1 Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
2 Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA
3 Cardiovascular Research Center, Massachusetts General Hospital East, Charlestown, MA 02129, USA
4 Howard Hughes Medical Institute and Department of Pharmacology, University of Washington School of Medicine, Seattle, WA 98185 USA
* Present address: Department of Cell Biology, Georgetown University Medical School, Washington, DC 20007, USA
Present address: Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB T2N 4N1, Canada
Present address: Biology Department, Texas A&M University, College Station, TX 77843, USA
Author for correspondence (e-mail: flyingfish{at}nih.gov)
Accepted 1 April 2002
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SUMMARY |
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Movies available on-line
Key words: Acvrl1, Hereditary hemorrhagic telangiectasia, Endothelium, Angiogenesis, Zebrafish, violet beauregarde
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INTRODUCTION |
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A number of ligand/receptor pairs have been identified that help to coordinate vasculogenesis and angiogenesis. These include vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2); angiopoietins 1 and 2 and the Tie2 receptor; ephrin-B2 and EphB4; and platelet-derived growth factor B (PDGFB) and PDGF receptor ß. Of these, only VEGF and VEGFR2 are required for both vasculogenesis and angiogenesis, whereas the rest play roles in angiogenic processes and/or perivascular sheath formation (for review, see Roman and Weinstein, 2000). TGFß family signaling is also important in blood vessel development, although the precise ligand/receptor pairs and their specific roles are not well established. TGFß family ligands bind to a heterodimeric complex consisting of a type II and a type I receptor, both of which are transmembrane serine/threonine kinases (for review, see Massague et al., 2000
). Ligand binding stimulates the type II receptor to phosphorylate the type I receptor, which in turn phosphorylates a receptor-specific Smad. This phosphorylated Smad dimerizes with a common partner Smad (Smad4), forming a complex that translocates to the nucleus and directly regulates gene transcription. TGFß family ligands can be divided into two groups based on Smad specificity: TGFßs, activins, and nodals signal through Smad2 and Smad3, whereas bone morphogenetic proteins (BMPs) signal through Smad1, Smad5, and Smad8.
The importance of TGFß family signaling in vessel development is illustrated by the following observations. Targeted deletion of Tgfb1 impairs yolk sac vasculogenesis but has no effect on vasculogenesis or angiogenesis within the embryo proper (Dickson et al., 1995). A similar extraembryonic vascular phenotype, characterized by dilated vessels that exhibit poor endothelial cell adhesion and impaired vascular smooth muscle development, results from targeted disruption of TGFß receptor II (Tgfbr2) or TGFß receptor I (Tgfbr1), which together constitute the canonical receptor pair for TGFß1 (Larsson et al., 2001
; Oshima et al., 1996
). In addition to defects in yolk sac vasculogenesis, angiogenic defects are observed within the embryo proper in Tgfbr2 null mice (R. J. L., unpublished observation) and within the embryonic portion of the placenta in Tgfbr1 null mice (Larsson et al., 2001
). Furthermore, disruption of endoglin (Eng), an accessory receptor that binds TGFßs, activins, and BMPs (Barbara et al., 1999
), impairs primitive plexus remodeling and vascular smooth muscle differentiation both in the yolk sac and within the embryo proper (Li et al., 1999
). Targeted disruption of Acvrl1, a TGFß type I receptor that signals through Smad1/5/8 (Chen and Massague, 1999
), or disruption of Smad5 itself, results in extraembryonic and intraembryonic vessel dilation, defects in vascular plexus remodeling, and impaired vascular smooth muscle differentiation (Oh et al., 2000
; Urness et al., 2000
; Yang et al., 1999
). The ligand involved in this Acvrl1-Smad1/5/8 pathway is not clear but may be TGFß1, which has been shown to bind Acvrl1 and TGFßRII (Lux et al., 1999
; Oh et al., 2000
) and to stimulate Smad1 phosphorylation (Yue et al., 1999
) in vitro. However, no downstream targets of TGFß1-stimulated Acvrl1 signaling have been identified.
In humans, mutations in ENG and ACVRL1 are responsible for the autosomal dominant vascular dysplasias, hereditary hemorrhagic telangiectasia (HHT) type 1 and type 2, respectively (Johnson et al., 1996; McAllister et al., 1994
), which together occur with a frequency of 1 in 10,000 (McDonald et al., 2000
). These diseases present clinically in a similar manner, with symptoms including epistaxis (recurrent nosebleeds), mucocutaneous telangiectases (superficial vascular dilations that present as small red spots), and arteriovenous malformations (AVMs) (Guttmacher et al., 1995
). Large AVMs, particularly in brain and lung, can lead to stroke if severe shunting or rupture occurs. The basis for the localized nature of these defects is not known, although it has been suggested that the appearance of pathological lesions is precipitated by some independent, site-specific event (Guttmacher et al., 1995
). The age of onset and expressivity of these diseases are highly variable and seem to depend on both genetic and epigenetic factors.
In the current study, we present a new vertebrate model for HHT2: the zebrafish mutant, violet beauregarde (vbg). In homozygous vbg mutants, the majority of blood flow is confined to a small number of dilated cranial vessels that contain more than twice as many endothelial cells as their wild-type counterparts. The vbg locus maps to linkage group (LG) 23 and encodes Acvrl1, which is expressed predominantly in the endothelium of those cranial vessels that are dilated in vbg mutants. As Acvrl1 ligand(s) and downstream targets of Acvrl1 signaling are presently ill-defined, the embryological, molecular and molecular genetic techniques afforded by the zebrafish system make the vbg mutant a valuable model for dissecting the molecular pathway by which Acvrl1 directs embryonic vessel formation, which in turn could lead to insight into the molecular mechanisms responsible for HHT2 pathogenesis.
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MATERIALS AND METHODS |
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Microangiography and confocal imaging
Microangiography was performed as described (Weinstein et al., 1995). For confocal microscopy, embryos were anesthetized using 0.016% Tricaine (Sigma), mounted on depression slides using 5% methylcellulose (Sigma), and Z-series of frame-averaged optical sections were generated using a Radiance 2000 confocal microscope (Bio-Rad). Two-dimensional and three-dimensional projections were generated using MetaMorph (Universal Imaging) software.
Nuclei counting
The basal communicating artery and posterior connecting segments (Isogai et al., 2001) were imaged by confocal microscopy at 2 and 3 days post-fertilization (dpf) in embryos derived from vbgft09e/+;TG(fli1:nEGFP)y7 incrosses. Cell number within these vessels was analyzed in 12 wild-type and 12 mutant embryos by counting nuclei on three-dimensional, rotating projections using the Manually Count Objects function in MetaMorph. Data were analyzed by repeated measures ANOVA.
Meiotic and physical mapping
Embryos used for meiotic mapping were digested overnight at 50°C in 100 µl of buffer containing 10 mM Tris, pH 8.0; 50 mM EDTA; 200 mM NaCl; 0.5 mg/ml proteinase K; and 0.5% SDS. Digests were spun through Sephacryl-S400 (Amersham Pharmacia), and the resulting preparation of genomic DNA was used for PCR. SSLP mapping was performed as described previously (Knapik et al., 1998) except primers were not radiolabeled and PCR reactions were resolved on 3% MetaPhor agarose gels (FMC). YAC clones were identified from DNA pools by PCR as described by the supplier (Invitrogen) and isolated in agarose plugs and sized by standard techniques. For PCR and end rescue, agarose plugs were equilibrated in TE, pH 8.0, and YAC DNA was purified using the Qiaquick gel extraction kit (Qiagen). YAC ends were rescued by digesting this preparation with either BamHI or SpeI and self-ligating to form transformable plasmids. BAC clones were identified from DNA pools by PCR as described by the supplier (Incyte Genomics), and prepared using Nucleobond columns (Clontech). Sequencing was performed using BigDye reagents on an ABI310 capillary sequencer (Perkin-Elmer). PCR primers designed against non-repetitive regions of YAC and BAC DNA shown to be present on LG23 by radiation hybrid mapping (Hukriede et al., 1999
) were used to establish physical contigs and to look for polymorphisms for use in meiotic mapping. To find polymorphisms, PCR products amplified from genomic DNA from individual wild-type, heterozygous and mutant embryos (genotype based on flanking markers) were sequenced. Single nucleotide polymorphisms were assayed as restriction fragment length polymorphisms (RFLPs), when possible, or were converted to RFLPs using derived cleaved amplified polymorphic sequence (dCAPS) analysis (Neff et al., 1998
). One novel SSLP (bac156CA) was identified in BAC156f04 as described previously (Knapik et al., 1998
).
Cloning of zebrafish acvrl1 cDNA
A tblastn search uncovered a 3' zebrafish EST, zehn1109, with homology to human ACVRL1. 5' SMART RACE (Clontech) was performed using first strand cDNA synthesized from total RNA from 24 hpf embryos and a zehn1109-specific primer, 5'-CCTCTGGTGCCATGTATCGCTTG-3'. Analysis revealed two RACE products that diverged five bases 5' of the putative start codon (see Results). cDNA was synthesized from RNA obtained from wild-type, vbgy6/vbgy6, and vbgft09e/vbgft09e embryos using SuperscriptII (Life Technologies), and PCR was performed using PLATINUM Pfx DNA polymerase (Life Technologies) and primers specific to the 5' UTR of the major RACE product (f9: 5'-TTGCCGCCCGTTATGAGAAT-3') and the 3' UTR of zehn1109 (r9: 5'-TCGGTGGAGCCTAAGGACAAGAAG-3'). A single product was obtained, cloned into pCRII-TOPO (Invitrogen), and sequenced. The GT transversion found in vbgy6 was assayed by dCAPS analysis using the following primers: 5'-CACGGTCCAACTAAGGCATGAAAACACCTT-3', 5'-GTGTGCTATGGCTGGTTTG-3'. The forward primer ends just 5' to the mutation and contains a single mismatch (underlined) that creates a BsaJI site in the wild-type sequence.
In situ hybridization and immunohistochemistry
Whole-mount in situ hybridization was performed as described previously (Hauptmann and Gerster, 1994). For acvrl1 detection, a PCR fragment was amplified from 24 hour cDNA (5'-GGCCCTGGGTCTCGTCTT-3' and 5'-AACCCCATCTTACCCTCACTTTAC-3') and cloned into pCRII-TOPO (Invitrogen). Other probes used were tie1 (Lyons et al., 1998
), gata2 (Detrich et al., 1995
), and ephrin-b2 (kindly provided by M. Tsang). Whole-mount immunohistochemistry was performed using the Vectastain Elite ABC kit (Vector Laboratories) as described by Westerfield (Westerfield, 1995
). Rabbit polyclonal phospho-Smad1 antibody (Cell Signaling Technology) was used at 1:1500. Mouse monoclonal myc antibody (Babco) was used at 1:2000.
Expression constructs and morpholinos
The acvrl1 pCRII-TOPO clones described above (wild-type and vbgy6 alleles) were used as templates in a PCR using primer f9 extended with a BamHI site (5'-TATAGGATCCTTGCCGCCCGTTATGAGAATAC-3'), and primer r9 extended with a ClaI site (5'-TATAATCGATCGAGGTCCAGTTTAAGCTTGTCTATG-3'). The resulting product was digested with BamHI and ClaI and cloned into BamHI/ClaI-digested pCS2+ upstream of a six-myc tag (pCS2+MT) (Rupp et al., 1994). Activated, myc-tagged constructs were generated by PCR using the QuikChange kit (Stratagene), acvrl1-pCS2+MT clones, and the following complementary primers: 5'-GCAGAGGACCATGGCGCGAGATATCTCTCTGGTTGAGTG-3', and 5'-CACTCAACCAGAGAGATATCTCGCGCCATGGTCCTCTGC-3'. These primers contain two mismatches (underlined), converting glutamine 193 (CAG) to aspartic acid (GAT). Dominant-negative, myc-tagged constructs were similarly generated using complementary primers 5'-GAAAGTGTGGCTGTCAGGATTTTCTCCTCTCGTGATG-3', and 5'-CATCACGAGAGGAGAAAATCCTGACAGCCACACTTTC-3'. These primers contain a single mismatch (underlined), converting lysine 221 (AAG) to arginine (AGG). All constructs were verified by sequencing. Capped mRNA was synthesized from NotI-digested constructs using mMessage mMachine with SP6 RNA polymerase (Ambion). Morpholino-modified antisense oligonucleotides (Gene Tools) used were standard control (5'-CCTCTTACCTCAGTTACAATTTATA-3'); MO1 (5'-CTGCGAGCATCACTGAAGCCTTC-3'), which targets +3 to +25 of the coding region of acvrl1; and MO2 (5'-CTCATTACTCAAACATAGAAGTGTA-3'), which targets 95 to 71 of the major acvrl1 splice variant containing noncoding exon1. Capped mRNAs and morpholinos were injected into 1- to 4-cell embryos either into a single blastomere or into the streaming yolk cytoplasm, just beneath the blastomeres, as described previously (Westerfield, 1995
).
Luciferase reporter assays
P19 cells maintained in DMEM with 10% fetal bovine serum and antibiotics were transiently transfected with Smad2/3-responsive pA3-lux or Smad1/5/8-responsive pTLX2-lux (luciferase reporters) and the indicated activated TGFß type I receptor constructs by calcium phosphate precipitation as described (Macias-Silva et al., 1998). One day following transfection, cells were placed in 0.2% serum in DMEM, and luciferase activity was determined using the Dual-Luciferase Reporter Assay System (Promega). For each experiment, transfections were performed in triplicate, and each well was assayed for luciferase activity in duplicate. Experiments were repeated at least three times with similar results. Data are expressed in relative luciferase units and were compared by Cochrans t-test.
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RESULTS |
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Cranial vessel enlargement in vbg mutants stems from an increase in endothelial cell number
In order to determine whether cranial vessel dilation in vbg mutants involves endothelial cell hypertrophy or an increase in endothelial cell number, we counted nuclei in the vascular triangle comprising the basal communicating artery and posterior connecting segments. These vessels were chosen for analysis because they are consistently dilated in vbg mutants and are easily imaged in TG(fli1:nEGFP)y7 embryos. (Strong expression of the fli1:nEGFP transgene in pharyngeal arch mesenchyme precludes visualization of endothelial nuclei in the first arch artery and the internal carotid artery/caudal division.) Within this vessel triangle at 2-2.25 dpf, wild-type embryos have 32.9±1.0 (mean±s.e.m., n=12) endothelial nuclei (Fig. 2A), whereas vbg mutants have 69.8±2.6 (mean±s.e.m., n=12) endothelial nuclei (Fig. 2B). This greater than 2-fold increase in endothelial cell nuclei in vbg mutants persisted at 3 dpf, at which time endothelial cell number was statistically unchanged compared to 2 dpf (data not shown).
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Conceptual translation of the largest open reading frame of zehn1109 reveals a 499 amino acid protein with 55% identity to human and mouse Acvrl1 (Fig. 4). Homology to mammalian Acvrl1 is highest (75%) within the C-terminal kinase domain (from L196). Within this domain, the L45 loop (S254-S261), which confers Smad specificity to TGFß type I receptors (Chen et al., 1998), contains only one conservative substitution. In contrast, the N-terminal ligand binding domain encoded by zehn1109 shares only 17% identity with mammalian Acvrl1, most of which can be accounted for by ten cysteines that are common to all TGFß type I receptors. Despite the lack of homology in the ligand binding domain, we strongly believe that this gene is the zebrafish ortholog of mammalian Acvrl1 (see Discussion).
Sequencing of acvrl1 cDNA synthesized from vbgy6 mutant embryos uncovered a point mutation within the kinase domain creating a leucine-to-phenylalanine substitution (L249F; Fig. 4). This leucine, which lies five residues N-terminal to the L45 loop, is conserved in all TGFß type I receptors, although it has not previously been ascribed a specific function. When assayed for this polymorphism, each vbgy6 mutant embryo recombinant at either of the closest flanking genomic markers was shown to possess the mutant genotype. Furthermore, sequencing of the vbgft09e allele revealed a point mutation converting a tyrosine to a stop codon (Y88Stop; Fig. 4). This mutation stops translation within the ligand binding domain and therefore would be expected to produce a nonfunctional protein.
To confirm that mutations in acvrl1 are responsible for the vbg phenotype, we attempted to phenocopy this mutant using morpholino-modified antisense oligonucleotides (morpholinos) (Summerton, 1999). Morpholinos have been used in zebrafish to phenocopy a number of early mutations (Nasevicius and Ekker, 2000
), and their stability allows translation inhibition relatively late in development. When injected into 1- to 4-cell wild-type embryos, a total of 22.5 ng of a control morpholino had no effect on blood flow or vascular architecture at 2.25 dpf (Fig. 5A). In contrast, 22.5 ng of a 2:1 mixture of acvrl1 morpholinos directed against coding sequence (MO1) and noncoding exon1 (MO2) increased cranial blood flow relative to control in 86% (114/132) of embryos at 2.25 dpf. A 15 ng dose of morpholino mixture produced fewer embryos (11/50) with obvious phenotypes. Analysis of a random sample of affected embryos by confocal microangiography revealed a range of phenotypes, from essentially complete phenocopy of vbg mutants (Fig. 5B,C) to focal cranial vessel dilations (Fig. 5D). Affected vessels included the basal communicating artery, posterior connecting segments, basilar artery, and primordial hindbrain channel, each of which can be dilated in vbg mutants.
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The specificity of zebrafish Acvrl1 for the Smad1/5/8 pathway was confirmed in cultured cells. In P19 cells, luciferase transcription from the Smad2/3-responsive A3 promoter is induced by human TGFBR1*, but not human ACVRL1*, human BMPR1B* or zebrafish acvrl1* (Fig. 8A). In contrast, luciferase transcription from the Smad1/5/8-responsive TLX2 promoter is induced by human ACVRL1*, human BMPR1B*, and zebrafish acvrl1* but not by human TGFBR1* (Fig. 8B). These results confirm in vivo findings that zebrafish Acvrl1 signals through Smad1/5/8 and also demonstrate the inability of this receptor to signal through Smad2/3.
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DISCUSSION |
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Although the zebrafish gene residing at the vbg locus is highly homologous to mammalian Acvrl1 in the kinase domain and in particular in the L45 loop, it shows little homology to any TGFß type I receptor in the ligand binding domain: only ten cysteines that are common to all TGFß type I receptors are conserved. However, we feel that it is appropriate to call the vbg gene acvrl1 for a number of reasons. First, human ACVRL1 is located on chromosome 12q13 (Johnson et al., 1996), which shows conserved synteny with the region surrounding the vbg locus on zebrafish LG23 (Postlethwait et al., 1998
). Second, like mammalian Acvrl1 (Johnson et al., 1996
; Oh et al., 2000
; Roelen et al., 1997
; Urness et al., 2000
), the vbg gene is expressed predominantly if not exclusively in endothelial cells, and vbg disruption results in vascular defects. Third, both mammalian Acvrl1 (Chen and Massague, 1999
) and the vbg gene product signal through the Smad1/5/8 pathway, and not the Smad2/3 pathway. And fourth, a lack of homology within the ligand binding domain has also been observed for the zebrafish TGFß type I receptors, Alk8 (Mintzer et al., 2001
; Payne et al., 2001
; Yelick et al., 1998
) and Taram-A (Renucci et al., 1996
). Based on conserved synteny, L45 loop sequence, and Smad specificity, Alk8 and Taram-A seem to be orthologs of mammalian Acvr1 and Acvr1b, respectively.
While the premature stop codon in the ligand binding domain generated by the vbgft09e mutation most likely produces a nonfunctional Acvrl1 protein, the consequence of the L249F substitution produced by the vbgy6 mutation is not self-evident. However, the resulting phenotype, which is indistinguishable from vbgft09e, suggests that L249 is critical for Acvrl1 protein function. Although in vivo assays using an activated form of the vbgy6 allele of acvrl1 suggest that it retains some activity, the artificial activation of this protein, allowing it to function independently of ligand and type II receptor, makes its true in vivo efficacy difficult to gauge. Activity of the non-activated vbgy6 allele could not be assayed because injection of either wild-type or vbgy6 mutant acvrl1 mRNA produced no phenotype in wild-type embryos (data not shown). The lack of effect of injection of mRNA encoding a TGFß type I receptor has been previously reported (Bauer et al., 2001) and suggests that pathway activity is limited by ligand or type II receptor.
The three vbg alleles analyzed in this study exhibit similar vascular phenotypes characterized by dilated cranial vessels that carry the bulk of blood flow. The cause of this vessel dilation is an increase in the number of endothelial cells within affected vessels, suggesting that Acvrl1 signaling may inhibit endothelial cell proliferation and promote vessel stabilization. While there is some in vivo evidence that TGFß family signaling in general (and Acvrl1 signaling specifically) plays a role in vessel stabilization via vascular smooth muscle cell recruitment and differentiation (Larsson et al., 2001; Li et al., 1999
; Oh et al., 2000
; Oshima et al., 1996
; Urness et al., 2000
; Yang et al., 1999
), the dilated first aortic arch, internal carotid artery, and caudal division of the internal carotid artery in vbg mutants normally express the vascular smooth muscle cell marker, sm22
, at 2 dpf (data not shown). These data suggest that in zebrafish, the acvrl1 mutation-induced increases in endothelial cell number and vessel caliber are not correlated with vascular smooth muscle deficits. A similar phenomenon has recently been reported as a result of misexpression of Smad7, an inhibitory Smad that blocks signaling mediated by all receptor-specific Smads (Vargesson and Laufer, 2001
).
The cross-species conservation of acvrl1 expression pattern and function suggests that the mechanism underlying vessel dilation in zebrafish vbg mutants is most likely similar to that underlying vessel dilation - the first step in telangiectasia and AVM formation (Braverman et al., 1990) - in HHT2 patients. Thus, although the enlarged cranial vessels in vbg mutants are not precursors of AVMs per se (the malformations seen in vbg mutants appear to be normal, primitive connections that are aberrantly retained), an understanding of their etiology should lead to insight into the mechanism of HHT2-associated vascular lesions. Although it has been proposed (Urness et al., 2000
) that the proximal event leading to AVMs in an Acvrl1 null mouse is loss of arterial identity, as assayed by loss of arterial ephrinB2 expression, vessel dilation in these mice (E8.0) precedes the onset of normal arterial ephrinB2 expression (E8.25) (Wang et al., 1998b
) and AVM formation (E8.5), suggesting that in mice, too, the earliest manifestation of loss of Acvrl1 expression is vessel dilation. It should be noted that vbg mutants express ephrinB2 normally in the dorsal aorta (data not shown); expression in cranial arteries, which are dilated in vbg mutants, could not be assessed by whole-mount in situ hybridization because of intense staining of other cranial structures.
While it is clear that the genetic lesion responsible for HHT2 is a mutation in ACVRL1, the age of onset, location, and severity of clinical manifestations of this autosomal dominant disorder vary greatly among heterozygous carriers. While it remains possible that loss of heterozygosity plays a role in determining lesion location, this phenomenon could not be demonstrated in lesions associated with the related disorder, HHT1 (Bourdeau et al., 2000a). Thus, in both diseases, additional genetic and/or environmental factors most likely come into play. While preliminary analysis of vbgy6/+ fish has revealed no obvious superficial telangiectases or hemorrhages, it remains possible that such defects might manifest in certain genetic backgrounds [as in Eng haploinsufficiency in mice (Bourdeau et al., 1999
; Bourdeau et al., 2000b
)], or that mutagenesis screening might help to define genetic modifiers that render acvrl1 haploinsufficiency symptomatic in zebrafish as well as in humans. Mutagenesis screens might also produce complementing mutations that may lead to discovery of upstream or downstream components of the Acvrl1 signaling pathway. Furthermore, vbg mutants should prove useful in describing the consequences of acvrl1 deficiency in terms of endothelial cell behavior, as vessel formation can be documented in real time in transgenic lines such as TG(fli1:nEGFP)y7. Thus, like a number of other zebrafish models of human diseases (Brownlie et al., 1998
; Childs et al., 2000
; Wang et al., 1998a
), this zebrafish model of HHT2 provides a powerful tool that should complement established mouse models in the study of disease etiology and the development of treatment strategies.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Barbara, N. P., Wrana, J. L. and Letarte, M. (1999). Endoglin is an accessory protein that interacts with the signaling receptor complex of multiple members of the transforming growth factor-ß superfamily. J. Biol. Chem. 274, 584-594.
Bassing, C. H., Yingling, J. M., Howe, D. J., Wang, T., He, W. W., Gustafson, M. L., Shah, P., Donahoe, P. K. and Wang, X. F. (1994). A transforming growth factor ß type I receptor that signals to activate gene expression. Science 263, 87-89.[Medline]
Bauer, H., Lele, Z., Rauch, G. J., Geisler, R. and Hammerschmidt, M. (2001). The type I serine/threonine kinase receptor Alk8/Lost-a-fin is required for Bmp2b/7 signal transduction during dorsoventral patterning of the zebrafish embryo. Development 128, 849-858.
Berg, J. N., Gallione, C. J., Stenzel, T. T., Johnson, D. W., Allen, W. P., Schwartz, C. E., Jackson, C. E., Porteous, M. E. and Marchuk, D. A. (1997). The activin receptor-like kinase 1 gene: genomic structure and mutations in hereditary hemorrhagic telangiectasia type 2. Am. J. Hum. Genet. 61, 60-67.[Medline]
Bourdeau, A., Cymerman, U., Paquet, M. E., Meschino, W., McKinnon, W. C., Guttmacher, A. E., Becker, L. and Letarte, M. (2000a). Endoglin expression is reduced in normal vessels but still detectable in arteriovenous malformations of patients with hereditary hemorrhagic telangiectasia type 1. Am. J. Pathol. 156, 911-923.
Bourdeau, A., Dumont, D. J. and Letarte, M. (1999). A murine model of hereditary hemorrhagic telangiectasia. J. Clin. Invest. 104, 1343-1351.
Bourdeau, A., Faughnan, M. E. and Letarte, M. (2000b). Endoglin-deficient mice, a unique model to study hereditary hemorrhagic telangiectasia. Trends Cardiovasc. Med. 10, 279-285.[Medline]
Braverman, I. M., Keh, A. and Jacobson, B. S. (1990). Ultrastructure and three-dimensional organization of the telangiectases of hereditary hemorrhagic telangiectasia. J. Invest. Dermatol. 95, 422-427.[Abstract]
Brownlie, A., Donovan, A., Pratt, S. J., Paw, B. H., Oates, A. C., Brugnara, C., Witkowska, H. E., Sassa, S. and Zon, L. I. (1998). Positional cloning of the zebrafish sauternes gene: a model for congenital sideroblastic anaemia. Nature Genet. 20, 244-250.[Medline]
Chen, J. N., van Bebber, F., Goldstein, A. M., Serluca, F. C., Jackson, D., Childs, S., Serbedzija, G. N., Warren, K. S., Mably, J. D., Lindahl, P. et al. (2001). Genetic steps to organ laterality in zebrafish. Comp. Funct. Genet. 2, 60-68.
Chen, Y. G., Hata, A., Lo, R. S., Wotton, D., Shi, Y., Pavletich, N. and Massague, J. (1998). Determinants of specificity in TGF-ß signal transduction. Genes Dev. 12, 2144-2152.
Chen, Y. G. and Massague, J. (1999). Smad1 recognition and activation by the ALK1 group of transforming growth factor-ß family receptors. J. Biol. Chem. 274, 3672-3677.
Childs, S., Weinstein, B. M., Mohideen, M. A., Donohue, S., Bonkovsky, H. and Fishman, M. C. (2000). Zebrafish dracula encodes ferrochelatase and its mutation provides a model for erythropoietic protoporphyria. Curr. Biol. 10, 1001-1004.[Medline]
Detrich, H. W., 3rd, Kieran, M. W., Chan, F. Y., Barone, L. M., Yee, K., Rundstadler, J. A., Pratt, S., Ransom, D. and Zon, L. I. (1995). Intraembryonic hematopoietic cell migration during vertebrate development. Proc. Natl. Acad. Sci. USA 92, 10713-10717.[Abstract]
Dickson, M. C., Martin, J. S., Cousins, F. M., Kulkarni, A. B., Karlsson, S. and Akhurst, R. J. (1995). Defective haematopoiesis and vasculogenesis in transforming growth factor-ß1 knock out mice. Development 121, 1845-1854.
Driever, W., Solnica-Krezel, L., Schier, A. F., Neuhauss, S. C., Malicki, J., Stemple, D. L., Stainier, D. Y., Zwartkruis, F., Abdelilah, S., Rangini, Z. et al. (1996). A genetic screen for mutations affecting embryogenesis in zebrafish. Development 123, 37-46.
Guttmacher, A. E., Marchuk, D. A. and White, R. I., Jr (1995). Hereditary hemorrhagic telangiectasia. N. Engl. J. Med. 333, 918-924.
Hauptmann, G. and Gerster, T. (1994). Two-color whole-mount in situ hybridization to vertebrate and Drosophila embryos. Trends Genet. 10, 266.[Medline]
Hild, M., Dick, A., Rauch, G. J., Meier, A., Bouwmeester, T., Haffter, P. and Hammerschmidt, M. (1999). The smad5 mutation somitabun blocks Bmp2b signaling during early dorsoventral patterning of the zebrafish embryo. Development 126, 2149-2159.
Hukriede, N. A., Joly, L., Tsang, M., Miles, J., Tellis, P., Epstein, J. A., Barbazuk, W. B., Li, F. N., Paw, B., Postlethwait, J. H. et al. (1999). Radiation hybrid mapping of the zebrafish genome. Proc. Natl. Acad. Sci. USA 96, 9745-9750.
Isogai, S., Horiguchi, M. and Weinstein, B. M. (2001). The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development. Dev. Biol. 230, 278-301.[Medline]
Johnson, D. W., Berg, J. N., Baldwin, M. A., Gallione, C. J., Marondel, I., Yoon, S. J., Stenzel, T. T., Speer, M., Pericak-Vance, M. A., Diamond, A. et al. (1996). Mutations in the activin receptor-like kinase 1 gene in hereditary haemorrhagic telangiectasia type 2. Nature Genet. 13, 189-195.[Medline]
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. F. (1995). Stages of embryonic development in the zebrafish. Dev. Dynam. 203, 253-310.[Medline]
Kishimoto, Y., Lee, K. H., Zon, L., Hammerschmidt, M. and Schulte-Merker, S. (1997). The molecular nature of zebrafish swirl: BMP2 function is essential during early dorsoventral patterning. Development 124, 4457-4466.
Knapik, E. W., Goodman, A., Ekker, M., Chevrette, M., Delgado, J., Neuhauss, S., Shimoda, N., Driever, W., Fishman, M. C. and Jacob, H. J. (1998). A microsatellite genetic linkage map for zebrafish (Danio rerio). Nature Genet. 18, 338-343.[Medline]
Larsson, J., Goumans, M. J., Sjostrand, L. J., van Rooijen, M. A., Ward, D., Leveen, P., Xu, X., ten Dijke, P., Mummery, C. L. and Karlsson, S. (2001). Abnormal angiogenesis but intact hematopoietic potential in TGF-ß type I receptor-deficient mice. EMBO J. 20, 1663-1673.
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.
Lekven, A. C., Helde, K. A., Thorpe, C. J., Rooke, R. and Moon, R. T. (2000). Reverse genetics in zebrafish. Physiol. Genomics 2, 37-48.
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.
Lux, A., Attisano, L. and Marchuk, D. A. (1999). Assignment of transforming growth factor ß1 and ß3 and a third new ligand to the type I receptor ALK-1. J. Biol. Chem. 274, 9984-9992.
Lyons, M. S., Bell, B., Stainier, D. and Peters, K. G. (1998). Isolation of the zebrafish homologues for the tie-1 and tie-2 endothelium-specific receptor tyrosine kinases. Dev. Dyn. 212, 133-140.[Medline]
Macias-Silva, M., Hoodless, P. A., Tang, S. J., Buchwald, M. and Wrana, J. L. (1998). Specific activation of Smad1 signaling pathways by the BMP7 type I receptor, ALK2. J. Biol. Chem. 273, 25628-25636.
Maeno, M., Mead, P. E., Kelley, C., Xu, R. H., Kung, H. F., Suzuki, A., Ueno, N. and Zon, L. I. (1996). The role of BMP-4 and GATA-2 in the induction and differentiation of hematopoietic mesoderm in Xenopus laevis. Blood 88, 1965-1972.
Massague, J., Blain, S. W. and Lo, R. S. (2000). TGFß signaling in growth control, cancer, and heritable disorders. Cell 103, 295-309.[Medline]
McAllister, K. A., Grogg, K. M., Johnson, D. W., Gallione, C. J., Baldwin, M. A., Jackson, C. E., Helmbold, E. A., Markel, D. S., McKinnon, W. C., Murrell, J. et al. (1994). Endoglin, a TGF-ß binding protein of endothelial cells, is the gene for hereditary haemorrhagic telangiectasia type 1. Nature Genet. 8, 345-351.[Medline]
McDonald, J. E., Miller, F. J., Hallam, S. E., Nelson, L., Marchuk, D. A. and Ward, K. J. (2000). Clinical manifestations in a large hereditary hemorrhagic telangiectasia (HHT) type 2 kindred. Am. J. Med. Genet. 93, 320-327.[Medline]
Mintzer, K. A., Lee, M. A., Runke, G., Trout, J., Whitman, M. and Mullins, M. C. (2001). lost-a-fin encodes a type I BMP receptor, Alk8, acting maternally and zygotically in dorsoventral pattern formation. Development 128, 859-869.
Mullins, M. C., Hammerschmidt, M., Kane, D. A., Odenthal, J., Brand, M., van Eeden, F. J., Furutani-Seiki, M., Granato, M., Haffter, P., Heisenberg, C. P. et al. (1996). Genes establishing dorsoventral pattern formation in the zebrafish embryo: the ventral specifying genes. Development 123, 81-93.
Nasevicius, A. and Ekker, S. C. (2000). Effective targeted gene knockdown in zebrafish. Nature Genet. 26, 216-220.[Medline]
Neff, M. M., Neff, J. D., Chory, J. and Pepper, A. E. (1998). dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics. Plant J. 14, 387-392.[Medline]
Oh, S. P., Seki, T., Goss, K. A., Imamura, T., Yi, Y., Donahoe, P. K., Li, L., Miyazono, K., ten Dijke, P., Kim, S. et al. (2000). Activin receptor-like kinase 1 modulates transforming growth factor-ß1 signaling in the regulation of angiogenesis. Proc. Natl. Acad. Sci. USA 97, 2626-2631.
Oshima, M., Oshima, H. and Taketo, M. M. (1996). TGF-ß receptor type II deficiency results in defects of yolk sac hematopoiesis and vasculogenesis. Dev. Biol. 179, 297-302.[Medline]
Payne, T. L., Postlethwait, J. H. and Yelick, P. C. (2001). Functional characterization and genetic mapping of alk8. Mech. Dev. 100, 275-289.[Medline]
Postlethwait, J. H., Yan, Y. L., Gates, M. A., Horne, S., Amores, A., Brownlie, A., Donovan, A., Egan, E. S., Force, A., Gong, Z. et al. (1998). Vertebrate genome evolution and the zebrafish gene map. Nature Genet. 18, 345-349.[Medline]
Renucci, A., Lemarchandel, V. and Rosa, F. (1996). An activated form of type I serine/threonine kinase receptor TARAM-A reveals a specific signalling pathway involved in fish head organiser formation. Development 122, 3735-3743.
Roelen, B. A., van Rooijen, M. A. and Mummery, C. L. (1997). Expression of ALK-1, a type 1 serine/threonine kinase receptor, coincides with sites of vasculogenesis and angiogenesis in early mouse development. Dev. Dyn. 209, 418-430.[Medline]
Roman, B. L. and Weinstein, B. M. (2000). Building the vertebrate vasculature: research is going swimmingly. BioEssays 22, 882-893.[Medline]
Rupp, R. A., Snider, L. and Weintraub, H. (1994). Xenopus embryos regulate the nuclear localization of XMyoD. Genes Dev. 8, 1311-1323.[Abstract]
Summerton, J. (1999). Morpholino antisense oligomers: the case for an RNase H-independent structural type. Biochim. Biophys. Acta 1489, 141-158.[Medline]
Urness, L. D., Sorensen, L. K. and Li, D. Y. (2000). Arteriovenous malformations in mice lacking activin receptor-like kinase-1. Nature Genet. 26, 328-331.[Medline]
Vargesson, N. and Laufer, E. (2001). Smad7 misexpression during embryonic angiogenesis causes vascular dilation and malformations independently of vascular smooth muscle cell function. Dev. Biol. 240, 499-516.[Medline]
Wang, H., Long, Q. M., Marty, S. D., Sassa, S. and Lin, S. (1998a). A zebrafish model for hepatoerythropoietic porphyria. Nature Genet. 20, 239-243.[Medline]
Wang, H. U., Chen, Z. F. and Anderson, D. J. (1998b). Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell 93, 741-753.[Medline]
Weinstein, B. M., Stemple, D. L., Driever, W. D. and Fishman, M. C. (1995). gridlock, a localized heritable vascular patterning defect in the zebrafish. Nature Med. 11, 1143-1147.
Westerfield, M. (1995). The Zebrafish Book. Eugene: University of Oregon Press.
Wieser, R., Wrana, J. L. and Massague, J. (1995). GS domain mutations that constitutively activate TßR-I, the downstream signaling component in the TGF-ß receptor complex. EMBO J. 14, 2199-2208.[Abstract]
Yang, X., Castilla, L. H., Xu, X., Li, C., Gotay, J., Weinstein, M., Liu, P. P. and Deng, C. X. (1999). Angiogenesis defects and mesenchymal apoptosis in mice lacking SMAD5. Development 126, 1571-1580.
Yelick, P. C., Abduljabbar, T. S. and Stashenko, P. (1998). zALK-8, a novel type I serine/threonine kinase receptor, is expressed throughout early zebrafish development. Dev. Dyn. 211, 352-361.[Medline]
Yue, J., Hartsough, M. T., Frey, R. S., Frielle, T. and Mulder, K. M. (1999). Cloning and expression of a rat Smad1: regulation by TGFß and modulation by the Ras/MEK pathway. J. Cell Physiol. 178, 387-396.[Medline]