Probing the brain’s earliest development, with a detour into rare childhood cancers

In early brain development there is an increase in ribosomes, contained in these nucleoli
Nucleoli, the structures in the cell nucleus that manufacture ribosomes, are enlarged in very early brain development, indicating an increase in ribosome production. Here, a 3D reconstruction of individual nucleoli. (Kevin Chau, Boston Children’s Hospital)

In our early days as embryos, before we had brains, we had a neural fold, bathed in amniotic fluid. Sometime in the early-to-mid first trimester, the fold closed to form a tube, capturing some of the fluid inside as cerebrospinal fluid. Only then did our brains begin to form.

In 2015, a team led by Maria Lehtinen, PhD, Kevin Chau, PhD and Hanno Steen, PhD, at Boston Children’s Hospital, showed that the profile of proteins in the fluid changes during this time. They further showed that these proteins “talk” to the neural stem cells that form the brain.

In new research just published in the online journal eLife, Lehtinen and Chau shed more light on this little-known early stage of brain development.

Proteins: Controlling the means of production

Studying embryonic mice, Chau isolated neural stem cells at different stages of development. He then sequenced the RNAs that were being produced, reflecting which genes were being turned on (expressed) or turned off. He found that just after neural tube closure, genes that encode the cell’s protein-making machinery were down-regulated. As a result, the production of ribosomes, the structures in cells whose job it is to build proteins, decreased. So did overall protein levels in the adjacent fluid. As the ribosome-related genes turned off, other genes, such as those related to neuron specialization and migration, turned on.

early brain development - enriched gene categories before and after neural tube closure
(Kevin Chau)

“With the formation of the brain, the machinery that produces proteins in cells is rapidly down-regulated,” says Lehtinen. “This hasn’t been shown previously. Many studies start at the point where the brain has already formed.”

Lehtinen thinks this is partly due to technical limitations in the past. The tissues are very small, and the changes occur on a scale of hours. Her team was able to take advantage of special micro-dissection tools and new small-animal imaging technologies not widely available 10 years ago.

“This very early time window is critical,” she says. “The early brain forms the scaffolding for what’s to come. We believe our work has the potential to uncover new mechanisms disrupted in neurodevelopmental conditions, a lot of which involve protein synthesis.”

The pediatric cancer connection

One particular down-regulated gene stood out: c-Myc. Chau’s experiments showed that c-Myc is highly expressed in the neural tube tissue just before it closes. After closure, the gene completely turns off. When he shut down Myc in mice during the period before neural tube closure — using chemical inhibitors or by deleting the gene — fewer ribosomes were made.

Interestingly, c-Myc is a well-known and potent oncogene. Reviewing the cancer literature, Lehtinen and Chau learned that when a healthy cell becomes cancerous because of c-Myc, the synthesis of ribosomes ramps up — the opposite of what happens during neural tube closure.

In further work, led by Lehtinen and Morgan Shannon, the team deliberately kept c-Myc turned on after tube closure, crossing strains of mice in such a way that the gene’s expression was increased only in neural stem cells. This increased the production of ribosomes. But as the mice got older, there were unexpected side effects, as reported in the American Journal of Pathology.

“A research associate in our lab told me, ‘you need to come take a look these mice,’” says Lehtinen.

The eyes of the mice appeared doubled in size. Working with neuropathologists at Boston Children’s, the team identified ciliary body medulloepithelioma, an extremely rare eye tumor of early childhood, affecting the tissue that produces fluid for the eye. The mice also turned out to have tumors in the choroid plexus, the brain tissue that produces cerebrospinal fluid. It, too, mirrored a rare childhood tumor.

The yin-yang of Myc

Lehtinen, Shannon and colleagues then wanted to study tumor samples from human patients. Tapping a tissue bank at Boston Children’s, they examined samples from 15 children with choroid plexus tumors. In the subset of tumor samples with the highest proliferation rate, c-Myc was turned on, even though it usually is silent in the choroid plexus. Samples with greater c-Myc expression showed increased cell proliferation, indicating more aggressive disease. Features of the tumors were similar to those in the mice.

The rarity of both tumors has hindered progress in understanding their origins, says Lehtinen. So has the lack of good animal models that faithfully recapitulate these diseases. The c-Myc-expressing mice provide such a model, allowing further study of both cancer and early brain development.

Myc is thought of as oncogene, but in the early brain it’s really critical. If you don’t have enough, you have major problems in neurodevelopment. If you have too much, you can have pediatric cancers,” says Lehtinen. “Being in a hospital research community — with RNA sequencing, radioactivity assays, small animal imaging, tissue banks and clinical neuropathologists to confirm diagnoses — is what’s made it possible to put together these studies. They open a lot of opportunities to start unpacking what’s going on.”