Neuron heterogeneity in the developing mouse brain

Deeply hidden in the brain, the tiny habenula is increasingly associated with different psychological roles. High-scale cellular heterogeneity may explain that multifunctionality. Here, we describe how single-cell RNA sequencing helps describe habenula diversity and function.

The habenula is a tiny structure in the brain that is believed to be involved in the regulation of mood and motivation. It is located just above the thalamus, deep within the brain.

Rather little was known about the habenula until relatively recent studies put it in the limelight. In the past fifteen years, studies have linked the habenula to the brain’s response to rewards and punishments. It is suspected to play a role in our sleep, eating behavior, and stress response. And its dysfunction correlates with afflictions such as depression, schizophrenia, bipolar disorder, anxiety, and addiction.

It’s intriguing that the habenula’s pocket-sized group of neurons could underpin such a large number of conditions in health and disease. Neuroscientists from the Brain Center at University Medical Center Utrecht suspect the tiny organ’s neuronal heterogeneity might account for that.

To investigate that hypothesis, the team examined mice habenula in single-cell resolution at key time points in their embryonic development. Lieke van de Haar et al. published their study in Cell Reports.

Sequencing multiple developmental stages

The researcher collected tissue from crucial stages in mouse habenula development. The habenula begins to develop during the embryo stage. A group of cells called the neural tube forms and begins to differentiate into the various regions of the brain. From 11 to 18.5 days after fertilization, the habenula takes its shape. Historically, the habenula is divided into the medial and lateral habenula. Many more functional subtypes might be present, though. Habenula neurons still change around synapses and connect to other neurons after birth. So, the researchers took samples at five moments during development plus three moments after birth.

This timeline of mouse brain development, shows the habenula location and shape at embryonic days E11, E12, E13, E15, and E18, the period of post-mitotic habenula neuron. These are also the sampling timepoints for this study.
Timeline of the key moments during mouse development on which the researchers collected habenula samples. Source: Lieke van de Haar et al. (2022), license: CC-BY 4.0.

They could separate habenula cells from the rest of the brain by making them specifically fluorescent and detecting that with flow cytometry (FACS). Then, they employed the single-cell sequencing platform SORT-seq. SORT-seq is based on 384-well cell-capture plates and integrates well with flow cytometry (FACS) cell isolation. For the study of atypical cells available in low quantities, SORT-seq provides the compatibility and flexibility needed. It resulted in the gene expression profile of 5,756 neurons across eight development stages.

Main findings

With that data, the researchers were able to identify which habenula subtypes exist during development. They detected fourteen cell clusters.

Neuron subtypes in the habenula

Guided by cell markers published in earlier studies, the researchers could label the clusters. Some seemed to be progenitor cells or a specific state of immature habenula neurons. Others stand out by the dominance of a type of neurotransmitter, such as acetylcholine or substance P.

When they related the cell clusters of developing habenulas with those of adult mice, they could label them based on their eventual anatomical location as well. There are neurons that make up the (dorsal, superior, ventral, and lateral) parts of the medial habenula and neurons that make up the parts of the lateral habenula. Still, the data showed that gene expression was diverse even within neuron clusters. Mouse habenulas seem to have very high levels of heterogeneity, indeed.

tSNE visualization of the 14 detected habenula cell clusters. In the plot on the left, cells are colored per clustered. On the right, they are colored by the age of the mouse brain samples
tSNE visualization of the fourteen detected habenula (Hb) cell clusters. Clusters 12 and 13 are progentior cell-like (PC), clusters 6 and 8 are immature habenula (iHb) cells. All other clusters carry names referring to their final location in the habenula. In the plot on the left, cells are colored per cluster. On the right, they are colored by the age of the mouse brain samples. Source: Lieke van de Haar et al. (2022), license: CC-BY 4.0

Genes that drive developmental pathways

The team could also find out when the neurons became heterogeneous and how specific gene expression patterns drove them down one developmental path or another.

They found out that specific clusters were present between 11 and 18 days after fertilization but disappeared after birth. These progenitor cells were at the root of the habenula developmental trajectory. Neuron subtype diversity, as it appears in adult mouse habenula, establishes at the fourth day after birth.

Based on the single-cell RNA data, the team could infer the trajectory along which embryonic progenitors develop into adult habenula cells. For example, progenitor cells seem to develop into a type of immature neuron before taking one of two paths. Then they either become part of the medial habenula or the lateral habenula. The researchers could also identify which genes or gene expression patterns played a role. For example, they saw that decreasing expression of the gene Cntn2 was a part of how progenitors develop into immature neurons. For another example, the expression of Calb2 indicated dorsal medial habenula cells, while Unc5d marked lateral habenula cells.

The left figure shows the development trajectory of habenula neurons, placed over the tSNE visualization of single-cell RNA data. On the right, the lineage tree is the same, but abstract, and associated pathway-driving genes are named: Cntn2 drives progenitor cell to immature neuron, Unc5d drives the lateral Habenula path, Calb2 the dorsal medial habenula progenitors, Chat the ventral medial habenula progenitors, Tac1 the dorsal medial habenula superior, Calb1 the dorsal medial habenula dorsal, Spon1 the ventral medial habenula lateral, Synpr the ventral medial habenula ventral.
The left figure shows the development trajectory of habenula neurons, placed over the tSNE visualization of the figure above. The right figure shows a lineage tree of the same trajector and names the suggested developmental pathway-driving genes. Source: Lieke van de Haar et al. (2022), CC-BY 4.0.

Within two of the fourteen clusters, the researchers detected a particularly interesting population of neurons that express the gene Cartpt. Mulitple studies associated the gene with a wide range of conditions, including energy homeostasis and anti-depressive capacity.

The Utrecht researchers expanded their experimental toolkit with immunostaining and electrophysiological analysis—well-established tests in neuroscience. This helped them verify that the Cartpt-population was functionally distinct from other habenula cells.

Interestingly, their axons reach into a part of the brain called the dorsal interpeduncular nucleus. Though the role of this brain area isn’t completely understood, it’s correlated with depressive-like symptoms. So, it’s possible that certain habenular cell subtypes hold important information for understanding or treating this mental illness.The researchers could indeed link genetic indications for psychiatric traits to particular neuron subtypes. For example, two habenula immature neuron clusters were related to genetic risk factors for depression. This research in mouse brains points toward a possible connection, which is far from understood but creates an opportunity for further study.

Impact of the results

Until fairly recently, neurobiological techniques were not powerful enough to investigate the habenula with adequate detail. Now, this and other studies have been able to study the developing habenula at single-cell resolution. What’s revealed is an exceedingly heterogeneous organ with many likely roles in neurological health and disease that remain to be unraveled.

Single-cell sequencing at different developmental time points brought out the gene expression patterns behind habenula growth as well as a better understanding of neuron subtypes. The researchers also mapped out the gene expression patterns of neurons and their progenitors at multiple stages of brain development.

You can find the full paper here:

Molecular signatures and cellular diversity during mouse habenula development

The authors performed SORT-seq on mouse habenular neurons at critical developmental stages, instructed by detailed 3D anatomical data. The data reveal cellular and molecular trajectories during embryonic and postnatal development, leading to different habenular subtypes. Among these, the Cartpt+ neuron subtype was studied in-depth and successfully linked to specific molecular and functional properties.

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