Journal: Biology Open

Article Title: Lateral line placodes of aquatic vertebrates are evolutionarily conserved in mammals

doi: 10.1242/bio.031815

Figure Lengend Snippet: Neurogenesis in the lateral line placodes of Q-VD-OPh-treated mice. (A,C,F,G) Reconstructions of serially sectioned mouse embryos (section interval evaluated=10 µm) show ectoderm (light grey), otic vesicle with detachment site (dark grey), epibranchial placodes (orange), lateral line placodes (blue), immunopositive cells (brown dots), developing cranial nerves (partly numbered brown contours in G). (B,D,E) Micrographs taken from serially sectioned mouse embryos. (A) Sox2 + cells are present in epibranchial placodes and, to a much lesser extent, in lateral line placodes (embryo #021). (B) Sox2 + mantle (m) and support (s) cells in a neuromast primordium of a middle lateral line placode. Note the apical cavity (arrowhead). (C) Ngn1 + cells are present in epibranchial placodes and, to a much lesser extent, in lateral line placodes (embryo #039, mirror-imaged right side). (D) Ngn1 + neuroblasts in an anterodorsal lateral line placode. (E,F) Ngn2 + neuroblasts are present in epibranchial placodes, but absent from lateral line placodes (embryo #001). (G) Tubb3 + neurons constitute the anlagen of the cranial nerves 7 and 8 (VII/VIII), 9 (IX), 10 (X), 11 (XI) and 12 (XII). Tubb3 + candidates for vestigial lateralis ganglia (asterisks) reside in close proximity to the middle lateral line placode (embryo #014). All micrographs were adjusted for brightness (including slight gamma changes), colour balance, and sharpness. Scale bars: 5 µm. ad, m, p, anterodorsal, middle, and posterior lateral line placode, respectively; e1, e2, e3, epibranchial placodes 1, 2, 3, respectively; ot, otic anlage; ov, optic vesicle; Q-VD-OPh, pan-caspase inhibitor; wec, whole embryo culture.

Article Snippet: Primary antibodies included: mouse anti-Atonal homolog 1 (Atoh1; Atoh1 supernatant, Developmental Studies Hybridoma Bank, Iowa City, USA, lot 6/20/13, RRID: AB_10805299; 1:50, overnight, 4°C) ( ; ), mouse anti-β-Tubulin-III (Tubb3; clone SDL.3D10, T8660, Sigma-Aldrich, lot 073K4835, RRID: AB_477590; 1:8000, overnight, 4°C) , rabbit anti-cleaved caspase-3 (9661; Cell Signaling Technology, lot 37, RRID: AB_2341188; 1:8000, overnight, 4°C) ( , ), mouse anti-Islet 1 (Isl1; 39.4D5 ascites fluid, Developmental Studies Hybridoma Bank, lot 2/12/09, RRID: AB_2314683; 1:2000, overnight, 4°C) , goat anti-Neurogenin1 (Ngn1; sc-19231, Santa Cruz Biotechnology, lot C1215, RRID: AB_2298242; 1:100, 4 h, 37°C) , mouse anti-Neurogenin2 (Ngn2; clone 7G4, MAB3314, R&D Systems, Minneapolis, USA, lot WWI01, RRID: AB_2149520; 1:20,000, overnight, 4°C) , goat anti-Neurogenic differentiation 1 (NeuroD1; sc-1084, Santa Cruz Biotechnology, lot A0616, RRID: AB_630922; 1:800, 2 h, 37°C) , rabbit anti-Paired homeobox (Pax) 2 (71-6000; Thermo Fisher Scientific, lot 1117672A, RRID: AB_2533990; 1:4000, overnight, 4°C) , mouse anti-Pax8 (ACI 438; Biocare Medical, Pacheco, USA, lot 051712, RRID: AB_10922964; 1:100, overnight, 4°C) , rabbit anti-phospho-Histone H3 (pHH3; 9701, Cell Signaling Technology, lot 13, RRID: AB_331535; 1:1000, 2 h, 37°C) , rabbit anti-Sine oculis homeobox 1 (Six1; HPA001893, Atlas Antibodies, Stockholm, Sweden, lot A00939, RRID: AB_1079991; 1:800, overnight, 4°C) , mouse anti-Six1 (clone CLO185, AMAb90544, Atlas Antibodies, lot 02852, RRID: AB_2665581; 1:100, overnight, 4°C) that, compared to rabbit anti-Six1, was raised against the same immunogen and shows identical reactivities (present study), mouse anti-Sex determining region Y-box (Sox) 2 (clone 245610, MAB2018, R&D Systems, lots KGQ0311111, KGQ0315062, RRID: AB_358009; 1:200, overnight, 4°C) , goat anti-Sox10 (sc-17342, Santa Cruz Biotechnology, lot L2908, RRID: AB_2195374; 1:800, overnight, 4°C) , mouse anti-Sox10 (sc-365692, Santa Cruz Biotechnology, lot I0516, RRID: AB_10844002; 1:1600, overnight, 4°C) that, compared to goat anti-Sox10, produced identical staining patterns in mice (present study) and rats , and rabbit anti-T-box transcription factor (Tbx) 3 (42-4800, Thermo Fisher Scientific, lot SI256120, RRID: AB_2533526; 1:200, 2 h, 37°C) ( ).

Techniques: Embryo Culture

Journal: Scientific Reports

Article Title: Extremely low-frequency electromagnetic fields facilitate proliferation and functional differentiation in spinal neural stem cells

doi: 10.1038/s41598-025-14738-x

Figure Lengend Snippet: ELF-EMFs upregulate the expression of pro-neuronal genes and activate T-type calcium channels. A , B Quantification of NeuroD1 ( n = 4), NeuroG1 ( n = 4), NeuroG2 ( n = 4), and NeuroD2 ( n = 4) mRNA levels in spinal cord-derived NSCs after exposure to ELF-EMFs at different intensities. Values are normalized to the 0 mT control group. C Protein levels of NeuroD1 and NeuroG1 in spinal cord-derived NSCs after exposure to ELF-EMFs at different intensities, with β-actin used as a loading control. Original blots are presented in Supplementary Fig. 1B. D Quantification of NeuroD1 ( n = 6) and NeuroG1 ( n = 6) protein levels in spinal cord-derived NSCs after exposure to ELF-EMFs at different intensities. Values are normalized to the 0 mT control group. E , F Quantification of Ca v 1.2 ( n = 5), Ca v 1.3 ( n = 5), Ca v 3.1 ( n = 5), Ca v 3.2 ( n = 5) and Ca v 3.3 ( n = 5) mRNA levels in spinal cord-derived NSCs after exposure to ELF-EMFs at different intensities. Values are normalized to the 0 mT control group. G Protein levels of Ca v 3.1 , Ca v 3.2 and Ca v 3.3 in spinal cord-derived NSCs after exposure to ELF-EMFs at different intensities, with β-actin used as a loading control. Original blots are presented in Supplementary Fig. 1C. H Quantification of Ca v 3.1 ( n = 3), Ca v 3.2 ( n = 3) and Ca v 3.3 ( n = 3) protein levels in spinal cord-derived NSCs after exposure to ELF-EMFs at different intensities. Values are normalized to the 0 mT control group. I Representative calcium current traces recorded at −30 mV and current density-voltage (I-V) curves of NSC-differentiated neurons after exposure to ELF-EMFs at different intensities (0 mT, black, n = 5; 0.5 mT, green, n = 5; 1 mT, red, n = 5). J Representative calcium current traces recorded at −30 mV and current density-voltage (I-V) curves in NSC-differentiated neurons at 1 mT (1 mT, purple, n = 6; 1 mT + TTA-P2, 1 mT with 2 µM TTA-P2 in the bath solution, blue, n = 6; 1 mT [washout], after 10-min washout with TTA-P2-free solution, light blue, n = 6). K Statistics for peak current density of NSC-differentiated neurons.

Article Snippet: NeuroG1 , Rabbit , Proteintech , 66642-1-Ig , WB.

Techniques: Expressing, Derivative Assay, Control

Journal: PLoS ONE

Article Title: Extremely Low-Frequency Electromagnetic Fields Promote In Vitro Neuronal Differentiation and Neurite Outgrowth of Embryonic Neural Stem Cells via Up-Regulating TRPC1

doi: 10.1371/journal.pone.0150923

Figure Lengend Snippet: Primers used in real-time PCR analyses.

Article Snippet: Mouse anti-mouse TRPC1 (1:1000, Santa Cruz, USA), rabbit anti-mouse NeuroD (1:500, Santa Cruz, USA), goat anti-mouse Ngn1 (1:500, Santa Cruz, USA), and mouse anti-mouse GAPDH (1:5000, Abcam, USA) were used as primary antibodies.

Techniques: Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Extremely Low-Frequency Electromagnetic Fields Promote In Vitro Neuronal Differentiation and Neurite Outgrowth of Embryonic Neural Stem Cells via Up-Regulating TRPC1

doi: 10.1371/journal.pone.0150923

Figure Lengend Snippet: eNSCs were cultured in differentiation medium and exposed to ELF-EMF for 3 days. The mRNA expression of pro-neuronal genes ( A ) and Hes genes ( B ) was analyzed by real-time PCR. The mRNA expression of both NeuroD and Ngn1 was increased after ELF-EMF exposure. ** p < 0.01 vs. sham groups. ( C ) Representative western blotting bands of the NeuroD and Ngn1 protein expression. Full-length blots are presented in . ( D, E ) Statistical results of western blotting results. * p < 0.01 vs. sham groups. ( F ) Representative images of NeuroD and Ngn1 staining. For all experiments, the data are from five independent experiments and are presented as the mean ± SEM.

Article Snippet: Mouse anti-mouse TRPC1 (1:1000, Santa Cruz, USA), rabbit anti-mouse NeuroD (1:500, Santa Cruz, USA), goat anti-mouse Ngn1 (1:500, Santa Cruz, USA), and mouse anti-mouse GAPDH (1:5000, Abcam, USA) were used as primary antibodies.

Techniques: Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining

Journal: PLoS ONE

Article Title: Tcf3 Represses Wnt–β-Catenin Signaling and Maintains Neural Stem Cell Population during Neocortical Development

doi: 10.1371/journal.pone.0094408

Figure Lengend Snippet: A,A’ , In situ hybridization of coronal sections of the E11.5 ( A ) and E14.5 ( A’ ) mouse cortex for the Tcf3 mRNA. B–H” , Coronal sections of E14.5 neocortex immunostained as indicated. VZ, ventricular zone; SVZ, subventricular zone; IMZ, intermediate zone; CP, cortical plate ( B ). Higher magnification of VZ in C–G are shown in C’–G’ . Scale bars, 100 µm ( A,A’,B ) and 20 µm ( C–G’ ). The percentages of Tcf3 + Pax6 − cells, Tcf3 + Pax6 + cells and Tcf3 − Pax6 + cells among either Tcf3 + or Pax6 + cells in the VZ were determined by immunostaining ( H , below). The percentages of Tcf3 + Tbr2 − cells, Tcf3 + Tbr2 + cells and Tcf3 − Tbr2 + cells and those of Tcf3 + Neurog1 − cells, Tcf3 + Neurog1 + cells and Tcf3 − Neurog1 + cells are shown in H’,H” . Venn diagram of these percentages ( H-H” , above). I , dissociated cells from E14.5 neocortices of Nestin-d4-Venus transgenic mouse were sorted into Nestin-d4-Venus -, Nestin-d4-Venus +, Nestin-d4-Venus ++, and Nestin-d4-Venus +++ fractions by using FACS (see also ). The mRNA level of Tcf3 in each fractions was determined by qPCR analysis. Data represents mean ± SEM ( H-H”, I ).

Article Snippet: Antibodies used in this study were: goat antibodies to Tcf3 (#1, M-20, Santa Cruz) 1∶1000, Neurog1 (A-20, Santa Cruz) 1∶200, Neurog2 (Santa Cruz) 1∶500, mouse antibodies to βIII-tubulin (TuJ1, Covance) 1∶1000, Tcf3/4 (#2, Upstate) 1∶1000, Sox2 (R&D) 1∶100, chick antibody to GFP (Abcam) 1∶2000, and rabbit antibodies to GFP (MBL) 1∶1000, Pax6 (Chemicon) 1∶1000, Tbr2 (Chemicon) 1∶1000.

Techniques: In Situ Hybridization, Immunostaining, Transgenic Assay

Journal: PLoS ONE

Article Title: Tcf3 Represses Wnt–β-Catenin Signaling and Maintains Neural Stem Cell Population during Neocortical Development

doi: 10.1371/journal.pone.0094408

Figure Lengend Snippet: A , Chromatin complex was immunoprecipitated from E11.5 neocortical lysates with anti-Tcf3. The immunoprecipitates were subjected to qPCR analysis. B,C , NPCs were infected with a retrovirus encoding control, Tcf3 shRNA #1 or Tcf3 shRNA #2 and incubated with FGF2 for 3 d. Cells were cultured for another 6 h in the presence (undifferentiated condition) or absence (differentiated condition) of FGF2. The mRNA levels of Tcf3 ( B ) and Neurog1 ( C ) were determined by qPCR analysis. Data obtained in differentiated condition are shown in B,C . Similar results were obtained in undifferentiated condition (not shown). D , E11.5 NPCs were infected with a retrovirus encoding control or Tcf3 and incubated with FGF2 for 3 d. The level of Neurog1 mRNA was determined by qPCR analysis. E,F , NPCs were infected with a retrovirus encoding control, Tcf3 shRNA #1 ( E ), Tcf3 shRNA #2 ( E ) or Tcf3 ( F ) as C,D . Then the cells were incubated in the presence of FGF2 for 3 d. The level of N-myc mRNA was determined by qPCR analysis. B–F , Data are normalized with GAPDH mRNA (arbitrary unit). A–F , Data represents mean ± SEM.

Article Snippet: Antibodies used in this study were: goat antibodies to Tcf3 (#1, M-20, Santa Cruz) 1∶1000, Neurog1 (A-20, Santa Cruz) 1∶200, Neurog2 (Santa Cruz) 1∶500, mouse antibodies to βIII-tubulin (TuJ1, Covance) 1∶1000, Tcf3/4 (#2, Upstate) 1∶1000, Sox2 (R&D) 1∶100, chick antibody to GFP (Abcam) 1∶2000, and rabbit antibodies to GFP (MBL) 1∶1000, Pax6 (Chemicon) 1∶1000, Tbr2 (Chemicon) 1∶1000.

Techniques: Immunoprecipitation, Infection, Control, shRNA, Incubation, Cell Culture

Journal: Development (Cambridge, England)

Article Title: Otx2 cell-autonomously determines dorsal mesencephalon versus cerebellum fate independently of isthmic organizing activity.

doi: 10.1242/dev.102954

Figure Lengend Snippet: Fig. 2. Loss of Otx2 remarkably affects identity and differentiation of m1 progenitors. Immunohistochemistry experiments performed at E11, E12.5 and E13.5 on Dbx1ICre/+ and Dbx1ICre/+;Otx2flox/flox embryos with Pou4f1 and Otx2 (A-C′), Ngn1 and Dbx1 (D-F′), Zic1 and Pax7 (G-I′), Mash1 and Helt (J-L′), Gata2 and Pou4f1 (M-O′), Lhx1 and Pou4f1 (P-R′), Lhx1 and Gad65 (S,S′) or vGlut2 and Pou4f1 (T,T′). The white arrows in D′-F′ and J′-L′ point respectively to the expression of Dbx1 and Ngn1, and Helt and Mash1 in the m1b sub-domain; the yellow arrows in D′-F′ point to the Ngn1 residual expression in the m1a sub- domain; and the white arrows in S′ and T′ respectively to Lhx1+ Gad65+ GABAergic and Pou4f1+ vGlut2+ glutamatergic neurons generated in the m1a and m1b sub-domains. Dashed lines delineate regions as marked in A-C′. m1, mesencephalic domain 1; m1a, mesencephalic sub-domain 1a; m1b, mesencephalic sub- domain 1b.

Article Snippet: 4F2), Pax6 (1:100; Developmental Studies Hybridoma Bank, concentrated), calbindin (1:100; Swant, CB300), parvalbumin (1:300; Sigma, P3088), GFAP (1:200; Chemicon, MAB3402) and Meis2 (1:150; Abnova, H00004212-H01); antibodies raised in goat were against Otx2 (1:100; R&D Systems, AF1979), Ngn1 (1:50; Santa Cruz Biotechnology, sc19231), calbindin (1:100; R&D Systems, AF3320), Sox2 (1:400; R&D Systems, AF2018), Lhx1 (C20) (1:100; Santa Cruz Biotechnology, sc19341); antibodies against β-Gal (1:1500; Abcam, ab9361) and 5′-bromo2′-deoxyuridine (BrdU) (1:1500; Novus Biologicals, NB500-169) were raised in chicken and rat, respectively.

Techniques: Immunohistochemistry, Expressing, Generated

Journal: bioRxiv

Article Title: Proneural genes form a combinatorial code to diversify neocortical neural progenitor cells

doi: 10.1101/2023.07.29.551096

Figure Lengend Snippet: (A,B) UMAP plot of scRNA-seq data from cortical tissue collected between E10 and P4 (GSE153162) , showing cell clusters associated with time points (A) and cell types (B). (C-G) Distribution of cell types expressing Neurog1 alone (green bars), Neurog2 alone (pink bars), co-expressing Neurog1 and Neurog2 (purple bars) or not expressing Neurog1 or Neurog2 (grey bars). Data is depicted as the total % of each cell population expressing the proneural genes (C), or separated into cell types, including apical NPCs (D), basal NPCs (E), Cajal-Retzius neurons (F) and immature and migrating neurons (G). (H) Feature plots depict the cortical populations expressing Neurog1 and Neurog2 and canonical markers of different cortical cell types. Astro, astrocyte; CR, Cajal-Retzius; DL, deep-layer; IN, interneuron; IPC, intermediate progenitor cell; L, layer; NPC, neural progenitor cell; oligo, oligodendrocyte; RG, radial glia; UL, upper layer. See also Figure S1 and S2.

Article Snippet: Neurog1 C-CreKI transgenic mice were generated by Cyagen using homologous recombination in embryonic stem cells and were produced on a BL6/J background (Jackson Labs).

Techniques: Expressing

Journal: bioRxiv

Article Title: Proneural genes form a combinatorial code to diversify neocortical neural progenitor cells

doi: 10.1101/2023.07.29.551096

Figure Lengend Snippet: A-D) Monocle3 lineage trajectory analysis of Neurog1 + , Neurog2 + and double + cells collected between E10-P4 (A), showing annotation of states 1-5 (B), a pseudotime trajectory of these states (C), and the distribution of each NPC population in each state (D). (E,F) Mapping marker expression onto the pseudotime trajectory plots (E) and showing expression levels graphically (F). State 1 and 2 are predominated by apical NPC markers, state 3 and the minor state 4 populations are mainly IPCs, and state 5 are IPCs and newborn neurons. Neurog1 + (green bars), Neurog2 + (pink bars) and co-expressing (purple bars) cells predominate in earlier pseudotime states (F). See also Figure S3.

Article Snippet: Neurog1 C-CreKI transgenic mice were generated by Cyagen using homologous recombination in embryonic stem cells and were produced on a BL6/J background (Jackson Labs).

Techniques: Marker, Expressing

Journal: bioRxiv

Article Title: Proneural genes form a combinatorial code to diversify neocortical neural progenitor cells

doi: 10.1101/2023.07.29.551096

Figure Lengend Snippet: (A,B) RC2 and BLBP immunostaining of the aRG scaffold in E18.5 cortices revealed Neurog1;Neurog2 DKO aRG basal processes are disorganized and do not reach the pial surface (*), and more minor aberrant terminations of the basal endfeet in Neurog2 KOs (arrowheads) (A). Schematic representation of defects in radial glial guides in Neurog1/Neurog2 KO and DKO cortices (B). (C-H) Analysis of Sox9 + NPCs (C), Pax6 + NPCs (D), Eomes + NPCs (E), Tbr1 + neurons (F), Bcl11b + neurons (G) and Satb2 + neurons (H) in E18.5 wild-type, Neurog1 -/- , Neurog2 -/- and Neurog1 -/- ;Neurog2 -/- cortices. Graphs show quantification of markers + cells/field (N=3, n=9 for all genotypes). Scale bars are 25 μm in A and 100 μm in C-H. Blue is DAPI counterstain. NPC markers were only counted in the germinal zone. Data represent the mean ± s.e.m, p-values: ns - not significant, <0.05 *, <0.01 **, <0.001 ***, by one-way ANOVA with Tukey correction for multiple comparisons. cp, cortical plate; gz, germinal zone; mz, marginal zone; ig, infragranular layers; iz, intermediate zone; sg, subgranular layers; svz, subventricular zone; vz, ventricular zone. See also Figure S4.

Article Snippet: Neurog1 C-CreKI transgenic mice were generated by Cyagen using homologous recombination in embryonic stem cells and were produced on a BL6/J background (Jackson Labs).

Techniques: Immunostaining

Journal: bioRxiv

Article Title: Proneural genes form a combinatorial code to diversify neocortical neural progenitor cells

doi: 10.1101/2023.07.29.551096

Figure Lengend Snippet: (A) Neurog1 C-CreKI ;Neurog2 N-CreKI (split-Cre) strategy. (B,C) RNAscope in situ hybridization of Neurog1/C-Cre and Neurog2/N-Cre (B) and Neurog1/Neurog2 (C). (D,E) Co-labeling of zsGreen with Sox9, Pax6, Eomes, Tbr1 and Reelin in E12.5 split-Cre;Rosa-zsGreen cortices. (D). Quantification of zsGreen co-expression at E12.5 (N=3, n=9) (E). (F-I) Lineage tracing in E15.5 split-Cre;zsGreen cortices, showing co-labeling of zsGreen with Sox9 in low and high magnification images (F). Co-localization of zsGreen and Neurog1 mRNA in low magnification and higher magnification images (G). Co-labeling of zsGreen with Tbr1, Bcl11b and Satb2 in E15.5 split-Cre;zsGreen cortices (H). Quantification of of zsGreen co-expression at E15.5 (N=3, n=9) (I). (J-L) Co-labeling of zsGreen with Sox9, Pax6, Eomes, Tbr1, Bcl11b, and Satb2 in E18.5 split-Cre;zsGreen cortices (J). Quantification of zsGreen-NPC (K) and -neuronal (L) marker co-expression. (N=3, n=9 for all). (A-L) Blue is DAPI counterstain. Scale bars are 100 μm. Data represent the mean ± s.e.m, ch, cortical hem; dp, dorsal pallium; lge, lateral ganglionic eminence; lp, lateral pallium; mge, medial ganglionic eminence; nc, neocortex; pp, preplate; str, striatum; svz, subventricular zone; th, thalamus; vz, ventricular zone. See also Figure S5.

Article Snippet: Neurog1 C-CreKI transgenic mice were generated by Cyagen using homologous recombination in embryonic stem cells and were produced on a BL6/J background (Jackson Labs).

Techniques: RNAscope, In Situ Hybridization, Labeling, Expressing, Marker

Journal: bioRxiv

Article Title: Proneural genes form a combinatorial code to diversify neocortical neural progenitor cells

doi: 10.1101/2023.07.29.551096

Figure Lengend Snippet: (A) GRN associated with E12.5 CD15 + Neurog2 + cortical NPCs, showing predicted target genes (purple nodes), from . Predicted changes in gene expression (teal nodes) caused by in silico ‘Neurog1 KO’ (no change, this study), Neurog2 (teal nodes, as generated in ) and ‘Neurog1;Neurog2 DKO’, predicting four new target genes (teal nodes). (B-D) Co-expression of zsGreen with Bcl11b (B), Neurod2 (C) and Nhlh2 (D) in P150 split-Cre;Rosa-zsGreen cortices, with higher magnification images to the right. Blue is DAPI counterstain. Scale bars in B-D are 400μm on the left and 100μm on the right. ig, infragranular; iz, intermediate zone; pp, preplate. sg, supragranular; v-svz, ventricular subventricular zone. See also Figure S6.

Article Snippet: Neurog1 C-CreKI transgenic mice were generated by Cyagen using homologous recombination in embryonic stem cells and were produced on a BL6/J background (Jackson Labs).

Techniques: Gene Expression, In Silico, Generated, Expressing