Journal: Science Advances

Article Title: Switch of serotonergic descending inhibition into facilitation by a spinal chloride imbalance in neuropathic pain

doi: 10.1126/sciadv.abo0689

Figure Lengend Snippet: ( A ) GFP (green) and PAX2 (red) immunolabeling in the DH. Scale bar, 100 μm. Inset: high magnification showing contacts between Pax2 and 5-HT fibers. Scale bar, 50 μm. ( B ) GFP (green) and Tlx3 (red) immunolabeling in the DH. Scale bar, 100 μm. Inset: high magnification, low or no contact. Scale bar, 25 μm. ( C ) After 3D reconstruction (see Materials and Methods) quantification of contacts between cell bodies (Pax2 and Tlx3) and 5-HT fibers. ( C ) Contacted cells are significantly higher for Pax2(+) than for Tlx3(+) in both males and females [males (blue): 86.2 ± 4.6% for Pax2 and 15.5 ± 3.8% for Tlx3, P < 0.001, Mann-Whitney; females (red): 76.3 ± 2.9% for Pax2 and 8.3 ± 2.2% for Tlx3, P < 0.001, Mann-Whitney]. ( D ) Immunostaining against TPH2 in slices of lumbar spinal cord of GAD67::GFP mice. Scale bar, 25 μm. GABA neurons [GFP(+) in green, stars] or fibers in DH are contacted by TPH2-positive putative synaptic button (in red, white arrows). ( E ) Expression of Myc-tag in synaptic 5-HT terminals in DH following injections of an AAV-synaptophysin-Myc. Immunoreactivity is present in the deeper lamina of the DHSC. Scale bar, 100 μm. Higher magnification shows 5-HT axonic button (white arrow). Scale bar, 20 μm. ( F ) Myc(+) synaptic buttons are in direct contact with GAD67-positive cells and fibers in epet::cre/ GAD67::GFP mice. Scale bar, 20 μm. Inset (top): Myc(+) button on GAD(+) cell bodies. Inset (bottom): Myc(+) button on GAD(+) on GAD(+) fibers.

Article Snippet: For molecular identifications of the spinal network, spinal cord sections were incubated free-floating in 0.1 M PBS containing Triton X-100 (0.3%), bovine serum albumin (1%; Sigma-Aldrich), guinea pig anti-TLX3 antibody (1:1000; a gift from T. Müller), goat anti-Pax2 antibody (1:300; Bio-techne), rabbit anti-Myc antibody (1:500; Euromedex), rabbit anti-TPH2 antibody (1:1000; Bio-techne), and rabbit anti-GAD65/67 (G5163, Sigma-Aldrich) in combination with chicken anti-GFP antibody (1:1000; Averlabs) overnight at 4°C.

Techniques: Immunolabeling, MANN-WHITNEY, Immunostaining, Expressing

Journal: Blood

Article Title: PHF6 regulates hematopoietic stem and progenitor cells and its loss synergizes with expression of TLX3 to cause leukemia

doi: 10.1182/blood-2018-07-860726

Figure Lengend Snippet: Loss of PHF6 synergizes with ectopic TLX3 expression to drive leukemogenesis. (A) Kaplan-Meier survival curve of host mice transplanted with Phf6-deleted or Phf6 control cells expressing MSCV-empty-GFP (empty-GFP) or MSCV-Tlx3-GFP (Tlx3-GFP) retrovirus. n = 11 Phf6-control;Tlx3-GFP, n = 10 Phf6lox/Y;Tie2-creTg/+;Tlx3-GFP, n = 6 Phf6-control;empty-GFP, and n = 6 Phf6lox/Y;Tie2-creTg/+;empty-GFP from 4 donors per genotype. Data were analyzed using the Gehan–Breslow–Wilcoxon test. (B) Cell surface phenotype of Tlx3-GFP tumors of indicated Phf6 genotype with comparison with wild-type spleen cells, showing expression of CD19 and B220. (C) Plots showing no GFP expression in normal B cells of a wild-type spleen and GFP+CD19+B220neg cells in Tlx3-GFP transplants. (B-C) The phenotype and quantification ± standard error of the mean are representative of all analyzed Tlx3-GFP tumors (n = 3 Phf6-control;Tlx3-GFP, n = 10 Phf6lox/Y;Tie2-creTg/+;Tlx3-GFP).

Article Snippet: Tlx3 construct Primers 5′-AGTTTCAGTGCGACTGGAGG-3′ and 5′-GGCAGCGATCCGTAGCTAC-3′ were used to amplify the Tlx3 coding complementary DNA sequence (National Center for Biotechnology Information accession number {"type":"entrez-nucleotide","attrs":{"text":"AK141870.1","term_id":"74202605","term_text":"AK141870.1"}} AK141870.1 ), which was cloned into the murine stem cell virus (MSCV)-IRES-GFP plasmid and confirmed by sequencing.

Techniques: Expressing, Control, Comparison

Journal: Nucleic Acids Research

Article Title: Prediction of DNA binding motifs from 3D models of transcription factors; identifying TLX3 regulated genes

doi: 10.1093/nar/gku1228

Figure Lengend Snippet: Experimental validation of predicted human TLX3 binding sequences. Left panel: Prediction of binding preferences for the human TLX3 TF. The top three predicted binding sequences, which were used for EMSA assays, are displayed as well as the consensus binding motif. The sequence highlighted in blue showed binding to TLX3 in the EMSA assay. Right panel: Results of the EMSA assay using sequence #2 (predicted sequence highlighted in blue), referred to as probe. Lane 1: Negative control (probe only). Lane 2: Biotinylated probe. Lane 3: Cold probe (competition assay). Lane 4: Scrambled probe. The yellow circle marks the shifted, bound motif with TLX3.

Article Snippet: According to The Human Protein Atlas , TLX3 protein expression is observed in epithelial cells (squamous, glandular and transitional), hematopoietic, endocrine, messenchimal and other types of cells (Figure and Supplementary Table S9).

Techniques: Biomarker Discovery, Binding Assay, Sequencing, Negative Control, Competitive Binding Assay

Journal: Nucleic Acids Research

Article Title: Prediction of DNA binding motifs from 3D models of transcription factors; identifying TLX3 regulated genes

doi: 10.1093/nar/gku1228

Figure Lengend Snippet: Predicted function of the TLX3 TF. (A) Protein expression levels of TLX3 as reported in the Human Protein Atlas . The tissue types were broadly grouped and the percent of observed expression levels were calculated for the tested subtissues within each category. Detailed expression levels in subtissues are presented in Supplementary Table S9. (B) Ingenuity pathway analysis of observed targets genes of TLX3 obtained with the TF2DNA predicted binding motif. The figure shows the five most significantly enriched networks in the physiological system development and function category. Sphere sizes are proportional (logarithmic scale) to the amount of genes populating the category. The TLX3 target genes that were enriched within this category are listed in Supplementary Table S12.

Article Snippet: According to The Human Protein Atlas , TLX3 protein expression is observed in epithelial cells (squamous, glandular and transitional), hematopoietic, endocrine, messenchimal and other types of cells (Figure and Supplementary Table S9).

Techniques: Expressing, Binding Assay

Journal: bioRxiv

Article Title: Cadherins orchestrate specific patterns of perisomatic inhibition onto distinct pyramidal cell populations

doi: 10.1101/2023.09.28.559922

Figure Lengend Snippet: a, Constructs of the viral Cre - and FlpO -dependent oEnvA/oTVA and oEnvB/oTVB constructs. b, Multiplex monosynaptic tracing strategy. A FlpO -expressing retrograde AAV was injected into the pons (Pn) of P2-3 Tlx3-Cre mice. A cocktail of Cre -dependent oG and TVB-BFP expressing AAV, together with FlpO -dependent oG and TVA-mGFP AAVs were injected into the S1. Three weeks later, EnvA-RVΔG-tdTomato and EnvB-RVΔG-H2B:GFP were co-injected into the S1. c, Schematic of the spatial distribution of receptor-expressing cells, starter cells and input cells for L5 IT and L5 ET populations. Note that L5 IT cells present in L5a express the Cre recombinase and can be infected with the TVB-receptor (magenta triangle) and/or EnvB (green and magenta triangle). L5 ET cells present in L5b express the FlpO recombinase and can be infected with the TVA-receptor (green outlined triangle) and/or EnvA (green and red triangle). Inputs from L5 IT (green circle) and L5 ET (red circle) neurons can then be mapped across the different cortical layers. d, Schematic representation of L5 IT and L5 ET starter cells and their direct inputs expressing the different viruses. e, Confocal images illustrating the spatial distribution of L5 IT starter cells. Top panel: L5 IT starter cells (white arrowhead) co-expressing TVB-BFP (magenta) and EnvB-H2B:GFP (green) were intermingled in L5 with TVB-BFP+ (asterisk) and EnvB-H2B:GFP-expressing cells. Bottom panel: Confocal image illustrating a L5 IT starter cell. f, Confocal images illustrating the spatial distribution of L5 ET starter cells. Top panel: L5 ET starter cells (white arrowhead) co-expressing TVA-mGFP (green) and EnvA-tdTomato (red) could be detected in L5b, among TVA-mGFP expressing cells and EnvA-tdtTomato expressing cells (asterisk). g, Confocal images illustrating the distribution of L5 IT (green) and L5 ET (red) input cells across a cortical column. L5 IT inputs were labelled with a nuclear GFP while L5 ET inputs expressed a cytosolic tdTomato. h, Fraction of L5 IT and L5 ET starter cells (n = 3 mice). i, Number of L5 IT and L5 ET total inputs (n = 3 mice). j, Number of L5 IT and L5 ET inputs per starter cell (n = 3 mice). Data are represented as mean ± s.e.m. Scale bars, 50 μm (e, f, g), 25 μm (e, f), 5 μm (g).

Article Snippet: Tlx3 Cre and Nex Cre were maintained in a C57BL/6 background (Charles River Laboratories).

Techniques: Construct, Multiplex Assay, Expressing, Injection, Infection