BMP signalling differentially regulates distinct haematopoietic stem cell types

Abstract

Adult haematopoiesis is the outcome of distinct haematopoietic stem cell (HSC) subtypes with self-renewable repopulating ability, but with different haematopoietic cell lineage outputs. The molecular basis for this heterogeneity is largely unknown. BMP signalling regulates HSCs as they are first generated in the aorta-gonad-mesonephros region, but at later developmental stages, its role in HSCs is controversial. Here we show that HSCs in murine fetal liver and the bone marrow are of two types that can be prospectively isolated--BMP activated and non-BMP activated. Clonal transplantation demonstrates that they have distinct haematopoietic lineage outputs. Moreover, the two HSC types differ in intrinsic genetic programs, thus supporting a role for the BMP signalling axis in the regulation of HSC heterogeneity and lineage output. Our findings provide insight into the molecular control mechanisms that define HSC types and have important implications for reprogramming cells to HSC fate and treatments targeting distinct HSC types.

ARTICLE

Received 21 Jan 2015 | Accepted 10 Jul 2015 | Published 18 Aug 2015

Mihaela Crisan1,2, Parham Solaimani Kartalaei1,2, Chris Vink1,2, Tomoko Yamada-Inagawa1, Karine Bollerot1, Wilfred van IJcken3, Reinier van der Linden1, Susana M.Chuva de Sousa Lopes4, Rui Monteiro4,Christine Mummery4 & Elaine Dzierzak1,2

Adult haematopoiesis is the outcome of distinct haematopoietic stem cell (HSC) subtypes with self-renewable repopulating ability, but with different haematopoietic cell lineage outputs. The molecular basis for this heterogeneity is largely unknown. BMP signalling regulates HSCs as they are rst generated in the aorta-gonad-mesonephros region, but at later developmental stages, its role in HSCs is controversial. Here we show that HSCs in murine fetal liver and the bone marrow are of two types that can be prospectively isolatedBMP activated and non-BMP activated. Clonal transplantation demonstrates that they have distinct haematopoietic lineage outputs. Moreover, the two HSC types differ in intrinsic genetic programs, thus supporting a role for the BMP signalling axis in the regulation of HSC heterogeneity and lineage output. Our ndings provide insight into the molecular control mechanisms that dene HSC types and have important implications for reprogramming cells to HSC fate and treatments targeting distinct HSC types.

DOI: 10.1038/ncomms9040 OPEN

BMP signalling differentially regulates distinct haematopoietic stem cell types

1 Department of Cell Biology, Erasmus MC Stem Cell Institute, Erasmus Medical Center, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands. 2 University of Edinburgh, Centre for Inammation Research, Queens Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, UK. 3 Center for Biomics, Erasmus Medical Center, Wytemaweg 80, 3015 CN Rotterdam, Netherlands. 4 Department of Anatomy and Embryology, Leiden University Medical Center, Building 2, Einthovenweg 20, 2333 ZC Leiden, The Netherlands. Correspondence and requests for materials should be addressed to E.D. (email: mailto:[email protected]

Web End [email protected] ).

NATURE COMMUNICATIONS | 6:8040 | DOI: 10.1038/ncomms9040 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 1

& 2015 Macmillan Publishers Limited. All rights reserved.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9040

Understanding the signalling pathways and mechanisms by which haematopoietic stem cells (HSCs) sustain their robust homoeostatic and regenerative characteristics is

important for disease treatments that may differentially affect HSC subtypes. Long-term repopulating HSC subtypes have been dened by their haematopoietic lineage outputmyeloid lymphoid balanced (Bala) HSCs, myeloid-biased (My) HSCs and lymphoid-biased (Ly) HSCs. The clonal composition of the HSC compartment is age-dependent14. Bala-HSCs are found throughout ontogeny, Ly-HSCs predominate in young individuals and My-HSCs predominate in older individuals24. The molecular basis for HSC subtypes is hindered by the lack of prospective isolation. Since bone morphogenetic protein (BMP) affects the development of HSCs in the embryo, HSC subtypes may be differentially controlled by the BMP signalling pathway. It is known that high concentrations of BMP4 maintain proliferation and repopulating activity of human cord blood HSCs5. However, it has also been shown that conditional inactivation of BMPR1a increases the number of bone marrow (BM) HSCs6, that the canonical BMP pathway is dispensable in embryonic day 14 (E14) fetal liver (FL) and adult BM7 and that inhibition of Smad-dependent BMP signalling enhances homing and engraftment of BM HSCs8. Since the role of BMP in HSC regulation is as yet uncertain, we examined whether HSCs in the embryo, fetus and adult are directly responsive to BMP signalling and the relationship of BMP signalling to HSC heterogeneity.

Here we show in BMP responsive element (BRE) GFP transgenic mouse embryos9 that all HSCs emerging in vivo in the aorta-gonad-mesonephros (AGM) region are BMP activated. In contrast, HSCs in murine FL and BM are of two typesBMP activated and non-BMP activated. The initially high proportion of BMP-activated HSCs decreases through ontogeny, and is surpassed by HSCs that are non-responsive to BMP. Clonal transplantation of the two BM HSC types demonstrates that HSC lineage output correlates with the state of BMP activation. Moreover, the two HSC types differ in intrinsic genetic programs thus supporting a role for the BMP signalling axis in the regulation of HSC heterogeneity and lineage output.

ResultsAll AGM HSCs in vivo are BMP-activated. The localized production of BMP4 in the AGM1012 and BMPR2 expression by enriched AGM haematopoietic progenitors and stem cells (HPSCs)10 suggests that BMP may act directly on HSCs. We examined the BMP activation status of HSCs during ontogeny in BREGFP transgenic mice. GFP is expressed in BREGFP mice when BMP and the BMP receptor signal through Smad1/5 to activate transcription from the BRE sequence (Fig. 1a). GFP expression denotes BMP activation at the moment of cell observation/isolation and does not represent previous BMP activation history. Importantly, all BREGFP-expressing cells also express pSmad1/5/8 (ref. 13). Three-dimensional imaging of whole-mount immunostained transgenic E10.5 embryos shows that BMP-activated (GFP ) cells are predominantly distributed in the ventral aspect of the aorta (lumenal hematopoietic, endothelial and underlying mesenchymal cells; Fig. 1b). HPSCs are known to reside in the haematopoietic clusters closely associated with the aorta1416. Some cluster cells are GFP (Fig. 1c), suggesting heterogeneity in BMP activation within the

HPSC compartment at the time of HSC generation. To test the BMP activation status of HSCs, E11 AGM GFP and GFP cells were sorted (6.23.1%(means.d.) of E11 AGM cells are

GFP; Fig. 1d, Supplementary Fig. 1A) and transplanted into adult irradiated recipient mice. All AGM HSCs were found in the

BMP-activated fraction (Fig. 1e): 3 out of 7 recipients receiving

GFP AGM cells were long term, high level repopulated (410% chimerism) by donor cells, whereas no recipients (0 out of 7)

receiving GFP AGM cells were donor engrafted (P 0.05), even

when high embryo equivalents of cells were injected. These data show that BMP signalling is activated in HSCs when they are rst detected in the AGM.

Two HSC types can be prospectively isolated from FL and BM . Examination of the BMP activation status of the cells in the BRE GFP E14 FL and adult BM revealed that 3.70.5% (means.d.) and 5.5%1.8 (means.d.) of cells, respectively, were GFP (Fig. 2a,c; Supplementary Fig. 1A). When sorted BREGFP E12

E14 FL or adult BM cells were transplanted, HSCs were found in both fractions, GFP and GFP . Recipient mice were found to be long-term, high level repopulated (Fig. 2b,d) and secondary transplantation of BM from primary recipients showed that the HSCs self-renew (Fig. 2b,d). Thus, the FL and BM contain two types of HSCsBMP activated and non-BMP activated. The percentage of phenotypic HSCs (LSK-SLAM; Lin Sca1 cKit

CD150 CD48 ) in E14 FL is signicantly higher in the BMP-activated fraction (73%) than in the non-BMP-activated fraction (27%; n 5, T-test Po0.001; Fig. 2e,f). In contrast, in the BM, the

majority (92%) of the phenotypic LSK-SLAM HSCs is non-BMP activated (Fig. 2g,h). BMP activation as detected by BREGFP expression allows prospective isolation of the two HSC types to enable study of their molecular and functional characteristics.

The expression proles of the BMP-activated and non-activated HSCs as examined by RNA sequencing show distinct genetic programs (Fig. 3). FL and BM LSK-SLAM GFP

HSCs express Bmpr genes, whereas GFP LSK-SLAM HSCs do not (Fig. 3a). Smad genes are expressed in both fractions.

Genes upregulated in the GFP FL and BM HSC fractions are signicantly enriched in BMPR2 downstream targets, conrming BMP signalling activation (Fig. 3b). Moreover, other categories/gene sets were found to be differentially expressed between the two HSC types. For example, sets common to FL and BM upregulated in the BMP-activated HSCs are signicantly enriched for genes involved in homoeostasis and metabolism, and MYC and STAT5B target genes (Fig. 3c). Upregulated sets in the non-BMP-activated HSCs are signicantly enriched for genes involved in haematopoietic system development, NFKB1, SP1 and NFE2 target genes (Fig. 3d; see gure legend for false discovery rate corrected Fisher exact test P values). As some of these transcription factors affect normal and malignant haematopoietic cells and specic haematopoietic lineages, the distinct programs may inuence the functional characteristics of the two HSC types.

BM HSC output correlates with BMP activation status. Limiting dilution and clonal transplantations were performed to examine the frequency and functional properties of BMP-activated and non-activated HSCs. We found that a high frequency of FL GFP cells are HSCs (1 out of 180), whereas only 1 out of 20,545 FL GFP cells is a HSC (Fig. 4a;

Supplementary Table 1). Given that the FL contains a mean of12.7 106 cells, of which 3.7% are GFP, our data demonstrate

that 81% of FL HSCs are BMP activated, with 19% being non-BMP activated (Fig. 4b). This corresponds well with the percentages of GFP and GFP cells found in the

FL LSK-SLAM phenotypic HSC compartment. In contrast, the non-BMP-activated HSC number is signicantly higher in the adult BM (Fig. 4c,d). One in 10,053 BM GFP cells and 1 in 17,760 BM GFP cells is an HSC (Supplementary Table 2).

Taking into account an average of 2 107 BM cells per mouse

(four long bones), of which 5.5% are GFP , we nd that 9% of

2 NATURE COMMUNICATIONS | 6:8040 | DOI: 10.1038/ncomms9040 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

& 2015 Macmillan Publishers Limited. All rights reserved.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9040 ARTICLE

a b

BMP4

Cell membrane

pSmad1/5

Co-Smad4

Dorsal

CD31

cKit

GFP

u

c

e

s

u

l

Mesenchyme

N

Aorta

BRE

BRE GFP

Ventral

c

d

e

*

GFP DAPI

E11 AGM

5.9%

Haematopoietic

Cluster

SSC-A

% Chimerism

100

80

60

40

20

0

GFP+ GFP

27

Endothelial cell

30 m

GFP

Figure 1 | Aorta-gonad-mesonephros HSCs are BMP activated. (a) Scheme showing activation of the canonical BMP signalling pathway through phospo-Smad1/5 binding of the double BMP responsive element (BRE) and the resulting transcription of GFP. (b) Three-dimensional whole-mount image of an E10.5 immunostained BREGFP mouse embryo (37 somite pairs). Anti-CD31 (magenta), cKit (red) and GFP (green) antibody staining shows the predominantly ventral distribution of GFP cells in various cell types within the aorta and underlying mesenchymal cells. (c) High-magnication image of a transverse section of an E11 BREGFP dorsal aorta stained with anti-GFP-antibody and 4,6-diamidino-2-phenylindole (DAPI). Intra-aortic hematopoietic clusters contain GFP and GFP cells. Some endothelial cells are GFP. (d) Representative FACS plot with side scatter on the y axis showing percentage of E11 AGM GFP cells (see Supplementary Fig. 1A for control). (e) Percentage donor cell chimerism in the peripheral blood of adult irradiated transplant recipients at 4 months after injection of E11 AGM GFP and GFP cells (24.5 embryo equivalents (ee) of AGM cells injected per mouse; n 3

independent transplantation experiments). *P 0.05 by z-Test for proportions.

BM HSCs are BMP activated and 91% are non-BMP activated. This is in correspondence with the percentages of GFP and

GFP cells found in the BM LSK-SLAM phenotypic HSC compartment. The inversed percentages of BMP-activated HSCs in the BM as compared with the FL are likely due to time- and/or niche-related ontogenic changes.

Considering this shift in the number of BMP-activated HSCs with developmental time and the data of others showing that there are changes in the clonal composition of the HSC compartment associated with lineage output2, we tested the lineage output of BMP-activated and non-activated HSCs. Clonal transplantations of GFP and GFP HSCs from FL and adult

BM (injected cell number was calculated by limiting dilution experiments) were performed and the peripheral blood lineage output data at 4 months post-transplantation was analysed according to Cho et al.2 (Supplementary Fig. 1B). Only primary recipients that were reconstituted in both lymphoid and myeloid lineages were considered. Our data in the FL show slight differences in the lineage output composition of HSCs, with more Bala-HSCs in the BMP-activated fraction. However, this did not reach signicance (Supplementary Fig. 1C,D; Supplementary Table 3). In contrast, BMP-activated and non-BMP-activated HSCs in the BM were signicantly different in terms of HSC lineage output composition (P 0.007).

Bala-HSCs were signicantly enriched in the BMP-activated HSC fraction: 7 out of 17 in the GFP as compared with 0 out of 15 in the GFP fraction (P 0.005) (Fig. 4e; Supplementary

Table 3). My-HSCs were signicantly enriched in the non-BMP-activated fraction: 7 out of 15 in GFP compared with 2 out of 17 in the GFP (P 0.031; Fig. 4f; Supplementary

Table 3). Ly-HSCs were equally distributed (8 out of 17 and 8 out of 15; P 0.727) (Fig. 4e,f; Supplementary Table 3).

Thus, prospective isolation of predominantly balanced and myeloid-biased HSCs is achieved in the BM by cell sorting based on BMP activation.

The two HSC types differ in their intrinsic molecular programs. Previously, it has been suggested that common lymphoid progenitor cells (CLPs) derived from clonal transplantation of lymphoidmyeloid HSCs are poised for expression of lymphoid regulator genes17. We examined whether the expression of such lymphoid genes are already present in LSK-SLAM HSCs and are associated with BMP activation status. Whereas 88% of GFP BM

HSCs have enhanced lymphoid differentiation potential (Bala Ly), this potential is present in only 53% of HSCs in the

GFP fraction (Supplementary Table 3). Moreover, RNA sequencing data showed that the GFP BM HSCs have higher

NATURE COMMUNICATIONS | 6:8040 | DOI: 10.1038/ncomms9040 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 3

& 2015 Macmillan Publishers Limited. All rights reserved.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9040

a

b

E14 FL

Adult BM

6.0%

Cells

transplanted

Number of

injected cells

10 repop/transplanted

(% PB chimerism)

10 repop/transplanted

(% PB chimerism)

20 repop/transplanted

(% PB chimerism)

20 repop/transplanted

(% PB chimerism)

FL

GFP+

E12

E13

E14

3.3104

4.8104

3.3104

5.6104

3.01041.7104

3/3 (100)

2/2 (100)

3/3 (100)

SSC-A SSC-A Lin CD48

3/4 (15,100,100)

3/4 (23,85,85)

GFP E12

E13

E14

1.2106

2.6106

1.8106

3/3 (100)

2/2 (100)

3/3 (100)

3.6%

GFP

GFP

c

d

Cells

transplanted

GFP

Number of

injected cells

BM

GFP+

Adult

Adult

2.0104 3/3 (5,6,28)

3/3 (82,86,87)

5/5 (100)

2/2 (62,100) 2/2 (10, 92)

2.0106

1.7106

1.0106

5/5 (100)

2/2 (100)

2/2 (100)

2/3 (100,100)

1/2 (30)

0.5106 3/3 (82,86,87) 1/3 (100)

e

f

100

E14 FL

Adult BM

Lin CD48

cKit

% LSK SLAM/E14 FL

% LSK SLAM/adult BM

80

**

LSK

LSK

Cell number

Cell number

37.1% 75.3%

60 40 20

0 GFP+ GFP

cKit Sca1 CD150 GFP

g

h

100

**

80

Cell number

Cell number

30.5% 9.8%

GFP

60 40 20

cKit

cKit Sca1

CD150

0 GFP+ GFP

Figure 2 | The fetal liver (FL) and bone marrow contain two distinct HSC types. Representative FACS plots showing percentage of GFP cells with side scatter shown on the y axis in the (a) E14 FL and (c) adult bone marrow (BM) of BREGFP mice (see Supplementary Figure S1A for controls).

Transplantation results of adult irradiated recipients injected with GFP and GFP cells sorted from (b) E12, E13 or E14 FL (n 3) or (d) adult BM (n 4).

Secondary irradiated adult recipients received BM cells, unsorted (3 106) or GFP (1.5 105) and GFP (0.8, 1.8 and 11.8 106) sorted cells from

repopulated primary recipients. Data from all secondary recipients were pooled. Peripheral blood (PB) donor chimerism is shown for all recipients. Representative FACS plots showing the sorting strategy for LSK-SLAM GFP and GFP cells from (e) E14 FL and (g) adult BM. Gates are shown for the

Lin- and LSK fractions. Histogram analysis shows percentages of CD150 cells in the LSK fractions and the percentages of GFP cells in the LSK-SLAM fractions of FL and BM. Grey curves show control non-transgenic LSK SLAM cells. Percentages of LSK SLAM cells that are GFP (black) or GFP (grey) as found in (f) E14 FL (n 3) and (h) adult BM (n 3). **Po0.001 T-test.

Fragments per kilobase of transcript per million mapped reads (FPKM) values for Ikaros (Ikzf1), E2A (Tcf3) and Flt3 genes than GFP HSCs, supporting a pro-lymphoid transcriptional programme for BM BMP-activated HSCs. This is not observed in the FL where similar proportions of HSCs with enhanced lymphoid differentiation potential exist in both GFP (96%)

and GFP (97%) fractions (Supplementary Table 3). FPKM values of the lymphoid genes are comparable in these fractions (Supplementary Fig. 1E). These data suggest that the BM microenvironment promotes a pro-lymphoid gene programme in subset of HSCs activated by the BMP signalling pathway. These results on HSC lineage output, together with the clear differences in the genetic programs between BMP-activated and non-BMP-activated HSCs, suggest the BMP signalling axis as an underlying basis for HSC heterogeneity.

DiscussionThe BMP activation marker that we used in this study represents a robust method by which functionally distinct types of HSCs can be prospectively enriched. Other methods previously used such as label retention18, ckit expression levels19, Hoechst dye efux levels20 and CD229 provide limited separation17. It was shown that TGFb1 differentially controls Ly- and My-HSCs20. However, the variation between the individual HSC clones found in this study suggests that HSC subtype segregation is not absolute. Indeed, as we show here only a subset of Ly-HSCs are controlled by the BMP pathway. Interestingly, TGFb1 appears to be a general stimulatory factor for My-HSC proliferation20. This is in line with our study showing that in BM, in absolute number, there are signicantly more My-HSCs in the non-BMP-activated fraction and these are likely to be controlled by the TGFb pathway.

4 NATURE COMMUNICATIONS | 6:8040 | DOI: 10.1038/ncomms9040 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

& 2015 Macmillan Publishers Limited. All rights reserved.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9040 ARTICLE

a b

BMPR2

1.8

FPKM

Bmpr2

6

Smad1

FPKM

1.2

4

0.6

2

0

ND

ND ND

ND

Bmpr1a

0

nd

nd

FL

FL

BM

BM

FL

FL

BM

BM

0.4

25

0.3

20

Smad4

15

FPKM

0.2

FPKM

10

0.1

5

0

ND

ND

ND

ND

0

FL

FL

BM

BM

FL

FL

BM

BM

1

0.8

Bmpr1b

12

15 Smad5

FPKM

0.6

9

0.4

FPKM

6

0.2

3

ND

ND ND

ND ND

ND ND

ND

FL

0 FL BM

BM

FL

0 FL BM

BM

GFP+

GFP

GFP

FL

BM FL

Low High

BM

NFE2 (human)

BM FL

GFP+

GFP

c d

Abnormal homeostasis

BM FL

Abnormal haematopoietic system

BM

GFP+ GFP GFP+ GFP

GFP+ GFP

GFP+ GFP

GFP+ GFP

FL

BM

FL

BM FL

BM

FL

BM

FL

GFP+

STAT5B (mouse)

BM FL

NFKB1 (mouse)

BM FL

SP1 (mouse)

BM FL

Abnormal metabolism

BM FL

FL

BM

MYC (mouse)

BM FL

GFP+ GFP

GFP+ GFP

BM

FL

BM

FL

BM

FL

Low High

Figure 3 | Transcriptome differences between BMP-activated and non-BMP-activated FL and BM HSCs. (a) FPKM values for BMP receptor and Smad genes expressed by GFP and GFP LSK SLAM sorted HSCs from E14 FL and adult BM. ND, not detected. (bd) Gene list enrichment analysis on genes with more than two fold difference in expression level between GFP and GFP LSK SLAM sorted cells from FL and BM using Enrichr web-application (threshold false discovery rate (FDR) 0.01). GFP high genes are enriched in (b) BMPR2 gene targets (FDRo1.0e 13; Gene Expression Omnibus (GEO)

Kinase perturbation gene sets). Gene lists available on request. (c) Also, overrepresented in GFP high genes are genes involved in homoeostasis (FDR 9.8e 4; Mouse Genome Informatics (MGI) mammalian phenotype term MP0001764), metabolism (FDR 5.4e 4; MGI mammalian phenotype

term MP0005266), MYC (FDR 2.7e 4; TRANSFAC and JASPAR Position Weight Matrix (PWM) genesets), and STAT5B (FDR 2.7e 4; TRANSFAC

and JASPAR PWM gene sets); (d) GFP high genes are enriched for NFE2 (FDR 9.6e 3; TRANSFAC and JASPAR PWM gene sets), NFKB1

(FDR 2.7e 3; TRANSFAC and JASPAR PWM gene sets), SP1 (FDR 2.7e 3; TRANSFAC and JASPAR PWM gene sets), and haematopoietic system

(FDR 5.2e 7; MGI mammalian phenotype term MP0002396)-related genes.

The ability to prospectively identify the distinct HSC types based on BMP activation and directly measure the clonal composition of the HSC pool highlights the importance of the BMP signalling axis in directing the intrinsic HSC programme during ontogeny. MYC and STAT5b target genes that are enriched in the BMP-activated HSCs are, respectively, known to be important in the balance between differentiation and self-renewal, and for conferring efcient lymphomyeloid repopulation and quiescence properties21,22. Of the (NFE2, SP1 and NFKB1) targets upregulated in the non-BMP-activated HSCs, NFE2 is often overexpressed in the myeloproliferative disorders, which spontaneously transform to acute myeloid leukaemia and polycythemia23. SP1 and NFKB1 are known to regulate HSC specication and are required for normal HSC function and differentiation24,25. Interestingly, aged HSCs express high levels of NFKB3,26. This is in line with our data showing that the non-BMP-activated BM HSCs are high in NFKB and are

predominantly myeloid biased. These transcriptomic proles together with further studies should provide insight into how HSC clonal composition may be regulated.

Our nding of the existence of non-BMP-activated HSCs now provides an explanation for the absence of a haematopoietic phenotype when the canonical BMP signalling pathway is impaired68. In the absence of BMP-activated HSCs, the non-activated HSCs are not only sufcient to assume the normal haematopoietic function, but may also expand. The earliest AGM HSCs are BMP activated and as development progresses and HSCs reside in other tissues, there is a shift in the ratio of HSC types from being predominantly BMP activated in the FL to being predominantly non-BMP activated in the adult BM. This implies quantitative changes in BMP-producing niches during the migration process from the AGM (BMPhigh) to FL and eventually multiple BM niches (osteoblastic, perivascular and neural) with variable BMP levels (BMPhigh versus BMPlow)

NATURE COMMUNICATIONS | 6:8040 | DOI: 10.1038/ncomms9040 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 5

& 2015 Macmillan Publishers Limited. All rights reserved.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9040

a

b

100

37

10

0

100

100

% negative recipients

% negative recipients % Negative recipients

% LTR- HSCs/E14 FL frac. % LTR-HSCs/adult BM frac.

80

37

60 40 20

1 in 180

1 in 20,545

10 0 0.1 0.4

0.2 0.3

10 20 30 40

Number 103 of FL GFP+ cells injected

Number 103 of FL GFP cells injected

0 GFP+ GFP

0 GFP+ GFP

c

d

100

37

100

37

100

% Negative recipients

80

60 40 20

1 in 10,053

1 in 17,760

10 0 50

10

20

30

40

10 0 20

5

10

15

Number 103 of BM GFP+ cells injected Number 103 of BM GFP cells injected

e f

BM GFP+

% Donor type cells (lineage)

BM GFP

100

0

100

0

% GM

% BT

% Donor type cells (lineage)

80

80

60

40

20

60

40

20

*Bala

Ly

My

Ly

**My

g

GFP+ BM LSK SLAM

GFP BM LSK SLAM

FPKM

103

102

101

100

Flt3

Ikzf1

Tcf3 Sfpi1

Gata3

Bcl11a

Figure 4 | Relative percentages and lineage output of the BMP-activated and non-BMP-activated HSCs. Limiting dilution transplantation experiments show the frequencies of HSCs in GFP and GFP cell fractions from (a) E14 FL and (c) adult BM. See Supplementary Tables 1 and 2 for detailed results.

Bar graphs showing percentages of long-term repopulating (LTR) HSCs that are GFP (black) or GFP (grey) in (b) E14 FL (n 5) and (d) adult BM

(n 5) as calculated from limiting dilution transplantations. Summary of lineage output analysis of repopulated recipient mice injected with BM (e) GFP

and (f) GFP cells at the clonal HSC level (n 3). Sorted GFP and GFP adult BM cells from 1432-week-old BREGFP (Ly5.2) mice were injected

intravenously into Ly5.1 irradiated (9 Gy) recipients and donor chimerism measured at 4 months post transplantation. Peripheral blood was analysed for donor Ly5.2 cell marker in granulocytemacrophage (GM) lineages (Gr-1 and CD11b) and B- and T-cell lineages (B220 and CD3) by ow cytometry. Per cent donor contribution and donor percentage of GM and B Twere calculated according to Cho et al.2, to reveal HSC type in each recipient. P values were

calculated by Fisher exact test between the GFP and the GFP fractions (P 0.007) and by two sample test for proportion (w2) between balanced

(*P 0.005), My (**P 0.031) and Ly-HSCs (P 0.727) of the two groups, GFP and GFP. (g) Fragments per kilobase of transcript per million mapped

reads (FPKM) values for selected lymphoid genes expressed by GFP and GFP LSK SLAM sorted HSCs from adult BM.

or intrinsic changes in HSC BMP receptor expression with developmental time. Future studies should examine the specic niches supporting the two HSC types in the FL and BM for BMP production, and examine whether other signalling molecules regulate the non-BMP-activated HSCs. Deciphering the

behaviour and lineage output of HSCs during ontogeny, understanding whether the two HSC types are interchangeable or have different origins and investigating the underlying intrinsic and extrinsic mechanisms that dictate these changes are of importance for ensuring that HSCs created through

6 NATURE COMMUNICATIONS | 6:8040 | DOI: 10.1038/ncomms9040 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

& 2015 Macmillan Publishers Limited. All rights reserved.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9040 ARTICLE

reprogramming strategies will be capable of generating all the desired cell types, and for understanding why specic haematological malignancies are prevalent in paediatric versus elderly patients27. Our ndings may be of particular interest to drug resistance in acute myeloid leukaemia patients28, raising the question of whether a combination of drugs affecting the distinct HSC types would be more efcient to control blood-related disorders.

Methods

Mice and embryo generation. Mice were bred and housed at Erasmus MC. BRE GFP transgenic mice9 were maintained in C57BL/6 background. Two independently derived transgenic lines were used in this study. Data from line 1 are shown. Matings were set-up between heterozygous BRE GFP transgenic male and wild-type C57BL/6 female mice. The day of vaginal plug detection was designated as E0. All animal procedures were approved by the local ethics committee and performed in compliance with Standards for Care and Use of Laboratory Animals.

Whole-mount immunostaining and immunohistochemistry. Whole-mount embryo immunostaining and three-dimensional imaging were performed as described previously16,29. Embryos were xed for 20 min with 2% PFA/PBS at 4 C; dehydrated in graded concentrations of methanol; stained with primary antibodies-unconjugated rabbit anti-GFP (MBL, 1:2000), biotinylated rat anti-CD31 (BD, 1:500) and subsequently with rat anti-cKit (BD, 1:500) antibodies in blocking buffer ON at 4 C; washed; incubated with secondary antibodies goat anti-rabbit IgG-Alexa Fluor488 (Invitrogen, 1:1,000), goat anti-rat IgG-Alexa Flour647 (Invitrogen, 1:5,000) and donkey Streptavidin-Cy3, (Jackson ImmunoResearch, 1:1,000); made transparent in BABB; and analysed with a confocal microscope (Zeiss LSM 510META JNI, Plan-Neouar 10/0.3, Eppiplan-Neouar 20/

0.50). Three-dimensional reconstructions were generated from z-stack with LSM Image Browser (Zeiss) and Amira (VISAGE IMAGING).

Flow cytometry. AGMs were dissected with needles under the microscope as previously described30 and tissues were dissociated in PBS/FCS/PS with collagenase type I (Sigma, 0,125%) in water bath at 37 C for 45 min. Cells were further mechanically separated by ushing with a pipette, washed with PBS/FCS/PS and centrifuged at 1,000 r.p.m. for 10 min at 4 C. Pellets were resuspended in PBS/FCS/ PS. E14 FL were mechanically disrupted until a homogeneous cell suspension was obtained. Adult BM was harvested from tibias and femurs by ushing with PBS/FCS/PS through a 1-ml syringe with a 25-G needle. Ficoll fractionation of BM cells was performed at 2,000 r.p.m., 20 min at room temperature. Mononuclear cells were harvested and washed with PBS/FCS/PS followed by centrifugation at 1,000 r.p.m., 10 min at 4 C. Viable cells were counted by Trypan Blue exclusion (Sigma, 0.4%).

FL and adult BM LSK/SLAM cell stainings included LinPE cocktail (anti-CD3/ 1:400, anti-B220/1:2000, anti-Ter119/1:400, anti-Ly6c/1:4000 antibodies), anti-Sca1PE-Cy7(1:800), anti-cKitAPC-Alexa Fluor780(1:600) or BV421(1:500), anti-CD48PE(1:500) and anti-CD150APC(1:500) antibodies. Anti-CD11b-PE antibody (1:2,000) was added in the Lin staining for adult BM. Cells were analysed on a FACSAria SORP or FACSAria III (BD) with FloJo software. Dead cell exclusion was with Hoechst 33258 (1:10,000, Molecular Probes). Lineage output was tested on primary recipient peripheral blood at 4 months post transplantation using myeloid lineage (GM) markers, anti-CD11b PE and anti-Ly6c PE antibodies, lymphoid lineage (B T) markers, anti-CD3APC(1:400) and anti-B220APC(1:2,000)

antibodies and the donor marker with anti-CD45.2 FITC(1:500) antibody. All antibodies used were from BD Pharmingen except the anti-CD150 antibody (eBiolegend). Haematopoietic lineage output in peripheral blood of clonal transplant recipients (4 months post transplantation) was considered balanced when theB Tvalue/GMvalue/ was 416, myeloid-biased when the ratio was 04 and

lymphoid-biased when the ratio was 416.

Transplantation assay. Single cell suspensions were injected intravenously into female ((129SV C57BL/6)F1 or Ly5.1) mice (36 months old) irradiated with 9 Gy

(split dose, 137Cs source). A total of 2 105 recipient background spleen cells were

co-injected. BM cells from primary recipients were injected into irradiated (9 Gy, split dose) secondary recipients. For (129SV C57BL/6) F1 recipients, peripheral

blood peripheral blood chimerism was quantied by DNA PCR for gfp and calculated by DNA normalization (myoD) and comparison with gfp contribution controls with Image Quant software. Mice showing 410% donor chimerism were considered repopulated. For Ly5.1 recipients, peripheral blood chimerism was quantied by CD45.1 and CD45.2 ow cytometric analysis. Mice with 41% CD45.2 CD45.1

donor chimerism were considered repopulated. Secondary recipients received either 3 106 unsorted BM cells or GFP (1.5 105 cells per mouse) and GFP

(0.8 11.8 106 cells per mouse) from the reconstituted primary recipients. For

lineage output analysis, clonal HSC were transplanted based on frequencies found in the limiting dilution experiments. Briey, GFP and GFP fractions from the BM and FL were rst injected at different cell numbers in irradiated recipients and the

repopulation activity measured at 4 months post injection. Using the following parameters, cell number transplanted and number of mice repopulated per number of mice transplanted, frequencies of HSCs were calculated with the L-Calc Software (StemCellTechnology). Subsequent transplants were performed with the cell numbers containing 1 HSC.

RNA preparation and RNA sequencing. RNA was isolated from E14 and adult BM with the mirVana miRNA Kit and prepared according to SMARTER protocol31 for the Illumina HiSeq2000 sequencer. The sequencing depth (unique mapped reads) was for BM (GFP 1.21e07; GFP 1.11e07) and for the FL (GFP 1.22e07; GFP

9.63e06). Sequences were mapped to the mouse (NCBI37/mm9) genome and FPKMs were calculated using Bowtie (v2.2.3), TopHat (v2.0.12) and Cufinks (v2.2.1). Differential expression was analysed using Cuffquant with fragment-bias and multi-read corrections and normalized across all samples using Cuffnorm with geometric library-size normalization32. Genes are high in the BMP-activated HSC fraction (GFP ) when FPKM in GFP 42 FPKM GFP in both BM and FL

LSK-SLAM cells, and high in the non-BMP-activated fraction (GFP ) when FPKM in GFP 42 FPKM GFP in both BM and FL LSK-SLAM cells. For gene

list enrichment analysis, genes were applied to Enrichr web application33. Gene sets with enrichment false discovery rate of 0.01 were selected and heatmaps (row scaled) were generated with GFP high or GFP high genes present in given enriched genelists.

References

1. Geiger, H., de Haan, G. & Florian, M. C. The ageing haematopoietic stem cell compartment. Nat. Rev. Immunol. 13, 376389 (2013).

2. Cho, R. H., Sieburg, H. B. & Muller-Sieburg, C. E. A new mechanism for the aging of hematopoietic stem cells: aging changes the clonal composition of the stem cell compartment but not individual stem cells. Blood 111, 55535561 (2008).

3. Benz, C. et al. Hematopoietic stem cell subtypes expand differentially during development and display distinct lymphopoietic programs. Cell Stem Cell 10, 273283 (2012).

4. Verovskaya, E. et al. Heterogeneity of young and aged murine hematopoietic stem cells revealed by quantitative clonal analysis using cellular barcoding. Blood 122, 523532 (2013).

5. Bhatia, M. et al. Bone morphogenetic proteins regulate the developmental program of human hematopoietic stem cells. J. Exp. Med. 189, 11391148 (1999).

6. Zhang, J. et al. Identication of the haematopoietic stem cell niche and control of the niche size. Nature 425, 836841 (2003).

7. Singbrant, S. et al. Canonical BMP signaling is dispensable for hematopoietic stem cell function in both adult and fetal liver hematopoiesis, but essential to preserve colon architecture. Blood 115, 46894698 (2010).

8. Khurana, S. et al. SMAD signaling regulates CXCL12 expression in the bone marrow niche, affecting homing and mobilization of hematopoietic progenitors. Stem Cells 32, 30123022 (2014).

9. Monteiro, R. M. et al. Real time monitoring of BMP Smads transcriptional activity during mouse development. Genesis. 46, 335346 (2008).

10. Durand, C. et al. Embryonic stromal clones reveal developmental regulators of denitive hematopoietic stem cells. Proc. Natl Acad. Sci. USA 104, 2083820843 (2007).

11. Kaimakis, P., Crisan, M. & Dzierzak, E. The biochemistry of hematopoietic stem cell development. Biochim. Biophys. Acta 1830, 23952403 (2013).

12. Marshall, C. J., Kinnon, C. & Thrasher, A. J. Polarized expression of bone morphogenetic protein-4 in the human aorta-gonad-mesonephros region. Blood 96, 15911593 (2000).

13. Moya, I. M. et al. Stalk cell phenotype depends on integration of Notch and Smad1/5 signaling cascades. Dev. Cell 22, 501514 (2012).

14. de Bruijn, M. F. et al. Hematopoietic stem cells localize to the endothelial cell layer in the midgestation mouse aorta. Immunity 16, 673683 (2002).

15. Taoudi, S. & Medvinsky, A. Functional identication of the hematopoietic stem cell niche in the ventral domain of the embryonic dorsal aorta. Proc. Natl Acad. Sci. USA 104, 93999403 (2007).

16. Yokomizo, T. & Dzierzak, E. Three-dimensional cartography of hematopoietic clusters in the vasculature of whole mouse embryos. Development 137, 36513661 (2010).

17. Oguro, H., Ding, L. & Morrison, S. J. SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors. Cell Stem Cell 13, 102116 (2013).

18. Kiel, M. J. et al. Haematopoietic stem cells do not asymmetrically segregate chromosomes or retain BrdU. Nature 449, 238242 (2007).

19. Grinenko, T. et al. Clonal expansion capacity denes two consecutive developmental stages of long-term hematopoietic stem cells. J. Exp. Med. 211, 209215 (2014).

20. Challen, G. A., Boles, N. C., Chambers, S. M. & Goodell, M. A. Distinct hematopoietic stem cell subtypes are differentially regulated by TGF-beta1. Cell Stem Cell 6, 265278 (2010).

NATURE COMMUNICATIONS | 6:8040 | DOI: 10.1038/ncomms9040 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 7

& 2015 Macmillan Publishers Limited. All rights reserved.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9040

21. Laurenti, E. et al. Hematopoietic stem cell function and survival depend on c-Myc and N-Myc activity. Cell Stem Cell 3, 611624 (2008).

22. Wang, Z. & Bunting, K. D. STAT5 in hematopoietic stem cell biology and transplantation. JAKSTAT 2, e27159 (2013).

23. Kaufmann, K. B. et al. A novel murine model of myeloproliferative disorders generated by overexpression of the transcription factor NF-E2. J. Exp. Med. 209, 3550 (2012).

24. Gilmour, J. et al. A crucial role for the ubiquitously expressed transcription factor Sp1 at early stages of hematopoietic specication. Development 141, 23912401 (2014).

25. Stein, S. J. & Baldwin, A. S. Deletion of the NF-kappaB subunit p65/RelA in the hematopoietic compartment leads to defects in hematopoietic stem cell function. Blood 121, 50155024 (2013).

26. Chambers, S. M. et al. Aging hematopoietic stem cells decline in function and exhibit epigenetic dysregulation. PLoS Biol. 5, e201 (2007).

27. Rodriguez-Abreu, D., Bordoni, A. & Zucca, E. Epidemiology of hematological malignancies. Ann. Oncol. 18(Suppl 1): i3i8 (2007).

28. Zahreddine, H. A. et al. The sonic hedgehog factor GLI1 imparts drug resistance through inducible glucuronidation. Nature 511, 9093 (2014).

29. Yokomizo, T., Ng, C. E., Osato, M. & Dzierzak, E. Three-dimensional imaging of whole midgestation murine embryos shows an intravascular localization for all hematopoietic clusters. Blood 117, 61326134 (2011).

30. Robin, C. & Dzierzak, E. Hematopoietic stem cell enrichment from the AGM region of the mouse embryo. Methods Mol. Med. 105, 257272 (2005).

31. Ramskold, D. et al. Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nat. Biotechnol. 30, 777782 (2012).

32. Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufinks. Nat. Protoc. 7, 562578 (2012).

33. Chen, E. Y. et al. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics 14, 128 (2013).

Acknowledgements

We thank our lab colleagues and Derk ten Berge for critical discussions of our data and

Danny Huylebroeck for comments on the manuscript. This work was supported by

EMBO LongTerm Fellowship (ALTF 260-2009), ZonMW Dutch Medical Research

Council (VENI 916-12-088, VICI 911-09-036), NIRM (Dutch Innovation Grant

FES0908), NIH (RO37 DK54077), Erasmus MC Fellowship (103.494) and Landsteiner

Society for Blood Research (1109).

Author contributions

The study was designed and developed by E.D. and M.C. M.C., C.V., T.Y.I., and K.B.

performed research. W.v.I. performed RNA sequencing and P.S.K. analysed and

interpreted the RNAseq data and part of the ow cytometry data. R.v.d.L. collected data.

R.M., S.d.S. and C.M. provided reagents. M.C. and E.D. designed the experiments,

analysed and interpreted the data and wrote the manuscript.

Additional information

Accession codes: The RNA-seq data have been deposited in the Gene Expression

Omnibus (NCBI) database with accession code GSE70132.

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

Web End =http://www.nature.com/

http://www.nature.com/naturecommunications

Web End =naturecommunications Competing nancial interests: The authors declare no competing nancial interests. Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/

Web End =http://npg.nature.com/

http://npg.nature.com/reprintsandpermissions/

Web End =reprintsandpermissions/

How to cite this article: Crisan, M. et al. BMP signalling differentially regulates distinct

hematopoietic stem cell types. Nat. Commun. 6:8040 doi: 10.1038/ncomms9040 (2015).

This work is licensed under a Creative Commons Attribution 4.0

International License. The images or other third party material in this

article are included in the articles Creative Commons license, unless indicated otherwise

in the credit line; if the material is not included under the Creative Commons license,

users will need to obtain permission from the license holder to reproduce the material.

To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

Web End =http://creativecommons.org/licenses/by/4.0/

8 NATURE COMMUNICATIONS | 6:8040 | DOI: 10.1038/ncomms9040 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

& 2015 Macmillan Publishers Limited. All rights reserved.