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Activation of the TGFβ pathway impairs endothelial to haematopoietic transition


Özge Vargel, Yang Zhang, Kinga Kosim, Kerstin Ganter, Sophia Foehr, Yannicka Mardenborough, Maya Shvartsman, Anton J. Enright, Jeroen Krijgsveld and Christophe Lancrin.

Scientific Reports. 2016 Feb 19.

The endothelial to haematopoietic transition (EHT) is a key developmental process where a drastic change of endothelial cell morphology leads to the formation of blood stem and progenitor cells during embryogenesis. As TGFβ signalling triggers a similar event during embryonic development called epithelial to mesenchymal transition (EMT), we hypothesised that TGFβ activity could play a similar role in EHT as well. We used the mouse embryonic stem cell differentiation system for in vitro recapitulation of EHT and performed gain and loss of function analyses of the TGFβ pathway. Quantitative proteomics analysis showed that TGFβ treatment during EHT increased the secretion of several proteins linked to the vascular lineage. Live cell imaging showed that TGFβ blocked the formation of round blood cells. Using gene expression profiling we demonstrated that the TGFβ signalling activation decreased haematopoietic genes expression and increased the transcription of endothelial and extracellular matrix genes as well as EMT markers. Finally we found that the expression of the transcription factor Sox17 was up-regulated upon TGFβ signalling activation and showed that its overexpression was enough to block blood cell formation. In conclusion we showed that triggering the TGFβ pathway does not enhance EHT as we hypothesised but instead impairs it.

 

GFI1 proteins orchestrate the emergence of haematopoietic stem cells through recruitment of LSD1


Roshana Thambyrajah*, Milena Mazan*, Rahima Patel, Victoria Moignard, Monika Stefanska, Elli Marinopoulou, Yaoyong Li, Christophe Lancrin, Thomas Clapes, Tarik Möröy, Catherine Robin, Crispin Miller, Shaun Cowley, Berthold Göttgens, Valérie Kouskoff and Georges Lacaud.

Nature Cell Biology. 2016 Jan;18:21-32. Epub 2015 Nov 30.
PubMed

*These authors contributed equally to the work.

In vertebrates, the first haematopoietic stem cells (HSCs) with multi-lineage and long-term repopulating potential arise in the AGM (aorta-gonad-mesonephros) region. These HSCs are generated from a rare and transient subset of endothelial cells, called haemogenic endothelium (HE), through an endothelial-to-haematopoietic transition (EHT). Here, we establish the absolute requirement of the transcriptional repressors GFI1 and GFI1B (growth factor independence 1 and 1B) in this unique trans-differentiation process. We first demonstrate that Gfi1 expression specifically defines the rare population of HE that generates emerging HSCs. We further establish that in the absence of GFI1 proteins, HSCs and haematopoietic progenitor cells are not produced in the AGM, revealing the critical requirement for GFI1 proteins in intra-embryonic EHT. Finally, we demonstrate that GFI1 proteins recruit the chromatin-modifying protein LSD1, a member of the CoREST repressive complex, to epigenetically silence the endothelial program in HE and allow the emergence of blood cells.

 

GFI1 and GFI1B control the loss of endothelial identity of hemogenic endothelium during hematopoietic commitment.


Lancrin C, Mazan M, Stefanska M, Patel R, Lichtinger M, Costa G, Vargel O, Wilson NK, Möröy T, Bonifer C, Göttgens B, Kouskoff V, Lacaud G..
Blood. 2012 Jul 12;120(2):314-22. Epub 2012 Jun 5.
PubMed

Recent studies have established that during embryonic development hematopoietic progenitors and stem cells are generated from hemogenic endothelium precursors through a process termed endothelial to hematopoietic transition (EHT). The transcription factor RUNX1 is essential for this process but its main downstream effectors remain largely unknown. Here we report the identification of Gfi1 and Gfi1b as direct targets of RUNX1 and critical regulators of EHT. GFI1 and GFI1B are able to trigger, in the absence of RUNX1, the down-regulation of endothelial markers and the formation of round cells, a morphological change characteristic of EHT. Conversely, blood progenitors in Gfi1 and Gfi1b deficient embryos maintain the expression of endothelial genes. Moreover, those cells are not released from the yolk sac and disseminated into embryonic tissues. Taken together, our findings demonstrate a critical and specific role of the GFI1 transcription factors in the first steps of the process leading to the generation of hematopoietic progenitors from hemogenic endothelium.

 

Identification and characterization of a novel transcriptional target of RUNX1/AML1 at the onset of hematopoietic development.


Ferreras, C., Lancrin, C., Lie-A-Ling, M., Kouskoff, V. & Lacaud, G.
Blood. 2011 Jul 21;118(3):594-7. Epub 2011 Apr 15.
PubMed

Although the critical requirement for the transcription factor RUNX1/AML1 at the onset of hematopoietic development is well established, little is known about its transcriptional targets at this pivotal stage of blood development. Using microarrays, we identified the uncharacterized gene AI467606 as a gene whose expression level is dramatically reduced in the absence of RUNX1. We further demonstrated by chromatin immunoprecipitation and promoter assay a direct regulation of its transcription by RUNX1. Using a bacterial artificial chromosome transgenic approach, we established that AI467606 is expressed during the development of the hematopoietic system in vivo and in vitro and that its expression is detected within the CD41(+) population and marks definitive hematopoietic potential. Similarly in the adult mouse all hematopoietic cell lineages, except mature erythrocytes, express AI467606. Taken together these findings indicate that AI467606 is a novel transcriptional target of RUNX1/AML1 at the onset of hematopoietic development that is extensively expressed within the hematopoietic system.

 

The sequential expression of CD40 and Icam2 defines progressive steps in the formation of blood precursors from the mesoderm germ layer.


Pearson, S., Lancrin, C., Lacaud, G. & Kouskoff, V.
Stem Cells. 2010 Jun;28(6):1089-98.
PubMed

During embryogenesis, the hematopoietic program is specified from the mesodermal germ layer through the formation of hemangioblast. This precursor gives rise to a hemogenic endothelium that later on matures to generate primitive and definitive hematopoietic precursors. A lack of specific cell surface markers to identify cells with discrete developmental potential is a major hurdle in the quest to further understand the cellular and molecular program governing blood formation. In the present study, we identify CD40 and Icam2, two markers typically associated with the adult immunological compartment, as expressed at the earliest stages of blood specification both in vitro and in vivo. Using in vitro serum-free culture conditions that support the efficient and directed differentiation of embryonic stem cells, we show that the sequential expression of CD40 and Icam2 delineate a transition in the acquisition of the blood potential from hemangioblast to hemogenic endothelium leading to the formation of primitive and definitive hematopoietic progenitors. CD40 is transiently expressed at the onset of blood development and marks first the hemangioblast then the hemogenic endothelium but is no longer expressed on fully committed hematopoietic precursors within the fetal liver. In contrast, Icam2 is first expressed on the hemogenic endothelium and its expression persists on fetal liver hematopoietic progenitors. Taken together, our data identify novel cell surface markers allowing us to further refine our understanding of the events marking progressive hematopoietic commitment from the mesoderm germ layer.

Blood cell generation from the hemangioblast.


Lancrin, C., Sroczynska, P., Serrano, A.G., Gandillet, A., Ferreras, C., Kouskoff, V. & Lacaud, G.
J Mol Med. 2010 Feb;88(2):167-72. Epub 2009 Oct 25.
PubMed

Understanding how blood cells are generated is important from a biological perspective but also has potential implications in the treatment of blood diseases. Such knowledge could potentially lead to defining new conditions to amplify hematopoietic stem cells (HSCs) or could translate into new methods to produce HSCs, or other types of blood cells, from human embryonic stem cells or induced pluripotent stem cells. Additionally, as most key transcription factors regulating early hematopoietic development have also been implicated in various types of leukemia, understanding their function during normal development could result in a better comprehension of their roles during abnormal hematopoiesis in leukemia. In this review, we discuss our current understanding of the molecular and cellular mechanisms of blood development from the earliest hematopoietic precursor, the hemangioblast, a precursor for both endothelial and hematopoietic cell lineages.

The differential activities of Runx1 promoters define milestones during embryonic hematopoiesis.


Sroczynska, P., Lancrin, C., Kouskoff, V. & Lacaud, G.
Blood. 2009 Dec 17;114(26):5279-89. Epub 2009 Oct 26.
PubMed

The transcription factor RUNX1/AML1 is a master regulator of hematopoietic development. Its spatiotemporal expression is tightly regulated during embryonic development and is under the control of 2 alternative promoters, distal and proximal. Despite the functional significance of Runx1, the relative and specific activities of these 2 promoters remain largely uncharacterized. To investigate these activities, we introduced 2 reporter genes under the control of the proximal and distal promoters in embryonic stem cell and transgenic mouse lines. Our study reveals that both in vitro and in vivo the proximal Runx1 isoform marks a hemogenic endothelium cell population, whereas the subsequent expression of distal Runx1 defines fully committed definitive hematopoietic progenitors. Interestingly, hematopoietic commitment in distal Runx1 knockout embryos appears normal. Altogether, our data demonstrate that the differential activities of the 2 Runx1 promoters define milestones of hematopoietic development and suggest that the proximal isoform plays a critical role in the generation of hematopoietic cells from hemogenic endothelium. Identification and access to the discrete stages of hematopoietic development defined by the activities of the Runx1 promoters will provide the opportunity to further explore the cellular and molecular mechanisms of hematopoietic development.

Early chromatin unfolding by RUNX1: a molecular explanation for differential requirements during specification versus maintenance of the hematopoietic gene expression program.


Hoogenkamp, M.*, Lichtinger, M.*, Krysinska, H.*, Lancrin, C.*, Clarke, D., Williamson, A., Mazzarella, L., Ingram, R., Jorgensen, H., Fisher, A., Tenen, D.G., Kouskoff, V., Lacaud, G. & Bonifer, C.
Blood. 2009 Jul 9;114(2):299-309. Epub 2009 Apr 1.
* These authors contributed equally to this study.
PubMed

At the cellular level, development progresses through successive regulatory states, each characterized by their specific gene expression profile. However, the molecular mechanisms regulating first the priming and then maintenance of gene expression within one developmental pathway are essentially unknown. The hematopoietic system represents a powerful experimental model to address these questions and here we have focused on a regulatory circuit playing a central role in myelopoiesis: the transcription factor PU.1, its target gene colony-stimulating-factor 1 receptor (Csf1r), and key upstream regulators such as RUNX1. We find that during ontogeny, chromatin unfolding precedes the establishment of active histone marks and the formation of stable transcription factor complexes at the Pu.1 locus and we show that chromatin remodeling is mediated by the transient binding of RUNX1 to Pu.1 cis-elements. By contrast, chromatin reorganization of Csf1r requires prior expression of PU.1 together with RUNX1 binding. Once the full hematopoietic program is established, stable transcription factor complexes and active chromatin can be maintained without RUNX1. Our experiments therefore demonstrate how individual transcription factors function in a differentiation stage-specific manner to differentially affect the initiation versus maintenance of a developmental program.

The haemangioblast generates haematopoietic cells through a haemogenic endothelium stage.


Lancrin, C., Sroczynska, P., Stephenson, C., Allen, T., Kouskoff, V. & Lacaud, G.
Nature. 2009 Feb 12;457(7231):892-5. Epub 2009 Jan 28.
PubMed

It has been proposed that during embryonic development haematopoietic cells arise from a mesodermal progenitor with both endothelial and haematopoietic potential called the haemangioblast. A conflicting theory instead associates the first haematopoietic cells with a phenotypically differentiated endothelial cell that has haematopoietic potential (that is, a haemogenic endothelium). Support for the haemangioblast concept was initially provided by the identification during mouse embryonic stem cell differentiation of a clonal precursor, the blast colony-forming cell (BL-CFC), which gives rise to blast colonies with both endothelial and haematopoietic components. Although recent studies have now provided evidence for the presence of this bipotential precursor in vivo, the precise mechanism for generation of haematopoietic cells from the haemangioblast still remains completely unknown. Here we demonstrate that the haemangioblast generates haematopoietic cells through the formation of a haemogenic endothelium intermediate, providing the first direct link between these two precursor populations. The cell population containing the haemogenic endothelium is transiently generated during BL-CFC development. This cell population is also present in gastrulating mouse embryos and generates haematopoietic cells on further culture. At the molecular level, we demonstrate that the transcription factor Tal1 (also known as Scl; ref. 10) is indispensable for the establishment of this haemogenic endothelium population whereas the core binding factor Runx1 (also known as AML1; ref. 11) is critical for generation of definitive haematopoietic cells from haemogenic endothelium. Together our results merge the two a priori conflicting theories on the origin of haematopoietic development into a single linear developmental process.

Extrathymic hemopoietic progenitors committed to T cell differentiation in the adult mouse.


Arcangeli, M.L., Lancrin, C., Lambolez, F., Cordier, C., Schneider, E., Rocha, B. & Ezine, S.
J Immunol. 2005 Feb 15;174(4):1980-8.
PubMed

The role of the thymus in T cell commitment of hemopoietic precursor is yet controversial. We previously identified a major T cell progenitor activity in precursor cells isolated from bone marrow-derived spleen colonies. In this study, we characterize the properties of these pre-T cells. We demonstrate that they have unique phenotype and can be generated in a total absence of any thymic influence. Indeed, even when studied at the single-cell level, extrathymic T cell-committed precursors express T cell-specific genes. Moreover, these cells are not committed to a particular T cell differentiation pathway because they can generate both extrathymic CD8alphaalpha+ intraepithelial lymphocytes and thymus-derived conventional thymocytes. We also compared these pre-T cells with fully T cell-committed thymic progenitors. When tested in vitro or by direct intrathymic transfer, these cells have a low clonogenic activity. However, after i.v. transfer, thymus repopulation is efficient and these precursors generate very high numbers of peripheral T cells. These results suggest the existence of extra steps of pre-T cell maturation that improve thymus reconstitution capacity and that can be delivered even after full T cell commitment. Consequently, our studies identify a source of extrathymic progenitors that will be helpful in defining the role of the thymus in the earliest steps of T cell differentiation.

Major T cell progenitor activity in bone marrow-derived spleen colonies.


Lancrin, C., Schneider, E., Lambolez, F., Arcangeli, M.L., Garcia-Cordier, C., Rocha, B. & Ezine, S.
J Exp Med. 2002 Apr 1;195(7):919-29.
PubMed

Common lymphoid progenitors (CLP) are generated in adult bone marrow (BM), but the intermediate steps leading to T cell commitment are unknown, and so is the site at which this commitment occurs. Here, we show that colonies arising in the spleen 12 days after BM injection harbor T cell precursors that are undetectable in BM. These precursors did not generate myeloid cells in vivo but repopulated the thymus and the peripheral T cell compartment much faster than did CLP. Two lineage negative (Lin(-)) subpopulations were distinguished, namely CD44(+) Thy1(-) cells still capable of natural killer generation and transient low-level B cell generation, and T cell-restricted CD44(-) Thy1(+) cells. At a molecular level, frequency of CD3epsilon and preTalpha mRNA was very different in each subset. Furthermore, only the CD44(-) Thy1(+) subset have initiated rearrangements in the T cell receptor beta locus. Thus, this study identifies extramedullary T cell progenitors and will allow easy approach to T cell commitment studies.