• Contributions of embryonic hsc-independent hematopoiesis to organogenesis and the adult hematopoietic system

      Neo, W H; Lie-A-Ling, Michael; Fadlullah, Muhammad Z H; Lacaud, Georges; Stem Cell Biology Group, Cancer Research UK Manchester Institute, The University of Manchester, Macclesfield (2021)
      During ontogeny, the establishment of the hematopoietic system takes place in several phases, separated both in time and location. The process is initiated extra-embryonically in the yolk sac (YS) and concludes in the main arteries of the embryo with the formation of hematopoietic stem cells (HSC). Initially, it was thought that HSC-independent hematopoietic YS cells were transient, and only required to bridge the gap to HSC activity. However, in recent years it has become clear that these cells also contribute to embryonic organogenesis, including the emergence of HSCs. Furthermore, some of these early HSC-independent YS cells persist into adulthood as distinct hematopoietic populations. These previously unrecognized abilities of embryonic HSC-independent hematopoietic cells constitute a new field of interest. Here, we aim to provide a succinct overview of the current knowledge regarding the contribution of YS-derived hematopoietic cells to the development of the embryo and the adult hematopoietic system.
    • Early human hemogenic endothelium generates primitive and definitive hematopoiesis in vitro.

      Garcia-Alegria, E; Menegatti, S; Fadlullah, Muhammad Z H; Menendez, P; Lacaud, Georges; Kouskoff, Valerie; Developmental Haematopoiesis Group, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester M13 9PT, UK (2018)
      The differentiation of human embryonic stem cells (hESCs) to hematopoietic lineages initiates with the specification of hemogenic endothelium, a transient specialized endothelial precursor of all blood cells. This in vitro system provides an invaluable model to dissect the emergence of hematopoiesis in humans. However, the study of hematopoiesis specification is hampered by a lack of consensus in the timing of hemogenic endothelium analysis and the full hematopoietic potential of this population. Here, our data reveal a sharp decline in the hemogenic potential of endothelium populations isolated over the course of hESC differentiation. Furthermore, by tracking the dynamic expression of CD31 and CD235a at the onset of hematopoiesis, we identified three populations of hematopoietic progenitors, representing primitive and definitive subsets that all emerge from the earliest specified hemogenic endothelium. Our data establish that hemogenic endothelium populations endowed with primitive and definitive hematopoietic potential are specified simultaneously from the mesoderm in differentiating hESCs.
    • HDAC1 and HDAC2 modulate TGF-β signaling during endothelial-to-hematopoietic transition.

      Thambyrajah, Roshana; Fadlullah, Muhammad Z H; Proffitt, Martin; Patel, Rahima; Cowley, S; Kouskoff, Valerie; Lacaud, Georges; CRUK Stem Cell Biology Group, CRUK Manchester Institute, 555 Wilmslow Road, Manchester M20 4GJ, UK (2018-04-10)
      The first hematopoietic stem and progenitor cells are generated during development from hemogenic endothelium (HE) through trans-differentiation. The molecular mechanisms underlying this endothelial-to-hematopoietic transition (EHT) remain poorly understood. Here, we explored the role of the epigenetic regulators HDAC1 and HDAC2 in the emergence of these first blood cells in vitro and in vivo. Loss of either of these epigenetic silencers through conditional genetic deletion reduced hematopoietic transition from HE, while combined deletion was incompatible with blood generation. We investigated the molecular basis of HDAC1 and HDAC2 requirement and identified TGF-β signaling as one of the pathways controlled by HDAC1 and HDAC2. Accordingly, we experimentally demonstrated that activation of this pathway in HE cells reinforces hematopoietic development. Altogether, our results establish that HDAC1 and HDAC2 modulate TGF-β signaling and suggest that stimulation of this pathway in HE cells would be beneficial for production of hematopoietic cells for regenerative therapies.
    • The hemogenic competence of endothelial progenitors is restricted by Runx1 silencing during embryonic development.

      Eliades, Alexia; Wareing, Sarah; Marinopoulou, Elli; Fadlullah, Muhammad Z H; Patel, Rahima; Grabarek, J; Plusa, B; Lacaud, Georges; Kouskoff, Valerie; Cancer Research UK Stem Cell Hematopoiesis Group, Cancer Research UK Manchester Institute, The University of Manchester, Manchester M20 4BX (2016-06-07)
      It is now well-established that hematopoietic stem cells (HSCs) and progenitor cells originate from a specialized subset of endothelium, termed hemogenic endothelium (HE), via an endothelial-to-hematopoietic transition. However, the molecular mechanisms determining which endothelial progenitors possess this hemogenic potential are currently unknown. Here, we investigated the changes in hemogenic potential in endothelial progenitors at the early stages of embryonic development. Using an ETV2::GFP reporter mouse to isolate emerging endothelial progenitors, we observed a dramatic decrease in hemogenic potential between embryonic day (E)7.5 and E8.5. At the molecular level, Runx1 is expressed at much lower levels in E8.5 intra-embryonic progenitors, while Bmi1 expression is increased. Remarkably, the ectopic expression of Runx1 in these progenitors fully restores their hemogenic potential, as does the suppression of BMI1 function. Altogether, our data demonstrate that hemogenic competency in recently specified endothelial progenitors is restrained through the active silencing of Runx1 expression.
    • Interplay between SOX7 and RUNX1 regulates hemogenic endothelial fate in the yolk sac.

      Lilly, Andrew J; Costa, Guilherme; Largeot, Anne; Fadlullah, Muhammad Z H; Lie-A-Ling, Michael; Lacaud, Georges; Kouskoff, Valerie; Stem Cell Hematopoiesis, Cancer Research UK Manchester Institute, The University of Manchester, Wilmslow road, M20 4BX (2016-10-17)
      Endothelial to hematopoietic transition (EHT) is a dynamic process involving the shutting down of endothelial gene expression and switching on of hematopoietic gene transcription. Whilst the factors regulating EHT in hemogenic endothelium (HE) of the dorsal aorta have been relatively well studied, the molecular regulation of yolk sac HE remains poorly understood. Here, we show that SOX7 inhibits the expression of RUNX1 target genes in HE, whilst having no effect on RUNX1 expression itself. We establish that SOX7 directly interacts with RUNX1 and inhibits its transcriptional activity. Through this interaction we demonstrate that SOX7 hinders RUNX1 DNA binding as well as the interaction between RUNX1 and its cofactor CBFβ. Finally, we show by single cell expression profiling and immunofluorescence that SOX7 is broadly expressed across the RUNX1(+) yolk sac HE population compared with SOX17. Collectively, these data demonstrate for the first time how direct protein-protein interactions between endothelial and hematopoietic transcription factors regulate contrasting transcriptional programs during HE differentiation and EHT.
    • Investigating the importance of B cells and antibodies during Trichuris muris infection using the IgMi mouse

      Sahputra, R.; Murphy, E. A.; Forman, R.; Mair, I.; Fadlullah, Muhammad Z H; Waisman, A.; Muller, W.; Else, K. J.; Division of Infection, Immunity and Respiratory Medicine, Lydia Becker Institute for Immunology, The University of Manchester, Manchester, UK. (2020)
      The IgMi mouse has normal B cell development; its B cells express an IgM B cell receptor but cannot class switch or secrete antibody. Thus, the IgMi mouse offers a model system by which to dissect out antibody-dependent and antibody-independent B cell function. Here, we provide the first detailed characterisation of the IgMi mouse post-Trichuris muris (T. muris) infection, describing expulsion phenotype, cytokine production, gut pathology and changes in T regulatory cells, T follicular helper cells and germinal centre B cells, in addition to RNA sequencing (RNA seq) analyses of wild-type littermates (WT) and mutant B cells prior to and post infection. IgMi mice were susceptible to a high-dose infection, with reduced Th2 cytokines and elevated B cell-derived IL-10 in mesenteric lymph nodes (MLN) compared to controls. A low-dose infection regime revealed IgMi mice to have significantly more apoptotic cells in the gut compared to WT mice, but no change in intestinal inflammation. IL-10 levels were again elevated. Collectively, this study showcases the potential of the IgMi mouse as a tool for understanding B cell biology and suggests that the B cell plays both antibody-dependent and antibody-independent roles post high- and low-dose T. muris infection. KEY MESSAGES: During a high-dose T. muris infection, B cells are important in maintaining the Th1/Th2 balance in the MLN through an antibody-independent mechanism. High levels of IL-10 in the MLN early post-infection, and the presence of IL-10-producing B cells, correlates with susceptibility to T. muris infection. B cells maintain gut homeostasis during chronic T. muris infection via an antibody-dependent mechanism. Keywords: B cells; IgMi mouse; Interleukin-10; Intestinal pathology; Th1/Th2; Trichuris muris.
    • Mouse RUNX1C regulates pre-megakaryocytic/erythroid output and maintains survival of megakaryocyte progenitors.

      Draper, Julia E; Sroczynska, P; Leong, Hui Sun; Fadlullah, Muhammad Z H; Miller, Crispin J; Kouskoff, Valerie; Lacaud, Georges; Cancer Research UK Stem Cell Biology Group, Cancer Research UK Manchester Institute, The University of Manchester (2017-05-10)
      RUNX1 is crucial for the regulation of megakaryocyte specification, maturation and thrombopoiesis. Runx1 possesses two promoters: the distal P1 and proximal P2 promoters. The major protein isoforms generated by P1 and P2 are RUNX1C and RUNX1B respectively, which differ solely in their N-terminal amino acid sequences. RUNX1C is the most abundantly expressed isoform in adult hematopoiesis, present in all RUNX1-expressing populations, including the cKit(+) hematopoietic stem and progenitor cells (HSPCs). RUNX1B expression is more restricted, being highly expressed in the megakaryocyte lineage but downregulated during erythropoiesis. We generated a Runx1P1 knock-in of RUNX1B, termed P1-MRIPV This mouse line lacks RUNX1C expression but has normal total RUNX1 levels, solely comprising RUNX1B. Utilizing this mouse line we establish a specific requirement for the P1-RUNX1C isoform in megakaryopoiesis, which cannot be entirely compensated for by RUNX1B overexpression. P1 knock-in Megakaryocyte Progenitors (MkP) have reduced proliferative capacity and undergo increased cell death, resulting in thrombocytopenia. P1 knock-in Pre-Megakaryocyte/Erythroid Progenitors (PreMegEs) demonstrate an erythroid-specification bias, evident from increased erythroid colony-forming ability and decreased megakaryocyte output. At a transcriptional level, multiple erythroid-specific genes are upregulated and megakaryocyte-specific transcripts are downregulated. In addition, pro-apoptotic pathways are activated in P1 knock-in PreMegEs, presumably accounting for the increased cell death in the MkP compartment. Unlike in the conditional adult Runx1 null models, megakaryocytic maturation is not affected in the P1 knock-in mice, suggesting that RUNX1B can regulate endomitosis and thrombopoiesis. Therefore, despite the high degree of structural similarity, RUNX1B and RUNX1C isoforms have distinct and specific roles in adult megakaryopoiesis.
    • A novel prospective isolation of murine fetal liver progenitors to study in utero hematopoietic defects.

      Draper, Julia E; Sroczynska, Patrycja; Fadlullah, Muhammad Z H; Patel, Rahima; Newton, Gillian; Breitwieser, Wolfgang; Kouskoff, V; Lacaud, Georges; Cancer Research UK Stem Cell Biology Group, Cancer Research UK Manchester Institute, Manchester Cancer Research Centre, The University of Manchester, Manchester (2018)
      In recent years, highly detailed characterization of adult bone marrow (BM) myeloid progenitors has been achieved and, as a result, the impact of somatic defects on different hematopoietic lineage fate decisions can be precisely determined. Fetal liver (FL) hematopoietic progenitor cells (HPCs) are poorly characterized in comparison, potentially hindering the study of the impact of genetic alterations on midgestation hematopoiesis. Numerous disorders, for example infant acute leukemias, have in utero origins and their study would therefore benefit from the ability to isolate highly purified progenitor subsets. We previously demonstrated that a Runx1 distal promoter (P1)-GFP::proximal promoter (P2)-hCD4 dual-reporter mouse (Mus musculus) model can be used to identify adult BM progenitor subsets with distinct lineage preferences. In this study, we undertook the characterization of the expression of Runx1-P1-GFP and P2-hCD4 in FL. Expression of P2-hCD4 in the FL immunophenotypic Megakaryocyte-Erythroid Progenitor (MEP) and Common Myeloid Progenitor (CMP) compartments corresponded to increased granulocytic/monocytic/megakaryocytic and decreased erythroid specification. Moreover, Runx1-P2-hCD4 expression correlated with several endogenous cell surface markers' expression, including CD31 and CD45, providing a new strategy for prospective identification of highly purified fetal myeloid progenitors in transgenic mouse models. We utilized this methodology to compare the impact of the deletion of either total RUNX1 or RUNX1C alone and to determine the fetal HPCs lineages most substantially affected. This new prospective identification of FL progenitors therefore raises the prospect of identifying the underlying gene networks responsible with greater precision than previously possible.
    • RUNX1B expression is highly heterogeneous and distinguishes megakaryocytic and erythroid lineage fate in adult mouse hematopoiesis

      Draper, Julia E; Sroczynska, Patrycja; Tsoulaki, Olga; Leong, Hui Sun; Fadlullah, Muhammad Z H; Miller, Crispin J; Kouskoff, Valerie; Lacaud, Georges; Cancer Research UK Stem Cell Biology Group, Cancer Research UK Manchester Institute, The University of Manchester, Manchester, United Kingdom (2016-01)
      The Core Binding Factor (CBF) protein RUNX1 is a master regulator of definitive hematopoiesis, crucial for hematopoietic stem cell (HSC) emergence during ontogeny. RUNX1 also plays vital roles in adult mice, in regulating the correct specification of numerous blood lineages. Akin to the other mammalian Runx genes, Runx1 has two promoters P1 (distal) and P2 (proximal) which generate distinct protein isoforms. The activities and specific relevance of these two promoters in adult hematopoiesis remain to be fully elucidated. Utilizing a dual reporter mouse model we demonstrate that the distal P1 promoter is broadly active in adult hematopoietic stem and progenitor cell (HSPC) populations. By contrast the activity of the proximal P2 promoter is more restricted and its upregulation, in both the immature Lineage- Sca1high cKithigh (LSK) and bipotential Pre-Megakaryocytic/Erythroid Progenitor (PreMegE) populations, coincides with a loss of erythroid (Ery) specification. Accordingly the PreMegE population can be prospectively separated into "pro-erythroid" and "pro-megakaryocyte" populations based on Runx1 P2 activity. Comparative gene expression analyses between Runx1 P2+ and P2- populations indicated that levels of CD34 expression could substitute for P2 activity to distinguish these two cell populations in wild type (WT) bone marrow (BM). Prospective isolation of these two populations will enable the further investigation of molecular mechanisms involved in megakaryocytic/erythroid (Mk/Ery) cell fate decisions. Having characterized the extensive activity of P1, we utilized a P1-GFP homozygous mouse model to analyze the impact of the complete absence of Runx1 P1 expression in adult mice and observed strong defects in the T cell lineage. Finally, we investigated how the leukemic fusion protein AML1-ETO9a might influence Runx1 promoter usage. Short-term AML1-ETO9a induction in BM resulted in preferential P2 upregulation, suggesting its expression may be important to establish a pre-leukemic environment.
    • TIAM1 antagonizes TAZ/YAP both in the destruction complex in the cytoplasm and in the nucleus to inhibit invasion of intestinal epithelial cells.

      Diamantopoulou, Zoi; White, Gavin R M; Fadlullah, Muhammad Z H; Dreger, M; Pickering, K; Maltas, J; Ashton, Garry; MacLeod, R; Baillie, G; Kouskoff, Valerie; et al. (2017-05-08)
      Aberrant WNT signaling drives colorectal cancer (CRC). Here, we identify TIAM1 as a critical antagonist of CRC progression through inhibiting TAZ and YAP, effectors of WNT signaling. We demonstrate that TIAM1 shuttles between the cytoplasm and nucleus antagonizing TAZ/YAP by distinct mechanisms in the two compartments. In the cytoplasm, TIAM1 localizes to the destruction complex and promotes TAZ degradation by enhancing its interaction with βTrCP. Nuclear TIAM1 suppresses TAZ/YAP interaction with TEADs, inhibiting expression of TAZ/YAP target genes implicated in epithelial-mesenchymal transition, cell migration, and invasion, and consequently suppresses CRC cell migration and invasion. Importantly, high nuclear TIAM1 in clinical specimens associates with increased CRC patient survival. Together, our findings suggest that in CRC TIAM1 suppresses tumor progression by regulating YAP/TAZ activity.