Lymphatic origins in the optically clear zebra fish

Raquel Harper

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 GFP.

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 vessel.

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.