Mapping the consequence of Notch1 proteolysis in vivo with NIP-CRE

Abstract

The four highly conserved Notch (N) receptors receive short-range signals that control many biological processes during development and in adult vertebrate tissues. The involvement of Notch1 signaling in tissue self-renewal is less clear, however. We developed a novel genetic approach N₁IP-CRE (Notch1 Intramembrane Proteolysis) to follow, at high resolution, the descendents of cells experiencing Notch1 activation in the mouse. By combining N₁IP-CRE with loss of function analysis, Notch activation patterns were correlated with function during development, self-renewal, and malignancy in selected tissues. Identification of many known functions of Notch1 throughout development validated the utility of this approach. Importantly, novel roles for Notch1 signaling were identified in heart, vasculature, retina and in the stem cell compartments of self-renewing epithelia. We find that the probability of Notch1 activation in different tissues does not always indicate a requirement for this receptor and that gradients of Notch1 activation are evident within one organ. These findings highlight an underappreciated layer of complexity of Notch signaling in vivo. Moreover, NIP-CRE represents a general strategy applicable for monitoring proteolysis-dependent signaling in vivo.

Introduction

Notch signaling controls spatial patterning and cell fate decisions throughout the animal kingdom (Artavanis-Tsakonas et al., 1999). The Notch genes encode large, single transmembrane receptors. Interaction between Notch receptors and ligands results in a conformational change followed by two proteolytic steps. First, the ectodomain is shed by an ADAM metalloprotease. Next, a presenilin-dependent enzyme called γ-secretase cleaves the receptor within its transmembrane domain. The freed intracellular domain enters the nucleus where it interacts with the transcriptional repressor RBP-J to mediate transcriptional activation of target genes (the “canonical” pathway; for a review (Mumm and Kopan, 2000)). While the possibility of proteolysis-independent Notch activity remains, the majority of Notch mediated signals in all metazoans depends on proteolysis (Fortini, 2002; Huppert et al., 2000; Schroeter et al., 1998), where Notch signaling regulates the balance between self-renewal and commitment in ectodermal (Yoon and Gaiano, 2005), mesodermal (Radtke et al., 2004b) and endodermal (Schonhoff et al., 2004) lineages. At present, however, we lack a comprehensive, high-resolution view of Notch1 proteolysis/activation patterns during embryogenesis or in adult vertebrate tissues.

Methods that reveal Notch pathway activity in an unbiased manner rely on the use of antibodies specific for cleaved Notch1 (α-VLLS) proteins (Cheng et al., 2003; Tokunaga et al., 2004) or the use of Notch-responsive reporter mice (Duncan et al., 2005; Ohtsuka et al., 2006; Souilhol et al., 2006). These have been informative, but have several limitations: i. the artificial nature of reporter transgenes may leave some Notch activity unreported (Ohtsuka et al., 2006; Souilhol et al., 2006); ii. Because much of Notch activity is mediated by the same DNA binding protein (RBP-J), target-based reporters are not receptor-specific; a critical deficiency if different Notch receptors perform distinct functions. iii. existing reporters only provide a snap-shot of pathway activity; iv. target-based reporters may respond to input from other signaling pathways (Ohtsuka et al., 2006); and v. each reflects only part of the Notch transcriptome (Ong et al., 2006).

Here we present a novel Cre recombinase approach (NIP-CRE), exploiting the requirement for receptor proteolysis to visualize cellular lineages experiencing Notch1 proteolysis. We provide evidence that this correlates with Notch1 activation. This approach should be widely applicable to the remaining Notch receptors and any biological process involving proteolysis of tethered, non-nuclear proteins.

Results

To generate a genetic sensor of Notch1 proteolysis in vivo we replaced the mouse Notch1 intracellular domain (NICD1), immediately downstream of the transmembrane domain, with the site–specific recombinase Cre (Fig. 1a and Supplementary Fig. S1a) such that the Cre activity is now governed by ligand-induced proteolysis of the Notch1 transmembrane domain tether. In cre-reporter strains such Rosa26-R (R26R; (Soriano, 1999)), Notch activation is visualized by β-galactosidase expression (Fig. 1b, d and e). Because Notch1 proteolysis releases Cre that leads to a cell- heritable expression of lacZ, Notch1 signaling in actively cycling stem/progenitor cells will mark all their descendents (i.e. producing a “clone”), whereas Notch1 activation in transit amplifying or differentiating cells will result in small clones (2–4 cells) or in salt and pepper patterns of individually labeled cells (Fig. 1c).

Fig. 1. Strategy of Cre-mediated lineage tracing of N1 activity.

A) Schematic diagram depicting the mouse Notch1 protein. Indicated are the extracellular domain containing a signal peptide (SP) and 36 EGF-like repeats, the three LIN-NOTCH repeats (LNR), the Transmembrane domain (TMD), and the RAM domain, seven Ankyrin repeats and the PEST and Transcriptional activation domain (Tad-PEST). S2 and S3 indicate the ADAM metalloprotease dependent cleavage and the γ-secretase dependent cleavage at Val1744, respectively. Using gene-targeting Cre-recombinase was inserted immediately downstream of Val1744 at R1752. Interaction of N1::cre receptors in vivo with Notch DSL ligands results in S2 and S3 proteolytic cleavages and release from Cre recombinase from the plasma membrane. B) Cre recombinase can irreversibly activate the ubiquitously expressed R26R reporter and permanently mark cells with lacZ expression in vivo. C) If N1::cre is activated in a stem cell, all surviving descendents appear blue; when Notch1 is activated in progenitors or differentiated cells, a mixture of blue and white cells will appear in any given tissue. D,E) Sagittal view of whole mount X-gal staining of E14.5 N1::cre;R26R embryos showing identical patterns of widespread labeling of several tissues. Black arrows indicate strong thymic staining in both embryos and white arrows indicate dorsal aorta (D) and umbilical artery (E). Original magnification 10x.

To test the fidelity of this system, we conducted cell-based transfection experiments with truncated Notch1-Cre fusion proteins (N1ΔE::cre), which are ligand-independent, constitutive substrates of γ-secretase (Kopan et al., 1996). Immunoblotting with a cleavage-specific Notch1 antibody (αVLLS) determined that cleavage of N1ΔE::cre by γ-secretase occurred at the identical amino-acid position to wild type Notch1 (Val1744). Moreover, Cre recombinase itself was not a substrate for proteolysis; release of Cre from N1ΔE::cre required Presenilin activity (Supplementary Fig., S2). This indicated that the Notch1-Cre fusion protein behaves similarly to wild type Notch1.

Using gene targeting in mouse embryonic stem cells we engineered a Notch1:cre fusion allele (N1::cre; Fig. 1a, Supplementary Fig. S1a and b). To minimize concerns that this allele may act as a dominant negative modifier of Notch signaling (Huppert et al., 2005), N1::cre mRNA was engineered to be less stable than the wild type N1 allele by including the exogenous late SV40 polyadenylation signal (Supplementary Fig., S1c). Two independent N1::cre mouse lines were derived from gene targeted ES cells that were healthy and fertile indicating absence of dominant negative effects as a consequence of competition for ligands. Furthermore, N1::cre homozygous embryos die at E9.5, confirming that this is a null N1 allele (Conlon, 1995); not shown))

N1::cre^tg/+; R26R^tg/+ (henceforth, N1::cre) embryos and adult tissues display remarkably consistent patterns of lacZ activation, indicating non-random proteolysis patterns of N1::cre (compare Fig. 1d and e) validating this approach. Here we report a survey of all three germ layers, identifying novel aspects of Notch1 signaling by comparing the clonal patterns of Notch1 activity with the behavior of ubiquitous lacZ expressing ROSA26 (Gt(ROSA)26Sor), Notch1-deficient ES cells (N1^+/+:N1^Δ1/Δ1;Rosa26-lacZ^tg/+) in chimeric mice (described in (Hadland et al., 2004)).

Ectoderm

Epidermis/appendages

The epidermis and the hair follicles are maintained via activation of two separate populations of multipotent adult stem cells. Notch1 has been suggested to play a key role in asymmetric epidermal stem cell division, in promoting epidermal differentiation and in maintaining hair follicle architecture (Blanpain and Fuchs, 2006). Whether Notch1 signaling is involved in epidermal stem cell renewal is less well defined. Starting as early as E11.5, N1::cre marked cell populations are observed in the apical ectodermal ridge (AER) of fore- and hind-limbs where Notch1 is required (Pan et al., 2005). At E12.5, lacZ staining becomes more prominent in the AER (Fig. 2a), expanding laterally between the dorsal and ventral surfaces of the epidermis (Fig. 2c), reminiscent of Notch activity in NAS mice which contain a transgenic, Notch pathway reporter composed of multimerized RBP-J binding sites driving lacZ (Souilhol et al., 2006). Later in development, lacZ staining is detected exclusively in supra-basal epidermal cells (Fig. 2c). Transit amplifying cells, which reside in the basal layer and undergo several rounds of cell division, were expected to have experienced Notch1 activation yet none were detected (Lowell et al., 2000). As epidermal morphogenesis proceeded (E14.5 onwards), lacZ-labeled cells remained confined to the supra-basal layer (Fig. 2d) and their abundance increased until virtually all supra-basal cells were LacZ positive by E16.5 (Fig. 2e). Immunostaining with α-VLLS antibodies (Fig. 2b) identified ongoing Notch proteolysis and activation only in supra-basal cells during these stages ((Pan et al., 2004; Pan et al., 2005)), consistent with a role for Notch1 in promoting differentiation (Okuyama et al., 2004). Strikingly, the adult epidermis demonstrated an almost complete absence of N1::cre marked clones (Fig. 2f); this provides independent confirmation that epidermal stem cells did not experience Notch1 activation. Infrequently, labeled subpopulations of cells were detected within adult hair follicles localized to the bulge region (Fig. 2f) where both epidermal and melanocyte stem cells reside (Moriyama et al., 2006). Control experiments demonstrated that lack of cell labeling was not due to lack of R26R reporter activity or to poor recombination frequency at the R26R locus in this tissue (Fig. 2g and (Vooijs, 2001)). Cre-induced epidermal-specific deletion of Notch1 does not affect cell fate selection or differentiation of epidermal progenitors until after weaning (Nicolas et al., 2003; Pan et al., 2004). In agreement, we observed that Notch1-deficient ES cells contribute to the epidermis and to the hair follicle throughout life without any apparent developmental defect (Fig. 2h). Our analysis argues that Notch1 activation does not play a role in the control of “stemness” within the epidermal stem cell niche in mice, but may promote differentiation in their descendents. Furthermore, it demonstrates that the frequency of Notch activation in an organ is dynamic; changing as animal’s age.

Fig. 2. Fate of N1 activated cells in the epidermal lineages.

A) At E12.5, lacZ is first activated in the AER (arrow) of the fore- and hind limb buds and the epidermis between the dorsal (C) and ventral surface. B) Nuclear α-VLLS staining demonstrating Notch1 activation in suprabasal nuclei of the epidermis at E12.5 (arrow). D) N1::cre marks only supra-basal cells in the epidermis at E14.5 (D) and at E16.5 (E) where the majority of supra-basal cells in the skin are labeled, but most of the follicular epidermis remains negative. In adult skin (F), staining was mostly absent in the epidermis; lacZ labeled cells were present only in the differentiated cells of the hair follicle and the bulge (arrow, and high magnification inset). G) Germ-line deleted R26R mice label all keratinocytes. H) Notch1 is not required for skin development since N1^−/−; Rosa26-lacZ^tg/+ cells efficiently contribute to all epidermal structures in adult mice. Original magnification A, 40x, C, D and E 20x and F,G and H 10x. Dashed line (B–E) delineates epidermis and dermis.

Neurons

Deletion of Notch1 or the downstream effector Hes genes leads to precocious neuronal differentiation, but may also directly promote glial cell fates (Gaiano and Fishell, 2002). In the retina Notch signaling regulates cell cycle exit, apoptosis and differentiation (Silver and Rebay, 2005) of neurons, but whether it is also involved in maintaining the earliest retinal progenitors is less clear (Jadhav et al., 2006). Genetic marking techniques have shown that all neurons and Müller glia are derived from a common multipotent progenitor (Turner and Cepko, 1987). In N1::cre mice scattered labeling of the retinal neuroepithelium was first observed at E14.5 coincident with the presence of NICD1 (Fig. 3a and not shown). In adults, robust cell labeling was observed throughout all retinal layers in both eyes (Fig. 3b and c). These data are in agreement with the dynamic expression patterns of the Notch-regulated Hes genes in the developing neuroepithelium and in the adult retina (Ohtsuka et al., 2006), and are consistent with a role for Notch1 activation in the earliest retinal progenitors identified by random clonal analysis (Turner and Cepko, 1987).

Fig. 3. Fate of N1 activated cells in neuronal lineages.

A) N1::cre labels retinal progenitors (arrows) at E14.5. B,C) In the adult retina ((L) left, (R) right eye), clones derived from retinal progenitors that experienced N1 activation contain all retinal cell types in both eyes. D) Adult cerebellum of N1::cre mice showing labeling of molecular layer (ML) and granular layer (GL) but little in the Purkinje layer (PL); note vascular staining (arrow) throughout. E) α-VLLS staining (red) identifies N1 signaling in ventral neural tube progenitors at E11.5. F) N1::cre marks similar progenitors at E12.5 in the ventral neural tube. G) Wild type ES cells efficiently contribute to all regions of the neural tube whereas N1-deficient, R26-lacZ tagged cells (H) are excluded from ventral region. I) Dashed line in E, G and H marks the presumptive border between neuronal progenitors and committed or differentiated motor neurons. Counter stain Neutral Red (A,B,C,D, F), DAPI (E), Haematoxylin (G,H). Original magnification A–H 20x. FP; floor plate, DA; dorsal aorta.

Notch1 has been shown to promote differentiation of V2 interneurons at the expense of motor neurons in the embryonic neural tube (Yang et al., 2006) and is essential for proper neuron and glial formation within the neural tube (Lutolf et al., 2002). At E11.5 α-VLLS staining identifies a pool of progenitors with ongoing Notch1 signaling in the ventral part of the hindbrain neural tube (Fig. 3e), partially overlapping with N1::cre labeling at E12.5 (Fig. 3f). In contrast, Notch-deficient cells displayed a bias against contribution to ventral progenitors (multiple sections from 3 embryos examined). The absence of Notch1-deficient cells in this small series could suggest the existence of lateral interactions with wild type cells (Fig. 3g and h), similar to that reported for Notch2 in the roof plate (Kadokawa and Marunouchi, 2002); While this possibility will be followed up in with larger cohorts, it tentatively suggests the interesting possibility that Notch1 activation may contribute to both specification of motor neuron progenitors and, later, to their differentiation.

In the postnatal cerebellum, glial cells and granular neurons were frequently labeled, whereas the Purkinje cells were rarely labeled (multiple embryos examined with LacZ and GFP reporters, Fig. 3d and Fig. S5-I). This is in contrast to NAS mice that specifically labeled Purkinje cells (Souilhol et al., 2006) likely reflecting the activity of other Notch receptors. This observation demonstrates the utility of NIP-CRE: Notch1 has been shown to be essential for Purkinje cell differentiation (Lutolf et al., 2002), NIP-CRE suggests this role is non-cell autonomous. While this possibility is still under investigation, these observations suggest that Notch1 activation in vertebrates can occur in neuroblasts (retina) or in specific populations of differentiating neurons (granular neurons, interneurons) where Notch1 selects a specific differentiation program.

Mesoderm

Vasculature

The vascular system is the first organ to function in the vertebrates and comprised of arteries and veins that are anatomically, functionally and molecularly distinct. Notch receptors and ligands are expressed in all endothelial lineages and implicated in several inherited syndromes with vascular involvement (Gridley, 2003). Notch1 deficient mice fail to form a proper vasculature, which results in their lethality by E9.5 (Huppert et al., 2000; Krebs et al., 2000).

As early as E12.5, endothelial cells within the dorsal aorta become labeled (Fig. 4a) and this pattern is expanded during embryogenesis until most arterial endothelium is marked. In contrast, venous endothelium was negative (Fig. 4a). At E14.5, endothelium in the aorta intercostal arteries was labeled (Fig. 4c). Likewise, umbilical arteries are labeled (Fig. 4e) while the umbilical veins and maternal vasculature was negative (Fig. 4e, E16.5). Staining for NICD1 confirmed that Notch1 signaling is active in arterial endothelial cells at these stages (Fig. 4d). Unlike the epidermis, staining of arterial endothelial cells persisted throughout adulthood (Fig. 4g, S5h), indicating that a Notch1 signal was either continuously active in these cells during tissue renewal or that all arterial endothelial cells were derived from primitive arterial endothelial precursors that experienced Notch1 proteolysis. Interestingly, the venous endothelium of adult mice contained labeled cells suggesting that these cells are only exposed to Notch1 signaling postnatally (Fig. 4h). Notch1 and Notch4 act redundantly in arterial vasculature (Krebs et al., 2000). To ask if Notch1 is required for arterial identity, we analyzed the vasculature in Notch1-deficient chimeric mice. N1-deficient cells rarely contribute to arterial vasculature in the yolk-sac labeled by N1::cre (compare Fig. 4e and f). Furthermore, N1-deficient cells show a strong bias against contribution to the arterial endothelium in the dorsal aorta (DA) (Fig. 4a and data not shown; the entire aorta examined in serial section in 2 embryos, sample sections examined from 4 additional N1−/− embryos). In the same embryos, N1-deficient cells efficiently contribute to the endothelium of the Posterior Cardinal Vein (PCV; Fig. 4b), a pattern complementary to the Notch1 fate map. Control N1^+/+;R26-lacZ^tg/+ cells show no bias and contributed to yolk-sac, DA and PCV (not shown). It is important to note that NAS transgenic reporters fail to identify Notch activity in yolk-sac (Souilhol et al., 2006). Our combined clonal and functional analysis argues for a non-redundant, cell autonomous requirement for Notch1 in establishing arterial identity.

Fig. 4. Fate of N1 activated cells vasculature.

A) N1::cre activity marks the endothelial lining of the dorsal aorta (DA) but not of adjacent posterior cardinal vein (PCV) at E12.5. B) In contrast, Notch1-deficient cells populate the PCV but do not contribute to the DA. C) At E14.5, complete labeling of the endothelial lining of the aorta and intrasomitic arteries (arrow) is observed consistent with NICD presence (D) as detected with α-VLLS staining (arrow) of endothelial cells. E) Whole mount X-gal staining of yolk sac of E16.5 N1::cre embryo showing labeling of umbilical arteries, whereas maternal vasculature and veins are unlabeled. F) Complete lack of contribution from Notch1-deficient, R26-lacZ marked cells to the yolk sac vasculature (small arrow). Normal contribution is seen to the capillary plexus (fat white arrow). N1 activity in adult vasculature labels endothelial cells and few smooth muscle cells (arrow) in the arteries (G) and in veins (H). Original magnification A,B,C,F; 20x, D,EG,H; 40 x.

Heart

The earliest indication of β-gal activity in N1::cre mice was detected in endocardial cells of the outflow tract and left and right ventricles at day 10.5 p.c. (Fig. 5a and b). As heart development proceeds, a single row of marked endocardial cells lined the future aortic valves (AV), as well as the outflow tract of the left ventricle and the brachiocephalic artery at E14.5 (Fig. 1d and e, Fig. 5c and d). This pattern is maintained and expanded during embryonic development, and at E16.5, the majority of endocardial cells in the embryonic heart were labeled. Notably, the X-gal-marked endocardial cells lining the valves and myocardium are receiving a Notch1 signal at E14.5, as shown by α-VLLS staining (Fig. 5e and f). In contrast, cardiomyocytes were neither labeled by N1::cre nor by α-VLLS. In adults, endocardial staining persists in addition to complete labeling of the endothelial vasculature (including veins and coronary arteries; Fig. 4g and h, 5h). Likewise, virtually complete labeling of all heart valves (mitral, tricuspid, pulmonic and aortic) was observed, indicating Notch1 activation occurred in their progenitors (Fig. 5g and h and not shown). In chimeric hearts, Notch1-deficient cells readily contributed to the cardiomyocyte lineage where N1::cre is inactive, indicating that Notch1 is not essential in this lineage (compare Fig. 5i and j). As with the embryonic dorsal aorta, preliminary chimera analysis suggests a cell autonomous requirement for Notch1 the endothelial linings of the coronary and in endocardium and since Notch1 deficient cells were excluded from these cell types. (Fig. 5j). The complementary patterns of N1::cre activity and Notch1 function highlight the predictive power of this approach to identify the derivatives of cells with a developmental requirement for Notch1. The demonstration for a developmental role of Notch1 in valve development offers hope that the mouse can serve as a model for human NOTCH1 haploinsufficiency, which was recently associated with aortic valve disease (Garg et al., 2005).

Fig. 5. Fate of N1 activated cells in the endocardial lineages.

A) Labeling of the heart in whole mount (E10.5) and after dissection in B). N1 activity results in lacZ labeling within the outflow tract (OFT, arrow) and the ventricles (not shown). C,D) Exclusive endocardial staining of the heart, the outflow tract and the lining of the valves (arrow) at E14.5. Myocardial cells are not stained. E,F) N1::cre labeled endocardial cells continuously receive a N1 signal (arrow) at E14.5, as shown by α-VLLS staining. G,H) In the adult heart, most endocardial cells retain label; the endothelial lining of the valve (arrow) and the arteries (H, arrow), where Notch1 is required, are also labeled (J). Compare complementary staining patterns of endocardial cells (arrow) and cardiomyocytes in I) N1::cre and J) N1−/−;N1+/+;R26lacZ chimeric hearts, respectively. Note that Notch1 is not active (I) in cardiomyocyte cells and also not required (J). Original magnification, A;16x, B 50x, C,G 10x,D, H20x, I,J;63x

Endoderm

Intestine

All four Notch receptors and several Notch ligands are expressed in the embryonic and adult gut (van Es et al., 2005b). Conditional disruption of the common downstream effector of all four mammalian Notch proteins, Rbpsuh (RBP-J), in adult intestine leads to massive differentiation of proliferative crypt cells into post (mitotic goblet cells, (van Es et al., 2005b) reminiscent of Hes1 deletion (Jensen et al., 2000). Currently, it is not known which of the four Notch receptors are critical in suppressing secretory differentiation of crypt progenitors. Here we address the role of Notch1 in this process. Notch1 mRNA expression was confined to the proliferative crypt compartment of the small intestine of adult mice (Supplementary Fig. S5k and (van Es et al., 2005b)) overlapping with NICD1 staining (Fig. 6i).

Fig. 6. Fate of N1 activated cells in the intestinal lineages.

A) N1::cre is not activated in the embryonic gut at E14.5. B,C) Abundant activity of N1::cre in the adult duodenum labels the complete crypt-villus axis. Within blue crypts, all cells appear labeled, suggesting monoclonality (B) but polyclonal villi show alternating patterns of Notch1 activity. Immuno identification of N1::cre descendents as enterocytes by Alkaline Phosphatase activity (D) goblet cells by PAS staining (E) endocrine cells by Synaptophysin staining (F, inset) and Paneth cells (G) by lysozyme staining (arrow). H) Infrequent labeling of single goblet cells suggests Notch1 signaling may also occur in (committed) differentiated intestinal epithelium. I) α-VLLS staining identifies Notch1 signaling in crypt progenitors and in few scattered goblet cells within the villus (also see Fig. S5-J). N1 VLLS staining is nuclear, the precipitate in the cytoplasm of goblet cells may be an artifact. J) Notch1 is expressed in spontaneous adenomas from Apc^min/+ (arrow) and adenomas from Apc^min/+:N1::cre mice are labeled, indicating N1 activation in cells sustaining Apc mutation (K). The normal villus epithelium does not express Notch1 but N1::cre marks this lineage (K, arrow). Notch1-deficient ES cells contribute efficiently to the adult intestinal epithelium of chimeric mice; they preferentially differentiate towards the secretory lineages at the expense of enterocytes. Note the significant increase in mucin-producing goblet cells in X-gal stained villi and crypts compared to unstained wild type intestine. (L) Control Notch1 wild type-R26-lacZ chimeric intestines show no preference. Quantitation of the differentiation defect observed in Notch1-deficient intestines by combining immunohistochemical staining for differentiated cell types with X-gal staining to identify N1-deficient cells expressed as absolute numbers with sdev (N) and as a ratio of N1-KO/N1-wild type (O). Note significant increase in all secretory lineages in the absence of Notch1 at the expense of enterocytes. The intestines of mice composed of wild type-R26-lacZ cells showed normal contribution and no defects (not shown). Original magnification A; 4x, B–G, J–M 20x; H,I 40x.

Whereas during embryonic gut development no labeling of epithelial cells cell was observed (Fig. 6a), the adult small intestine of N1::cre mice displays significant labeling along the cephalocaudal axis (Supplemental Fig. S3). Most proximal segments (duodenum) displayed a high frequency of N1::cre activation (Fig. 6b–g), whereas more distal segments (e.g., ileum, not shown) contained only a few labeled crypt-villus structures. A similar labeling pattern was detected in the colonic epithelium of N1::cre, albeit to a lesser extent (not shown). The reduction in N1::cre activation distally was not a consequence of lack of R26R activity in these cells (Supplementary Fig. S3, and not shown). Double Immuno- and histochemical staining of X-gal-stained tissues from N1::cre mice for differentiation markers indicated that all four epithelial types (i.e., goblet cells, Paneth cells, enteroendocrine cells and enterocytes) were lacZ marked (Fig. 6d–g). Lineage tracing identified uniformly labeled monoclonal crypts feeding labeled cells into adjacent, chimeric (polyclonal) villi (e.g. Fig. 6c). Within blue crypts, no unlabeled cells were detected, strongly suggesting that Notch1 activation occurred in a stem cell. Interestingly we also observed scattered X-gal labeled cells in villi (i.e. Fig. 6h). Goblet cells were also positive for α-VLLS immunoreactivity (Fig. 6i and S5l), suggesting that, in addition to its possible function in stem cells Notch1 signaling may also contribute to goblet cell differentiation (Zecchini et al., 2005), reminiscent of a role reported for Wnt-β-catenin-TCF signaling (van Es et al., 2005a).

To ask if Notch1 activation was essential for stem cell maintenance, we analyzed the contribution of Notch1-deficient cells to adult chimeric small intestine. Surprisingly, this analysis showed that Notch1 was not essential to the maintenance of intestinal crypt progenitors, despite its robust expression and activation (Fig. 6l and m). We did, however, observe a significant increase in the acquisition of the secretory cell fates at the expense of enterocytes throughout the cephalocaudal axis in the absence of Notch1 (Fig. 6n and o). These effects were mild, compared to those seen in RBP-J-deficient animals (van Es et al., 2005b) indicating that regulation of the enterocyte-secretory fate-switch requires Notch1, but that Notch1 acts redundantly with another Notch receptor in stem cell maintenance in the crypt epithelium. The graded activity of N1::cre observed along the cephalocaudal axis is in contrast with a constant requirement for Notch1 activity throughout the entire intestine, consistent with the interpretation that N1::cre also reports ligand density, revealing a higher-order organization not previously appreciated.

Colorectal cancer results from mutational activation of the Wnt pathway, most commonly due to the loss of the tumor suppressor gene APC(Bienz and Clevers, 2000). Consequently, mice carrying a mutated APC allele (Apc^min/+) develop intestinal adenomas that require an activated Notch pathway for their survival(van Es et al., 2005b). Notch1 and downstream Hes genes are also expressed and activated in adenomas that spontaneously arise in Apc^min/+ mice (Fig. 6j, and(van Es et al., 2005b)). To investigate whether the same target cell population that sustains Apc mutation in Apc^min/+ mice experienced Notch1 activation, we analyzed Apc^min/+, N1::cre compound mice. We observed that lacZ expression throughout entire dysplastic atypical foci and adenomas, suggesting that Notch1 activation was an early event during colorectal tumor formation (Fig. 6k). This analysis highlights the utility of N1::cre mice to mark cancer stem cells and will facilitate screening for tumors where Notch1 activation is an early event and inhibition of Notch signaling may have therapeutic benefit for the treatment of these cancers.

Discussion

Here we present the groundwork for high resolution in vivo mapping of vertebrate Notch1 activation. Although most if not all Notch1 activity depends on intramembrane proteolysis and monitored by our NIP-Cre approach, a different strategy will be needed to report proteolysis-independent functions of Notch1. We observed that the N1::cre fate-maps are highly reproducible between mice from two independent ES lines and, in many tissues, corresponds to known expression patterns of Notch target genes and reporter strains (Ohtsuka et al., 2006; Souilhol et al., 2006). This indicates that N1::cre activation patterns are not stochastic, reflecting authentic Notch1 activity. Significantly, our genetic labeling method combined with functional analysis identified important roles for Notch1 not appreciated in these transgenic reporters. In addition to confirming known activation patterns, N1::cre revealed aspects of Notch biology not yet appreciated. In these tissues, we correlated N1::cre activation with function by comparing our results to the published literature and by following the fate of lacZ marked Notch1-deficient ES cells during development and self-renewal. The survey described here demonstrates that Notch1 is activated in derivatives of all three germ layers and in each; Notch1 has both redundant and non-redundant functions.

First, and unexpectedly, the dependence on Notch1 function does not correlate with the probability of its activation. Limited activation of Notch1 in this particular reporter does not necessarily imply lack of an important function (for example, the somite: see supplemental figure S5c and (Huppert et al., 2005)). High levels of Notch1 activation correlate well with its essential role in T-cell development, and, as we show here, in arterial and endocardial/valve development; however, high levels of activation do not necessarily indicate an essential role (for example, the intestinal stem cell). The mechanistic basis for this observation is not understood; however, an essential role for canonical Notch signals in intestinal ES cells is demonstrated by the impact of γ-secretase inhibitors or loss of RBP-J (van Es et al., 2005b).

Second, a requirement for Notch1 activity was revealed even when other Notch receptors are present. For example, Notch4 is expressed in the arterial endothelial cells, but only Notch1 is essential (Limbourg et al., 2005). Interestingly, postnatally venous endothelial cells also became labeled. This finding would be consistent with a novel role for Notch1 during maintenance of venous endothelial cells that is distinct from the cell-fate choices mediated by Notch1 during the specification of arterial vs. venous identity. Recently, it was demonstrated that vein identity is controlled by the orphan receptor COUPTFIII repressing Notch signaling (You et al., 2005). Our results suggest that such repression may be relieved after establishing venous endothelial fate to permit Notch1 activity.

Third, whereas the notion emerging from expression profiling experiments suggests a common role for Notch1 in maintaining stemness (Ivanova et al., 2002; Ramalho-Santos et al., 2002), a more complex view surfaces from the fate map encompassing the four stem cell compartments surveyed here. Notch1 is highly activated in differentiating keratinocytes during an early developmental window, but the adult epidermis and hair follicles emerge from a progenitor population that did not experience Notch1 activation, do not contain NICD1, and are not labeled with HES-gfp or NAS, general reporters of Notch pathway activity. Loss of function analysis confirms that epidermal stem cells are not depleted when Notch1 is absent. High levels of Notch1 activation are observed in the intestine but Notch1 is not required for maintenance of this niche either, indicating that other Notch receptors may act non-redundantly there or that Notch1 is redundant with other Notch receptors. In contrast, during endothelial/endocardial development, a strict correlation between Notch1 activation and function was observed (Limbourg et al., 2005), despite the presence of Notch4. In the vertebrate nervous system, Notch1 ligands have been suggested to play a key, γ-secretase-dependent role in stem cell survival, but a role for a Notch receptor in this process was not demonstrated (Androutsellis-Theotokis et al., 2006). While N1::cre is active in early progenitor/stem cells within the retina and the ventral neural tube, N1::cre activation is not evident in a stem cell contributing to the cortex (data not shown) or the cerebellum. Likewise, hematopoietic stem cell labeling could not be assessed directly in this reporter strain, but B cells were derived from progenitors lacking LacZ activity (Supplemental Fig. 4b and c). In both cases, either the low probability of Notch1 activation explains the lack of label or Notch1 does not function the CNS and in the definitive HSC in the manner suggested. One implication of our finding is that HSC may emerge from dorsal aorta endothelial cells before they experience high Notch activation or that definitive HSC emerge from another, non-endothelial origin. Taken together, our data do not support a global assignment of Notch1 function in stem cells and demonstrate a more common function in the differentiating descendents.

Fourth, the lower expression of N1::cre compared to the wild type Notch1 allele (Supplementary Fig. S1) is fortuitous as it permits mosaic analysis. In tissues exposed to high Notch1 ligand, N1::cre is easily activated. In contrast, in tissues with low levels of Notch1 ligand, N1::cre ineffectively competes with the wild type allele and only a few cells are marked. We therefore propose that the N1::cre map reflects ligand densities as well as recording the consequences of Notch1 activation (Fig. 7). Based on this assumption, the somite, pancreas, hematopoietic/CNS and epidermal/bulge stem cells may all have a low probability of Notch1 activation, perhaps because functional ligand is scarce, whereas arterial endothelial cells, T-cells, endocardial cells, retinal progenitors and the more proximal small intestinal stem cells have a high probability of Notch activation. Our analysis provides in vivo experimental support for the existence of context-dependent thresholds for Notch1 activation and that Notch signaling functions in a dose-dependent manner (Guentchev and McKay, 2006). The molecular bases for these differences are not clear but may reflect the abundance of functional ligand, controlled by ubiquitination and endocytosis (Schweisguth, 2004).

Fig. 7. Ligand density and N1::cre genetic mapping.

Shown is a hypothetical model explaining the relationship between the strength of Notch signaling as a function of ligand concentration. The probability to identify a marked lineage in tissues within N1::cre mice is correlated with Notch1 function revealed by loss of function phenotypes (lof) and the ability to detect activated Notch1 protein by immunostaining (see Table 1).

Finally by comparing the timing of LacZ activity with the presence of NICD1 one can appreciate that lineage labeling in N1::cre appears to be delayed by several days. Furthermore, N1 activity in some lineages may be left unreported in these mice because of non-uniform reporter expression or because of poor Cre-mediated recombination at the R26R locus (Vooijs, 2001), although this appears not to be true for the epidermis. Obviously, lineages that undergo apoptosis in response to Notch1 activation require other methods for detection (Yang et al., 2004). Our results in the venous endothelium and the intestine suggest that, similar to the fly peripheral nervous system and the mouse hematopoietic system (Radtke et al., 2004a), Notch1 signaling may be utilized in a recurrent fashion to influence multiple cell fate decisions. Further refinement of the approach presented here employing hormone-inducible N1::cre alleles will allow the interrogation of consecutive uses of the Notch1 signaling pathway in any given cell type under physiological conditions and in disease processes.

Materials and Methods

Mouse manipulations

Homologous recombination was used to replace the Notch1 intracellular domain with Cre recombinase using the same strategy used to generate processing deficient-Notch1 mice (Huppert et al., 2000). Details are presented in supplementary information. N1::cre mice on a C57Bl6/J background were crossed with R26R/+ reporter mice (Soriano, 1999) to obtain embryo’s or tissues for histological analysis.

Histology

X-gal staining

Embryos or tissues were dissected in ice-cold PBS-MgCl₂ (2mM), fixed at 4 °C in 4% PFA and processed for X-gal staining as described (Hogan, 1994). Stained embryos or tissues were postfixed and embedded in paraffin. For cryo-sections, following fixation tissues were equilibrated at 4 °C, o/n in 30% sucrose-PBS MgCl₂, rinsed in PBS and embedded in OCT compound (Miles Scientific). Sections (5μM–15μM) were processed for X-gal staining as above.

In Situ Hybridization

Dissected intestines were immediately fixed in 4% neutral buffered formalin o/n at 4°C and further processed for paraffin embedding. RNA in situ hybridization on sections was performed as described (Gregorieff et al., 2005; van Es et al., 2005b).

Immunohistochemistry

For α-VLLS staining (Val1744, Cell signaling), 4% PFA fixed tissues were embedded in paraffin, dewaxed blocked with Methanol/H₂O₂ and antigens were retrieved in citrate buffer (pH 6.0). Sections were blocked in 3% BSA/PBS and incubated with primary rabbit polyclonal antibody Val1744 (2 days at 4°C followed by o/n incubation at ambient temperature). After washing, antibodies were visualized with DAB (Powervision, DAKO). Sections were counterstained with neutral red or Hematoxylin.

Table 1.

Correlation between N1::cre fate-map, α-VLLS staining and Notch1 loss of function in tissues.

Tissue	N1::cre	LOF	α-VLLS
Intestine	+	Mu	+
Vasculature	+	Mu	+
Endocardium	+	Mu	+
CNS	+	Mu	+
Epidermis	+/−	WT	+
Kidney	+/−−	WT	−*
Somites	+/−−	Mu	+/−
Pancreas	+/−−	Wt	−

LOF; loss of function, + staining, +/− some staining, +/−− very infrequent clones, − no staining

^*Note abundant staining throughout the vasculature of kidney but only infrequent staining of proximal tubules and no staining of podocytes where α-VLLS is positive but no effect of loss of N1 function is found (see text for details).