Different parts of people’s brains help keep them alive and make them human. The forward-most portion—the forebrain—executes cognitive functions, giving people the capability to understand poetry, science, and themselves. The hindbrain, which contains most of the brainstem, controls breathing, heart rate, consciousness, and sleep.1
Degeneration of hindbrain neurons causes diseases like spinal muscular atrophy and amyotrophic lateral sclerosis, drawing research interest in the structure. However, scientists have struggled to grow mammalian hindbrain cells in vitro. Now, researchers may finally know why.
By studying mouse brains, scientists discovered that the forebrain and hindbrain develop from distinct progenitor cells.2 Their findings, published in Nature Neuroscience, offer deeper insights into brain development and provide a model to study disorders that affect the hindbrain.
The researchers observed two separate developmental origins of the brain in 9.5-day-old mouse embryos. Otx2-expressing cells (blue) are present in the front of the brain, specifically the forebrain and midbrain. The cells stained red are present in the hindbrain, which originates from a different developmental source than the rest of the brain. This red-stained cell population also extends into the spinal cord, which runs throughout the body.
Loh Laboratory / Stanford Medicine
“We’ve shown for the first time that the front of the brain arises from a totally different progenitor cell than the back of the brain,” said Stanford University developmental biologist and study coauthor Kyle Loh, in a press release. “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.”
Distinct Progenitors Give Rise to Different Parts of the Brain
Loh and his team examined the neural ectoderm—the germ layer that gives rise to the nervous system—in seven-day-old mouse embryos. In situ hybridization and single-cell RNA sequencing indicated the presence of two distinct cell populations within the germ layer.
The cells expressing the transcription factor orthodenticle homeobox 2 (Otx2) occurred toward the anterior, or front side of the brain. Classical developmental fate maps indicated that these were destined to form the forebrain. Conversely, cells towards the back expressed the transcription factor gastrulation brain homeobox 2 (Gbx2) and appeared to give rise to the hindbrain.
Loh and his team then carried out lineage tracing by labeling Gbx2-expressing cells red in seven-day-old embryos. They observed that the label accumulated in the hindbrain, but not the forebrain, of 18-day-old embryos, indicating that the progenitor cells did indeed give rise to this brain part.
Next, the researchers developed a strategy to coax human pluripotent stem cells—including embryonic stem cells and induced pluripotent stem cells (iPSCs)—into forebrain and hindbrain progenitors using growth factors. High-throughput sequencing of these differentiated cells revealed distinct transcriptional and chromatin landscapes; the researchers also saw that these progenitor cells only responded to their respective growth factors, offering clues into their potential to eventually form different parts of the brain.
Unlocking the Recipe for Growing Hindbrain Neurons
Equipped with this information, the researchers differentiated human pluripotent stem cells to form hindbrain motor neurons. These showed the hallmarks of this hindbrain neuron population, including electrophysiological activity and gene expression.
“Previous attempts to make hindbrain neurons likely tried to coax forebrain and midbrain progenitors into hindbrain cells, which our study shows is not possible,” said study coauthor Rayyan Jokhai, a graduate student in Loh’s lab, in the press release.
Finally, Loh and his team wondered whether such compartmentalization of progenitors existed in other species. Analyzing previously published data revealed separate Otx+ anterior neural ectoderm and Gbx+ posterior neural ectoderm populations in macaques, chickens, and zebrafish. In situ staining indicate that acorn worms—which diverged from vertebrate species nearly 550 million years ago—also contained mutually exclusive forebrain- and hindbrain-specific progenitor cell populations.
“I was surprised at our findings because the word ‘brain’ implies a contiguous organ that likely has a singular origin,” Jokhai said. “But even 500 million years ago, there were these separate neural systems, which now almost operate as one, which is very cool.”
- Philippidou P, Dasen JS. Hox genes: Choreographers in neural development, architects of circuit organization. Neuron. 2013;80(1):12-34.
- Jokhai RT, et al. Two parallel neural ectoderm progenitors contribute to the developing brain. Nat Neurosci. 2026.
