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A new lymphoid-primed progenitor marked by Dach1 downregulation identified with single cell multi-omics

Abstract

A classical view of blood cell development is that multipotent hematopoietic stem and progenitor cells (HSPCs) become lineage-restricted at defined stages. Linc-Kit+Sca-1+Flt3+ cells, termed lymphoid-primed multipotent progenitors (LMPPs), have lost megakaryocyte and erythroid potential but are heterogeneous in their fate. Here, through single-cell RNA sequencing, we identify the expression of Dach1 and associated genes in this fraction as being coexpressed with myeloid/stem genes but inversely correlated with lymphoid genes. Through generation of Dach1–GFP reporter mice, we identify a transcriptionally and functionally unique Dach1–GFP subpopulation within LMPPs with lymphoid potential with low to negligible classic myeloid potential. We term these ‘lymphoid-primed progenitors’ (LPPs). These findings define an early definitive branch point of lymphoid development in hematopoiesis and a means for prospective isolation of LPPs.

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Fig. 1: scRNA-seq reveals heterogeneous subpopulations within LMPPs.
Fig. 2: Dach1–GFP expression among HSPC subpopulations.
Fig. 3: Dach1–GFP defined populations change with age.
Fig. 4: scRNA-seq reveals that Dach1–GFP levels demarcate a unique putative lymphoid-primed progenitor.
Fig. 5: LPPs are enriched for lymphoid potential, with negligible myeloid potential.
Fig. 6: LPPs generate lymphoid but not myeloid cells in vivo.
Fig. 7: Revised model of hematopoiesis.

Data availability

Raw sequencing data are available from GEO under accession numbers GSE136341 and GSE136225. Processed data from these analyses are available from https://github.com/LuyiTian/Dach1_analysis_script. The Haemopedia database can be accessed at http://haemosphere.org/. The Immgen database is available from immgen.org.

Code availability

Code from these analyses is available from https://github.com/LuyiTian/Dach1_analysis_script.

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Acknowledgements

We thank the WEHI flow cytometry facility, the Single Cell Open Research Endeavour, S. Wilcox for genomics support and Animal Bioservices for animal husbandry. This work was supported by the National Health & Medical Research Council, with fellowships to S.H.N. (0516782), J.E.B (0637403), C.d.G (1035229), S.L.N. (1155342) and W.S.A (1058344); project grants (1062820, 1124812, 1085765, 1060179, 1122783); a program grant (1113577), the Australian Research Council’s special initiative Stem Cells Australia, an Australian Cancer Research Fund, Victorian State Government Infrastructure Support, Australian Government NHMRC IRIIS and CSL Limited.

Author information

Affiliations

Authors

Contributions

D.Z. developed the analogous CEL-Seq2 protocol and performed scRNA-seq with the WEHI Single Cell Open Research Endeavour. L.T. with M.R. performed bioinformatics analysis, and C.G. performed the hematopoietic expression survey. Most in vivo and in vitro experiments were performed by J.S. and S.T. with assistance from A.P.N. and L.D. (myeloid colony assays), S.L.N, M.P.M. and J.T.J. (lymphoid colony assays) and D.S.L. (FACS characterization). Dach1–GFP reporter mice were generated by W.A., with initial characterization by J.S., K.A.F. and J.E.B. CRISPR construct generation and validation were performed by A.J., A.H. and M.D.M. S.H.N. designed and supervised the study and wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Shalin H. Naik.

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The authors declare no competing interests.

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Editor recognition statement Jamie D. K. Wilson was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Dach1 expression amongst HSPCs.

Haematopoietic mRNA expression survey for Dach1 from a) Haemopedia Mouse RNA-Seq dataset53 and b) Immgen ULI RNA-Seq dataset19, as derived from haemosphere.org20. Expression shown is log2 transcripts per million (tpm) and cells are coloured by lineage.

Extended Data Fig. 2 Dach1 deficiency does not overtly affect haematopoiesis.

a, c-Kit-enriched cells from the BM of CD45.1 (WT) and CD45.2 (Cas9-Tg) mice were transduced with lentivirus expressing guide RNAs (Cherry+) that were b) previously validated by western blot to delete Dach1 expression in AML246 cells. These cells were co-injected into sub-lethally irradiated CD45.1/CD45.2 recipient mice and cellular output was analysed at different time-points; c) FACS data from days 20 and 55 is shown. d, For each cell type, the ratio of Cherry+/Cherry between donor WT and donor Cas9 Tg is shown at multiple times after transplantation as indicated (mean ± S.E.M, 1 experiment). No significant differences in this ratio between WT and Cas9 Tg were observed. Number of mice for each guide RNA and time point is shown.

Extended Data Fig. 3 scRNA-seq gating parameters.

a, Gating parameters of cells from scRNA-seq in Fig. 4. c-Kit+Sca-1+ LSKs, which contained IL-7R+ lymphoid progenitors, were index sorted from Dach1-GFP reporter mice. b, gating parameters based on the index sorted cells for the indicated HSPC subpopulations used for Fig. 4e–i. c, UMAP with all trajectories inferred by slingshot over cells annotated by transcriptional clusters from Fig. 4a.

Extended Data Fig. 4 Cytometry and gating strategy for limiting dilution assays.

Phenotype of progeny from limiting dilution experiments in Fig. 5b. Flt3hi, Flt3int, Dach1-GFP+ and LPPs were isolated and seeded in a) OP9-DL1 cultures to allow T cell generation and b) OP9 cultures to allow B cell generation. Progeny from wells were assessed after 3 weeks and 2 weeks respectively by flow cytometry. One representative well from the row seeded with the lowest dilution of cells is shown. The top row of panel a) and b) is the gating strategy used for all wells.

Extended Data Fig. 5 Flt3hi versus Flt3int separation of LMPPs does not separate myeloid and lymphoid fate.

a, Dach1+, Flt3hi, Flt3int LMPPs and LPPs were sorted from Dach1-GFP reporter mice (CD45.2+) as per Fig. 4a, then transferred into separate groups of sub-lethally irradiated recipients (CD45.1+). b, Myeloid and lymphoid output of donor-derived LMPP fractions is shown by FACS at day 13 and day 28 post transplantation from the blood of recipient mice. Numbers indicate percentage of cells from parent gate. c, Histograms show the percentage of contribution of donor-derived (CD45.2+) cells to differentiated lineages from spleen at day 13 post-transplantation. n = 4 mice from 2 experimental repeats (mean ± S.E.M). Two-tailed unpaired T-test * p < 0.01, ** p < 0.001, *** p < 0.0001. d, Differentiated lineages were also analysed from blood of recipient mice at different time points. Shown is the contribution of donor-derived CD45.1+ cells to each indicated cell type. n = 4 mice from 2 experimental repeats (mean ± S.E.M).

Supplementary information

Reporting Summary

Supplementary Table 1

Seurat-defined significant marker genes.

Supplementary Table 2

Full list of antibodies used for flow cytometry.

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Amann-Zalcenstein, D., Tian, L., Schreuder, J. et al. A new lymphoid-primed progenitor marked by Dach1 downregulation identified with single cell multi-omics. Nat Immunol 21, 1574–1584 (2020). https://doi.org/10.1038/s41590-020-0799-x

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