Outline of the characterization of mRNA spatial distribution within single neurons

A comparison of gene-specific mRNA densities across probe combinations and neuronal compartments. A) A violin plot of mRNA densities (number of detected mRNAs per pixel area of neuronal compartment) across the triplet probe combinations. Probe combinations are shown in different colors. B) A scatter plot of dendrite mRNA density vs. soma mRNA density. The dashed line represents the linear regression line of best fit. C) A violin plot of compartmental density ratio across genes. Pairwise Kolmogorov–Smirnov test was performed. Each asterisk represents a distinct level of statistical significance: one asterisk indicates adjusted p-value < 0.05, two asterisks denote adjusted p-value < 0.01, and three asterisks represent adjusted p-value < 0.001.

Characterizing the spatial distribution of dendritically localized mRNAs. A) Histograms of graph distances from the soma of dendritically localized mRNAs plotted per gene. Binwidth is 1 µm. B) Violin plot of Jensen-Shannon distances of mRNA distance distribution comparing two different neurons probed for the same gene (red) and mRNA distance distributions comparing different genes probed within the same neuron (blue). Distance values closer to 0 represent a higher degree of similarity between distributions. C) Histogram of mRNAs’ distances from the soma specific to each spatial patterning cluster. D) Bar graph representing the proportion of distance distributions from each gene that was assigned to each spatial pattern cluster. Genes are represented in separate colors.

Characterizing the spatial distribution of mRNAs within single dendrites to account for dendrite length. A) Histograms of normalized graph distances from the soma of dendritically localized mRNAs plotted per gene. B) Histogram of mRNAs’ normalized distances from the soma specific to each spatial patterning cluster. C) Violin plot of dendrite lengths of dendrites assigned to each spatial patterning cluster identified. D) Bar graph representing the proportion of distance distributions from each gene that was assigned to each spatial pattern cluster. Genes are represented in separate colors.

Analysis of dendritic morphology and mRNA spatial distributions using machine learning approaches. A) A schematic representation of the CNN model utilizing binary masks, Z-stacks, and maximum projection images of dendrites, integrated with one-hot encoded gene identity vectors, to predict cluster labels associated with mRNA distribution. B) Confusion matrices for the CNN classifier showing the performance on training and test datasets, highlighting the challenge of achieving high accuracy due to the unbalanced nature of the data. C) GradCAM visualizations demonstrating the convolutional layer activations associated with the various predicted clusters, which suggest patterns but do not conclusively identify specific morphological features across layers.

Simulation of passive transport in dendrites by random walk. Histograms of A) raw distances and B) normalized distances from the soma for simulated mRNAs. Each panel represents a snapshot at a specific time elapsed from the start of the simulation.

Colocalization frequencies of different gene combinations. Each cell presents the mean percentage of transcripts detected from transcripts from the row-labeled gene detected within 250 nm of transcripts from the column-labeled gene. Gene combinations of probes ordered in the same color channel could not be tested and left blank.

Characterizing colocalization frequencies of dendritically localized mRNAs at various search radii. Scatter plots present summarized Ripley’s K (number of samples with observed Ripley’s K larger than the 95th percentile subtracted by the number of samples with observed Ripley’s K was lower than the 5th percentile) at each radius. Each panel represents A) genes in analyses of self-colocalization or B) gene combinations in analyses of multi-gene colocalization. N represents the number of dendrite-gene/dendrite-gene combination pairs used in each analysis. C) Diagram of spatial organization of dendritically localized mRNAs derived from Ripley’s K analysis.