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First published online January 10, 2007
doi: 10.1242/10.1242/dev.02733

Development 134, 535-544 (2007)
Published by The Company of Biologists 2007
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Mapping the consequence of Notch1 proteolysis in vivo with NIP-CRE

Marc Vooijs1,2,*,{ddagger}, Chin-Tong Ong1, Brandon Hadland1, Stacey Huppert1,{dagger}, Zhenyi Liu1, Jeroen Korving2, Maaike van den Born2, Thaddeus Stappenbeck3, Yumei Wu1, Hans Clevers2 and Raphael Kopan1,{ddagger}

1 Department of Molecular Biology and Pharmacology and the Department of Medicine, Division of Dermatology, Washington University Medical School, St Louis, MO 63110, USA.
2 Netherlands Institute for Developmental Biology, Hubrecht Laboratory, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands.
3 Department of Pathology & Immunology, Washington University School of Medicine, St Louis, MO 63110, USA.


Figure 1
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  		Fig. 1. Strategy of Cre-mediated lineage tracing of Notch1 activity. (A) Schematic 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 (TM) and the Ram domain, seven ankyrin repeats and the PEST and transcriptional activation domain (Tad-PEST). S2 and S3 indicate Adam metalloprotease-dependent cleavage and the {gamma}-secretase dependent cleavage at Val1744, respectively. Using gene-targeting, Cre recombinase was inserted immediately downstream of Val1744 at Arg1752. Interaction of N1::cre receptors in vivo with Notch DSL ligands results in S2 and S3 proteolytic cleavages and release of 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 and white arrows indicate dorsal aorta (D) and umbilical artery (E). Magnification: 10x.

 

Figure 2
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  		Fig. 2. Fate of Notch1-activated cells in the epidermal lineages. (A,C) At E12.5, lacZ is first activated in the AER of the fore- and hind limb buds (A, arrows) and the epidermis between the dorsal and ventral surface (C, arrows). (B) Nuclear {alpha}-VLLS staining demonstrating Notch1 activation in suprabasal nuclei of the epidermis at E12.5 (arrows). (D,E) 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. (F) In adult skin, staining was mostly absent in the epidermis; lacZ-labeled cells were present only in the differentiated cells of the hair follicle and the bulge (arrowhead, and high magnification inset). (G) Germ-line-deleted R26R mice label all keratinocytes. (H) Notch1 is not required for skin development because N1-/-; Rosa26-lacZtg/+ cells efficiently contribute to all epidermal structures in adult mice. Dashed line (B-E) delineates epidermis and dermis. Magnification: A, 40x; B,C,D,E, 20x; F,G,H, 10x.

 

Figure 3
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  		Fig. 3. Fate of Notch1-activated cells in neuronal lineages. (A) N1::cre labels retinal progenitors (arrows) at E14.5. (B,C) In the adult retina, clones derived from retinal progenitors that experienced Notch1 activation contain all retinal cell types in both eyes. (B) Left eye, L; (C) Right eye, R. (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 (arrowhead) throughout. (E) {alpha}-VLLS staining (red) identifies Notch1 signaling in ventral neural tube progenitors at E11.5. (F) N1::cre marks similar progenitors at E12.5 in the ventral neural tube. (G,H) Wild-type ES cells (G) efficiently contribute to all regions of the neural tube whereas Notch1-deficient, R26 lacZ-tagged cells (H) are excluded from ventral region. Dotted line in E,G,H marks the presumptive border between neuronal progenitors and committed or differentiated motor neurons. Counter stain: Neutral Red (A-D,F), DAPI (E), Haematoxylin (G,H). FP, floor plate; DA, dorsal aorta. Magnification: A, 10x; B-H, 20x.

 

Figure 4
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  		Fig. 4. Fate of Notch1-activated cells in the 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) By contrast, Notch1-deficient cells populate the PCV but do not contribute to the DA. (C,D) At E14.5, complete labeling of the endothelial lining of the aorta and intrasomitic arteries is observed (C, arrowhead) consistent with NICD presence as detected with {alpha}-VLLS staining (D, arrowhead) 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 not labeled. (F) Complete lack of contribution from Notch1-deficient, R26 lacZ-marked cells to the yolk sac vasculature (arrow). A normal contribution is seen to the capillary plexus (arrowhead). (G,H) N1 activity in adult vasculature labels endothelial cells and a few smooth muscle cells (arrowhead) in the in the arteries(G) and veins(H). Magnification: A,B,C,F 20x; D,E,G,H, 40x

 

Figure 5
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  		Fig. 5. Fate of Notch1-activated cells in the endocardial lineages. (A,B) Labeling of the heart (E10.5) in whole-mount (A) and after dissection (B). Notch1 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 (arrowhead) at E14.5. Myocardial cells are not stained. BA, branchiocephalic artery. (E,F) N1::cre-labeled endocardial cells continuously receive a Notch1 signal (arrowheads) at E14.5, as shown by {alpha}-VLLS staining. (G,H) In the adult heart, most endocardial cells retain label; the endothelial lining of the valve and the arteries (H, arrows), where Notch1 is required, are also labeled. (I,J) Comparison of the complementary staining patterns of endocardial cells (arrowhead) and cardiomyocytes in (I) N1::cre and (J) N1-/-;N1+/+;R26 lacZ chimeric hearts, respectively. Note that Notch1 is not active in cardiomyocyte cells(I) and also not required(J). Magnification: A, 16x; B, 50x; C,G,10x; E,F,40x; I,J 63x.

 

Figure 6
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  		Fig. 6. Fate of Notch1-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 (C). (D-G) 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 by lysozyme staining (G, arrowhead). (H) Infrequent labeling of single goblet cells (arrowhead) suggests Notch1 signaling may also occur in (committed) differentiated intestinal epithelium. (I) {alpha}-VLLS staining identifies Notch1 signaling in crypt progenitors (black arrowhead) and in a few scattered goblet cells (white arrowheads) within the villus (see also Fig. S5J in the supplementary material). Notch1 {alpha}-VLLS staining is nuclear; the precipitate in the cytoplasm of goblet cells may be an artifact. (J) Notch1 is expressed in spontaneous adenomas from Apcmin/+ mice (arrowhead) and in adenomas from Apcmin/+:N1::cre mice, indicating Notch1 activation in cells sustaining Apc mutation. (K) The normal villus epithelium does not express Notch1 but N1::cre marks this lineage (arrowhead). (L) 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 mucinproducing goblet cells in X-Gal-stained villi and crypts compared with unstained wild-type intestine. (M) Control Notch1 wild-type R26-lacZ chimeric intestines show no preference to differentiate towards the secretory lineages. (N,O) Quantitation of the differentiation defect observed in Notch1-deficient intestines by combining immunohistochemical staining for differentiated cell types with X-Gal staining to identify Notch1-deficient cells, expressed as absolute numbers with s.d. (N) and as a ratio of Notch1-knockout/Notch1-proficient to 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 a normal contribution and no defects (not shown). Magnification: A, 4x; B-G,J-M,20x; H,I, 40x.

 

Figure 7
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  		Fig. 7. Notch ligand density and N1::cre genetic mapping. A hypothetical model to explain the relationship between the strength of Notch signaling and fate mapping as a function of ligand concentration. The probability of identifying a marked lineage in tissues within N1::cre mice is correlated with Notch1 function as revealed by loss-of-function phenotypes and the ability to detect activated Notch1 protein by immunostaining (see Table 1).

 

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© The Company of Biologists Ltd 2007