Single Cell Transcriptomics and Spatial Localization in Mouse Neurons

Neurons are complex cells whose primary functions are to send and receive electrochemical signals to and from other neurons in their connected network. Though their primary functions are similar, neurons are spatially and functionally specialized across a wide array of neuronal subtypes. Characterizing the molecular contents and its variations within and across various neuronal subtypes can not only elucidate how these cell types interact with one another, but also elucidate how their molecular contents contribute to their functionality. To better understand the interplay between molecular composition and neuronal molecular function, I used single-cell RNA sequencing to characterize the dental primary afferent (DPA) neurons, a subset of the trigeminal ganglion that innervates the dental pulp and plays a significant role in dental hypersensitivity; I found that the DPA neurons were heterogenous and primarily composed of a subtype of neuron which had molecular characteristics of both proprioceptive and nociceptive neurons, which may partly explain how these neurons elicit a pain response to innocuous sensory stimuli, such as a light touch.Additionally, neurons are highly polarized cells with molecularly and functionally distinct subcellular compartments, which require complex molecular organization, partly mediated by mRNA localization. Consequently, tremendous efforts have been made to identify which mRNA species are present and enriched in the soma and dendrites. However, these studies do not describe the spatial distribution and fine scale localization of the mRNA within the subcellular compartment. Here, I utilize single molecule RNA FISH along with novel analytical techniques to characterize the spatial distribution of six previously uncharacterized genes using single molecule fluorescence in situ hybridization at high spatial resolution. I identified common patterns of spatial distributions and examined how morphological features of the dendrite might affect these patterns, as well as the spatial relationships of transcripts within the dendrites. Lastly, to address tradeoffs in spatial resolution and transcriptomic throughput in current methodologies to study mRNA localization, I developed a new technology that can physico-chemically guide the placement of the neurons and the growth of their projections. I used this technology to perform subcellular microdissections on neurons to compare the sub-compartmental transcriptomes of proximal and distal sections of neuronal dendrites. From these experiments, I discovered that localization is not broadly uniform along the length of the dendrites, with sets of genes preferring proximal or distal sections of the dendrites, speaking to potential molecular and functional differences between the two compartments.

Author dc:creator

• Kim, Jihoon

Advisor dc:contributor.advisor

• Kim, Junhyong

• Neuroscience and Neurobiology
• Bioinformatics
• Engineering

Language dc:language.iso

en

Repository record dc:identifier.uri

https://repository.upenn.edu/handle/20.500.14332/60482

OAI identifier oai:identifier

oai:repository.upenn.edu:20.500.14332/60482