Supplementary Material for: Comparative Single Cell Transcriptome Analysis of c-Met Receptor Expressing and Non-Expressing Projection Neurons in the Developing Frontal and Visual Cortices

Introduction. Single cell transcriptomic analyses in adult mice show that cortical projection neuron (PN) subclasses exhibit heterogenous gene expression profiles that reflect their projection targets, and laminar and areal positions. Further analyses revealed that PNs within the same subclass also exhibit transcriptomic heterogeneity. Recent evidence suggests that differences in maturation state reflect one source of this heterogeneity. The MET receptor tyrosine kinase, a regulator of synapse maturation, is expressed in a subpopulation within cortical PN subclasses, providing an experimental model to address transcriptomic heterogeneity within developing PN subclasses. Methods. Single cell RNA sequencing and smFISH were used to identify transcriptomic differences between Met+ and Met- PN populations in the mouse visual and frontal cortices during the early phase of synapse formation and dendritic growth. Results. Analyses confirmed enrichment of Met in select PN subclasses and further identified astrocytes as the major source of its ligand, Hgf. No genes were expressed uniquely in Met+ or Met- PNs within a subclass; rather, there were graded differences in gene expression between the populations. While the identity of differentially expressed genes varied between subclass and cortical area, there was a consistent overrepresentation of genes associated with axon growth, as well as synapse structure, development and function, with a subset associated with the MET interactome. Further, compared to Met- PNs, expression differences in genes associated with maturation indicate less mature excitatory synapses and spines in the Met+ population at this age. Conclusion. The current findings provide support for the hypothesis that Met+ PNs are in a less mature state than Met- PNs within the same subclass. Further, the data are consistent with converging lines of biochemical and electrophysiological evidence that MET contributes to asynchronous maturation of developing cortical circuits.