Mouse Fbw7/Sel-10/Cdc4 Is Required for Notch Degradation during Vascular Development

Mammalian Fbw7 (also known as Sel-10, hCdc4, or hAgo) is the F-box protein component of an SCF (Skp1-Cul1-F-box protein-Rbx1)-type ubiquitin ligase, and the mouse Fbw7 is expressed prominently in the endothelial cell lineage of embryos. We generated mice deficient in Fbw7 and found that the embryos died in utero at embryonic day 10.5-11.5, manifesting marked abnormalities in vascular development. Vascular remodeling was impaired in the brain and yolk sac, and the major trunk veins were not formed. In vitro para-aortic splanchnopleural explant cultures from Fbw7-/- embryos also manifested an impairment of vascular network formation. Notch4, which is the product of the proto-oncogene Int3 and an endothelial cell-specific mammalian isoform of Notch, accumulated in Fbw7-/- embryos, resulting in an increased expression of Hey1, which encodes a transcriptional repressor that acts downstream of Notch signaling and is implicated in vascular development. Expression of Notch1, -2, or -3 or of cyclin E was unaffected in Fbw7-/- embryos. Mammalian Fbw7 thus appears to play an indispensable role in negative regulation of the Notch4-Hey1 pathway and is required for vascular development. Mammalian Fbw7 (also known as Sel-10, hCdc4, or hAgo) is the F-box protein component of an SCF (Skp1-Cul1-F-box protein-Rbx1)-type ubiquitin ligase, and the mouse Fbw7 is expressed prominently in the endothelial cell lineage of embryos. We generated mice deficient in Fbw7 and found that the embryos died in utero at embryonic day 10.5-11.5, manifesting marked abnormalities in vascular development. Vascular remodeling was impaired in the brain and yolk sac, and the major trunk veins were not formed. In vitro para-aortic splanchnopleural explant cultures from Fbw7-/- embryos also manifested an impairment of vascular network formation. Notch4, which is the product of the proto-oncogene Int3 and an endothelial cell-specific mammalian isoform of Notch, accumulated in Fbw7-/- embryos, resulting in an increased expression of Hey1, which encodes a transcriptional repressor that acts downstream of Notch signaling and is implicated in vascular development. Expression of Notch1, -2, or -3 or of cyclin E was unaffected in Fbw7-/- embryos. Mammalian Fbw7 thus appears to play an indispensable role in negative regulation of the Notch4-Hey1 pathway and is required for vascular development. The Notch signaling pathway is evolutionarily conserved and essential both for pattern formation during development and for execution of a wide variety of cell fate decisions. In Caenorhabditis elegans, the Notch homolog LIN-12 ensures that only one of two undifferentiated gonadal cells develops into an anchor cell, whereas the other cell becomes a ventral uterine precursor cell (1Wu G. Hubbard E.J. Kitajewski J.K. Greenwald I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15787-15791Crossref PubMed Scopus (51) Google Scholar, 2Hubbard E.J. Wu G. Kitajewski J. Greenwald I. Genes Dev. 1997; 11: 3182-3193Crossref PubMed Scopus (203) Google Scholar). Given that relatively small differences in the activity of the Notch signaling pathway result in marked differences in cell behavior, this pathway is strictly regulated by various mechanisms (3Artavanis-Tsakonas S. Rand M.D. Lake R.J. Science. 1999; 284: 770-776Crossref PubMed Scopus (4951) Google Scholar). The pathway is activated by interaction of the receptor Notch with Delta-like ligands presented on the surface of adjacent cells, which results in proteolytic cleavage of Notch and the consequent release of its intracellular domain (ICD) 1The abbreviations used are: ICD, intracellular domain; PBS, phosphate-buffered saline; HA, hemagglutinin; ES, embryonic stem; E, embryonic day; P-Sp, para-aortic splanchnopleural; CDK, cyclin-dependent kinase. from the plasma membrane into the cytosol. The Notch ICD then migrates to the nucleus, forms a complex with the transcriptional suppressor CBF1 (also known as Su(H) or RBP-Jκ), and acts as a transcriptional activator of Notch target genes. Several recent studies have highlighted the importance of Notch signaling in vascular development. Endothelial cell precursors differentiate and coalesce into a network of uniformly sized primitive blood vessels, a process known as vasculogenesis, both in the embryo and in extraembryonic tissues such as the yolk sac during the early stages of mammalian vascular development. This primary vascular plexus is then remodeled by angiogenesis, a process that includes the sprouting, branching, splitting, and differential growth of vessels and that results in the formation of the large and small vessels of the mature vascular system (4Gale N.W. Yancopoulos G.D. Genes Dev. 1999; 13: 1055-1066Crossref PubMed Scopus (698) Google Scholar, 5Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4867) Google Scholar). In mice, several Notch isoforms are expressed in endothelial cells of the embryonic vasculature (6Reaume A.G. Conlon R.A. Zirngibl R. Yamaguchi T.P. Rossant J. Dev. Biol. 1992; 154: 377-387Crossref PubMed Scopus (198) Google Scholar), and both Notch1 loss-of-function mutant embryos and Notch1-Notch4 double mutant embryos manifest severe defects in angiogenic vascular remodeling (7Krebs L.T. Xue Y. Norton C.R. Shutter J.R. Maguire M. Sundberg J.P. Gallahan D. Closson V. Kitajewski J. Callahan R. Smith G.H. Stark K.L. Gridley T. Genes Dev. 2000; 14: 1343-1352PubMed Google Scholar). Expression of an activated form of Notch4 also results in abnormal vessel structure and patterning (8Uyttendaele H. Ho J. Rossant J. Kitajewski J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5643-5648Crossref PubMed Scopus (245) Google Scholar). Furthermore, Notch signaling has been shown to control an arterial versus venous cell fate decision (9Zhong T.P. Childs S. Leu J.P. Fishman M.C. Nature. 2001; 414: 216-220Crossref PubMed Scopus (467) Google Scholar, 10Zhong T.P. Rosenberg M. Mohideen M.A. Weinstein B. Fishman M.C. Science. 2000; 287: 1820-1824Crossref PubMed Scopus (358) Google Scholar, 11Lawson N.D. Scheer N. Pham V.N. Kim C.H. Chitnis A.B. Campos-Ortega J.A. Weinstein B.M. Development. 2001; 128: 3675-3683Crossref PubMed Google Scholar). A role for the Notch pathway in vascular homeostasis has also been suggested by the observation that the human degenerative vascular disease CADASIL is caused by missense mutations in the Notch3 gene (12Joutel A. Corpechot C. Ducros A. Vahedi K. Chabriat H. Mouton P. Alamowitch S. Domenga V. Cecillion M. Marechal E. Maciazek J. Vayssiere C. Cruaud C. Cabanis E.A. Ruchoux M.M. Weissenbach J. Bach J.F. Bousser M.G. Tournier-Lasserve E. Nature. 1996; 383: 707-710Crossref PubMed Scopus (1711) Google Scholar). The activity of the Notch pathway is regulated by the ubiquitin-proteasome system of protein degradation (13Lai E.C. Curr. Biol. 2002; 12: R74-R78Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Protein ubiquitylation is mediated by several enzymes that act in concert (14Weissman A.M. Nat. Rev. Mol. Cell Biol. 2001; 2: 169-178Crossref PubMed Scopus (1262) Google Scholar, 15Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6959) Google Scholar). A ubiquitin-activating enzyme, with ATP as a substrate, catalyzes the formation of a thioester bond between itself and ubiquitin, and it then transfers the activated ubiquitin to a ubiquitin-conjugating enzyme. Certain ubiquitin-conjugating enzymes transfer ubiquitin directly to the protein substrate, whereas others require the participation of a third component, termed a ubiquitin ligase, to achieve this effect. Mammalian Fbw7 (also known as Sel-10, hCdc4, or hAgo) (16Koepp D.M. Schaefer L.K. Ye X. Keyomarsi K. Chu C. Harper J.W. Elledge S.J. Science. 2001; 294: 173-177Crossref PubMed Scopus (659) Google Scholar, 17Moberg K.H. Bell D.W. Wahrer D.C. Haber D.A. Hariharan I.K. 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Biochemical evidence has suggested that the ICDs of both Notch1 (24Gupta-Rossi N. Le Bail O. Gonen H. Brou C. Logeat F. Six E. Ciechanover A. Israel A. J. Biol. Chem. 2001; 276: 34371-34378Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 25Oberg C. Li J. Pauley A. Wolf E. Gurney M. Lendahl U. J. Biol. Chem. 2001; 276: 35847-35853Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 26Wu G. Lyapina S. Das I. Li J. Gurney M. Pauley A. Chui I. Deshaies R.J. Kitajewski J. Mol. Cell. Biol. 2001; 21: 7403-7415Crossref PubMed Scopus (277) Google Scholar) and Notch4 (2Hubbard E.J. Wu G. Kitajewski J. Greenwald I. Genes Dev. 1997; 11: 3182-3193Crossref PubMed Scopus (203) Google Scholar, 26Wu G. Lyapina S. Das I. Li J. Gurney M. Pauley A. Chui I. Deshaies R.J. Kitajewski J. Mol. Cell. Biol. 2001; 21: 7403-7415Crossref PubMed Scopus (277) Google Scholar) as well as presenilin (1Wu G. Hubbard E.J. Kitajewski J.K. Greenwald I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15787-15791Crossref PubMed Scopus (51) Google Scholar) and cyclin E (16Koepp D.M. Schaefer L.K. Ye X. Keyomarsi K. Chu C. Harper J.W. Elledge S.J. Science. 2001; 294: 173-177Crossref PubMed Scopus (659) Google Scholar, 17Moberg K.H. Bell D.W. Wahrer D.C. Haber D.A. Hariharan I.K. Nature. 2001; 413: 311-316Crossref PubMed Scopus (374) Google Scholar, 18Strohmaier H. Spruck C.H. Kaiser P. Won K.A. Sangfelt O. Reed S.I. Nature. 2001; 413: 316-322Crossref PubMed Scopus (503) Google Scholar) are targets for ubiquitylation mediated by mammalian Fbw7. The human FBW7 gene has also been shown to be mutated in breast and cell H. Spruck C.H. Kaiser P. Won K.A. Sangfelt O. Reed S.I. Nature. 2001; 413: 316-322Crossref PubMed Scopus (503) Google Scholar, 25Oberg C. Li J. Pauley A. Wolf E. Gurney M. Lendahl U. J. Biol. 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The was by with a of that the The of of with and were and for the and mutant Fbw7 cells were into and resulting were with The of the mutant was by were to mutant of embryos, was from the yolk sac at to and by with the and of cells the mutant Fbw7 were with a that encodes the in to the of the was by and the of Fbw7 mutant cells, the cells were then with the with and and by and In and were with in phosphate-buffered in and with a at a of In was with the The Fbw7 to of the mouse Fbw7 was with a was as M. Nat. Cell Biol. PubMed Scopus Google Scholar) with and E were with with an and were with a differential in and were with in and then with in was as N. S. Y. K. S. S. H. M. T. K. Dev. 1997; PubMed Scopus Google Scholar). The Hey1, and to the of the were with a embryos were for in and was used at a of were with and were with a and in at were and to as S. M. K. M. M. K. H. H. K. K. R.A. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar) with E and as well as with J.A. J. 2000; PubMed Scopus Google Scholar), and In vitro for activity with cyclin or cyclin E were as K. N. M. A. T. N. I. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) with of embryos or of protein for and of Notch in expression both with the and at its and by the gene was by into and Fbw7-/- cells were with a for with the at its with for the or cells were at a of cells well in and for was then to and the cells were at the for of the of the by with or In of and explant on cells and were as N. T. S. Y. T. Y. M. T. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, N. Y. T. N.W. Yancopoulos G.D. T. 2001; 98: PubMed Scopus Google Scholar). In for the was in with in for at with PBS, and for at with in to The cultures were then for at with and in to at with the was with and were with and The cultures were with a of generated mice deficient in Fbw7. The Fbw7 gene was in mouse embryonic cells by of the S. S. K. N. K. 2001; PubMed Scopus Google Scholar) with a mice were generated from the of to was The thus to be in the In that Fbw7 was to cells of the endothelial cells of and endothelial cells of the and major trunk was not in the large veins of embryos at embryonic day and The Fbw7 gene was not expressed in the of embryos at it is expressed in the brain (16Koepp D.M. Schaefer L.K. Ye X. Keyomarsi K. Chu C. Harper J.W. Elledge S.J. Science. 2001; 294: 173-177Crossref PubMed Scopus (659) Google Scholar, S. S. K. N. K. 2001; PubMed Scopus Google Scholar). was in Fbw7-/- embryos Vascular in the at which the Fbw7 becomes embryos from at various Fbw7-/- embryos been by were at in the was in mutant embryos were and in and and impaired of the formation with the that the between adjacent was and was in with embryos and R.A. A.G. Rossant J. Development. PubMed Google Scholar, Le A. R. Nature. 2000; PubMed Scopus Google Scholar) and M. J. A. Nature. 1997; 386: PubMed Scopus Google Scholar) embryos severe abnormalities of of in Fbw7-/- embryos. E, and and Fbw7-/- and embryos at were to in with for and and and and and or and The of was increased in Fbw7-/- embryos, the in with that in embryos. The vascular of Fbw7-/- embryos were complex were of embryos. We the vessels of and Fbw7-/- embryos with to a of endothelial The vascular in the brain of embryos to form in Fbw7-/- embryos the vascular and vessels in the brain not or Fbw7-/- embryos also manifested and as a result of the of the endothelial of the abnormal The in vascular remodeling was in the yolk sac resulting in the formation of large and small vessels, in the yolk sac of embryos the vascular plexus to form at in Fbw7-/- embryos, it of the surface of the yolk sac at that Fbw7 is essential for the remodeling of Furthermore, formation of the major the was impaired in Fbw7-/- embryos and that the vascular of Fbw7-/- embryos was not a of a in embryonic in vitro para-aortic splanchnopleural explant which the growth of endothelial cells N. T. S. Y. T. Y. M. T. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, N. Y. T. N.W. Yancopoulos G.D. T. 2001; 98: PubMed Scopus Google Scholar). from and Fbw7-/- embryos were with cells and then with The formation of an vascular network in the was in the Fbw7-/- that the of vascular development in Fbw7-/- embryos is to a in endothelial cells or not to a of embryonic of Notch4 in that the of Fbw7-/- mice is to of mutant or mice in which Notch signaling is that of the Notch ICD, a target for is for the abnormal vascular development in Fbw7-/- embryos. We the expression of of which are to be for vascular development (7Krebs L.T. Xue Y. Norton C.R. Shutter J.R. Maguire M. Sundberg J.P. Gallahan D. Closson V. Kitajewski J. Callahan R. Smith G.H. Stark K.L. Gridley T. Genes Dev. 2000; 14: 1343-1352PubMed Google Scholar, H. Ho J. Rossant J. Kitajewski J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5643-5648Crossref PubMed Scopus (245) Google Scholar), in Fbw7-/- embryos. The of the ICDs of not to be by the Fbw7 whereas that of was increased in Fbw7-/- embryos The of in embryos to be between that in and that in Fbw7-/- embryos. the of generated cells in which both Fbw7 were and then both cells and cells with expression for or The of degradation of was in and Fbw7-/- cells, whereas the of was in Fbw7-/- cells in cells thus that Fbw7 is required for of not for that of Expression of an Fbw7 mutant that the and F-box was shown G. Lyapina S. Das I. Li J. Gurney M. Pauley A. Chui I. Deshaies R.J. Kitajewski J. Mol. Cell. Biol. 2001; 21: 7403-7415Crossref PubMed Scopus (277) Google Scholar) to result in of both and in We with cells, and found that the expression of the of not that of the for this with to is the in the cell be a We also found that the of presenilin not between and Fbw7-/- embryos not Expression of in the expression of of the and downstream of the Notch signaling by in the of and in Fbw7-/- embryos was to that in embryos, the of was increased in the vessels of Fbw7-/- embryos with that in the vessels of embryos. The of in Fbw7-/- embryos thus to increased expression of and a consequent on vascular development. Expression of E in that and evidence in that cyclin E is a target for ubiquitylation and (16Koepp D.M. Schaefer L.K. Ye X. Keyomarsi K. Chu C. Harper J.W. Elledge S.J. Science. 2001; 294: 173-177Crossref PubMed Scopus (659) Google Scholar, 17Moberg K.H. Bell D.W. Wahrer D.C. Haber D.A. Hariharan I.K. 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Genes 1997; 2: PubMed Scopus Google Scholar, H. G. Wu G. D. Kitajewski J. Development. 1996; PubMed Google Scholar). Vascular remodeling to in Fbw7-/- embryos have been in mice Notch1, both Notch1 and Notch4 (7Krebs L.T. Xue Y. Norton C.R. Shutter J.R. Maguire M. Sundberg J.P. Gallahan D. Closson V. Kitajewski J. Callahan R. Smith G.H. Stark K.L. Gridley T. Genes Dev. 2000; 14: 1343-1352PubMed Google Scholar), Y. U. K. Y. M. Gridley T. H. W. Y. Nature. PubMed Scopus Google Scholar), or C. S. S. S. Yancopoulos G.D. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) not in embryos (7Krebs L.T. Xue Y. Norton C.R. Shutter J.R. Maguire M. Sundberg J.P. Gallahan D. Closson V. Kitajewski J. Callahan R. Smith G.H. Stark K.L. Gridley T. Genes Dev. 2000; 14: 1343-1352PubMed Google Scholar), that Notch1 for the of of In increased expression of Notch4 vascular as is in mice a for an activated form of Notch4 (8Uyttendaele H. Ho J. Rossant J. Kitajewski J. Proc. Natl. Acad. Sci. U. 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