1 Program in Human Molecular Biology and Genetics, University of Utah, Building
533 Room 4220, 15 N 2030 East, Salt Lake City, Utah 84112, USA
2 Department of Medicine, University of Utah, 4C104 SOM, 30 N 1900 East, Salt
Lake City, Utah 84132, USA
3 Department of Molecular Genetics and Microbiology, Duke University Medical
Center, 268 CARL Building, Box 3175, Durham, North Carolina 27710, USA
Authors for correspondence (e-mail:
march004{at}mc.duke.edu
or
dean.li{at}hmbg.utah.edu)
Accepted 2 December 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Ccm1, Gene targeting, Mice, Angiogenesis, Stroke, Epilepsy, Notch, Efnb2
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recently it has been shown that a monogenic form of CCM is linked to
loss-of-function mutations in the CCM1 locus on chromosome 7q, which
encodes the KRIT1 protein (Craig et al.,
1998; Laberge-le Couteulx et
al., 1999
; Marchuk et al.,
1995
; Moriarity et al.,
1999
; Rigamonti et al.,
1988
; Sahoo et al.,
1999
). KRIT1 is an intracellular protein with ankyrin repeats and
a FERM domain (Serebriiskii et al.,
1997
; Zawistowski et al.,
2002
), initially cloned on the basis of a yeast two-hybrid
interaction with KREV1/RAP1a, a small RAS family GTPase
(Serebriiskii et al., 1997
).
Subsequently, additional 5' sequences were found that extend the open
reading frame (Sahoo et al.,
2001
; Zhang et al.,
2000
). The longer protein did not interact with KREV1/RAP1a, but
instead showed strong interaction with integrin cytoplasmic domain associated
protein-1-alpha (ICAP1
) (Faisst and
Gruss, 1998
; Zawistowski et
al., 2002
; Zhang et al.,
2001
), which binds a similar NPXY motif on both KRIT1 and
ß1-integrin (Zawistowski et al.,
2002
; Zhang et al.,
2001
). In addition, KRIT1 interacts with the plus ends of
microtubules, suggesting a possible role in microtubule targeting
(Gunel et al., 2002
). Although
these biochemical data have provided useful insights into KRIT1 function in
vitro, the in vivo role of KRIT1 remains unclear, particularly with respect to
vascular disease.
Using in situ hybridization, others have reported that Ccm1 is
expressed ubiquitously until E10.5, at which point the expression starts to
become restricted to neural and epithelial tissues
(Denier et al., 2002;
Kehrer-Sawatzki et al., 2002
).
These studies suggest that Ccm1 may play a role in neural development
or function, and have led some authors to suggest that Ccm1 does not
play a role in cardiovascular development
(Kehrer-Sawatzki et al.,
2002
). The neural and epithelial expression of Ccm1 in
adulthood suggests that cavernous malformations may be the result of primary
neural defects.
Human vascular malformation syndromes, such as CCM, affect the junction
between veins and arteries. Arteries and veins form separately but follow
parallel trajectories and can be distinguished on the basis of molecular
markers, even prior to the onset of flow at E8.5
(Ji et al., 2003;
McGrath et al., 2003
;
Wang et al., 1998
). Recent
studies in zebrafish have demonstrated a genetic pathway governing arterial
identity, in which the expression of vascular endothelial growth
factor (Vegf) is necessary for the expression of arterial
markers and morphology (Lawson et al.,
2002
). Specifically, Vegf is necessary for the expression
of Notch family members in arteries, and Notch signaling, in
turn, is required for the arterial expression of ephrin B2
(Efnb2) and correct arterial-venous relationships
(Lawson et al., 2001
). A
specific role for this genetic pathway in arterial identity has not yet been
confirmed in mammalian systems.
The expanding link between human vascular disorders such as hereditary
hemorrhagic telangiectasia (Li et al.,
1999; Park et al.,
2003
; Sorensen et al.,
2003
; Urness et al.,
2000
) and CADASIL (Gridley,
2001
; Joutel et al.,
1996
) to molecular pathways implicated in arterial identity and
vascular patterning suggests that Ccm1 may have an essential role in
arterial development. Here we demonstrate, using murine gene targeting, that
Ccm1 is required for vascular development. Our data indicate that
Krit1-associated vascular defects are not secondary to disrupted neural
patterning. We also demonstrate impaired arterial identity in embryos lacking
Ccm1, and provide evidence that Krit1 lies upstream of Notch
signaling in the vasculature in mice and humans. Our studies suggest that
mutations in Ccm1 impact vascular development and disease by
disrupting a genetic pathway important in establishing arterial identity.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In situ hybridization
We carried out in situ hybridization at 70°C as previously described
(Urness et al., 2000). Probes
were generated by in vitro transcription of appropriate plasmids. We were
provided plasmids for Hand1, Hand2 (Dr Deepak Srivastava),
Dll4 (Dr John Shutter, Amgen), Notch4 (Dr Thomas Gridley)
and Efnb2 (Dr David Anderson). Using RT PCR on mouse cDNA with
appropriate primers (sequences available upon request) we generated probe
templates for Ccm1, IRES-lacZ, Nppa, Six3, Otx2, Fgf8, Gbx2, Shh, Pax7,
Nkx2-2, Ntn1, Brachyury and Vegfa. A probe for MafB was
generated by in vitro transcription of an EST (IMAGE: 2811217).
Immunohistochemistry
Mouse tissues were studied with antibodies to Pecam (clone MEC13.3, BD
Biosciences) and -smc actin (clone 1A4, DAKO). Human tissues were
studied with antibodies to PECAM (clone JC70A, DAKO) and NOTCH4 (polyclonal
H-225, SantaCruz).
In vivo proliferation and apoptosis
Frozen sections were stained first with Pecam antibody (clone MEC13.3, BD
Biosciences), followed by antibodies against phosphohistone H3 or cleaved
caspase 3 (Cell Signaling Technology). Finally, appropriate fluorescent
secondary antibodies (Jackson ImmunoResearch) were applied. Slides were
counterstained with DAPI (Molecular Probes).
Total endothelial nuclei and mitotic endothelial nuclei were counted from the dorsal aorta, branchial arch arteries, and vitelline arteries, and data were labeled as originating from either caudal or rostral regions. All sections distal to the sinus venosus of the heart were considered caudal. The mean percentages of rostral or caudal mitotic nuclei for each of three separate experiments were compared using an unpaired t-test (InStat, by GraphPad software).
Confocal immunofluorescence
Embryos were prepared for confocal immunofluorescent detection of PECAM
antigen as previously described (Drake and
Fleming, 2000). Images were obtained using an Olympus
FluoViewTM 300 confocal microscope (University of Utah Cell Imaging
Core).
India ink injection
Ink injections were carried out in E8.5-E9.5 embryos as previously
described (Urness et al.,
2000). Following ink injection, yolk sacs were removed and embryos
were photographed directly.
ß-Galactosidase staining
Mice heterozygous for Efnb2 expressing a tau-lacZ
transgene under the control of the Efnb2 locus were previously
generated and described (Wang et al.,
1998). We crossed this transgene into Ccm1 heterozygous
mice. Double heterozygous (both Ccm1 and Efnb2) male mice
were mated with Ccm1 heterozygous females to generate embryos
heterozygous for Efnb2 with all possible Ccm1 genotypes.
Genotyping was performed with allele-specific primers (sequences available
upon request). Staining for ß-galactosidase expression was performed with
X-gal as previously described (Sorensen et
al., 2003
). Littermates homozygous for Ccm1 and wild type
at the Efnb2 locus were used to control for the negligible embryonic
ß-galactosidase expression from the Ccm1 IRES-lacZ
construct.
Real-time quantitative RT-PCR
RNA was isolated from single E8.8 (13 somite) embryos (RNAqueous 4PCR kit,
Ambion) and used as template to make random primed cDNA (RetroScript kit,
Ambion). Assays for Efnb2, Dll4, Notch4 and Vegfa were
obtained, as well as for Ccm1 as a control for genotype
(Assays-on-Demand, Applied Biosystems), and used according to the
manufacturer's instructions on an Applied Biosystems 7900HT thermal cycler
(University of Utah Genomics Core Facility). Transcripts were normalized in
relation to Gapdh expression (rodent GAPDH, Applied Biosystems).
Comparisons were made between three separate pairs of wild-type and homozygous
mutant embryos, and reported in relation to wild-type expression (samples run
in triplicate).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
To obtain a more complete view of the vasculature, we performed whole-mount confocal immunofluorescence using Pecam antibodies (Fig. 4A-D). The enlargement of the distal dorsal aortae was again observed (arrow in Fig. 4B); however, in contrast to the extensive vascular dilatation, we observed a discrete region of vascular narrowing in the first branchial arch artery and adjacent proximal dorsal aorta that develops at E8.5 (Fig. 4D). All Ccm1-/- embryos at E8.5 have this narrowing; however, the extent and severity of involvement is variable.
|
We further characterized the time-course of this narrowing with a developmental series of embryo cross-sections stained for Pecam. These studies indicated that the proximal aorta and the first branchial arch artery formed normally at E8.0 (Fig. 5A,B). At E8.5, the first branchial arch artery had failed to enlarge (Fig. 5C,D). At E9.0, the vestige of the first arch artery and adjacent aorta remained severely narrowed (Fig. 5E,F), and the formation of the second branchial arch artery was abnormal (Fig. 4G,H; and data not shown). We postulated that this narrowing might be due to increased vascular apoptosis, yet we found no immunological evidence of increased cell death at E8.5 (data not shown). We also did not observe differential endothelial proliferation by immunostaining against phosphorylated histone H3 in this discrete region of the embryo, although this method may lack sufficient power to detect a difference in such a small number of cells. Thus, in addition to vascular dilatation, we observed narrowing of the branchial arch arteries and rostral dorsal aorta in Ccm1-/- embryos at E8.5.
|
Neuronal patterning proceeds normally in Ccm1-/- embryos
Concurrent with early vascular development, the embryonic neural tube
becomes organized and structured in both anteroposterior (AP) and dorsoventral
(DV) aspects as an early step in neural development
(Briscoe and Ericson, 2001;
Liu and Joyner, 2001
). The
neural expression of Ccm1 and the CNS predilection of cavernous
malformations led us to investigate neural patterning in
Ccm1-/- embryos. In order to determine whether vascular
defects occur secondary to neural defects in mice lacking Ccm1, we
used RNA in situ hybridization to study the AP and DV organization of the
developing CNS at E8.5. From anterior to posterior, the CNS at this stage can
be divided into the forebrain, midbrain and hindbrain. The hindbrain itself
can be subdivided further into 8 rhombomeres
(Liu and Joyner, 2001
).
Molecular markers can also distinguish the midbrain-hindbrain junction, or
isthmus, which has an important organizer function
(Liu and Joyner, 2001
). The
homeobox gene Six3 (Fig.
6A,B) is expressed in the forebrain at E8.5
(Oliver et al., 1995
). The
transcription factor Otx2 (Fig.
6C,D) is also expressed in the forebrain and extends into the
midbrain up to the isthmus, where expression abruptly stops
(Liu and Joyner, 2001
). The
secreted molecule Fgf8 (Fig.
6E,F) has a domain of expression restricted to the isthmus at the
midbrain-hindbrain junction (Liu and
Joyner, 2001
). The homeobox gene Gbx2
(Fig. 6G,H), which helps
establish the posterior boundary of the isthmus, is then expressed throughout
the hindbrain (Liu and Joyner,
2001
). Within the hindbrain the leucine zipper transcription
factor MafB (Fig.
6I,J) is limited in expression to rhombomeres 5 and 6
(Grapin-Botton et al., 1998
).
Along the AP axis of the neural tube, the secreted protein Shh
(Fig. 6K,L) is expressed in the
notochord and floor plate where it performs an inductive role
(Briscoe and Ericson, 2001
).
The homeobox gene Pax7 (Fig.
6M,N) is expressed in the dorsal neural tube
(Fu et al., 2003
;
Mansouri et al., 1996
). The
homeobox gene Nkx2-2 (Fig.
6O,P) is expressed in the lateral floor plate
(Charrier et al., 2002
;
Fu et al., 2003
). The axon
guidance molecule Netrin1 (Fig.
6Q,R) is expressed in the medial floor plate as well as in
adjacent somites (Serafini et al.,
1996
). No alterations in the normal expression pattern of these
genes were observed in embryos lacking Ccm1. We conclude that AP and
DV patterning of the neural tube in Ccm1-/- embryos is
intact at the onset of vascular defects. These studies indicate that the
vascular developmental defects observed in Ccm1-/- embryos
are not secondary to a primary neural developmental defect.
|
|
Disruption of a pathway governing arterial identity
A genetic pathway has been described in zebrafish placing Efnb2
expression genetically downstream of Notch gene signaling.
Notch signaling, in turn, is genetically downstream of Vegfa
in the control of arterial identity
(Lawson et al., 2001;
Lawson et al., 2002
). The
expression of the arterial-specific mammalian Notch genes,
Dll4 and Notch4 was significantly downregulated in
Ccm1-/- embryos by E8.5
(Fig. 8A-D), prior to the gross
appearance of the mutant phenotype and prior to the onset of circulation. The
expression of the pan-endothelial markers Pecam
(Fig. 4A,B) and Kdr
(data not shown) remained unperturbed. We observed similar intensity and
distribution of expression of Vegfa
(Fig. 8E,F) in
Ccm1-/- embryos, by in situ hybridization, compared with
wild type. The downregulation of arterial markers with intact expression of
Vegfa in mice lacking Ccm1 was confirmed and quantified by
real-time quantitative RT-PCR (Fig.
8G). These results would suggest that Ccm1 lies
genetically downstream of Vegf in the control of Notch
signaling and arterial morphogenesis.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Role of Ccm1 in development
Ccm1 is essential for angiogenesis
Vasculogenesis, the de novo formation of endothelial tubes from angioblast
precursors (Risau, 1997), is
intact in Ccm1-/- mice, as the vascular pattern is formed
correctly by E8.0. In this study, we demonstrate that Ccm1 is
essential for angiogenesis, the process of vessel maturation and network
remodeling wherein new vessels arise from preexisting vessels
(Risau, 1997
). Mice lacking
Ccm1 die by E11 with prominent vascular defects associated with
inappropriate angiogenic remodeling. Arteries in Ccm1-/-
embryos develop morphologic defects starting at E8.5, with marked enlargement
of the caudal aorta and the vessels of the cephalic mesenchyme. We observed
increased endothelial proliferation in the dilated aorta of the caudal embryo
compared with wild type. This dilatation and proliferation is not a
consequence of increased flow as demonstrated by a lack of circulation of
India ink when injected into the cardiac ventricle. Narrowing of the branchial
arch arteries and the proximal aortae is also present at E8.5. Cardiac
development appears entirely normal until E9.5, when generalized developmental
arrest occurs in Ccm1-/- mice. We conclude that
Ccm1 is required for angiogenesis.
Neural morphology and neural patterning develop normally in mice lacking Ccm1
The neural and epithelial expression of Ccm1 in adulthood suggests
that cerebral cavernous vascular malformations may be secondary to neural
defects. Our results indicate that loss of Ccm1 expression results in
primary vascular defects in the embryo. Vascular defects in mice lacking
Ccm1 are uniformly present at E8.5. We demonstrate that the
anteroposterior and dorsoventral patterning of embryos lacking Ccm1
is normal at E8.5, based on the expression of a broad panel of genes. The
optic and otic anlagen are both present, and no morphologic neural tube
defects are observed despite vascular enlargement throughout the embryo. These
results suggest that endothelial cells require Ccm1. To determine
whether Ccm1 expression is necessary within the endothelial cell
itself, or whether endothelial cells depend upon interactions with adjacent
cells expressing Ccm1, further experiments using conditional
mutagenesis will be important.
Ccm1 is required to establish arterial identity
Arteries are distinguished from veins even at the earliest stages of
development and prior to the onset of circulation. This distinction is first
recognized on the basis of a unique set of molecular markers that label
arteries and not veins. We examined the expression of the known arterial
markers, Efnb2, Notch4 and Dll4, and found significant
downregulation of the arterial expression of all three genes by in situ
hybridization and by real-time quantitative RT-PCR. The extravascular
expression of Efnb2 from somites, nephrogenic cord and hindbrain
remains intact in Ccm1-/- embryos, suggesting a specific
effect of Ccm1 on arterial identity. These data demonstrate that
arterial specification of endothelial tubes is disrupted in
Ccm1-/- mice.
Recently, much progress has been made in understanding the process by which
arteries are distinguished from veins. This process is best understood in the
zebrafish, a model organism particularly well suited to the establishment of
genetic pathways (Fishman,
2001). In the zebrafish, loss of Notch signaling leads to a
reduction of arterial Efnb2 expression, and abnormal arterial-venous
connections as seen angiographically
(Lawson et al., 2001
). The
expression of Vegf was shown to be necessary for Notch signaling and
Efnb2 expression (Lawson et al.,
2002
). This genetic pathway has not been confirmed in mammalian
systems, in part because of the relative difficulty in genetically
manipulating mice compared with zebrafish. In our studies, we found no
detectable disruption of Vegf expression in mice lacking
Ccm1. These data suggest that Ccm1 lies genetically
downstream of Vegf, and indicates that it is genetically upstream of
Notch and Efnb2 signaling in the control of arterial
specification.
In mammalian systems, it has been demonstrated that Notch
signaling is important for vascular development
(D'Amore and Ng, 2002;
Gridley, 2001
;
Krebs et al., 2000
;
Leong et al., 2002
). The
combined loss of the Notch1 and Notch4 receptors leads to
severe vascular defects including narrowed or collapsed dorsal aortae
(Krebs et al., 2000
). The
vascular defects observed in Notch1 and Notch4 double-mutant
embryos are more severe than those observed in mice lacking Notch1
alone, whereas mice lacking Notch4 show no phenotype
(Krebs et al., 2000
). These
studies have led others to hypothesize that genetic interactions between
Notch4 and Notch1 are important for angiogenic vascular
remodeling (Krebs et al.,
2000
). Our studies support a crucial role for Notch
signaling in angiogenesis, and suggest that disruption of Notch4,
with its probable ligand Dll4, may contribute to the vascular
phenotype observed in Ccm1-/- mice. These studies lead to
a hypothesis that is readily tested: whereas disruption of Notch4 may
lead to no phenotype, ablating the endothelial expression of both
Notch4 and Dll4 in mice will result in a severe vascular
phenotype reminiscent of Ccm1-/- mice.
Insights into the role of CCM1 in disease
Impaired arterial identity associated with human cavernous malformations
The disruption of the Notch signaling pathway in
Ccm1-/- mice led us to postulate that a similar reduction
in NOTCH expression may be important in the pathogenesis of human cavernous
malformations. Our studies of affected individuals with known mutations in
CCM1 (Marchuk et al.,
1995; Sahoo et al.,
1999
) show that NOTCH4 is significantly reduced in arterioles
associated with CCM lesions. We were unable to test the expression of
EFNB2 and DLL4 because neither reliable antibodies, nor
frozen sections for in situ hybridization from these affected individuals are
available. We observe additional parallels between the human lesions and mice
lacking Ccm1. Both exhibit enlarged, thin-walled vessels that lack
vascular smooth muscle support. In both instances, the enlarged vessels do not
opacify well angiographically, suggesting that human lesions are isolated from
a significant arterial supply. We demonstrate that cavernous lesions in mice
lacking Ccm1 are isolated from arterial inflow by vascular narrowing
proximal to the lesion. This suggests that similar narrowing may be present in
human lesions. The decrease in NOTCH4 and these other parallels lead us to
further suggest that impaired arterial specification may contribute to the
etiology of CCM.
This characterization of Ccm1-/- mice exemplifies the
efficacy of studying vascular malformation genes to better understand arterial
development. We have previously characterized mice lacking the two genes known
to cause Hereditary Hemorrhagic Telangiectasia, an autosomal dominant vascular
dysplasia caused by loss-of-function mutations in either endoglin
(Eng) (Li et al.,
1999) or activin receptor-like kinase 1 (Acvrl1)
(Urness et al., 2000
).
Affected individuals develop enlarged arteriovenous channels that bypass
capillary beds, and are prone to rupture
(Guttmacher et al., 1995
).
Mice lacking Acvrl1 and Eng fail to maintain distinct
arterial and venous beds, developing similar arteriovenous shunts
(Sorensen et al., 2003
;
Urness et al., 2000
). These
three examples suggest that a natural mutagenesis screen has occurred in
humans, in which the readout of vascular malformations suggests disruption of
genes involved in arterial and venous development.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
* These authors contributed equally to this work
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arias, E. and Smith, B. L. (2003). Deaths: preliminary data for 2001. Natl. Vital Stat. Rep. 51, 1-44.
Barrow, J. R., Thomas, K. R., Boussadia-Zahui, O., Moore, R.,
Kemler, R., Capecchi, M. R. and McMahon, A. P. (2003).
Ectodermal Wnt3/betacatenin signaling is required for the establishment and
maintenance of the apical ectodermal ridge. Genes Dev.
17,394
-409.
Brenner, R. M., Slayden, O. D., Rodgers, W. H., Critchley, H.
O., Carroll, R., Nie, X. J. and Mah, K. (2003).
Immunocytochemical assessment of mitotic activity with an antibody to
phosphorylated histone H3 in the macaque and human endometrium.
Hum. Reprod. 18,1185
-1193.
Briscoe, J. and Ericson, J. (2001). Specification of neuronal fates in the ventral neural tube. Curr. Opin. Neurobiol. 11,43 -49.[CrossRef][Medline]
Charrier, J. B., Lapointe, F., Le Douarin, N. M. and Teillet, M. A. (2002). Dual origin of the floor plate in the avian embryo. Development 129,4785 -4796.[Medline]
Craig, H. D., Gunel, M., Cepeda, O., Johnson, E. W., Ptacek, L.,
Steinberg, G. K., Ogilvy, C. S., Berg, M. J., Crawford, S. C., Scott, R. M. et
al. (1998). Multilocus linkage identifies two new loci for a
mendelian form of stroke, cerebral cavernous malformation, at 7p15-13 and
3q25.2-27. Hum. Mol. Genet.
7,1851
-1858.
D'Amore, P. A. and Ng, Y. S. (2002). Won't you be my neighbor? Local induction of arteriogenesis. Cell 110,289 -292.[Medline]
Del Curling, O., Jr, Kelly, D. L., Jr, Elster, A. D. and Craven, T. E. (1991). An analysis of the natural history of cavernous angiomas. J. Neurosurg. 75,702 -708.[Medline]
Denier, C., Gasc, J., Chapon, F., Domenga, V., Lescoat, C., Joutel, A. and Tournier-Lasserve, E. (2002). Krit1/cerebral cavernous malformation 1 mRNA is preferentially expressed in neurons and epithelial cells in embryo and adult. Mech. Dev. 117, 363.[CrossRef][Medline]
Drake, C. J. and Fleming, P. A. (2000).
Vasculogenesis in the day 6.5 to 9.5 mouse embryo.
Blood 95,1671
-1679.
Faisst, A. M. and Gruss, P. (1998). Bodenin: a novel murine gene expressed in restricted areas of the brain. Dev. Dyn. 212,293 -303.[CrossRef][Medline]
Fishman, M. C. (2001). Genomics. Zebrafish -
the canonical vertebrate. Science
294,1290
-1291.
Fu, H., Qi, Y., Tan, M., Cai, J., Hu, X., Liu, Z., Jensen, J. and Qiu, M. (2003). Molecular mapping of the origin of postnatal spinal cord ependymal cells: evidence that adult ependymal cells are derived from Nkx6.1+ ventral neural progenitor cells. J. Comp. Neurol. 456,237 -244.[CrossRef][Medline]
Grapin-Botton, A., Bonnin, M. A., Sieweke, M. and Le Douarin, N.
M. (1998). Defined concentrations of a posteriorizing signal
are critical for MafB/Kreisler segmental expression in the hindbrain.
Development 125,1173
-1181.
Gridley, T. (2001). Notch signaling during
vascular development. Proc. Natl. Acad. Sci. USA
98,5377
-5378.
Gunel, M., Laurans, M. S., Shin, D., DiLuna, M. L., Voorhees,
J., Choate, K., Nelson-Williams, C. and Lifton, R. P. (2002).
KRIT1, a gene mutated in cerebral cavernous malformation, encodes a
microtubule-associated protein. Proc. Natl. Acad. Sci.
USA 99,10677
-10682.
Guttmacher, A. E., Marchuk, D. A. and White, R. I., Jr
(1995). Hereditary hemorrhagic telangiectasia. N.
Engl. J. Med. 333,918
-924.
Hendzel, M. J., Wei, Y., Mancini, M. A., Van Hooser, A., Ranalli, T., Brinkley, B. R., Bazett-Jones, D. P. and Allis, C. D. (1997). Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106,348 -360.[CrossRef][Medline]
Ji, R. P., Phoon, C. K., Aristizabal, O., McGrath, K. E., Palis,
J. and Turnbull, D. H. (2003). Onset of cardiac function
during early mouse embryogenesis coincides with entry of primitive
erythroblasts into the embryo proper. Circ. Res.
92,133
-135.
Joutel, A., Corpechot, C., Ducros, A., Vahedi, K., Chabriat, H., Mouton, P., Alamowitch, S., Domenga, V., Cecillion, M., Marechal, E. et al. (1996). Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia. Nature 383,707 -710.[CrossRef][Medline]
Kehrer-Sawatzki, H., Wilda, M., Braun, V. M., Richter, H. P. and Hameister, H. (2002). Mutation and expression analysis of the KRIT1 gene associated with cerebral cavernous malformations (CCM1). Acta Neuropathol. (Berl.) 104,231 -240.[Medline]
Krebs, L. T., Xue, Y., Norton, C. R., Shutter, J. R., Maguire,
M., Sundberg, J. P., Gallahan, D., Closson, V., Kitajewski, J., Callahan, R.
et al. (2000). Notch signaling is essential for vascular
morphogenesis in mice. Genes Dev.
14,1343
-1352.
Laberge-le Couteulx, S., Jung, H. H., Labauge, P., Houtteville, J. P., Lescoat, C., Cecillon, M., Marechal, E., Joutel, A., Bach, J. F. and Tournier-Lasserve, E. (1999). Truncating mutations in CCM1, encoding KRIT1, cause hereditary cavernous angiomas. Nat. Genet. 23,189 -193.[CrossRef][Medline]
Lawson, N. D., Scheer, N., Pham, V. N., Kim, C. H., Chitnis, A.
B., Campos-Ortega, J. A. and Weinstein, B. M. (2001). Notch
signaling is required for arterial-venous differentiation during embryonic
vascular development. Development
128,3675
-3683.
Lawson, N. D., Vogel, A. M. and Weinstein, B. M. (2002). sonic hedgehog and vascular endothelial growth factor act upstream of the Notch pathway during arterial endothelial differentiation. Dev. Cell 3,127 -136.[Medline]
Leong, K. G., Hu, X., Li, L., Noseda, M., Larrivee, B., Hull,
C., Hood, L., Wong, F. and Karsan, A. (2002). Activated
Notch4 inhibits angiogenesis: role of beta 1-integrin activation.
Mol. Cell. Biol. 22,2830
-2841.
Li, D. Y., Sorensen, L. K., Brooke, B. S., Urness, L. D., Davis,
E. C., Taylor, D. G., Boak, B. B. and Wendel, D. P. (1999).
Defective angiogenesis in mice lacking endoglin.
Science 284,1534
-1537.
Liu, A. and Joyner, A. L. (2001). Early anterior/posterior patterning of the midbrain and cerebellum. Annu. Rev. Neurosci. 24,869 -896.[CrossRef][Medline]
Lynch, J. K., Hirtz, D. G., DeVeber, G. and Nelson, K. B.
(2002). Report of the National Institute of Neurological
Disorders and Stroke workshop on perinatal and childhood stroke.
Pediatrics 109,116
-123.
Mansouri, A., Stoykova, A., Torres, M. and Gruss, P.
(1996). Dysgenesis of cephalic neural crest derivatives in
Pax7-/- mutant mice. Development
122,831
-838.
Marchuk, D. A., Gallione, C. J., Morrison, L. A., Clericuzio, C. L., Hart, B. L., Kosofsky, B. E., Louis, D. N., Gusella, J. F., Davis, L. E. and Prenger, V. L. (1995). A locus for cerebral cavernous malformations maps to chromosome 7q in two families. Genomics 28,311 -314.[CrossRef][Medline]
McGrath, K. E., Koniski, A. D., Malik, J. and Palis, J.
(2003). Circulation is established in a stepwise pattern in the
mammalian embryo. Blood
101,1669
-1676.
Moriarity, J. L., Clatterbuck, R. E. and Rigamonti, D. (1999). The natural history of cavernous malformations. Neurosurg. Clin. N. Am. 10,411 -417.[Medline]
Nechiporuk, A. and Keating, M. T. (2002). A
proliferation gradient between proximal and msxb-expressing distal blastema
directs zebrafish fin regeneration. Development
129,2607
-2617.
Oliver, G., Mailhos, A., Wehr, R., Copeland, N. G., Jenkins, N.
A. and Gruss, P. (1995). Six3, a murine homologue of the sine
oculis gene, demarcates the most anterior border of the developing neural
plate and is expressed during eye development.
Development 121,4045
-4055.
Otten, P., Pizzolato, G. P., Rilliet, B. and Berney, J. (1989). A propos de 131 cas d'angiomes caverneux (cavernomes) du S.N.C. repérés par l'analyse rétrospective de 24 535 autopsies. Neurochirurgie 35,128 -131.
Park, K. W., Morrison, C. M., Sorensen, L. K., Jones, C. A., Rao, Y., Chien, C. B., Wu, J. Y., Urness, L. D. and Li, D. Y. (2003). Robo4 is a vascular-specific receptor that inhibits endothelial migration. Dev. Biol. 261,251 -267.[CrossRef][Medline]
Rigamonti, D., Hadley, M. N., Drayer, B. P., Johnson, P. C., Hoenig-Rigamonti, K., Knight, J. T. and Spetzler, R. F. (1988). Cerebral cavernous malformations. Incidence and familial occurrence. N. Engl. J. Med. 319,343 -347.[Abstract]
Risau, W. (1997). Mechanisms of angiogenesis. Nature 386,671 -674.[CrossRef][Medline]
Robinson, J. R., Awad, I. A. and Little, J. R. (1991). Natural history of the cavernous angioma. J. Neurosurg. 75,709 -714.[Medline]
Robinson, J. R., Jr, Awad, I. A., Masaryk, T. J. and Estes, M. L. (1993). Pathological heterogeneity of angiographically occult vascular malformations of the brain. Neurosurgery 33,547 -554.[Medline]
Ruhrberg, C., Gerhardt, H., Golding, M., Watson, R., Ioannidou,
S., Fujisawa, H., Betsholtz, C. and Shima, D. T. (2002).
Spatially restricted patterning cues provided by heparin-binding VEGF-A
control blood vessel branching morphogenesis. Genes
Dev. 16,2684
-2698.
Sahoo, T., Johnson, E. W., Thomas, J. W., Kuehl, P. M., Jones,
T. L., Dokken, C. G., Touchman, J. W., Gallione, C. J., Lee-Lin, S. Q.,
Kosofsky, B. et al. (1999). Mutations in the gene encoding
KRIT1, a Krev-1/rap1a binding protein, cause cerebral cavernous malformations
(CCM1). Hum. Mol. Genet.
8,2325
-2333.
Sahoo, T., Goenaga-Diaz, E., Serebriiskii, I. G., Thomas, J. W., Kotova, E., Cuellar, J. G., Peloquin, J. M., Golemis, E., Beitinjaneh, F., Green, E. D. et al. (2001). Computational and experimental analyses reveal previously undetected coding exons of the KRIT1 (CCM1) gene. Genomics 71,123 -126.[CrossRef][Medline]
Serafini, T., Colamarino, S. A., Leonardo, E. D., Wang, H., Beddington, R., Skarnes, W. C. and Tessier-Lavigne, M. (1996). Netrin-1 is required for commissural axon guidance in the developing vertebrate nervous system. Cell 87,1001 -1014.[Medline]
Serebriiskii, I., Estojak, J., Sonoda, G., Testa, J. R. and Golemis, E. A. (1997). Association of Krev-1/rap1a with Krit1, a novel ankyrin repeat-containing protein encoded by a gene mapping to 7q21-22. Oncogene 15,1043 -1049.[CrossRef][Medline]
Sorensen, L. K., Brooke, B. S., Li, D. Y. and Urness, L. D. (2003). Loss of distinct arterial and venous boundaries in mice lacking endoglin, a vascular-specific TGFbeta coreceptor. Dev. Biol. 261,235 -250.[CrossRef][Medline]
Tybulewicz, V. L., Crawford, C. E., Jackson, P. K., Bronson, R. T. and Mulligan, R. C. (1991). Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene. Cell 65,1153 -1163.[Medline]
Urness, L. D., Sorensen, L. K. and Li, D. Y. (2000). Arteriovenous malformations in mice lacking activin receptor-like kinase-1. Nat. Genet. 26,328 -331.[CrossRef][Medline]
Wang, H. U., Chen, Z. F. and Anderson, D. J. (1998). Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell 93,741 -753.[Medline]
Zawistowski, J. S., Serebriiskii, I. G., Lee, M. F., Golemis, E. A. and Marchuk, D. A. (2002). KRIT1 association with the integrin-binding protein ICAP-1: a new direction in the elucidation of cerebral cavernous malformations (CCM1) pathogenesis. Hum. Mol. Genet. 11,389 -396.[CrossRef][Medline]
Zhang, J., Clatterbuck, R. E., Rigamonti, D. and Dietz, H. C. (2000). Cloning of the murine Krit1 cDNA reveals novel mammalian 5' coding exons. Genomics 70,392 -395.[CrossRef][Medline]
Zhang, J., Clatterbuck, R. E., Rigamonti, D., Chang, D. D. and
Dietz, H. C. (2001). Interaction between krit1 and icap1alpha
infers perturbation of integrin beta1-mediated angiogenesis in the
pathogenesis of cerebral cavernous malformation. Hum. Mol.
Genet. 10,2953
-2960.
Related articles in Development: