Fine-tuning Notch1 activation by endocytosis and glycosylation

Seminars in Immunology 15 (2003) 99–106

Fine-tuning Notch1 activation by endocytosis and glycosylation Ute Koch, Julie S. Yuan, James A. Harper, Cynthia J. Guidos∗ Program in Developmental Biology, Department of Immunology, University of Toronto, Hospital for Sick Children Research Institute, Rm 8104, 555 University Avenue, Toronto, Ont., Canada M5G 1X8

Abstract Recent studies have shown that disruption of Notch1 signaling in lymphocyte progenitors (LP) inhibits T cell development and promotes B cell development in the thymus. Conversely, inappropriate activation of Notch1 in LP inhibits B cell development and causes ectopic T cell development in the bone marrow. These observations imply that Notch1 activation must be spatially regulated to ensure that LP generate B cells in the bone marrow and T cells in the thymus. However, Notch ligands are expressed in both tissues. Studies in flies and worms have revealed that Notch activation is extremely sensitive to small changes in the amount of receptor or ligand expressed, and defined multiple mechanisms that limit Notch activation to discrete cells at specific times during development. Here, we describe how some of these mechanisms might regulate Notch activity in LP during the T/B lineage decision. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: T/B lineage commitment; Lymphocyte progenitors; Endocytosis; Glycosyltransferases

1. Notch receptors and ligands The Notch pathway is highly conserved in mammals, and inherited or acquired mutations in Notch receptors, ligands, or regulators cause a plethora of developmental defects, complex disease syndromes, and oncogenesis in humans and mice [1]. Notch receptors and Notch ligands are transmembrane proteins that contain many cysteine-rich EGF-like repeats in their extracellular (EC) domains. Notch ligands also contain a characteristic sequence motif termed DSL near their N-termini that is required for binding Notch proteins. The biochemical basis of Notch signaling has been recently reviewed [2], so will only be described briefly here. Receptor–ligand interactions induce proteolytic cleavage events that liberate the Notch intracellular domain (NIC ) from the plasma membrane and allow its transport to the nucleus. NIC then nucleates the assembly of a complex containing a CSL (CBF1, Su(H), LAG-1) transcription factor as well as transcriptional co-activators that induce the expression of the major effectors of the Notch pathway: Hairy/Enhancer of Split (HES) genes. Drosophila has one Notch protein and two structurally related Notch ligands, Delta and Serrate. However, mammals have four Notch proteins (Notch1–4), three Delta-like (Dll) ligands (Dll-1, Dll-3, and Dll-4), two Serrate-like ligands ∗ Corresponding

author. Tel.: +1-416-813-5026; fax: +1-416-813-8823. E-mail address: [email protected] (C.J. Guidos).

(Jagged-1 and Jagged-2), and seven HES genes. Genetic studies suggest that Delta and Serrate have both redundant and non-redundant functions in flies [3,4], but the degree to which different receptors, ligands, and HES genes have unique versus overlapping functions in mammals is not yet clear.

2. Notch signaling regulates binary cell fate decisions The Notch signaling pathway is widely used to regulate cell fate choices during development of invertebrate and vertebrates. Perhaps its most well-studied function is to regulate the choice between two alternative cell fates during neurogenesis. Typically, Notch signals inhibit progenitors from adopting a neuronal (primary) fate by default. In some contexts, this inhibition appears to be transient, and is thought to maintain the competence of progenitors to respond to later inductive signals that promote a secondary non-neuronal fate [5]. Accordingly, loss-of-function mutations in Notch pathway genes cause premature neuronal differentiation in mice [6–8]. In addition to inhibiting neuronal differentiation, Notch signals may also promote the self-renewal of neuronal stem cells in the mouse brain [9–11]. However, in other contexts, Notch signaling both inhibits the primary fate and instructively induces the secondary fate [12–15]. Notch signaling plays a similar role in determining how lymphocyte progenitors (LP) choose between the T or B cell

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fates in the thymus and bone marrow. First, Notch1-deficient LP cannot generate T lineage cells in the thymus or intestinal epithelium [16,17], suggesting that the Notch1 pathway is essential for the development and/or survival of committed T cell progenitors. Second, retroviral transduction of LP derived from adult bone marrow with N-1IC , a constitutively active form of Notch1, suppresses B cell development but induces ectopic development of CD4/CD8 double positive (DP) T cell precursors in the bone marrow in a thymus-independent manner [18]. Finally, two different strategies have revealed that an essential function of Notch1 is to suppress B cell development in the thymus. First, transgenic expression of Lunatic Fringe in immature thymocytes can potently inhibit Notch1 activation in LP, causing them to develop into B cells, rather than T cells in the thymus [19]. Similarly, ectopic expression of Deltex1 can also induce LP to generate B cells in the thymus [20]. Second, LP lacking Notch1 generate B cells, but not T cells when injected into a host thymus [21]. Interestingly, the magnitude of intrathymic B cell development appeared to be substantially greater when Notch1 was inhibited by Lunatic Fringe than when Notch1 was deleted from LP and other hematopoietic cells. This difference implies that Lunatic Fringe might regulate the activity of other Notch receptors as B cells develop in the thymus. Consistent with this notion, Notch signals have been shown to regulate late stages of B cell maturation in lymphoid follicles [22]. Collectively, these studies show that LP choose the B cell fate by default (in the absence of Notch1 signaling), and that Notch1 activation is required to inhibit B cell commitment and direct LP toward the secondary T cell fate in the thymus. Moreover, these studies demonstrated that contrary to prevailing paradigms, the thymic microenvironment can support all stages of B cell development when LP are prevented from activating Notch1. These data strongly suggest that LP are prevented from activating Notch1 while they reside in the bone marrow, but are induced to activate Notch1 when they enter the thymus. Consistent with the latter idea, all of the Notch ligands are expressed in thymic stromal cells or in one of the major thymocyte subsets [23], but it is not yet clear whether all five Notch ligands can efficiently activate Notch1 in LP. Jagged-2 is clearly not essential, since Jagged-2-deficient mice make normal numbers of T cells in the thymus [24]. Interestingly, a recent study showed that expression of Dll-1, but not Jagged-1, in the S-17 bone marrow stromal cell line can activate Notch and inhibit B cell production from human LP in vitro [25]. It will thus be important to determine whether Dll-1 and Jagged-1 play distinct roles in regulating the T/B lineage decision in vivo. However, Dll-1, Dll-3 and Jagged-1 are all essential for murine embryogenesis [26–28], so determining their essential functions in lymphocyte development will require conditional inactivation of each gene, perhaps in combinations of two or more. In addition, Notch receptors and ligands are expressed in the bone marrow and fetal liver, but their pattern of expression in specific stromal cell types and LP have not been reported. Finally, specific stro-

mal cell types and microenvironmental niches that support B cell development in vivo have not been identified, and this is a pre-requisite for designing conditional targeting strategies.

3. Regulation of Notch activation by inductive versus lateral signaling Two distinct types of cell–cell interaction regulate Notch-dependent cell fate choices in invertebrates (Fig. 1) [29,30]. Inductive signaling involves Notch–ligand interactions between distinct cell types, and regulates development of wings and eyes in flies. Signals through the EGF receptor have recently been shown to regulate Delta expression in these contexts [31]. An alternative mode of cell–cell interaction, termed lateral signaling (also called lateral inhibition or lateral specification) spatially regulates Notch activation in the developing nervous system of invertebrates to ensure that adjacent precursor cells adopt distinct cell fates. Lateral signaling involves interactions between equivalent precursors that initially express equal amounts of Notch receptor and ligand. Thus, each precursor can both send and receive Notch signals, and under normal circumstances, each precursor has an equal chance of adopting the primary or secondary cell fate (Fig. 2). However, mosaic analyses in flies and worms have shown that if a precursor has only one functional Notch allele, it will nearly always adopt the primary fate, whereas its neighbor expressing wild-type levels of Notch will be biased towards adopting the secondary fate [30]. Conversely, if one precursor has only one functional Delta allele, it will be heavily biased towards adopting the secondary fate, whereas its neighbor will preferentially adopt the primary fate. The key feature of lateral specification is that the amount of Notch activity in one precursor will influence the fate of neighboring precursor cells. How can such small differences in the amount of Notch receptor or ligand initially expressed affect the outcome of binary cell fate decisions so dramatically? Multiple studies suggest that Notch activation induces a transcriptional feedback loop to amplify these initially small differences [29,30]. Thus, activation of Notch signaling increases expression of Notch but decreases expression of Delta. This precursor will then be more likely to receive than send Notch signals, causing a further increase in Notch and decrease in Delta, until finally it adopts the secondary cell fate. In some cases, this feedback loop appears to get started by random fluctuations in the amount of Notch or Delta expressed by a particular precursor. In other cases, the precursor’s ability to send or receive Notch signals is intrinsically or extrinsically biased by expression of a Notch modulator. These include Numb, Neuralized, and Fringe, and their functions in regulating Notch signaling will be described in detail below. Surprisingly, in addition to Delta’s non-autonomous role in sending Notch signals to adjacent precursor cells, cell-autonomous receptor–ligand interactions (i.e. taking place within a single cell) also occur during lateral signaling.

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Fig. 1. Inductive vs. lateral signaling. Inductive signaling operates between non-equivalent cells that differentially express Notch and Notch ligands. Precursors that interact with the sending cell will activate Notch and adopt the secondary fate, whereas those that do not interact with the sending cell will fail to activate Notch and adopt the primary fate. In lateral signaling, precursor cells express both receptor and ligand and can thus signal to each other. Subtle differences in the amount of Notch activity in different cells become amplified by a feedback loop such that one cell expresses more ligand and the adjacent cell expresses more Notch. Ultimately, cells with more ligand are prevented from activating Notch and become sending cells that efficiently activate Notch in adjacent cells, diverting them to the secondary fate.

Fig. 2. Effects of haploinsufficiency for Notch or Delta on lateral signaling. See text for details.

Genetic studies have documented that Delta can inhibit the activation of Notch in the same precursor that expresses it [32–34]. Thus, a precursor expressing more Delta than Notch will be relatively harder to activate and be biased towards become a sending cell. Ultimately, ligand-dominant precursors will adopt the primary fate and activate Notch in neighboring precursors to divert them to the secondary fate. This cell-autonomous function of Delta may be due to the formation of Delta–Notch heteromeric complexes within the same cell [35].

4. Endocytosis and glycosylation fine-tune the expression and function of Notch receptors and ligands The genetic experiments described earlier reveal that Notch signaling is exquisitely sensitive to small changes in the amount of receptor or ligand expressed. While Notch activation by lateral signaling clearly plays an important role in transcriptionally regulating the amounts of receptor and ligand expressed, genetic screens have identified a

plethora of other gene products that modulate Notch signaling through post-transcriptional and post-translational pathways. Endocytosis plays a particularly critical role, as first revealed by studies showing that Dynamin function is required in both the sending and receiving cell for Notch-mediated lateral signaling to occur [36]. Below we discuss recent studies showing that Numb and Neuralized function as endocytic regulators that affect the outcome of lateral signaling by fine-tuning the amount of receptor or ligand expressed at the cell surface. We also discuss the functions of Fringe, a glycosyltransferase that alters the efficiency of Notch activation by Delta versus Serrate during inductive signaling in flies. Finally, will discuss data from our lab showing that Fringe can regulate Notch1 activation in LP by lateral signaling. 4.1. Numb Genetic studies in Drosophila have shown that Numb acts cell-autonomously and upstream of Notch to suppress its activity in regulating the development of external sensory organs and some neuronal lineages of the central nervous

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system [37]. Sensory organ precursor cells divide four times, giving rise to sensory neurons and non-neuronal support cells. After each division, Notch-dependent lateral signaling between the daughter cells ensures that they adopt distinct fates. As the precursor prepares to divide, Numb protein becomes asymmetrically localized and eventually segregates into only one of the two daughter cells. The importance of this asymmetric cell division was revealed by genetic experiments showing that loss-of-function Numb mutations cause two non-neuronal cells to develop from each cell division. In contrast, when Numb is ectopically expressed such that both daughter cells inherit the protein, they both adopt the neuronal fate. Thus, the differential expression of Numb in the two daughter cells determines their cell fate. Structurally, Numb resembles an adapter or scaffold protein that possesses an N-terminal phosphotyrosine binding (PTB) domain and a proline-rich C-terminal region [38]. In vitro studies have shown that Numb binds directly to NIC [39–41]. The C-terminal half of the PTB domain and the N-terminus of Numb are required to inhibit Notch [42]. Numb also has two motifs associated with endocytic proteins: an Eps 15 homology binding motif [43] and two binding sites for ␣-adaptin, a component of the AP-2 clathrin adaptor complex. Accordingly, mammalian Numb (mNumb) localizes to clathrin coated pits and early endosomes, and over-expression of fragments of Numb can block internalization of the EGFR and transferrin receptors [44], implying a role for Numb in intracellular vesicle trafficking events. The asymmetric localization of Numb is dependent on its interaction with ␣-adaptin, and mutations in the latter that prevent its asymmetric localization cause phenotypes similar to those seen in Numb mutants [45]. Moreover, ␣-adaptin, like Numb, functions genetically upstream of Notch. Given that Numb also binds NIC , one possibility is that Numb links Notch to AP-2, targeting Notch for endocytosis and thus reducing the amount capable of interacting with Delta at the cell surface. However, this model is hard to reconcile with the observation that endocytosis plays a positive role in Notch signaling [36]. Thus, another scenario is that endocytosis is required for proteolytic cleavage of NIC , and that Numb subsequently targets endocytosed NIC for proteosomal destruction, preventing it from translocating to the nucleus. Consistent with this notion, mNumb can associate with two E3 ubiquitin ligases, Mdm2 [46] and LNX [47]. In addition, two other ubiquitin ligases, Suppressor of deltex/Itch and SEL-10 can target NIC for ubiquitination and degradation [48]. It will clearly be challenging to develop an integrated understanding of the mechanisms by which so many different proteins fine-tune the amount and/or localization of NIC in receiving cells. The phenotypes of mNumb-deficient mice are consistent with the notion that it inhibits Notch during embryonic neurogenesis [49,50]. Furthermore, ectopic expression of mNumb reduces Notch1 target gene expression in immature thymocytes, indicating that mNumb can antagonize Notch1 signaling in vivo [51]. However, thymocyte development,

cell cycle and survival were unperturbed by transgenic over-expression of mNumb. This observation is consistent with the fact that Notch1 is not essential for T cell development after the CD4/CD8 double negative 4 (DN4) thymocyte stage. Given that essential roles for Notch1 have been defined during T versus B cell lineage specification and at the DN2/3 thymocyte stage [52], modulating mNumb levels at earlier stages of hematopoiesis may suggest a role for it in lymphocyte development. Ultimately, this possibility will need to be evaluated by conditionally inactivating mNumb in LP, but the related Numblike gene could well be functionally redundant [53]. 4.2. Neuralized Neuralized was first identified over 20 years ago as a recessive loss of function mutation giving rise to embryonic hypertrophy of the nervous system, similar to that observed in Notch mutants [54]. Drosophila Neuralized (Dneur) is expressed in a subset of tissues regulated by Notch signaling in early development, such as developing neuronal precursor cells. Mouse Neuralized is expressed in neural tissues, the skeletal system, sensory and internal organs undergoing epithelial interactions [55]. It localizes to the cytosolic side of the plasma membrane via two internal conserved motifs [55–57]. Neuralized functions as a conserved C-terminal RING finger domain that ubiquitinates the cytoplasmic tail of Delta, targeting it for endocytosis and proteosomal degradation [56,58–60]. The level of Neuralized activity is critical in Notch activation. Decreasing Neuralized activity by deletion of the RING domain reduces ubiquitination of Delta, leading to its accumulation at the cell surface. Conversely, increased Neuralized activity results in Delta being endocytosed more rapidly. Surprisingly, however, both manipulations interfere with Notch activation [55,56,58]. These apparently diametric activities can be reconciled by realizing that Neuralized, like Delta, can act both autonomously (in receiving cells) or non-autonomously (in sending cells) to regulate Notch activity during lateral signaling. If Neuralized decreases Delta expression on the surface of sending cells, they will be less able to activate Notch in adjacent receiving cells. In contrast, reduced Neuralized activity in receiving cells would increase Delta, autonomously inhibiting Notch. It is also possible that Neuralized acts directly on Notch in receiving cells [61]. An alternate and unusual mechanism of non-autonomous Neuralized regulation has also been proposed [55,62]. This model is based on the observation that the extra-cellular domain of Notch (NEC ) can be internalized into the sending cell upon binding to Delta [62]. This trans-endocytosis appears to be induced following Neuralized-dependent ubiquitination of Delta. As mentioned earlier, Notch activation is genetically dependent on intact endocytosis in both sending and receiving cells [36]. Thus, the proteolytic cleavage of Notch may require a conformational change that is induced during trans-endocytosis of NEC and Delta into the sending

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cell. In the receiving cell, the final proteolytic cleavage of NIC may occur within an endosome en route to the nucleus via retrograde microtubular motors [63]. While the detailed mechanisms remain to be fully elucidated, it is clear that endocytosis plays a major role in Notch activation during lateral signaling. 4.3. Fringe Fringe was first identified by genetic studies in Drosophila which suggested that it spatially restricts Notch activation to cells along the dorsal–ventral boundary of the primordial wing and eye [64]. In the Drosophila wing imaginal disc, all cells initially express Notch. Cells in the dorsal compartment (that will give rise to the top of the wing) express Serrate, whereas ventral cells (fated to become the bottom of the wing) express Delta. Fringe is co-expressed with Serrate in dorsal cells, where it acts cell-autonomously to inhibit Notch activation by Serrate and to potentiate Notch activation by Delta [65,66]. Consequently, cells on the ventral side of the boundary can only respond to Serrate and those on the dorsal side can only respond to Delta. In this fashion, Fringe alters the sensitivity of Notch for Delta versus Serrate and restricts Notch activation to cells along the dorsal–ventral border, inducing them to develop into the wing margin. Fringe similarly regulates Notch activation in the eye imaginal disc. To date, three mammalian Fringe proteins have been identified: Lunatic Fringe, Manic Fringe and Radical Fringe. Mammalian Fringes are expressed in several tissues where Notch-dependent patterning events occur as well as where Notch–ligand expression boundaries are found [67,68]. Mammalian Fringes have also been detected in lymphoid tissues including bone marrow [69] and various thymic populations (J. Tan, U. Koch, C. Guidos, unpublished observations). Analyses of Lunatic Fringe-null mice suggests that it critically regulates Notch1 activation during embryonic somitogenesis [70,71], whereas Radical Fringe has no essential function in embryogenesis [72]. Fringe proteins were recently shown to be Golgi-localized ␤1,3,-N-acetylglycosylaminyl transferases that modify the extracellular domain of the Notch receptor, explaining the cell-autonomous function of Fringe in flies [73–76]. Several Notch EGF repeats are modified by the addition of fucose onto serine and threonine residues [77], and Fringe proteins catalyze the addition of N-acetylglucosamine to these O-fucose modifications. The direct linkage of O-fucose to proteins as well as the Fringe-catalyzed elongation of O-fucose is quite rare, as only 11 different proteins from six different families have been shown to have this type of modification [78]. The biochemical mechanism by which Fringe affects Notch activation by Delta versus Serrate is not well understood. One report suggested that Fringe-mediated glycosylation of NEC alters its ability to bind Delta [74], but this was not seen in another study [73]. A more recent study suggests that Lunatic Fringe inhibits the cell-autonomous

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formation of Notch–Delta or Notch–Serrate complexes within the endoplasmic reticulum or Golgi [35]. To investigate the potential for Lunatic Fringe to modulate Notch1 activation in the thymus, we transgenically expressed it in T lineage-committed cortical thymocytes that also express Notch1. Surprisingly, we found that Lunatic Fringe had no cell-autonomous effect on thymocytes. Instead, transgenic thymocytes non-autonomously induced Tg− LP to develop into B cells, rather than T cells, in the thymus [19]. Two independent genetic approaches revealed that transgenic Lunatic Fringe altered the T/B lineage decision by modulating Notch1 signaling in LP. First, we showed that transgenic Lunatic Fringe preferentially promoted B cell development from LP with a single functional Notch1 allele (N1+/− ) relative to LP with two functional Notch1 alleles (N1+/+ ) in the same thymic lobe. Since prior studies demonstrated exquisite gene dosage sensitivity of Notch signaling, these data strongly suggest that Lunatic Fringe inhibits Notch1 activation in LP. In addition, we showed that retroviral transduction of LP with N1IC precluded them from adopting the B cell fate in response to Lunatic Fringe. Thus, these data demonstrate that Lunatic Fringe can act upstream of Notch1 to regulate the T/B cell fate choice of LP. The observation that Lunatic Fringe acts non-autonomously to influence Notch1 signaling in LP may suggest that Fringe proteins can modify Notch ligands, which also have EGF repeats with the consensus sequence for O-fucosylation. Indeed, a recent biochemical study showed that Delta and Serrate ligands, as well as Dll-1 and Jagged-1 can be glycosylated in vitro by purified Fringe [78]. While the effect of Fringe-mediated glycosylation on the function of Notch ligands has not yet been defined, these in vitro studies clearly suggest that Fringe could act non-autonomously to influence Notch signaling. Two genetic studies in Drosophila are consistent with a non-autonomous function for Fringe. First, Notch activation by ectopically expressed Delta or Serrate during Drosophila bristle development is inhibited by Fringe expression [66]. Second, co-expression of Serrate and Fringe in the Drosophila wing disc enhanced Serrate-dependent signaling [34]. Further studies will likely uncover important roles for Fringe proteins in regulating the functions of Notch ligands in vivo.

5. Inductive versus lateral Notch signaling in the T/B lineage decision As mentioned earlier, Notch receptors and multiple Notch ligands are expressed by thymic stromal cells as well as by different thymocyte subsets. Moreover, Notch1, Notch2, Dll-1, Jagged-1, and Jagged-2 are expressed by primitive hematopoietic progenitors, thymic epithelium, and bone marrow stromal cells in humans and mice [23]. Thus, either lateral or inductive Notch signaling (or perhaps both) regulate the T/B lineage decision. In support of lateral signaling, we have shown that the ability of N1+/+ LP to be inhibited

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Fig. 3. Communication between Notch1+/+ and Notch1+/− LP can affect the T/B lineage decision. (A) Data are summarized from Koch et al. [19]. Differing mixtures of N1+/+ LP inhibit N1+/− LP were injected into the thymus of Lunatic Fringe (L-Fng) transgenic mice. The production of T and B cells from each LP donor was assessed 3 weeks later. The results showed that N1+/+ LP predominantly chose the B cell fate when injected alone (left). However, many fewer N1+/+ LP became B cells when a small number of N1+/− LP were co-injected with N1+/+ LP (middle). Finally, hardly any N1+/+ LP became B cells when a large number of N1+/− LP were co-injected (right). These experiments demonstrate L-Fng can bias LP towards the B cell fate, and that the amount of Notch1 activity in one LP will influence the T/B lineage choice of neighboring LP, consistent with a lateral signaling mechanism. (B) These observations can be explained as follows: (1) L-Fng preferentially inhibits Notch1 activation in N1+/− LP. (2) Once inhibited, these N1+/− LP become sending cells which ultimately adopt the 1◦ B cell fate. (3) N1+/− LP activate Notch in adjacent N1+/+ LP, rendering them insensitive to L-Fng and allowing them to adopt the 2◦ T cell fate.

by Lunatic Fringe was influenced by the frequency of N1+/− LP co-injected into the thymus (Fig. 3A). Strikingly, even a small number of N1+/− LP profoundly inhibited N1+/+ LP from choosing the B cell fate when co-injected into the same Lunatic Fringe Tg+ thymic lobe (Fig. 3A). Thus, the choice of T/B cell fate by an individual LP can be influenced by the degree of Notch1 activity in neighboring cells, and Lunatic Fringe can strongly bias LP towards the B cell fate in the thymus. These data are consistent with the predictions of the lateral signaling model, and suggest that there is a dynamic and competitive element to the way in which Notch1–ligand interactions regulate the T/B lineage decision in this system (Fig. 3B). We suggest that Lunatic Fringe inhibits Notch1 activation more rapidly in N1+/− than in N1+/+ LP. Once inhibited, N1+/− LP may become sending cells which promote Notch1 activation in neighboring N1+/+ LP, rendering them insensitive to Lunatic Fringe.

6. Speculations Approaches to defining the roles of lateral versus inductive Notch signaling in the T/B cell fate decision are currently hampered by several unresolved issues. First, the Notch ligand(s) required for inducing LP to adopt the T cell fate in vivo have not been defined. Second, it is not known whether T cell commitment normally takes place in the fetal liver/bone marrow, or only after LP migrate to the thymus. While it is clear that LP can activate Notch1 when artifi-

cially placed in the thymus, it remains formally possible that they can also activate Notch1 prior to arriving in the thymus. This notion is consistent with the identification of committed T cell progenitors in fetal liver [79] and fetal blood [80]. Moreover, committed T cell progenitors have been observed in the bone marrow of congenitally athymic nude mice [81]. The extrathymic generation of these committed T cell progenitors has not yet been shown to be Notch1-dependent, but it would be surprising if it were Notch1-independent. Therefore, these data suggest that LP can sometimes activate Notch1 outside the thymus, and we must consider the possibility that LP may activate Notch1 by multiple mechanisms and in different microenvironments. For example, LP that adopt the primary B cell fate in the fetal liver/bone marrow may become sending cells that can activate Notch1 in adjacent LP, inducing them to adopt the T cell fate while still residing in those tissues. However, if this lateral Notch signal is weak or not sustained, it may simply prevent LP from becoming B cells, allowing them to respond to inductive Notch1 signals once they enter the thymus. Given the paucity of B cells in the normal thymus, it seems unlikely that lateral signaling is the primary mechanism for inducing LP to adopt the T cell fate in the thymus. Nonetheless, it may be an important back-up mechanism to ensure that rare LP that become B cells (because they failed to activate Notch1 upon entering the thymus) prevent their neighbors from adopting the same incorrect fate. Thus, lateral and inductive signaling may both have important roles in ensuring that B and T cells develop in different tissues. This notion is not without precedent, as the development of the Drosophila

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compound eye is sequentially regulated by lateral followed by inductive Notch signaling.

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Acknowledgements This work was supported by a grant from the Canadian Institutes of Health Research. In addition, C.G. is supported by a CIHR Scientist Award, U.K. was supported by post-doctoral fellowships by the National Cancer Institute of Canada with funds from the Canadian Cancer Society and the Leukemia Research Fund, and J.S.Y. is supported by a RESTRACOMP studentship from the Hospital for Sick Children.

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