Single-cell analysis of mixed-lineage states leading to a binary cell fate choice

In the paper

“Similarly, clustering of LKCD34+ cells recreated the entire developmental ordering with granulocytic precursors at one end of the continuum (Extended Data Fig. 6b).”

In the paper

“Genetic perturbations and ChIP-Seq revealed Irf8 and Gfi1 as key components of counteracting myeloid-gene-regulatory networks.”

In the paper

“Thus, during steady-state myelopoiesis a prevalent mixed-lineage state is encountered that expresses HSCP and myeloid progenitor genes (Ctsg, Mpo, and Elane), while displaying molecular priming of erythrocytic, megakaryocytic, granulocytic and monocytic potentials.”

In the paper

“Strikingly, similar binding patterns for Gfi1 and Irf8 were seen on the Irf8, Klf4 and Zeb2 genes (Extended Data Fig. 8a).”

In the paper

“Notably, Gfi1 and Irf8, which are required for normal granulopoiesis and monopoiesis, respectively9,11,12,24,25, displayed strong partitioning within granulocyte- versus monocyte-specified cells (Fig. 1f).”

In the paper

“Gene expression patterns of Csf1r, Flt3 and Cx3cr1 suggested that both CMP and GMP contain macrophage/dendritic cell precursors (MDP: CX3CR1+CD115+CD135+)18, which was confirmed by flow cytometry (Extended Data Fig. 6d–f).”

What the sources say

• Not checked yetcites [18] Auffray, C. et al. CX3CR1+ CD115+ CD135+ common macrophage/DC precursors and the role of CX3CR1 in their response to inflammation. J. Exp. Med. 206, 595–606 (2009) source

In the paper

“Clustering of Pearson-correlation coefficients for ICGS-delineated TF-gene pairs (Fig. 1d, e, Extended Data Fig. 6g–j), revealed three distinct regulatory states within GMPs (Fig. 1e).”

In the paper

“Analysis of GMPs using an Irf8-GFP reporter (Fig. 3a) and CD115 (Csf1r) revealed two major Irf8-GMP subpopulations (IG1 and IG3) and a minor intermediate (IG2) (Fig. 3b).”

In the paper

“Conversely Gfi1 transcripts were anti-correlated with Irf8 and Csf1r (Fig. 3c).”

In the paper

“Importantly, loss of either TF reduced the heterogeneity of genomic states manifested at the single-cell level (Fig. 2a).”

In the paper

“GO-Elite pathway enrichment assigned cellular identities to these states; HSCP-1 (Hematopoietic Stem Cell Progenitor), HSCP-2, Meg (Megakaryocytic), Eryth (Erythrocytic), Multi-Lin* (Multi-Lineage Primed), MDP (Monocyte-Dendritic cell precursor), Mono (Monocytic), Gran (Granulocytic) and Myelocyte (myelocytes and metamyelocytes).”

In the paper

“Flow cytometry analysis revealed two major Gfi1-GMP intermediates (GG2, GG3) and a minor population (GG1) (Fig. 3f).”

In the paper

“The shared genomic regions manifested increased H3K4me2 upon Gfi1 loss (Fig. 2c) or Lsd1-inhibition, correlating with enhanced monocytic potential (Extended Data Fig. 7d, e).”

In the paper

“It also revealed rare metastable intermediates that had collapsed the HSCP program and expressed low levels of the myeloid determinants, Irf8 and Gfi19–13.”

In the paper

“Notably, the Multi-Lin* population co-expressed the transcription factors (TFs) Gata2, Meis1, PU.1 (Spi1) and C/EBPα, the latter two are key regulators of myelopoiesis20,21.”

In the paper

“The analysis captured prevalent mixed-lineage intermediates that manifested coincident expression of hematopoietic stem cell/progenitor (HSCP) and myeloid progenitor genes.”

In the paper

“Expression of Csf1r protein and transcripts was strongly correlated with Irf8 (Fig. 3b, c).”

In the paper

“Strikingly, the unbiased ICGS analysis inferred a developmental order in agreement with the experimentally determined hematopoietic sequence19 (Fig. 1b, bottom).”

What the sources say

• Not checked yetcites [19] Orkin, S. H. & Zon, L. I. Hematopoiesis: an evolving paradigm for stem cell biology. Cell 132, 631–644 (2008) source

In the paper

“ICGS resolved nine hierarchically-ordered cellular states (Fig. 1b) that encompassed all those delineated above.”

In the paper

“Intersection of the Gfi1 and Irf8 peaks revealed shared regions that were deemed accessible in GMPs27 based on ATAC-Seq27 (Fig. 2b, c).”

What the sources say

• Not checked yetcites [27] Lara-Astiaso, D. et al. Immunogenetics. Chromatin state dynamics during blood formation. Science 345, 943–949 (2014) source

In the paper

“Furthermore, loss of Irf8 or Gfi1 reciprocally perturbed the expression of transcription factors that were associated with the monocytic (Klf4, Zeb2, Irf5) and granulocytic (Per3, Ets1) regulatory states, respectively (Fig. 2a, Extended Data Fig. 7b, c).”

In the paper

“They also manifested infrequent and variable-amplitude expression of megakaryocytic, erythroid, granulocytic and monocytic genes (Fig. 1c).”

In the paper

“Two were demarcated by TFs involved in granulocyte (e.g., Cebpe, Gfi1) or monocyte (e.g., Irf8, Klf4) specification10–12,22.”

What the sources say

• Not checked yetcites [10] Tamura, T., Nagamura-Inoue, T., Shmeltzer, Z., Kuwata, T. & Ozato, K. ICSBP directs bipotential myeloid progenitor cells to differentiate into mature macrophages. Immunity 13, 155–165 (2000) source
• Not checked yetcites [11] Karsunky, H. et al. Inflammatory reactions and severe neutropenia in mice lacking the transcriptional repressor Gfi1. Nat. Genet. 30, 295–300 (2002) source
• Not checked yetcites [12] Hock, H. et al. Intrinsic requirement for zinc finger transcription factor Gfi-1 in neutrophil differentiation. Immunity 18, 109–120 (2003) source
• Not checked yetcites [22] Yamanaka, R. et al. Impaired granulopoiesis, myelodysplasia, and early lethality in CCAAT/enhancer binding protein epsilon-deficient mice. Proc. Natl Acad. Sci. USA 94, 13187–13192 (1997) source

In the paper

“The third encompassed HSCP TFs, Gata2 and Meis1, along with signal-induced TFs, Jun, Fos and Egr1.”

In the paper

“Combined loss of these two determinants “trapped” the metastable transition state.”

In the paper

“Flow cytometric analysis of lineage negative Cx3CR1-GFP+ mouse bone marrow cells confirms the presence of phenotypic CD135+(Flt3), CD115+(Csf1r) MDP in CMP and GMP gates.”

In the paper

“Multi-Lin* intermediates expressing HSCP genes induce robust myeloid progenitor gene expression and transcripts for alternate lineage genes.”

In the paper

“In rare cells, HSCP gene expression wanes with the simultaneous acquisition of CFU-G and CFU-M potentials.”

In the paper

“Gfi1−/− GMPs show a significant increase in cell cycle-related gene expression compared to wildtype or Irf8−/− GMPs.”

In the paper

“One group was enriched for HSCP genes including Gata1, Gata2, Egr1, FosB and Jun (Fig. 4a).”

In the paper

“A remarkable feature of this mixed-lineage state is its prevalence and apparent stability in spite of mixing of alternate lineage determinants.”

In the paper

“Activation of a mitotic gene expression program in developmentally distinct cell populations.”

In the paper

“Gfi1−/− GMP demonstrate significantly increased HSC and cell-cycle-associated gene expression.”

In the paper

“Importantly GG1s were enriched for bipotential cells (CFU-GM) as well as those undergoing lineage specification (CFU-G and CFU-M; Fig. 3h, Extended Data Fig. 9a).”

In the paper

“Macrophage-dendritic precursors (MDP) and nascent dendritic cells within myeloid progenitor gates.”

In the paper

“The rare cells within the GMP gate that are undergoing monocyte versus neutrophil specification have collapsed multipotential gene expression program and manifest a metastable mixed-lineage transcriptional state involving low-level expression of both Irf8 and Gfi1.”

In the paper

“Notably the Irf8−/−Gfi1−/− GMPs were more tightly correlated as a group than the IG2s.”

In the paper

“These were eosinophilic progenitors based on CFU assays, cytospins and flow cytometry (Extended Data Fig. 10a–h)30.”

What the sources say

• Not checked yetcites [30] Voehringer, D., van Rooijen, N. & Locksley, R. M. Eosinophils develop in distinct stages and are recruited to peripheral sites by alternatively activated macrophages. J. Leukoc. Biol. 81, 1434–1444 (2007) source

In the paper

“Pseudotemporal ordering of all 382 hematopoietic progenitors along the 9 identified Monocle cell states. These states were identified from the original four annotated flow cytometric gates, based on 5,110 Monocle identified genes (p<0.01).”

In the paper

“Analysis of their genomic states revealed that they, like the IG2s, were primarily distributed between the monocytic and granulocytic specified cells (Fig. 4b, Extended Data Fig. 10l), underscored by quantitative indexing of monocytic and granulocytic signature genes (Fig. 4c).”

In the paper

“Four clusters of cells could be delineated within GG1s (Supplementary Information, Extended Data Fig. 9b–d).”

In the paper

“Expression of the HSCP module in GMPs is associated with CFU-GM potential.”

In the paper

“The rare GG1 cells expressed intermediate levels of both transcription factors (Fig. 3g).”

In the paper

“Notably, myeloid priming occurs in cells which express the TFs PU.1 and/or C/EBPα32.”

What the sources say

• Not checked yetcites [32] Jung, S. et al. Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Mol. Cell. Biol. 20, 4106–4114 (2000) source

In the paper

“Thus hematopoietic intermediates, which express a multipotential program (HSCP1, HSCP2) span the LSK, CMP and GMP flow cytometric gates.”

In the paper

“We were able to identify both prevalent and rare mixed-lineage genomic states that are encountered during myelopoiesis (Fig. 5).”

In the paper

“HOPACH clustering of the same genes and cells from ED Fig. 9b with arrows indicating the GG1 and 16 GG1-like cells identified in the other sort gates.”

In the paper

“Gfi1 (green), Irf8 (blue) and Cebpα (red) ChIP-Seq and RNA-Seq tracks illustrating co-regulation at select loci.”

In the paper

“Bi-potential GG1 cells comprise transcriptionally distinct progenitor populations”

In the paper

“Nearly all the GG1-derived IL5 + SCF CFU are positive for eosinophil markers.”

In the paper

“G3-tetracycline-inducible promoter-driven Gfi1 allele (G3- Gfi1-IRES-Venus eYFP = “G3GV”) results in granulocytic differentiation.”

In the paper

“GG1 and IG1 cells that are enriched for CFU-GM also preferentially express HSCP1-and HSCP2-cluster genes.”

In the paper

“GMP subpopulations enriched for CFU-GM also contain eosinophil-granulocyte progenitors.”

In the paper

“Hierarchical clustering using genes in panel b that are expressed in GG1 cells (TPM>1) identifies four distinct sub clusters.”

In the paper

“Back-gating of sorted Irf8-GFP GMP subpopulations (e; IG1, IG2, IG3) or Gfi1-GFP GMP subpopulations (f; GG1, GG2, GG3) showing that all populations are phenotypically GMP (CD16/32high CD34high).”

In the paper

“Heat map showing the expression of a subset of genes (214) associated with Gfi1 and Irf8 shared ChIP-Seq peaks. All displayed genes are significantly differentially expressed (p<0.05, Benjamini Hochberg adjusted) among at least one of the four comparisons (Irf8−/− versus WT; Irf8−/− versus Irf8-high WT; Gfi1−/− versus WT; Gfi1−/− versus Gfi1-high WT).”

In the paper

“Thus Gfi1 antagonizes the specification of the monocytic-dendritic program in GMPs by repressing enhancers activated by PU.1-Irf8.”

In the paper

“Exclusion of cell-cycle genes improved predictions of developmental states (Supplementary Information, Extended Data Fig. 6a–c).”

In the paper

“Thus Gfi1 likely represses the Irf8, Klf4 and Zeb2 genes by interrupting positive regulation by PU.1 and Irf8, similar to its antagonism of PU.1 on the Spi1 gene29.”

What the sources say

• Not checked yetcites [29] Spooner, C. J., Cheng, J. X., Pujadas, E., Laslo, P. & Singh, H. A recurrent network involving the transcription factors PU.1 and Gfi1 orchestrates innate and adaptive immune cell fates. Immunity 31, 576–586 (2009) source

In the paper

“Thus, we were able to assign genomic states at a single cell level to well-known myeloid intermediates, CFU-M, CFU-G and CFU-GM.”

In the paper

“The genomic states of these latter groups suggested they represented mixed-lineage intermediates poised for binary cell fate choice.”

In the paper

“Thus using reporters for reciprocally expressed TFs we were able to distinguish bipotential cells, their lineage-committed progeny and rare intermediates poised to undergo binary cell-fate choice.”

In the paper

“Higher amplitude Irf8 expression appears necessary for the latter.”

In the paper

“We propose that coincident expression of counteracting regulatory network components manifests as dynamic instability34.”

What the sources say

• Not checked yetcites [34] Buettner, F. et al. Computational analysis of cell-to-cell heterogeneity in single-cell RNA-sequencing data reveals hidden subpopulations of cells. Nat. Biotechnol. 33, 155–160 (2015) source

In the paper

“Counter-acting functions of Irf8 and Gfi1 in myeloid cell fate choice”