Lymphatic origins in the optically clear zebra fish
Two-photon time-lapse imaging shows lineage of lymphatic cells
An important step in tumor progression is the incorporation of blood
vessels to supply the tumor with blood from the host. Researchers are working to
find a way to stop the growth and progression of tumors by interfering with that
step. This requires understanding how vessels develop and grow. Because recent
evidence shows that tumors use the lymphatic system to spread to secondary locations,
knowing the lymphatic vessels’ origin could help in the development of tumor-attacking
drugs as well as in the promotion of healthy blood vessel growth.
The migration and lineage of lymphatic endothelial cells in zebra
fish (shown here) were traced recently using two-photon time-lapse microscopy.
The foundation of the lymphatic system has been
difficult to study because available animal models, such as mice, cannot provide
optical images with high resolution. Brant M. Weinstein and his colleagues from
the National Institutes of Health in Bethesda, Md., and from Iwate Medical University
in Morioka, Japan, therefore tried observing the lymphatic system of the zebra fish
because of its optically clear embryo and because it is easy to breed and yields
good genetic mapping results.
They wanted to be sure that the zebra
fish shares many of the important characteristics of the lymphatic vessels found
in other higher vertebrates. They hoped to trace the migration and lineage of individual
lymphatic endothelial cells by using two-photon time-lapse imaging.
Researchers have discovered that the zebra fish’s lymphatic
vessels are not connected to their blood vessels. The red box (a) shows the region
of the trunk imaged in b-e and g; the blue box shows the region for f. Confocal
images b-d show zebra fish (green) injected with microspheres (red). The dorsal
aorta (large arrow) and posterior cardinal vein (asterisk) in b and d are labeled
with microspheres, but the thoracic duct (small arrow) is not. When the thoracic
duct (small arrow) was injected with microspheres (c), the dorsal aorta (large arrow)
was not labeled. The scientists injected rhodamine dextran into the tail of a zebra
fish (e), and the dye entered the thoracic duct (small arrow) but not the neighboring
dorsal aorta (large arrow). Images f and g show rhodamine-dextran-labeled vessels
(red) between the blood vessels (green).
As reported in the June issue of Nature
Medicine, the researchers obtained confocal images of a 4-day-old postfertilization
zebra fish using a Bio-Rad confocal imaging system. They used fli1:EGFP transgenic
zebra fish in which the endothelial cells of vessels had been tagged with enhanced
The images showed the obvious two blood
vessels in the trunk — the dorsal aorta and the posterior cardinal vein —and
revealed an additional thin vessel between the two blood vessels, where the thoracic
duct, a lymphatic vessel, is found in other vertebrates.
To ascertain whether the third vessel
was lymphatic, the researchers examined whether the tube expressed lymphatic genes
such as those found in mammals. They found that two were expressed in the third
vessel. For further examination, they injected inhibitors for genes essential for
lymphatic formation in mammals. As expected, the third vessel in the zebra fish
was completely lost, while the blood vessels appeared normal.
They also wanted to determine whether
zebra fish keep their lymphatic and blood vascular systems separate, as do other
higher vertebrates. They injected 0.02-mm fluorescent microspheres into the zebra
fish’s blood vascular system and found that the microspheres did not show
up in the thoracic duct and, when injected directly into the duct, did not enter
the blood vessels. These results proved that the blood vessels and lymphatic vessels
are not connected.
Because the lymphatic system lacks
red blood cells, the investigators used another transgenic zebra fish in which red
blood cells were tagged with the fluorescent protein DsRed. They bred this fish
with the fli:EGFP fish to generate a red/green double transgenic line of
fish. Two-photon microscopy revealed the red blood cells in the zebra fish’s
blood vessels, but not in the potential thoracic duct. Even after 24 hours of imaging,
no red blood cells entered the third vessel, indicating that it was probably a lymphatic
And as a final test of its similarity
to other vertebrates, the researchers injected rhodamine-dextran, which is known
to concentrate in lymphatic vessels, into the tail of a zebra fish. Within minutes,
the dye entered the thoracic duct but was never detected in the adjacent blood vessels.
Once they were satisfied that the zebra
fish could be used as an effective model for studying lymphatic development, they
sought the origins of this system by long-term multiphoton time-lapse imaging of
transgenic fish. According to Weinstein, standard confocal imaging creates too much
photodamage to the samples and doesn’t allow the vessels to develop properly
in the long time-lapse imaging experiments. And other imaging techniques could not
provide sufficient tissue penetration, even in these fairly clear animals.
They placed the fish inside a continuous-flow
home-built chamber to allow oxygenated water to flow throughout without moving the
zebra fish embryos during the long-term imaging. The entire microscope stage was
then encapsulated in a black plastic box created for the experiment. Weinstein said
the box allowed them to lower the emission and increase the sensitivity of their
imaging system without encountering too much background noise.
Using 960-nm two-photon time-lapse
imaging of fli1:EGFP transgenic zebra fish with pulsed laser emission from
a Ti:sapphire laser manufactured by Spectra-Physics, they collected image stacks
every 10 minutes with 60 to 80 planes per stack at a spacing of 2 μm. They
discovered that the thoracic duct emerges adjacent to the dorsal aorta and grows
across the fish’s trunk to join with other segments.
Two-photon time-lapse imaging of zebra fish shows a lymphatic sprout (red) emerging next to
the dorsal aorta (a) and the lymphatic vessel growing across the fish’s trunk
(b). Reprinted from Nature Medicine with permission of the researchers.
In another image sequence, they discovered
the source of lymphatic endothelial cells for the thoracic duct. They imaged transgenic
zebra fish with previously transferred vascular promoter genes, which helped direct
GFP to endothelial cell nuclei, and collected long time-lapse sequences (three or
more days) of the zebra fish.
They traced any nucleus found in the
thoracic duct at the end of the sequence backward in time to discover its origin
and found that the lymphatic endothelial cells came from the parachordal vessel,
a vascular vessel that emerges from the posterior cardinal vein. These results indicate
that primitive lymphatic vessels have a venous origin, as first proposed more than
100 years ago but not directly visualized.
Weinstein believes that the zebra fish
may be a good source for further studies of the lymphatic and blood vascular systems
as well as for possible research in drug therapy. To further their understanding
of how vessels develop, the investigators plan to explore with two-photon time-lapse
imaging the process of how lumens (the cavities within blood vessels) form.
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