Reader TTS

ABSTRACT It is well established that haematopoietic stem and progenitor cells (HSPCs) are generated from a transient subset of specialized endothelial cells termed haemogenic, present in the

yolk sac, placenta and aorta, through an endothelial-to-haematopoietic transition (EHT). HSPC generation via EHT is thought to be restricted to the early stages of development. By using

experimental embryology and genetic approaches in birds and mice, respectively, we document here the discovery of a bone marrow haemogenic endothelium in the late fetus/young adult. These

cells are capable of de novo producing a cohort of HSPCs in situ that harbour a very specific molecular signature close to that of aortic endothelial cells undergoing EHT or their immediate

progenies, i.e., recently emerged HSPCs. Taken together, our results reveal that HSPCs can be generated de novo past embryonic stages. Understanding the molecular events controlling this

production will be critical for devising innovative therapies. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS

OPTIONS Access through your institution Access Nature and 54 other Nature Portfolio journals Get Nature+, our best-value online-access subscription $29.99 / 30 days cancel any time Learn

more Subscribe to this journal Receive 12 print issues and online access $209.00 per year only $17.42 per issue Learn more Buy this article * Purchase on SpringerLink * Instant access to

full article PDF Buy now Prices may be subject to local taxes which are calculated during checkout ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our

FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS MAPPING HUMAN HAEMATOPOIETIC STEM CELLS FROM HAEMOGENIC ENDOTHELIUM TO BIRTH Article 13 April 2022 IDENTIFICATION OF A

RETINOIC ACID-DEPENDENT HAEMOGENIC ENDOTHELIAL PROGENITOR FROM HUMAN PLURIPOTENT STEM CELLS Article 28 April 2022 HAEMATOPOIETIC STEM AND PROGENITOR CELL HETEROGENEITY IS INHERITED FROM THE

EMBRYONIC ENDOTHELIUM Article Open access 17 July 2023 DATA AVAILABILITY Microarray data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO)

under accession codes GSE122857, GSE133804 and GSE133812. All other data supporting the findings of this study are available from the corresponding author on reasonable request. REFERENCES *

Sugimura, R. et al. Haematopoietic stem and progenitor cells from human pluripotent stem cells. _Nature_ 545, 432–438 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar  * Lis, R.

et al. Conversion of adult endothelium to immunocompetent haematopoietic stem cells. _Nature_ 545, 439–445 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar  * Jaffredo, T.,

Gautier, R., Eichmann, A. & Dieterlen-Lievre, F. Intraaortic hemopoietic cells are derived from endothelial cells during ontogeny. _Development_ 125, 4575–4583 (1998). Article  CAS 

PubMed  Google Scholar  * Zovein, A. C. et al. Fate tracing reveals the endothelial origin of hematopoietic stem cells. _Cell Stem Cell_ 3, 625–636 (2008). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Chen, M. J., Yokomizo, T., Zeigler, B. M., Dzierzak, E. & Speck, N. A. Runx1 is required for the endothelial to haematopoietic cell transition but not

thereafter. _Nature_ 457, 887–891 (2009). Article  CAS  PubMed  PubMed Central  Google Scholar  * Kissa, K. & Herbomel, P. Blood stem cells emerge from aortic endothelium by a novel type

of cell transition. _Nature_ 464, 112–115 (2010). Article  CAS  PubMed  Google Scholar  * Swiers, G., Rode, C., Azzoni, E. & de Bruijn, M. F. A short history of hemogenic endothelium.

_Blood Cells Mol. Dis._ 51, 206–212 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Boisset, J. C. et al. In vivo imaging of haematopoietic cells emerging from the mouse

aortic endothelium. _Nature_ 464, 116–120 (2010). Article  CAS  PubMed  Google Scholar  * Mikkola, H. K. & Orkin, S. H. The journey of developing hematopoietic stem cells. _Development_

133, 3733–3744 (2006). Article  CAS  PubMed  Google Scholar  * Dzierzak, E. & Speck, N. A. Of lineage and legacy: the development of mammalian hematopoietic stem cells. _Nat. Immunol._

9, 129–136 (2008). Article  CAS  PubMed  PubMed Central  Google Scholar  * Yokomizo, T. & Dzierzak, E. Three-dimensional cartography of hematopoietic clusters in the vasculature of whole

mouse embryos. _Development_ 137, 3651–3661 (2010). Article  CAS  PubMed  PubMed Central  Google Scholar  * Boisset, J. C. & Robin, C. On the origin of hematopoietic stem cells:

progress and controversy. _Stem Cell Res._ 8, 1–13 (2012). Article  CAS  PubMed  Google Scholar  * Drevon, C. & Jaffredo, T. Cell interactions and cell signaling during hematopoietic

development. _Exp. Cell Res._ 329, 200–206 (2014). Article  CAS  PubMed  Google Scholar  * Dejana, E., Hirschi, K. K. & Simons, M. The molecular basis of endothelial cell plasticity.

_Nat. Commun._ 8, 14361 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar  * Thambyrajah, R. et al. New insights into the regulation by RUNX1 and GFI1(s) proteins of the

endothelial to hematopoietic transition generating primordial hematopoietic cells. _Cell Cycle_ 15, 2108–2114 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Blaser, B. W.

& Zon, L. I. Making HSCs in vitro: don’t forget the hemogenic endothelium. _Blood_ 132, 1372–1378 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  * Ottersbach, K.

Endothelial-to-haematopoietic transition: an update on the process of making blood. _Biochem. Soc. Trans._ 47, 591–601 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  *

Dzierzak, E. & Bigas, A. Blood development: hematopoietic stem cell dependence and independence. _Cell Stem Cell_ 22, 639–651 (2018). Article  CAS  PubMed  Google Scholar  * Pardanaud,

L. et al. Two distinct endothelial lineages in ontogeny, one of them related to hemopoiesis. _Development_ 122, 1363–1371 (1996). Article  CAS  PubMed  Google Scholar  * Pouget, C., Gautier,

R., Teillet, M. A. & Jaffredo, T. Somite-derived cells replace ventral aortic hemangioblasts and provide aortic smooth muscle cells of the trunk. _Development_ 133, 1013–1022 (2006).

Article  CAS  PubMed  Google Scholar  * Yvernogeau, L., Auda-Boucher, G. & Fontaine-Perus, J. Limb bud colonization by somite-derived angioblasts is a crucial step for myoblast

emigration. _Development_ 139, 277–287 (2012). Article  CAS  PubMed  Google Scholar  * Ambler, C. A., Nowicki, J. L., Burke, A. C. & Bautch, V. L. Assembly of trunk and limb blood

vessels involves extensive migration and vasculogenesis of somite-derived angioblasts. _Dev. Biol._ 234, 352–364 (2001). Article  CAS  PubMed  Google Scholar  * Christ, B., Huang, R. &

Scaal, M. Amniote somite derivatives. _Dev. Dyn._ 236, 2382–2396 (2007). Article  CAS  PubMed  Google Scholar  * McGrew, M. J. et al. Efficient production of germline transgenic chickens

using lentiviral vectors. _EMBO Rep._ 5, 728–733 (2004). Article  CAS  PubMed  PubMed Central  Google Scholar  * McNagny, K. M. et al. Thrombomucin, a novel cell surface protein that defines

thrombocytes and multipotent hematopoietic progenitors. _J. Cell Biol._ 138, 1395–1407 (1997). Article  CAS  PubMed  PubMed Central  Google Scholar  * Thornton, M. A. & Poncz, M.

Characterization of the murine platelet αIIb gene and encoded cDNA. _Blood_ 94, 3947–3950 (1999). Article  CAS  PubMed  Google Scholar  * Yvernogeau, L. et al. An in vitro model of hemogenic

endothelium commitment and hematopoietic production. _Development_ 143, 1302–1312 (2016). CAS  PubMed  Google Scholar  * Lassila, O., Eskola, J., Toivanen, P. & Dieterlen-Lievre, F.

Lymphoid stem cells in the intraembryonic mesenchyme of the chicken. _Scand. J. Immunol._ 11, 445–448 (1980). Article  CAS  PubMed  Google Scholar  * Jaffredo, T., Gautier, R., Brajeul, V.

& Dieterlen-Lièvre, F. Tracing the progeny of the aortic hemangioblast in the avian embryo. _Dev. Biol._ 224, 204–214 (2000). Article  CAS  PubMed  Google Scholar  * Taoudi, S. et al.

Progressive divergence of definitive haematopoietic stem cells from the endothelial compartment does not depend on contact with the foetal liver. _Development_ 132, 4179–4191 (2005). Article

  CAS  PubMed  Google Scholar  * Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L. & Luo, L. A global double-fluorescent Cre reporter mouse. _Genesis_ 45, 593–605 (2007). Article  CAS 

PubMed  Google Scholar  * Sorensen, I., Adams, R. H. & Gossler, A. DLL1-mediated Notch activation regulates endothelial identity in mouse fetal arteries. _Blood_ 113, 5680–5688 (2009).

Article  PubMed  CAS  Google Scholar  * 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, 102–116 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Kiel, M. J., Yilmaz, O. H., Iwashita, T., Terhorst, C. & Morrison, S. J. SLAM

family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. _Cell_ 121, 1109–1121 (2005). Article  CAS  PubMed  Google Scholar  *

Langfelder, P. & Horvath, S. WGCNA: an R package for weighted correlation network analysis. _BMC Bioinformatics_ 9, 559 (2008). Article  PubMed  PubMed Central  CAS  Google Scholar  *

Richard, C. et al. Endothelio-mesenchymal interaction controls _runx1_ expression and modulates the _notch_ pathway to initiate aortic hematopoiesis. _Dev. Cell_ 24, 600–611 (2013). Article

  CAS  PubMed  PubMed Central  Google Scholar  * Pardanaud, L. & Dieterlen-Lievre, F. Manipulation of the angiopoietic/hemangiopoietic commitment in the avian embryo. _Development_ 126,

617–627 (1999). Article  CAS  PubMed  Google Scholar  * Noden, D. M. Embryonic origins and assembly of blood vessels. _Am. Rev. Respir. Dis._ 140, 1097–1103 (1989). Article  CAS  PubMed 

Google Scholar  * Pudliszewski, M. & Pardanaud, L. Vasculogenesis and angiogenesis in the mouse embryo studied using quail/mouse chimeras. _Int. J. Dev. Biol._ 49, 355–361 (2005).

Article  CAS  PubMed  Google Scholar  * Nguyen, P. D. et al. Haematopoietic stem cell induction by somite-derived endothelial cells controlled by _meox1_. _Nature_ 512, 314–318 (2014).

Article  CAS  PubMed  Google Scholar  * Qiu, J. et al. Embryonic hematopoiesis in vertebrate somites gives rise to definitive hematopoietic stem cells. _J. Mol. Cell Biol._ 8, 288–301

(2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Palis, J. Hematopoietic stem cell-independent hematopoiesis: emergence of erythroid, megakaryocyte and myeloid potential in

the mammalian embryo. _FEBS Lett._ 590, 3965–3974 (2016). Article  CAS  PubMed  Google Scholar  * Chen, M. J. et al. Erythroid/myeloid progenitors and hematopoietic stem cells originate from

distinct populations of endothelial cells. _Cell Stem Cell_ 9, 541–552 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Christensen, J. L., Wright, D. E., Wagers, A. J. &

Weissman, I. L. Circulation and chemotaxis of fetal hematopoietic stem cells. _PLoS Biol._ 2, E75 (2004). Article  PubMed  PubMed Central  Google Scholar  * Beaudin, A. E. et al. A

transient developmental hematopoietic stem cell gives rise to innate-like B and T cells. _Cell Stem Cell_ 19, 768–783 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Plein,

A., Fantin, A., Denti, L., Pollard, J. W. & Ruhrberg, C. Erythro-myeloid progenitors contribute endothelial cells to blood vessels. _Nature_ 562, 223–228 (2018). Article  CAS  PubMed 

PubMed Central  Google Scholar  * Chevallier, A., Kieny, M. & Mauger, A. Limb–somite relationship: origin of the limb musculature. _J. Embryol. Exp. Morphol._ 41, 245–258 (1977). CAS 

PubMed  Google Scholar  * Christ, B., Jacob, H. J. & Jacob, M. Experimental analysis of the origin of the wing musculature in avian embryos. _Anat. Embryol._ _(__Berl__)_ 150, 171–186

(1977). Article  CAS  Google Scholar  * Hamburger, V. & Hamilton, H. L. A series of normal stages in the development of the chick embryo. _J. Morphol._ 88, 49–92 (1951). Article  CAS 

PubMed  Google Scholar  * Boisset, J. C., Andrieu-Soler, C., van Cappellen, W. A., Clapes, T. & Robin, C. Ex vivo time-lapse confocal imaging of the mouse embryo aorta. _Nat. Protoc._ 6,

1792–1805 (2011). Article  CAS  PubMed  Google Scholar  * Srinivas, S. et al. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. _BMC Dev. Biol._ 1,

4 (2001). Article  CAS  PubMed  PubMed Central  Google Scholar  * Engleka, K. A. et al. Insertion of Cre into the _Pax3_ locus creates a new allele of _splotch_ and identifies unexpected

_Pax3_ derivatives. _Dev. Biol._ 280, 396–406 (2005). Article  CAS  PubMed  Google Scholar  * Huang, D. W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large

gene lists using DAVID bioinformatics resources. _Nat. Protoc._ 4, 44–57 (2009). Article  CAS  Google Scholar  * Huang, D. W. et al. Extracting biological meaning from large gene lists with

DAVID. _Curr. Protoc. Bioinformatics_ 13, 13.11 (2009). Google Scholar  Download references ACKNOWLEDGEMENTS We apologize to those investigators whose important work we were unable to cite

or describe due to space constraints. We thank D. Traver for critical reading of the manuscript and S. Gournet for help in image preparation. We are indebted to R. Adams for his sharing of

the Tg(Cdh5Cre/ERT2) transgenic mouse line and to S. Germain for providing us with the founders. We thank the Cell Imaging and Flow Cytometry facility of the IBPS (Paris, France) for access

and technical support in confocal image acquisition and the Genom’IC platform at Cochin Institute, Paris for their invaluable help with transcriptomic samples treatment. This work was

supported by CNRS, UPMC, Fondation Les Treilles (L.Y.), an EMBO short-term fellowship (L.Y.), MERI and FRM PhD fellowships (H.K.) and grants from FRM (DEQ20100318258) and an ANR/CIRM joint

grant (ANR/CIRM 0001–02) in T.J.’s laboratory. The production of the GFP+ chicken embryos was supported by grants from BBSRC and the Wellcome Trust. Part of the work and L.Y. were supported

by a European Research Council grant (ERC, project no. 220-H75001EU/HSCOrigin-309361) and a TOP subsidy from NWO/ZonMw (912.15.017) in C.R.’s laboratory. AUTHOR INFORMATION Author notes *

Laurent Yvernogeau Present address: Hubrecht Institute, Utrecht, Netherlands * Michèle Souyri Present address: UMRS 1131, Institut Universitaire d’Hématologie, Hôpital Saint Louis, Paris,

France * These authors contributed equally: Rodolphe Gautier, Laurence Petit. AUTHORS AND AFFILIATIONS * IBPS, Laboratoire de Biologie du Développement CNRS UMR7622, Inserm U1156, Sorbonne

Université, Paris, France Laurent Yvernogeau, Rodolphe Gautier, Laurence Petit, Hanane Khoury, Pierre Charbord, Michèle Souyri & Thierry Jaffredo * Institut Mondor de Recherches

Biomédicales, Créteil, France Frédéric Relaix * Institut Jacques Monod, Paris, France Vanessa Ribes * The Roslin Institute and R(D)SVS, University of Edinburgh, Edinburgh, UK Helen Sang *

Hubrecht Institute, Utrecht, Netherlands Catherine Robin * Regenerative Medicine Center, University Medical Center Utrecht, Utrecht, Netherlands Catherine Robin Authors * Laurent Yvernogeau

View author publications You can also search for this author inPubMed Google Scholar * Rodolphe Gautier View author publications You can also search for this author inPubMed Google Scholar *

Laurence Petit View author publications You can also search for this author inPubMed Google Scholar * Hanane Khoury View author publications You can also search for this author inPubMed 

Google Scholar * Frédéric Relaix View author publications You can also search for this author inPubMed Google Scholar * Vanessa Ribes View author publications You can also search for this

author inPubMed Google Scholar * Helen Sang View author publications You can also search for this author inPubMed Google Scholar * Pierre Charbord View author publications You can also

search for this author inPubMed Google Scholar * Michèle Souyri View author publications You can also search for this author inPubMed Google Scholar * Catherine Robin View author

publications You can also search for this author inPubMed Google Scholar * Thierry Jaffredo View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS

L.Y. and T.J. conceived the project. L.Y. and T.J. designed the experiments on chicken embryos, performed chicken somite grafts and interpreted data. L.Y. performed chicken grafts, carried

out immunohistological analysis and performed FACS analysis. R.G. performed chicken graft analysis. L.P. performed all mouse work, including transplantation studies, and analysed the data.

H.S. provided GFP transgenic chickens. V.R. and F.R. provided the Pax3CRE X Rosa mTmG mice and critical associated materials. H.K. analysed the YFP+ transcriptome versus the YFP−

transcriptome. P.C. analysed transcriptomes, performed network analyses and interpreted data. M.S. designed Pax3CRE X ROSAmTmG and the initial VECAD-Cre ERT X Rosa 26 approaches, analysed

the data and provided the additional mouse embryonic and BM adult transcriptomes. C.R. and L.Y. designed and supervised the experiments on adult chickens and the live imaging on sections.

T.J. wrote the manuscript. L.Y. and C.R. provided significant input for data analyses, findings, interpretations and manuscript writing. CORRESPONDING AUTHOR Correspondence to Thierry

Jaffredo. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to

jurisdictional claims in published maps and institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 PSM REMOVAL EXPERIMENT. A, E2 (15-somite stage, HH11/12) wild-type chicken embryo

submitted to a PSM ablation. White arrows located the regions of transverse histological analysis at two representative anterior and posterior levels displayed in 1 and 2. MEP21 (red) and

DAPI (blue) staining showed that only the PSM was removed without disturbing the surrounding tissues (10 chicken embryos). B, Whole mounts of two PSMs isolated from six E2 (15-somite stage,

HH11/12) wild-type embryos and stained with DAPI (blue), MEP21 (red) and CD45 (green) antibodies, showing that PSMs do not contain any ECs or HCs after dissection. Two independent examples

are shown (#1, #2) (6 chicken embryos). C, Mid trunk, thick transverse section of an E3 chicken embryo stained with MEP21 (red) and CD45 (green) antibodies shown as positive control of the

immunostainings. Of note, PSMs (B) and thick slice (C) were stained at the same time, using the same antibody solutions. D, Representative FACS analysis on single PSMs isolated from 8

independent 13-15 somite stage embryos showing no contamination by haematopoietic (CD45) or endothelial (MEP21) cells. Controls are two whole embryos without PSMs showing the presence of

CD45+ haematopoietic and MEP21+ endothelial cells. Ao, Aorta; Ec, Ectoderm; En, Endoderm; Nc, Notochord; NT, Neural Tube; PSM, Presomitic mesoderm; So, Somatopleura; Sp, Splanchnopleura.

Scale bars, 200µm in A, 25μm in A1, A2; 50μm in B and C. EXTENDED DATA FIG. 2 GFP+CD45+ HAEMATOPOIETIC CELLS IN CIRCULATING BLOOD OF THE GRAFTED OR CONTROL EMBRYOS WITH AGE. A-F, CD45

expression in the sample (left) and GFP plot in the CD45+ population (right). A-D, chimeric embryos. A, E4 embryo. B, E8 embryo. C, E12 embryo. D, E16 embryo. Of note, only E16 embryos show

GFP+ cells within the CD45+ fraction. E, E12 GFP transgenic embryo. F, E12 WT embryo. 23 chicken embryos analysed. See Extended Data Table 1 for details. Data representative of 4 independent

experiments. EXTENDED DATA FIG. 3 GFP+ SOMITE-DERIVED CELLS CONTRIBUTE TO E16/E17 BONE MARROW CD45+ HAEMATOPOIETIC PRODUCTION. A-C, Representative fluorescent cross section of the limb bone

showing the co-distribution of GFP+ sinusoid cells and CD45+ cells. B, Enlargement of the bone marrow showing GFP+CD45+ cells lining the sinusoids. C, Enlargement of the frame in B, showing

the double positive cells. D-L, Series of confocal cross sections separated by 1µm, showing a GFP+CD45+ cell (white arrow). D, G, J, CD45 staining. E, H, K, GFP signal. F, I, L, Merged

CD45, GFP and DAPI. Right and bottom banners correspond to YZ and XZ projections of the confocal image, respectively. Experiments were repeated 16 times with similar results (see Extended

Data Table 1 for details). Confocal analysis was performed 5 times with similar results. Scale bars in A, 200µm; in B, 40µm; in C, 15µm; in D-L, 20µm. EXTENDED DATA FIG. 4 FLOW CYTOMETRY

ANALYSIS REVEALS THE PRESENCE OF ENDOTHELIAL AND HAEMATOPOIETIC CELLS EXPRESSING GFP IN THE E16-GRAFTED CHICKEN BM. Distribution of CD45, MEP21, CD41, c-KIT, and CD3 populations in grafted

(A, B) or wild-type (C, D) BM mononucleated cells. A, n = 12 independently grafted animals were used for CD45, CD41, c-KIT and CD3 expression and n = 8 for MEP21. B, Representative flow

cytometry analysis of an E16/E17 grafted BM showing that GFP+ cells derived from the PSM graft contributed to all lineages. Numbers in green indicate the percentage of GFP+ cells in each

population (that is in CD45+, MEP21+, CD41+, c-KIT+ and CD3+ populations). C, n = 3 independent WT animals were used for CD45, CD41, c-KIT expression analysis, n = 2 animals were used for

MEP21 and CD3 expression analysis. D, Representative flow cytometry analysis of an E16/E17 WT BM. Data in B representative of 4 independent experiments, 3 in D. Error bars are mean ± SEM.

Source data EXTENDED DATA FIG. 5 PSM-DERIVED CELLS PROVIDE HAEMATOPOIETIC PRECURSORS ABLE TO COLONIZE THE SECONDARY HAEMATOPOIETIC ORGANS OF E16/E17 GRAFTED CHICKENS. A, F, I, Global views

of the thymus (A), spleen (F) and bursa of Fabricius (I) of an E16 chicken colonized by GFP+ PSM-derived grafted cells. A, Most of the thymus lobes were colonized by GFP+ cells (3 chickens).

B, Immunostaining (with DAPI) of a thymus transverse section. C-D, Higher magnification of the frame in (B). GFP (C) and merge of DAPI and GFP (D). E, Flow cytometry analysis of an E16/E17

thymus (2 chickens) revealed the contribution of GFP+ PSM-derived cells to the whole thymus and more precisely to the T lineage (GFP+CD4+). G, Higher magnification of the frame in (F),

showing dispersed GFP+ cells in the spleen (2 chickens). H, Flow cytometry analysis of the spleen, showing the contribution of GFP+ PSM-derived cells to the whole spleen and more precisely

to the CD45+ haematopoietic lineage (2 chickens). J, Higher magnification of the frame in (I) showing dispersed GFP+ cells in the bursa (2 chickens). K, Flow cytometry analysis showing that

the bursa of Fabricius, located far away from the PSM graft, was also colonized by PSM-derived haematopoietic progenitors that derived from the graft (2 chickens). Drawings are shown on the

left side of the organ pictures to locate the thymus, spleen and bursa of Fabricius in an E16/E17 chicken. Scale bars in A, B, F, I: 100µm. EXTENDED DATA FIG. 6 FLOW CYTOMETRY ANALYSIS

REVEALS THE PRESENCE OF ENDOTHELIAL AND HAEMATOPOIETIC CELLS EXPRESSING GFP IN ADULT BM GRAFTED CHICKENS. Distribution of CD45, MEP21, CD41, c-KIT, CD3 and KUL1 populations in grafted (A, B)

and wild-type (C, D) adult BM mononucleated cells. A, Grafted chickens. CD45 (n = 7), MEP21 (n = 4), CD41 (n = 7), c-KIT (n = 7), CD3 (n = 7) and KUL1 (n = 6) independent chickens were used

for the analysis. B, Representative multilineage flow cytometry analysis of a grafted adult BM showing that GFP+ cells derived from the PSM graft contributed to all lineages. Numbers in

green indicate the percentages of GFP+ cells in each population (that is in CD45+, MEP21+, CD41+, c-KIT+, CD3+ and KUL1+ populations). C, Wild-type animals. CD45 (n = 5), CD41 (n = 4), c-KIT

(n = 5), CD3 (n = 4) and KUL1 (n = 4); independent animals were used for the analysis. n = 2 animals were used for MEP21 expression analysis. D, Representative multilineage flow cytometry

analysis of a wild-type adult BM showing the absence of GFP staining. Error bars represent mean ± SEM. Data in B and D representative of 4 independent experiments. Source data EXTENDED DATA

FIG. 7 PAX3+ SOMITE-DERIVED CELLS DO NOT CONTRIBUTE TO THE AORTA HAEMATOPOIEISIS BUT CONTRIBUTE TO THE ENDOTHELIAL AND HAEMATOPOIETIC LINEAGES IN THE LATE FETAL BM. A, Transverse section

through the aorta of an E10.5 Pax3 GFP+ mouse embryo at the mid-trunk level. CD31 (red, left), Pax3-GFP (green, middle) double staining counterstained with DAPI (blue). Right panel

represents the merge (5 mice). B, Cross section of a Pax3KICRE; ROSA GFP embryo at E8.5 showing that this model tags the somite and recapitulates the Pax3 GFP expression at the same stage (5

mice). C, Flow cytometry analysis of the endothelial and haematopoietic lineages in the BM of Pax3-Cre/mTmG fetuses at E19-E21. n = 3 fetuses from 3 independent experiments. D, Flow

cytometry analysis showing GFP+ cell (that is somite-derived cell) contribution to the different endothelial and haematopoietic populations. Mouse somite-derived cells mostly contributed to

fetal/new-born BM CD3+, Sca1+c-Kit+ and CD144+CD45+ cell populations. n = 3 foetuses from 3 independent experiments. E, Representative GFP expression in mononucleated BM cells. F,

Representative flow cytometry analysis of the mononucleated BM cells revealed GFP+ cells mainly in the CD144+CD45+ population. G, Flow cytometry analysis showing the presence of GFP+ cells

in the Sca1+c-KIT+ population (that is HSPC population). H, Negative controls are non-recombined fetuses. Data represent mean ± SEM. Data in E-H representative of 2 independent experiments.

Scale bars 350µm in A; 200µm in B. Source data EXTENDED DATA FIG. 8 YFP EXPRESSION ON NEGATIVE CONTROLS AND YFP TRACING FOLLOWING INDUCTION IN VE-CADHERIN+ CELLS AT DIFFERENT TIME POINTS

AFTER BIRTH. A, Flow cytometry analysis for the presence of YFP+ cells in the CD45+ fraction of the BM immediately after the last tamoxifen injection. Representative new-born mice injected

with tamoxifen at post-natal days 1, 2, and 3 and analysed for the expression of CD45 and YFP at day 4. No CD45+ YFP+ cells were found indicating the absence of contamination by

haematopoietic progenitors expressing VE-Cadherin (11 animals in 3 independent experiments). The gating strategy is shown for mouse #1. B, C, BM from a representative non-induced mouse at 27

post-natal days. B, Representative flow cytometry analysis showing the absence of YFP expression in mononucleated BM cells and in the CD144−CD45+, CD144+CD45+ and CD144+CD45− cell fractions

(4 animals in 2 independent experiments). C, Representative flow cytometry analysis of the mononucleated BM cells showing the absence of YFP expression in the Sca1+c-KIT+ population (that

is HSPC population) and in the different HSC, HPC and MPP populations (2 independent experiments). D, Scheme showing the activation of YFP in VE-Cadherin+ (CDH5) cells by tamoxifen injection

at different time points after birth (coloured arrows). E, Analysis of the percentages of total YFP+ cells, and YFP+ cells in CD144+CD45− and LSK populations at 21 days post tamoxifen

injection. Of note, the x axis values represent the age of the mice from the time of tamoxifen injection. n = 3 mice for d1, n = 6 for d10, n = 7 for d21, n = 7 for d36, n = 5 for d68. Error

bars are mean ± SEM. Data in A-C representative of 2 independent experiments. Source data EXTENDED DATA FIG. 9 HAEMOGENIC POTENTIAL OF BM ECS AND TRANSCRIPTOME CHARACTERISATION OF THE YFP+

AND YFP− LSK CELLS. A, representative flow cytometry profile showing the gates used to isolate the CD144+CD45− and the CD144−CD45+ populations. Image representative of 7 mice. B-E,

representative pictures of the CD144−CD45+ (B) and CD144+CD45− (C, D) cells from 8-day old mouse bone marrow in culture in the endothelial/haematopoietic medium. A high number of floating

cells are present in the CD144−CD45+ cells fraction (B). Flat adherent cells (in C) and round, floating, haematopoietic-like cells (arrows in C and D) are present in the CD144+CD45− cell

fraction after 4 days of culture. Image representative of 3 experiments. Bar = 10µm. E, F, representative FACS analysis of the CD45+ populations generated after 4 days of culture from the

CD144−CD45+ (E) and the CD144+CD45− (F) cell populations. Image representative of 3 experiments. G, PCA with the entire set of mRNAs (30,922 genes) as variables and the basic set of YFP+

(green) and YFP− (red) LSK cells as observations. The two types of transcriptomes were strongly separated (3 biological replicates per population). H, Major GO categories given by DAVID for

the gene sets up-regulated in YFP+ (green) and YFP− (red) LSK cells. I, Hierarchical clustering obtained with the 23 samples (3 biological replicates per population except for HC BM and LSK

CD150+ BM where quadruplicates were used) as observations and the gene set of 2056 DEGs (986 up, 1070 down) as variables was generated from the PCA displayed in Fig. 6a, bottom panel. Branch

organisation reflects the association between the different samples displayed on the PCA. Scale Bars: 50µm in B, C, D. EXTENDED DATA FIG. 10 LONG-TERM REPOPULATION ANALYSIS IN THE

PERIPHERAL BLOOD OF RECIPIENTS TRANSPLANTED EITHER WITH YFP− OR YFP+ LSK CELLS. A, B, Flow cytometry analysis of circulating blood from 6 (A) and 4 (B) C57Bl/6-CD45.1 recipient mice at 11

weeks injected with 2,000 YFP− (A) or YFP+ (B) LSK cells (C57Bl/6-CD45.2), respectively. Mice recipients transplanted with YFP− LSK cells displayed a more robust reconstitution than the ones

transplanted with YFP+ LSK cells. Of note, two mice in the LSK YFP+ series died before two weeks post-injection. C, Back-gating within the GFP+ population of the mononucleated bone marrow

cells from mouse #d at 14 weeks of reconstitution. Dot plot representation of flow cytometry multilineage analysis showing Gr1 (granulocytes), CD11b (macrophages/monocytes), B220 and CD19 (B

cells), CD4 (T cells), CD8 (T cells), Ter119 and CD71 (red cells), CD41 and CD61 (megakaryocytes) staining. The YFP population was more prominent in B220, CD19, CD71, Gr1 and CD8 fractions.

Data in A-C representative of 2 independent experiments. SUPPLEMENTARY INFORMATION REPORTING SUMMARY SUPPLEMENTARY VIDEO 1 Whole-mount staining of E16 chicken bone marrow after GFP+ PSM

graft. The bone marrow was stained with anti-MEP21 (endothelial marker, red), anti-CD45 (haematopoietic marker, blue) and anti-GFP (green, which revealed grafted PSM-derived cells)

antibodies. The marrow was heavily colonized by GFP+ PSM-derived cells that contributed to the whole bone marrow vascularization (_n_ = 3). SUPPLEMENTARY VIDEO 2 A GFP+ endothelial cell

co-expressing CD45 as revealed by confocal analysis of an E16-grafted BM. Series of confocal sections separated by 1 µm, showing a GFP+CD45+ cell (white arrow). Panels: upper left, DAPI;

upper right, CD45; lower left, GFP; lower right, merged. Right and bottom banners correspond to _y–z_ and _x–z_ projections of the confocal image, respectively (_n_ = 5 in three independent

embryos). SUPPLEMENTARY VIDEO 3 GFP+ endothelial cells co-expressing CD41 are integrated in the vascular endothelium of E16-grafted BM. Series of confocal sections of 1 µm, showing a

GFP+CD41+ cell integrated in the GFP+ vascular endothelium (_n_ = 8 in three independent embryos). SUPPLEMENTARY VIDEO 4 Emergence of CD41+ haematopoietic precursors from GFP+ endothelial

cells in the bone marrow. A, Time-lapse live imaging of a transversal E16 bone marrow slice showing the emergence of a GFP+ cell from the endothelium of a blood vessel. B, After time-lapse

imaging (A) the section was stained again with anti-CD41-PE antibodies. The newly emerged GFP+ cell expressed the haematopoietic marker CD41. Of note, this cell was CD41- at the beginning of

imaging. Time is in hours and minutes (_n_ = 2 independent experimental animals with five different sections per slide). Scale bar, 15 µm. SUPPLEMENTARY VIDEO 5 Whole-mount staining of an

E16 chicken thymus after GFP+ PSM graft. The thymus was stained with anti-MEP21 (endothelial marker, red), anti-CD45 (haematopoietic marker, blue) and anti-GFP (green, which revealed grafted

PSM-derived cells) antibodies. The thymus was colonized by GFP+ PSM-derived cells, some of the lobes being more colonized than others. GFP+ cells could colonize secondary haematopoietic

organs that are located far from the grafting site. (_n_ = 2 independent experimental animals). EXTENDED DATA TABLE 1 Number of grafted chickens, time points of analyses and type of analyses

performed. This table summarizes the number of chicken samples according to the age at analysis; the organs or tissues analysed, that is, the thymus, bone marrow, spleen; the kind of

analysis applied, that is, FACS, immunostaining, confocal imaging or videos; and the number of cases analysed for each time point. EXTENDED DATA TABLE 2 List of anti-mouse and anti-chicken

antibodies. List of anti-mouse (first) and anti-chicken (second) antibodies used in the study, including the conjugated fluorochrome when applicable, clone of origin, cell specificity,

supplier and catalogue number. SOURCE DATA SOURCE DATA FIG. 2 Statistical source data SOURCE DATA FIG. 4 Statistical source data SOURCE DATA FIG. 5 Statistical source data SOURCE DATA FIG. 6

Statistical source data SOURCE DATA EXTENDED DATA FIG. 4 Statistical source data SOURCE DATA EXTENDED DATA FIG. 6 Statistical source data SOURCE DATA EXTENDED DATA FIG. 7 Statistical source

data SOURCE DATA EXTENDED DATA FIG. 8 Statistical source data RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Yvernogeau, L., Gautier, R., Petit, L. _et

al._ In vivo generation of haematopoietic stem/progenitor cells from bone marrow-derived haemogenic endothelium. _Nat Cell Biol_ 21, 1334–1345 (2019).

https://doi.org/10.1038/s41556-019-0410-6 Download citation * Received: 06 November 2018 * Accepted: 23 September 2019 * Published: 04 November 2019 * Issue Date: November 2019 * DOI:

https://doi.org/10.1038/s41556-019-0410-6 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not

currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative