Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Hematopoietic stem cell fate is established by the Notch–Runx pathway Caroline Erter Burns,1 David Traver,2 Elizabeth Mayhall,1 Jennifer L. Shepard,1 and Leonard I. Zon1,3 1Stem Cell Program and Division of Hematology/Oncology Children’s Hospital and Dana Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachusetts 02115, USA; 2Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, California 92093-0380, USA Identifying the molecular pathways regulating hematopoietic stem cell (HSC) specification, self-renewal, and expansion remains a fundamental goal of both basic and clinical biology. Here, we analyzed the effects of Notch signaling on HSC number during zebrafish development and adulthood, defining a critical pathway for stem cell specification. The Notch signaling mutant mind bomb displays normal embryonic hematopoiesis but fails to specify adult HSCs. Surprisingly, transient Notch activation during embryogenesis via an inducible transgenic system led to a Runx1-dependent expansion of HSCs in the aorta-gonad-mesonephros (AGM) region. In irradiated adults, Notch activity induced runx1 gene expression and increased multilineage hematopoietic precursor cells approximately threefold in the marrow. This increase was followed by the accelerated recovery of all the mature blood cell lineages. These data define the Notch–Runx pathway as critical for the developmental specification of HSC fate and the subsequent homeostasis of HSC number, thus providing a mechanism for amplifying stem cells in vivo. [Keywords: Stem cell; Notch; Runx; AGM; zebrafish; irradiation] Supplemental material is available at http://www.genesdev.org. Received May 25, 2005; revised version accepted July 25, 2005. In vertebrates, the adult hematopoietic system is com- binds integral membrane ligands (Delta and Jagged) to posed of distinct cell lineages that undergo progressive transmit signals between cells in direct contact. Juxta- differentiation from multipotent hematopoietic stem crine signaling generally occurs between neighbors that cells (HSCs) (Durand and Dzierzak 2005). The clinical arise from a common precursor and share similar devel- importance of HSCs in transplantation protocols raises opmental potential (Lewis 1998). Notch is best known the significance of understanding their anatomical origin for its role in a process termed “lateral inhibition,” a and fate potential. Hematopoiesis arises during two em- model explaining how one cell is selected from a group of bryonic phases: a brief primitive wave that predomi- equivalent precursors to adopt an alternative fate (Green- nantly generates erythrocytes, followed by a definitive wald and Rubin 1992). In this scenario, activation of the wave that produces long-term hematopoietic stem cells Notch receptor in a particular cell inhibits ligand pro- (LT-HSCs) capable of reconstituting the blood system for duction by that cell (the “signal-receiving cell”). Con- life. Definitive hematopoiesis occurs, at least in part, comitantly, cells producing high levels of ligand (the in the aorta–gonad–mesonephros (AGM) region, where “signal-emitting cell”) force their neighbors to activate LT-HSCs are associated with the ventral aortic wall. Al- Notch and produce less ligand. Lateral inhibition though much is known about the cellular and functional through the Notch pathway modulates lineage decisions properties of HSCs in mammals, relatively little is un- in related cells by inhibiting one fate and promoting an- derstood about the genetic pathways regulating their in- other (Bray 1998; Lewis 1998). duction, expansion, and homeostasis during embryogen- The mechanism by which Notch determines cell fate esis and adulthood. One pathway known to play a fun- has been studied extensively in a variety of organisms damental role in regulating a variety of cell fate decisions and has led to the following model in which the Notch in progenitors of various organ systems is the Notch sig- receptor undergoes three successive cleavage events to naling pathway. become active. Notch is first processed in the Golgi net- notch encodes a single pass glycoprotein receptor that work to produce a functional transmembrane receptor. Once bound to its ligand, the second cleavage event oc- curs, which generates two products, the Notch extra- 3Corresponding author. cellular domain (NECD) and a membrane-bound acti- E-MAIL [email protected]; FAX (617) 730-0222. Article published online ahead of print. Article and publication date are vated form (NEXT). The final cleavage event, mediated at http://www.genesdev.org/cgi/doi/10.1101/gad.1337005. by presenilin-dependent ␥ secretase activity, occurs GENES & DEVELOPMENT 19:2331–2342 © 2005 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/05; www.genesdev.org 2331 Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Burns et al. within the NEXT fragment and results in the release of tant expansion of vascular fates (Mandal et al. 2004). the Notch intracellular domain (NICD), which trans- These results indicate that Notch signaling is important locates to the nucleus and acts as a transcriptional regu- in the early generation of blood precursors across widely lator (Le Borgne and Schweisguth 2003). divergent phyla. The downstream target genes of Notch Post-translational modification, specifically by addi- signaling in stem cells, however, remain to be deter- tion of ubiquitin peptides, plays a critical role in regu- mined. lating Notch–Delta activity (Lai 2002b). Drosophila neu- Here, we used classic genetic experiments to reveal a ralized was shown to encode an E3 ligase that ubiqui- hierarchical molecular network leading to the induction tylates and promotes the endocytosis of the Notch ligand and expansion of hematopoietic stem and progenitor Delta (Deblandre et al. 2001; Lai et al. 2001; Pavlopoulos cells in the zebrafish embryo and adult. We show that et al. 2001; Yeh et al. 2001). In vertebrate systems, how- the Notch signaling mutant mind bomb develops nor- ever, Neuralized is not essential for lateral inhibition, mal embryonic hematopoiesis but fails to specify defini- suggesting that other E3 ligases may modify Delta. mind tive HSCs normally found associated with the ventral bomb, a neurogenic mutant isolated in several zebrafish aortic wall. Using an inducible transgenic system, we mutagenesis screens (Haffter et al. 1996; Schier et al. found that brief Notch stimulation greatly expanded 1996; Golling et al. 2002), encodes a highly conserved HSC number in the AGM. Morpholino knock-down of and previously uncharacterized E3 ligase that functions Runx1 function completely abolished this increase, similarly to Neuralized (Itoh et al. 2003; Chen and Casey showing for the first time that Runx1 is a downstream Corliss 2004). Mind bomb is required cell nonautono- effector of Notch signaling in HSC induction. In the mously for Notch signaling and lateral inhibition by con- adult, we examined the effect of activated Notch on mar- trolling Delta protein trafficking (Itoh et al. 2003; Chen row recovery following irradiation. Compared with con- and Casey Corliss 2004). trol siblings, the percentage of multilineage hematopoi- Notch receptors and their ligands are expressed in he- etic precursors increased approximately threefold, fol- matopoietic cells and have been implicated in regulating lowed by mature myeloid and lymphoid cells. The HSC induction and lineage cell fate decisions (Ohishi expanded precursor population showed transcriptional et al. 2003). In murine cell culture, constitutive Notch1 up-regulation of the runx1, lmo2, and scl genes, all mark- expression in hematopoietic progenitor and stem cells ers of stem and progenitor cells. These findings define established immortalized cell lines able to generate prog- the Notch–Runx pathway as critical during both embryo- eny with either lymphoid or myeloid characteristics genesis and adulthood to maintain HSC homeostasis. both in vitro and in long-term mouse reconstitution as- says (Varnum-Finney et al. 2000, 2003). Retroviral acti- vation of Notch1 in recombination activating gene-1 Results (RAG-1)-deficient mouse stem cells resulted in an in- Mind bomb, the ubiquitin ligase for Notch ligands, crease of HSCs due to decreased differentiation in vivo is required for HSC specification (Stier et al. 2002). Similar results were observed when overexpressing the Notch effector gene HES-1 in cell To assess the requirement for the Notch pathway in de- lines (Kunisato et al. 2003). Increased numbers of finitive HSC induction, a null allele of the Notch signal- Notch1-expressing HSCs were documented in mice with ing mutant mind bomb (mib) was analyzed. Both c-myb expanded osteoblastic niche cells that present Notch li- and runx1 transcripts mark emerging definitive hemato- gands in the bone marrow microenvironment (Calvi et poietic stem and progenitor cells that are normally con- al. 2003). More recently, the Notch signaling pathway fined to the ventral wall of the zebrafish dorsal aorta was shown to be active in native adult HSCs and down- (Burns et al. 2002; Traver and Zon 2002; Gering and Pa- regulated in differentiating progeny (Duncan et al. 2005). tient 2005). mind bomb mutants lack aortic c-myb and Taken together, activation of Notch in marrow-derived runx1 expression