Is The Brain Actually Two Separate Organs?

Forebrain and Hindbrain Appear to Have Separate Origins, Stanford Study Finds

Anatomy textbooks draw the brain as a single organ, one wrinkled mass wired together from front to back. New research led by Stanford Medicine suggests that picture hides a split dating back to the earliest stages of embryonic development. Two regions, the part of the brain that handles thinking and the part that keeps a person breathing, eating and waking up each morning, appear to grow from two different families of starter cells. In lab experiments, those families seem to lock in their jobs almost immediately.

For more than 70 years, a leading idea in developmental biology held that one early group of cells builds the entire brain. In 1952, embryologist P. D. Nieuwkoop proposed that this early tissue carries the potential to form every brain region, and some lab methods for turning stem cells into brain cells still assume that model. In the new study, published in Nature Neuroscience, researchers report that two separate progenitors emerge side by side in the embryo: one destined for the forebrain and midbrain, the other for the hindbrain. In the paper, the authors write that they “postulate the brain is a composite organ.”

A practical payoff comes with the finding. Scientists have had far more success growing forebrain cells in the lab than cells from the hindbrain, the region at the base of the skull that forms most of the brainstem. Using their new roadmap, the Stanford team grew human hindbrain motor neurons, which could give researchers a better way to study diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS).

Brain Development Begins With Two Starter Groups in Mice

Brains begin forming during gastrulation, when a ball of identical cells reorganizes into the basic layers of a body. In mice, that happens about a week after conception. Researchers led by graduate students Rayyan Jokhai and Carolyn Dundes, working in the lab of developmental biologist Kyle Loh, started by studying mouse embryos 7.5 days old.

Among the early neural cells, they found two groups that did not overlap. Cells toward the front switched on a gene called Otx2. Cells toward the back switched on a different gene, Gbx2. To see where each group ended up, the team used lineage tracing, a genetic technique that permanently tags cells with a glowing marker so their descendants can be followed later, much like dyeing a single strand of yarn and finding it again in a finished sweater.

After tagging the Gbx2 cells, researchers found the glowing descendants only in the hindbrain, even in mouse embryos close to birth. They were almost entirely absent from the forebrain and midbrain.

A second experiment cast a wider net. This time the team randomly tagged scattered neural cells in 16 mouse embryos with one of three colors, without regard to which gene each cell used. Of 494 cell clusters they later found, about 63% sat in the forebrain or midbrain and roughly 33% sat in the hindbrain. Only about 4% straddled the boundary. By the end of gastrulation, then, most early neural cells in the mouse were already headed to one side of the brain or the other.

Human Stem Cells That Refused to Switch Sides

Lineage tracing shows where cells go under normal conditions. It cannot show whether they could go somewhere else if pushed. To test that, the team turned to human pluripotent stem cells, lab-grown cells that can become almost any tissue in the body.

Standard lab recipes for making brain cells block three chemical signals. In the new work, the Stanford group found that this recipe produces only the front-of-brain type. Adding two more signals, a growth factor called FGF and retinoic acid (a compound made from vitamin A), produced the back-of-brain type instead. Both types appeared within two days, across four separate stem cell lines.

Next came the stress test. Researchers bathed each cell type in chemical signals meant to steer it toward forebrain, midbrain or hindbrain, then counted how many cells took on each identity. Front-type cells readily became forebrain (about 95% carried a forebrain marker) and midbrain (about 82%). Back-type cells almost completely refused, at 0.5% and 0.7%. Results flipped for hindbrain: roughly 97% of back-type cells made the switch, compared with fewer than 2% of front-type cells. Growing the two types together in one dish changed little, though a few back-type cells did pick up forebrain or midbrain markers.

To learn why the cells were so stubborn, the team examined chromatin, the packaging that decides which genes a cell can easily reach and which stay tucked away. Each cell type had its own packaging from the start. When back-type cells were given forebrain signals, they opened up hindbrain genes instead. Based on those results, the authors propose that a cell’s ability to form either region is settled within the first two days of stem cell growth, even though producing actual neurons in the lab typically takes weeks or months.

“In stem cell biology, people are always fixated with creating the end cell type, like the neuron,” Jokhai said in a statement. “But it’s important to begin at the earliest stages of embryonic development.”

Lab-Grown Hindbrain Neurons for ALS and SMA Research

Starting from the back-type cells, the researchers grew motor neurons matching a specific stretch of the hindbrain (segments 5 and 6) that, in the body, helps control swallowing. These neurons carried the molecular ID tags of that region, made the machinery for acetylcholine, the chemical messenger motor neurons use to signal muscles, and showed few signs of being spinal cord cells. They also behaved like working neurons, firing electrical signals when stimulated with current or with light and producing spontaneous bursts of calcium activity. Front-type cells, meanwhile, gave rise to several forebrain neuron types, including hypothalamus-like cells that make POMC, a protein involved in suppressing appetite.

According to the paper, neurons from this part of the hindbrain could serve as a platform to study SMA, ALS and other diseases in which impaired swallowing can lead to choking and death. Brainstem tissue cannot be taken from living patients, so a lab-grown supply fills a real gap. “Our discovery means that we can now grow neurons from the back of the brain, the hindbrain, in a petri dish and study their functions,” Loh said.

An Ancient Split in Brain Development

Next, the team asked whether the pattern shows up in other animals. They found separate front and back neural cell groups in early embryos of macaque monkeys, chickens and zebrafish. An identical split also appeared in the acorn worm, a small seafloor animal whose lineage shares a common ancestor with vertebrates from about 550 million to 600 million years ago.

That spread across species hints that the two-part pattern is very old. According to the authors, it may predate chordates, the broader animal group that includes vertebrates, arising roughly 550 million to 600 million years ago. “Our research suggests that evolution took two existing neural systems and pushed them together spatially,” Loh said. Jokhai admitted the result caught him off guard: “I was surprised at our findings because the word ‘brain’ implies a contiguous organ that likely has a singular origin.”

Open questions remain. A single “pan-brain” cell could still exist for a very short window before the split, and the origin of the cerebellum is unresolved; it may come from a third type of early cell. Most important, the evidence that the two cell types cannot switch sides comes from lab dishes, not living embryos.

Even so, the study offers a concrete reason hindbrain cells have been so hard to grow: many labs may have started with cells already committed to the front of the brain. In daily life the brain works as one organ, but its construction appears to begin with two separate crews, and researchers hoping to grow brainstem cells may need to recruit the right one from day one.

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