Review articles
Notch signaling in hematopoiesis and lymphopoiesis: lessons from Drosophila Freddy Radtke,* Anne Wilson, and H. Robson MacDonald
Summary The evolutionarily conserved Notch signaling pathway regulates a broad spectrum of cell fate decisions and differentiation processes during fetal and postnatal life. It is involved in embryonic organogenesis as well as in the maintenance of homeostasis of self-renewing systems. In this article, we review the role of Notch signaling in the hematopoietic system with particular emphasis on lymphocyte development and highlight the similarities in Notch function between Drosophila and mammalian differentiation processes. Recent studies indicating that aberrant NOTCH signaling is frequently linked to the induction of T leukemia in humans will also be discussed. BioEssays 27:1117–1128, 2005. ß 2005 Wiley Periodicals, Inc.
Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland. *Correspondence to: Freddy Radtke, Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Chemin des Boveresses 155, 1066 Epalinges, Switzerland. E-mail:
[email protected] DOI 10.1002/bies.20315 Published online in Wiley InterScience (www.interscience.wiley.com).
Abbreviations: CNS, central nervous system; PNS, peripheral nervous system; SOP sensory organ precursor; NLS, nuclear localization signal; TACE, tumor-necrosis factor a converting enzyme; NIC, Notch intracellular domain; CSL, name of the Notch signaling mediating transcription factor derived from different species, CBF-1 in humans, Supressor of hairless in Drosophila and Lag1 in Caenorhabditis elegans, also known as RBP-J in the mouse; Hes, hairy enhancer of split, Herp, hairy enhancer of split related protein; Nrarp, Notchregulated ankyrin repeat protein; Pre-T-a, pre T cell receptor alpha; MINT, Msx2-interaccting nuclear target protein, YS, yolk sac; AGM, aorta-gonad-mesonephros; BM bone marrow; HSCs, hematopoietic stem cells; SP, side population; MAML1, mastermind like protein; TCRb, T cell receptor beta; MZB, marginal zone B cells, FoB, follicular zone B cells; DCs, dendritic cells; T-ALL, T cell actue lymphoblastic leukemia; TAN1, translocation associated Notch homolog, HD, heterodimerization domain, ANK, ankyrin repeats, PM, plasma membrane; CR, cysteine-rich, EGF-like, epidermal growth factor-like, TAD transactivation domain, COR, co-repressors; LP, lymphoid progenitors; MP, myeloid progenitors.
BioEssays 27:1117–1128, ß 2005 Wiley Periodicals, Inc.
Introduction The Notch signaling pathway is highly conserved in evolution and is found in organisms as diverse as worms and humans. At the beginning of the 20th century, Thomas Hunt Morgan and colleagues described notches at the margin of wing blades of fruit flies (Drosophila melanogaster).(1) These notches were found to be the result of a partial loss of function (haploinsufficiency) of the Notch gene encoding for a single transmembrane receptor that was cloned in the mid 1980s.(2,3) Notch proteins and their corresponding ligands regulate many cell fate decision and differentiation processes during development.(4) They are also involved in apoptosis, proliferation and border formation. As most of our initial knowledge of the Notch pathway is derived from studies in worms and flies, we will first introduce some basic concepts of Notch function derived from experiments in invertebrates before addressing the role of Notch signaling in hematopoiesis and lymphocyte development. These early studies are very important since interpretation of many of the results obtained in vertebrate organs such as the hematopoietic system are based on long held concepts established in invertebrates. Paradigms of Notch function In Drosophila, Notch was shown to influence cell fate choices within a group of cells having equipotent developmental potential. For example, in the Drosophila central nervous system (CNS), as well as in the peripheral nervous system (PNS), Notch signaling specifies a single cell amongst a cluster of cells in the ectoderm (proneural cluster), all with the same potential to become neuroblasts in the CNS(5) or sensory organ precursor cells (SOPs) in the PNS.(6,7) The cell within the proneural cluster that expresses the highest levels of Notch ligand triggers Notch signaling in the Notch receptorexpressing surrounding cells. These Notch signal-receiving cells are inhibited from becoming neuroblasts (in the CNS) or SOPs (in the PNS). Conversely, in the absence of Notch signaling, all of the cells within a proneural cluster become neuroblasts (or SOPs), designated as a neurogenic phenotype(8,9) (Fig. 1). Since this cell fate specification process occurs between neighboring cells, it is referred to as lateral inhibition or lateral specification. Although these results were interpreted to mean that Notch signaling specifies one
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Figure 1. Notch regulates cell fate specification of the peripheral nervous system in the fruit fly. A: Schematic view of a mechanosensory bristle of the fruit fly. B: The role of Notch during selection of sensory organ precursor cells (SOP) from a proneural cluster and their subsequent cell fate decisions during development of a mechanosensory bristle. Notch signaling controls multiple binary cell fate choices of the SOP cell, the IIa and IIIb progenitors to give rise to a hair, a socket, a neuron and a sheath cell. N, Notch signal receiving cell.
particular cell fate from a pool of cells that can adopt at least two different fates, they also fostered the common view that one function of Notch signaling is to inhibit differentiation and thereby maintain uncommitted precursor cells. In this case, cells not receiving a Notch signal will differentiate, while cells receiving a Notch signal will not. This view was further reinforced by studies in the frog in which injection of RNA coding for the Notch ligand Delta1 into a Xenopus embryo at the two-cell stage inhibited neuron production.(10) In contrast, injection of RNA coding for a dominant negative form of Delta1 caused excess production of neurons.(11) Similarly, introduction of a dominant active form of Notch into the vertebrate P19 cell line (which under certain conditions can differentiate into neuronal lineages and muscle progenitors) inhibited differentiation.(12) Although these latter experiments are consistent with the idea that Notch plays a role in inhibition of differentiation of progenitor cells, they are at best suggestive. Nevertheless they have fostered the common view that Notch functions as progenitor or stem cell gate-keeper. The best-studied role of Notch, however, is the regulation of binary cell fate choices. A classical example of this is observed in the Drosophila PNS. Once a SOP cell has been specified out of the proneural cluster in the ectoderm, it divides into two cells termed IIa and IIb. These cells divide a second time to give rise to either hair and socket cells (derived from IIa), or neuron and sheath cells (derived from IIb), which participate in the
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formation of sensory bristles. Loss- and gain-of-function studies during this differentiation process show that Notch signaling is required both at the first cell division to specify the IIa cell formed from a bi-potent SOP, and, in the second round of cell division, for specification of the socket cell from the IIa cell or the sheath cell from the IIb cell(13) (Fig. 1). These results showing that Notch signaling regulates the acquisition of distinct fates in daughter cells of SOPs led to the more general concept that Notch may control binary cell fate choices from bipotent progenitor cells. A third proposed function of Notch signaling between developmentally related cell types would be to induce or enhance terminal differentiation. This has been shown in both human(14) and mouse skin, as well as in primary keratinocytes where Notch signaling induces early differentiation markers and cell cycle arrest by Waf1.(15) In summary, Notch signaling can regulate and influence cell fate decisions in at least three different ways: (1) Maintenance of undifferentiated progenitors, (2) regulation of binary cell fate decisions, and (3) induction of terminal differentiation. Notch receptors and signaling Drosophila has one Notch receptor activated by two different transmembrane-bound ligands called Serrate and Delta, while Caenorhabditis elegans (C. elegans) contains two receptors (Glp-1 and Lin-12) and two ligands (Apx-1 and
Review articles
Lag-2).(4) In contrast, mammals such as mice and humans have four receptors (Notch1-4), and five ligands: Jagged1 and Jagged2 (homologues of Serrate) and Delta-like 1, 3 and 4 (homologues of Delta) (Fig. 2). Notch receptors are synthesized as precursor proteins that are cleaved during transport to the cell surface where they are expressed as heterodimers. Their extracellular domains contain between 29 and 36 epidermal growth factor-like (EGF) repeats followed by three cysteine-rich Notch/Lin12 (LIN) repeats. While the amino terminal EGF-like repeats bind ligand DSL (Delta, Serrate, Lag-2) domains, the function of the LIN repeats is to prevent signaling in the absence of ligand. The cytoplasmic domains of Notch receptors, which convey the signal to the nucleus, harbor multiple protein–protein interaction domains such as the RAM domain and the CDC10 repeats, which bind downstream effector molecules, two nuclear localization signals (NLS), and a PEST sequence involved in regulating protein stability. While Notch1 and Notch2 also contain a transcriptional transactivation domain, such domains have not been described yet for Notch3 and Notch4. Ligand binding to the extracellular domain of Notch receptors triggers two proteolytic cleavages within the receptor. The first is mediated by the ADAM protease TACE (tumor-necrosis factor aconverting enzyme), which cleaves the receptors close to the transmembrane domain. The extracellular Notch domain is ‘transendocytosed’ by the ligand-expressing neighboring cell.(16) A second cleavage, mediated by the g-secretase activity of the multiprotein complex of presenilins, occurs within the transmembrane domain. The liberated cytoplasmic domain (NIC) translocates to the nucleus and binds the transcription factor CSL (CBF1 in humans, RBP-J in mice, Suppressor of hairless in Drosophila, Lag1 in C. elegans), converting it from a transcriptional repressor into a transcriptional activator by displacing corepressor complexes(17–19) and recruiting coactivators such as mastermind-like proteins (MAMLs),(20,21) which interact with CBP/p300 proteins.(22) To date only a few Notch target genes have been identified, some of which are utilized in multiple tissues while others seem to be tissue specific. The basic helix–loop–helix transcription factors of the Hairy enhancer of split (Hes) family such as Hes1 and Hes5,(23) the related Herp (Hes-related repressor protein) transcription factor family,(24) the cell cycle regulator Cdkn1a,(15) Nrarp (Notch-regulated ankyrin repeat protein),(25) Deltex1(26) and Ptcra (Pre-T-a)(27) have been reported as Notch target genes. An additional level of complexity stems from the fact that Notch signaling can be regulated by several modulators that act at extracellular, cytoplasmic or nuclear levels. Fringe proteins belong to a family of glycosyl transferases that add N-acetylglucosamine to certain EGF-like repeats of Notch receptors, thereby promoting Notch signaling in response to Delta ligands and inhibiting Jagged-mediated signaling.(28,29) Examples of cytoplasmic modulators of Notch signaling
include Deltex(30) and Numb(31) while Nrarp (Notch-regulated ankyrin repeat protein) and MINT (Msx2-interacting nuclear target protein) are nuclear proteins both of which seem to negatively regulate Notch signaling. How Nrarp inhibits Notch signaling(32) is currently unknown, whereas MINT has been shown to compete with NIC for binding to CSL.(33) Notch, a gatekeeper of hematopoietic stem cells? The wide expression patterns of Notch receptors and their corresponding ligands (reviewed in Ref. 34) within the adult hematopoietic system suggest an important role for Notch signaling during adult hematopoiesis. Such a role has already been confirmed during embryonic hematopoiesis.(35) The first hematopoietic cells in the mouse embryo appear in the extraembryonic yolk sac (YS) around E7.5, often referred to as primitive hematopoiesis. During development, YS hematopoiesis shifts, first to a site within the embryo called the aorta– gonad–mesonephros (AGM), then to the fetal liver and finally to the adult bone marrow (BM).(36) Hematopoietic stem cells (HSCs) within the AGM region contain long-term repopulating activity as they can reconstitute the entire hematopoietic system of an adult mouse. These fetal HSCs are likely to be derived from a bi-potential hemangioblast that gives rise to both endothelial and hematopoietic cells.(37,38) Recently hematopoietic cells have been shown to bud from the endothelial cell layer in the murine midgestation dorsal aorta,(39) supporting the view that the first long-term repopulating hematopoietic cells originate from hemangioblasts. Hirai and colleagues showed that, while Notch1 signaling is dispensable for primitive hematopoiesis within the extraembryonic YS, it is essential for the reconstitution activity of fetal HSCs found in the AGM region.(35) In addition, blocking Notch signaling with g-secretase inhibitors (which block the proteolytic release of NIC) suppresses in vitro hematopoiesis in explant cultures derived from the E9.5 AGM region, but not from the E10.5 AGM region, a time point when HSCs already exist.(35) During the onset of definitive hematopoiesis in the embryo Notch1/CSL signaling activates the expression of Gata2,(40) which has previously been shown to be an essential transcription factor for hematopoiesis.(41) These results strongly suggest that Notch1 signaling is necessary for development of HSCs during a very narrow window of time, but not for their maintenance at later stages of embryonic development. It is therefore tempting to speculate that Notch1 signaling functions in a way analogous to the binary cell fate specification of SOP cells by influencing the lineage choice of a bi-potential progenitor cell (Fig. 1). If this were true, the bipotential progenitor would be the hemangioblast that has to choose between hematopoietic and endothelial cell fates. In the absence of Notch1 signaling, hemangioblasts would fail to adopt the hematopoietic cell fate and would differentiate into endothelial cells by default (Fig. 3). Although endothelial cell
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Figure 2. Notch receptors/ligands and signaling. A: Notch receptors and ligands. While Drosophila has one Notch receptor (dNotch), vertebrates have four (Notch1–4). The extracellular domain of the receptors contain between 29 and 36 EGF-like repeats (36 in dNotch, Notch1 and 2, 34 in Notch3 and 29 in Notch4) involved in ligand binding, followed by three cysteine-rich Notch/LIN12 (LIN) repeats. The LIN domain prevents ligand-independent activation of the receptor, and is followed by the heterodimerization domain (HD). The cytoplasmic part of the receptor contains two protein–protein interaction domains, the RAM (R) domain, six ankyrin repeats (ANK), two nuclear localization signals (NLS), a transcriptional transactivation domain (TAD) and a PESTsequence. dNotch can be activated by two different ligands, Delta and Serrate while the vertebrate receptors can be activated by at least five ligands (Jagged1 and 2 (homologs of Serrate), and Delta1, 3 and 4 (homologs of Delta)). The common feature of these two ligand families is an N-terminal structure called DSL (Delta, Serrate and Lag). Both type of ligands contain EGF-like repeats in the extracellular domain, but only Serrate, Jagged1 and Jagged2 harbor an additional cysteinerich (CR) sequence downstream of the EGF-like repeats. PM, plasmamembrane. B) Notch signaling. Notch receptors are synthesized as precursor proteins and cleaved in two during transport to the cell surface where they are expressed as heterodimers. Notch signaling is initiated by ligand binding which induces two subsequent proteolytic cleavages, the first mediated by TACE (tumor-necrosis factor aconverting enzyme) near the transmembrane domain, while the second cleavage which occurs within the transmembrane domain is mediated by the g-secretase activity of presenilins. The liberated cytoplasmic portion of the receptor (NIC) translocates to the nucleus and heterodimerizes with the transcription factor CSL. Binding of NIC to CSL leads to transcriptional activation by displacement of co-repressors (COR) and simultaneous recruitment of coactivators such as Mastermind-like proteins (MAML) that interact with P300.
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Figure 3. Notch triggered cell fate specification within the hematopoietic system. A schematic model analogous to Figure1 showing how Notch influences multiple, potentially binary cell fate decisions during hematopoietic and lymphocyte development. This includes the generation of hematopoietic stem cells (HSCs) within the aorta-gonad-mesonephros (AGM) region of the developing embryo, the T versus B lineage decision of lymphoid progenitors (LP) and ab versus gd T cell development within the thymus as well as marginal zone B (MZB) versus follicular B (FoB) cell development within the spleen. N (Notch signal receiving cell), BM (bone marrow), MP (myeloid progenitor).
fate specification appears to occur independently of Notch in this particular instance, it is clear that Notch plays other essential roles during vasculogenesis (reviewed in Ref. 42). Whether Notch signaling is also important for the cell fate specification of HSCs in adult BM is currently unknown. However, several lines of evidence support the possibility that Notch signaling might be necessary for adult HSC maintenance. First, expression of a dominant active form of the Notch receptor (NIC) in murine BM progenitors can lead to increased hematopoietic stem cell self-renewal in vivo,(43) or to immortalization of hematopoietic progenitor cells with myeloid and lymphoid differentiation potential.(44) Second, coculture assays in which murine or human HSCs are incubated in the presence of immobilized or soluble Notch ligands, or together with ligand-expressing feeder cells can maintain or even enhance HSC self-renewal.(45–51) This latter process is possibly mediated by the Notch target gene Hes1, since Hes1 gain-of-function studies have shown maintenance of HSCs ex vivo. In addition, transplantation of HSCs overexpressing Hes1 resulted in increased numbers of cells with ‘‘side population’’ (SP) activity, characterized by the active efflux of the DNA dye Hoechst 33342. Long-term hematopoietic repopulating activity has been shown to be concentrated within BM SP cells.(52) More recently, Reya and colleagues have
knocked down Notch signaling in vitro using either a retroviral transduction approach to overexpress dominant negative forms of CSL or the transcriptional co-activator MAML1, or by using g-secretase-specific inhibitors. All three approaches resulted in enhanced differentiation of HSCs in vitro. In other in vivo experiments, HSCs expressing a dominant negative CSL were unable to reconstitute the long-term HSC pool in transplanted recipients.(53) Despite this large body of evidence, which is mostly interpreted according to the idea that Notch signaling inhibits differentiation and is therefore necessary for HSC maintenance, there is no clear-cut genetic loss of Notch function model supporting this hypothesis. In particular, neither conditional inactivation of the CSL gene, which mediates Notch signaling of all Notch receptors, nor conditional loss of function of the Notch1 or the Notch2 genes in adult BM cells result in any HSC phenotype, even in stringent repopulation assays where BM cells deficient for Notch signaling components have to compete with wild-type cells. Recently, maintenance of BM HSC numbers has been postulated to be mediated by Jagged1-expressing osteoblasts located in the BM stem cell niche. Osteoblast-specific expression of the activated parathyroid hormone-related protein receptor resulted in an increase in the number of osteoblasts, which correlated with an increase in the number
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of functional HSCs.(54) Since osteoblasts showed evidence of Notch1 activation in vivo,(54) these results were interpreted to mean that Jagged1-expressing osteoblasts regulate HSC homeostasis through Notch1 signaling. However, conditional inactivation of the Jagged1 gene in BM progenitors and/or BM stromal cells does not impair HSC self-renewal or differentiation into any blood lineage,(55) suggesting that Jagged1 is not essential for HSC hematopoiesis. Although physiological roles for Notch signaling in adult HSC maintenance and/or self-renewal appear rather unlikely the potential of Notch to expand HSCs in vitro could still be important in the exploration of specific therapeutic goals. Notch and T cell fate specification The best-established role for Notch signaling in the hematopoietic system is the essential function of Notch1 in T cell lineage commitment. Inducible inactivation of the Notch1 gene in BM progenitors results in a block in thymic T cell development at or before the earliest intrathymic precursor stage. As a consequence of the Notch1 deficiency, immature B cells develop in the thymus from incoming BM progenitors suggesting that Notch1 instructs an early lymphoid progenitor to adopt a T versus B cell fate.(56,57) Similar results have been obtained by inducible inactivation of the CSL gene in BM progenitors.(58) These nearly identical phenotypes, coupled with the fact that CSL mediates signaling of all Notch receptors strongly suggest that Tcell lineage commitment is mediated by Notch1/CSL-dependent signaling in a non-redundant manner despite expression of other Notch receptors in BM and/or thymic progenitors.(34) In agreement with these results, no T cell phenotype is observed after inducible inactivation of Notch2 in the hematopoietic system,(59) and to date no hematopoietic phenotypes have been reported for Notch3(60) or Notch4(61) gene targeted mice suggesting that T cell fate specification is a non-redundant function of Notch1. The role of Notch signaling in specifying T cell fate has also been deduced from reciprocal gain-of-function studies. First, transduction of BM progenitors with a retrovirus containing a ligand-independent dominant active form of the Notch1 receptor (NIC), led to ectopic Tcell development in the BM concomitant with a block in B cell development.(62) This particular phenotype is not intrinsic to the cytoplasmic domain of Notch1 since similar results have been obtained with the cytoplasmic domain of Notch2(63) or Notch4.(64) Similarly, retroviral transduction of BM progenitors with the Notch ligands Delta1 or Delta 4 also results in ectopic T cell development and a simultaneous block in B cell development.(65 –67) This phenotype could be explained by continuous activation of endogenous Notch receptors expressed on hematopoietic progenitor cells within the BM compartment. Another set of experiments supporting the essential role of Notch signaling for Tcell fate specification is based on the use of negative modulators of the Notch pathway such as
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Fringe,(68) Deltex(69) or Nrarp,(32) or a dominant negative form of the transcriptional coactivator MAML.(70) Expression of any one of these modulators in either BM progenitors or immature thymocytes also results in a block of Tcell development as well as in ectopic thymic B cell development. Surprisingly overexpression of the Numb gene (a negative modulator of the Notch pathway in Drosophila) had no effect on T cell development.(71) Although overexpression of these modifiers can interfere with Notch signaling, their physiological role within the hematopoietic systems remains to be elucidated. Collectively these gain- and loss-of-function studies clearly demonstrate that Notch1 signaling is necessary and sufficient for T cell lineage commitment. Two models have been proposed to explain how Notch regulates T cell lineage commitment. The first model is again adapted from the function of Notch in influencing binary cell fate decisions from a bi-potential precursor, such as described previously for the development of mechanosensory bristles (Fig. 1). According to this model, Notch1 would regulate the commitment of a bi-potential T/B precursor (Fig. 3). In the absence of Notch1 signaling, bi-potential lymphoid progenitors are not instructed towards the Tcell lineage and thus adopt a B cell fate in the thymus by default. As this has not been demonstrated at the single cell level, an alternative scenario in which cells entering the thymus are not bi- or (multi-) potent but represent a mixed population of progenitors with separate lineage potential cannot be formally excluded. If this were the case, Notch1 signaling would instruct an already T lineage committed early lymphoid progenitor to further differentiate towards the T cell lineage and simultaneously suppress the development of a separate B cell precursor. Despite this caveat, the simplest explanation is that Notch1 regulates the fate of a bi-potential lymphoid progenitor cell entering the thymus. In addition, these reciprocal gain- and loss-of-function studies support the concept that B cell and Tcell development must be compartmentalized, with B cell development occurring in the BM while the vast majority of T cell development takes place in the thymus. In order to allow B cell development in the BM, Notch signaling must be absent or negatively regulated, despite the fact that some Notch receptors and ligands are expressed on BM progenitors and stroma.(34) One possible explanation for this paradox is that hematopoietic progenitors downregulate Notch receptors as soon as they are engaged towards the B cell lineage. Evidence supporting such a possibility stems from studies on the B lineage commitment factor Pax5, which represses transcription of the Notch1 gene in B cell progenitors. Indeed, forced Pax5 expression promotes B cell development at the expense of T cell development while all other blood lineages develop normally.(72,73) An alternative hypothesis is that induction of the Tcell lineage commitment program would be restricted to a particular family of Notch ligands. In this scenario, only Delta-
Review articles
mediated and not Jagged-mediated Notch signaling would be able to induce T cell fate specification. Normal B cell development would occur as long as Delta expression is restricted to the thymus or to areas within the BM where B cell development does not take place. Such a model is supported by ectopic expression studies for Delta1 and Delta4 (see above), in which T cell development is induced in the BM compartment concomitantly with a block in B cell development. To date such ectopic expression experiments using ligands from the Jagged family have not been reported. Further evidence that Delta and Jagged ligands elicit qualitatively different Notch signals and thereby drive hematopoietic progenitors into different developmental programs is derived from in vitro studies using stromal cells expressing different Notch ligands. While Delta1- or Delta4-expressing stroma can drive HSCs into the T cell lineage, HSCs grown on Jagged-expressing stromal cells develop into B cells.(74–76) In agreement with these in vitro studies, Jagged2 gene targeted mice have apparently normal ab Tcell development and only a minor decrease in gd T cells,(77) while inducible inactivation of Jagged1 has no effect on T cell development.(55) Collectively these observations would appear to exclude an important physiological role for Jagged family members during T cell development, thus raising the question of which of the Delta ligands is involved. Surprisingly, T cell development in mice with an inducible inactivation of Delta1 was not perturbed(76) suggesting that either Delta4 must be the crucial ligand or that Delta1 and Delta4 can compensate for each other during Tcell lineage commitment. Further gene targeting studies are needed to address this issue. Notch and intra-thymic T cell development Following commitment to the T cell lineage, immature pro-T cells face two more lineage decisions. The first choice is between the ab and gd T cell fates while a further decision has to be made within the ab Tcell lineage to either differentiate into CD4þ helper T cells or CD8þ cytotoxic T cells. The original suggestion that Notch signaling influences the ab versus gd Tcell fate decision(78) has been challenged by two conditional loss-of-function approaches where either Notch1 or CSL was inactivated in immature thymocyte progenitors. Loss of Notch1 in developing thymocytes had no effect on gd T cell numbers or phenotype,(79) whereas loss of CSL resulted in a moderate 2-fold increase in gd T cell numbers and an accelerated turn-over rate as measured by BrdU incorporation.(80) While suggestive that Notch signaling may influence the rate of production of gd T cells, these data appear to exclude a critical role for Notch in ab versus gd T cell lineage commitment. Although Notch1 deficiency in immature thymocyte progenitors does not influence gd T cell development, it does result in a partial block in ab T cell development. Notch1deficient thymocytes are defective in TCRb gene rearrange-
ment. While Db to Jb rearrangement appears to be normal, rearrangement of Vb gene segments to DJb is perturbed.(79) Decreased amounts of Vb-germline transcripts have been observed in Notch1-deficient progenitors suggesting that the chromatin is not accessible to the recombination machinery.(81) Thus Notch signaling may control chromatin accessibility of Vb genes in the TCRb locus. The absence of Notch1 in immature ab lineage thymocytes results in an additional phenotype. Under normal conditions, those ab lineage pre-T cells that cannot form a functional pre-TCR complex are eliminated by apoptosis. However Notch1-deficient pre-Tcells lacking an in-frame TCRb chain accumulate in the thymus. Thus Notch1 appears to have two functions within the ab lineage, the first of which is linked to the successful rearrangement of TCRb while the second involves the elimination of thymocytes that fail to assemble a functional pre-TCR complex. A similar phenotype is observed in mice in which the CSL gene is inactivated in immature thymocytes suggesting that Notch1-dependent functions within the ab lineage are CSL dependent.(80) The final intrathymic cell fate decision is made by ab Tcells as CD4þCD8þ (DP) thymocytes must choose to adopt either a CD4þ T helper- or a CD8þ cytotoxic-T cell fate. Several gainof-function studies have linked Notch signaling to this lineage decision. Survival, maturation and influencing binary cell fate decisions were among the suggested roles for Notch signaling at this particular stage of thymocyte development.(26,82–85) However, since constitutive Notch signaling within the thymus causes T cell leukemias, the results were very difficult to interpret. In contrast, conditional inactivation of either Notch1(86) or the CSL(80) gene in DP thymocytes does not result in any detectable skewing towards either the CD4 or CD8 lineage clearly indicating that Notch signaling is dispensable for this cell fate decision. In this review, we will not discuss the role of Notch during peripheral T cell function. The interested reader is referred to several recently published reviews on this subject.(87–90) Notch and B cell development While Notch1 is essential for T cell lineage specification, Notch2 is important during B cell development. The Notch receptor that is predominantly expressed in B cells is Notch2, while Notch1 and Notch3 are expressed in thymocytes and Notch4 is barely detectable in lymphocytes.(59,91) Expression levels of Notch2 increase with B cell maturation and are highest in splenic B cells suggesting a role for Notch signaling in peripheral B cell development and/or function.(59) Indeed, conditional inactivation of the Notch2 gene leads to loss of a particular splenic B cell subset located on the margin of the B cell follicle at the blood–lymphoid interface, known as marginal zone B (MZB) cells.(59) MZB cells respond to blood-borne viral and bacterial agents. Their rapid activation and differentiation into antibody-secreting plasma cells helps to bridge the gap
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between innate and adaptive immunity, the latter of which is mainly effected by follicular B cells (FoB).(92,93) Loss of MZB cells is also observed in mice in which the CSL gene is inactivated in B cells demonstrating that Notch2 signals via CSL to specify the MZB cell fate.(94) The development of immature B cells in the BM compartment of both the Notch2and the CSL conditional gene-targeted mice appears to be normal suggesting that Notch signaling is dispensable for early B cell development.(59,94) Loss of MZB cells in conditional gene-targeted CSL mice is accompanied by a moderate increase in splenic FoB cells. A reciprocal splenic phenotype has been observed in MINT gene-targeted mice.(33) MINT is a negative modulator of Notch signaling, which is more abundantly expressed in FoB cells compared to MZB cells. MINT-deficient mice show an increase in MZB cell numbers with a concomitant reduction of FoB cells. These reciprocal phenotypes have led to the suggestion that Notch signaling influences the commitment of a bi-potential splenic B cell progenitor that has to choose between the MZB and FoB cell lineages (Fig. 3). To date it is unclear whether MZB cells and FoB cells are derived from a common progenitor since a skew towards FoB cells in the absence of MZB cells has not been observed in Notch2 conditional gene-targeted mice.(59) Further investigation is required to clarify this discrepancy. Most of our knowledge concerning Notch signaling and lymphocyte development is derived from conditional genetargeted mice for Notch receptors (Notch1 and Notch2) or the CSL transcription factor. Much less is known about the physiological roles of Notch ligands. Whether ligand-receptor specificity exists in vivo or whether different ligands elicit qualitatively different responses when binding to the same Notch receptor is currently unclear. In this context, it is interesting to note that conditional inactivation of Delta1 in the hematopoietic system (using the interferon inducible MxCre system) resulted in the selective loss of MZB cells in the spleen while Tcell development appeared to be normal.(76) As mentioned above, identical MZB cell phenotypes have been observed in conditional gene-targeted mice for Notch2 and CSL indicating that Delta1-mediated Notch2/CSL signaling specifies MZB cell lineage commitment in a non-redundant fashion in vivo. However it is unclear whether Notch signaling occurs between homologous or heterologous cell types in order to specify this particular B cell population. This question was addressed by B-cell-specific inactivation of Delta1 using the CD19-Cre system.(76) In this case, MZB cells were not lost suggesting that Delta1–Notch2-mediated signaling must occur between heterologous cells. Dendritic cells (DCs) were suggested to mediate Notch2 signaling on B cell progenitors based on the fact that DCs expressing Delta1 are found in close proximity to MZB cells at the margins of B cell follicles.(33) However, this interpretation awaits further experimental confirmation. In summary, these experiments demonstrate that MZB cells are specified by a unique Notch receptor–
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ligand pair. Whether another specific receptor–ligand pair also exists for Tcell fate specification will certainly be addressed in the near future. Notch and neoplasia The first linking of Notch with T cell neoplasia was made in the late 1980s(95) and early 1990s(96) with the cloning and sequencing of a t (7;9) chromosomal translocation in a small subset of patients suffering from T cell acute lymphoblastic leukemia (T-ALL). This chromosomal translocation juxtaposes the C-terminal region of EGF repeat 34 of the human NOTCH1 gene to the TCRb enhancer, which leads to the aberrant expression of a truncated, dominant active and ligandindependent form of Notch1. This truncated Notch1 gene is also known as Notch1-IC and has been named TAN1 for translocation-associated Notch homolog.(96) The causative role of this truncated protein in disease development has been confirmed in mouse models, as mice in which hematopoietic BM progenitors express TAN1 proteins develop T cell leukemia.(97) Similarly, constitutive expression of Notch1IC(26,82) or Notch3-IC(98) in immature thymocytes leads to the development of T cell leukemia suggesting that the oncogenic potential of the Notch pathway is not restricted to Notch1. Furthermore T cell malignancies such as lymphoproliferative disease(66) and Tcell leukemia can also be induced by constitutive expression of the Delta4 ligand in BM progenitors.(65) Athough all these aberrant gain-of-function studies show that Notch can indeed be oncogenic, to date the hematopoietic oncogenic potential seems to be restricted to T cell leukemias since no myeloid leukemias have been reported. This suggests that Notch signaling on its own cannot induce tumorigenesis but that Notch must cooperate with a T cell-specific signal in order to cause these Tcell malignancies. Indeed Notch-IC- expressing BM progenitors derived from either Rag2/ or Lcp2/ (SLP76-deficient) mice do not develop Tcell leukemias.(99) Both of these gene-targeted mice exhibit a developmental block during early Tcell differentiation because these genes are required for pre-TCR signaling, an essential component of ab T cell development. In contrast, expression of a transgenic TCR in Rag2-deficient mice restored the oncogenic potential of Notch-IC strongly suggesting that Notch-IC must cooperate with signals either mediated by the pre-TCR or the TCR itself to render T cells tumorigenic.(99) The molecular details of Notch-induced tumorigenesis are still not well understood and need further investigation. Experimentally Notch1 can collaborate with c-Myc,(100) E2APBX1(101) and Ikaros.(102) Notch3 downregulates tumor suppressor E2A activity(103) and activates protein kinase C which leads to activation of the NF-kB pathway.(104) Although, in mouse models, the association of Notch and T cell leukemia has been widely demonstrated, the rare frequency of the t(7,9) translocation in humans (less than 1% of all T-ALL patients) questions the clinical importance of these
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findings. However recent studies by Aster and colleagues show that NOTCH signaling is one of the key players in all TALL subtypes.(105) This group screened and identified a number of T-ALL cell lines that undergo G0/G1 arrest when cultured in the presence of g-secretase inhibitors (which inhibit cleavage of Notch receptors upon ligand-mediated activation). Subsequent sequencing of the NOTCH1 gene in these cell lines identified two domains that were frequently mutated, one of which is the extracellular heterodimerization domain (HD) and the other is the PEST domain, which is involved in regulating turnover of the protein. Analysis of 96 pediatric primary T-ALL tumors revealed that 55% of these samples had at least one mutation in the HD or the PEST domain, with approximately 20% of tumors having a mutation in both domains. Importantly transcriptional reporter assays confirmed that NOTCH1 mutations within these tumor samples correlated with increased Notch activity, which could be suppressed by g-secretase inhibitors. Mutations in the HD domain lead to a 3- to 9-fold increase and mutations in the PEST domain to an approximately 2-fold increase in Notch activity. However mutations in cis (in the HD and the PEST domain simultaneously) resulted in a 20- to 40-fold increase in Notch activity showing a striking synergistic effect. How these point mutations in cis lead to synergistic Notch-mediated transcriptional activation is mechanistically currently un-
known. Nevertheless these data push Notch from being marginally involved in T-ALL to being one of the major players, and highlight it as a potential therapeutic target for T cell malignancies.
Concluding remarks Notch signaling controls multiple cell fate decisions and differentiation processes during fetal hematopoiesis and adult lymphocyte development. Many of the crucial paradigms of Notch function such as maintenance of undifferentiated progenitors or the control of binary cell fate decisions have been established in invertebrates (Fig. 1). By analogy, many of the lineage specification events controlled by Notch in the mammalian hematopoietic system have been interpreted as binary cell fate decisions in analogy to the concept originally shown in Drosophila. For example, in the embryo, Notch signaling in hemangioblasts would specify hematopoietic versus endothelial cell fates. In the adult, further Notchmediated lineage decisions such as T versus B, ab T versus gd T and MZB versus FoB could be viewed as binary cell fate decisions (Fig. 3). However, an alternative interpretation is that Notch signaling promotes previously specified differentiation processes as has been shown for ab Tcell development or the skin.
Figure 4. Chromosomal translocations and mutations within the human NOTCH1 gene cause T cell malignancies. A: The t(7;9) chromosomal translocation in human Tcell acute lymphoblastic leukemia (T-ALL) patients is characterized by juxtaposition of the 30 portion of the human NOTCH1 gene into the T cell receptor b (TCRb) locus. This leads to expression of truncated NOTCH1 transcripts and consequent production of dominant active, ligand-independent forms of the NOTCH1 receptor causing T-ALL. This rare event occurs in less than 1% of all T-ALL patients. B: Schematic diagram of the full-length human NOTCH1 protein. Indicated are ‘hot spots’ of mutations found in more than 50% of T-ALL patients(105) HD, heterodimerization domain; P, Pest domain.
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Another commonly used analogy derived from invertebrates is the ‘gate-keeper’ function of Notch (maintenance of the undifferentiated state) that has been postulated for hematopoietic stem cells or BM progenitor cells. Although experimentally Notch signaling has been partially associated with preventing BM progenitors from differentiation, to date we have no clearcut genetic evidence to support this notion. However, it has recently become clear that aberrant Notch signaling in humans due to activating mutations in the Notch1 receptor plays a key role in the development of T-ALL (Fig. 4). Questions concerning specific Notch target genes, mechanistic insights into activating Notch mutations, ligand–receptor specificity, the physiological function of different Notch modifiers and cross talk between Notch and other signaling pathways remain to be investigated. Answers to such questions will surely expand our limited understanding of cell fate decisions and may ultimately lead to new strategies of how to fight against cancer. Acknowledgments We thank Pierre Dubied for preparation of the figures, and apologize to those whose work was not cited due to space limitations. References 1. Morgan TH. 1917. The theory of the gene. Am Nat 51:513–544. 2. Wharton KA, Johansen KM, Xu T, Artavanis-Tsakonas S. 1985. Nucleotide sequence from the neurogenic locus notch implies a gene product that shares homology with proteins containing EGF-like repeats. Cell 43:567–581. 3. Kidd S, Kelley MR, Young MW. 1986. Sequence of the notch locus of Drosophila melanogaster: relationship of the encoded protein to mammalian clotting and growth factors. Mol Cell Biol 6:3094–3108. 4. Artavanis-Tsakonas S, Rand MD, Lake RJ. 1999. Notch signaling: cell fate control and signal integration in development. Science 284: 770–776. 5. Artavanis-Tsakonas S, Delidakis C, Fehon RG. 1991. The Notch locus and the cell biology of neuroblast segregation. Annu Rev Cell Biol 7:427–452. 6. Furukawa T, Maruyama S, Kawaichi M, Honjo T. 1992. The Drosophila homolog of the immunoglobulin recombination signal-binding protein regulates peripheral nervous system development. Cell 69:1191–1197. 7. Schweisguth F, Posakony JW. 1992. Suppressor of Hairless, the Drosophila homolog of the mouse recombination signal-binding protein gene, controls sensory organ cell fates. Cell 69:1199–1212. 8. Poulson DF. 1937. Chromosomal deficiencies and the embryonic development of Drosophila Melanogaster. PNAS 23:133–137. 9. Knust E, Campos-Ortega JA. 1989. The molecular genetics of early neurogenesis in Drosophila melanogaster. Bioessays 11:95–100. 10. Coffman CR, Skoglund P, Harris WA, Kintner CR. 1993. Expression of an extracellular deletion of Xotch diverts cell fate in Xenopus embryos. Cell 73:659–671. 11. Chitnis A, Henrique D, Lewis J, Ish-Horowicz D, Kintner C. 1995. Primary neurogenesis in Xenopus embryos regulated by a homologue of the Drosophila neurogenic gene Delta. Nature 375:761–766. 12. Nye JS, Kopan R, Axel R. 1994. An activated Notch suppresses neurogenesis and myogenesis but not gliogenesis in mammalian cells. Development 120:2421–2430. 13. Jan YN, Jan LY. 1994. Genetic control of cell fate specification in Drosophila peripheral nervous system. Annu Rev Genet 28: 373–393.
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