Multiple cell and neuron types are essential for the assembly and function of complex nervous systems. Diverse neuron types develop distinct morphologies and functions during development. While morphology is the traditional way of identifying neuron types, a better understanding of how neuronal networks are assembled can now be achieved by relying on experimental and computational methods that analyse morphology, gene expression, and developmental trajectory and timing simultaneously. In this study, Wendy Xueyi Wang, PhD student at the University of Toronto and the Hospital for Sick Children Research Institute, presents a multimodal diversification map of the cerebellar molecular layer interneurons (MLIs), a heterogeneous inhibitory interneuron population that derives from a common progenitor population. This study provides new insight into the specialisation of neuron cells and their integration in functional networks.
Over 130 years ago, Santiago Ramón y Cajal took advantage of the intriguingly heterogeneous and accessible organization of the cerebellar MLIs to propose and substantiate the neuron doctrine, the concept that the nervous system is made of individual cells, or neurons, instead of being connected through continuity such as in the vascular system. In homage to the original master, the researchers re-examined MLI diversity using genetic and computational methods of modern single-cell biology.
In the cerebellum, molecular layer interneurons (MLIs) can be broadly qualified, based on their morphology as basket cells (so named as they contain cellular structures which resemble baskets) or stellate cells (with a more star like shape). As many intermediate forms exist, it was unknown whether the two populations represented truly distinct cell types. Using a combination of genetic, histological and computational methods, the researchers defined the diversity of mature MLIs based on morphology and gene expression. These analyses reveal examples of both discrete and continuous heterogeneity for each criteria. Interestingly, they did not find direct correlation between MLI morphological and transcriptional identities – that is, neurons with similar shapes did not necessarily express the same genes. Instead, they identified, for the first time, an example cell-type displaying graded transitions in gene expression between morphologically discrete neurons, as well as graded transitions in neuronal morphology within transcriptionally discrete neurons. This demonstrates that neither morphology nor gene expression alone can be used to fully annotate interneuron diversity. Through a novel application of pseudotime trajectory inference to neuronal morphology, they further defined the early emergence of discrete MLI morphological types, days prior to upregulation of subtype-specific expression of marker genes. Altogether, these studies present a multimodal map of MLI diversification and offer a framework that is broadly applicable for defining the developmental trajectory of interneuron populations.
Understanding how diverse neurons are assembled into circuits requires a framework for describing cell-types and their developmental trajectories. Studies at maturity have shown that multimodal approaches are needed to delineate the repertoire of neuronal cell-type diversity. However, similar methods have not been extended to interrogate the steps by which neuronal progenitors are programmed to acquire their adult forms and functions.
About Wendy Xueyi Wang
In this two-author publication which encapsulates the majority of her PhD research in the laboratory of Dr. Julie Lefebvre at the Hospital for Sick Children, Wendy Xueyi Wang conducted 100% of the experiments and analyses. Initiation of the project idea and writing of the manuscript was performed in collaboration with her advisor and last author, Dr. Julie Lefebvre. Dr. Wang is now a postdoctoral fellow at the Broad Institute of MIT and Harvard, and Harvard University.
Funding sources
This work was supported by an Ontario Graduate scholarship to Wendy Xueyi Wang, as well as funding from a Sloan Fellowship in Neuroscience, NSERC discovery grant, and CIHR project grant to Dr. Julie Lefebvre.
Scientific article
Wang, W.X. and Lefebvre, J.L. Morphological pseudotime ordering and fate mapping reveal diversification of cerebellar inhibitory interneurons. Nature Communications. 13, Article number: 3433 (2022)
https://www.nature.com/articles/s41467-022-30977-2
