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Stem Cells, Vol. 19, No. 6, 543-552, November 2001
© 2001 AlphaMed Press

The Notch Receptor and Its Ligands Are Selectively Expressed During Hematopoietic Development in the Mouse

Liberty Walkera, Anne Carlsonb, Hongying Tina Tan-Pertelb, Gerry Weinmastera,c, Judith Gassona,b,c

a UCLA Molecular Biology Institute, Los Angeles, California, USA;
b Division of Hematology-Oncology, Department of Medicine, UCLA School of Medicine, Los Angeles, California, USA;
c Department of Biological Chemistry, UCLA School of Medicine; Los Angeles, California, USA

Key Words. Notch1 • Notch2 • Jagged1 • Delta1 • Hematopoiesis • Fetal

Judith C. Gasson, Ph.D., Jonsson Comprehensive Cancer Center, 8-684 Factor, UCLA, Box 9517821, Los Angeles, California 90095-1781, USA. Telephone: 310-825-5268; Fax: 310-206-5553; e-mail: jgasson{at}mednet.ucla.edu


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Members of the Notch family of transmembrane receptors are found on primitive hematopoietic precursors, and Notch ligand expression has been demonstrated on the surface of stromal cells, suggesting a role for Notch signaling in mammalian blood cell development. The current report examines the expression of Notch receptors and their ligands in murine hematopoietic tissues to determine: A) which blood cell lineages in the adult are influenced by Notch activity, and B) whether fetal hematopoiesis in the embryo involves the Notch pathway. In the adult mouse, a combination of flow cytometry, immunohistochemistry and Northern analysis was used to examine Notch receptor or ligand expression in bone marrow and spleen. In the embryo, Northern analysis and in situ hybridization were used to characterize Notch receptor and ligand expression in fetal liver on embryonic day 12 (E12) through E17, an active period encompassing both erythropoiesis and granulopoeisis. Flow cytometry demonstrated the presence of Notch1 and Notch2 receptors on bone marrow-derived myeloid cells but not on erythroid cells positive for the marker, Ter-119. In situ hybridization of E12 through E17 fetal liver demonstrated widespread expression of Jagged1 and Delta1 in a pattern similar to but less abundant than that of the erythropoietin receptor. Taken together with earlier functional results, the current expression data suggest a role for Notch activity in establishing definitive hematopoiesis in fetal liver, as well as a selective use of Notch signaling in adult erythropoiesis and granulopoiesis. Notch receptors in the adult are most likely utilized by early erythroid precursors and intermediate-stage granulocytes, but not by terminally differentiating cells of either subset.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Signaling through the Notch cell surface receptor is a highly conserved mechanism of cell fate specification widely used during embryonic development in both vertebrates and invertebrates. When Notch interacts with one of its ligands on the surface of an adjacent cell, the receptor is proteolytically processed, and nuclear translocation of the receptor's intracellular domain results in transcriptional regulation of lineage-specific genes [1–3].

In vertebrates, gene duplication has led to a diverse repertoire of Notch-related molecules. Four Notch genes [4], four Delta genes [5–8], and two Serrate-like or Jagged genes [9–11] have been described. Notch function has been studied extensively in mouse models, where it is involved in early pattern formation of the embryo [12, 13], as well as fate specification of tissues as diverse as brain, muscle, pancreas, skin and kidney [14, 15].

Hematopoiesis is also regulated by Notch signaling [16]. Notch ligands have been identified in both human [17] and murine [18, 19] bone marrow stroma, while Notch receptors have been found in many blood cell types, including developing T cells [20, 21], B cells [22, 23] and myeloblasts [22, 24–26]. Notch has also been identified in primitive hematopoietic precursors that lack lineage-specific antigens [19, 22, 27], suggesting a role for the Notch pathway in early stem cell populations.

Notch activation during blood cell maturation has generally been associated with enhanced survival and protection against apoptosis. For example, Notch function is important to early thymocyte expansion [28, 29] and rescue from apoptosis [30, 31]. Moreover, activated forms of Notch enhance the survival of developing T cells committed to the CD8 [32] and the {gamma}{delta} lineages [33]. In myelopoiesis, Notch signaling promotes granulocytic differentiation [24, 34] and reduces cell proliferation [35, 36], but it also protects against apoptosis [24] and enhances the survival of primitive multipotent precursors [18, 19, 36].

To date, hematopoietic Notch expression studies have focused on isolated precursors or progenitors and the stromal cells that support them. Outside the thymus [10, 20, 37], few studies have examined the expression of Notch receptors and their ligands in intact hematopoietic organs such as bone marrow [22, 38] and fetal liver. Hematopoiesis is an unusual developmental system, in that it requires stem cell maintenance, along with progenitor cell commitment, proliferation, and differentiation in both the adult and the embryo. The present study addresses whether the emerging role for Notch signaling in adult hematopoiesis is complemented by a role for the receptor in the early development of hematopoietic tissue. Isolated mouse bone marrow cells were used to identify those subsets expressing Notch1 and Notch2 receptors in the adult, while murine fetal liver was used to characterize expression of Notch ligands in the developing embryo. The results support a model whereby Notch signaling is involved in establishing definitive hematopoiesis in the developing embryo. Together with previously published functional data [24, 36], the current results also suggest that the Notch receptor enhances stem cell maintenance in adult bone marrow, where it is selectively used by early erythroid precursors and intermediate-stage granulocytes, but not by terminally differentiating cells of either subset.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Flow Cytometry
Bone marrow cells (106 per sample) were blocked with anti-CD32 monoclonal antibody (mAb) (PharMingen; San Diego, CA; http://www.pharmingen.com) and incubated for 30 minutes with phycoerythrin (PE)-conjugated mAbs to one of the following lineage markers: B220, CD11b, GR-1, or Ter-119 (PharMingen). After gentle fixation in 0.5% paraformaldehyde and permeabilization in 70% ethanol, cells were allowed to rehydrate at 37°C before blocking again with anti-CD32 mAb. Cells were then incubated with 0.4 µg of affinity-purified 93-4 antiserum [39] to the intracellular domain of rat Notch1, 1 µg of affinity-purified 93-7 antiserum to the intracellular domain of rat Notch2 [40], or an equivalent amount of normal rabbit IgG. After incubation with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Caltag; Burlingame, CA; http://www.caltag.com), cells were analyzed on a FACScan flow cytometer, using Cell Quest software (Becton Dickinson; Mountain View, CA; http://www.bd.com). Notch- or FITC-positive staining was examined on the lineage-committed or PE-positive cells in each sample.

Lineage-negative precursors were separated from committed bone marrow progenitors by incubating unfractionated bone marrow cells with a cocktail of unconjugated rat antibodies to the above lineage markers prior to incubation with a magnetic bead-conjugated goat anti-rat IgG antibody (Miltenyi; Burlingame, CA; http://www.miltenyibiotec.com). Labeled cells were retained on a Vario-MACS column (Miltenyi), while unbound cells were eluted and restained with an allophycocyanin (APC)-conjugated anti-rat IgG antibody. Subsequent Notch labeling was performed as described above, and Notch- or FITC-positive staining was visualized on APC-negative cells.

Northern Blot Analysis
Poly (A)+ RNA was extracted from fresh adult BALB/C spleens, splenocyte subpopulations, or the S194 cell line (kindly provided by Dr. Robert Hyman, Salk Institute; San Diego, CA). Splenocyte populations expressing surface immunoglobin (Ig+) were separated from Ig– populations on sterile plastic dishes coated with anti-Ig antibody. Northern blot analysis for Notch1 and Notch2 expression was performed as previously described [41]. Methylene blue staining verified equivalent RNA transfer to nylon membranes. Random-primed 32P-radiolabeled probes were prepared from cDNA inserts isolated from SN6 [14] to identify rat Notch1 transcripts, and from pooled cDNA inserts isolated from H10, H10-6, and RSC-3 [15] to detect rat Notch2 mRNA.

Embryonic RNA was extracted in STAT-60 reagent (Tel-Test Inc.; Friendswood, TX) from CD1 mouse fetal livers snap-frozen in liquid nitrogen. Approximately 15 µg of total RNA per sample were separated on a 1% agarose formaldehyde gel before transfer to Hybond nitrocellulose (Amersham-Pharmacia Biotech; Piscataway, NJ; http://www.apbiotech.com). Ethidium bromide staining was used to normalize sample loading. Blots were hybridized overnight in Ultrahyb (Ambion; Austin, TX) at 60°C with 32P-labeled riboprobe for Notch1, Notch2, Jagged1, or Delta1. The regions of cDNA used as templates for riboprobe synthesis have been previously described [42]. After low and high stringency washes, blots were cleared with 1 µg/ml RNase A before exposure to film.

Immunohistochemistry
Femurs from 8-week-old female CD1 mice were fixed overnight at 4°C in 4% paraformaldehyde/phosphate-buffered saline (PBS), and decalcified at 4°C in 0.1M TRIS pH 7.4 containing 1M EDTA and 7.5% polyvinylpyrrolidone (molecular weight 360,000) (Sigma-Aldrich; St. Louis, MO; http://www.sigma-aldrich.com). Samples were then cryoprotected in 10% sucrose/2% paraformaldehyde/PBS, followed by 20% sucrose/PBS, before being frozen in optimum cutting temperature embedding medium (VWR Scientific; Ontario, CA; http://www.vwrsp.com) and stored at –70°C. Frozen sections (12-µm) were thaw-mounted on Superfrost-Plus slides (Fisher Scientfic; Tustin, CA; http://www2.fishersci.com/main.jsp), post-fixed in 4% paraformaldehyde/PBS, quenched in 3% H2O2, and blocked with 5% normal donkey serum prior to incubation with C20 goat anti-Jagged1 antibody (Santa Cruz Biotechnology; Santa Cruz, CA; http://www.santacruzbiotechnology.com) or an equivalent amount of normal goat IgG.

Sections were subsequently incubated in biotinylated donkey anti-goat IgG (Jackson Labs; West Grove, PA; http://www.jax.org), followed by peroxidase-conjugated avidin-biotin complex reagent (Vector Labs; Burlingame, CA; http://www.vectorlabs.com) and the chromogenic substrate, diaminobenzidine (Vector Labs). Sections were lightly counterstained in aqueous hematoxylin (Biomeda; Foster City, CA; http://www.biomeda.com), and photographed on an Olympus IX 50 microscope. Images were prepared for publication with Photoshop 5.0 software.

In Situ Hybridization
Mouse embryos were fixed for 24 hours in 4% paraformaldehyde/PBS, then cryoprotected, embedded, and sectioned, as detailed above. In situ hybridization was performed as described previously [42]. Briefly, 12-µm frozen sections were hybridized with digoxigenin-labeled sense or antisense riboprobes (Roche Molecular Biochemicals; Indianapolis, IN) for rat Jagged1, Delta1, or the mouse erythropoietin (Epo) receptor (a generous gift from Hong Wu, University of California at Los Angeles). Bound probes were detected using alkaline phosphatase-conjugated sheep anti-digoxigenin antibody (Roche Molecular Biochemicals) and a nitroblue tetrazolium/BCIP (5 bromo-4 chloro-3-indolyl-phosphate) chromogenic substrate. Slides were coverslipped in aqueous mounting medium, and images were prepared as described above.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Notch Receptors are Widely Expressed on Hematopoietic Precursors
To assess the hematopoietic lineages influenced by Notch interactions, flow cytometry was used to characterize endogenous Notch expression in various subsets of committed progenitors harvested from normal bone marrow. Fluorescence-activated cell sorter analysis revealed Notch1 and Notch2 expression in myeloid cells positive for the markers CD11b (Fig. 1Go) and Gr-1 (data not shown), but not in erythroid cells positive for marker Ter-119 (Fig. 1Go). Precursors lacking expression of any lineage markers also expressed Notch1 and Notch2 receptors (Fig. 1Go). These data suggest that although the Notch receptor may play an extended role in granulocytic and monocytic differentiation, it probably is not involved in the later stages of erythroid maturation after the appearance of Ter-119. Bone marrow cells positive for the B220 marker also expressed Notch1 and Notch2, suggesting a role for Notch signaling in B cell development as well (Fig. 1Go). Northern analysis of adult spleen (Fig. 2Go) showed that Notch2, in particular, is highly expressed on the S194 myeloma cell line and on Ig+ splenocytes, suggesting that mature B cells in the periphery also make use of Notch signaling.



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  		Figure 1. FACS analysis shows Notch receptor expression on myeloid and lymphoid but not erythroid bone marrow progenitors. Hematopoietic antigens were labeled with PE-conjugated antibodies, while Notch1 and Notch2 proteins were identified using rabbit antisera 93-4 and 93-7, respectively, followed by FITC-conjugated goat anti-rabbit IgG. Cells were gated for the presence or absence of hematopoietic markers, then examined for Notch staining intensity. Histograms of cells stained with isotype-matched control antibodies (in gray) are displayed simultaneously with Notch stained cells (in bold). Lin– = lineage marker-negative.

 


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  		Figure 2. Northern blot analysis of adult mouse spleen indicates that Notch2 is highly expressed on mature Ig+ B cells. Ig+ splenocytes were separated from Ig– populations by adherence to anti-Ig-coated plates. S194 in lane 1 is a non-Ig-producing mutant of an IgA myeloma.

 
Jagged1 is Expressed in Adult Bone Marrow
Immunohistochemistry of intact adult bone marrow revealed significant Jagged1 staining (Fig. 3AGo). Minimal background staining was visible with an isotype-matched control antibody (Fig. 3BGo). Jagged1 expression has been previously demonstrated in primary bone marrow stroma [17–19, 36] and in bone marrow stromal lines [17, 19, 36]. The morphology of the positively stained cells in the sample presented here is not reticular, fibroblastic, or adipocyte-like, but rather, could be that of either a hematopoietic progenitor or a mature macrophage.



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  		Figure 3. Jagged1 immunohistochemistry of adult mouse femoral bone marrow. Frozen sections from decalcified bone were stained with goat anti-rat Jagged1 C20 antibody (A) or goat IgG (B), followed by biotinylated donkey anti-goat Ig, peroxidase-conjugated avidin and diaminobenzidine as chromogenic substrate. Positively stained cells do not have reticular or fibroblast-like morphology, but appear to be hematopoietic. Photomicrographs were taken at 100x magnification.

 
Notch Receptors and Ligands are Expressed in Fetal Liver
Northern blot analysis of fetal liver during definitive hematopoiesis from embryonic day 12 (E12) through E17 revealed full-length Notch1 and Notch2 transcripts, as well as Jagged1 and Delta1 expression (Fig. 4Go). Ethidium bromide staining of the 28S ribosomal RNA band is displayed below the riboprobe-stained lanes as a measure of sample loading. After normalizing for variation in sample loading, expression of both Jagged1 and Delta1 appeared to peak at E13, but subsequently declined.



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  		Figure 4. Northern blot analysis of Notch and its ligands in fetal liver during hematopoiesis. Total RNA was extracted from isolated murine liver on E12 through E17. 32P-labeled riboprobes identified full-length transcripts in each case. Ethidium bromide staining of each gel is displayed below the riboprobe-stained lanes as a measure of loading equivalence.

 
In situ hybridization was then used to determine whether any of the Notch ligand signal seen by Northern blot analysis could be found in hematopoietically relevant regions of the developing liver. Analysis of serial sections with digoxigenin-labeled antisense riboprobes revealed widespread patterns of Notch ligand expression similar to that of the Epo receptor during the onset of definitive hematopoiesis in the liver at E12 (Fig. 5Go). Positive cells (deep purple) were abundant, and peri-sinusoidal in sections stained for the Epo receptor (Figs. 5A and 5DGo), Jagged1 (Fig. 5BGo) or Delta1 (Fig. 5EGo), supporting a role for Notch signaling in the maturation of erythroid progenitors. Sample positive regions are indicated in Figure 5Go with arrowheads. Signal specificity was confirmed by staining with equivalent amounts of sense probe (Figs. 5C and 5FGo). Little nonspecific staining was detected in either the Jagged1 (Fig. 5CGo) or Delta1 (Fig. 5FGo) sense controls.



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  		Figure 5. In situ hybridization of murine fetal liver on E12. Frozen sections were stained with digoxigenin-labeled antisense riboprobes for the Epo receptor (A, D), Jagged1 (B), and Delta1 (E). Black arrows indicate sample areas where Epo receptor and Notch ligand staining are prominent. Control panels using sense probes for Jagged1 (C) and Delta1 (F) reveal minimal staining. All photomicrographs were taken at 20x magnification. Key: s = sinus; er = mature erythrocytes.

 
Widespread Epo receptor staining was still evident in the livers of older E16 embryos (Figs. 6A and 6DGo), even though erythropoiesis is declining and granulopoeisis has begun to predominate in the liver at this stage. Widespread patterns of Jagged1 (Fig. 6BGo) and Delta1 (Fig. 6EGo) expression were also detected in these animals (see arrowheads), consistent with a role for ligand-mediated Notch signaling in the maturation of granulocytic progenitors as well. Although Notch ligand expression was strong in fetal liver at E12 (Fig. 5Go), expression of both Jagged1 and Delta1 appeared to decline by E16 (Fig. 6Go), corroborating the expression detected by Northern analysis.



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  		Figure 6. In situ hybridization of murine fetal liver on E16. Frozen sections were stained with digoxigenin-labeled antisense riboprobes for the Epo receptor (A, D), Jagged1 (B), and Delta1 (E). Black arrows indicate sample areas where Epo receptor and Notch ligand staining are prominent. Control panels using sense probes for Jagged1 (C), and Delta1 (F) reveal minimal staining. Note chararcteristic Delta1 staining of the metanephric tubule (mt) in the kidney. All photomicrographs were taken at 20x magnification. Scale bar = 50 µm. Key: k = kidney, s = sinus, sk = skin.

 
To monitor probe specificity and validate specific staining of the liver, documented ligand staining patterns were confirmed in other tissues, such as neural tube, kidney and vasculature. For example, Delta1 is highly expressed in the metanephric tubule (mt, Fig. 6EGo), while a specific signal in this structure was not detected with sense control probe (Fig. 6FGo). In fact, specific staining in E16 sections was not detected with sense control probes for either Jagged1 (Fig. 6CGo) or Delta1 (Fig. 6FGo).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Widespread expression of Notch family members on the surface of bone marrow-derived hematopoietic cells suggests an ongoing role for Notch signaling in the continuous blood cell production of adults. Notch family members are expressed on the surface of primitive hematopoietic precursors [19, 22, 27, 36], implying that Notch signaling is involved very early in mammalian blood cell development.

Notch interactions have been implicated by a number of research groups in the survival of early multipotent precursors. For example, precursors exposed to Jagged ligands demonstrate enhanced survival of primitive long-term culture initiating cells [36] or generate increased numbers of mixed lineage colonies compared to controls [18, 19, 43]. Moreover, an activated form of Notch1 can immortalize primitive bone marrow precursors still capable of generating both lymphoid and myeloid progeny [44]. Thus, Notch signaling in uncommitted precursors leads to enhanced survival, while preserving multilineage potential. Such a role for Notch activation in early hematopoiesis is indicated at the top of Figure 7Go, which illustrates, for selected hematopoietic lineages, those stages at which Notch signaling may be involved.



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  		Figure 7. Proposed sites of action for Notch signaling in selected hematopoietic lineages. Current evidence suggests that Notch expression is restricted to the shaded cell types. Notch signaling promotes the survival of multipotent stem cells, as well as the formation of erythroid colonies and granulocytic intermediates. Notch is not expressed in erythroblasts, and probably not in mature neutrophils. Since the Notch receptor may inhibit early B cell development, its activity is probably restricted to more mature B cells (see text).

 
Notch signaling clearly has an impact on lineage-committed hematopoietic cells. Notch1 and Notch2 receptors are expressed on primary murine bone marrow cells positive for the granulocytic and monocytic markers, Gr-1 and CD11b (Fig. 1Go). In addition, the murine myeloblast cell line, 32D, which is positive for Gr-1, also expresses Notch family members [24, 26]. Moreover, activated forms of Notch in these cells promote the intermediate steps of granulocytic differentiation, but inhibit terminal differentiation in response to G-CSF [17, 24, 34]. In monocytes, Notch1 and Notch2 are expressed at high levels; in particular, on peripheral and bone marrow-derived human CD14+ cells [22]. Activation of the Notch receptor in these cells induces apoptosis rather than macrophage differentiation in response to macrophage colony-stimulating factor (M-CSF). In summary, Notch signaling may play an extended role in granulocytic and monocytic development (as indicated in Fig. 7Go), but most likely it is not utilized by terminally differentiating neutrophils or macrophages.

Notch activation may also play a role early in erythropoiesis, but not in later stages. Elegant expression studies in intact bone marrow have identified Notch1 expression in primitive erythroid cells, but not in more mature erythroid cells such as acidophilic normoblasts [22]. Moreover, CD34+ cells exposed to Jagged1 in the presence of stem cell factor yield increased BFU-E compared to controls [36]. On the other hand, neither Notch1 nor Notch2 are expressed on Ter-119+ erythroid progenitors (Fig. 1Go), and activated Notch1 suppresses erythroid differentiation in the erythroleukemic cell line, K562 [45]. Thus, Notch signaling may enhance erythroid progenitor survival up to the colony-forming unit, erythroid stage prior to the appearance of Ter-119 [46]. However, it is likely to be downregulated during the intermediate and terminal differentiation of maturing erythroblasts (as indicated in Fig. 7Go).

The appearance in this study of Notch1 and Notch2 on bone marrow-derived cells and on Ig+ splenocytes is consistent with other reports demonstrating Notch1 or Notch2 expression in the murine spleen [15], on peripheral blood B cells in humans [22], and on human fetal B cell precursors [23]. Interestingly, recent evidence suggests an inhibitory role for Notch signaling in B cell development, an effect that appears to be reciprocal to its role in T cells [47]. Overexpression [48] and gene inactivation [28] studies suggest that Notch activity promotes the commitment of a common lymphoid precursor to the T cell lineage at the expense of B cell development. The expression of Notch receptors on B220+ cells in the bone marrow reported here suggests that either such cells are sequestered in a microenvironment that does not allow ligand binding, or that Notch signaling plays a role in later developmental stages of B cell maturation after lineage commitment, as postulated in Figure 7Go.

The staining of intact adult murine bone marrow suggests that in addition to expressing Notch receptors, hematopoietic precursors may express the ligand Jagged1. Bone marrow and thymic stroma have proven rich in expression of the Jagged1 [17–19, 36, 37] and Delta1 [49] genes. On the other hand, Jagged1 has been identified in myeloid colony-forming cells [25], while Jagged2 has been amplified from thymocytes [37], and from long- and short-term repopulating hematopoietic stem cells [50]. These data, along with our own, imply that Notch signaling during hematopoiesis may be mediated, not only by heterotypic interactions between precursors and stromal support cells, but also by homotypic interactions between hematopoietic precursors themselves.

Hematopoiesis is not restricted to the bone marrow, but is a lifelong developmental process with complex origins in the embryo (Fig. 8Go). Primitive erythropoiesis in the yolk sac prevails in the mouse until approximately E9, when colony-forming cells start to appear in the embryo proper in the aorta-gonad-mesonephros (AGM) region [51–54]. Because all the Notch and Notch ligand knockouts created to date survive at least until E9.5 with no obvious defects in yolk sac erythropoiesis [12, 13, 55–57], Notch interactions are less likely to play a significant role in primitive hematopoiesis. Therefore, this report focuses on embryonic Notch expression during definitive hematopoiesis in fetal liver.



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  		Figure 8. Sites of hematopoiesis during development. Bold text represents those tissues where Notch interactions probably play a role in normal hematopoietic development. Outlined text represents those tissues where evidence has not yet been acquired indicating that hematopoiesis makes use of the Notch pathway. AGM = aorta-gonad-mesonephros.

 
Northern blot analysis demonstrated expression of both Delta1 and Jagged1 in liver from E12 through E17, an active period during which definitive erythropoiesis emerges and peaks, followed by a wave of granulopoiesis [58, 59]. Because previous work has shown that Jagged1 is expressed in the arterial endothelial cells ([60] and G. Weinmaster, unpublished observations) and the biliary epithelium [61] of human fetal liver, it was of interest to determine whether any of the Notch ligand signal seen by Northern blot analysis was present in hematopoietically relevant regions of the developing liver. In situ hybridization studies revealed both Jagged1 and Delta1 expression in a pattern similar to that of the Epo receptor, consistent with the previously identified role for Notch signaling in promoting the survival of early erythroid precursors [36]. Moreover, the expression of Jagged1 and Delta1 in older embryos (E15-E17) confirms the previously identified role for Notch interactions in the maturation of granulocytic progenitors as well [24, 34]. Nonetheless, both Northern blot analysis and in situ hybridization demonstrated a peak in Notch ligand expression by E13 in fetal liver, which significantly declined by E17, consistent with the gradual transfer of hematopoiesis to the bone marrow, beginning on E15 [62]. These results support a model (Fig. 8Go) whereby Notch signaling is involved in establishing definitive hematopoiesis in the fetal liver as early as E12, just as the liver is being colonized by hematopoietic precursors emerging from the AGM region of the embryo [52] and persisting at least until E17.

Taken together, the current data imply a role for Notch signaling in establishing definitive hematopoiesis in fetal liver, as well as a selective use of Notch signaling in the adult in both myelopoiesis and lymphopoiesis. In the future, it will be of interest to determine whether this pattern of Notch usage in adult hematopoiesis is similar to or different from that used by maturing blood cells during fetal development.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We thank Rubina Ismail for assistance with Northern blot analysis, Enca Montecino-Rodriguez for assistance with flow cytometry, Claire Lindsell for assistance with in situ hybridization, Karen Lyons and Sharon Sampogna for assistance with immunohistochemistry and bone decalcification, and Wendy Aft for excellent manuscript preparation. Figures were prepared with the assistance of the staff in the Biomedical Technology Research and Instructional Productions facility at UCLA.

This work was supported in part by National Institutes of Health grants R01 CA40163 (J.C.G.) and P01 32737 (J.C.G.), and a fellowship from the UCLA Training Program in Biotechnology 5T32 GM08375 (L.W.).


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received August 20, 2001; accepted for publication August 21, 2001.




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