Journal: medRxiv

Article Title: A non-coding variant at 2p24.2 confers susceptibility to non-syndromic cleft lip and palate through LLPS-dependent regulation of MYCN

doi: 10.64898/2026.04.07.26350283

Figure Lengend Snippet: (A and B) FOXP2 overexpression upregulates MYCN . (A) Quantification of mRNA (RT-qPCR) and (B) protein levels (western blot) for FOXP2 and MYCN expression in cNCCs transfected with a FOXP2 overexpression vector (OE-FOXP2) or an empty vector control (OE-Mock). Data normalized to GAPDH . Mean ± SD ( n = 3). Unpaired two-tailed Student’s t-test. (C) Predicted FOXP2-binding motif. Schematic representation of the predicted FOXP2-binding sequence including the rs4263114 SNP within Enh- MYCN . (D and E) FOXP2 preferentially binds the non-risk allele at rs4263114. (D) Anti-FOXP2 chromatin immunoprecipitation (ChIP)-qPCR assay validating FOXP2 enrichment at the Enh- MYCN locus in heterozygous (T/G) cNCCs. Normal IgG served as the negative control. Mean ± SD ( n = 3). Two-way ANOVA (Sidak’s post-hoc test). (F) The risk allele abolishes FOXP2-mediated transactivation of Enh- MYCN . Relative luciferase activity of Enh- MYCN reporter constructs harboring either the non-risk (T) or risk (G) allele in HEPM cells co-transfected with OE- FOXP2 or OE-Mock. Mean ± SD (n = 3). One-way ANOVA (Tukey’s post-hoc test).

Article Snippet: For immunoprecipitation, the sheared chromatin was incubated with 2 μg of either anti-FOXP2 antibody (Novus Biologicals, NB100-55411, CO, USA) or normal rabbit IgG (Cell Signaling Technology, 2729S).

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Expressing, Transfection, Plasmid Preparation, Control, Two Tailed Test, Binding Assay, Sequencing, Chromatin Immunoprecipitation, ChIP-qPCR, Negative Control, Luciferase, Activity Assay, Construct

Journal: medRxiv

Article Title: A non-coding variant at 2p24.2 confers susceptibility to non-syndromic cleft lip and palate through LLPS-dependent regulation of MYCN

doi: 10.64898/2026.04.07.26350283

Figure Lengend Snippet: (A and B) FOXP2 contains a prominent intrinsically disordered region (IDR). Bioinformatic prediction of the FOXP2 IDR sequence using (A) PONDR and (B) PLAAC, providing the structural basis for phase separation. (C) The risk allele disrupts the formation of FOXP2 nuclear condensates. Representative immunofluorescence images of FOXP2 in cNCCs harboring heterozygous (T/G) or homozygous risk (G/G) alleles at rs4263114. Droplet-like FOXP2 condensates are prominent in T/G cells but show a diffuse distribution in G/G cells. Scale bars, 20 μm (left) and 5 μm (right). (D and E) FOXP2 condensates show highly dynamic liquid-like properties. (D) Representative time-lapse fluorescence recovery after photobleaching (FRAP) images of EGFP-FOXP2 overexpressing HEPM cells. Scale bar, 5 μm. (E) Quantification of normalized fluorescence recovery intensity over time ( n = 3 independent experiments). (F) LLPS is required for the allele-specific regulatory activity of Enh- MYCN . Dual-luciferase reporter assay comparing the activity of Enh- MYCN constructs harboring the non-risk (T) or risk (G) allele in HEPM cells upon treatment with 1.5% 1,6-hexanediol (1,6-HD) to chemically disrupt LLPS. Mean ± SD ( n = 3). Unpaired two-tailed Student’s t-test. (G-I) Overexpression of the FOXP2-IDR domain rescues phase separation and MYCN expression in risk-allele cNCCs. (G) Immunofluorescence staining of FOXP2 showing restoration of nuclear condensates in homozygous risk (G/G) cNCCs after FOXP2-IDR overexpression (OE-IDR) versus a Mock-vector baseline (OE-Mock). Scale bar, 10 μm. (H) RT-qPCR and (I) western blot analyses of FOXP2 and MYCN mRNA levels, and their corresponding protein levels, in OE-Mock and OE-IDR G/G cNCCs. Data normalized to GAPDH . For (H) Mean ± SD ( n = 3). Two-way ANOVA (Sidak’s post-hoc test).

Article Snippet: For immunoprecipitation, the sheared chromatin was incubated with 2 μg of either anti-FOXP2 antibody (Novus Biologicals, NB100-55411, CO, USA) or normal rabbit IgG (Cell Signaling Technology, 2729S).

Techniques: Sequencing, Immunofluorescence, Fluorescence, Activity Assay, Luciferase, Reporter Assay, Construct, Two Tailed Test, Over Expression, Expressing, Staining, Plasmid Preparation, Quantitative RT-PCR, Western Blot

Journal: The Journal of Comparative Neurology

Article Title: Immature Excitatory Neurons in the Postnatal Ferret Paralaminar Nuclei and Their Relationship to the Amygdala Across Species

doi: 10.1002/cne.70155

Figure Lengend Snippet: Molecular characterization of Dcx + cells in the juvenile ferret PL . (A and B) Immunofluorescence for Dcx, CoupTFII, and Tbr1 in coronal ferret brain sections at P30 (A) and P67 (B). (C) Immunofluorescence for Dcx + cells in the PL at P30 and P67 showing co‐localization (arrows) or absence of co‐localization (arrowheads) with Tbr1, CoupTFII, or Ctip2. Dcx cells at both ages do not exhibit co‐localization with FoxP2 or Satb2. (D) Quantification of the percentage of Dcx + Tbr1 + and Dcx + CoupTFII + cells. (E) Pie charts showing the percentage of Dcx + cells positive for only one, both, or neither transcription factor at P30, P40, or P67. (F) Representative images of Dcx + Tbr1 + CoupTFII + cells (arrows) and Dcx + Tbr1 − CoupTFII − cells (arrowheads) at P30 and P40. (G) Schematic sagittal section of the ferret brain indicating the locations of the intercalated nuclei (I) and the PL. (H and I) Insets show FoxP2 expression in the intercalated nuclei (I), which are largely Dcx negative at these ages. (J) In the PL (bottom), many Dcx + cells are CoupTFII + and Tbr1 + , but FoxP2 negative. Scale bars: 100 µm (A, B, H, J) and 20 µm (C, F, H magnification, I). BLA, basolateral amygdala; D, dorsal; I, intercalated nuclei; L, lateral; PL, paralaminar nuclei.

Article Snippet: FoxP2 , Goat , 1:500 , IF , Santa Cruz Biotechnology, Cat# 517261; lot # G2216; RRID:AB_2721204 , Inhibitory neurons.

Techniques: Immunofluorescence, Expressing

Journal: Neurobiology of disease

Article Title: Aberrant medial ganglionic eminence (MGE) GABAergic neurogenesis contributes to Huntington’s disease pathogenesis

doi: 10.1016/j.nbd.2026.107297

Figure Lengend Snippet: Dlx1 + arkypallidal GABAergic projection neurons, but not prototypical neurons, are increased in the PND13 BACHD Globus Pallidus. (A/A’–D/D′) Representative immunofluorescence images of the Globus Pallidus from PND13 mice stained for the prototypical neuron marker, PV + cells (A/A’), the arkypallidal neuron marker, Foxp2 (B/B′), EGFP driven by the Dlx1 promoter (C/C′), and combined Foxp2/Dlx1 double labeling (D/D′). (E–H) Quantification of PV + prototypical (E), all Foxp2 + arkypallidal cells (F), Foxp2 + /Dlx1 + arkypallidal cells (G), and Foxp2 + /Dlx1 − cells (H). Data are presented as box-and-whisker plots showing median, interquartile range, and minimum/maximum values. Individual data points represent biological replicates. Statistical comparisons were performed using unpaired Student’s t -test; p < 0.05 was considered significant. Scale bars: 200 μm (A, D).

Article Snippet: For BrdU immunodetection, heat-mediated antigen retrieval was performed in citrate buffer containing 0.05% Tween-20 for 30 min at 100 °C, followed by DNA denaturation in 2 N HCl at 37 °C for 40 min and neutralization in 0.1 M sodium borate buffer (pH 8.0) for 20 min. Primary antibodies included: ChAT (goat anti-ChAT, 1:1000, Sigma-Aldrich, #ab144), BrdU (mouse anti-BrdU, 1:500, R&D systems, #MAB7225), Foxp2 (sheep anti-Foxp2, 1:200, R&D Systems, #AF5647), GFP (chicken anti-GFP, 1:1000, Abcam, #ab13970), Lhx6 (mouse anti-Lhx6, 1:200, Santa Cruz Biotechnology, #sc-271,433), Mafb (rabbit anti-Mafb, 1:200, Cell Signaling Technology, #30919), Nkx2–1 (mouse anti-Nkx2–1, 1:100, Sigma-Aldrich, #MAB5460), NPY (rabbit anti-NPY, 1:500, Cell Signaling Technology, #D745A), phospho-Histone H3 (Rabbit anti-pHH3, 1:300, Cell Signaling Technology, #9701), PV (mouse anti-PV, 1:300, Sigma-Aldrich, #P3086), Satb1 (mouse anti-Satb1, 1:200, Santa Cruz Biotechnology, #sc-376,096), Sox3 (Goat anti-Sox3, 1:500, R&D Systems, #AF2569), SST (rabbit anti-SST, 1:1000, BMA Biomedicals, #T-4103), and VIP (mouse anti-VIP, 1:200, R&D Systems, #576721).

Techniques: Immunofluorescence, Staining, Marker, Labeling, Whisker Assay

Journal: Neurobiology of disease

Article Title: Aberrant medial ganglionic eminence (MGE) GABAergic neurogenesis contributes to Huntington’s disease pathogenesis

doi: 10.1016/j.nbd.2026.107297

Figure Lengend Snippet: The E12.5 BACHD subpallium displays selective alterations of GABAergic precursor cells. Representative immunofluorescence images of the lateral ganglionic eminence [LGE] and ventral pallium [VP] (A–D, A’–D′), the primordium of the globus pallidus (GP; F–G, F′–G’), and the medial ganglionic eminence (MGE) germinative zone (H/H′). Tissue sections were immunostained for Lhx6 (A/A′), Satb1 (B/B′), EGFP driven by the Dlx1 promoter (C/C′), merged staining in A to C (D/D′), Lhx6/Dlx1 (E/E′), Nkx2–1/Dlx1 (F/F′), Lhx6/Foxp2/Dlx1 (G/G′), and VZ/SVZ Nkx2–1/Dlx1 (H/H′). Quantification analyses included LGE mantle: Lhx6 + (I), Satb1 + /Lhx6 + (J), and Mafb + (K); VP: EGFP + driven by the Dlx1 promoter (L), Lhx6 + cells (M), Satb1 + /Lhx6 + (N), and Mafb + (O); Globus Pallidus (GP): Nkx2–1 + (P) and Foxp2 + /Lhx6 + (Q) cells; and MGE VZ/SVZ Nkx2–1 + progenitors (R). Data are presented as box-and-whisker plots showing the median, interquartile range, and minimum/maximum values. Individual data points represent biological replicates. Statistical comparisons were performed using Mann-Whitney U test (I,J,Q) and unpaired Student’s t -test (K, L-P, R); p < 0.05 was considered significant. Scale bars: 50 μm.

Article Snippet: For BrdU immunodetection, heat-mediated antigen retrieval was performed in citrate buffer containing 0.05% Tween-20 for 30 min at 100 °C, followed by DNA denaturation in 2 N HCl at 37 °C for 40 min and neutralization in 0.1 M sodium borate buffer (pH 8.0) for 20 min. Primary antibodies included: ChAT (goat anti-ChAT, 1:1000, Sigma-Aldrich, #ab144), BrdU (mouse anti-BrdU, 1:500, R&D systems, #MAB7225), Foxp2 (sheep anti-Foxp2, 1:200, R&D Systems, #AF5647), GFP (chicken anti-GFP, 1:1000, Abcam, #ab13970), Lhx6 (mouse anti-Lhx6, 1:200, Santa Cruz Biotechnology, #sc-271,433), Mafb (rabbit anti-Mafb, 1:200, Cell Signaling Technology, #30919), Nkx2–1 (mouse anti-Nkx2–1, 1:100, Sigma-Aldrich, #MAB5460), NPY (rabbit anti-NPY, 1:500, Cell Signaling Technology, #D745A), phospho-Histone H3 (Rabbit anti-pHH3, 1:300, Cell Signaling Technology, #9701), PV (mouse anti-PV, 1:300, Sigma-Aldrich, #P3086), Satb1 (mouse anti-Satb1, 1:200, Santa Cruz Biotechnology, #sc-376,096), Sox3 (Goat anti-Sox3, 1:500, R&D Systems, #AF2569), SST (rabbit anti-SST, 1:1000, BMA Biomedicals, #T-4103), and VIP (mouse anti-VIP, 1:200, R&D Systems, #576721).

Techniques: Immunofluorescence, Staining, Whisker Assay, MANN-WHITNEY