Spatial Transcriptomics Technologies Research Articles

Modeling zero inflation is not necessary for spatial transcriptomics

BackgroundSpatial transcriptomics are a set of new technologies that profile gene expression on tissues with spatial localization information. With technological advances, recent spatial transcriptomics data are often in the form of sparse counts with an excessive amount of zero values.ResultsWe perform a comprehensive analysis on 20 spatial transcriptomics datasets collected from 11 distinct technologies to characterize the distributional properties of the expression count data and understand the statistical nature of the zero values. Across datasets, we show that a substantial fraction of genes displays overdispersion and/or zero inflation that cannot be accounted for by a Poisson model, with genes displaying overdispersion substantially overlapped with genes displaying zero inflation. In addition, we find that either the Poisson or the negative binomial model is sufficient for modeling the majority of genes across most spatial transcriptomics technologies. We further show major sources of overdispersion and zero inflation in spatial transcriptomics including gene expression heterogeneity across tissue locations and spatial distribution of cell types. In particular, when we focus on a relatively homogeneous set of tissue locations or control for cell type compositions, the number of detected overdispersed and/or zero-inflated genes is substantially reduced, and a simple Poisson model is often sufficient to fit the gene expression data there.ConclusionsOur study provides the first comprehensive evidence that excessive zeros in spatial transcriptomics are not due to zero inflation, supporting the use of count models without a zero inflation component for modeling spatial transcriptomics.

Cell type identification in spatial transcriptomics data can be improved by leveraging cell-type-informative paired tissue images using a Bayesian probabilistic model.

Spatial transcriptomics technologies have recently emerged as a powerful tool for measuring spatially resolved gene expression directly in tissues sections, revealing cell types and their dysfunction in unprecedented detail. However, spatial transcriptomics technologies are limited in their ability to separate transcriptionally similar cell types and can suffer further difficulties identifying cell types in slide regions where transcript capture is low. Here, we describe a conceptually novel methodology that can computationally integrate spatial transcriptomics data with cell-type-informative paired tissue images, obtained from, for example, the reverse side of the same tissue section, to improve inferences of tissue cell type composition in spatial transcriptomics data. The underlying statistical approach is generalizable to any spatial transcriptomics protocol where informative paired tissue images can be obtained. We demonstrate a use case leveraging cell-type-specific immunofluorescence markers obtained on mouse brain tissue sections and a use case for leveraging the output of AI annotated H&E tissue images, which we used to markedly improve the identification of clinically relevant immune cell infiltration in breast cancer tissue. Thus, combining spatial transcriptomics data with paired tissue images has the potential to improve the identification of cell types and hence to improve the applications of spatial transcriptomics that rely on accurate cell type identification.

Reference-free cell\xa0type deconvolution of multi-cellular pixel-resolution spatially resolved transcriptomics data

Recent technological advancements have enabled spatially resolved transcriptomic profiling but at multi-cellular pixel resolution, thereby hindering the identification of cell-type-specific spatial patterns and gene expression variation. To address this challenge, we develop STdeconvolve as a reference-free approach to deconvolve underlying cell types comprising such multi-cellular pixel resolution spatial transcriptomics (ST) datasets. Using simulated as well as real ST datasets from diverse spatial transcriptomics technologies comprising a variety of spatial resolutions such as Spatial Transcriptomics, 10X Visium, DBiT-seq, and Slide-seq, we show that STdeconvolve can effectively recover cell-type transcriptional profiles and their proportional representation within pixels without reliance on external single-cell transcriptomics references. STdeconvolve provides comparable performance to existing reference-based methods when suitable single-cell references are available, as well as potentially superior performance when suitable single-cell references are not available. STdeconvolve is available as an open-source R software package with the source code available at https://github.com/JEFworks-Lab/STdeconvolve.

DestVI identifies continuums of cell types in spatial transcriptomics data.

Most spatial transcriptomics technologies are limited by their resolution, with spot sizes larger than that of a single cell. Although joint analysis with single-cell RNA sequencing can alleviate this problem, current methods are limited to assessing discrete cell types, revealing the proportion of cell types inside each spot. To identify continuous variation of the transcriptome within cells of the same type, we developed Deconvolution of Spatial Transcriptomics profiles using Variational Inference (DestVI). Using simulations, we demonstrate that DestVI outperforms existing methods for estimating gene expression for every cell type inside every spot. Applied to a study of infected lymph nodes and of a mouse tumor model, DestVI provides high-resolution, accurate spatial characterization of the cellular organization of these tissues and identifies cell-type-specific changes in gene expression between different tissue regions or between conditions. DestVI is available as part of the open-source software package scvi-tools ( https://scvi-tools.org ).

Spatial Transcriptomic Analysis of Acute Heart Rejection Model

<h3>Purpose</h3> Heart transplantation (HTx) is sometimes the only treatment for end-stage cardiac disease, but survival after HTx is limited. Understanding the molecular mechanisms behind allograft injury, dysfunction, and failure, are critical in improving the patients' outcome. Rapidly evolving single cell and spatial transcriptomic technologies have opened a new field of precision to diagnostics and therapeutics of cardiovascular diseases. Here we studied the cellular mechanisms of acute rejection in a rat heart transplant model with spatial transcriptomics methods. <h3>Methods</h3> We investigated the effects of acute rejection in heterotopic rat heart transplantation model with syngeneic (DA-DA) and major MHC-mismatched allogeneic (DA-WF) recipients. The recipient rats were sacrificed 5 days after transplantation, and graft cross-sections were analyzed with the Visium Spatial Gene Expression platform (10xGenomics). Raw sequencing reads were processed using SpaceRanger. All main data analysis steps were performed using Seurat. Clusters were identified based on the expression of several known cell markers. <h3>Results</h3> From all samples together, a total of 17 clusters of spots were identified. Our analysis suggests that two of the clusters presented mainly cardiomyocytes, one cluster of smooth muscle cells, two clusters of fibroblasts, 11 immune cells and one cluster of erythrocytes. Endothelial cells did not form a unique cluster. Syngeneic HTx alone induced marked transcriptomic changes in 5 clusters based on differential gene expression analysis, mainly increasing the proportion of clusters with certain types of macrophages. Few clusters remained unchanged between the allogenic and the syngeneic groups. Allogenic group had four unique clusters representing mainly different immune cell populations: granulocytes, lymphocytes and a type of macrophages that were not present in other samples. One cluster consisted of erythrocytes from hemorrhagic area. <h3>Conclusion</h3> Acute rejection changes the cellular composition of the heart muscle dramatically and induces transcriptomic changes in the structural cells of the heart. Our spatial transcriptomic analysis provides novel transcriptomic data on cellular changes occurring following acute rejection. This method allows us to study the cells and mechanisms involved in rejection and look for potential targets for drug development and therapeutics.

Analyzing Spatial Transcriptomics Data Using Giotto.

Spatial transcriptomic technologies have been developed rapidly in recent years. The addition of spatial context to expression data holds the potential to revolutionize many fields in biology. However, the lack of computational tools remains a bottleneck that is preventing the broader utilization of these technologies. Recently, we have developed Giotto as a comprehensive, generally applicable, and user-friendly toolbox for spatial transcriptomic data analysis and visualization. Giotto implements a rich set of algorithms to enable robust spatial data analysis. To help users get familiar with the Giotto environment and apply it effectively in analyzing new datasets, we will describe the detailed protocols for applying Giotto without any advanced programming skills. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Getting Giotto set up for use Basic Protocol 2: Pre-processing Basic Protocol 3: Clustering and cell-type identification Basic Protocol 4: Cell-type enrichment and deconvolution analyses Basic Protocol 5: Spatial structure analysis tools Basic Protocol 6: Spatial domain detection by using a hidden Markov random field model Support Protocol 1: Spatial proximity-associated cell-cell interactions Support Protocol 2: Assembly of a registered 3D Giotto object from 2D slices.

Spatial transcriptomics and the kidney.

The application of spatial transcriptomics technologies to the interrogation of kidney tissue is a burgeoning effort. These technologies share a common purpose in mapping both the expression of individual molecules and entire transcriptomic signatures of kidney cell types and structures. Such information is often superimposed upon a histologic image. The resulting datasets are readily merged with other imaging and transcriptomic techniques to establish a spatially anchored atlas of the kidney. This review provides an overview of the various spatial transcriptomic technologies and recent studies in kidney disease. Potential applications gleaned from the interrogation of other organ systems, but relative to the kidney, are also discussed. Spatial transcriptomic technologies have enabled localization of whole transcriptome mRNA expression, correlation of mRNA to histology, measurement of in situ changes in expression across time, and even subcellular localization of transcripts within the kidney. These innovations continue to aid in the development of human cellular atlases of the kidney, the reclassification of disease, and the identification of important therapeutic targets. Spatial localization of gene expression will complement our current understanding of disease derived from single cell RNA sequencing, histopathology, protein immunofluorescence, and electron microscopy. Although spatial technologies continue to evolve rapidly, their importance in the localization of disease signatures is already apparent. Further efforts are required to integrate whole transcriptome and subcellular expression signatures into the individualized assessment of human kidney disease.

Deconvolution Tactics and Normalization in Renal Spatial Transcriptomics.

The kidney is composed of heterogeneous groups of epithelial, endothelial, immune, and stromal cells, all in close anatomic proximity. Spatial transcriptomic technologies allow the interrogation of in situ expression signatures in health and disease, overlaid upon a histologic image. However, some spatial gene expression platforms have not yet reached single-cell resolution. As such, deconvolution of spatial transcriptomic spots is important to understand the proportion of cell signature arising from these varied cell types in each spot. This article reviews the various deconvolution strategies discussed in the 2021 Indiana O’Brien Center for Microscopy workshop. The unique features of Seurat transfer score methodology, SPOTlight, Robust Cell Type Decomposition, and BayesSpace are reviewed. The application of normalization and batch effect correction across spatial transcriptomic samples is also discussed.

Cell2location maps fine-grained cell types in spatial transcriptomics.

Spatial transcriptomic technologies promise to resolve cellular wiring diagrams of tissues in health and disease, but comprehensive mapping of cell types in situ remains a challenge. Here we present сell2location, a Bayesian model that can resolve fine-grained cell types in spatial transcriptomic data and create comprehensive cellular maps of diverse tissues. Cell2location accounts for technical sources of variation and borrows statistical strength across locations, thereby enabling the integration of single-cell and spatial transcriptomics with higher sensitivity and resolution than existing tools. We assessed cell2location in three different tissues and show improved mapping of fine-grained cell types. In the mouse brain, we discovered fine regional astrocyte subtypes across the thalamus and hypothalamus. In the human lymph node, we spatially mapped a rare pre-germinal center B cell population. In the human gut, we resolved fine immune cell populations in lymphoid follicles. Collectively, our results present сell2location as a versatile analysis tool for mapping tissue architectures in a comprehensive manner.

Principles of Spatial Transcriptomics Analysis: A Practical Walk-Through in Kidney Tissue.

Spatial transcriptomic technologies capture genome-wide readouts across biological tissue space. Moreover, recent advances in this technology, including Slide-seqV2, have achieved spatial transcriptomic data collection at a near-single cell resolution. To-date, a repertoire of computational tools has been developed to discern cell type classes given the transcriptomic profiles of tissue coordinates. Upon applying these tools, we can explore the spatial patterns of distinct cell types and characterize how genes are spatially expressed within different cell type contexts. The kidney is one organ whose function relies upon spatially defined structures consisting of distinct cellular makeup. Thus, the application of Slide-seqV2 to kidney tissue has enabled us to elucidate spatially characteristic cellular and genetic profiles at a scale that remains largely unexplored. Here, we review spatial transcriptomic technologies, as well as computational approaches for cell type mapping and spatial cell type and transcriptomic characterizations. We take kidney tissue as an example to demonstrate how the technologies are applied, while considering the nuances of this architecturally complex tissue.

Spatial Transcriptomics to define transcriptional patterns of zonation and structural components in the mouse liver

Reconstruction of heterogeneity through single cell transcriptional profiling has greatly advanced our understanding of the spatial liver transcriptome in recent years. However, global transcriptional differences across lobular units remain elusive in physical space. Here, we apply Spatial Transcriptomics to perform transcriptomic analysis across sectioned liver tissue. We confirm that the heterogeneity in this complex tissue is predominantly determined by lobular zonation. By introducing novel computational approaches, we enable transcriptional gradient measurements between tissue structures, including several lobules in a variety of orientations. Further, our data suggests the presence of previously transcriptionally uncharacterized structures within liver tissue, contributing to the overall spatial heterogeneity of the organ. This study demonstrates how comprehensive spatial transcriptomic technologies can be used to delineate extensive spatial gene expression patterns in the liver, indicating its future impact for studies of liver function, development and regeneration as well as its potential in pre-clinical and clinical pathology.

Gastrointestinal mucosal biopsies in Parkinson's disease: beyond alpha-synuclein detection.

Alpha-synuclein deposits, the pathological hallmarks of Parkinson's disease, are consistently found in the gastrointestinal tract of parkinsonian subjects. These observations have raised the potential that endoscopically obtainable mucosal biopsies can aid to a molecular diagnosis of the disease. The possible usefulness of mucosal biopsies is, however, not limited to the detection of alpha-synuclein, but also extends to other essential aspects underlying pathophysiological mechanisms of gastrointestinal manifestations in Parkinson's disease. The aim of the current review is to provide an appraisal of the existing studies showing that gastrointestinal biopsies can be used for the analysis of enteric neuronal and glial cell morphology, intestinal epithelial barrier function, and gastrointestinal inflammation in Parkinson's disease. A perspective on the generation of organoids with GI biopsies and the potential use of single-cell and spatial transcriptomic technologies will be also addressed.

Spatial deconvolution of HER2-positive breast cancer delineates tumor-associated cell type interactions

In the past decades, transcriptomic studies have revolutionized cancer treatment and diagnosis. However, tumor sequencing strategies typically result in loss of spatial information, critical to understand cell interactions and their functional relevance. To address this, we investigate spatial gene expression in HER2-positive breast tumors using Spatial Transcriptomics technology. We show that expression-based clustering enables data-driven tumor annotation and assessment of intra- and interpatient heterogeneity; from which we discover shared gene signatures for immune and tumor processes. By integration with single cell data, we spatially map tumor-associated cell types to find tertiary lymphoid-like structures, and a type I interferon response overlapping with regions of T-cell and macrophage subset colocalization. We construct a predictive model to infer presence of tertiary lymphoid-like structures, applicable across tissue types and technical platforms. Taken together, we combine different data modalities to define a high resolution map of cellular interactions in tumors and provide tools generalizing across tissues and diseases.

Exploring tissue architecture using spatial transcriptomics.

Deciphering the principles and mechanisms by which gene activity orchestrates complex cellular arrangements in multicellular organisms has far-reaching implications for research in the life sciences. Recent technological advances in next-generation sequencing- and imaging-based approaches have established the power of spatial transcriptomics to measure expression levels of all or most genes systematically throughout tissue space, and have been adopted to generate biological insights in neuroscience, development andplant biology as well asto investigate a range of disease contexts, including cancer. Similar to datasets made possible by genomic sequencing and population health surveys, the large-scale atlases generated by this technology lend themselves to exploratory data analysis for hypothesis generation. Here we review spatial transcriptomic technologies and describe the repertoire of operations available for paths of analysis of the resulting data. Spatial transcriptomics can also be deployed for hypothesis testing using experimental designs that compare time points or conditions-including genetic or environmental perturbations. Finally, spatial transcriptomic data are naturally amenable to integration with other data modalities, providing an expandable framework for insight into tissue organization.

CoSTA: unsupervised convolutional neural network learning for spatial transcriptomics analysis

BackgroundThe rise of spatial transcriptomics technologies is leading to new insights about how gene regulation happens in a spatial context. Determining which genes are expressed in similar spatial patterns can reveal gene regulatory relationships across cell types in a tissue. However, many current analysis methods do not take full advantage of the spatial organization of the data, instead treating pixels as independent features. Here, we present CoSTA: a novel approach to learn spatial similarities between gene expression matrices via convolutional neural network (ConvNet) clustering.ResultsBy analyzing simulated and previously published spatial transcriptomics data, we demonstrate that CoSTA learns spatial relationships between genes in a way that emphasizes broader spatial patterns rather than pixel-level correlation. CoSTA provides a quantitative measure of expression pattern similarity between each pair of genes rather than only classifying genes into categories. We find that CoSTA identifies narrower, but biologically relevant, sets of significantly related genes as compared to other approaches.ConclusionsThe deep learning CoSTA approach provides a different angle to spatial transcriptomics analysis by focusing on the shape of expression patterns, using more information about the positions of neighboring pixels than would an overlap or pixel correlation approach. CoSTA can be applied to any spatial transcriptomics data represented in matrix form and may have future applications to datasets such as histology in which images of different genes are from similar but not identical biological sections.

StPlus: a reference-based method for the accurate enhancement of spatial transcriptomics.

MotivationSingle-cell RNA sequencing (scRNA-seq) techniques have revolutionized the investigation of transcriptomic landscape in individual cells. Recent advancements in spatial transcriptomic technologies further enable gene expression profiling and spatial organization mapping of cells simultaneously. Among the technologies, imaging-based methods can offer higher spatial resolutions, while they are limited by either the small number of genes imaged or the low gene detection sensitivity. Although several methods have been proposed for enhancing spatially resolved transcriptomics, inadequate accuracy of gene expression prediction and insufficient ability of cell-population identification still impede the applications of these methods.ResultsWe propose stPlus, a reference-based method that leverages information in scRNA-seq data to enhance spatial transcriptomics. Based on an auto-encoder with a carefully tailored loss function, stPlus performs joint embedding and predicts spatial gene expression via a weighted k-nearest-neighbor. stPlus outperforms baseline methods with higher gene-wise and cell-wise Spearman correlation coefficients. We also introduce a clustering-based approach to assess the enhancement performance systematically. Using the data enhanced by stPlus, cell populations can be better identified than using the measured data. The predicted expression of genes unique to scRNA-seq data can also well characterize spatial cell heterogeneity. Besides, stPlus is robust and scalable to datasets of diverse gene detection sensitivity levels, sample sizes and number of spatially measured genes. We anticipate stPlus will facilitate the analysis of spatial transcriptomics.Availability and implementationstPlus with detailed documents is freely accessible at http://health.tsinghua.edu.cn/software/stPlus/ and the source code is openly available on https://github.com/xy-chen16/stPlus.

Abstract 2696: Spatially resolved whole transcriptome molecular investigation of FFPE triple positive breast cancer tumors using the Visium platform

Abstract Triple-positive breast cancer (TPBC) is a rare but aggressive form of human epidermal growth factor receptor 2 (HER2) positive cancer. It accounts for fewer than 10% of all new breast cancer diagnoses in the USA annually. TPBC routinely exhibits elevated levels of HER2, along with oncogenic cell surface estrogen receptors (ER) and progesterone receptors (PR), but the underlying phenotype and survival rates vary. To better understand TPBC heterogeneity, we examined the tumor microenvironment by characterizing the spatial distribution of cellular transcriptomes. Spatial transcriptomics technology combines the benefits of histological techniques with the massive throughput and discovery power of RNA sequencing, addressing the limitations of traditional pathological examination. However, standard clinical workflows collect formalin-fixed paraffin-embedded (FFPE) tissue, which can significantly damage molecules such as RNA, making investigation of the underlying biology at the transcriptomic level intractable. We utilized the 10x Genomics Visium Spatial Gene Expression Solution for FFPE tissue to analyze and resolve tumorigenic profiles of serial sections of TPBC samples. This assay incorporates ~5,000 barcoded, spatially encoded capture probes in spots on a slide onto which tissue is placed, imaged, and permeabilized. Native RNA is detected and quantified using an FFPE compatible whole transcriptome probeset. Imaging and sequencing data are processed, resulting in a spatially resolved transcriptional readout. Expression profiles and spatially mapped clustering data from FFPE sections processed using the whole transcriptome probeset highly correlated with data from fresh-frozen tissue. The expression signatures also aligned with pathologist annotations combined with immunofluorescent staining to demarcate the tumor, stromal, and immune compartments as well as HER2, PR, and ER. These data were paired with single nucleus RNA-seq (Chromium Single Cell 3' assay), generating cell type expression profiles giving insight into cell-type co-localization. Taken together, these results demonstrate that whole transcriptome profiling of FFPE tissues using the Visium platform provide a powerful complement to traditional histopathological methods. This technology allows analysis of the whole transcriptome across tissue sections with high sensitivity and specificity, paired with morphological context and protein co-detection. The pairing of the whole transcriptome profile of TPBC FFPE samples at both the single nucleus and spatial level provides a comprehensive understanding of the tumor architecture. This in-depth knowledge, enabled by Visium for FFPE tissues, can provide unprecedented insights into tumor biology, disease progression, predictive biomarkers, drug response and resistance, and development of therapeutic targets. Citation Format: Stephen R. Williams, Mesruh Turkekul, Aleksandra Jurek, Marco Mignardi, Paulius Mielinis, Neil I. Weisenfeld, Marlon Stoeckius, James Chell. Spatially resolved whole transcriptome molecular investigation of FFPE triple positive breast cancer tumors using the Visium platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2696.

Single-cell spatial transcriptomic analysis reveals common and divergent features of developing postnatal granule cerebellar cells and medulloblastoma

BackgroundCerebellar neurogenesis involves the generation of large numbers of cerebellar granule neurons (GNs) throughout development of the cerebellum, a process that involves tight regulation of proliferation and differentiation of granule neuron progenitors (GNPs). A number of transcriptional regulators, including Math1, and the signaling molecules Wnt and Shh have been shown to have important roles in GNP proliferation and differentiation, and deregulation of granule cell development has been reported to be associated with the pathogenesis of medulloblastoma. While the progenitor/differentiation states of cerebellar granule cells have been broadly investigated, a more detailed association between developmental differentiation programs and spatial gene expression patterns, and how these lead to differential generation of distinct types of medulloblastoma remains poorly understood. Here, we provide a comparative single-cell spatial transcriptomics analysis to better understand the similarities and differences between developing granule and medulloblastoma cells.ResultsTo acquire an enhanced understanding of the precise cellular states of developing cerebellar granule cells, we performed single-cell RNA sequencing of 24,919 murine cerebellar cells from granule neuron-specific reporter mice (Math1-GFP; Dcx-DsRed mice). Our single-cell analysis revealed that there are four major states of developing cerebellar granule cells, including two subsets of granule progenitors and two subsets of differentiating/differentiated granule neurons. Further spatial transcriptomics technology enabled visualization of their spatial locations in cerebellum. In addition, we performed single-cell RNA sequencing of 18,372 cells from Patched+/− mutant mice and found that the transformed granule cells in medulloblastoma closely resembled developing granule neurons of varying differentiation states. However, transformed granule neuron progenitors in medulloblastoma exhibit noticeably less tendency to differentiate compared with cells in normal development.ConclusionIn sum, our study revealed the cellular and spatial organization of the detailed states of cerebellar granule cells and provided direct evidence for the similarities and discrepancies between normal cerebellar development and tumorigenesis.

SpatialDWLS: accurate deconvolution of spatial transcriptomic data

Recent development of spatial transcriptomic technologies has made it possible to characterize cellular heterogeneity with spatial information. However, the technology often does not have sufficient resolution to distinguish neighboring cell types. Here, we present spatialDWLS, to quantitatively estimate the cell-type composition at each spatial location. We benchmark the performance of spatialDWLS by comparing it with a number of existing deconvolution methods and find that spatialDWLS outperforms the other methods in terms of accuracy and speed. By applying spatialDWLS to a human developmental heart dataset, we observe striking spatial temporal changes of cell-type composition during development.

Spatial Transcriptomics of Supraoptic Nucleus and Paraventricular Nucleus of the Hypothalamus

Magnocellular neurosecretory cells (MNCs) in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus release oxytocin (OXY) and arginine vasopressin (AVP) into the peripheral circulation, playing a key role in the regulation of fluid and electrolyte homeostasis. Several groups have investigated transcriptomics in these regions; however, the data lack spatial context important for better understanding physiological functions of genes. Our study leverages spatially-resolved transcriptomics to visualize the relationship between cells, their gene expression profiles, and their relative locations within a tissue sample. Here, we use a novel spatial transcriptomics technology, Visium Spatial Gene Expression, manufactured by 10x Genomics to obtain spatially-resolved gene expression data for the SON and PVN of an adult male Sprague-Dawley rat. Briefly, a fresh frozen brain was sectioned at 10μm thickness to collect four sections (~4x4mm) containing SON and PVN regions. The tissue was then mounted and fixed on the capture areas of a Visium slide containing probes that bind mRNA. Next, hematoxylin and eosin (H&E) staining and high-resolution imaging of the slide was performed prior to tissue permeabilization. The permeabilization releases mRNA from cells that then binds to capture probes on the slide beneath the tissue. cDNA is then synthesized from captured mRNA from which sequencing libraries are prepared. Sequence data is then visualized, using 10x Genomics’ Space Ranger and Loupe Browser applications, to overlay spatial gene expression data with our H&E stained images. Foremost, our results show that gene cluster analysis successfully differentiated myelinated fiber tracts from nuclei and identified several distinct neuronal populations. Gene clusters overlaying fiber tracts contain glia-specific markers (e.g., Mbp and Gfap) and those overlaying SON and PVN regions contain neuron-specific markers (e.g., Syn1 and Nrgn). These data also have the resolution to resolve OXY- and AVP-specific clusters in the SON via targeted gene analysis, with anatomical distribution consistent with immunohistochemistry results for the regions. Additionally, our results show clustering of SON with magnocellular PVN, while parvocellular PVN was a different cluster. Differential expression analysis of these two clusters revealed relevant genes for the distinct regions and neuronal subpopulations in the top 20 globally distinguishing genes (e.g., Avp and Oxt for SON and magnocellular PVN cluster and Trh for parvocellular PVN cluster). This spatially-resolved transcriptomic data allows us to obtain whole transcriptomic data without sacrificing morphological context (e.g., SON and PVN regions). Future spatial transcriptomic studies can be used to investigate changes in MNC gene expression in terms of sex differences and disease models that can potentially inform cellular mechanisms involved in pathophysiology.