1 Laboratory of Molecular Genetics, NICHD, NIH, Bethesda, MD 20892, USA
2 Department of Anatomy, School of Medicine, Iwate Medical University, Morioka,
Japan
Author for correspondence (e-mail:
flyingfish{at}nih.gov)
Accepted 21 July 2003
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SUMMARY |
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Key words: Zebrafish, Transgenics, Intersegmental vessels, Vascular development
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Introduction |
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The blood vessels of the developing trunk are ideal for studying the cues
and mechanisms guiding vascular patterning during development. The vascular
anatomy of the developing trunk is both reproducible in gross anatomy from
animal to animal, and characteristically conserved in its basic plan with some
species-specific variations (Fig.
1). All vertebrates possess longitudinal axial vessels (dorsal
aorta and posterior cardinal vein) that form by vasculogenesis, or the
co-migration and coalescence of angioblast progenitor cells originating in the
trunk lateral mesoderm to form vessels de novo
(Risau and Flamme, 1995).
There is also a conserved network of secondary vessels including
dorsoventrally aligned intersegmental vessels at the vertical myotomal
boundaries between somites, and longitudinal parachordal vessels to either
side of the notochord. These secondary vessels are believed to form via
angiogenesis, or the sprouting and growth of new vessels from preexisting
vessels, although their formation has not been examined in detail. Secondary
angiogenic trunk vessels form in metameric units along the trunk, making them
ideal for efficient descriptive survey of developmental mechanisms and
well-controlled experimental analysis. In this study we use multiphoton
time-lapse imaging to examine the formation of the trunk angiogenic vascular
network in living Tg(fli1:EGFP)y1 transgenic
zebrafish embryos. We find that these vessels form by a novel two-step
process. Based on our observations, we propose a model for how genetically
programmed assembly of vessel tracts is combined with flow dynamic regulation
of vessel interconnections to assemble a network with both defined and
conserved anatomy and optimized hemodynamic properties. We also discuss
possible broader implications of our results for mechanisms of vascular
network formation.
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Materials and methods |
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Microscopy
Transmission videomicroscopic imaging of zebrafish embryos and larvae was
performed using a Zeiss Axioplan 2 compound microscope equipped with a Dage
SIT-68 camera and an S-VHS recorder. Confocal microscopic imaging of
Tg(fli1:EGFP)y1 zebrafish embryos and larvae was
performed using a Radiance 2000 imaging system (BioRad). Standard confocal
imaging of EGFP (used for some of the single, isolated images) was performed
using the 480 nm laser emission supplied by a Krypton-Argon laser. Multiphoton
imaging of EGFP (used for time-lapse sequences and most images in figures) was
performed using 950 nm pulsed mode-locked laser emission from a tunable
Ti-Sapphire laser (Tsunami laser, Spectra Physics). Time-lapse imaging was
performed with the minimal necessary laser power, and development of imaged
vessels was not significantly delayed compared with the vessels in adjacent
unimaged regions of the trunk.
Embryos were held for time lapse analysis in an imaging chamber prepared from a modified 60 mm petri dish. The embryo medium was prepared with tricaine (0.016%) to inhibit movement of the embryo, and with PTU (0.002%) when non-albino mutant embryos after 3 dpf were imaged, to prevent pigment development. Embryos held in this way maintained heartbeat and robust circulation throughout the imaging period (up to 24 hours). Stacks of frame-averaged (5 frames) confocal optical slices were collected digitally, at 1.67 to 20 minute intervals (as noted) for time-lapse sequences. 2D or 3D reconstructions of image data were prepared using the Lasersharp (BioRad) or Metamorph (Universal Imaging) software packages. The images shown in this paper are single-view 2D reconstructions of collected image z-series stacks, reconstructed at a single angle of zero degrees. 3D reconstructions and raw image stacks of single images, and Quicktime timelapse movie sequences, are available for viewing online at http://dev.biologists.org/supplemental
Quantitative analysis of intersegmental vessel arterial-venous (AV)
identity
Arterial-venous identity was determined for each one of the intersegmental
vessels in each of six different albino zebrafish larvae on days 2, 3, 4, 5, 6
and 7 post-fertilization. These data can be accessed in Tables S1-S6 at
http://dev.biologists.org/supplemental
(follow the `numerical data' link). Assignment of artery or vein identity was
made based on two criteria: first, direction of flow of blood cells transiting
the segment; and second, whether the segment was visibly joined ventrally to
the posterior cardinal vein or to the dorsal aorta. If blood cells could not
be observed transiting through the segment and/or a link to the dorsal aorta
or posterior cardinal vein could not be verified, vessel identity was recorded
as `undetermined' (no entry present). A final `definitive' or `composite'
arterial or venous assignment was made if an intersegmental vessel maintained
a solely venous or solely arterial identity on at least three of these days
and was otherwise of undetermined identity, or if the intersegmental
maintained its identity from day 4 onwards. When intersegmental vessels on
both right and left sides were functioning, AV identity of each was determined
and listed. When only one intersegmental was functioning, the particular side
that vessel was present on could not be definitively determined using the
microscopic assay employed, and the vessel position was listed as unknown. The
definitive or composite vessel identities are listed for each fish in the raw
data tables and are compiled together in Table S7 at
http://dev.biologists.org/supplemental.
Our assignment criteria take into account that intersegmental vessels initiate
blood cell circulation asynchronously, even as late as day 4 or even 5, and
that intersegmental vessels also occasionally temporarily stop carrying blood
cell flow altogether, resuming circulation at a later point. Using the data on
AV identities in these six fish, we quantified AV identity in nearest- or
next-nearest neighboring intersegments to determine the correlation between
vessel identities in spatially juxtaposed intersegments, and also performed a
statistical analysis of the data set (see Tables S8 and S9 at
http://dev.biologists.org/supplemental).
The results of these calculations are presented graphically in
Fig. 8.
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Results |
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Primary angiogenic sprouts emerge from the dorsal aorta
As described previously (Fouquet et al.,
1997), the dorsal aorta condenses as a distinct cord of angioblast
cells at the trunk midline beginning at approximately the 15 somite stage
(16.5 hours), developing an open (patent) lumen by about 28 somites (23 hpf),
with circulation initiating shortly thereafter. In contrast to the axial
vessels, the later-forming intersegmental arteries, intersegmental veins and
parachordal vessels are believed to form by angiogenesis
(Childs et al., 2002
), or the
sprouting and growth of new vessels from preexisting vessels. To determine the
mechanism by which these blood vessels form, we used multiphoton
laser-scanning microscopy (Denk and
Svoboda, 1997
) of living
Tg(fli1:EGFP)y1 zebrafish embryos. Our findings
are summarized schematically in Fig.
8, and detailed in the remainder of this paper.
Beginning at 20 hpf, pairs of endothelial sprouts emerge bilaterally
from the dorsal aorta adjacent to the vertical boundaries between myotomes
(Fig. 2A,B) (Movies 2, 3 at
http://dev.biologists.org/supplemental/).
These sprouts emerge solely from the DA - no sprouts emerge from the posterior
cardinal vein at this stage. We designate these first sprouts `primary' to
differentiate them from later-appearing `secondary' sprouts. The terms
`arterial' and `venous' are not used because the eventual functional identity
of primary vessels can be either (see below). Primary sprouts grow dorsally
between the somites and notochord and then between the somites and neural
tube, tracking along vertical myotomal boundaries
(Fig. 2C-E). They grow in a
saltatory fashion with numerous active, filopodia rapidly extending and
retracting in all directions around the elongating vessels, particularly near
the dorsalmost leading extension (Fig.
2C and Movies 3-5). Processes frequently extend up to tens of
µm and then retract in successive frames of time-lapse sequences collected
at 5 or even 1-3 minute intervals. As growing intersegmental vessels approach
the dorsolateral roof of the neural tube (at approximately 28 hpf) they divide
into two major branches that turn caudally and rostrally
(Fig. 2E,F; Movies 4, 5 at
http://dev.biologists.org/supplemental/),
elongate, and then fuse together with branches from adjacent segments to form
the bilateral dorsal longitudinal anastomotic vessels
(Fig. 2G). By 1.5 dpf, two
completed lattices of endothelial vessels are present on each side of the
trunk (Fig. 2H), composed
entirely of primary intersegmental vessel segments that emerged from the
dorsal aorta. Our observations as well as previous work suggest that each of
the primary segments is composed of three linked endothelial cells (S.I. and
B.M.W., unpublished) (Childs et al.,
2002
). In the completed primary network, endothelial cell bodies
are located approximately: (1) at the DLAV-primary segment junction, (2) at
the level of the parachordal vessels and (3) at the dorsal aorta-primary
segment junction. We have also observed that the formation of the primary
network described here occurs with little or no cell division, with cells
migrating to their positions from the dorsal aorta. There is undoubtedly
additional cell division at later stages of development, but we have not
examined these later stages in detail to observe when and how this occurs.
Dorsally, the two parallel dorsal longitudinal anastomotic vessels are only
sparsely linked by filopodial connections and are essentially distinct and
separate vessels at this stage (Fig.
2I). Throughout this entire period, and often even after
completion of the primary intersegmental vessel lattice, few of these primary
intersegmental vessels have actually formed open lumens. Most remain as cords
or strands of endothelial cells. Where lumens are present, they are usually
found first in the ventralmost regions of the vessels, proximal to the dorsal
aorta (Fig. 2F,G).
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We find that the primary vessel network forms normally in TG(fli1:egfp)y1 embryos mutant for sih which lack blood circulation (Fig. 5A). Primary sprouts emerge from the DA, elongate and branch to form two complete lattices, including two continuous DLAV. The timing and dynamics of primary vessel lattice formation are similar in sih mutant animals and their phenotypically wild-type siblings. Secondary intersegmental vessel sprouts appear at the proper time in mutant animals (Fig. 5B), and, as in wild-type animals, many sprouts contribute to the parachordal system (Fig. 5C). The connection of secondary sprouts to primary segments cannot be definitively assayed in the absence of blood flow, but it is not obviously evident in sih mutants. The formation of additional, supernumerary vessels in the trunks of sih mutants is not observed even at 3 dpf, although enlargement of dorsal regions of the intersegmentals and the dorsal longitudinal anastomotic vessels is observed (Fig. 5D).
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Arterial-venous identity of the intersegmental vessels and
interconnection of the network
The primary vascular network forms in essentially the same manner in every
myotomal segment, but the eventual functional fate of these vessels varies.
Depending on whether or not a functional connection is made to a secondary
sprout, approximately half of the primary segments eventually become part of
intersegmental veins, while the remainder become intersegmental arteries. As
previously noted (Isogai et al.,
2001), the anteroposterior sequence of intersegmental arteries and
intersegmental veins in the zebrafish trunk does not appear regularly ordered
(e.g. artery, vein, artery, vein, artery, vein, etc.). Furthermore, with the
exception of the first five pairs of vessels the arterial or venous identity
of intersegmental vessels along the trunk differs in every individual animal
(Isogai et al., 2001
). To
examine whether the arrangement of intersegmental arteries and intersegmental
veins is in fact random, we performed a statistical analysis of the pattern of
intersegmental arteries and veins in the trunks of six different
embryos/larvae. A detailed description of this analysis and the resulting data
are provided in the Materials and methods section and in the web supplement at
http://dir.nichd.nih.gov/lmg/uvo/ISVdata.html.
This analysis revealed that while the pattern is not regular, it is also not
random. There is a highly significant bias toward preserving hemodynamic
balance between adjacent intersegmental arteries and intersegmental veins (see
web supplement at
http://dir.nichd.nih.gov/lmg/uvo/ISVdata.html,
bottom of the web page). In other words, veins tend to be surrounded by
arteries while arteries tend to be surrounded by veins.
To examine how early patterns of secondary sprout emergence and
interconnection relate to the later AV identity of trunk intersegmental
vessels, we imaged all of the trunk vessels on the left side of two different
embryos (a total of 26 intersegments) at approximately 1.8-2.2 dpf, then
scored the final AV identity of the same intersegmental vessels at 7 dpf. The
results are diagrammed in Fig.
6, and the corresponding images are available at
http://dir.nichd.nih.gov/lmg/uvo/ISVhome.html.
When a secondary sprout forms a root for the parachordal system, the adjacent
primary segment almost always becomes an intersegmental artery. At 2.2
dpf a parachordal root was found adjacent to 10/13 future intersegmental
arteries but only 1/13 future intersegmental veins. The presence of
parachordal sprouts emanating from an intersegmental vessel at the level of
the horizontal myoseptum is also strongly correlated with a venous fate for
that vessel. 12/13 future intersegmental veins were connected to the
parachordal system by 2.2 dpf, whereas only 1/13 future intersegmental
arteries were connected at the same time point. The one exceptional
intersegmental artery (noted with an asterisk in
Fig. 6) possessed only a thin
connection to the parachordal system at 2.2 dpf, and did have an adjacent
parachordal root like other future intersegmental arteries. The results of
this survey suggested that there is a more or less binary fate choice for
secondary sprouts between serving as parachordal roots or connecting to the
primary network, and that this is predictive of future intersegmental
identity. Does this choice depend on flow dynamics?
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Discussion |
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Our studies show that circulatory flow appears to play a minimal role in
the gross anatomical patterning of trunk blood vessels (i.e. the positioning
of vessel tracts relative to other tissues and organs), and in the formation
of the primary vessel network in particular. We used sih mutants to
examine to what extent flow-based cues guide trunk vessel formation and
patterning. sih mutants lack a heartbeat and have no blood
circulation, although they appear normal in most other respects
(Stainier et al., 1996). Gross
anatomical patterning of the early trunk vessels is relatively unaltered in
sih mutants. Primary sprouts emerge, elongate and form bilateral
lattices of vessels in mutants with morphology and kinetics similar to
wild-type embryos. Secondary sprouts also emerge and contribute to parachordal
vessel formation as in wild-type embryos. Previously published reports have
also indicated that subjecting developing zebrafish embryos to hypoxic and
hyperoxic conditions and disrupting hemoglobin transport does not appreciably
alter early trunk vascular patterning
(Pelster and Burggren, 1996
).
These results support the view we have previously put forward
(Weinstein, 1999
) that
`hard-wired' genetic cues play a preeminent role in the defining the overall
anatomical architecture of early, major blood vessels in the trunk, and most
likely other locales as well. The nature of the cues that determine the
pattern of the primary angiogenic vessels of the trunk remains to be
determined.
Although flow dynamics do not appear to strongly influence the gross
anatomical structure of the trunk angiogenic network, they may play a crucial
role in determining and/or refining the pattern of connections between vessels
that allows this network to function properly. Trunk intersegmental vessel AV
identity is not fixed until after secondary sprout emergence and connection.
Primary segments that acquire robust connections to secondary segments become
intersegmental veins, whereas those that do not become intersegmental
arteries. Although there is not a regularly alternating or reproducible
distribution of intersegmental arteries and veins along the trunk, there is a
strong bias toward maintaining a local balance between arterial feed and
venous return in the intersegmental vessel system. The simplest explanation
for how this bias could be generated is that flow dynamics determine this
choice once patent connections begin to be made between primary and secondary
segments and circulation begins. Based on our observations we suggest a model
for determination of secondary sprout fate and intersegmental vessel AV
identity based on four `rules'. First, formation of the primary network and
emergence of secondary sprouts is genetically programmed and fixed, as we have
noted above. Second, secondary sprout connection to primary segments occurs
stochastically. Third, a crucial caveat to the second rule is that blood flow
through a primary vessel segment strongly inhibits the adjacent secondary
segment from connecting to it. We have previously noted for many different
developing vessels that the initiation of blood flow through a developing
angiogenic vessel correlates with a dramatic reduction in its dynamic activity
[see Movie 5 by Lawson and Weinstein
(Lawson and Weinstein, 2002)],
and we have observed the same phenomenon in the primary vascular network (this
work and S.I., unpublished). Fourth, a patent vessel segment with little or no
blood flow will eventually undergo regression. This has also been previously
noted by other investigators in other systems and is likely to be a general
feature of developing blood vessels.
Fig. 8B shows how we propose these four rules act together to generate a hemodynamically balanced intersegmental vessel network. The primary angiogenic network forms in a defined, programmed pattern, as noted above, but there is no circulation through this initial network as it has no venous return route. As it emerges, the first secondary sprout to form a patent connection to a primary segment (segment 1 in Fig. 8B) provides a venous return route for the adjacent primary segments 2 and 2', permitting robust blood to begin flowing through all three vessels (Fig. 8B, part ii). Blood flow through 2 and 2'prevents secondary sprouts from connecting to these segments, `fixing' their identity as arteries. However, little or no blood flows through the more distant segments 3 and 3'as a result of venous flow beginning in segment 1 (S.I., unpublished) so secondary sprouts are able to connect to these segments. Once this connection is made, venous blood flow begins through 3 and 3'as a result of their proximity to 2 and 2' (Fig. 8B, part iii), which in turn initiates arterial blood flow in segments 4 and 4'. Flow through 4 and 4'prevents secondary sprout connection to these vessels and fixes their identity as arterial, as for 2 and 2'. Robust venous flow through segments 1, 3, and 3'reduces blood flow through the (primary) connections these vessels still retain to the dorsal aorta, and with time these connections regress and disappear. In order to generate a strictly alternating pattern of intersegmental arteries and veins throughout the entire trunk, secondary sprout connection would have to initiate at only a single primary segment in the trunk and propagate outward from this point in a temporal wave. This violates the first rule and is contrary to our observations of actual patterns of secondary sprout emergence (data not shown). But, as we have noted, the pattern of intersegmental vessels in the zebrafish trunk is neither regularly alternating nor reproducible from animal to animal, but is biased toward balanced flow as would be the case.
This model can also account for the existence and persistence of `dual-connection' segments (Fig. 7). As secondary vessel connection is stochastic, and does initiate at multiple points throughout the trunk, primary segments will occasionally find themselves surrounded by a relative balance between arterial and venous hemodynamic forces even after a patent connection to a secondary sprout is established. With a dual connection ventrally and no flow dorsally, blood will as a matter of course flow directly from dorsal aorta to posterior cardinal vein as shown in Fig. 7A. If both connections possess robust blood flow, neither one will regress and the dual connection will persist. This state of affairs will continue until shifts in surrounding hemodynamic forces result in initiation of robust arterial or venous flow through dorsal portions of the vessel. This will reduce or eliminate flow through one of the two ventral connections, leading to its regression and to the assumption of a definitive venous or arterial intersegmental identity. Observation of dual-connected segments supports this interpretation. Almost all of these segments lack dorsal blood flow at 2 dpf. Examination of the intersegmental vessels surrounding dual connected segments reveals that in almost every case there is a relative balance of arterial and venous blood flow (data not shown) and that these segments resolve in approximately equal numbers to form intersegmental arteries and intersegmental veins, although this resolution can take an extended period of time..
The two-step model that we have proposed has many appealing features. It
allows for effective interplay between genetically programmed patterning cues
and flow dynamics, ensuring that a vascular network will be both properly
positioned within the context of the embryo as a whole and wired together for
optimal hemodynamic function. It also provides for a remarkably
self-assembling and self-correcting system that ensures venous drainage is
provided for arterial blood vessels. In addition, it is potentially adaptable
to many different vascular beds, as it relies upon simple and widely
applicable properties of developing vessels. There is in fact ample evidence
in the scientific literature to suggest that other vessels beside the initial
trunk network might form by a similar two-step process during development [see
Weinstein and Lawson (Weinstein and
Lawson, 2002) for discussion of additional evidence for sequential
assembly of arterial and venous vascular components]. With the experimental
tools available in the zebrafish, further analysis of trunk vessel formation
should permit the testing of the validity of this model and eventually
elucidate the nature of the genetic and hemodynamic cues that direct vascular
network assembly.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Uniformed Services University of the Health Sciences,
Bethesda, MD 20892, USA
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