Nancy A. Speck

Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukaemia.

Familial platelet disorder with predisposition to acute myelogenous leukaemia (FPD/AML, MIM 601399) is an autosomal dominant disorder characterized by qualitative and quantitative platelet defects, and propensity to develop acute myelogenous leukaemia (AML). Informative recombination events in 6 FPD/AML pedigrees with evidence of linkage to markers on chromosome 21q identified an 880-kb interval containing the disease gene. Mutational analysis of regional candidate genes showed nonsense mutations or intragenic deletion of one allele of the haematopoietic transcription factor CBFA2 (formerly AML1) that co-segregated with the disease in four FPD/AML pedigrees. We identified heterozygous CBFA2 missense mutations that co-segregated with the disease in the remaining two FPD/AML pedigrees at phylogenetically conserved amino acids R166 and R201, respectively. Analysis of bone marrow or peripheral blood cells from affected FPD/AML individuals showed a decrement in megakaryocyte colony formation, demonstrating that CBFA2 dosage affects megakaryopoiesis. Our findings support a model for FPD/AML in which haploinsufficiency of CBFA2 causes an autosomal dominant congenital platelet defect and predisposes to the acquisition of additional mutations that cause leukaemia.

Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter

Haematopoietic stem cells (HSCs) are the founder cells of the adult haematopoietic system, and thus knowledge of the molecular program directing their generation during development is important for regenerative haematopoietic strategies. Runx1 is a pivotal transcription factor required for HSC generation in the vascular regions of the mouse conceptus—the aorta, vitelline and umbilical arteries, yolk sac and placenta. It is thought that HSCs emerge from vascular endothelial cells through the formation of intra-arterial clusters and that Runx1 functions during the transition from ‘haemogenic endothelium’ to HSCs. Here we show by conditional deletion that Runx1 activity in vascular-endothelial-cadherin-positive endothelial cells is indeed essential for intra-arterial cluster, haematopoietic progenitor and HSC formation in mice. In contrast, Runx1 is not required in cells expressing Vav1, one of the first pan-haematopoietic genes expressed in HSCs. Collectively these data show that Runx1 function is essential in endothelial cells for haematopoietic progenitor and HSC formation from the vasculature, but its requirement ends once or before Vav is expressed.

Of lineage and legacy - the development of mammalian hematopoietic stem cells

The hematopoietic system is one of the first complex tissues to develop in the mammalian conceptus. Of particular interest in the field of developmental hematopoiesis is the origin of adult bone marrow hematopoietic stem cells. Tracing their origin is complicated because blood is a mobile tissue and because hematopoietic cells emerge from many embryonic sites. The origin of the adult mammalian blood system remains a topic of lively discussion and intense research. Interest is also focused on developmental signals that induce the adult hematopoietic stem cell program, as these may prove useful for generating and expanding these clinically important cell populations ex vivo. This review presents a historical overview of and the most recent data on the developmental origins of hematopoiesis.

Runx1 Expression Marks Long-Term Repopulating Hematopoietic Stem Cells in the Midgestation Mouse Embryo

Hematopoietic stem cells (HSCs) are first found in the aorta-gonad-mesonephros region and vitelline and umbilical arteries of the midgestation mouse embryo. Runx1 (AML1), the DNA binding subunit of a core binding factor, is required for the emergence and/or subsequent function of HSCs. We show that all HSCs in the embryo express Runx1. Furthermore, HSCs in Runx1(+/-) embryos are heterogeneous and include CD45(+) cells, endothelial cells, and mesenchymal cells. Comparison with wild-type embryos showed that the distribution of HSCs among these various cell populations is sensitive to Runx1 dosage. These data provide the first morphological description of embryonic HSCs and contribute new insight into their cellular origin.

The CBFβ Subunit Is Essential for CBFα2 (AML1) Function In Vivo

Abstract The CBFβ subunit is the non-DNA-binding subunit of the heterodimeric core-binding factor (CBF). CBFβ associates with DNA-binding CBFα subunits and increases their affinity for DNA. Genes encoding the CBFβ subunit ( CBFB ) and one of the CBFα subunits ( CBFA2 , otherwise known as AML1 ) are the most frequent targets of chromosomal translocations in acute leukemias in humans. We and others previously demonstrated that homozygous disruption of the mouse Cbfa2 ( AML1 ) gene results in embryonic lethality at midgestation due to hemorrhaging in the central nervous system and blocks fetal liver hematopoiesis. Here we demonstrate that homozygous mutation of the Cbfb gene results in the same phenotype. Our results demonstrate that the CBFβ subunit is required for CBFα2 function in vivo.

Definitive hematopoietic stem cells first develop within the major arterial regions of the mouse embryo.

The aorta–gonad–mesonephros (AGM) region is a potent hematopoietic site within the mammalian embryo body, and the first place from which hematopoietic stem cells (HSCs) emerge. Within the complex embryonic vascular, excretory and reproductive tissues of the AGM region, the precise location of HSC development is unknown. To determine where HSCs develop, we subdissected the AGM into aorta and urogenital ridge segments and transplanted the cells into irradiated adult recipients. We demonstrate that HSCs first appear in the dorsal aorta area. Furthermore, we show that vitelline and umbilical arteries contain high frequencies of HSCs coincident with HSC appearance in the AGM. While later in development and after organ explant culture we find HSCs in the urogenital ridges, our results strongly suggest that the major arteries of the embryo are the most important sites from which definitive HSCs first emerge.

Core-binding factors in haematopoiesis and leukaemia

Core-binding factors (CBFs) are a class of haematopoietic transcription factors that are crucial for the regulation of haematopoietic ontogeny, and are frequent targets of mutation and gene rearrangement in human leukaemia. So, what are the functions of CBFs during development, and what are the functional consequences of CBF mutations in leukaemia? Synergy between these convergent lines of enquiry has furthered our understanding of both normal and malignant haematopoiesis.

Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor.

Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major determinant of the thymic disease specificity of Moloney virus genetically maps to the conserved viral core motif in the Moloney virus enhancer. Point mutations introduced into the core site significantly shifted the disease specificity of the Moloney virus from thymic leukemia to erythroid leukemia (N.A. Speck, B. Renjifo, E. Golemis, T.N. Fredrickson, J.W. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We previously reported the purification of core-binding factors (CBF) from calf thymus nuclei (S. Wang and N.A. Speck, Mol. Cell. Biol. 12:89-102, 1992). CBF binds to core sites in murine leukemia virus and T-cell receptor enhancers. Affinity-purified CBF contains multiple polypeptides. In this study, we sequenced five tryptic peptides from two of the bovine CBF proteins and isolated three cDNA clones from a mouse thymus cDNA library encoding three of the tryptic peptides from the bovine proteins. The cDNA clones, which we call CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6, encode three highly related but distinct proteins with deduced molecular sizes of 22.0, 21.5, and 17.6 kDa that appear to be translated from multiply spliced mRNAs transcribed from the same gene. CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6 do not by themselves bind the core site. However, CBF beta p22.0 and CBF beta p21.5 form a complex with DNA-binding CBF alpha subunits and as a result decrease the rate of dissociation of the CBF protein-DNA complex. Association of the CBF beta subunits does not extend the phosphate contacts in the binding site. We propose that CBF beta is a non-DNA-binding subunit of CBF and does not contact DNA directly.

Failure of Embryonic Hematopoiesis andLethal Hemorrhages in Mouse Embryos Heterozygousfor a Knocked-In Leukemia Gene CBFB–MYH11

The fusion oncogene CBFB-MYH11 is generated by a chromosome 16 inversion in human acute myeloid leukemia subtype M4Eo. Mouse embryonic stem (ES) cells heterozygous for this oncogene were generated by inserting part of the human MYH11 cDNA into the mouse Cbfb gene through homologous recombination (knock-in). Chimeric mice were leukemia free, but the ES cells with the knocked-in Cbfb-MYH11 gene did not contribute to their hematopoietic tissues. Mouse embryos heterozygous for Cbfb-MYH11 lacked definitive hematopoiesis and developed multiple fatal hemorrhages around embryonic day 12.5. This phenotype is very similar to that resulting from homozygous deletions of either Cbfb or Cbfa2 (AML1), consistent with a dominant negative function of the Cbfb-MYH11 fusion oncogene. An impairment of primitive hematopoiesis was also observed, however, suggesting a possible additional function of Cbfb-MYH11.

Core-binding factors in hematopoiesis and immune function.

Core binding factors are heterodimeric transcription factors containing a DNA binding Runx1, Runx2, or Runx3 subunit, along with a non DNA binding CBFβ subunit. All four subunits are required at one or more stages of hematopoiesis. This review describes the role of Runx1 and CBFβ in the initiation of hematopoiesis in the embryo, and in the emergence of hematopoietic stem cells. We also discuss the later stages of hematopoiesis for which members of the core binding factor family are required, as well as the recently described roles for these proteins in autoimmunity.