Fig 1: Notch receptor-ligand interactions mediate EV internalization selectively by neurons(A) Proteinase K (PK) treatment reduces the apparent Notch1 and Notch2 molecular weight by ~10 kDa, consistent with removal of the extracellular portion of NotchICD. No intact CD81 was detected in the PK-treated EVs presumably due to multiple cleavage events, while Sdcbp was unaffected by PK treatment.(B) Schematic illustrating the arrangement of Notch1, Notch2, CD81, and Sdcbp in EVs.(C) Representative ICC images showing Notch ligands Jag1, Jag2, Dll1, and Dll4 colocalize with internalized neuronal EVs, labeled with CFSE. Top, neuronal soma, scale bar, 10 μm; bottom, dendrites, scale bar, 5 μm(D) Quantification of the percentage of CFSE-labeled EVs colocalized with the indicated Notch ligands in neuronal soma. One-tailed Student’s t test, n = 12–15 from two cultures. NS, not significant. *p value (Jag1 vs. Jag2) = 0.0204, **p value (Jag1 vs. Dll1) = 0.0082, **p value (Jag1 vs. Dll4) = 0.0051, p value (Jag2 vs. Dll1) = 0.2947, p value (Jag2 vs. Dll4) = 0.2476, p value (Dll1 vs. Dll4) = 0.4396.(E) Left, CFSE labeled EVs that are internalized by primary cultured rat hippocampal neurons remain generally intact and punctate. Right, neurons fail to internalize PK-treated EVs. Scale bar, 10 μm.(F) Following a 60-min incubation of primary cultured rat hippocampal neurons with EVs, there is a notable increase in the levels of activated Notch1, activated Notch2, and Hes1 proteins. This effect is not observed with EVs treated with DeltaMAX or with DeltaMAX treatment alone.(G–I) Quantifications of (F). One-tailed Student’s t test, n = 4 biological. (G) ***p value (Veh vs. EV) = 0.0002, ***p value (EV vs. DeltaMAX -treated EV) = 0.0004, NS p value (DeltaMAX -treated EV vs. DeltaMAX) = 0.4453. (H) **p value (Veh vs. EV) = 0.0043, **p value (EV vs. DeltaMAX-treated EV) = 0.0042, NS p value (DeltaMAX-treated EV vs. DeltaMAX) = 0.4927. (I) ***p value (Veh vs. EV) = 0.0004, **p value (EV vs. DeltaMAX-treated EV) = 0.0011, NS p value (DeltaMAX -treated EV vs. DeltaMAX) = 0.2677. See also Figures S2 and S3.
Fig 2: MMD-Dll4 pathway expression in RPE-choroid tissues of laser-induced CNV. RPE-choroid tissues were resected from mice exhibiting laser-induced CNV. (A) The expression of MMD, Dll1 and Dll4 was determined by western blotting, with β-actin as a control. Each experiment was repeated three times. Statistical analysis was performed using ImageJ software. (B) Reverse transcription-quantitative PCR was performed, and the expression of MMD, Dll4 and Dll1 was normalized to β-actin. Data are expressed as the mean ± SD (n=3 mice per group). *P<0.05 and **P<0.01 as indicated. Dll1, delta-like 1; Dll4, delta-like 4; IL-4, adenovirus-expressed IL-4 vector; LacZ and LacZ, adenovirus-expressed β-galactosidase; MMD, monocyte to macrophage differentiation-associated; ns, no significance; RPE, retinal pigment epithelium; WT, wild-type.
Fig 3: Foxc1 mutant VZ neural progenitors have elevated Notch signaling and are rescued by retinoic acid treatment(A–C) Representative images (A) and fluorescence intensity quantification (B and C) of Dll1 (yellow) and Notch1 (magenta) immunolabeling in E14 control and Foxc1-mutant neocortex exposed to control or retinoic acid (RA)-supplemented diets.(D and E) Representative images (D) and fluorescence intensity quantification (E) of NICD (magenta) immunolabeling in E14 control and Foxc1-mutant neocortical VZ exposed to control or RA-supplemented diets.(F) Tracks depict ATAC peaks near the Hes1, Hes5, and Notch1 locus in control and Foxc1 mutants (−/+RA), with fold change in peak.Dotted lines outline the ventricular surface. Means for each biological replicate (n = 3 embryos for each genotype treatment) are represented by colored shapes and corresponding technical replicates (n = 3 images per embryo) are in gray matching shapes; lines show the mean. **p < 0.01 and ***p < 0.001.
Fig 4: Spatial transcriptomic profiling of Foxc1 mutants identifies increased Notch signaling pathway in neural progenitors(A and B) Forebrain clusters from E14 control and Foxc1-mutant tissue sections were selected (A), and clusters were annotated and represented as a UMAP split by sample (controls or Foxc1 KOs, n = 2) (B).(C) The number of spots representing cellular populations were quantified for each condition.(D) From spatial data, comparison of control and Foxc1-KO AP and IP clusters for “neurogenic” and “expansion” genes.(E) GSEA between control and Foxc1-KO AP and IP clusters showed a decrease in the pathways: neuron development, cell differentiation, cellular developmental process, system development, and multicellular organism development.(F) Differential gene expression analysis between controls and Foxc1 KOs using Ingenuity Pathway Analysis (IPA) showed the most significantly overlapped biological pathway, “development of neurons” (Z score = −4.005, p = 3.44E−49) and the predicted regulator Notch1 (increased 1.62-fold). IPA revealed that Notch1 signaling in Foxc1 KOs is increased with downstream Notch targets upregulated (Hes1, Hey1, Hes5, and Sox2).(G) Dot plots show Dll1, Notch1, Notch2, and Sox2 expression is increased in Foxc1-KO clusters relative to controls.(H and I) NICHES analysis was used to represent spatial sites of ligand receptor signaling in spatial transcriptomic data (H), suggesting that Dll1-Notch1/2 signaling had stronger signaling interactions in the cortical VZ and the VZ of the ganglionic eminences.
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