Journal: Cell & Bioscience

Article Title: NOTCH2NLC GGC repeat expansions cause retinal neurodegeneration in neuronal intranuclear inclusion disease mouse model

doi: 10.1186/s13578-026-01542-x

Figure Lengend Snippet: Expression of NOTCH2NLC-polyG in the retina of NIID mice. A – F Co-immunostaining on retinas of NIID and control mice using anti-Flag (indicating NOTCH2NLC-polyG) with anti-VSX2 ( A ), anti-PAX6 ( B ), anti-RBPMS ( C ), anti-Rhodopsin ( D ), anti-Arrestin C ( E ), or anti-RPE65 ( F ). Red: VSX2 ( A ), PAX6 ( B ), RBPMS ( C ), Rhodopsin ( D ), Arrestin C ( E ) or RPE65 ( F ); Green: NOTCH2NLC-polyG; Blue: DAPI. Scale bar = 50 μm. The yellow dashed box indicates the region shown at higher magnification. G The percentage of cells co-expressing PolyG and VSX2, PAX6 or RBPMS. H The quantification of the number of VSX2-, PAX6-, and RBPMS-positive cells in NIID and control mice. Data are presented as mean ± SEM. N = 6 per group, ** P = 0.0012 (PAX6), ** P = 0.0021 (RBPMS), ns = no significance, two-tailed t-test. I Transmission electron microscopy images of intranuclear inclusions in the INL and RGC layer of the retinas of NIID mice. The yellow dashed box indicates the region shown at higher magnification. The yellow arrows indicate the round-shaped, filamentous, non-membranous intranuclear inclusions. Scale bar = 1 μm

Article Snippet: Primary antibodies used in this study include: RBPMS (Thermo Fisher Scientific, PA5-31231, 1/300), VSX2 (Proteintech, 25825-1-AP, 1/300), PAX6 (Abcam, ab195045, 1/300), Rhodopsin (Proteintech, 30438-1-AP, 1/200), Arrestin C (Proteintech, 85067-1-AP, 1/200), Flag (Sigma, F1804, 1/300), Flag (Thermo Fisher Scientific, PA5-31231, 1/300), Tuj1 (Sigma, MAB5564, 1/300), GFAP (Cell Signaling Technology, 3670S, 1/300), IBA1 (Wako, 019-19741, 1/300), GFP (Thermo Fisher Scientific, MA5-15256, 1/300).

Techniques: Expressing, Immunostaining, Control, Two Tailed Test, Transmission Assay, Electron Microscopy

Journal: Scientific Reports

Article Title: Preconditioning the Initial State of Feeder-free Human Pluripotent Stem Cells Promotes Self-formation of Three-dimensional Retinal Tissue

doi: 10.1038/s41598-019-55130-w

Figure Lengend Snippet: Preconditioning and d0-SAG treatment promote self-formation of 3D-neuroepithelium from Ff-hiPSCs. (a) Timing for preconditioning of Ff-hiPSCs (Preconditioning) and SAG-treatment on day 0 (d0-SAG). ( b ) Bright-field view of Ff-hiPSC-1231A3-derived aggregates and controls on day 6. Note that SAG treatment on day 0 (d0-SAG) improves aggregate growth compared with untreated controls (Control). ( c ) Immunostaining for Oct3/4 (green) in preconditioned Ff-hiPSC-1231A3 cells and control cells on day 0. ( d ) Bright-field view of preconditioned Ff-hiPSC-1231A3-derived aggregates and controls on day 9. Note that preconditioning with SB and LDN improves aggregate growth compared with untreated controls (Control). ( e ) Percentages of aggregates with a neuroepithelium on day 17 derived from preconditioned Ff-hiPSC-1231A3 cells and untreated controls in combination with or without d0-SAG treatment. Data represent mean ± SEM of three independent experiments (each experiment with 48 aggregates; counted aggregates with neuroepithelium, Control = 0, d0-SAG = 20, Pre: SB + SAG = 61, Pre: SB + SAG plus d0-SAG = 124. * p < 0.05. ** p < 0.01. ANOVA followed by post-hoc Tukey’s test. ( f ) Time-lapse imaging of a SB + SAG-preconditioned-Ff-hiPSC-derived aggregate and a control. ( g ) Immunostaining of preconditioned-Ff-hiPSC-derived aggregates on day 23. Red, Chx10 (upper panels). Green, Rx (lower panels). Blue, nuclear staining with DAPI. Scale bars represent 200 µm in ( b,c,d,f,g ). Similar results were obtained in three independent experiments.

Article Snippet: The qPCR primers used in this study were as follows (TaqMan Probes; Thermo Fisher Scientific): gapdh (Hs02758991_g1), ACTB (Hs01060665-g1), Rx (Rax; Hs00429459_m1), Chx10 (Vsx2; Hs01584047_m1), Pax6 (Hs00240871_m1), Sox1 (Hs01057642-s1), Sox2 (Hs01053049_s1), Oct3/4 (POU5F1; Hs04260367_gH), Nodal (Hs00415443-m1), ID1 (Hs03676575-s1), Gli1 (Hs00171790_m1), Patched-1 (PTCH1; Hs00181117-m1), Gata4 (Hs00171403-m1), Gata6 (Hs00232018-m1), Hand1 (Hs02330376-s1).

Techniques: Derivative Assay, Control, Immunostaining, Imaging, Staining

Journal: Scientific Reports

Article Title: Preconditioning the Initial State of Feeder-free Human Pluripotent Stem Cells Promotes Self-formation of Three-dimensional Retinal Tissue

doi: 10.1038/s41598-019-55130-w

Figure Lengend Snippet: Preconditioning promotes self-formation of 3D-retina from multiple hiPSC and hESC lines in various conditions. ( a,b ) Comparison of NR epithelium-inducing efficiency between preconditioned cells and untreated controls using the hiPSC-C3 line established with Sendai virus vectors. ( a ) Bright-field view of hiPSC-C3 cells in feeder-free culture on day 0. ( b ) Bright-field view (left) and immunostaining for Chx10 (right, red) and nuclear staining with DAPI (right, blue) of aggregates on day 22. ( c,d ) Comparison of NR epithelium-inducing efficiency between preconditioned cells and untreated controls using the hESC-KhES-1 line harboring a Venus-knock-in reporter for Rx ( Rx ::Venus line). ( c ) Bright-field view of hESC-KhES-1 cells on day 0. ( d ) Bright-field view with expression of Rx:: Venus of aggregates (green, left), and immunostaining for Chx10 (left, red) and Rx (right, green) nuclear staining with DAPI (blue) of aggregates on day 18. (e,f ) Comparison of NR epithelium-inducing efficiency between preconditioned cells and untreated controls using the hiPSC-1231A3 line cultured on LM511-E8 matrix in Essential8 medium. ( e ) Bright-field view of hiPSCs cultured in Essential8 medium. ( f ) Bright-field view (left) and immunostaining for Chx10 (left, red) and Rx (right, green) and nuclear staining with DAPI (blue) of aggregates on day 18. (g , h ) Comparison of telencephalic tissue-inducing efficiency between preconditioned cells and untreated controls using the hiPSC-1231A3 line. ( g ) Scheme of differentiation culture. ( h ) Left: Bright-field view of controls and SB + SAG-preconditioned iPSC-derived aggregates. Right: Preconditioned aggregates immunostained for FoxG1 (red) and nuclear staining with DAPI (blue) on day 27 (left), and immunostained for Pax6 (white), Ctip2 (red), and Tbr1 (green) on day 60 (right). Scale bars represent 100 µm ( a,c,e ) and 200 µm ( b,d,f,h ).

Article Snippet: The qPCR primers used in this study were as follows (TaqMan Probes; Thermo Fisher Scientific): gapdh (Hs02758991_g1), ACTB (Hs01060665-g1), Rx (Rax; Hs00429459_m1), Chx10 (Vsx2; Hs01584047_m1), Pax6 (Hs00240871_m1), Sox1 (Hs01057642-s1), Sox2 (Hs01053049_s1), Oct3/4 (POU5F1; Hs04260367_gH), Nodal (Hs00415443-m1), ID1 (Hs03676575-s1), Gli1 (Hs00171790_m1), Patched-1 (PTCH1; Hs00181117-m1), Gata4 (Hs00171403-m1), Gata6 (Hs00232018-m1), Hand1 (Hs02330376-s1).

Techniques: Comparison, Virus, Immunostaining, Staining, Knock-In, Expressing, Cell Culture, Derivative Assay

Journal: Scientific Reports

Article Title: Preconditioning the Initial State of Feeder-free Human Pluripotent Stem Cells Promotes Self-formation of Three-dimensional Retinal Tissue

doi: 10.1038/s41598-019-55130-w

Figure Lengend Snippet: Preconditioning of Ff-hiPSCs affects the proportions of NR and RPE. Comparison of 3D-retina from hiPSCs preconditioned with SB + SAG or LDN + SAG. Ff-hiPSCs (LPF11 line) were preconditioned with SB + SAG or LDN + SAG for 24 h and then differentiated into 3D-retina using the preconditioning, BMP, and induction-reversal methods. (a) Scheme. ( b ) Immunostaining for retinal markers in aggregates on day 18. Immunostaining for Rx (green, left), Pax6 (purple, left), and Chx10 (red, right) and nuclear staining with DAPI (blue, right) are shown. ( c ) Bright-field view of aggregates on day 91. Note that SB + SAG-preconditioned Ff-hiPSCs-aggregates tend to form NR-RPE-conjugated two-domain aggregates. Orange arrows represent NR-RPE-conjugated two-domain aggregates. ( d ) Percentages of the three types of aggregates on day 91. Orange bars represent NR-RPE-conjugated two-domain aggregates. Data are represented as mean ± SEM ( n = 4 experiments, with 12 aggregates per experiment). ( e ) Immunostaining for retinal markers (left) and merged bright-field view (right) of frozen sections of aggregates on day 91. Immunostaining for Crx (green) and Chx10 (red) and staining with DAPI (blue) are shown. Note that the pigmented cells in the bright-field view are the RPE. Orange arrows represent NR-RPE-conjugated two-domain aggregates. ( f ) Comparison of NR epithelium on day 91 derived from cells preconditioned with SB + SAG or LDN + SAG. Immunostaining for retinal markers and nuclear staining with DAPI in aggregates on day 91 are shown. Left: Crx (green), Chx10 (red), and DAPI (blue). Middle: Recoverin (green), Chx10 (red), and Pax6 (blue). Right: NRL (green) and RXRG (purple). ( g ) Comparison of the proportions of multilayered stratified NR epithelium on day 91 derived from cells preconditioned with SB + SAG or LDN + SAG. Data are represented as mean ± SEM ( n = 10 aggregates). ** p < 0.01. Student’s t -test. Similar results were obtained in three independent experiments. Scale bars represent 200 µm in ( b,c,e ) and 100 µm ( f ).

Article Snippet: The qPCR primers used in this study were as follows (TaqMan Probes; Thermo Fisher Scientific): gapdh (Hs02758991_g1), ACTB (Hs01060665-g1), Rx (Rax; Hs00429459_m1), Chx10 (Vsx2; Hs01584047_m1), Pax6 (Hs00240871_m1), Sox1 (Hs01057642-s1), Sox2 (Hs01053049_s1), Oct3/4 (POU5F1; Hs04260367_gH), Nodal (Hs00415443-m1), ID1 (Hs03676575-s1), Gli1 (Hs00171790_m1), Patched-1 (PTCH1; Hs00181117-m1), Gata4 (Hs00171403-m1), Gata6 (Hs00232018-m1), Hand1 (Hs02330376-s1).

Techniques: Comparison, Immunostaining, Staining, Derivative Assay

Journal: Scientific Reports

Article Title: Preconditioning the Initial State of Feeder-free Human Pluripotent Stem Cells Promotes Self-formation of Three-dimensional Retinal Tissue

doi: 10.1038/s41598-019-55130-w

Figure Lengend Snippet: Preconditioned Ff-hiPSCs self-form a multilayered NR epithelium and differentiate into rod and cone photoreceptors. Ff-hiPSC-1231A3 cells were preconditioned with Pre: SB + SAG, treated with d0-SAG and differentiated into 3D-retina. ( a ) Bright-field view of NR-RPE-conjugated two domain aggregate (turnip-shaped) on day 70 derived from SB + SAG-preconditioned Ff-hiPSC-1231A3 cells. (b–g ) Immunostaining for retinal markers and nuclear staining with DAPI in SB + SAG-preconditioned Ff-hiPSC-1231A3-derived 3D-retina on day 178. ( b ) Crx (green), Chx10 (red), and Pax6 (blue). ( c ) Calbindin (green), Rhodopsin (red), and DAPI (blue). ( d ) Crx (green), Chx10 (red), and DAPI (blue). ( e ) Recoverin (green) and DAPI (blue). ( f ) Rhodopsin (red) and S-opsin (light blue). ( g ) L/M-opsin (green) and DAPI (blue). Scale bars represent 100 µm in all panels.

Article Snippet: The qPCR primers used in this study were as follows (TaqMan Probes; Thermo Fisher Scientific): gapdh (Hs02758991_g1), ACTB (Hs01060665-g1), Rx (Rax; Hs00429459_m1), Chx10 (Vsx2; Hs01584047_m1), Pax6 (Hs00240871_m1), Sox1 (Hs01057642-s1), Sox2 (Hs01053049_s1), Oct3/4 (POU5F1; Hs04260367_gH), Nodal (Hs00415443-m1), ID1 (Hs03676575-s1), Gli1 (Hs00171790_m1), Patched-1 (PTCH1; Hs00181117-m1), Gata4 (Hs00171403-m1), Gata6 (Hs00232018-m1), Hand1 (Hs02330376-s1).

Techniques: Derivative Assay, Immunostaining, Staining

Journal: Scientific Reports

Article Title: Preconditioning the Initial State of Feeder-free Human Pluripotent Stem Cells Promotes Self-formation of Three-dimensional Retinal Tissue

doi: 10.1038/s41598-019-55130-w

Figure Lengend Snippet: Transplantation of Ff-hiPSC-derived 3D-retina into RD-nude rats. Immunostaining of rat eyes transplanted with hiPSC-derived 3D-retina. Ff-hiPSC-1231A3 cells were preconditioned with Pre: SB + SAG, treated with d0-SAG and differentiated into 3D-retina. 3D-retina on day 58 was dissected and transplanted into the subretinal space of RD-nude rats. The rat retinas were fixed at 259 days after transplantation (317 days after initiation of differentiation). ( a ) Merged image of bright-field view and immunostaining data shown in ( b ). ( b–j ) Immunostaining for retinal markers and nuclear staining with DAPI. ( b ) Rhodopsin (green), Chx10 (red), and DAPI (blue). ( c,d ) Rhodopsin (green), Chx10 (red), PKCalpha (white), and DAPI (blue). ( e ) L/M-opsin (green) and DAPI (blue). ( f ) Rhodopsin (red) and S-opsin (light blue). ( g–i ) NRL (green), Recoverin (red), HuNu (white), and DAPI (blue). ( j ) Maximum projection image of Z-stacks stained for Rhodopsin (green), PKCalpha (red), and DAPI (blue). Note that Rhodopsin + rods are in direct contact with PKCalpha + bipolar cells, which have axons toward the basal side of the host retina. Similar results were obtained in four eyes. Scale bars represent 1000 µm in ( a,b ), 100 µm in ( c–f ), and 10 µm in ( g–j ). INL, inner nuclear layer; GCL, ganglion cell layer.

Article Snippet: The qPCR primers used in this study were as follows (TaqMan Probes; Thermo Fisher Scientific): gapdh (Hs02758991_g1), ACTB (Hs01060665-g1), Rx (Rax; Hs00429459_m1), Chx10 (Vsx2; Hs01584047_m1), Pax6 (Hs00240871_m1), Sox1 (Hs01057642-s1), Sox2 (Hs01053049_s1), Oct3/4 (POU5F1; Hs04260367_gH), Nodal (Hs00415443-m1), ID1 (Hs03676575-s1), Gli1 (Hs00171790_m1), Patched-1 (PTCH1; Hs00181117-m1), Gata4 (Hs00171403-m1), Gata6 (Hs00232018-m1), Hand1 (Hs02330376-s1).

Techniques: Transplantation Assay, Derivative Assay, Immunostaining, Staining

Journal: bioRxiv

Article Title: Gene regulatory mechanisms guiding bifurcation of inhibitory and excitatory neuron lineages in the anterior brainstem

doi: 10.1101/2025.01.26.634960

Figure Lengend Snippet: A. Chromatin accessibility per cell group (normalized signal), Ensembl gene track (Genes), scATACseq features (Feat.), feature linkage to gene (Links with the LinkPeaks abs(zscore) >2) and nucleotide conservation (Cons.) within +/-50 kbp region around Tal1 TSS. Violin plots on the right show the expression levels of Tal1 and Pdzk1ip1 per cell group. B. Spline-smoothed z-score transformed heatmaps of chromatin accessibility at Tal1 -linked scATACseq features ( Tal1 cCREs) in the single cells of rV2 GABAergic and glutamatergic lineages with RNA expression (sliding window mean(width=6) smoothed) of Tal1 , Gata2 , Gad1 and Slc17a6 as column covariable (top). Cells on the x-axis are first grouped per cell group and then ordered by the pseudotime (bottom) within each group. C. Same as in A, but for the Vsx2 locus. D. Accessibility of Vsx2 cCREs in the rV2 GABAergic and glutamatergic lineages, shown as in (B). The expression levels of Vsx2 , Tal1 , Gad1 , and Slc17a6 are shown above the heatmaps.

Article Snippet: The following primary antibodies were used: rabbit anti-Gata2 (Abcam, ab109241), 1:80 rabbit anti-Gata3 (Boster, M00593), rabbit anti-Tal1 (Abcam, ab75739), rabbit anti-Vsx2 (BioSite, 25825-1-AP-20), rabbit anti-Ebf1 ( ) rabbit anti-Insm1 (BioSite, ASJ-IO4DE3-50), rabbit anti-Tead2 (Biorbyt, orb382464), rabbit anti-IgG (Cell Signalling, 86652/66362) and rabbit anti-H3K4me3 (Cell Signalling, 86652/9751).

Techniques: Expressing, Transformation Assay, RNA Expression

Journal: bioRxiv

Article Title: Gene regulatory mechanisms guiding bifurcation of inhibitory and excitatory neuron lineages in the anterior brainstem

doi: 10.1101/2025.01.26.634960

Figure Lengend Snippet: A. Schematic explaining the strategy of identifying the targets of Tal1 , Gata2 and Gata3 selector TFs. Within the TAD containing a gene, a feature overlapping a CUT&Tag peak for the selector TF, and a footprint for the TF at a position with weighted mean conservation score > 0.5 are found. Genes linked to features fulfilling these conditions are considered target genes. For linkage, the Spearman correlation-based LinkPeaks score between the feature targeted by the selector TF and expression of the gene is required to be >2 (positive effect link) or <-2 (negative effect link) with a p-value <0.01. B. Number of target genes and the overlap between Gata2 , Gata3 and Tal1 target genes. C. GSEA of Tal1 targets. Mouse genes are ranked by the difference in the expression in GA1-2 vs GL1-2 cell groups (log2 avg FC). Tal1 target genes are indicated with black lines. In leading edges, the GA1-2-enriched target genes are highlighted in blue and GL1-2-enriched genes with red. Scatterplots show the expression of the target genes in both edges. D-F. Characterization of Tal1 target genes. D. Count of target features by the positive and negative effect link. E. Count of target genes by the nearest linked feature, stratified by the nearest feature distance bins as indicated. F. The variability of target gene expression in rV2 lineage cell clusters, stratified by the nearest feature distance bins. G. Top terms in CellMarker gene set database using the list of Tal1 target genes with exp>0.5 (log1p) in GA1-2 or GL1-2 cell groups or both. H. GSEA of Gata2 targets, as in (C). I-L. Characterization of Gata2 target genes, as in (D-G). M. GSEA of Gata3 targets, as in (C). N-Q. Characterization of Gata3 target genes, as in (D-G). R. Venn diagram of selector TF target genes that show specificity to rV2 glutamatergic neurons (defined as avg(exp in GA)<0.5 (log1p) AND avg(exp in GL)>0.5 (log1p), and see Methods). Genes in every overlap category are listed. Vsx2 target genes that show specificity to rV2 glutamatergic neurons (n=3) are separately listed. S. Venn diagram of selector TF target genes that show specificity to rV2 GABAergic neurons (defined as avg(exp in GL)<0.5 (log1p) AND avg(exp in GA)>0.5 (log1p)). Genes in every overlap category are listed. Vsx2 target genes that show specificity to rV2 GABAergic neurons (n=2) are separately listed. T. Proposed gene regulatory network guiding the GABA-vs glutamatergic fate selection in the rV2. Arrows represent positive regulation and were drawn when the regulator and the target expression were co-expressed in the same cell group. Blunt arrows represent negative regulation and were drawn when the regulator and the target expression were found in different cell groups (GA1-2 vs GL1-2) and not in the same cell group together.

Article Snippet: The following primary antibodies were used: rabbit anti-Gata2 (Abcam, ab109241), 1:80 rabbit anti-Gata3 (Boster, M00593), rabbit anti-Tal1 (Abcam, ab75739), rabbit anti-Vsx2 (BioSite, 25825-1-AP-20), rabbit anti-Ebf1 ( ) rabbit anti-Insm1 (BioSite, ASJ-IO4DE3-50), rabbit anti-Tead2 (Biorbyt, orb382464), rabbit anti-IgG (Cell Signalling, 86652/66362) and rabbit anti-H3K4me3 (Cell Signalling, 86652/9751).

Techniques: Expressing, Targeted Gene Expression, Selection

Journal: bioRxiv

Article Title: Gene regulatory mechanisms guiding bifurcation of inhibitory and excitatory neuron lineages in the anterior brainstem

doi: 10.1101/2025.01.26.634960

Figure Lengend Snippet: A. Normalized scATACseq signal in +/-50 kb region of Gata2 (A) TSS, in the rV2 lineage single-cell clusters at top. Below, the Ensembl gene models (Genes), the linkage of features to Gata2 or Gata3 (Links), the scATAC features (Feat.; cCREs are shown in blue), and by-nucleotide conservation of DNA across vertebrate species (Cons.) is shown. Violin plots show the distribution of Gata2 (A) RNA expression (log1p) in single cell clusters. B. Smoothed heatmaps of the accessibility of the Gata2 cCREs in the rV2 GABAergic and glutamatergic cell lineages (GABA, GLUT). Cluster identities are shown on top of heatmaps. Cells are ordered by the pseudotime value (Pseudotime). cCREs within the TAD bounds are shown on feature heatmaps. RNA expression (log1p) of Gata2 , Vsx2 , Gad1 and Slc17a6 is shown above the heatmaps. C. scATACseq signal in the rV2 lineage single-cell clusters and genomic features in +/-50 kb region of Gata3 TSS, similar to (A). Violin plots show the Gata3 RNA expression (log1p) in single cells of rV2 cell clusters. Gata3 gene ATG is located +11 kb from the TSS, the position is indicated in Genes view. D. Smoothed heatmaps of the accessibility of the Gata3 cCREs in the rV2 GABAergic and glutamatergic cell lineages (GABA, GLUT). RNA expression (log1p) of Gata3 , Tal1 , Gad1 and Slc17a6 is shown above the heatmaps.

Article Snippet: The following primary antibodies were used: rabbit anti-Gata2 (Abcam, ab109241), 1:80 rabbit anti-Gata3 (Boster, M00593), rabbit anti-Tal1 (Abcam, ab75739), rabbit anti-Vsx2 (BioSite, 25825-1-AP-20), rabbit anti-Ebf1 ( ) rabbit anti-Insm1 (BioSite, ASJ-IO4DE3-50), rabbit anti-Tead2 (Biorbyt, orb382464), rabbit anti-IgG (Cell Signalling, 86652/66362) and rabbit anti-H3K4me3 (Cell Signalling, 86652/9751).

Techniques: RNA Expression

Journal: bioRxiv

Article Title: Gene regulatory mechanisms guiding bifurcation of inhibitory and excitatory neuron lineages in the anterior brainstem

doi: 10.1101/2025.01.26.634960

Figure Lengend Snippet: A. ATAC features within +/-50 kb of the Tal1 gene. Tal1 cCREs are shown in blue. CUT&Tag, CUT&Tag consensus peaks indicating Tal1, Gata2, Gata3, Vsx2, Ebf1, Insm1, and Tead2 binding in the E12.5 mouse r1. B-D. Footprinting based TF binding activity in the features at +1 kb, +23 kb and +40 kb of the Tal1 TSS. Feature linkage p to Tal1 is indicated by asterisks. Footprint scores at conserved TFBSs are shown for progenitors (PRO1-2), common precursors (CO1-2), GABAergic precursors (GA1-2) and glutamatergic precursors (GL1-2). In each dotplot, the strength of footprint at TFBS in the feature is shown as colour (Footprint score, average of cell group) and the expression of the TF gene in dot size (log1p). Average feature accessibility in cell groups is shown at the right. TFBS names (Hocomoco v12) are shown at the top and the TF gene names (mouse) are shown at the bottom of the dotplot. The red arrowhead in (B) indicates the conserved Gata2 TFBS at –37 bp position required for the neural expression of Tal1 (see also Supplementary Table 4). E. Overlap of the TFs with footprints in the cCREs of Tal1 , Gata2 , and Gata3 in the common precursors of rV2 lineages (CO1-2) and in the rV2 GABAergic precursors (GA1-2). Venn diagrams show the number of TFs with a footprint detected in Tal1 , Gata2 , or Gata3 -linked features (Fp in cCRE = 1) and with their gene expression > 1.2 (log1p) in the analysed cell group. The TFs associated with the cCREs of all three selector genes in common precursors (CO1-2) and GABAergic precursors (GA1-2) are listed. Blue text: 19 TFs interact with cCREs of Tal1 , Gata2 and Gata3 in the CO1-2 cells. Some of these TFs continue to be expressed and interact with the Tal1 , Gata2 and Gata3 genes in the GA1-2 cells. 22 TFs interact with Tal1 , Gata2 and Gata3 in GA1-2 cells. Green text, TFs found associated with two selector genes in CO1-2 (green in the Venn diagram of CO1-2) and associated with all three selector genes in the GA1-2. Black text, TFs co-regulating the selector TFs in GA1-2 and not expressed in the CO1-2 cells. § The probability of finding n overlapping genes considering all mouse genes equally is p<1e-6. *,** The collective minimum statistical significance of feature to gene links for selector genes Tal1, Gata2 and Gata3 cCREs for the given TF is shown as: *p-value<0.05; **p-value<0.01 (with LinkPeaks z-score above 2 or below -2).

Article Snippet: The following primary antibodies were used: rabbit anti-Gata2 (Abcam, ab109241), 1:80 rabbit anti-Gata3 (Boster, M00593), rabbit anti-Tal1 (Abcam, ab75739), rabbit anti-Vsx2 (BioSite, 25825-1-AP-20), rabbit anti-Ebf1 ( ) rabbit anti-Insm1 (BioSite, ASJ-IO4DE3-50), rabbit anti-Tead2 (Biorbyt, orb382464), rabbit anti-IgG (Cell Signalling, 86652/66362) and rabbit anti-H3K4me3 (Cell Signalling, 86652/9751).

Techniques: Binding Assay, Footprinting, Activity Assay, Expressing

Journal: bioRxiv

Article Title: Gene regulatory mechanisms guiding bifurcation of inhibitory and excitatory neuron lineages in the anterior brainstem

doi: 10.1101/2025.01.26.634960

Figure Lengend Snippet: A. Vsx2 gene with its protein-coding regions and associated chromatin features. B. CUT&Tag. Consensus peaks of CUT&Tag signal with Gata2, Gata3, Tal1, Vsx2, Ebf1, Insm1 and Tead2 antibodies in E12.5 mouse r1. C-E. TF binding activity in the Vsx2 -associated features at +20.5 kb, -61 kb and –68.8 kb of Vsx2 TSS according to ATAC-footprinting analysis. In each feature, the strength of footprint (averaged over the cell group shown at the side) at TFBS in the feature is shown as colour and the expression of the TF gene in dot size. Feature accessibility in cell groups is shown at the right (Accessibility dotplot). TFBS names are shown at the top and the TF gene names (mouse genes) are shown under the dotplot. F. Overlap of the TFs interacting with the cCREs of Vsx1 and Vsx2 in the common precursors of rV2 lineages (CO) and in the rV2 glutamatergic precursors (GL). Common regulators Vsx1 and Vsx2 in CO (n=18) and in GL (n=21) are listed next to venn diagrams. The 18 TFs regulating both Vsx1 and Vsx2 in CO cells are listed in blue text. § The probability of finding n overlapping genes considering all mouse genes equally is p<1e-6. *,** The collective minimum statistical significance of feature-to-gene links for selector genes Vsx1 and Vsx2 cCREs for the given TF is shown as: *p-value<0.05; **p-value<0.01 (with LinkPeaks z-score above 2 or below -2).

Article Snippet: The following primary antibodies were used: rabbit anti-Gata2 (Abcam, ab109241), 1:80 rabbit anti-Gata3 (Boster, M00593), rabbit anti-Tal1 (Abcam, ab75739), rabbit anti-Vsx2 (BioSite, 25825-1-AP-20), rabbit anti-Ebf1 ( ) rabbit anti-Insm1 (BioSite, ASJ-IO4DE3-50), rabbit anti-Tead2 (Biorbyt, orb382464), rabbit anti-IgG (Cell Signalling, 86652/66362) and rabbit anti-H3K4me3 (Cell Signalling, 86652/9751).

Techniques: Binding Assay, Activity Assay, Footprinting, Expressing

Journal: PLoS Genetics

Article Title: Vsx2 Controls Eye Organogenesis and Retinal Progenitor Identity Via Homeodomain and Non-Homeodomain Residues Required for High Affinity DNA Binding

doi: 10.1371/journal.pgen.1002924

Figure Lengend Snippet: (A) ClustalW alignment of the homeodomain and adjacent 60 amino acids in select VSX orthologs and the most similar non-VSX proteins in mice. Only the VSX sequences have a discernable CVC domain. The positions of the orJ , R200Q , and R227W mutations are shown. (B) Left panel: EMSA with in vitro translated VSX2, VSX2 [R200Q] , and VSX2 [R227W] proteins and [ 32 ] P-labeled P3 oligo (see for sequence). Top right panel: Extended exposure reveals weak binding by VSX2 [R227W] . Bottom right panel: Western blot of in vitro translated proteins with VSX2 antibody (Lys, control lysate; -, P3 probe only). (C) Schematic shows five putative Vsx2 binding sites (Hx-6 – Hx-10) in the proximal promoter region (∼0.3 kb) of D-Mitf . Carats and dashed line marks the region of PCR amplification in the ChIP assay shown below schematic (primer set 13; ). Arrowhead points to sequence-verified ChIP product. (D) Luciferase assays in P0 primary retinal cells transfected with the indicated expression vectors ( x -axis) and ∼2.2 kb of the D-Mitf promoter region (pGL3P- DMitf ). (E) The Hx-9 site was mutated in pGL3P-m DMitf to eliminate DNA binding at that site. Reporter assays were normalized to empty vector controls (white bars). (F) CAT assays in HEK293 cells transfected with the X4G2CAT reporter and VSX2 variants fused to the LexA DNA binding domain. Gal4-Hsf1 was included to stimulate high basal reporter activity . ** P≤0.01; *** P≤0.001.

Article Snippet: Vsx2 R200Q chimeric mice were generated at inGenious Targeting Laboratory (Stony Brook, NY) and Vsx2 R227W chimeric mice were generated at the University of Utah Gene Targeting and Transgenic Core Facility (See and for details on gene targeting strategy).

Techniques: In Vitro, Labeling, Sequencing, Binding Assay, Western Blot, Control, Amplification, Luciferase, Transfection, Expressing, Plasmid Preparation, Activity Assay

Journal: PLoS Genetics

Article Title: Vsx2 Controls Eye Organogenesis and Retinal Progenitor Identity Via Homeodomain and Non-Homeodomain Residues Required for High Affinity DNA Binding

doi: 10.1371/journal.pgen.1002924

Figure Lengend Snippet: (A–D) Mice homozygous for the orJ , R200Q , and R227W alleles had smaller eyes than wild-type by E11.5. (E–H) At E14.5, overall embryonic development was unaffected in the mutants, but the failure of the mutant eyes to keep pace with the growth of the wild-type eye was evident. Eye growth in the R227W mutant also failed to keep pace with the orJ and R200Q mutants. (I–L) Dissected E17.5 eyes (right eyes rotated 90°) show similar reductions in eye size in orJ and R200Q homozygotes whereas the reduction in eye size of R227W homozygotes was the most severe. (M–P) VSX2 immunohistochemistry in E12.5 retinas. VSX2 protein was not detected in the orJ retina, confirming it as an expression null. VSX2 [R200Q] and VSX2 [R227W] were expressed similarly to VSX2 [wt] , although to a reduced extent in peripheral retina. Dashed lines bound retinas. (Q) ChIP assays with VSX2 antibody reacted with E12.5 native chromatin lysates from wild-type, R200Q , and R227W retinas and amplified using D-Mitf primer set 13 . Arrowhead denotes amplification product. Graph shows quantification results of ChIP-qPCR. Scale bars: 0.5 mm (E11.5); 5 mm (E14.5); 1 mm (E17.5).

Article Snippet: Vsx2 R200Q chimeric mice were generated at inGenious Targeting Laboratory (Stony Brook, NY) and Vsx2 R227W chimeric mice were generated at the University of Utah Gene Targeting and Transgenic Core Facility (See and for details on gene targeting strategy).

Techniques: Mutagenesis, Immunohistochemistry, Expressing, Amplification, ChIP-qPCR

Journal: PLoS Genetics

Article Title: Vsx2 Controls Eye Organogenesis and Retinal Progenitor Identity Via Homeodomain and Non-Homeodomain Residues Required for High Affinity DNA Binding

doi: 10.1371/journal.pgen.1002924

Figure Lengend Snippet: (A–H) Merged images of cryosections showing DAPI staining (blue) and melanogenic pigmentation (white) for each of the indicated genotypes and ages. Arrowheads in H point to aberrant pigmentation in peripheral retina asterisk denotes ectopic periocular mesenchyme (POM) in vitreal cavity. (I–P) Expression patterns of the neuronal differentiation marker class III β-Tubulin (TUBB3). Neurogenesis lagged behind wild-type and to a similar extent in the orJ and R200Q retinas, but did not initiate in the R227W retina. (Q–T) Merged images of cryosections showing DAPI staining (blue) and melanogenic pigmentation (white) for each of the indicated genotypes at E17.5. The R227W retina was aberrantly pigmented, either partially (T (a) ) or completely (T (b) ). Arrowheads in T (a) and T (b) point to aberrant pigmentation in peripheral retina, arrows to central retinal regions, and asterisks to ectopic pigmentation in vitreal cavity. (U–W) Pigmented cells expressing VSX2 [R227W] were detected in pigmented retinal region. L, lens; RPE, retinal pigment epithelium. Scale bars: 100 µm (A–T), 20 µm (U–W).

Article Snippet: Vsx2 R200Q chimeric mice were generated at inGenious Targeting Laboratory (Stony Brook, NY) and Vsx2 R227W chimeric mice were generated at the University of Utah Gene Targeting and Transgenic Core Facility (See and for details on gene targeting strategy).

Techniques: Staining, Expressing, Marker

Journal: PLoS Genetics

Article Title: Vsx2 Controls Eye Organogenesis and Retinal Progenitor Identity Via Homeodomain and Non-Homeodomain Residues Required for High Affinity DNA Binding

doi: 10.1371/journal.pgen.1002924

Figure Lengend Snippet: (A) Wild-type, orJ/+ , R200Q/+ , and R227W/+ eyes were indistinguishable at P0. No significant differences in eye circumferences were detected. (B–E) Merged images of cryosections showing DAPI staining (blue) and melanogenic pigmentation (white) for each of the indicated genotypes at P0. (F–I) Expression of the retinal ganglion cell marker POU4F2 in wild-type or Vsx2 heterozygous retinas. (J) Eye circumference of orJ/R227W heterozygotes was intermediate to orJ and R227W homozygotes. (K–S) Expression of VSX2, CCND1, and TUBB3 in orJ , orJ/R227W , and R227W retinas at P0. VSX2 was detected in the orJ/R227W retina only. CCND1 and TUBB3 were detected in orJ or orJ/R227W retinas but not in R227W pigmented retina. (T) Genotype-phenotype correlation of Vsx2 alleles arranged by retinal phenotype. * P≤0.05 Scale bars: 1 mm (A, J); 100 µm (B–I, K–S).

Article Snippet: Vsx2 R200Q chimeric mice were generated at inGenious Targeting Laboratory (Stony Brook, NY) and Vsx2 R227W chimeric mice were generated at the University of Utah Gene Targeting and Transgenic Core Facility (See and for details on gene targeting strategy).

Techniques: Staining, Expressing, Marker

Journal: PLoS Genetics

Article Title: Vsx2 Controls Eye Organogenesis and Retinal Progenitor Identity Via Homeodomain and Non-Homeodomain Residues Required for High Affinity DNA Binding

doi: 10.1371/journal.pgen.1002924

Figure Lengend Snippet: (A–D) MITF expression at E12.5 for the indicated genotypes. The R227W retina expressed MITF at much higher levels compared to the orJ and R200Q mutants. (D′) Merged images of VSX2 [R227W] (red) and MITF (green) shows overlap in expression. The lack of VSX2 [R227W] expression in the peripheral retina corresponded to the highest levels of MITF. (E–H) OTX expression at E12.5 for the indicated genotypes. OTX expression was highest in the R227W retina. (H′) Merged images of VSX2 [R227W] (red) and OTX (green). Like MITF, OTX expression was highest in regions lacking VSX2 [R227W] . MITF and OTX were also expressed in RPE (outside lower dashed lines). (I–K) Relative mRNA expression levels of pan- Mitf (I), Otx1 and Otx2 (J), and the D- , H- , A- , J- , and B - Mitf isoforms (K) in E12.5 retinas of the indicated genotypes as determined by qRT-PCR. Samples were normalized to the expression level for each transcript in the orJ retina (white bars). * P≤0.05 Scale bar: 50 µm.

Article Snippet: Vsx2 R200Q chimeric mice were generated at inGenious Targeting Laboratory (Stony Brook, NY) and Vsx2 R227W chimeric mice were generated at the University of Utah Gene Targeting and Transgenic Core Facility (See and for details on gene targeting strategy).

Techniques: Expressing, Quantitative RT-PCR

Journal: PLoS Genetics

Article Title: Vsx2 Controls Eye Organogenesis and Retinal Progenitor Identity Via Homeodomain and Non-Homeodomain Residues Required for High Affinity DNA Binding

doi: 10.1371/journal.pgen.1002924

Figure Lengend Snippet: (A–F) Merged images of cryosections showing DAPI staining (blue) and melanogenic pigmentation (white) in P0 orj , R200Q , and R227W mice that were Mitf wild-type ( Mitf +/+ ; A–C) and mi heterozygous ( Mitf mi/+ ; D–F). Insets show whole eyes. The retina in C was completely transformed into pigmented tissue (bounded by dashed line) and ectopic POM was partially pigmented (asterisk). Eye size and retinal histology were restored to a comparable degree in all Vsx2 , mi compound mutants (D–F). Also notable in the R227W , mi compound mutant was the lack of POM in the vitreal chamber (asterisk in F). (G–L) TUBB3 staining at P0. In all cases, lamination patterns were restored in the compound mutants, indicating robust neurogenesis. Retinal tissue in I is bounded by the dashed lines. (M) The reduced eye size in the R227W mutant was partially rescued in the R227W ; Mitf mi/+ mutant at E12.5. (N) The expression of TUBB3 was detected in the R227W ; Mitf mi/+ retina at E13.5. (O) Mitf and Otx1 transcript levels were much lower in the R227W ; Mitf mi/+ retina (black bars) compared to the R227W mutant (white bars). (P) pan- Mitf transcript level in orJ; Mitf mi/+ retina was not lower than that in orJ retina. * P≤0.05; *** P≤0.001 Scale bars: 100 µm (A–F); 1 mm (insets); 50 µm (G–L); 0.5 mm (M); 100 µm (N).

Article Snippet: Vsx2 R200Q chimeric mice were generated at inGenious Targeting Laboratory (Stony Brook, NY) and Vsx2 R227W chimeric mice were generated at the University of Utah Gene Targeting and Transgenic Core Facility (See and for details on gene targeting strategy).

Techniques: Staining, Transformation Assay, Mutagenesis, Expressing

Journal: PLoS Genetics

Article Title: Vsx2 Controls Eye Organogenesis and Retinal Progenitor Identity Via Homeodomain and Non-Homeodomain Residues Required for High Affinity DNA Binding

doi: 10.1371/journal.pgen.1002924

Figure Lengend Snippet: (A) p27 mRNA expression in E12.5 retinas of the indicated genotypes as determined by qRT-PCR. Samples were normalized to orJ . Only the R227W retina was significantly different. (B) Luciferase activities from HEK293 cells transfected with the indicated expression vectors ( x- axes) and ∼1.1 kb of the p27 promoter region (pGL3B- p27 ). Graph I: H-MITF repressed reporter activity in a DNA binding-dependent manner. Graph II: VSX2 and VSX2 [R227W] enhanced reporter activity. Graph III: H-MITF combined with VSX2 [R227W] elicited a specific and synergistic increase in reporter activity that depended on DNA binding as revealed by the abrogated activity of the VSX2 [R200Q, R227W] double mutant (RQRW). Graph IV: Expression of the mi version of H-MITF had no effect on reporter activity resulting from VSX2 or its variants. (C) Schematic of p27 5′-intergenic region (∼1.1 kb). Positions of putative Mitf binding sites (M) and homeodomain core sequences (H) are shown. Positions are relative to p27 transcriptional start site. Position of primers that constitute p27 primer set 2 is also shown. Graphs show quantification of ChIP-qPCR assays using MITF or VSX2 antibodies reacted with E12.5 lysates from wild-type, R200Q and R227W retinas. MITF binding was detected in R200Q and R227W lysates. VSX2 binding was detected in R227W lysate. (D) Co-IPs of E12.5 R227W and R200Q retinal protein lysates with a negative control sheep IgG or VSX2 antibodies followed by western blot probed with MITF antibody (top panel). Co-IPs of HEK293 cells transfected with VSX2 or its variants (listed below images) plus H-MITF (middle panel) or its mi variant (bottom panel). IPs were performed with sheep IgG or VSX2 antibodies followed by western blot probed with MITF antibody. input refers to the 20% of whole protein lysate used for co-IP. (E) Luciferase assays in HEK293 cells transfected with the indicated expression vectors ( x- axes) and the pGL3B- HMitf . Left graph: effects of VSX2 and its variants on reporter activity were not statistically significant. Right graph: H-MITF repressed reporter activity, whereas OTX1 enhanced reporter activity. Reporter activity in cells co-expressing of OTX1 and H-MITF was significantly higher than the sum of the factors expressed individually (** associated with lines over bars). H-MITF [mi] enhanced reporter activity, but reporter activity in cells co-expressing OTX1 and H- MITF [mi] was not significantly different than the sum of the two factors expressed individually. * P≤0.05; ** P≤0.01; *** P≤0.001.

Article Snippet: Vsx2 R200Q chimeric mice were generated at inGenious Targeting Laboratory (Stony Brook, NY) and Vsx2 R227W chimeric mice were generated at the University of Utah Gene Targeting and Transgenic Core Facility (See and for details on gene targeting strategy).

Techniques: Expressing, Quantitative RT-PCR, Luciferase, Transfection, Activity Assay, Binding Assay, Mutagenesis, ChIP-qPCR, Negative Control, Western Blot, Variant Assay, Co-Immunoprecipitation Assay

Journal: PLoS Genetics

Article Title: Vsx2 Controls Eye Organogenesis and Retinal Progenitor Identity Via Homeodomain and Non-Homeodomain Residues Required for High Affinity DNA Binding

doi: 10.1371/journal.pgen.1002924

Figure Lengend Snippet: (A) During early eye development, Mitf is expressed in optic neuroepithelial cells (Mitf ONC ) in response to upstream activators. Vsx2 expression is activated in the newly specified retinal domain by upstream activators, which leads to repression of Mitf in RPCs (Mitf RPC ) and suppression of the pigmentation program. (B) In orJ mice, Mitf persists in RPCs because the VSX2 protein is absent, which increases the probability that pigmentation will occur. (C) In R200Q mice, VSX2 [R200Q] protein is present but unable to bind DNA, allowing Mitf to persist in RPCs, increasing the probability of pigmentation. (D) In R227W mice, VSX2 [R227W] protein is present and may still suppress the pathway that leads to pigmentation in orJ and R200Q RPCs, but its interaction with Mitf combined with its weak DNA binding activity engages a novel positive feedback loop that activates a robust pigmentation program. Our genetic data place Otx1 downstream of p27 and D-Mitf, but the mechanism causing its elevated expression is not clear.

Article Snippet: Vsx2 R200Q chimeric mice were generated at inGenious Targeting Laboratory (Stony Brook, NY) and Vsx2 R227W chimeric mice were generated at the University of Utah Gene Targeting and Transgenic Core Facility (See and for details on gene targeting strategy).

Techniques: Expressing, Binding Assay, Activity Assay

Journal: PLoS Genetics

Article Title: Vsx2 Controls Eye Organogenesis and Retinal Progenitor Identity Via Homeodomain and Non-Homeodomain Residues Required for High Affinity DNA Binding

doi: 10.1371/journal.pgen.1002924

Figure Lengend Snippet: (A) In response to RPC program activators, Vsx2 expression is initiated and newly produced protein interacts with preexisting MITF protein, preventing access to targets required for the pigmentation program. (B) Once VSX2 protein expression is established, it regulates Mitf activity by directly repressing Mitf transcription of isoforms such as D-Mitf and binds to MITF proteins produced from promoters that Vsx2 does not efficiently repress such as A-Mitf and possibly H-Mitf .

Article Snippet: Vsx2 R200Q chimeric mice were generated at inGenious Targeting Laboratory (Stony Brook, NY) and Vsx2 R227W chimeric mice were generated at the University of Utah Gene Targeting and Transgenic Core Facility (See and for details on gene targeting strategy).

Techniques: Expressing, Produced, Activity Assay

Journal: bioRxiv

Article Title: The neuronal architecture of autonomic dysreflexia

doi: 10.1101/2024.05.06.592781

Figure Lengend Snippet: a , Scheme illustrating our experimental protocol followed by single-nucleus RNA sequencing. Mice received upper-thoracic SCI. After 30 days, half of the mice underwent repetitive episodes of autonomic dysreflexia during 90 minutes. The lumbosacral spinal cord and the lower thoracic were dissected from the mice according to standard procedures. b , We obtained high-quality transcriptomes from 64,739 nuclei that were evenly represented across experimental conditions and spatial locations. c , Number of unique molecular identifiers (UMIs) per nucleus. Inset text shows the median number of UMIs. d , Number of genes detected per nucleus. Inset text shows the median number of genes detected. e , Proportion of mitochondrial counts per nucleus. Inset text shows the median proportion of mitochondrial counts. f , Number of UMIs quantified per nucleus in each major cell type of the mouse spinal cord. g , Number of genes detected per nucleus in each major cell type of the mouse spinal cord. h , Proportion of mitochondrial counts per nucleus in each major cell type of the mouse spinal cord. i , UMAP visualization of 64,739 nuclei colored by major cell type, segregated by the location of spinal cord tissues (L6, T12) and experimental conditions (SCI only, exposure to repeated episode of autonomic dysreflexia, AD). experimental condition. j , Proportions of nuclei from each major cell type depending on the location of spinal cord tissues and experimental conditions. k , UMAP visualization showing expression of key marker genes for the major cell types of the mouse spinal cord. l , UMAP visualization of 29,144 neuronal nuclei colored by neuronal subpopulations, split by experimental condition. m , UMAP visualization showing expression of key marker genes for the major neuronal subpopulation classifications of the mouse spinal cord. n , UMAP visualization and dendrograms showing cell type prioritizations assigned by Augur across the neuronal taxonomy of the lower thoracic ( Left ) and lumbosacral ( Right ) spinal cord. o , Photomicrographs of the lower thoracic and lumbosacral spinal cord after repetitive episodes of autonomic dysreflexia. Vsx2 ON neurons were labelled with immunohistochemistry. Long-distance projecting (Zfhx3, lumbosacral spinal cord) and locally-projecting (Nfib, lower thoracic spinal cord) were additionally colocalized with immunohistochemistry labelling of cFos.

Article Snippet: The sections were incubated with following primary antibody diluted in blocking solution at room temperature overnight: rabbit anti-cFos (1:500), chicken anti-vGlut1 (1:500), goat anti-Chat (1:100), rabbit anti-Chx10 (now known as Vsx2) (1:500, Synaptic Systems Gmbh).

Techniques: RNA Sequencing Assay, Expressing, Marker, Immunohistochemistry

Journal: bioRxiv

Article Title: The neuronal architecture of autonomic dysreflexia

doi: 10.1101/2024.05.06.592781

Figure Lengend Snippet: a , Schematic overview of the single-nucleus sequencing experiment. Uniform manifold approximation and projection (UMAP) visualization of 64,739 neuronal nuclei, colored by neuronal subpopulation identity. Middle , UMAP visualizations of neuronal subpopulations in the lower thoracic (top) and lumbosacral (bottom) spinal cord. Right , Ranking neuronal subpopulations most responsive to autonomic dysreflexia with Augur. b , Schematic overview of the neuronal architecture of autonomic dysreflexia, including the nodes (numbers) that are dissected anatomically and functionally in the subsequent panels. c , Whole spinal cord visualization of projections from SC Hoxa10::Zfhx3::Vsx2 neurons located in the lumbosacral spinal cord that project to SC Hoxa7::Nfib::Vsx2 neurons located in the lower thoracic spinal cord. Insets illustrate the synaptic-like appositions of Calca ON projections labeled with immunohistochemistry onto SC Hoxa10::Zfhx3::Vsx2 neurons in the lumbosacral spinal cord. d , Calca ON projections labeled with immunohistochemistry onto SC Hoxa10::Zfhx3::Vsx2 neurons in the lumbosacral spinal cord, including insets showing synaptic-like appositions. e , Bar plot reporting the severity of autonomic dysreflexia, quantified as the mean change in systolic blood pressure in response to colorectal distension before and after the ablation of Calca ON neurons located in the dorsal root ganglia in Calca Cre ::Advil FlpO ::iDTR mice (n = 5; independent samples t-test; t = -6.0; p-value = 0.0006). f , Severity of autonomic dysreflexia before and after chemogenetic silencing of Vsx2 ON neurons located in the lumbosacral spinal cord in Vsx2 Cre (n = 5; paired samples t-test; t = -9.47; p-value = 0.00069). g , Projections from SC Hoxa10::Zfhx3::Vsx2 in the lower thoracic spinal cord co-labeled with SC Hoxa7::Nfib::Vsx2 neurons and their local projections as well as immunohistochemical labelling of Chat ON . Insets show synaptic-like appositions from SC Hoxa10::Zfhx3::Vsx2 neurons onto SC Hoxa7::Nfib::Vsx2 , and synaptic-like appositions of projections from SC Hoxa7::Nfib::Vsx2 to Chat ON sympathetic preganglionic neurons located in the intermediolateral column. h , Severity of autonomic dysreflexia before and after chemogenetic silencing of Vsx2 ON neurons located in the lower thoracic spinal cord in Vsx2 Cre (n = 5; paired samples t-test; t = -9.39; p-value = 0.00072). i , Severity of autonomic dysreflexia before and after chemogenetic silencing of Chat ON neurons located in the lower thoracic spinal cord in Chat Cre mice (n = 5; paired samples t-test; t = -8.03; p-value = 0.00048).

Article Snippet: The sections were incubated with following primary antibody diluted in blocking solution at room temperature overnight: rabbit anti-cFos (1:500), chicken anti-vGlut1 (1:500), goat anti-Chat (1:100), rabbit anti-Chx10 (now known as Vsx2) (1:500, Synaptic Systems Gmbh).

Techniques: Sequencing, Labeling, Immunohistochemistry, Immunohistochemical staining

Journal: bioRxiv

Article Title: The neuronal architecture of autonomic dysreflexia

doi: 10.1101/2024.05.06.592781

Figure Lengend Snippet: a , Schematic overview of the neuronal architecture of autonomic dysreflexia. b , Zoom on the first node of the neuronal architecture of autonomic dysreflexia that involves the growth of projections from Calca ON neurons onto Vsx2 ON neurons with long-distance projections, named SC Hoxa10::Zfhx3::Vsx2 neurons. This growth was assessed on tissues collected 30 days after SCI in wild-type mice. c , Photomicrograph taken at L6 spinal segment from a mouse with an intact spinal cord and a mouse with a chronic SCI in which Calca ON axons were labelled with immunohistochemistry. d , Bar plots reporting the density of Calca ON axonal projections into the intermediate laminae of the spinal cord in uninjured mice and mice with chronic SCI (n = 4; independent samples t-test; t = 9.38; p-value = 0.000086). e , Overview of the experimental protocol to test the severity of autonomic dysreflexia after the ablation of Calca ON and PV ON neurons. To achieve the ablation of these neurons exclusively in the dorsal root ganglia, we used a Cre- and Flp-dependent strategy in Calca Cre ::Avil FlpO ::iDTR and PV Cre ::Avil FlpO ::iDTR mice that allowed the expression of diphtheria toxin receptors (DTR) in Calca ON and PV ON neurons located in the dorsal root ganglia, respectively. f , Pressor responses ( Left ; bold line represents mean trace ±standard error of mean (sem) for each group and individual line traces are from each mouse) and severity of autonomic dysreflexia ( Right ) measured by the change in systolic blood pressure during colorectal distension in mice without diphtheria toxin-induced ablation of either Calca ON neurons or PV ON neurons, mice with diphtheria toxin-induced ablation of Calca ON neurons and mice with diphtheria toxin-induced ablation of PV ON neurons (n = 5; independent samples t-test; t= -5.9998; p-value = 0.00064, independent samples t-test; t= -9.3261; p-value = 0.00014). g , Zoom on the second node of the neuronal architecture of autonomic dysreflexia that involves SC Hoxa10::Zfhx3::Vsx2 neurons projecting to the low thoracic spinal cord. An intersectional viral labelling strategy was used to label the axons of SC Hoxa10::Zfhx3::Vsx2 neurons located in the lumbosacral spinal cord and that establish projections in the lower thoracic spinal cord. Vsx2 Cre mice received SCI and were injected with Retro-AAV-DIO-FlpO into the lower thoracic spinal cord and AAV8-Con/Fon-EYFP into the lumbosacral spinal cord. h , Photomicrograph of the L6 spinal segment from a Vsx2 Cre mouse with an intact spinal cord and a Vsx2 Cre mouse with a chronic SCI that received intersectional viral tracing to label SC Hoxa10::Zfhx3::Vsx2 neurons. Axons from Calca ON were also labelled with immunohistochemistry. Insets show synaptic-like appositions from Calca ON axons onto SC Hoxa10::Zfhx3::Vsx2 neurons. i , The necessary role of SC Hoxa10::Zfhx3::Vsx2 neurons in autonomic dysreflexia was evaluated using Cre-dependent expression of Gi DREADDs in SC Hoxa10::Zfhx3::Vsx2 neurons. j , Photomicrograph showing the expression of DREADD (G i ) receptors in SC Hoxa10::Zfhx3::Vsx2 neurons. k , Left , changes in systolic blood pressure in response to colorectal distension (shared area). Bold line represents mean trace ±sem for each group and individual line traces are from each mouse) and severity of autonomic dysreflexia. Right , Severity of autonomic dysreflexia in Vsx2 Cre mice before and after chemogenetic silencing of Vsx2 ON neurons located in the lumbosacral spinal cord (n = 5; paired samples t-test; t = -9.47; p-value = 0.00069). l , The sufficient role of SC Hoxa10::Zfhx3::Vsx2 neurons in triggering autonomic dysreflexia was evaluated using optogenetic activation of SC Hoxa10::Zfhx3::Vsx2 neurons in Vsx2 Cre mice injected with AAV-Syn-flex-ChrimsonR-tdTomato into at the lumbosacral spinal cord. 30 days after SCI, blood pressure responses were monitored beat-by-beat using a blood pressure catheter inserted into the carotid artery. Red-shifted light was shined over the lumbosacral spinal cord for 60 seconds during each trial. m , Photomicrograph showing the expression of ChrimsonR in SC Hoxa10::Zfhx3::Vsx2 neurons. n , Left, Changes in systolic blood pressure in response to the photostimulation of SC Hoxa10::Zfhx3::Vsx2 neurons in mice with intact spinal cord and with chronic SCI. Bold line represents mean trace ±sem for each group and individual line traces are from each mouse) and blood pressure responses due to optogenetic activation of SC Hoxa10::Zfhx3::Vsx2 neurons. Right , Bar plots reporting mean changes in blood pressure in Vsx2 Cre mice with intact spinal cord and with SCI during optogenetic activation of SC Hoxa10::Zfhx3::Vsx2 neurons (n = 5; independent samples t-test; t = 5.14; p-value = 0.00496).

Article Snippet: The sections were incubated with following primary antibody diluted in blocking solution at room temperature overnight: rabbit anti-cFos (1:500), chicken anti-vGlut1 (1:500), goat anti-Chat (1:100), rabbit anti-Chx10 (now known as Vsx2) (1:500, Synaptic Systems Gmbh).

Techniques: Immunohistochemistry, Expressing, Injection, Activation Assay

Journal: bioRxiv

Article Title: The neuronal architecture of autonomic dysreflexia

doi: 10.1101/2024.05.06.592781

Figure Lengend Snippet: a , Zoom on the third node of the neuronal architecture of autonomic dysreflexia that involves SC Hoxa7::Nfib::Vsx2 neurons located in the lower thoracic spinal cord. b , Overview of intersectional viral tracing strategy to label projections from SC Hoxa10::Zfhx3::Vsx2 into the lower thoracic spinal cord concomitantly to the labelling of SC Hoxa10::Zfhx3::Vsx2 Step 1 , AAV5-hSyn-flex-tdTomato was infused into the lower thoracic spinal cord of Vsx2-Cre mice to label SC Hoxa7::Nfib::Vsx2 . Step 2 , Retro-AAV-DIO-FlpO was infused into the lower thoracic spinal cord and AAV8-Con/Fon-EYP into the lumbosacral spinal cord to label the projections from SC Hoxa10::Zfhx3::Vsx2 neurons located in the lumbosacral spinal cord and that project in the lower thoracic spinal cord. c , Photomicrographs of the lower thoracic spinal cord with intersectional viral tracing labelling projections from SC Hoxa10::Zfhx3::Vsx2 , SC Hoxa7::Nfib::Vsx2 neurons and their projections from a representative mouse with an intact spinal cord and mouse with SCI. d , Bar plots reporting the mean density of projections from SC Hoxa10::Zfhx3::Vsx2 neurons in the grey matter of the lower thoracic spinal cord in mice with an intact spinal cord and with chronic SCI (n = 5; independent samples t-test; t = -3.09; p-value = 0.0162). e , Whole spinal cord visualization of projections from SC Hoxa10::Zfhx3::Vsx2 neurons located in the lumbosacral spinal cord (red) and visualization of SC Hoxa7::Nfib::Vsx2 neurons (blue) located in the lower thoracic spinal cord in mice with chronic SCI. f , The necessary role of SC Hoxa7::Nfib::Vsx2 neurons in autonomic dysreflexia was evaluated using Cre-dependent expression of G i DREADDs in SC Hoxa7::Nfib::Vsx2 neurons. g , Photomicrograph showing the expression of G i DREADD receptors in SC Hoxa7::Nfib::Vsx2 neurons. h , Left , changes in systolic blood pressure in response to colorectal distension. Bold line represents mean trace ±sem for each group and individual line traces are from each mouse) and severity of autonomic dysreflexia. Right , Severity of autonomic dysreflexia in Vsx2 Cre mice before and after chemogenetic silencing of Vsx2 ON neurons located in the lower thoracic spinal cord (n = 5; paired samples t-test; t = -9.39; p-value = 0.00072). i , The sufficient role of SC Hoxa7::Nfib::Vsx2 neurons in autonomic dysreflexia was evaluated using optogenetic activation of SC Hoxa7::Nfib::Vsx2 neurons in Vsx2 Cre mice injected with AAV-Syn-flex-ChrimsonR-tdTomato into the lower thoracic spinal cord. 30 days after SCI, blood pressure responses were monitored beat-by-beat using a blood pressure catheter inserted into the carotid artery. Red-shifted light was shine over the lumbosacral spinal cord for 60 seconds during each trial. j , Photomicrograph showing the expression of ChrimsonR in SC Hoxa7::Nfib::Vsx2 neurons. k , Left , changes in systolic blood pressure in response to colorectal distension. Bold line represents mean trace ±sem for each group and individual line traces are from each mouse) and blood pressure responses due to optogenetic activation of SC Hoxa7::Nfib::Vsx2 neurons. Right , Blood pressure responses in Vsx2 Cre mice with intact spinal cord and with chronic SCI during optogenetic activation of SC Hoxa7::Nfib::Vsx2 neurons (n = 5; independent samples t-test; t = 15.4; p-value = 0.0000148). l , Zoom on the fourth node of the neuronal architecture of autonomic dysreflexia that involves Chat ON sympathetic preganglionic neurons. m , Overview of experimental protocol to label projections from SC Hoxa7::Nfib::Vsx2 neurons located in the lower thoracic spinal cord in Vsx2 Cre mice with SCI. Thirty days after SCI and viral tracing, the spinal cord tissues were collected and processed. n , Photomicrograph of the lower thoracic spinal cord from a mouse with an intact spinal cord and a mouse with chronic SCI in which the projections of SC Hoxa7::Nfib::Vsx2 were labelled concomitantly to the immunohistochemical labelling of Chat ON neurons. o , The necessary role of Chat ON neurons in autonomic dysreflexia was evaluated using Cre-dependent expression of G i DREADDs in Chat ON neurons. p , Photomicrograph illustrating the expression of G i DREADD receptors in SC Hoxa7::Nfib::Vsx2 neurons. q , As in h , for Chat ON neurons located in the lower thoracic spinal cord (n = 5; paired samples t-test; t = -8.03; p-value = 0.00048).

Article Snippet: The sections were incubated with following primary antibody diluted in blocking solution at room temperature overnight: rabbit anti-cFos (1:500), chicken anti-vGlut1 (1:500), goat anti-Chat (1:100), rabbit anti-Chx10 (now known as Vsx2) (1:500, Synaptic Systems Gmbh).

Techniques: Expressing, Activation Assay, Injection, Immunohistochemical staining

Journal: bioRxiv

Article Title: The neuronal architecture of autonomic dysreflexia

doi: 10.1101/2024.05.06.592781

Figure Lengend Snippet: a , Schematic overview of experiments to trigger pressor responses with EES in mice with SCI. b , Pressor response induced by continuous (40 Hz) EES in a mouse with SCI. c , Uniform manifold approximation and projection (UMAP) visualization of 21,098 neuronal nuclei, colored by neuronal subpopulation identity. Right , Identification of perturbation-responsive neuronal subpopulations with Augur. d-f , Schematic overview of the successive nodes constituting the neuronal architecture though which EES applied over the low thoracic spinal cord induces pressor responses. d , EES-induced pressor responses before and after the ablation of PV ON neurons located in the dorsal root ganglia in PV Cre ::Advil FlpO ::iDTR mice (n = 5; independent samples t-test; t = -5.41; p-value = 0.0043). e , EES-induced pressor responses before and after chemogenetic silencing of Vsx2 ON located in the lower thoracic spinal cord in Vsx2 Cre mice (n = 5; paired samples t-test; t = -4.21; p-value = 0.014). f , EES-induced pressor responses before and after chemogenetic silencing of Chat ON neurons located in the lower thoracic spinal cord in Chat Cre mice (n = 5; paired samples t-test; t = -7.07; p-value = 0.0021). g , Photomicrograph of the lower thoracic spinal cord demonstrating vGlut1 ON synaptic puncta and synaptic-like appositions from large-diameter afferent neurons onto SC Hoxa7::Nfib::Vsx2 neurons labelled with in situ hybridization ( Left ) or viral tract tracing ( Right ) in the lower thoracic spinal cord of PV Cre ::Advil FlpO ::tdTomato mice.

Article Snippet: The sections were incubated with following primary antibody diluted in blocking solution at room temperature overnight: rabbit anti-cFos (1:500), chicken anti-vGlut1 (1:500), goat anti-Chat (1:100), rabbit anti-Chx10 (now known as Vsx2) (1:500, Synaptic Systems Gmbh).

Techniques: In Situ Hybridization

Journal: bioRxiv

Article Title: The neuronal architecture of autonomic dysreflexia

doi: 10.1101/2024.05.06.592781

Figure Lengend Snippet: a , Schematic overview of the neuronal architecture through which EES induces pressor responses. b , Zoom on the first node of the neuronal architecture of EES-induced pressor responses that involves PV ON . c , Overview of the experimental protocol to test the involvement of afferent fibers from PV ON and Calca ON neurons in EES-induced pressor responses. To achieve the ablation of these neurons exclusively in the dorsal root ganglia, we used a Cre- and Flp-dependent strategy in Calca Cre ::Avil FlpO ::iDTR and PV Cre ::Avil FlpO ::iDTR mice that allowed the expression of diphtheria toxin receptors (DTR) in these specific neurons. d , EES-induced pressor responses ( Left ; bold line represents mean trace ±sem for each group and individual line traces are from each mouse) ( Right ) measured by the change in systolic blood pressure during EES in mice without any ablation, mice with diphtheria toxin-induced ablation of PV ON neurons and mice with diphtheria toxin-induced ablation of Calca ON neurons (n = 5; independent samples t-test; t= -5.4141; p-value = 0.0043, independent samples t-test; t= 6.3166; p-value = 0.0020). e , Overview of the experiment strategy to visualize large-diameter PV ON fibers in PV Cre ::Avil FlpO ::Ai9 (RCL-tdT) mice and confirmed that they established vGlut1 ON synaptic-appositions onto SC Hoxa7::Nfib::Vsx2 neurons. Thirty days after SCI, spinal cord tissues were collected and processed. f , Photomicrograph of the lower thoracic spinal cord showing vGlut1 synaptic puncta and synaptic-like appositions from large-diameter afferent neurons (PV Cre ::Advil FlpO ::Ai9 (RCL-tdT) mice) onto SC Hoxa7::Nfib::Vsx2 neurons labelled with in situ hybridization ( Left ) or viral tract tracing ( Right ). g , Photomicrograph of the lower thoracic spinal cord from a PV Cre ::Advil FlpO ::Ai9 (RCL-tdT) mouse combined with immunohistochemical labelling of ChatON neurons. h , Quantification of vGlut1 ON synaptic-appositions onto SC Hoxa7::Nfib::Vsx2 neurons and ChatON neurons in PV Cre ::Advil FlpO ::Ai9 (RCL-tdT) mice with an intact spinal cord and with a chronic SCI. i , Zoom on the second node of the neuronal architecture of EES-induced pressor responses that involves SC Hoxa7::Nfib::Vsx2 . The necessary role of SC Hoxa7::Nfib::Vsx2 neurons in EES-induced pressor response was evaluated using Cre-dependent expression of Gi DREADDs in SC Hoxa7::Nfib::Vsx2 neurons. j , EES-induced pressor responses ( Left ; bold line represents mean trace ±sem for each group and individual line traces are from each mouse) ( Right ) measured by the change in systolic blood pressure during EES in the same mice before and after chemogenetic silencing of Vsx2 ON neurons located in the lower thoracic spinal cord (n = 5; paired samples t-test; t = -4.21; p-value = 0.014). k , Zoom on the third node of the neuronal architecture of EES-induced pressor responses that involves Chat ON sympathetic preganglionic neurons. As in h , for Chat ON neurons located in the lower thoracic spinal cord. l , As in j , for Chat ON neurons in the lower thoracic spinal cord (n = 5; paired samples t-test; t = -7.07; p-value = 0.0021). m , Photomicrograph showing the expression of ChrimsonR in Vsx2 ON neurons and the tract resulting from the insertion of one electrode shank. n , Schematic overview of experiments to record the activity of SC Hoxa7::Nfib::Vsx2 during the application of EES and during episodes of autonomic dysre-flexia. o , Top , the waveforms display spikes and firing rate evoked by optogenetic stimulation of Vsx2 ON neurons by the application of continuous EES over the lower thoracic spinal cord, and by colorectal distention. Heatmap of neuronal clusters activated by EES, activated by EES and colorectal distension, activated by EES and tagged as Vsx2 ON neurons by optogenetic stimulation and EES, and activated by EES and colorectal distension and tagged as Vsx2 ON neurons activated by optogenetic stimulation.

Article Snippet: The sections were incubated with following primary antibody diluted in blocking solution at room temperature overnight: rabbit anti-cFos (1:500), chicken anti-vGlut1 (1:500), goat anti-Chat (1:100), rabbit anti-Chx10 (now known as Vsx2) (1:500, Synaptic Systems Gmbh).

Techniques: Expressing, In Situ Hybridization, Immunohistochemical staining, Activity Assay

Journal: bioRxiv

Article Title: The neuronal architecture of autonomic dysreflexia

doi: 10.1101/2024.05.06.592781

Figure Lengend Snippet: a , Schematic overview of autonomic neurorehabilitation and paradigm to quantify the severity of autonomic dysreflexia. b , Pressor responses ( Left ; individual mice and mean trace) and severity of autonomic dysreflexia ( Right ) in 5 with chronic SCI and 5 mice that underwent autonomic neurorehabilitation for 4 weeks, starting 1 week after SCI (independent samples t-test; t = -7.45; p-value = 0.00056). c , Schematic overview illustrating the competitive (overlapping) neuronal architectures of autonomic dysreflexia and EES-induced pressor responses, and their rearrangement after autonomic neurorehabilitation. d , vGlut1 ON synaptic puncta and synaptic-like appositions from SC Hoxa10::Zfhx3::Vsx2 neurons onto SC Hoxa7::Nfib::Vsx2 neurons in in mice with SCI and mice with SCI that underwent autonomic neurorehabilitation. ( top ) Bar plots reporting the mean density of axonal projections from SC Hoxa10::Zfhx3::Vsx2 neurons in the thoracic spinal cord in mice with SCI and mice with SCI that underwent autonomic neurorehabilitation (n = 5; independent samples t-test; t = 2.51; p-value = 0.0369). ( bottom ) (bottom) Bar plots reporting the mean number of vGlut1 ON synaptic puncta apposing SC Hoxa7::Nfib::Vsx2 neurons (n = 5; independent samples t-test; t = 4.44; p-value = 0.0055). e , Schematic overview of experiments in which EES was applied daily over the lumbosacral spinal cord of mice with SCI, and paradigm to quantify the severity of autonomic dysreflexia. f , As in b , for mice with SCi that were subjected to the daily application of EES over the lumbosacral spinal cord (n = 5; independent samples t-test; t = 5.82; p-value = 0.00070).

Article Snippet: The sections were incubated with following primary antibody diluted in blocking solution at room temperature overnight: rabbit anti-cFos (1:500), chicken anti-vGlut1 (1:500), goat anti-Chat (1:100), rabbit anti-Chx10 (now known as Vsx2) (1:500, Synaptic Systems Gmbh).

Techniques:

Journal: bioRxiv

Article Title: The neuronal architecture of autonomic dysreflexia

doi: 10.1101/2024.05.06.592781

Figure Lengend Snippet: a , Overview of the experimental protocol to deliver autonomic neurorehabilitation in mice with SCI. Step 1 . Mice received a complete transection of the spinal cord at the level of the T4 segment. Step 2 .Intersectional viral tracing by infusing Retro-AAV-DIO-FlpO into the lower thoracic spinal cord and AAV8-Con/Fon-EYP into the lumbosacral spinal cord to label SC Hoxa10::Zfhx3::Vsx2 neurons located in the lumbosacral spinal cord that project onto SC Hoxa7::Nfib::Vsx2 neurons located in the lower thoracic spinal cord. Step 3 . One week after SCI, electrodes were implanted over the T12 spinal segment to deliver EES. Step 4 . EES was applied for 30 minutes everyday for 4 weeks. Step 5 . F of autonomic dysreflexia was assessed during terminal experiments conducted in mice with chronic SCI and mice with chronic SCI that underwent autonomic neurorehabilitation. Step 6 . Spinal cord tissues were collected and processed. b , Changes in systolic blood pressure ( Left ; bold line represents mean trace ± sem for each group and individual line traces are from each animal) and severity of autonomic dysreflexia ( Right ) measured by the change in systolic blood pressure during colorectal distension in mice with and without autonomic neurorehabilitation (n = 5; independent samples t-test; t = -7.45; p-value = 0.00056). c , Left , Photomicrographs of the lower thoracic spinal cord in mice with chronic SCI and mice with chronic that underwent autonomic neurorehabilitation in which SC Hoxa10::Zfhx3::Vsx2 neurons located in the lumbosacral spinal cord were labelled with an intersection virus strategy concomitantly to the labelling of SC Hoxa7::Nfib::Vsx2 neurons. (Right) Photomicrographs of the lower thoracic spinal cord with intersectional viral labelling combined with immunohistochemical labelling of vGlut1 ON synapses in mice with chronic SCI and mice with chronic SCI that underwent autonomic neurorehabilitation. vGlut1ON synaptic puncta and synaptic-like appositions from SC Hoxa10::Zfhx3::Vsx2 neurons onto SC Hoxa7::Nfib::Vsx2 neurons in mice with chronic SCI and mice with chronic SCI that underwent autonomic neurorehabilitation. d , Left , Bar plots reporting the mean number of vGlut1 ON synaptic puncta apposing SC Hoxa7::Nfib::Vsx2 neurons (n = 5; independent samples t-test; t = 4.44; p-value = 0.0055), right , and the mean density of axonal projections from SC Hoxa10::Zfhx3::Vsx2 neurons in the grey matter of the lower thoracic spinal cord in mice with chronic SCI and mice with chronic SCI that underwent autonomic neurorehabilitation (n = 5; independent samples t-test; t = 2.51; p-value = 0.0369). e , As in a , for mice subjected to daily application of EES over the lumbosacral spinal cord. f , As in b , for mice subjected to daily application of EES over the lumbosacral spinal cord (n = 5; independent samples t-test; t = 5.82; p-value = 0.00070). g , As in c , for mice subjected to daily application of EES over the lumbosacral spinal cord. h , As in d , for mice subjected to daily application of EES over the lumbosacral spinal cord.

Article Snippet: The sections were incubated with following primary antibody diluted in blocking solution at room temperature overnight: rabbit anti-cFos (1:500), chicken anti-vGlut1 (1:500), goat anti-Chat (1:100), rabbit anti-Chx10 (now known as Vsx2) (1:500, Synaptic Systems Gmbh).

Techniques: Virus, Immunohistochemical staining

Journal: bioRxiv

Article Title: The neuronal architecture of autonomic dysreflexia

doi: 10.1101/2024.05.06.592781

Figure Lengend Snippet: a , Schematic overview of autonomic neurorehabilitation in rats. A wireless telemetry system was implanted chronically to acquire longitudinal recordings of haemodynamic parameters. An electronic dura mater ( e-dura ) designed to target the dorsal roots projecting to the T11, T12, and T13 spinal segments was then implanted over the hemodynamic hotspot to regulate blood pressure. b , Augmentation of systolic blood pressure within a target range using a proportional integral (PI) controller that adjusts the amplitude of EES in closed-loop. c , Line graph reporting the severity of autonomic dysreflexia that was assessed weekly using colorectal distension in rats with SCI and rats with SCI that were undergoing autonomic neurorehabilitation. Raw data and statistics provided in Supplementary Table 1 . d , Whole spinal cord visualization of projections from neurons located in the lumbosacral spinal cord that establish neurons into the lower thoracic spinal cord. e , Axonal projections and synaptic puncta from neurons located in the lumbosacral spinal cord and projecting to the lower thoracic spinal cord, shown in rats with SCI and rats with SCI that underwent autonomic neurorehabilitation. f , Density of axonal projections from lumbosacral neurons in the thoracic spinal cord before and after autonomic neurorehabilitation (n = 5; independent samples t-test; t = -4.03; p-value = 0.0081). g , Synaptic-like appositions from neurons located in the lumbosacral spinal cord onto SC Hoxa7::Nfib::Vsx2 neurons combined with the labelling of vGlut1 ON synaptic puncta form large-diameter afferent fibers in a rat with chronic SCI and a rat with SCI that underwent autonomic neurorehabilitation. h , Bar plots reporting the mean density of vGlut1 ON synaptic puncta onto SC Hoxa7::Nfib::Vsx2 neurons in rats with SCI and rats with SCI that underwent autonomic neurorehabilitation (n = 5; independent samples t-test; t = 12.71; p-value = 2.78e-06).

Article Snippet: The sections were incubated with following primary antibody diluted in blocking solution at room temperature overnight: rabbit anti-cFos (1:500), chicken anti-vGlut1 (1:500), goat anti-Chat (1:100), rabbit anti-Chx10 (now known as Vsx2) (1:500, Synaptic Systems Gmbh).

Techniques:

Journal: bioRxiv

Article Title: The neuronal architecture of autonomic dysreflexia

doi: 10.1101/2024.05.06.592781

Figure Lengend Snippet: a , Overview of the experimental protocol to deliver autonomic neurorehabilitation in rats with SCI. Step 1 . Rats received a severe contusion (380 Kdyn) of the spinal cord at the level of T3 segment. Step 2 . AAV-DJ-hSyn-flex-mGFP-2A-Synaptophysin-mRuby and an AAV-Cre were co-infused into the the L6 segment of the spinal cord to label the projections from neurons located in the lumbosacral spinal cord. Step 3 . A wireless telemeter recording system, including a blood pressure cannula inserted into the abdominal aorta and microelectrodes sutured over the sympathetic renal nerve, was implanted chronically to monitor hemodynamics and sympathetic nerve activity, respectively. Step 4 . Seven days after SCI, an an electronic dura mater (e-dura) designed to target the dorsal roots projecting to the T11, T12, and T13 spinal segments was implanted over the hemodynamic hotspot to regulate blood pressure. Step 5 . EES was applied for 30 minutes everyday during 6 weeks using a proportional-integral (PI) controller that adjusted the amplitude of EES in closed-loop s to augment the systolic blood pressure to a target range. Step 6 . The severity of autonomic dysreflexia, induced by colorectal distension, was assessed every week for 6 weeks. Step 7 . After 6 weeks of autonomic neurorehabilitation, a final assessment was performed to test the severity of autonomic dysreflexia in all groups, which included rats with intact spinal cord, rats with chronic SCI and rats with chronic SCI that underwent autonomic neurorehabilitation. Step 8 . Spinal cords were collected and processed. b , Changes in systolic blood pressure in response to colorectal distension ( Left ; bold line represents mean trace ± sem for each group and individual line traces are from each rat) and bar plots reporting the severity of autonomic dysreflexia ( Right ) measured by the change in systolic blood pressure during colorectal distension over the course of 6 weeks in rats with intact spinal cord, rats with chronic SCI and rats with chronic SCI that underwent autonomic neurorehabilitation. Raw data and statistics provided in Supplementary Table 1 . c , Whole spinal cord visualization of projections from neurons located in the lumbosacral spinal cord. d , Plots reporting density of axonal projections ( top ) and synaptic punta ( bottom ) from neurons located in the lumbosacral spinal cord into the grey matter of the lower thoracic spinal cord in rats with intact spinal cord, rats with chronic SCI and rats with chronic SCI that underwent autonomic neurorehabilitation. e , Micrographs of the lower thoracic spinal cord in which the axonal projections and synaptic puncta from neurons located in the lumbosacral spinal cord are labelled for the three groups of rats. f , Bar plots reporting the mean density of axonal projections and synaptic puncta from neurons located in lumbosacral spinal cord into the grey matter of the lower thoracic spinal cord for the three groups of rats. Raw data and statistics are provided in Supplementary Table 1 . g , Micrographs of the lower thoracic spinal cord in which axonal projections and synaptic puncta from neurons located in the lumbosacral spinal cord are labelled concomitantly to vGlut1 ON synapses from large-diameter afferents and Vsx2 ON neurons. The density of vGlut1 ON synapses onto Vsx2 ON neurons is reconstructed for a rat with chronic SCI and a rat with chronic SCI that underwent autonomic neurorehabilitation. h , Bar plots reporting the density of synaptic-like appositions from neurons located in the lumbosacral spinal cord onto Vsx2 ON neurons in rats with intact spinal cord, rats with chronic SCI, and rats with chronic SCI that underwent autonomic neurorehabilitation. Raw data and statistics are provided in Supplementary Table 1 . i , As in i, for vGlut2 ON synaptic puncta onto SC Hoxa7::Nfib::Vsx2 neurons. Raw data and statistic provided are in Supplementary Table 1 . j , Quantification of vGlut1 ON synaptic puncta from large-diamter afferents Vsx2 ON in rats with chronic SCI and rats with chronic SCI that underwent autonomic neurorehabilitation (n = 5; independent samples t-test; t = 12.71; p-value = 2.78e-06).

Article Snippet: The sections were incubated with following primary antibody diluted in blocking solution at room temperature overnight: rabbit anti-cFos (1:500), chicken anti-vGlut1 (1:500), goat anti-Chat (1:100), rabbit anti-Chx10 (now known as Vsx2) (1:500, Synaptic Systems Gmbh).

Techniques: Activity Assay

Journal: bioRxiv

Article Title: Independent evolution of holocentric centromeres in an early branching apicomplexan parasite

doi: 10.1101/2025.09.30.679541

Figure Lengend Snippet: (A) Phylogenetic relationship of C. parvum H3 and centromeric H3 (CENH3) histones and their corresponding top BLASTP hits in other eukaryotes, apicomplexan parasites, Saccharomyces cerevisiae , or Homo sapiens . Cyan stars represent annotated histone H3 genes. (B) Protein sequence alignment of H3 (cyan) and CENH3 (black) in C. parvum . Amino acids that are identical between at least two sequences are highlighted in grey. (C) Diagram of the targeting constructs designed to replace the endogenous tk locus with a second copy of either of the genes annotated as Histone H3-like proteins in C. parvum ( cgd3_2540 or cgd4_3220 ), a 3HA tag, and a Nluc-P2A-NeoR cassette. (D) Immunofluorescence staining of transgenic H3.1-3HA or (E) H3.2-3HA parasites. HCT-8 cells were infected with transgenic oocysts, fixed at 18 hpi, and stained with rat anti-HA and VVL-Biotin, followed by secondary antibodies Alexa Fluor 488 goat anti-rat IgG and Alexa Fluor 647 Streptavidin. Hoechst was used for nuclear staining. Images were acquired as Z-stacks using a Zeiss LSM-880 Laser Scanning Confocal microscope equipped with Airyscan (LSCM-A) and are presented with orthogonal views. Scale bars, 1 μm.

Article Snippet: 2) The 3HA-pLinker region was amplified from a TK-3HA-CENH3-Nluc-P2A-Neo R -TK plasmid (data not shown in this study), 3) the GBP CDS was amplified from C. parvum genomic DNA, 4) the pLinker-3Ty sequence was amplified from a tagging plasmid previously generated in our lab [ ], and 5) the P2A skip peptide sequence was generated as a gBlock Gene Fragment from Azenta.

Techniques: Sequencing, Construct, Immunofluorescence, Staining, Transgenic Assay, Infection, Microscopy

Journal: bioRxiv

Article Title: Independent evolution of holocentric centromeres in an early branching apicomplexan parasite

doi: 10.1101/2025.09.30.679541

Figure Lengend Snippet: (A) Diagram of the targeting construct designed to add a 3HA tag and Nluc-P2A-Neo R cassette to the C-terminus of the C. parvum CENH3 ( cgd4_2030 ). (B) HCT-8 cells were infected with CENH3-3HA C. parvum parasites, fixed at 22 hpi, and stained with rat anti-HA and VVL-Biotin, followed by secondary antibodies Alexa Fluor 488 goat anti-rat IgG and Alexa Fluor 647 Streptavidin followed by Hoechst staining. Images were acquired as Z-stacks using LSCM-A and are presented with orthogonal views. Scale bars, 1 μm. (C) Cartoon depiction corresponding to the microscopy image in panel (B). In Cryptosporidium , a late-stage meront contains eight nuclei that have recently completed cytokinesis to form mature merozoites. The CENH3-3HA signal within each nucleus displays a diffuse staining pattern in C. parvum . (D) HCT-8 cells were infected with CENH3-3HA T. gondii parasites, fixed at 24 hours post-infection (hpi), and stained with rat anti-HA and rabbit anti-aldolase (ALD) to visualize the cytosol, followed by secondary antibodies Alexa Fluor 488 goat anti-rat IgG and Alexa Fluor 568 anti-rabbit IgG. Hoechst was used for nuclear staining. Images were acquired as Z-stacks using LSCM-A and are presented with orthogonal views. Scale bars, 1 μm. (E) Cartoon depiction corresponding to the microscopy image in panel (D). In T. gondii tachyzoites, the CENH3-3HA signal within each nucleus displays a discrete staining pattern. (F) HCT-8 cells were infected with excysted C. parvum sporozoites for 2 h, then washed twice to remove extracellular parasites. Cultures were fixed at 30 min increments between 6-9 hpi to identify the first (trophozoites with one nucleus), second (early meronts with 2 nuclei), and third (mid-stage meronts with 4 nuclei) mitotic divisions during the first round of merogony by widefield microscopy. Scale bars, 1 μm. (G) Cultures were infected with CENH3-3HA C. parvum sporozoites for 2 h, washed, and fixed at 6.5 hpi to capture the first round of mitosis. Cultures were stained with rat anti-HA, rabbit anti-centrin-1, and VVL-Biotin, followed by secondary antibodies Alexa Fluor 488 goat anti-rat IgG, Alexa Fluor 568 goat anti-mouse IgG, and Alexa Fluor 647 Streptavidin, and lastly Hoechst. Parasites undergoing mitosis were identified by having two centrin-1 points per nucleus, whereas parasites in interphase had only one centrin-1 point. Images were acquired as Z-stacks using LSCM-A and are presented with orthogonal views. Scale bars, 1 μm.

Article Snippet: 2) The 3HA-pLinker region was amplified from a TK-3HA-CENH3-Nluc-P2A-Neo R -TK plasmid (data not shown in this study), 3) the GBP CDS was amplified from C. parvum genomic DNA, 4) the pLinker-3Ty sequence was amplified from a tagging plasmid previously generated in our lab [ ], and 5) the P2A skip peptide sequence was generated as a gBlock Gene Fragment from Azenta.

Techniques: Construct, Infection, Staining, Microscopy

Journal: bioRxiv

Article Title: Independent evolution of holocentric centromeres in an early branching apicomplexan parasite

doi: 10.1101/2025.09.30.679541

Figure Lengend Snippet: (A) The 3’ overhang single-stranded DNA at the end of telomeres contains repetitive sequences (GGTTTA) n in C. parvum that form G-quadruplex structures. (B) The G-quadruplex binding protein (GBP) is essential for forming and stabilizing these structures. The sequence of Cp GBP and its binding motif has been previously determined and the protein structure is modeled by Alphafold and ChimeraX . (C) Diagram of the targeting construct designed to add a 3HA tag and Nluc-P2A-Neo R cassette to the C-terminus of the G-quadruplex binding proteins gene ( GBP , cgd1_3530) (D) HCT-8 cells were infected with GBP-3HA parasites, fixed at 6.5 hpi to capture early meronts (2 nuclei), and stained with rat anti-HA and VVL-Biotin, followed by secondary antibodies Alexa Fluor 488 goat anti-rat IgG and Alexa Fluor 647 Streptavidin. Hoechst was used for nuclear staining. Images were acquired as Z-stacks using LSCM-A and are presented with orthogonal views and the scale bar is 1 μm. (E) Diagram of the targeting construct designed to replace the endogenous tk locus with a second copy of CENH3 N-terminally tagged with 3HA and GBP with a C-terminal 3xTy tag separated with a P2A split peptide. (F) Immunofluorescence staining of transgenic 3HA-CENH3-GBP-3Ty parasites. HCT-8 cells were infected with transgenic oocysts, fixed at 6.5 hpi to capture early meronts (2 nuclei), and stained with rat anti-HA, mouse anti-Ty, and VVL-Biotin followed by a secondary antibody stain containing Alexa Fluor 488 goat anti-rat IgG, Alexa Fluor 568 goat-anti mouse, and Alexa Fluor 647 Streptavidin. Hoechst was used for nuclear staining. Images were acquired as Z-stacks using LSCM-A and are presented with orthogonal views. Scale bars, 1 μm.

Article Snippet: 2) The 3HA-pLinker region was amplified from a TK-3HA-CENH3-Nluc-P2A-Neo R -TK plasmid (data not shown in this study), 3) the GBP CDS was amplified from C. parvum genomic DNA, 4) the pLinker-3Ty sequence was amplified from a tagging plasmid previously generated in our lab [ ], and 5) the P2A skip peptide sequence was generated as a gBlock Gene Fragment from Azenta.

Techniques: Binding Assay, Sequencing, Construct, Infection, Staining, Immunofluorescence, Transgenic Assay