Prefrontal Cortex Development of Diverse Functional Cells

 

Nature volume 555, pages 524–528 (22 March 2018)

A single-cell RNA-seq survey of the developmental landscape of the human prefrontal cortex

Suijuan Zhong, et.al.

State Key Laboratory of Brain and Cognitive Science, CAS Center for Excellence in Brain Science and Intelligence Technology (Shanghai), Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China

University of Chinese Academy of Sciences, Beijing, 100049, China

Beijing Advanced Innovation Center for Genomics, College of Life Sciences, Department of Obstetrics and Gynecology, Third Hospital, Peking University, Beijing, 100871, China

Obstetrics and Gynecology, Medical Center of Severe Cardiovascular of Beijing Anzhen Hospital, Capital Medical University, Beijing, 100029, China

Biomedical Institute for Pioneering Investigation via Convergence and Center for Reproductive Medicine, Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing, 100871, China

Peking–Tsinghua Center for Life Sciences, Peking University, Beijing, 100871, China

Beijing Institute for Brain Disorders, Beijing, 100069, China.

[paraphrase]

The mammalian prefrontal cortex comprises a set of highly specialized brain areas containing billions of cells and serves as the centre of the highest-order cognitive functions, such as memory, cognitive ability, decision-making and social behavior. Although neural circuits are formed in the late stages of human embryonic development and even after birth,    diverse classes of functional cells are generated and migrate to the appropriate locations    earlier in development. Dysfunction of the prefrontal cortex contributes to cognitive deficits and the majority of neurodevelopmental disorders; there is therefore a need for detailed knowledge of the development of the prefrontal cortex. However, it is still difficult to identify cell types in the developing human prefrontal cortex and to distinguish their developmental features. Here we analyse more than 2,300 single cells in the developing human prefrontal cortex from gestational weeks 8 to 26 using RNA sequencing. We identify 35 subtypes of cells in six main classes and trace the developmental trajectories of these cells. Detailed analysis of neural progenitor cells highlights new marker genes and unique developmental features of intermediate progenitor cells. We also map the timeline of neurogenesis of excitatory neurons in the prefrontal cortex and detect the presence of interneuron progenitors in early developing prefrontal cortex. Moreover, we reveal the intrinsic development-dependent signals that regulate neuron generation and circuit formation using single-cell transcriptomic data analysis. Our screening and characterization approach provides a blueprint for understanding the development of the human prefrontal cortex in the early and mid-gestational stages in order to systematically dissect the cellular basis and molecular regulation of prefrontal cortex function in humans.

The prefrontal cortex (PFC) covers the front of the frontal lobe of the brain in mammals and has important roles in memory, emotion, cognitive behaviour, decision-making and social behavior. To analyse the molecular features of cells in the PFC during human brain development, we obtained 2,309 single cells from human embryonic PFCs at gestational weeks (GW)8 to 26. To classify the major cell types in the developing PFC, we performed t-distributed stochastic neighbour embedding (t-SNE) analysis and identified six major clusters:    neural progenitor cells (NPCs),    excitatory neurons,    interneurons,    astrocytes,    oligodendrocyte progenitor cells (OPCs)    and microglia. Biological replicate samples were evenly distributed on the t-SNE plot. To further analyse the subclusters within each cell type, we used random forest analysis to segregate cells into 35 distinct subtypes. By analysing differentially expressed genes among the clusters, we identified SFRP1 and RBFOX1 as markers of NPCs and excitatory neurons and verified this by immunofluorescence. We found that NPCs,    excitatory neurons and interneurons were sub-clustered in a development-dependent manner. We then reconstructed the developmental time course and lineage relationships using Monocle analysis. Microglia and interneurons were excluded because microglia are mesoderm-derived cells, and interneurons are generated in the ganglionic eminence and migrate tangentially to the PFC. The remaining cells were distributed along pseudo-temporally ordered paths from NPCs to neurons (neuronal lineage) or to OPCs and astrocytes (glial lineage). Neurons developed from NPCs in early gestational weeks whereas OPCs and astrocytes differentiated from NPCs in later weeks.

The two main types of neurons in the brain are excitatory neurons and interneurons. Cortical interneurons originate from the ganglionic eminence and migrate tangentially to the neocortex, where excitatory neurons are generated.

Since excitatory neurons and interneurons    build up circuits cooperatively, we next investigated whether the development of these two types of neurons was synchronized. Excitatory neuron development peaked at GW16, whereas interneuron development peaked at GW26. We therefore selected neurons at GW16 and GW26 for pathway analysis by gene-set enrichment analysis (GSEA). Axon guidance signals, including axon attraction,   repulsion    and outgrowth, were more active in excitatory neurons than in interneurons at GW16; this was reversed at GW26. The neurotrophin signalling pathway, which has roles in neuronal differentiation and prevention of cell death, exhibited a similar pattern. Additionally, gene enrichment analysis showed that Notch signals were more involved in regulating biological activities of NPCs than those of neurons. Together, these results suggest that excitatory neurons mature earlier than interneurons, and that this maturation process is regulated by extrinsic and intrinsic signals.

Our single-cell-resolution data illustrate the complex diversity of cell types in the developing human PFC that underlies the sophisticated cognitive function of humans. Notably, we found new IPC markers that enabled us to identify diverse neuron subtypes that are likely to contribute to the cellular basis of elaborate circuit formation in humans. Some neurological disorders and social cognition deficits, such as autism spectrum disorder and schizophrenia, have been linked to an imbalance of excitatory and inhibitory neurons (E/I ratio) in the PFC. Our data indicate that interneurons appear and mature later than excitatory neurons, and that this pattern is intrinsically regulated. Thus, transcriptome profiling of thousands of single cells in the developing PFC is a powerful tool for investigating the mechanisms behind neurological diseases and exploring potential therapies.

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