Barcoded Connectomics

Uncovering brain architecture and variations using high throughput in situ sequencing

Goals and Approach

The brain is incredibly diverse on many levels. At the cellular level, myriad neuronal types are wired in diverse patterns, and this complex wiring provides the physical basis for our ability to think, feel, and act. This cellular level diversity extends to the population and species level, generating both diverse behaviors within a species of animals and distinct behaviors across species. How is the diversity in brain structures generated within and across cell types, across individuals, and across species? And how do these structural differences enable the behaviors that distinguish us from other species? We approach these questions from a developmental perspective. We reason that since circuits develop through both stringent genetic programs and stochastic and variable processes, understanding what circuit features are “variable” or “fixed” during development should provide insights into what kind of circuit variations are possible and how circuits can evolve across species.

To answer these questions, our team uses high-throughput in situ sequencing and barcoded connectomics techniques to interrogate circuit organization, development, and evolution. We use the mouse cortex as a model to understand the developmental trajectory of the wiring and organization of cell types, and how developmental mechanisms establish these organizations. In parallel, we compare mouse and non-human primates to understand how circuits evolve and specialize to enable new functions. Finally, we continue to develop in situ sequencing and barcoded connectomics techniques. These techniques already achieve unprecedented throughput in interrogating cell type and connectivity on a brain-wide scale. We aim to further improve the resolution of these techniques, make them orders of magnitude faster and cheaper, and provide easy access to these techniques to the broader research community.

Scientific Projects

What makes cortical areas distinct?

The cortex is separated into many areas with distinct functions. What makes cortical areas different, and what determines the identity of cortical areas? Combining developmental perturbation with high throughput in situ sequencing, we study how cortical areas differ from each other at the cellular level, and how development shapes area identity.

Development of cortical projections

Cortical neurons are incredibly diverse in their long-range projections, but this diversity in projections cannot simply be explained by transcriptomically defined neuronal types. How is this diversity achieved? We reason that any gene regulatory program that specifies projections likely occurs in development, and these patterns of gene expression may not persist in mature neurons. To overcome this transient gene expression problem, we systematically interrogate gene expression as projections are formed to uncover gene regulatory programs that specify cortical projections.

What makes the primate cortex special?

The primate cortex, compared to the mouse cortex, has undergone tremendous expansion both across layers and across areas. This expansion presumably involves replication and specialization of cell types. With additional cell types and cortical areas, how do they wire into existing brain structures? We use barcoded connectomics approaches to compare the cortex of non-human primates to the mouse cortex to understand how new cell types are wired, and how they relate to behavioral differences between the two species.

Next-gen in situ sequencing techniques

The high throughput and low cost of in situ sequencing and barcoded connectomics allows these techniques to be applied across many individuals on a brain-wide scale. The ability to interrogate many individuals opens up the possibility of going from a single reference brain atlas to understanding population variations. Associating structural variations with behavioral differences across individuals will allow us to infer the circuit basis of behaviors in an unbiased manner. With these goals in mind, we also focus on further developing in situ sequencing techniques to make it faster, cheaper, and easily accessible to non-expert labs. Separately, we develop next-generation barcoded connectomics techniques to improve the resolution at which projections and synapses are resolved.

Select Recent Publications

Chen, X.*^†, Fischer, S.^†, Rue, M.C.P^†, Zhang, A., Mukherjee, D., Kanold, P.O., Gillis, J.*, Zador, A.M.* (2023). Whole-cortex in situ sequencing reveals peripheral input-dependent cell type-defined area identity. bioRxiv 2022.11.06.515380. doi: https://doi.org/10.1101/2022.11.06.515380.
Zhang, A.^†, Jin, L.^†, Yao, S., Matsuyama, M., van Velthoven, C., Sullivan, H., Sun, N., Kellis, M., Tasic, B., Wickersham, I.R.*, Chen, X.* (2024). Rabies virus-based barcoded neuroanatomy resolved by single-cell RNA and in situ sequencing. eLife. 12:RP87866. doi: https://doi.org/10.7554/eLife.87866.3
Chen, Y.^†, Chen, X.^†, Baserdem, B., Zhan, H., Li, Y., Davis, M.B., Kebschull, J.M., Zador, A.M., Koulakov, A.A., Albeanu, D.F. (2022). High-throughput sequencing of single neuron projections reveals spatial organization in the olfactory cortex. Cell. 185(2), 4117-4134.e28. doi: 10.1016/j.cell.2022.09.038.
Muñoz-Castaneda, R.^†, Zingg, B.^†, Matho, K.S.^†, Chen, X.^†, Wang, Q.^†, et al. (2021). Cellular Anatomy of the Mouse Primary Motor Cortex. Nature. 598, 159-166. doi: 10.1038/s41586-021-03970-w.
Sun, Y.^†, Chen, X.*^†, Fischer, S., Lu, S., Gillis, J., Zador, A.M.* (2021). Integrating barcoded neuroanatomy with spatial transcriptional profiling reveals cadherin correlates of projections shared across the cortex. Nat Neurosci.24(6), 873-885. doi: 10.1038/s41593-021-00842-4.
Chen, X., Sun, Y., Zhan, H., Kebschull, JM., Fischer, S., Matho, K., Huang, ZJ., Gillis, J., and Zador, AM. (2019). High-throughput mapping of long-range neuronal projection using in situ sequencing. Cell. 179(3), 772-786. doi: 10.1016/j.cell.2019.09.023.
Chen, X.*, Sun, Y., Church, GM., Lee, JH., and Zador, AM. (2018). Efficient in situ barcode sequencing using padlock probe-based BaristaSeq. Nucleic Acids Res. 46(4), e22. doi: 10.1093/nar/gkx1206.