Fig 1: Identification of genes specific to area, time, and both area and time in the developing marmoset PFC(a) Schema for 10x Visium analysis of developing marmoset PFC. A representative image for the clusters of cortical layers for each region (mPFC, vPFC, dPFC) was presented in the registered spatial image at P16.(b) Confirmation of expression patterns for PFC stable area marker genes within PFC. ISH validated the enriched expression of area marker genes within PFC at P0 (CBLN4 in dPFC, PCDH17 in mPFC, and SYT17 in vmPFC and vPFC).(c) Confirmation of expression patterns for PFC temporal marker genes within PFC. ISH for THBS1 and INSYN2A validated the enriched expression at early developmental stage in the developing PFC (P0, 1M, 3M, and 6M).(d) Confirmation of expression patterns for PFC spatiotemporal marker genes within PFC. ISH for PRSS12, SLIT3, CHRD, FRZB, and CCN3 validated area-, time-, layer-specific expression in developing PFC. Each PFC region is indicated in the schema (i: mPFC, ii: dlPFC, iii: vmPFC, iv: dmPFC).(e) A dot plot showed distinct cortical layer marker genes in mPFC between mice and marmosets. Comparison of mPFC layer marker genes between P7 mice and P16 marmosets identified conserved and marmoset-specific layer marker genes in the developing mPFC. The upper panel displays conserved mPFC layer marker genes between species, while the lower panel displays marmoset-specific mPFC layer marker genes.
Fig 2: Marmoset specific genes control PFC neural circuits.(a) GO analysis from the comparison between P16 mPFC and 3M mPFC. The graphs show GO terms showing similarity to the category of “nervous system development” for biological process (BP).(b) ISH confirmed SLIT3 is highly expressed in marmoset mFPC at P0 not in mice mPFC at P6.(c) Schema of over expression of SLIT3 in mouse mPFC L2 neurons using IUE. Brains were harvested at P6 or 7, then DiI crystal was placed on MD of hemi-dissected brains to label MD neurons. Representative images showed co-electroporated YFP expression in mouse mPFC. Scale bars, 5 mm. Representative images showed Dil-labelled MD axons (white arrows) in MD regions and PFC in control and SLIT3-overexpressed brains. Scale bars, 1 mm. The mPFC regions are shown in the high-magnification images of the white dotted boxes. The white dashed lines indicate the border between white matters and gray matters. Scale bar, 100 mm.(d) Quantitative analysis of fluorescent intensities in mPFC of control and SLIT3-overexpression brains. The values normalized with control indicate the mean ± SEM. Mann-Whitney U test, **P < 0.01.(e) ISH showed Prss12 expression at P0 and P7 mouse mPFC. The surface of this part is marked by the blue dots.(f) ISH validated the layer 2-specific expression for PRSS12 in the mPFC of marmoset at P0. Additionally, a violin plot showed dynamic expression for PRSS12 during development in marmoset PFC from P16 to 6M.(g) Schema of over expression of PRSS12 in mouse mPFC L2 neurons using IUE. Brains were harvested at 3 weeks and co-electroporated YFP expression in cell bodies and fibers are observed in electroporation site, contralateral mPFC, BLA, and SNR. Scale bars, 200 mm.(h) Quantitative analysis of fluorescent intensities in each region. The values indicate the mean ± SEM. Mann-Whitney U test, *P < 0.05 and **P < 0.01.(i) The cartoon depicts the projection patterns from mPFC L2 neurons to contralateral mPFC, BLA, and SNR in mice and marmoset. In mice, mPFC L2 neurons project to the contralateral mPFC, BLA, SNR in control. However, PRSS12 overexpressing mouse mPFC L2 neurons showed decreased projection to BLA and SNR. Similarly, the marmoset A32 exhibits less projection to BLA and SNR compared to contralateral mPFC.(j) The scRNA-seq data from the human PFC exhibited similar expression patterns of PRSS12 compared to marmosets. PRSS12 is predominantly expressed in L2–3 cluster (highlighted by a black dashed circle in the left panel) during early developmental stages (highlighted by a black dashed circle in the right panel).
Supplier Page from Sino Biological, Inc. for Human PRSS12 Gene ORF cDNA clone expression plasmid