Journal: Frontiers in Neuroanatomy

Article Title: Transcription factor 4 expression in the developing non-human primate brain: a comparative analysis with the mouse brain

doi: 10.3389/fnana.2024.1478689

Figure Lengend Snippet: Validation of TCF4 antibody NCI-R159-6 (ab217668). A : Western blot analysis demonstrating the specificity of ab217668 for long (L), medium (M), and short (S) TCF4 isoforms with minimal background. B : TCF4 immunostaining in 2-week-old macaque brain using ab217668. B1 , shows that TCF4 staining concentrates in nuclei with minimal cytoplasmic or neuropil background. The arrow in B1 indicates the nucleus shown in B2–4 . B2–4 , higher magnification reveals a punctate nuclear staining pattern, with little to no signal in condensed DNA regions (arrowheads) and the nucleolus (arrow). C,D : Validation via shRNA-mediated Tcf4 knockdown in cultured mouse cortical neurons. C : Control: robust nuclear TCF4 staining in untransfected and scrambled shRNA-transfected neurons (arrowhead). D : Tcf4-targeting shRNA: absence of TCF4 staining in transfected neurons (arrowhead). Scale bars: B1 : 25 μm; B2,3 : 5 μm; C,D upper panel: 50 μm; C,D lower panel: 10 μm.

Article Snippet: On DIV3 (3 days in vitro ), neurons were transfected with 0.33 μg CamKIIα-tdTomato plasmid ( ) together with 0.66 μg of vector expressing scramble shRNA (SHC002) or a mixture of five Tcf4-targeting shRNA vectors (TRCN0000012093 - TRCN0000012097) developed by the RNAi Consortium (TRC; Broad Institute) using Lipofectamine2000 reagent (11668019, Invitrogen) at 2:1 ratio per well.

Techniques: Western Blot, Immunostaining, Staining, shRNA, Knockdown, Cell Culture, Control, Transfection

Journal: Frontiers in Neuroanatomy

Article Title: Transcription factor 4 expression in the developing non-human primate brain: a comparative analysis with the mouse brain

doi: 10.3389/fnana.2024.1478689

Figure Lengend Snippet: Details of commercial TCF4 antibodies evaluated in this study.

Article Snippet: On DIV3 (3 days in vitro ), neurons were transfected with 0.33 μg CamKIIα-tdTomato plasmid ( ) together with 0.66 μg of vector expressing scramble shRNA (SHC002) or a mixture of five Tcf4-targeting shRNA vectors (TRCN0000012093 - TRCN0000012097) developed by the RNAi Consortium (TRC; Broad Institute) using Lipofectamine2000 reagent (11668019, Invitrogen) at 2:1 ratio per well.

Techniques: Staining, Recombinant

Journal: Frontiers in Neuroanatomy

Article Title: Transcription factor 4 expression in the developing non-human primate brain: a comparative analysis with the mouse brain

doi: 10.3389/fnana.2024.1478689

Figure Lengend Snippet: Immunoreactivity to TCF4 in sagittal mouse brain sections at P0 through P60. From P0 to P60, TCF4 expression is primarily seen in the cortical plate and cerebellum. Initially concentrating in the hippocampal formation and isocortex, with a notable presence in the anterior olfactory nucleus (arrowhead) and pontine grey (arrow). TCF4 expression peaks around P2–P5. After that, it gradually decreases across all regions, stabilizing by P60 with the highest levels remaining in the cerebellum, hippocampal formation, cerebral cortex, and olfactory bulb. Scale bars = 1 mm.

Article Snippet: On DIV3 (3 days in vitro ), neurons were transfected with 0.33 μg CamKIIα-tdTomato plasmid ( ) together with 0.66 μg of vector expressing scramble shRNA (SHC002) or a mixture of five Tcf4-targeting shRNA vectors (TRCN0000012093 - TRCN0000012097) developed by the RNAi Consortium (TRC; Broad Institute) using Lipofectamine2000 reagent (11668019, Invitrogen) at 2:1 ratio per well.

Techniques: Expressing

Journal: Frontiers in Neuroanatomy

Article Title: Transcription factor 4 expression in the developing non-human primate brain: a comparative analysis with the mouse brain

doi: 10.3389/fnana.2024.1478689

Figure Lengend Snippet: Immunoreactivity to TCF4 in the developing mouse cerebellum. At P2, TCF4 is highly expressed in the external (arrowhead in A ) and internal granular layers (arrow in A ). Over time, expression decreases in the granule cell layer but increases in the molecular layer. Small cells in the Purkinje cell layer remain TCF4-positive throughout, while Purkinje cells are consistently TCF4-negative (arrowheads in E,F ). GL, granule cell layer; EGL, external granular layer; MCL, molecular cell layer; PCL, Purkinje cell layer. Scale bars: A–C : 200 μm; D–F : 50 μm.

Article Snippet: On DIV3 (3 days in vitro ), neurons were transfected with 0.33 μg CamKIIα-tdTomato plasmid ( ) together with 0.66 μg of vector expressing scramble shRNA (SHC002) or a mixture of five Tcf4-targeting shRNA vectors (TRCN0000012093 - TRCN0000012097) developed by the RNAi Consortium (TRC; Broad Institute) using Lipofectamine2000 reagent (11668019, Invitrogen) at 2:1 ratio per well.

Techniques: Expressing

Journal: Frontiers in Neuroanatomy

Article Title: Transcription factor 4 expression in the developing non-human primate brain: a comparative analysis with the mouse brain

doi: 10.3389/fnana.2024.1478689

Figure Lengend Snippet: Immunoreactivity to TCF4 in the developing mouse striatum and pallidum. TCF4 immunoreactivity is prominent in dense cell clusters (arrows in A ) and dispersed cells within the pallidum and caudoputamen. TCF4 expression diminishes rapidly during postnatal development. By P60, most cells in these regions exhibit weak TCF4 immunoreactivity, with only a few cells showing strong positivity (arrowheads in D ), albeit less intense than in the hippocampus. The arrow in C highlights the marked difference in staining intensity between the pyramidal cell layer of the hippocampal region and the adjacent caudoputamen. CP, caudoputamen; GP, globus pallidus; HP, hippocampus. Scale bars: A–C : 200 μm; D: 100 μm.

Article Snippet: On DIV3 (3 days in vitro ), neurons were transfected with 0.33 μg CamKIIα-tdTomato plasmid ( ) together with 0.66 μg of vector expressing scramble shRNA (SHC002) or a mixture of five Tcf4-targeting shRNA vectors (TRCN0000012093 - TRCN0000012097) developed by the RNAi Consortium (TRC; Broad Institute) using Lipofectamine2000 reagent (11668019, Invitrogen) at 2:1 ratio per well.

Techniques: Expressing, Staining

Journal: Frontiers in Neuroanatomy

Article Title: Transcription factor 4 expression in the developing non-human primate brain: a comparative analysis with the mouse brain

doi: 10.3389/fnana.2024.1478689

Figure Lengend Snippet: Immunoreactivity to TCF4 in the developing mouse hippocampal formation. A–E : overview of TCF4 expression in the developing hippocampus. F–I : closeup of the CA1 region. J–M : closeup of the dentate gyrus. P0–P2 hippocampal formation shows strong TCF4 expression throughout, especially in immature pyramidal cell and dentate gyrus granule cell layers. Expression peaks around P8, resembling mature patterns. At P28, TCF4 levels decrease but are still notable. Interestingly, small cells in the dentate gyrus sub-granular zone maintain high expression (arrowheads in M ), possibly representing precursor cells or immature neurons. CA1, CA3, fields of the hippocampus, DG, dentate gyrus; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bars: A – E : 200 μm; F – M : 50 μm.

Article Snippet: On DIV3 (3 days in vitro ), neurons were transfected with 0.33 μg CamKIIα-tdTomato plasmid ( ) together with 0.66 μg of vector expressing scramble shRNA (SHC002) or a mixture of five Tcf4-targeting shRNA vectors (TRCN0000012093 - TRCN0000012097) developed by the RNAi Consortium (TRC; Broad Institute) using Lipofectamine2000 reagent (11668019, Invitrogen) at 2:1 ratio per well.

Techniques: Expressing

Journal: Frontiers in Neuroanatomy

Article Title: Transcription factor 4 expression in the developing non-human primate brain: a comparative analysis with the mouse brain

doi: 10.3389/fnana.2024.1478689

Figure Lengend Snippet: Immunoreactivity to TCF4 in the developing mouse neocortex. A1 – F1 : Overview of TCF4 expression across the neocortex layers. A2 – F2 : Magnified view of the boxed region in A1 – F1 . At P0 and P2, all neocortical layers exhibit densely packed, strongly TCF4-positive cells. By P5, TCF4-positive cell density decreases, particularly in the more mature deep layers, with the emergence of cells showing lower TCF4 immunoreactivity (arrow in C2 ). At P8 and P10, strongly positive cells (arrowheads in D2 , E2 ) continue to decline in number, while lightly stained cells become more prevalent as the neocortex matures. By P60, TCF4 expression is markedly reduced overall, with only a small subset of cells remaining strongly positive (arrow in F2 ) amidst a larger population of weakly positive cells. Scale bars: A1–F1 : 100 μm; A2 – F2 : 25 μm.

Article Snippet: On DIV3 (3 days in vitro ), neurons were transfected with 0.33 μg CamKIIα-tdTomato plasmid ( ) together with 0.66 μg of vector expressing scramble shRNA (SHC002) or a mixture of five Tcf4-targeting shRNA vectors (TRCN0000012093 - TRCN0000012097) developed by the RNAi Consortium (TRC; Broad Institute) using Lipofectamine2000 reagent (11668019, Invitrogen) at 2:1 ratio per well.

Techniques: Expressing, Staining

Journal: Frontiers in Neuroanatomy

Article Title: Transcription factor 4 expression in the developing non-human primate brain: a comparative analysis with the mouse brain

doi: 10.3389/fnana.2024.1478689

Figure Lengend Snippet: Immunoreactivity to TCF4 in the developing mouse olfactory bulb. High TCF4 expression in the anterior olfactory nucleus, olfactory bulb glomerular layer, and developing internal plexus layer are seen at P0-P5. By P8, TCF4 staining in the olfactory bulb reached adult-like patterns, with strongly positive cells across all layers. AON, anterior olfactory nucleus; EPL, external plexiform layer; GlL, glomerular layer; GrL, Granular layer; IPL, internal plexiform layer; ML, mitral cell layer. Scale bars: A–E : 200 μm; F,G : 50 μm.

Article Snippet: On DIV3 (3 days in vitro ), neurons were transfected with 0.33 μg CamKIIα-tdTomato plasmid ( ) together with 0.66 μg of vector expressing scramble shRNA (SHC002) or a mixture of five Tcf4-targeting shRNA vectors (TRCN0000012093 - TRCN0000012097) developed by the RNAi Consortium (TRC; Broad Institute) using Lipofectamine2000 reagent (11668019, Invitrogen) at 2:1 ratio per well.

Techniques: Expressing, Staining

Journal: Frontiers in Neuroanatomy

Article Title: Transcription factor 4 expression in the developing non-human primate brain: a comparative analysis with the mouse brain

doi: 10.3389/fnana.2024.1478689

Figure Lengend Snippet: Immunoreactivity to TCF4 in the prenatal, infant, and young adult rhesus macaque brain. TCF4 staining in coronal sections from gestation day 151, 2-week-old, 3-month-old, and 5.5-year-old rhesus macaque brain. TCF4 expression is widespread throughout the brain, peaking prenatally and decreasing with age. It concentrates in the grey matter, with the strongest expression in the presubiculum and dentate gyrus of the hippocampal formation and neocortical layers II and IV. In contrast, the thalamus shows relatively lower levels of TCF4. DG, dentate gyrus; PRS, presubiculum; Scale bar: 0.5 cm.

Article Snippet: On DIV3 (3 days in vitro ), neurons were transfected with 0.33 μg CamKIIα-tdTomato plasmid ( ) together with 0.66 μg of vector expressing scramble shRNA (SHC002) or a mixture of five Tcf4-targeting shRNA vectors (TRCN0000012093 - TRCN0000012097) developed by the RNAi Consortium (TRC; Broad Institute) using Lipofectamine2000 reagent (11668019, Invitrogen) at 2:1 ratio per well.

Techniques: Staining, Expressing

Journal: Frontiers in Neuroanatomy

Article Title: Transcription factor 4 expression in the developing non-human primate brain: a comparative analysis with the mouse brain

doi: 10.3389/fnana.2024.1478689

Figure Lengend Snippet: Immunoreactivity to TCF4 in the infant (1 month) rhesus macaque brain. TCF4 expression in 4 coronal sections across the 1-month-old rhesus macaque brain. Staining is stronger in grey matter than white matter, with the cerebellum, hippocampal formation, and neocortex showing the highest expression throughout development. Subcortical regions show lower expression levels. Scale bars: 0.5 cm. HP, hippocampal formation (dentate gyrus, hippocampus, subiculum, presubiculum, parasubiculum); ITG, inferior temporal gyrus; LG, lateral geniculate nucleus; MTG, middle temporal gyrus; PCgG, posterior cingulate gyrus; PoG, postcentral gyrus; PrG, precentral gyrus; PRS, presubiculum; SMG, supramarginal gyrus; STG, superior temporal gyrus; Th, thalamus; WM, white matter. Scale bar: 0.5 cm.

Article Snippet: On DIV3 (3 days in vitro ), neurons were transfected with 0.33 μg CamKIIα-tdTomato plasmid ( ) together with 0.66 μg of vector expressing scramble shRNA (SHC002) or a mixture of five Tcf4-targeting shRNA vectors (TRCN0000012093 - TRCN0000012097) developed by the RNAi Consortium (TRC; Broad Institute) using Lipofectamine2000 reagent (11668019, Invitrogen) at 2:1 ratio per well.

Techniques: Expressing, Staining

Journal: Frontiers in Neuroanatomy

Article Title: Transcription factor 4 expression in the developing non-human primate brain: a comparative analysis with the mouse brain

doi: 10.3389/fnana.2024.1478689

Figure Lengend Snippet: Immunoreactivity to TCF4 in the developing macaque cerebellum. TCF4 immunoreactivity in the developing macaque cerebellum is consistently high, peaking prenatally. Intense staining is observed in granule cell precursors within the external granule cell layer (GD 151 to 3 months), migrating postmitotic granule cells with distinctive oblong shapes (arrowheads in A3 ), and mature granule cells in the granule cell layer. Deep cerebellar nuclei exhibit only weak staining (arrows in A1,B1 ). Small oblong immunopositive cells are visible in the arbor vitae from prenatal stages to 4 weeks of age (arrowheads in inset A2 ). During adolescence, granule cells maintain TCF4 immunoreactivity at reduced intensity, while scattered small immunopositive cells appear in the molecular layer (arrowheads in E2 ). Purkinje cells consistently lack TCF4 immunoreactivity across all ages studied (asterisks). abr: arbor vitae; EGL: External granule layer, GL: Granule cell layer, MCL: Molecular cell layer, PCL: Purkinje cell layer. Scale bars: A1 – E1 : 1 mm; A3 : 100 μm; B3 , B2 – E2 : 100 μm.

Article Snippet: On DIV3 (3 days in vitro ), neurons were transfected with 0.33 μg CamKIIα-tdTomato plasmid ( ) together with 0.66 μg of vector expressing scramble shRNA (SHC002) or a mixture of five Tcf4-targeting shRNA vectors (TRCN0000012093 - TRCN0000012097) developed by the RNAi Consortium (TRC; Broad Institute) using Lipofectamine2000 reagent (11668019, Invitrogen) at 2:1 ratio per well.

Techniques: Staining

Journal: Frontiers in Neuroanatomy

Article Title: Transcription factor 4 expression in the developing non-human primate brain: a comparative analysis with the mouse brain

doi: 10.3389/fnana.2024.1478689

Figure Lengend Snippet: Immunoreactivity to TCF4 in the developing macaque striatum. Prenatally, rare, strongly TCF4-positive cells (arrowheads) are scattered among many weakly stained cells (arrows). The number of strongly positive cells increases during development, forming a distinct, strongly TCF4-positive population (H and J). Scale bars: A,C,E,G,I : 500 μm; B,D,F,H,J : 100 μm.

Article Snippet: On DIV3 (3 days in vitro ), neurons were transfected with 0.33 μg CamKIIα-tdTomato plasmid ( ) together with 0.66 μg of vector expressing scramble shRNA (SHC002) or a mixture of five Tcf4-targeting shRNA vectors (TRCN0000012093 - TRCN0000012097) developed by the RNAi Consortium (TRC; Broad Institute) using Lipofectamine2000 reagent (11668019, Invitrogen) at 2:1 ratio per well.

Techniques: Staining

Journal: Frontiers in Neuroanatomy

Article Title: Transcription factor 4 expression in the developing non-human primate brain: a comparative analysis with the mouse brain

doi: 10.3389/fnana.2024.1478689

Figure Lengend Snippet: Immunoreactivity to TCF4 in the developing macaque hippocampal formation. A1 : Prenatally, the most prominent staining is observed in the dentate gyrus, followed by layer II of the presubiculum (arrowheads). A2 is a magnified view of the dentate gyrus region. The most intense staining is seen in the neuronal progenitor cells of the subgranular zone (arrowheads), followed by cells in the polymorphic layer and granule cells in the granule cell layer. A3 is a magnified view of the pyramidal cell layer. Most pyramidal neurons show strong TCF4 staining. A4 is a magnified view of the dentate gyrus polymorphic and granule cell layers. Intense TCF4 expression is seen in the cells within the subgranular zone and the elongated migrating cells in the polymorphic layer. B1–D1 are overviews of TCF4 expression in the still-developing infant hippocampus. B2–D2 are magnified views of the dentate gyrus. B3–D3 are magnified views of the pyramidal cell layer. B4–D4 are magnifications of the dentate gyrus polymorphic and granule cell layers. TCF4 staining in the infant hippocampus is similar to that seen prenatally. E1 is an overview of TCF4 expression in the young adult hippocampus. TCF4 staining is less than in the infant hippocampus but is still robust, especially in the presubiculum (arrowheads) and the dentate gyrus. E2 is a magnified view of the dentate gyrus. A small population of cells maintained high TCF4 levels, with more such cells in the polymorphic layer than in the granule cell layer. Other cells have a much lower but still robust TCF4 expression. E3 is a magnified view of the pyramidal cell layer. Most pyramidal neurons show robust TCF4 staining, although less intense than in the infant brain. E4 is a magnified view of the dentate gyrus polymorphic and granule cell layers. Most granule cells are robustly stained for TCF4, and a small population of granule cell are more strongly stained, as were cells in the polymorphic layer. DG, dentate gyrus; GCL, granule cell layer; PCL, pyramidal cell layer; PL, polymorphic layer; PRS, presubiculum; SGZ, subgranular zone. Scale bars: A1–E1 : 500 μm; A2–E2 : 100 μm; A3–E3 : 100 μm; A4–E4 : 40 μm.

Article Snippet: On DIV3 (3 days in vitro ), neurons were transfected with 0.33 μg CamKIIα-tdTomato plasmid ( ) together with 0.66 μg of vector expressing scramble shRNA (SHC002) or a mixture of five Tcf4-targeting shRNA vectors (TRCN0000012093 - TRCN0000012097) developed by the RNAi Consortium (TRC; Broad Institute) using Lipofectamine2000 reagent (11668019, Invitrogen) at 2:1 ratio per well.

Techniques: Staining, Expressing

Journal: Frontiers in Neuroanatomy

Article Title: Transcription factor 4 expression in the developing non-human primate brain: a comparative analysis with the mouse brain

doi: 10.3389/fnana.2024.1478689

Figure Lengend Snippet: Immunoreactivity to TCF4 in the developing macaque inferior temporal gyrus. At GD 151, neocortical TCF4 expression concentrates in layers II and IV. As the brain matures, the overall number of TCF4-positive cells decreases. However, in the young adult neocortex, a subset of cells continues to exhibit strong TCF4 staining. Scale bars: 50 μm.

Article Snippet: On DIV3 (3 days in vitro ), neurons were transfected with 0.33 μg CamKIIα-tdTomato plasmid ( ) together with 0.66 μg of vector expressing scramble shRNA (SHC002) or a mixture of five Tcf4-targeting shRNA vectors (TRCN0000012093 - TRCN0000012097) developed by the RNAi Consortium (TRC; Broad Institute) using Lipofectamine2000 reagent (11668019, Invitrogen) at 2:1 ratio per well.

Techniques: Expressing, Staining

Journal: Frontiers in Neuroanatomy

Article Title: Transcription factor 4 expression in the developing non-human primate brain: a comparative analysis with the mouse brain

doi: 10.3389/fnana.2024.1478689

Figure Lengend Snippet: Comparison of TCF4 expression patterns in developing mouse and macaque brains.

Article Snippet: On DIV3 (3 days in vitro ), neurons were transfected with 0.33 μg CamKIIα-tdTomato plasmid ( ) together with 0.66 μg of vector expressing scramble shRNA (SHC002) or a mixture of five Tcf4-targeting shRNA vectors (TRCN0000012093 - TRCN0000012097) developed by the RNAi Consortium (TRC; Broad Institute) using Lipofectamine2000 reagent (11668019, Invitrogen) at 2:1 ratio per well.

Techniques: Comparison, Expressing, Staining, Concentration Assay