Cardiovascular Research Center, Massachusetts General Hospital, and Harvard Medical School, 149 13th St. Charlestown, MA 02129, USA
* Present address: Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary AB, Canada, T2N 4N1
Present address: Department of Molecular, Cell and Developmental Biology, UCLA, Los Angeles, CA 90095, USA
Author for correspondence (e-mail: fishman{at}cvrc.mgh.harvard.edu)
Accepted 16 November 2001
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SUMMARY |
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We have performed lineage analysis, using laser activation of a caged dye and mosaic construction to determine the origin of cells that constitute the ISV. Individual angioblasts destined for the ISVs arise from the lateral posterior mesoderm (LPM), and migrate to the dorsal aorta, from where they migrate between somites to their final position in the ISVs and dorsal longitudinal anastomotic vessel (DLAV). Cells of each ISV leave the aorta only between the ventral regions of two adjacent somites, and migrate dorsally to assume one of three ISV cell fates. Most dorsal is a T-shaped cell, based in the DLAV and branching ventrally; the second constitutes a connecting cell; and the third an inverted T-shaped cell, based in the aorta and branching dorsally. The ISV remains between somites during its ventral course, but changes to run mid-somite dorsally. This suggests that the pattern of ISV growth ventrally and dorsally is guided by different cues.
We have also performed an ENU mutagenesis screen of 750 mutagenized genomes and identified one mutation, obd that disrupts this pattern. In obd mutant embryos, ISVs sprout precociously at abnormal sites and migrate anomalously in the vicinity of ventral somite. The dorsal extent of the ISV is less perturbed. Precocious sprouting can be inhibited in a VEGF morphant, but the anomalous site of origin of obd ISVs remains. In mosaic embryos, obd somite causes adjacent wild-type endothelial cells to assume the anomalous ISV pattern of obd embryos.
Thus, the launching position of the new sprout and its initial trajectory are directed by inhibitory signals from ventral somites. Zebrafish ISVs are a tractable system for defining the origins and fates of vessels, and for dissecting elements that govern patterns of vessel growth.
Key words: Zebrafish, Angiogenesis, Vascular pattern
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INTRODUCTION |
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Several growth factors have been shown to be crucial for normal angiogenesis in the embryo. Vascular endothelial growth factor, VEGF, is expressed in the vicinity of sprouting vessels, and its receptor (VEGF-R2/Flk-1/kdr) on the angioblasts and new vessels, and both are required for vasculogenesis and angiogenesis (Leung, 1989; Keck, 1989
; Shalaby, 1995
; Carmeliet, 1996
; Ferrara, 1996
). Angiopoietin 1 and angiopoietin 2, and their receptor Tie2 are reciprocally expressed in surrounding mesenchyme and early vessels, and appear to function in vascular remodeling and stabilization (Dumont, 1994
; Maisonpierre and Radziejewski, 1997
; Sato, 1995
; Suri et al., 1996
). Several EphB receptors and ephrinB ligands are expressed, some with arterial versus venous specificity, both on vessels and in surrounding tissues, and have been shown to be required for remodeling of angiogenesis (Adams, 1999
; Wang, 1998
).
In the zebrafish, cells in the lateral posterior mesoderm express endothelial and hematopoietic markers, suggesting they include bipotential precursors for both the hematopoietic and angioblastic lineages, termed hemangioblasts (Fouquet, 1997; Stainier et al., 1995
). By lineage tracking we have previously shown that the LPM includes precursors for the dorsal aorta and posterior cardinal vein (Zhong et al., 2001
).
We have focused on the origin and pathway guidance of the ISVs, vessels that connect the dorsal aorta to the DLAVs. The ISVs are particularly interesting because their pattern appears to be established primarily, unlike other vessels such as those of the yolk sac, brain or retina, which first form in a honeycomb-like plexus to be remodeled subsequently into more organized regularly branching larger and smaller vessels (Suri et al., 1996; Benjamin, 1998
). Thus, it appears that for ISVs there might be designated sites for branching and initial pathway guidance. By flk-1 labeling, ISVs appear to sprout from the aorta, beginning at the 24 somite stage (Fouquet, 1997
). In mutants that lack the dorsal aorta, such as the zebrafish mutant floating head, intersegmental arteries fail to form. No sprouts are observed in mutants that fail to undergo tubulogenesis of the aorta, such as sonic-you. However, sprouts form normally in the one eyed pinhead mutant, which has an artery but lacks a vein (Brown et al., 2000
). Thus, a well-formed aorta appears to be a prerequisite for ISV formation. It is not clear what guides the initial positioning of the sprouts. VEGF is needed for the outgrowth to occur: zebrafish exposed to low-dose VEGF morpholino antisense establish an aorta but not ISVs (Nasevicius et al., 2000
). The source of angioblasts for the ISVs has been examined in chick, where tissue transplants have suggested derivation from both the splanchnopleural mesoderm and the roof of the dorsal aorta (which is somitically derived) (Pardanaud, 1996
).
We have examined by lineage analysis, the source, cell fates and migratory trajectory of angioblasts that constitute the ISVs. Additionally, we have performed a genetic screen to identify mutations perturbing primary vascular pattern formation.
We find that the angioblasts for zebrafish ISVs arise in the lateral posterior mesoderm. Each sprout is fashioned from three cell types, all of which originate in the aorta and are deployed in a carefully choreographed manner. One cell remains ventrally in the aorta and sprouts a dorsally directed process, resembling a plumbers T junction; a second cell is a mirror image of this, based dorsally in the DLAV; and a third cell generates the tube in between. At no time do cells or sprouts of the sprout extend beyond the intersegmental boundaries, suggesting that there are strong repulsive guidance signals. One of these signals controls the site of take-off of the sprout, as revealed by the mutation, out of bounds, in which all angiogenic cell fates are generated, but sprout launching and ISV growth are not restricted to the intersegmental region.
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MATERIALS AND METHODS |
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Mosaic transgenics
A construct encoding the mouse tie-2 promotor and enhancer (Schlaeger, 1997) was digested with SalI and injected into single-cell stage wild-type and obd mutant embryos at a concentration of 200 ng/µl. Embryos were examined between 24 and 46 hours post fertilization (hpf). For sectioning, embryos were fixed in 4% PFA/PBT, permeabilized with cold methanol/acetone, and stained with a 1/2000 dilution of JL-8 antibody (Clontech). Staining was visualized with a goat anti-mouse Alexa Fluor 568 antibody (Molecular Probes).
Genetic screening and embryo staining
The screen for recessive ENU-induced mutants in TopLong fin strain zebrafish has already been described (Chen et al., 2001). To identify vascular patterning mutants, endogenous AP activity was assayed as before. To assay motoneuron pattern, a 1/300 dilution of znp-1 supernatant (Developmental Studies Hybridoma Bank) (Trevarrow et al., 1990
) was applied to embryos after fixation in 4% PFA and permeabilization in methanol acetone. Staining was visualized by diaminobenzidine (Vectastain). fli1, tie1, delta D, fgf8 or vegf expression (Brown et al., 2000
; Haddon et al., 1998
; Liang et al., 1998
; Lyons, 1998
; Reifers et al., 1998
) was visualized by in situ hybridization with digoxigenin labeled probe as described (Jowett and Lettice, 1994
). Signal was detected with an alkaline phosphatase-conjugated anti-digoxigenin antibody, and reaction with NBT/BCIP (Boehringer Mannheim).
Microangiography
Microangiography was performed as described (Weinstein et al., 1995) with the exception that 0.2 µM yellow-green polystyrene Fluospheres (Molecular Probes) in 1% bovine serum albumin (BSA) were sonicated for 2 minutes at full power before injection into the sinus venosus.
Transplantation
Dechorionated one-cell donor embryos were injected with tetramethylrhodamine biotin dextran (Molecular Probes). Cells from the animal pole were transplanted to the margin of mutant recipient embryos at the sphere stage of development, as this gave high numbers of transplanted cells in the somite.
Morpholino treatment
Dechorionated one-cell wild-type and mutant embryos were injected with VEGF morpholino antisense oligonucleotideA-1 (5'GTATCAAATAAACAACCAAGTTCAT-3') described previously (Nasevicius et al., 2000). Stock concentrations from 150 µM to 1 mM were injected.
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RESULTS |
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ISVs are arranged in an extremely regular array. In the medial-lateral plane, the ventral portion of the ISVs runs between the notochord and somite, and the dorsal portion between the neural tube and somite boundaries (Fig. 1A). The launching site of the ISV is in a small triangle of space bounded by two somites (Fig. 1C). Ventrally, the ISVs follow the chevron shape of the somite. More dorsally, when adjacent to the neural tube, the ISVs no longer run between somite borders, and rather adopt a straight course to the DLAV (Fig. 1B). In the vicinity of the notochord we do not observe cells or their processes outside of boundaries defined by the somite borders. The initiation of the angiogenic sprout, the processes of the cell, and at least the ventral part of the migratory path all appear to be constrained by somite borders.
In order to define the cellular architecture of the ISV, we labeled the angioblastic lineage with GFP under control of a tie-2 promotor, previously shown to be expressed in zebrafish endothelial cells (Motoike et al., 2000). We find that transient expression of the construct labels cells in a highly mosaic fashion, allowing analysis of individual cells. By examining mosaic embryos repetitively over 20 hours, we tracked the migration of individual angioblasts (Fig. 2). Beginning at 25 hpf, scattered GFP-expressing angioblasts are evident in the axial vessels and began to send projections dorsally into the somitic region. We followed the migration and fates of these cells as they assume positions in the ISVs and DLAV.
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Angioblasts for all components of the angiogenic sprout arise in the lateral posterior mesoderm
We wished to identify the origin, migratory path and cellular characteristics of the angioblasts that form the ISVs. Previously we have shown that precursors for the dorsal aorta and posterior cardinal vein can be labeled by uncaging a caged fluorescein marker in cells of the LPM using a laser (Zhong et al., 2001). To identify ISV precursors, we focused the laser to label five to ten cells of the LPM (Fig. 3A). We activated the LPM at different somite levels from anterior to posterior, mediolaterally, and in embryonic stages from seven somites up to 12 somites. We observed that only a small proportion of labeled cells were angioblasts, and most LPM cells contributed to other tissues, notably pronephric duct and blood.
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There is clearly an anteroposterior pattern in the LPM that is reflected in the ultimate vascular destination. Progeny assume positions in the vessels at approximately the same anteroposterior level as the LPM precursors that were activated, and they contribute to the vasculature between only one to two pairs of somites. The same three types of progeny are revealed regardless of the anteroposterior level, whether activated adjacent to the first or to the last-formed somites of a ten somite embryo. However, as the embryo develops to ten somites and beyond, angioblasts are more commonly revealed after labeling in the posterior regions of the LPM, suggesting that there is an anteroposterior gradient in the initiation of medial migration of angioblasts, such that the angioblasts adjacent to the more mature anterior somites migrate slightly earlier. This anterior to posterior maturation program is also suggested tie1 expression at the 12- to 14-somite stages (data not shown).
There does not appear to be a left-right preference in the tracking of LPM angioblasts. DLAVs and ISVs are labeled ipsilateral and contralateral to the LPM activation side. Labeled cells in the DLAV are also observed on both left and right sides (Fig. 3D). As these cells transit through the midline dorsal aorta before sprouting to form the derivatives, left-right laterality may be lost at the midline. Similar contralateral labeling of ISVs has also been observed in quail-chick transplantation studies (Ambler et al., 2001), although it has not been seen in other studies (Wilting, 1995
; Pardanaud et al., 1996
). By contrast, labeling of pronephric duct is exclusively limited to the side of the LPM labeling (Serluca and Fishman, 2001
) (this study).
In chick, the roof of the aorta and the ISVs have been shown to be mosaic, being derived from splanchnopleural mesoderm and somite when evaluated by quail-chick transplantation (Pardanaud et al., 1996). We labeled lateral, central and medial somite with the laser at the 11 somite stage of development. Although the somites were strongly labeled, we did not observe any endothelial cells deriving from labeled somite (n=0/63, data not shown). Thus the development of the ISV vasculature in zebrafish may be different from that of the chick, and appears to be exclusively derived from the LPM.
The sub-intestinal venous vessels (SIVs) we find to be outgrowths of the posterior cardinal vein. The SIVs develop in the ventral trunk of the embryo underneath the somites and grow over the yolk. They are spatially restricted to a segment of anterior trunk. By using the laser, LPM activation at the six somite level, which labeled the vein, also resulted in labeling of projections spreading over the yolk where they will form the SIVs (n=8/8).
The pattern of angiogenic vessels is abnormal in out of bounds
We performed a genetic screen to identify mutants with defects in vessel patterning (Chen et al., 2001). From 750 mutagenized genomes, we isolated one vessel patterning mutation, termed out of bounds (obd). This is a recessive and fully penetrant mutation with two non-complementing alleles (fs31-l and fv109-k). The mutation is embryonic lethal in more than 80% of homozygotes. However, some homozygous embryos do survive to adulthood and are fertile.
obd mutant embryos appear normal until midway through somitogenesis. At this time, around the 17-18 somite stage, the aorta of obd mutant embryos launches precocious sprouts, as observed by staining with fli1 (Fig. 4A). Sprouts in wild-type embryos do not appear until the 24S somite stage (Fouquet et al., 1997) and thus obd ISVs develop more than 3 hours in advance of the normal schedule. These precocious sprouts originate from abnormal positions along the aorta and traverse domains normally forbidden to sprouts in wild-type embryos. Interestingly, there is still approximately one vessel per somite in obd. Thus, neither their site of origin nor path of migration is restricted to the intersegmental space. By 24 hpf, the ISV sprouts of obd embryos appear chaotic compared with wild type, particularly in their ventralmost region. The dorsal portion of the ISVs appears far less affected, tending to run along a more-or-less straight path to the DLAV. obd ISVs are patent, and begin to carry blood flow at the same time as wild-type embryos. The pattern of vessels is highly disorganized, however (Fig. 4B): in transverse section, the ISVs may be found in any position along the AP axis of the somite (Fig. 4C).
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VEGF is normally expressed segmentally in the ventral-medial somite, adjacent to the notochord. The expression of VEGF in obd is normal (Fig. 8A). We find that injection of VEGF antisense morpholino into wild-type embryos does not diminish the apparent number of angioblasts, as assayed by tie1 in situ hybridization. It does, however, as noted previously (Nasevicius et al., 2000), completely inhibit ISV formation until 48 hpf. We find that there are several explanations for this effect. First, angioblast migration from the LPM to the midline is impaired (Fig. 8B). Although delayed, cells do eventually migrate to the position of artery and vein by the 19-somite stage. This dose completely prevents the formation of ISV sprouts in wild-type embryos. Additionally, precocious migration of angioblasts in obd is completely inhibited (Fig. 8C). Interestingly, obd sprouting is more sensitive to VEGF than is wild type and the suppression of precocious sprouting is evident at very low doses (as low as 150 µM).
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DISCUSSION |
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The lineage and migration of angioblasts
The lateral posterior mesoderm of the zebrafish provides angioblasts for all major vessels of the trunk, including the aorta and vein, which form by vasculogenesis, and the ISVs, SIVs and DLAVs, which form by angiogenesis. In the chick, transplantation experiments suggest that cells from the somite also provide angioblasts, subsequently populating the roof of the aorta and the ISVs (Wilting, 1995; Pardanaud et al., 1996
; Ambler et al., 2001
). DiI-labeled sclerotome cells in zebrafish also contribute to both aorta and ISVs (Morin-Kensicki and Eisen, 1997
). While not ruling out the possibility of a mosaic origin of trunk vessels in the zebrafish, in our experiments it is clear that the majority of the trunk vasculature is derived from cells of the LPM, via the axial vessels. It is certainly possible that there is a species difference in source of angioblasts. Among these various methods, the advantage of laser uncaging is its ability to label only a few cells with great precision.
There is a strong correlation between the anteroposterior position of a precursor in the zebrafish LPM, and that of the ISV site. This also is different from the long distances migrated by angioblasts in the chick after they are transplanted (Pardanaud et al., 1996). It is possible that the physical manipulations of transplantation may disturb some normal constraints, or it may be that somitically derived angioblasts in the chick are more invasive than LPM-derived angioblasts.
The generation of the ISV is by carefully choreographed cell migration. In our model (Fig. 9), the first cell to leave the aorta migrates dorsally between the somites, and at the DLAV assumes a T shape, with the junction becoming part of the DLAV. The next cell migrates between the somites until reaching the stem of the T, when it stops and seems to form a tube. The direct connection to the aorta is formed last, by extension of an inverted T stem, a mirror image of the cell at the DLAV. Using a mosaic transgenic marker, single labeled cells assume positions in the DLAV, ISVs or aorta without their neighboring cells being labeled. This strongly suggests that these sprouting vessels of the trunk are formed by migration of cells from the aorta with minimal or no proliferation. Hence, despite the fact that the ISV is generated as a sprout, it seems more appropriate to describe this process as vasculogenesis type II, meaning that there is migration of angioblasts followed by tubular formation, rather than using the traditional definition of angiogenesis, which includes cellular proliferation as well as migration (Risau, 1997). A similar vasculogenic type II origin of ISVs has been recently been suggested in a chick chimera model (Ambler et al., 2001
). This may be an important distinction, in that growth factors, such as VEGF, enhance cell division as well as directed migration, while others, such as angiopoietin 1, affect migration without inducing cell division (Koblizek et al., 1998
).
It is notable as well that regions of the ISV are reproducibly configured by endothelial cells with distinctive shapes. These are effectively different cell fates. How do they arise? It seems reasonable to speculate that during the ventral-to-dorsal migration, angioblasts encounter distinct signals, or different levels of a signal arrayed in a morphogenetic gradient that dictates the fate of the individual cell and the shape of the vessel. Perhaps also the establishment of contact with other angioblasts already in position in the nascent ISV would inhibit migration of further angioblasts. The position of nuclei in the ISV also appears stereotypical and may reflect guidance constraints.
obd is part of a patterning system for ventral ISV
What signals could direct ISV patterning? Hypoxia is detected via oxygen-sensitive elements in the HIF1 gene, and induces expression of VEGF and subsequent angiogenesis (Shweiki et al., 1992
). However, hypoxia cannot account for the exquisite patterning of ISVs. The zebrafish embryo does not depend upon circulation for oxygen delivery, and mutants without blood flow have a trunk vessel pattern that is indistinguishable from wild-type (F. Senluca and M. C. F., unpublished).
VEGF is, however, likely to be crucial to vasculogenesis, in supporting the migration of angioblasts from the LPM to form the dorsal aorta (Cleaver and Krieg, 1998). VEGF is also segmentally expressed in the trunk during somitogenesis and is clearly important to sprouting. Mice hemizygous for VEGF have few or no ISVs despite having an aorta (Carmeliet et al., 1996
; Haigh et al., 2000
). In zebrafish, morpholino antisense blocks ISV formation (Nasevicius et al., 2000
). Nonetheless, there is little evidence for a role for VEGF in patterning (Flamme et al., 1995
), and VEGF is expressed normally in obd. Additionally, we have shown that inhibition of VEGF translation during vessel sprouting does not affect pattern formation.
Angiogenesis, including of the intersomitic vessels, is abnormal in mice mutant for ephrinB2, EphB2/EphB3 or Eph B4 (Adams et al., 1999; Wang et al., 1998
; Gerety et al., 1999
). Overexpression of dominant negative EphB4 in frog also results in an ISV patterning defect (Helbling et al., 2000
). The ISV phenotype does not affect the localization of sprouting, and has been attributed to perturbation of remodeling rather than of initial patterning.
One gene that is crucial to the patterning per se is obd. We have examined some candidate genes for linkage to obd (ephrinA2, ephrinA5a, ephrinA5b, ephrinB2a, Eph zek1, Eph zek2, Eph zek3, Eph rtk4, Eph rtk5, Eph rtk6/EphA2/eck, Eph rtk7, Eph rtk8, tie1, tie2, angiopoietin 1, angiopoietin 2, vegf, flk1 and flt4; S. C., J. D. Mably, J. Chan and M. C. F., unpublished), but none of these genes is linked. Of course, this does not rule out the possibility that obd is an unmapped member of the ephrin family. Mutation of obd does not appear to affect generation of the normal aorta or posterior cardinal veins. Nor does it interfere with its ability to generate a sprout, to grow from aorta to DLAV or to become a functional vessel. Rather, it specifically perturbs the site of origin of the sprout along the aorta and the subsequent direction of growth. Normally the sprout is constrained to a region between the somites. Sprouts in obd grow in the same plane as in wild type, abutting either notochord or neural tube, but cross somite borders. This is not due to the accompanying precocity of sprouting, as sprouting can be delayed by VEGF antisense morpholinos while the pattern of vessels remains aberrant. Taken together, the precocity of sprouting, the abnormal launching site and the aberrant path of angiogenesis, all indicate that obd is part of a normal inhibitory system that guides vessel patterning along its ventral course. It is not clear what changes occur more dorsally, where ISVs normally no longer obey somite boundaries and where obd appears to be less crucial, The position of change in ISV pathway correlates with the notochord-neural tube interface, so it is conceivable that these tissues generate different guidance signals, either directly or via the somite. It is interesting that we have also discovered a mutation, fv055b, that causes increased dorsal branching of the ISV, but does not affect ventral pattern (S. C., D. M. G. and M. C. F., unpublished).
obd is also a key temporal regulator of migration. Normally, it appears to counter a VEGF-induced tendency to sprout at early stages. VEGF is expressed 3.5 hours before sprouting normally begins, and the precocious sprouting in obd mutant embryos can be suppressed by VEGF morpholinos. This balance of angiogenic promoting and inhibiting molecules is also important in determining tumor angiogenesis (Holash et al., 1999). It will be of interest to determine whether sprouting of vessels in other locales, both normal and pathological, generate vessels by accumulation of cells with distinctive fates, and how local guidance cues determine the site of sprouting.
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ACKNOWLEDGMENTS |
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