Researchers uncovered separate developmental origins for the front and back of the brain, opening a path to grow hindbrain neurons in vitro.
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The human brain is divided into regions that work in concert to sustain life and enable complex thought. The forebrain, situated at the front, handles cognitive tasks, ranging from scientific reasoning to self-awareness. Meanwhile, the hindbrain, which comprises much of the brainstem, is vital for autonomic functions such as regulating breathing, heart rate, sleep, and consciousness.
Degeneration of hindbrain neurons is linked to devastating conditions such as spinal muscular atrophy and amyotrophic lateral sclerosis (ALS). Consequently, scientists have long sought to study these cells, but growing mammalian hindbrain cells in vitro has proven exceptionally challenging. A breakthrough study now reveals the underlying reason.
By studying mouse embryos, researchers have discovered that the forebrain and hindbrain develop from entirely distinct progenitor cells. Published in Nature Neuroscience, these findings provide profound insights into brain development and offer a robust model for studying disorders that target 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
To investigate this division, Loh and his team examined the neural ectoderm—the embryonic germ layer that gives rise to the nervous system—in seven-day-old mouse embryos. Using in situ hybridization and single-cell RNA sequencing, the researchers identified two distinct cell populations within this layer.
Cells expressing the transcription factor orthodenticle homeobox 2 (Otx2) were localized toward the anterior, or front side, of the developing brain. Classical developmental fate maps indicated that these cells were destined to form the forebrain and midbrain. Conversely, cells toward the posterior expressed the transcription factor gastrulation brain homeobox 2 (Gbx2) and were slated to give rise to the hindbrain.
To confirm this, the team performed lineage tracing by labeling Gbx2-expressing cells in seven-day-old embryos. By the 18-day mark, the red label had accumulated specifically in the hindbrain and not the forebrain, confirming that these progenitor cells indeed give rise to the hindbrain.
Next, the researchers developed a method to coax human pluripotent stem cells—including embryonic stem cells and induced pluripotent stem cells (iPSCs)—into forebrain and hindbrain progenitors using specific growth factors. High-throughput sequencing revealed distinct transcriptional and chromatin landscapes for each cell type. Furthermore, the progenitor cells only responded to their respective growth factors, highlighting their specialized developmental potential.
Unlocking the Recipe for Growing Hindbrain Neurons
Armed with this molecular map, the researchers successfully differentiated human pluripotent stem cells into hindbrain motor neurons. These lab-grown cells displayed the hallmark electrophysiological activity and gene expression profiles characteristic of native hindbrain neurons.
“Previous attempts to generate 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, the team investigated whether this progenitor compartmentalization is unique to mammals. Analyzing previously published data, they found separate Otx+ anterior and Gbx+ posterior neural ectoderm populations in macaques, chickens, and zebrafish. In situ staining further revealed that acorn worms—which diverged from vertebrate species nearly 550 million years ago—also possess mutually exclusive forebrain- and hindbrain-specific progenitor cell populations.
“I was surprised by 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.”
