First-Ever Recording Of Blood Vessel Development During Organ Formation
A new microscope system that can take 3-D pictures of an embryonic mouse organ over 24 to 48 hours has shown Duke Medical Center researchers the first glimpse of the formation of blood vessels during development.
Among other things, a team lead by cell biologist Blanche Capel, Ph.D., has found a previously unknown mechanism in the formation of blood vessels that may help scientists better understand how a tumor rallies a blood supply to its aid.
Using mice that have blood vessel cells marked by green fluorescence, the Duke University cell biologists studied vessels that supply mouse gonads. These are the embryonic organs that give rise to ovaries or testes later in development.
The team studied gonads because they could remove and culture the gonad along with the nearby tissue that initially houses the major blood vessels. This way they could watch how the blood vessel system (vasculature) develops as the gonad changes into a testis or ovary.
The scientists' novel system for studying development using time-lapse microscopy and tiny samples of tissue shed new light on the dynamic process of organ formation. This system answered key questions about how the vasculature gets fitted into the organ as it forms, Capel said. Before this, scientists could only image one point in development at a time.
The striking new images became the cover story of the Proceedings of the National Academy of Sciences and were assembled into a time-lapse movie.
The research was funded by grants from the National Institutes of Health Heart, Lung and Blood Institute and the Lalor Foundation.
The Duke team was surprised by the vigorous cell movements involved in the development of male gonads. "In the male gonad, the major blood vessel in the adjacent tissue comes apart and the individual blood vessel cells move to a new location, and reassemble into new vessels inside the testis," Capel said. "This breakdown process represents a possible way for growing tumors to access a blood supply, by commandeering a mechanism similar to the ones organs use to recruit vessels into the tumor."
She pointed out that a blood supply is critical to a growing tumor, and this may be an important mechanism in the formation of blood vessels in tumors that scientists have not appreciated before. "That is an exciting finding," Capel said.
This imaging in 3-D over time was possible because Capel's laboratory already had developed a culture system for studying the organ in the lab. "We were positioned to convert that to a live imaging system when advances in microscopy became available at Duke University Medical Center," Capel explained. "The Duke Department of Cell Biology has an imaging facility that is really outstanding, and our chair, Brigid Hogan, has put a lot of energy into making sure it is state of the art. One of the authors on this paper, Tim Oliver, who manages this facility, helped us to get the imaging set up."
The organs were placed in small wells in an agar block designed to hold them still. The entire system was enclosed in a humidified and temperature-controlled chamber around the microscope. Scientists captured an image every 20 minutes for 24-48 hours, then later assembled the images in sequence to make movies.
It wasn't easy, Capel said. "We had to work a lot of kinks out of the system. For example, we were exposing the organ to a laser to detect the fluorescent vascular cells throughout the duration of the culture. But too much laser light damages cells. You need to create a bright enough fluorescence in the cells so that you don't have to turn the laser on such a high setting that it kills cells during the culture period."
This success with recording the growth of blood vessels has spurred the Capel lab team on to new projects. "Our goal now is to have different colored fluorescent markers for other types of cells in the organ. I hope we can simultaneously image the vessels and other cells as the vessels move into the organ, so we can see how they interact together as a functional organ is forming."