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(Stroke. 2001;32:6.)
© 2001 American Heart Association, Inc.

Expedited Publication

An Animal Model for the Molecular Genetics of CADASIL

Karl J. Fryxell, PhD; Marcus Soderlund, BS Theodor V. Jordan, MS

From the Department of Biology, George Mason University, Fairfax, Va.

Correspondence to Karl J. Fryxell, Department of Biology, MSN 3E1, George Mason University, Fairfax, VA 22030. E-mail kfryxell{at}gmu.edu

	Abstract

Top Abstract Introduction Both CADASIL and Drosophila... Notch Phenotypes Are Cell... The Structure and Molecular... Does CADASIL Result From... Antimorphic Actions of CADASIL... Conclusions References Introduction  References

 
Background—CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is an inherited condition that causes repeated small-scale strokes in adults. CADASIL is caused only by mutations in the human NOTCH3 gene that increase or decrease the number of cysteines within the epidermal growth factor (EGF) repeats of the NOTCH3 protein. Drosophila lethal-Abruptex is a similar condition because it is also caused only by mutations that increase or decrease the number of cysteines within the EGF repeat portion of the Notch protein.

Summary of Comment—Drosophila lethal-Abruptex and human CADASIL are precisely analogous at the molecular level, and both are genetically dominant. These precise similarities, together with the fact that the structure and function of Notch has been highly conserved throughout the animal kingdom, provide an animal model for the molecular and genetic aspects of human CADASIL. It also provides support for Spinner’s proposal that CADASIL results from dominant inhibition of the Notch pathway.

Conclusions—Because the phenotypes of Notch mutations are cell-autonomous, the symptoms of CADASIL indicate that adult vascular smooth muscle cells require the continuing function of the NOTCH3 pathway in the adult. For this reason, further analysis of the NOTCH3 pathway may provide more general insights into the biology of vascular smooth muscle cells. In the case of CADASIL, the powerful genetic tools available in Drosophila should help to facilitate future research.

Key Words: CADASIL • dementia, multi-infarct • Drosophila melanogaster • genetics

	Introduction

Top Abstract Introduction Both CADASIL and Drosophila... Notch Phenotypes Are Cell... The Structure and Molecular... Does CADASIL Result From... Antimorphic Actions of CADASIL... Conclusions References Introduction  References

 
CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) causes a type of stroke and dementia whose key features include recurrent subcortical ischemic events and diffuse while matter abnormalities on neuroimaging.^{1 2} At the behavioral level, CADASIL is associated with adult-onset symptoms (average age of onset is 45 years) that include migraine headaches, strokes, mood disorders, epileptic seizures, and progressive dementia.^{1 3 4 5 6} CADASIL can be distinguished from related disorders by the presence of a family history of autosomal dominant inheritance and skin biopsies that show frequent small lesions.⁷ Nevertheless, CADASIL is currently underdiagnosed because of its variable mode of presentation and recent characterization.

Pathological findings on autopsy of patients with CADASIL include multiple small, deep infarcts in the brain, a diffuse myelin loss and pallor of the hemispheric white matter, and a widespread vasculopathy of the small arteries penetrating the white matter.⁸ The arterial lesions occur throughout the arterial tree, including arteries and arterioles within muscle, skin,⁹ and peripheral nerve.¹⁰ These arterial lesions are neither arteriosclerotic nor amyloid,^{8 11} and affected families rarely have vascular risk factors or hypertension.⁴ Nevertheless, the arterial lesions are characterized by a marked thickening of the arterial wall associated with the proliferation of vascular smooth muscle cells and accumulation of extensive perivascular, eosinophilic, osmiophilic, periodic acid–Schiff positive (PAS⁺) deposits in the tunica media.^{8 10 11 12} Immunohistological staining patterns indicate that these deposits may contain collagen IV and smooth muscle myosin,¹¹ as would be expected after repeated cycles of smooth muscle cell degeneration and proliferation.

The clinical severity of symptoms in CADASIL is related to the extent of loss of white matter from the brain,¹³ which occurs progressively in CADASIL even without overt ischemic events.^{2 3} Given that white matter is particularly sensitive to partial anoxia,¹³ the diffuse loss of white matter is probably caused by widespread arteriolar dysfunction.

	Both CADASIL and Drosophila Lethal-Abruptex Are Specifically Caused by Unpaired Cysteines Within the Epidermal Growth Factor Repeat Domain of Notch

Top Abstract Introduction Both CADASIL and Drosophila... Notch Phenotypes Are Cell... The Structure and Molecular... Does CADASIL Result From... Antimorphic Actions of CADASIL... Conclusions References Introduction  References

 
Genetic linkage analysis^{14 15} and DNA sequencing⁶ have shown that CADASIL is caused by mutations in the human NOTCH3 gene. Moreover, CADASIL mutations are invariably caused by amino acid substitutions in the epidermal growth factor (EGF) repeat domain of the NOTCH3 protein that cause the gain or loss of a cysteine.^{16 17 18} One apparent exception, a recently discovered case of CADASIL caused by a DNA base substitution that leads to an RNA splicing defect,¹⁹ is the exception that proves the rule, because the aberrant RNA splicing in this case deletes 7 amino acids from the EGF repeat domain of the NOTCH3 protein, and one of the deleted amino acids is cysteine.¹⁹ Because all CADASIL mutant gene products have unpaired cysteines in the EGF repeat domain, the remaining cysteines may pair inappropriately or cross-link to other proteins,²⁰ leading to abnormal protein secondary structure in the EGF repeat domain, which comprises the majority of the extracellular portion of the Notch receptor.^{21 22 23}

Drosophila Abruptex alleles are a distinct phenotypic class of mutations within the Drosophila Notch gene. Abruptex alleles are classified as such, based on the presence of certain dominant visible phenotypes such as interrupted wing veins and a reduced number of dorsal bristles (Figure).^{24 25 26} At the molecular level, Abruptex alleles generally correspond to amino acid substitutions within the extracellular portion of the Drosophila Notch receptor, many or all of which alter the binding affinity of Notch for protein ligands such as Delta.^{26 27 28}

View larger version (45K): [in this window] [in a new window]   Figure 1. The Notch gene was named after mutant fruit flies with "notched wings" (left), who are typically heterozygous for a null allele (or deletion) of the Notch gene. Other mutations within this same fruit fly Notch gene cause the Abruptex phenotype, which includes interrupted wing veins and a reduced number of dorsal bristles (right). Lethal Abruptex alleles are analogous to the NOTCH3 mutations in patients with CADASIL. Redrawn after Reference 30.

Abruptex alleles have been further classified into 3 subcategories, based on classical genetic criteria: (1) viable enhancers of Notch mutations, (2) viable suppressors of Notch mutations, and (3) alleles that are lethal in combination with Notch mutations (ie, lethal when heterozygous over a null allele of Notch). On the basis of the classical genetic nomenclature of mutant gene functions,²⁹ these 3 subcategories of Abruptex alleles correspond to hypomorphs, hypermorphs, and antimorphs, respectively.^{24 25 26} Lethal-Abruptex alleles are said to be "antimorphs" because they partially block the function of the normal Notch receptor in heterozygotes. This point can be established readily in Drosophila because of our detailed knowledge of the phenotypic effects of various doses of genes at each step in the Drosophila Notch signaling pathway.^{30 31 32} Drosophila lethal-Abruptex alleles are lethal when homozygous as well as when heterozygous over a deficiency for the Notch gene.³⁰

All lethal-Abruptex alleles (sequenced to date) have amino acid substitutions that create or eliminate a cysteine within the EGF repeat domain of the Notch receptor, whereas all hypomorphic and hypermorphic Abruptex alleles have amino acid substitutions that neither create nor eliminate a cysteine.^{28 30} Therefore, the lethal-Abruptex phenotype is specifically caused by unpaired cysteines in the EGF repeat domain of the Notch protein. Unpaired cysteines presumably lead to inappropriate disulfide bonds within the Notch receptor, abnormal secondary structure in the extracellular domain of the Notch receptor, and perhaps cross-links to other proteins.²⁰

Of course, mutations may occur in other portions of the Drosophila Notch gene. However, these produce phenotypes that are quite distinct from the lethal-Abruptex phenotype. As an example, when a downstream frameshift is added to the Drosophila lethal-Abruptex allele Ax^59b, the result is a nonfunctional Notch protein that does not confer the Abruptex phenotype.²⁸ A similar situation probably exists for mammalian NOTCH3 genes. As an example, a mutation in the mouse Notch3 gene that does not involve cysteines produces phenotypes (embryonic lethality) quite different from CADASIL.³³

	Notch Phenotypes Are Cell-Autonomous

Top Abstract Introduction Both CADASIL and Drosophila... Notch Phenotypes Are Cell... The Structure and Molecular... Does CADASIL Result From... Antimorphic Actions of CADASIL... Conclusions References Introduction  References

 
Mutations in the Drosophila Notch gene, including Abruptex alleles, are expressed in a cell-autonomous fashion.^{23 31 34} In other words, individual cells that synthesize the mutant receptor exhibit the mutant morphology, whereas cells that do not express the mutant receptor are unaffected. This aspect of Notch function has been highly conserved: Notch acts as a cell-autonomous receptor that regulates cellular proliferation, differentiation, and developmental fate switching in a wide variety of tissues and animal species.^{21 23} However, Drosophila has one Notch gene, whereas mammals have 4 Notch genes. Each of the 4 mammalian Notch genes acts on a different subset of tissues.³⁵ This difference in the number and tissue specificity of Notch genes is presumably responsible for the differences in tissue specificity of Abruptex versus CADASIL.

In the case of CADASIL, cellular defects in arterial smooth muscle cells are caused by a NOTCH3 mutation. This suggests that human arterial smooth muscle cells autonomously express the morphological consequences of the defect in their own NOTCH3 signaling pathway. In prenatal rodents, the normal Notch3 gene is expressed during gastrulation and in the developing central nervous system.^{35 36 37} In postnatal rodents, Notch gene expression is generally downregulated in most tissues. However, the Notch3 gene of rats³⁸ and the NOTCH3 gene of humans²⁰ continue to be specifically expressed in adult vascular smooth muscle cells (Table), whereas the NOTCH1 and NOTCH2 genes continue to be expressed in a few other specific tissues.³⁸

View this table: [in this window] [in a new window]   Table 1. Comparison of Some Notch Genes and Phenotypes

	The Structure and Molecular Functions of Notch Have Been Highly Conserved

Top Abstract Introduction Both CADASIL and Drosophila... Notch Phenotypes Are Cell... The Structure and Molecular... Does CADASIL Result From... Antimorphic Actions of CADASIL... Conclusions References Introduction  References

 
When Notch genes from one animal phyla or tissue are expressed in another animal phyla or tissue, they have proven to be functional in all cases so far examined. For example, injection of mRNA encoding activated mouse Notch (ie, the isolated cytoplasmic domain) into frog embryos produced the same effects on embryonic development as injection of activated frog Notch (also known as Xotch).^{39 40} When frog Notch is expressed in cultured Drosophila cells, it binds specifically to the correct Drosophila ligands (Delta and Serrate) and even binds within the correct subdomain of the Notch receptor (EGF repeats 11 to 12, out of 36 EGF repeats).⁴¹

Evolutionary studies have shown that the arthropod and chordate lineages diverged during the original evolutionary radiation of bilaterally symmetrical animals.⁴² In other words, the common ancestor of flies and frogs was also the common ancestor of mammals, reptiles, echinoderms, mollusks, and nematodes. Moreover, the nematode Caenorhabditis elegans has 2 Notch genes (also known as lin-12 and glp-1), which are more diverged than any pair of mammalian Notch genes. In fact, lin-12 and glp-1 share only 50% amino acid identity with each other, they are expressed in different tissues, and they have different numbers of EGF repeats. Nevertheless, lin-12 and glp-1 have proven to be biochemically interchangeable in the sense that they activate the same signal transduction pathway,⁴³ and each can substitute for the other if expressed in the appropriate tissues.⁴⁴ Taken together, the available evidence suggests that the basic structure and molecular functions of the Notch receptor have probably been conserved since before the origin of vertebrates.

	Does CADASIL Result From a Notch Signaling Defect or a Protein Accumulation Problem?

Top Abstract Introduction Both CADASIL and Drosophila... Notch Phenotypes Are Cell... The Structure and Molecular... Does CADASIL Result From... Antimorphic Actions of CADASIL... Conclusions References Introduction  References

 
None of the >50 CADASIL alleles sequenced to date appear to represent a null allele (such as a frameshift or deletion) of NOTCH3. This led Spinner⁴⁵ to suggest that CADASIL may not arise from a loss of Notch signaling. However, extensive studies of Notch in flies, worms, frogs, and mice have shown that cells that synthesize the Notch receptor respond to Notch signaling with dramatic changes in proliferation, differentiation, or developmental fate switching, whereas cells that become refractory to the Notch pathway invariably stop synthesizing the Notch receptor.^{21 23 32 39 40 46 47 48} These rules hold even for Notch1 expression in postmitotic neurons in the mammalian brain.^{49 50} Because adult human vascular smooth muscle cells express NOTCH3 (see above), it follows that NOTCH3 signaling is likely to play a role in vascular smooth muscle cell proliferation and/or differentiation. Because cysteine substitutions in the EGF repeat domain of Notch are known to have a profound (dominant inhibitory) effect on Notch signaling in Drosophila (see above) and because the basic structure and molecular functions of Notch have been highly conserved (see above), it follows that cysteine substitutions in the EGF repeat domain of human NOTCH3 probably also have a profound (dominant inhibitory) effect on NOTCH3 signaling in patients with CADASIL. In other words, the comparison with Drosophila lethal-Abruptex argues strongly against Spinner’s first suggestion (that CADASIL is caused by protein accumulation, not loss of Notch signaling) but argues just as strongly in favor of Spinner’s second suggestion (that CADASIL is caused by a dominant inhibition of Notch signaling⁴⁵ ).

According to this view, the reason that patients with CADASIL with null alleles of NOTCH3 have not been found is that null alleles cause different symptoms, which are less severe in heterozygotes (ie, less severe than antimorphs) and are lethal in homozygotes. This is the same reason that null alleles of Drosophila Notch do not produce the lethal-Abruptex phenotype.³⁰ Moreover, there is ample precedent for our suggestion that a homozygous null allele of NOTCH3 would be lethal in humans, given that some Notch3 mutations in the mouse are embryonic lethals.³³

	Antimorphic Actions of CADASIL and Lethal-Abruptex Are Probably Related to Formation of Notch Heterodimers

Top Abstract Introduction Both CADASIL and Drosophila... Notch Phenotypes Are Cell... The Structure and Molecular... Does CADASIL Result From... Antimorphic Actions of CADASIL... Conclusions References Introduction  References

 
The Notch receptor exists on the cell surface as a heterodimer. This was originally inferred from classical genetic experiments with Drosophila Abruptex alleles^{25 51} and was later confirmed by biochemical studies in insect^{32 52} and mammalian cells.⁵³ Presumably, this explains why the Drosophila lethal-Abruptex gene product can partially block the function of the normal gene product: They interact physically with each other at the cell surface.

The extracellular domain of the Notch protein is constitutively cleaved during processing of the Notch protein in the Golgi apparatus. The cleavage site is located between the LIN-12 repeats (named after the nematode Notch gene in which they were discovered) and the transmembrane domain.^{20 53} The freed extracellular domain then binds to the remainder of the receptor (which includes the intracellular domain, the transmembrane segment, and a small extracellular peptide), forming a heterodimer.⁵³ The remaining extracellular peptide must contain the heterodimer binding site, which is likely to be compact because there are few conserved sequences within the remaining extracellular peptide (Dr Fryxell, unpublished observations). Binding of this peptide to the extracellular domain could easily be disrupted by inappropriate pairing of cysteines within the EGF repeat domain. Consistent with this view, recent studies have shown that the extracellular domain of NOTCH3 in patients with CADASIL fails to bind to the remainder of NOTCH3, is not recycled by reuptake into the cell as it normally is,²³ and consequently accumulates to high levels within the extracellular space near vascular smooth muscle cells.²⁰

Notch receptors, in general, are stimulated by binding to transmembrane proteins expressed by neighboring cells (such as Delta, Serrate, Jagged1, Jagged2, Delta-like1, Delta-like3, and so on).^{22 23 32 45} This implies a mechanism by which the release (and consequent accumulation) of the extracellular domain of NOTCH3 would produce dominant inhibition of NOTCH3 signaling—by competitive inhibition of binding to its protein ligand.⁴⁵ In conclusion, the comparison of Drosophila lethal-Abruptex with human CADASIL strengthens the case for dominant inhibition in both cases. In the case of Drosophila lethal-Abruptex, we have clear genetic evidence for dominant inhibition. In the case of human CADASIL, we have a precisely analogous defect at the molecular level plus clear biochemical evidence of a mechanism by which dominant inhibition is likely to occur.

	Conclusions

Top Abstract Introduction Both CADASIL and Drosophila... Notch Phenotypes Are Cell... The Structure and Molecular... Does CADASIL Result From... Antimorphic Actions of CADASIL... Conclusions References Introduction  References

 
In both human CADASIL and Drosophila lethal-Abruptex, cysteine substitutions within the EGF repeats of Notch cause a partial (antimorphic) block of function of the normal allele attributable to a disruption of the secondary structure of the extracellular domain of Notch, which may include disrupted heterodimer formation and inappropriate cross-links to other proteins. The genetic dominance of CADASIL is likely to result from antimorphic function in heterozygotes, whereas homozygotes probably never will be found because of recessive lethality.

The cell-autonomous phenotypes of Notch mutations, together with the histological evidence of cycles of smooth muscle cell proliferation and degeneration in CADASIL, indicate that mammalian arterial smooth muscle cells have a continuing requirement for NOTCH3 activity in the adult. Thus, the NOTCH3 signaling pathway also may play a role in the response of arterial smooth muscle cells to other types of injury and stress.

Finally, the molecular analogy between Drosophila lethal-Abruptex and human CADASIL may be of use in the development of potential treatments for CADASIL. The most obvious approach, namely gene therapy, has already been tested in flies, and the results are encouraging—additional copies of the normal Notch gene do reduce the symptoms of lethal-Abruptex.^{24 54}

	Acknowledgments

 
This work was supported by the College of Arts and Sciences of George Mason University, through an Indirect Cost Award and a Graduate Research Assistantship (both awarded to Dr Fryxell).

Received September 5, 2000; revision received November 6, 2000; accepted November 6, 2000.

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Top Abstract Introduction Both CADASIL and Drosophila... Notch Phenotypes Are Cell... The Structure and Molecular... Does CADASIL Result From... Antimorphic Actions of CADASIL... Conclusions References Introduction  References

 

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51. Foster G. Negative complementation at the Notch locus of Drosophila melanogaster. Genetics. 1975;81:99–120.[Abstract/Free Full Text]
52. Blaumueller CM, Qi H, Zagouras P, Artavanis-Tsakonas S. Extracellular cleavage of Notch leads to a heterodimeric receptor on the plasma membrane. Cell. 1997;90:281–291.[Medline] [Order article via Infotrieve]
53. Logeat F, Bessia C, Brou C, LeBail O, Jarriault S, Seidah NG, Israel A. The Notch1 receptor is cleaved constitutively by a furin-like convertase. Proc Natl Acad Sci U S A . 1998;95:8108–8112.
54. Sirén M, Portin P. Gene dosage studies of a temperature sensitive Abruptex mutation of the Notch locus of Drosophila melanogaster. Hereditas. 1989;110:175–178.

Editorial Comment

Joseph M. Verdi, PhD, Guest Editor Christopher J. Kubu, MSc, Guest Editor

Laboratory of Neural Stem Cell Biology, Robarts Research Institute, Department of Physiology, Program in Neuroscience, London, Ontario, Canada

	Introduction

Top Abstract Introduction Both CADASIL and Drosophila... Notch Phenotypes Are Cell... The Structure and Molecular... Does CADASIL Result From... Antimorphic Actions of CADASIL... Conclusions References Introduction  References

 
The Notch-mediated signaling pathway is evolutionarily conserved and controls cell fate determination and differentiation in several Drosophila and mammalian lineages. Both Drosophila and mammalian Notch are transmembrane receptor proteins for Delta and Delta-like ligands. To date, 4 distinct mammalian Notch genes have been identified in mammals. On activation by its ligand (Delta or Jagged), the intracellular domain of Notch is cleaved and transports the Suppressor of Hairless transcription factor (CBF1 in mammals) into the nucleus and acts as a transactivator for Enhancer of Split (HES in mammals) gene expression. Notch activation thus acts as a transducer of extrinsic signals into altered gene expression.

The role of Notch signaling in human disease is receiving great attention of late with the finding that presenilins, genes associated with early onset familial Alzheimer’s disease, are regulators of Notch signaling.^R1 This has led to the theoretical possibility that Notch signaling may underlie the cause of this autosomal dominant variant of the disease. Whereas this hypothesis is founded on strictly correlative data, alterations in Notch signaling are at the core of 2 autosomal dominant disorders called Alagille syndrome (Jagged mutant^R2 ) and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL^R3 ; see also references within the preceding [Fryxell] article).

Alagille syndrome is characterized by bile duct paucity and resultant liver disease in combination with cardiac, skeletal, ocular, and facial abnormalities. CADASIL results from mutations within the extracellular domain of the Notch 3 receptor that disrupt the normal cysteine patterning required for proper ligand binding and receptor activation. CADASIL is associated with a wide spectrum of symptomology, including migraines, mood disorders, epileptic seizures, progressive dementia, and stroke. The question in developmental biology and clinical science is how can we lean more about the roles Notch signaling plays in these disorders and how can we use this knowledge to develop new treatment regimes to treat human patients.

In a brilliantly written article, Dr Karl Fryxell and his research team make the strong case for using animal models of disease to elucidate potential targets for gene therapy or pharmaceutical intervention. The authors compare and contrast the effects of extracellular mutations in Notch in Drosophila (only one Notch species in Drosophila) and Notch 3 in mice to the human equivalent that results in CADASIL. The molecular and morphological correlates of extracellular Notch mutations in Drosophila and Notch 3 mutations in mice match the clinical consequences of CADASIL in human populations. What is particularly insightful about this article is that the authors take this correlative data and make a strong case for using the power of Drosophila genetics to gain insight into human disease. Specifically, the author suggests (and rightly so) that weak alleles of Notch (called Abruptex^R4 ) in Drosophila will serve as good models for studying CADASIL in humans and provide a viable model for testing gene replacement therapies to rescue the mutant phenotype. Also, genetic alterations to Notch 3 signaling in mice either by knock-out or transgenic technology will also be valued tools in learning more about this disease in particular and about smooth muscle development in general. The attempt by Fryxell et al to find new ways of integrating what is learned in these genetic experiments into the clinic to treat human suffering should be complimented, and studies in this vein should render both interesting and clinically relevant results. In particular, their idea of using wild-type Notch and components of the Notch signaling pathway as potential candidates for gene therapy is right on the mark. Taking this idea a step further, using the power of Drosophila genetics to identify other targets for either conventional pharmaceutical intervention or gene therapy would warrant consideration. One classic way to identify genes involved in a particular developmental pathway is called suppressor or enhancer screens. In Drosophila, this consists of taking individual fly lines, each with a random mutation within its genome, and breeding these flies with a weak Abruptex phenotype to determine whether mutation X exacerbates the Abruptex phenotype, rescues the phenotype, or does nothing. Those mutations that alter the Abruptex phenotype can be easily cloned and represent potential new targets for drug design or gene therapy. These powerful techniques, as pointed out by Fryxell and colleagues, are possible only in Drosophila with the speed and efficacy required.

Recently, Notch 3 has been shown to act more as a repressor of HES function than an activator.^R5 The conclusions drawn from the study of Fryxell and colleagues are enhanced by these observations. An increase in Notch extracellular domain may in fact sequester the available ligand required for normal signaling, making CADASIL still a dominant autosomal mutation, but gene therapy regimes and future experiments need to examine the actual regulation of HES in these studies. The authors point out that overexpression of normal Notch in Abruptex mutants rescues the phenotype.^R4 It is conceivable that Abruptex and Notch 3 mutants in mice may be rescued by restoration of Enhancer of Split (HES in mammals) levels and, ultimately, any of the downstream regulators identified in suppresser or enhancer of Abruptex genetic screens. If true, this opens the range of potential therapeutic targets for treatment of CADASIL.

It must be somewhat frustrating for Drosophila geneticists at times to have their organism referred to as "simple," given all the complex questions one can address by using this model. That, unfortunately, is still exactly the case anytime someone working on Drosophila, C elegans, and zebrafish genetics or development is introduced at seminars. The notion that these systems are simple is even scattered throughout textbooks concerning the subject. As more articles like the insightful and challenging one presented by Fryxell and colleagues appear, the bias or label should be erased. "Simple systems," indeed. Kudos, Dr Fryxell.

Received September 5, 2000; revision received November 6, 2000; accepted November 6, 2000.

	References

Top Abstract Introduction Both CADASIL and Drosophila... Notch Phenotypes Are Cell... The Structure and Molecular... Does CADASIL Result From... Antimorphic Actions of CADASIL... Conclusions References Introduction  References

 

1. Chan Y-M, Jan YN. Presenilins, processing of ß-amyloid precursor protein, and Notch signaling. Cell. 1999;23:201–204.
2. Jones EA, Clement-Jones M, Wilson DI. Jagged1 expression in human embryos: correlation with the Alagille syndrome phenotype. J Med Genet. 2000;37:663–668.[Abstract/Free Full Text]
3. Joutel A, Andreux F, Gaulis S, Domenga V, Cecillon M, Battail N, Piga N, Chapon F, Godfrain C, Tournier-Lasserve E. The ectodomain of Notch3 receptor accumulates within the cerebrovasculature of CADASIL patients. J Clin Invest. 2000;105:597–605.
4. Siren M, Portin P. Gene dosage studies of temperature sensitive Abruptex mutation of the Notch locus of Drosophila melanogaster. Hereditas. 1998;110:175–178.
5. Beatus P, Lundkvist J, Oberg C, Lendahl U. The Notch 3 intracellular domain represses Notch 1-mediated activation through Hairy/Enhancer of Split promoters. Development. 1999;126:3915–3935.[Abstract]

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