNA-seq. a PCA of Bcl11a +/+ HSCs based on the expression of 2212 cell cycle-related genes annotated in the GO, Cyclebase database and Oki et al. [19]. The loading plot of the first two principal components is shown. Each point represents one HSC. b Subgrouping of Bcl11a +/+ HSCs by PCA. The Bcl11a +/+ HSCs were grouped into five different clusters (C1-5) based on their proximity in the PCA loading plot as in (a). c Hierarchical clustering and Pearson correlation heatmap of Bcl11a +/+ HSCs. Correlation between Bcl11a +/+ HSCs was estimated by the Pearson correlation coefficients. The column and row colors shown above and to the left correspond to the five subgroups by PCA as in (b). d PCA loading plots of Bcl11a +/+ HSCs as in (a) with expression of selected cell cycle stage-specific genes overlaid. The expression level was calculated as log 10 (normalized counts +1). e The normalized enrichment scores of significantly enriched gene sets (p < 0.05, false discovery rate < 0.05) in the C3 cluster compared with the C1 cluster curated by the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. GSEA gene set enrichment analysis cellular subpopulations in the HSC compartment, and predict stem cell behavior, such as self-renewal capacity and differentiation potential in silico.
Reconstruction of cell cycle progression at the singlecell level provides a new avenue to study the molecular characteristics of quiescent and proliferative HSCs without additional staining or sorting. This advantage is illustrated by our demonstration of the direct comparison of quiescent and proliferative HSCs defined in the reconstruction by GSEA. Reported features of proliferative HSCs, such as activation of the DNA damage repair pathway and transition to oxidative metabolism, can be clearly identified from such an exercise.We noted that the fraction of proliferative cells classified by the transcriptomic approach is higher than the estimation by conventional BrdU staining in this study (~27 % versus 12 %). Others have reported an even lower fraction (<2 %) of cycling cells with alternative DNA content staining and Ki67 expression [35]. The discrepancy is not, in fact, unexpected; instead, the transcriptomic approach may provide a more sensitive staging as cell cycle progression requires further translational and post-translational regulation. Changes in the transcriptome are therefore one of the earliest events in entry to the cell cycle. We further extended the use of the reconstruction in predicting cell cycle phenotype in the Bcl11a −/− HSC compartment. Comparison of Bcl11a −/− and the control datasets predicted reduction of quiescence in the Bcl11a −/− HSC compartment, which was experimentally confirmed by BrdU staining. Gene expression correlation analysis revealed a correlative expression pattern of Bcl11a with multiple known quiescence regulators and signaling mediators. The analysis provided a basis for future mechanistic dissection of Bcll1a in the regulation of HSC self-renewal and quiescence.
We have previously shown that Bcl11a −/− HSCs are deficient in lymphoid reconstitution [9]. This defect could be caused by blockage of lymphoid commitment at the HSC level or selective elimination of lymphoidcompetent HSCs. The possibility to dissect subpopulations using single-cell RNA-seq data provides a new avenue to test these hypotheses.
Segregation of HSCs on the basis of hematopoietic gene expression by PCA revealed an interesting separation of Bcl11a −/− and Bcl11a +/+ HSC expression patterns with partial overlap (Fig. 6a, upper panel). To better dissect their hematopoietic characteristics, we subdivided all the HSCs sequenced in this study into four groups (H1-4) based on their location proximity. The H1 subgroup overwhelmingly consisted of Bcl11a +/+ HSCs. GSEA of the lineage signature showed that they are enriched with the self-renewal signature and lymphoid potential. Overlaying the PCA plot with cell cycle stage information, we found that the lymphoid-competent H1 HSCs contained all the G0/G1 Bcl11a +/+ HSCs (Fig. 6a,  lower panel). In contrast, the H2 subgroups contained both Bcl11a −/− and Bcl11a +/+ HSCs and the Bcl11a +/+ HSCs in H2 are predominantly proliferative. Few proliferative Bcl11a +/+ HSCs are also present in the H4 subgroup clustering with Bcl11a −/− HSCs. Compared with the H1 lymphoid-competent subgroup, the H3 and H4 subgroups showed marked enrichment of MEP and GMP progenitor signatures (Fig. 6d) and high expression of activation markers such as Cd48 and Cd34. The computational exercises and experimental data indicate that, firstly, the lymphoid-competent H1 subgroup is selectively depleted after Bcl11a deletion. Secondly, the dominance of G0/G1 phase HSCs and the self-renewal signature in the H1 subgroup compared with the H2 subgroup is associated with lymphoid competency and HSC self-renewal. Thirdly, the presence of mixed myeloidrestricted Bcl11a −/− and Bcl11a +/+ HSCs in the H2 and H4 subgroups suggests the pre-existence of myeloid-restricted Bcl11a −/− -like HSCs in the wild-type compartment. Fourthly, the clear MEP-and GMP-like signature distinction in the H3 and H4 subgroups suggests that the myeloerythroid-restricted Bcl11a −/− HSC compartment is constituted of two distinct progenitor-like subpopulations. Finally, the close relationship of the H3 and H4 subgroups with the H2 subgroup suggests that they may arise from activation and expansion of the latter subpopulations after Bcl11a deletion. Based on this evidence, we propose that Bcl11a serves two roles in the HSC compartment: in the (See figure on previous page.) Fig. 4 Bcl11a-deficient HSCs showed significant proliferative changes in the HSC compartment. a Distribution of Bcl11a −/− HSCs (purple) and cell cycle reconstructed-Bcl11a +/+ HSC clusters (C1-C5) in a PCA loading plot of the first two principal components. b Boxplots comparing the transcriptional activity of G0/G1 stage (C1 and C2) and S/G2/M stage (C3 and C4) HSCs in the Bcl11a +/+ and Bcl11a −/− datasets, estimated by the total number of read counts normalized by ERCC size factor per cell. c Violin plots of gene expression of selected cyclin genes, progenitor markers and cell cycle stage-associated genes in Bcl11a +/+ and Bcl11a −/− HSCs. The black dots represent the mean expression for each gene. The purple box marks the BrdU + fraction in the HSC compartment of different genotypes. FSC-A: Forward scatter area. g The dose-dependent changes in BrdU + cell number in the HSC compartment in different Bcl11a genotypes. HSCs were harvested and sorted from adult mouse bone marrow 5 days after tamoxifen-induced Bcl11a ablation. *p < 0.05, **p < 0.01, n = 3 mice for each group. Bcl11a +/− , CreERT2; Bcl11a +/flox (treated with tamoxifen); Bcl11a −/− , CreERT2; Bcl11a flox/flox (treated with tamoxifen). The error bar represented mean ± 1 standard deviation maintenance of lymphoid-competent HSCs and in the regulation of HSC self-renewal. Deletion of Bcl11a selectively eliminated lymphoid-competent HSCs, causing the lymphoid deficiency in the Bcl11a −/− compartment. The myeloerythroid restrictive phenotype of Bcl11a −/− HSCs is contributed by the residual surviving myelo-restricted subpopulations in the HSC compartment, which may have limited self-renewal potential.
The detection of Bcl11a −/− -like wild-type HSCs (H2 and H4 subgroups) in the wild-type compartment in our dataset raises interesting questions about their resemblance to the recently reported myeloid-restricted repopulating progenitors in the myeloid bypass model proposed by Yamamoto et al. [14]. In that study, HSCs were purified with a variant sorting scheme (LSK CD150 + 34 − 41 + ), which selectively enriched myeloid-biased HSCs [5,11,12,14]. Notably, Itga2b (CD41) was also found to be expressed predominantly in the H2 and H3 clusters in our dataset, supporting the mega-erythroid signature enrichment in these cells (Fig. 6b). Nonetheless, we did not observe clustered expression of Vwf in our dataset, which is suggested to mark platelet-primed primitive HSCs in mice [13]. The transcriptomic relationship of these variant HSC compartments with the Bcl11a −/− HSCs thus warrants further investigation.

Conclusions
We have successfully applied single-cell transcriptomic analysis in reconstructing the cell cycle process and dissecting the molecular heterogeneities of the rare HSC population. Our data reveal the diversity of lineagebiased subpopulations and the effect of Bcl11a in the maintenance of the HSC compartment. Bcl11a is essential in the maintenance of lymphoid-competent HSC survival and self-renewal.

Mice
Bcl11a flox/flox Rosa26 CreERT2/CreERT2 and Bcl11a eGFP/eGFP mice were previously reported [9]. F1 hybrid wild-type mice from C57BL/6 CD45.1 + crossed with 129S5 mice were used as recipients for transplantation of bone marrow or LSK cells. All mice used were from colonies maintained at the Research Support Facility of the Wellcome Trust Sanger Institute. Housing and breeding of mice and experimental procedures were according to the UK 1986 Animals Scientific Procedure Act and the Animal Welfare and Ethical Review Body of the Wellcome Trust Sanger Institute.

Cell-cycle analysis
Mice were given a single intraperitoneal injection of (BrdU (100 μg g −1 mouse weight) and then maintained on 0.8 mg/ml BrdU in the drinking water for 14 or 20 h before being euthanized. HSCs were double FACS-sorted from mouse bone marrow. BrdU incorporation was measured by flow cytometry using an APC BrdU Flow Kit (BD Pharmingen) according to the manufacturer's instructions.

Single-cell RNA-seq library preparation
Two thousands FACS-sorted Bcl11a +/+ and Bcl11a −/− HSCs pooled from eight and five mice, respectively, were loaded onto two separate 5-10 μm C1 Single-Cell Auto Prep integrated fluidic circuits (Fluidigm) and cell h Comparison of the cell numbers of myeloerythroid progenitors in different genotypes as in (g). Cells were harvested from the two femurs of each mouse (n = 4/genotype). The error bar represented mean ± 1 standard deviation capture was performed according to the manufacturer's protocol. Similarly, 1000 sBcl11a +/+ HSCs from 10 mice were loaded. Individual capture sites were inspected under light microscope to confirm the presence of single cells. The locations of empty capture sites and sites containing multiple cells or burst cells were noted for downstream quality control during data analysis. The lysis and reverse transcription mixes were then prepared with the SMARTer PCR cDNA Synthesis kit (Clontech) and the Advantage 2 PCR kit (Clontech) according to Fluidigm recommendations. The ERCC Spike-In Control Mix (1.0 μl in a 1:1000 dilution; Ambion) was added to the lysis mix to allow control of technical variation of the library preparation protocol. Lysis and cDNA reverse transcription and PCR were performed and cDNA harvested by the C1 Single-Cell Auto Prep system according to the manufacturer's settings. The success of cDNA preparation was confirmed by optimal DNA signal detected by a 2100 Bioanalyzer with High-sensitivity DNA chip (Agilent). Multiplex sequencing libraries were then prepared using the Nextera DNA Sample Preparation Kit and the Nextera Index Kit (Illumina) according to the recommendation in the C1 Single-Cell Auto Prep manual. The libraries of individual cells of each experiment group (Bcl11a +/+ /Bcl11a −/− ) were then pooled and sequenced on four lanes of a HiSeq 2500 (Illumina) to generate 100-bp paired-end reads through the Wellcome Trust Sanger Institute in-house sequencing pipeline. For the sBcl11a +/+ HSC library, samples from empty capture sites, abnormal cells or multiplets detected by microscopy were removed before library construction and were similarly sequenced on two lanes of a HiSeq 2500 (Illumina). The raw sequencing data can be found in the European Nucleotide Archive (sudy ERP010829).

Read alignment and quality control of dataset
Paired-end reads were aligned to the Mus musculus genome (Ensembl GRCm38.75 assembly) by GSNAP (version 2013-08-14) with the following parameter settings: 'gsnap -A sam -B 5 -t 8 -n 1 -Q -nofails' [51]. The quantification of expression was performed by the htseq-count module from the HTSeq package [52] with gene annotation from GTF files (Ensembl GRCm38.75 assembly) with parameter "-s no" in intersection-nonempty mode [53]. Cells with less than 500,000 counts in annotated features, expression of less than 3000 genes or a high percentage (>10 %) of counts mapping to mitochondrial genes were removed from subsequent analysis. Cells with reads detected on the Bcl11a locus chr11:24163146-24165166 were removed from the Bcl11a −/− dataset as they represent incomplete exon 4 deletion after tamoxifen induction. Significant outliers in PCA based on the whole transcriptome were removed from the merged dataset of Bcl11a +/+ , sBcl11a +/+ and Bcl11a −/− HSCs (Fig. 1d). Cells with anomalies, empty capture sites, or multiple cells at capture sites were removed prior to sequencing in the sBcl11a +/+ dataset. The quality control summary is detailed in Additional file 1.
Detection of highly variable genes and statistical analysis