Journal: bioRxiv

Article Title: Dynamic regulation of NeuroD1 expression level by a novel viral construct during astrocyte-to-neuron reprogramming

doi: 10.1101/2025.02.17.638625

Figure Lengend Snippet: The reprogrammed neurons by the ND1-124T-GFP construct can express markers of mature neurons and neuronal subtypes in long term cultures. ( A ) A live image of HA cell cultures infected by ND1-GFP retrovirus showing floating cell spheroids in the hanging drops of culture medium at 3 DPI. ( B ) Live images of the resulting cell spheroids that are attached to a monolayer of HA cells at 20 DPI. #, the core of the attached spheroid; Arrowhead, a HA cell infected by ND1-GFP that has migrated to periphery of the spheroid showing a typical neuronal morphology. At 30 DPI, the attached HA spheroid cultures infected by either ND1-GFP or ND1-124T-GFP were fixed and immunostained with the indicated mature neuronal markers (NeuN and/or Map2) along with NeuroD1 ( C ), the synaptic vesicle marker SV2 ( D ), the glutamatergic neuronal markers vGlut1 and HuD ( E ), and Ctip2 ( F ). ( G ) Expression levels of the markers were quantified by their cellular fluorescence intensities and compared. ( H ) The GABAergic interneuron markers GABA and GAD67 ( F ) were also analyzed and compared ( I ). Arrows, the marker+ cells. ns, not significant; ***, p < 0.005; ****, p < 0.001 by student t -test. Scale bars, 40μm.

Article Snippet: Briefly, fixed cell cultures were incubated with monoclonal antibodies against NeuroD1 (mouse IgG, 1:1000, Abnova), GFP (rat IgG2a, 1:400, BioLegend), HuD/ELAVL4 (mouse IgG, 1:200, Santa Cruz), GAD67 (mouse IgG, 1:200, Millipore), and NeuN (mouse IgG, 1:400, Millipore); polyclonal antibodies against GFP (chicken IgY, 1:400, Aves), mCherry/RFP (rabbit IgG, 1:500, Abcam), mCherry/RFP (chicken IgY, 1:400, Aves), Map2 (rabbit IgG, 1:400, Abcam), DCX (rabbit IgG, 1:500, Abcam), vGlut1 (guinea pig IgG, 1:200, Millipore), Ctip2 (guinea pig IgG, 1:200, Synapse Systems), anti-SV2 (rabbit, 1:2000, Developmental Studies Hybridoma Bank), GABA (rabbit IgG, 1:200, Calbiochem), and DCX (guinea pig IgG, 1:1000, Millipore), followed by appropriate species-specific secondary antibodies (Molecular Probes).

Techniques: Construct, Infection, Marker, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Imp1 acts as a dosage- and stage-dependent temporal rheostat orchestrating radial glial fate transitions and cortical morphogenesis

doi: 10.1101/2025.11.18.688993

Figure Lengend Snippet: (A,D,G) Representative images of control, continuous Imp1 overexpression, and T1 Imp1 conditions showing TEMPO reporters, Cux1/Ctip2 immunostaining and overlays. Boxed regions: Ctip2+ TEMPO neurons (dashed) or double-positive Cux1+/Ctip2+ TEMPO cells (solid). (B,E,H) High magnification images of boxed regions highlight CFP-/RFP-labeled neurons in layers V-VI colocalizing with Ctip2 (outlined arrowheads) or double-positive for both markers (solid arrowheads). (C,F,I) Quantification of marker expression in CFP+ and RFP+ neurons residing in layers V-VI. Following continuous or T1 Imp1 overexpression, neurons in deep layer maintain appropriate deep-layer molecular identities (predominantly Ctip2+), demonstrating that laminar distribution reflects bona fide fate specification changes rather than mislocalization. (C) In control conditions, CFP+: Ctip2 (78.31% ± 13.82%), Cux1 (2.93% ± 2.11%), double-negative (18.76% ± 11.73%). RFP+: Ctip2 (50.35% ± 17.01%), Cux1 (1.85% ± 1.85%), double-negative (46.76% ± 17.20%), double-positive (1.04% ± 1.04%). (F) Following continuous Imp1 overexpression, CFP+: Ctip2+ (71.18% ± 3.06%), Cux1+ (7.38% ± 4.93%), double-negative (16.06% ± 1.93%), double-positive (5.38% ± 1.80%). RFP+: Ctip2+ (58.92% ± 7.18%), Cux1+ (5.84% ± 3.01%), double-negative (5.10% ± 3.12%), double-positive (30.14% ± 11.98%). (I) In T1 Imp1 overexpression, CFP+: Ctip2+ (47.26% ± 4.88%), Cux1+ (14.63% ± 3.76%), double-negative (36.41% ± 5.64%), double-positive (1.68% ± 0.57%). RFP+: Ctip2+ (47.38% ± 13.91%), Cux1+ (9.17% ± 4.68%), double-negative (26.86% ± 5.03%), double-positive (16.59% ± 10.02%). Dashed lines: upper (II-IV), lower cortical layers (V-VI) and subplate zone (SPZ). Scale bars: (A,D,G) 100 µm and (B,E,H) 20 µm. Data show mean±SEM. Statistics: two-tailed unpaired Welch’s t-test (*P < 0.05, **P < 0.01, *** P < 0.001).

Article Snippet: Primary antibodies were applied overnight to amplify TEMPO signals or detect fate markers at 4°C: mouse anti-V5 (1:650, Thermo Fisher R96025) to detect CFP+ cells, rat anti-mCherry (1:500, Thermo Fisher M11217) or chicken anti-mCherry (1:500, AvesLabs MCHERRY AB_2910557) to detect RFP+ cells, rat anti-Ctip2 (1:100, Abcam [25B6] ab18465) to detect Ctip2+ cells, and rabbit anti-Cux1 (1:50, Proteintech 11733-1-AP) to detect Cux1+ cells.

Techniques: Control, Over Expression, Immunostaining, Labeling, Marker, Expressing, Two Tailed Test

Journal: Cell reports

Article Title: Cell-type-specific roles of FOXP1 in the excitatory neuronal lineage during early neocortical murine development

doi: 10.1016/j.celrep.2025.115384

Figure Lengend Snippet: (A) Violin plot demonstrating the expression levels of three representative DEGs by cell type at P0.5. Rora and Nrxn3 are downregulated whereas Kirrel3 is upregulated. (B and C) Representative RNA in situ hybridization (ISH) images for Rora (B) and Kirrel3 and Nrxn3 (C) confirm snRNA-seq findings in control and Foxp1 cKO mice at P0.5. Ctip2 was used to label L5–L6 cortical neurons. (D) The total numbers of pixels in each of the 4 bins were measured in the Ctip2 -expressing layer and normalized to the total nuclei in their respective bins. (E–G) Boxplots illustrate significant downregulation of Rora (E) and Nrxn3 (F) and upregulation of Kirrel3 (G) in the L5–L6 cortical layers of Foxp1 cKO mice compared to controls. Each dot in the boxplots represents normalized pixel counts from individual bins, with colors indicating data from different mice in each group. Significance was tested using a linear mixed model with genotype as the fixed factor and individual as the random factor, nested with sections, hemisphere, and bins. n = 3 mice per genotype, with 2–3 sections from each mouse. Scale bar: 100 μm.

Article Snippet: RNAscope probe Ctip2, C1 and C2 , ACD Bio-techne , Cat# 413271.

Techniques: Expressing, RNA In Situ Hybridization, Control

Journal: Cell reports

Article Title: Cell-type-specific roles of FOXP1 in the excitatory neuronal lineage during early neocortical murine development

doi: 10.1016/j.celrep.2025.115384

Figure Lengend Snippet: (A and B) EdU birth-dating experiment shows that neurons generated from progenitors dividing at E15.5 remain in the DLs in Foxp1 cKOs at higher levels than in controls throughout the cortex. Some of these cells are EdU+CUX1+, indicating they have a UL cell type identity and are CTIP2−. (B) Foxp1 cKO cortices are at (top) 20× and (bottom) 63× showing EdU+CUX1+CTIP2− cells in the L5–L6 region. (C) P7.5 brains stained for EdU that was pulsed at E15.5 have significantly more cells in the DLs in Foxp1 cKOs. No significant difference was found between EdU+ cells in the ULs between genotypes. n = 5/genotype. (D) P16.5 brains from E15.5 EdU-pulsed animals with CUX1 and CTIP2 to demarcate cortical layer boundaries at 103 resolution. (E) P16.5 brains with EdU cell quantification in ULs or DLs show a significant number of EdU+ cells located in the DLs in cKOs but not controls. No significant change was seen in the number of UL EdU+ cells. n = 5/genotype. EdU+ cells were CTIP2− despite being in L5–L6, suggesting the appropriate timing of neurogenesis at E15.5 for UL cells and a likely selective migration deficit that remains at P16.5. Mann-Whitney test. Mean ± SEM shown.

Article Snippet: RNAscope probe Ctip2, C1 and C2 , ACD Bio-techne , Cat# 413271.

Techniques: Generated, Staining, Migration, MANN-WHITNEY

Journal: Cell reports

Article Title: Cell-type-specific roles of FOXP1 in the excitatory neuronal lineage during early neocortical murine development

doi: 10.1016/j.celrep.2025.115384

Figure Lengend Snippet:

Article Snippet: RNAscope probe Ctip2, C1 and C2 , ACD Bio-techne , Cat# 413271.

Techniques: Recombinant, Electron Microscopy, RNAscope, Isolation, Software, Microscopy

Journal: bioRxiv

Article Title: Porphyromonas gingivalis outer membrane vesicles alter neuronal architecture and Tau phosphorylation in the embryonic mouse brain

doi: 10.1101/2024.09.03.611094

Figure Lengend Snippet: (A) Representative immunofluorescent images from sagittal sections of PFA fixed brains at GA 18.5 pup brains showing location of cortical layer markers Cux1, SatB2, and Ctip2. Approximate position of cortical layers 1-6 with the intraventricular zone (IZ) are shown in the PBS-DAPI image. (B) Quantification and comparison of cortical layer proteins stained with the marker antibodies. Intensities were averaged across three serial section per sample for statistical analysis. Cux1 (Student’s T-test p = 0.03) and SatB2 (students T-test p = 0.0001) were significantly reduced in the Pg-OMV group. Data are presented with the median and 95% CI.

Article Snippet: Sections were washed with PBS, blocked in PBS containing 0.1% tritonX- 100, and 10% donkey serum and probed with and primary antibodies to Cux1 (Protein Tech 11733-1-AP; diluted 1:300), SatB2 (Abcam ab92446; diluted 1:100), and Ctip2 (Abcam ab-18465; diluted 1:500).

Techniques: Comparison, Staining, Marker