Giant bubbles protect fish from scoliosis

Sea squirts, fish and mammals don't look much alike, but glimpse at their embryos and you probably couldn't tell them apart. Among other similarities, all sport a tube-like structure stretching from head to tail - the notochord - that serves as a backbone, before being replaced by the spine. New research now shows that mysterious bubble-like structures in the notochord are critical to make a straight spine.

In the centre of the notochord there are unusual cells packed with huge fluid-filled vacuoles (a sort of bubble) that press against a fibrous elastic wrapping. This makes the notochord stiff but also flexible, much like the sausage-shaped balloons used to make balloon animals. A tube like this can come handy when you don’t have a bony spine, for instance if you are a wiggly sea squirt larvae swimming away from a hungry predator.

Zebrafish embryo with the notochord marked by a green fluorescent protein.
(Credit: Kathryn Ellis/ Duke University Medical Center) 

Even though the notochord vacuoles were first described decades ago, they have remained a bit of a mystery. Michel Bagnat and colleagues at Duke University Medical Center are interested in understanding how these strange structures form, and what they are for. “The dogma was that the notochord is an evolutionary relic that falls apart and is replaced by the spine […] so there was not much interest,” Bagnat says. In vertebrates such as humans, the notochord eventually turns into the jelly that fills up the space between the spine vertebrae. Because it seemingly ‘disappears’, many scientists had brushed aside the possibility that the notochord might have other important functions besides its well-known roles in early embryogenesis (in making muscle and nerve cells for instance).

Bagnat’s team took advantage of the zebrafish (Danio rerio) photogenic qualities to study the notochord vacuoles. Zebrafish is a small aquarium fish widely used in research because it is transparent and grows fast, so it is possible to image cells and tissues developing live throughout embryogenesis. By making movies of zebrafish embryos, Bagnat and his colleagues Kathryn Ellis and Jennifer Bagwell showed in a new study how the notochord vacuoles start off as small bubbles, then fuse together and finally inflate until they fill up the cell. The researchers then used fluorescent markers to disclose the identity of the vacuoles. They discovered they are related to lysosomes - the cell’s rubbish bins where unwanted molecules and foreign invaders like viruses are destroyed - but have a completely different function. So what do the notochord vacuoles do?

Notochord vacuoles marked with a lysosome-specific protein (green).
(Credit: Kathryn Ellis/ Duke University Medical Center) 

Earlier studies with dissected frog notochords suggested that, as the vacuoles swell up, they somehow force the embryo to elongate, but these experiments had caveats because the right tools simply didn’t exist at the time. Bagnat’s team was now in a good place to test this in living fish. Disrupting the vacuoles (without affecting the rest of the notochord) using genetic tricks produced dwarf embryos, confirming the old study in frogs. However, something unexpected happened to these embryos. “We tried to raise those very short larvae and we saw that they developed kinks in the spine,” Bagnat says. This surprising finding showed that, contrary to what was previously believed, the notochord has a function later in development in making a straight spine. These fish without notochord vacuoles had the equivalent to human scoliosis, a spine deformity of unknown cause that affects over 2% of the adult population. But how do the vacuoles ensure the spine develops straight? “We think it is the internal pressure of the vacuoles that allows the axis of the embryo to remain straight,” Bagnat explains. He suggests that the notochord vacuoles act as an inflatable scaffold to build the spine.

Zebrafish embryos without vacuoles are short and develop kinked spines.
(Credit: Kathryn Ellis/ Duke University Medical Center) 

Richard Adams, a developmental biologist from the University of Cambridge (UK) who works on zebrafish tissue morphogenesis thinks studies like this could help us understand what causes human scoliosis “This study raises the possibility of a new underlying cause for this condition. […] The zebrafish provides a powerful model organism with which to rapidly test the roles of many such molecular pathways.”

In the most severe cases, scoliosis can be painful and debilitating, and patients might need corrective surgery. Scoliosis research is difficult in part because the spine is not readily accessible in mammalian animal models, such as mouse. But because zebrafish are transparent, researchers can follow spine formation in living animals, and see what goes wrong when they get a kink in the spine. And this is exactly what Bagnat’s team plans to do in the future “We think that we should be able to image scoliosis as it happens in the fish.”

Reference:

Ellis K., Bagwell J. & Bagnat M. (2013). Notochord vacuoles are lysosome-related organelles that function in axis and spine morphogenesis, The Journal of Cell Biology, 200 (5) 667-679. DOI: 10.1083/jcb.201212095

I wrote a short article about this study for ScienceNow that includes a movie of the vacuoles forming. You can read it here.