Spatial Mapping of Unique Cellular Populations as a Foundation for Functional Characterization

Expression of spatial molecular gradients is a defining feature in essentially every phase of cellular organ development. While much of the research regarding these gradients has been conducted in developing systems, there is strong evidence suggesting that spatial gradients play an important role in adulthood as well. A robust example of post-developmental gradients is seen in the liver, which is uniquely organized into metabolic zones that are characterized by the graded expression of gene markers, proteins, nutrients, and oxygen levels. This makes the liver an ideal organ to model studies of post-developmental gradients in more complex systems. Here, we establish methodology for studying gradients of expression via fluorescent in situ hybridization technique, multiplex RNAscope. In doing so, we also create the first spatial atlas of the ten Frizzled receptors, which are involved in canonical Wnt signaling, in adult mouse liver and, in parallel, utilize publicly available single-cell RNA sequencing data to define Frizzled receptor expression in various hepatic cell types, revealing surprising zonation of Frizzled receptor 6 (Chapter 2). Once established, we then translate this methodology to study molecular spatial gradients in the brain. For this, we turn to the striatum, given its heterogenous cellular composition of dopamine receptor-expressing neurons and their roles in a wide-range of neurodegenerative and psychiatric disease. In Chapter 3, using multiplex RNAscope, we elucidate striatal dopamine receptor expression of spiny projection neurons (SPNs) along both dorsal-ventral and rostral-caudal axes. We confirm the expression of SPNs that express only one dopamine receptor subtype versus SPNs that co-express multiple subtypes. These subpopulations are not only displayed in gradients along each axis but are also transcriptomically unique in a species-conserved manner. Further exploration of a unique SPN subpopulation that co-expresses D1 and D2 dopamine receptors reveals that these cells possess distinct electrophysiological qualities compared to SPNs that express the D1 receptor alone. Lastly, we show that these SPN subpopulations may uniquely contribute to genetic risk in a plethora of neurological-related conditions. Overall, this dissertation represents a foundation for studying post-developmental gradients and cell-type heterogeneity in adult systems for further characterization of their roles in both health and disease.