NIDCR scientists first to discover how embryonic salivary gland regulates its own innervation
Many of the estimated 280,000 Americ ans who have survived oral cancer treatments contend with disabling dry mouth as a result of radiation therapy aimed at their mouth and throat. For these oral cancer survivors, the mouth is dry because radiation damaged the salivary glands. A dry mouth makes it challenging to talk and eat, and makes tooth decay, painful ulcers, and other problems in the mouth much more likely. For some oral cancer survivors, the salivary glands may never work well again.
So that someday doctors will be able to repair or regenerate salivary glands, Matrix and Morphogenesis Section Chief and Senior Investigator Matthew P. Hoffman, BDS, PhD, has been identifying the cells, signals, and other factors that are key for the gland’s development. In a recent article in Developmental Cell, Hoffman and colleagues are the first to describe the cellular cross talk that promotes the innervation of the salivary gland. These findings are an important advance toward the goal of developing treatments that restore a cancer survivor’s ability to make saliva.
Studying complex living tissues
Mouse salivary gland on about 14th day after conception. Gland appears green and nerves appear red (from Developmental Cell2015).
For more than 65 years, scientists have been extracting salivary glands from mouse embryos and observing how they grow in living tissue culture. About 12 days after conception, a bud near the tongue forms in the embryo. This bud branches into three or four buds, and those buds continue branching until the embryonic gland looks like a cluster of grapes. Shortly after that, the solid cores hollow out so the gland can release saliva through ducts into the mouth.
About 40 years ago, it was discovered that while the branches and cavities of the gland are forming, nerves also develop and send out projections that weave around the gland to innervate it. By the 13th day after conception, researchers can separate the tiny embryonic nerves from the salivary gland (see image of red nerves overlaying a green salivary gland).
“As the salivary gland develops in the embryo, the different types of tissues are obviously communicating with each other,” said Hoffman. Using genetically modified mouse embryos to eavesdrop on the cross talk between the tissues, his team is identifying what drives the precise coordination.
Isolating progenitor cells
“For a cancer survivor who is unable to make much or any saliva, progenitor cells could be isolated from the patient’s body and then transplanted inside the diseased gland along with the factors they need to enhance or restore the ability to make saliva,” said Hoffman. Progenitor cells are precursor cells that have the potential to differentiate into many other cell types. Hoffman’s team is targeting a number of cell types, including K5+ (keratin-5-positive) progenitor cells, for potential use in salivary gland regeneration and repair. In adults, K5+ cells can be isolated from a patient’s skin, salivary gland, or other tissues.
In an embryo, the salivary gland needs a pool of K5+ cells in their precursor, undifferentiated state. If K5+ cells within the salivary gland all differentiate into other cells, the gland doesn’t grow and develop properly. Five years ago, Hoffman’s team published in Science their discovery that the nervous tissue maintains the K5+ cells within the gland in a precursor state. If the nerves are removed from an embryo, K5+ cells differentiate, the pool is depleted, and gland growth is impaired.
Discovering a novel interaction
“Many researchers are looking at stem cells or progenitor cells without taking into account nerve development and what may drive it,” said Hoffman. And yet, for glands to work properly, nerves are as essential as the blood vessels that supply oxygen and nutrients.
The experiments described in the Developmental Cell article represent 6 years of work. One of the article’s co-first authors, Wendy M. Knosp, PhD, was a postdoctoral fellow in Hoffman’s lab at the time the most recent experiments were conducted. The project at NIDCR was a continuation of work she began as a postdoc at the University of California San Francisco (UCSF) in the lab of Gail R. Martin, PhD. Coincidentally, the other co-first author, Sarah M. Knox, PhD, was a postdoctoral fellow in Hoffman’s lab at the time of the research and is now a UCSF assistant professor and continues investigations of gland development in her newly established lab.
“We already knew that signals from the nerves keep K5+ cells as precursor cells,” said Knosp, who is now a science policy analyst in NIDCR’s Office of Science Policy and Analysis. “But what we were surprised to find is that K5+ cells are producing the signals required for the initiation of innervation.” In other words, the cellular cross talk in an embryo is bidirectional. The nerve cells help the salivary gland to develop its branched architecture by maintaining the K5+ cells as precursor cells, and the K5+ cells within the salivary gland help the nerve cells survive and innervate the gland.
Using molecular techniques and living tissue cultures from genetically modified mouse embryos, Knosp was able to determine that the signals coming from the K5+ cells were proteins called Wnts. “When we treated the culture of fetal salivary glands with Wnt inhibitors, nerve development and branching were disrupted,” said Knosp. “Normally, more than half of the nerve cells wrap around the gland’s duct, but with the Wnt inhibitor, only about a quarter of the nerve cells were observed around the duct.” The image on the right shows the dramatic difference between normal nerve cell growth (on the left) and its disruption after Wnt inhibition (on the right).
Knosp’s experiments were confirmed using genetically modified mice that did not have nerves around the salivary gland. By studying these mice, she discovered that the gland did not produce the Wnt signals that drive innervation of the salivary gland. As a result, the gland did not form properly.
Knosp reasoned that what was true for salivary gland development would also be true for the growth of other glands. Because the key signals for innervation of the pancreas hadn’t been discovered yet, Knosp repeated the experiments with embryonic pancreases from the same genetically modified mice. Her hypothesis was confirmed: Wnt signals also drive innervation of the pancreas.
By showing that Wnt signals are necessary for nerve development, the study provides a new mechanism for the regulation of innervation. With the new information reported in Developmental Cell, researchers may be one step closer toward the goal of developing methods of regenerating or repairing complex glands.
- Knosp WM, Knox SM, Lombaert IMA, Haddox CL, Patel VN, and Hoffman MP. Submandibular parasympathetic gangliogenesis requires Sprouty-dependent Wnt signals from epithelial progenitors. Development Cell 2015; 32:667-677.
- Knox SM, Lombaert IM, Reed X, Vitale-Cross L, Gutkind JS, Hoffman MP. Parasympathetic innervation maintains epithelial progenitor cells during salivary organogenesis. Science. 2010;329(5999):1645-7. doi: 10.1126/science.1192046.