rabbit anti vsx2  (Proteintech)

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: 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: Cell stem cell

Article Title: Human brain organoids assemble functionally integrated bilateral optic vesicles.

doi: 10.1016/j.stem.2021.07.010

Figure Lengend Snippet: Figure 1. Generation of 3D brain organoids with primordial eye fields from human iPSCs (A) Schematic of human embryonic nervous system development. The neural tube (left) segregates into the fore-, mid- and hindbrain and spinal cord. The forebrain develops into the diencephalon, from which the optic cups invaginate laterally. Optic cups later form the eye. (B) Organoids with primitive eye fields with pigments (arrow). Arrow points to an invagination, an optic fissure-like structure. Scale bars, 1 mm (left panel) and 500 mm (right panel). Cell line F14536.2. (C) RAX-positive primordial eye field (magenta). Scale bars, 500 mm (whole organoid) and 50 mm (inset). Cell line IMR-90. n = 10 organoids from at least 4 batches. (D) Pax6-containing (green, left and center) and FOXG1-containing (green, right) ventricular zones. Scale bar, 50 mm. n = 21 organoids from at least 4 batches. Cell lines IMR-90 and Crx-iPS. (E–G) SOX2-positive (green) invaginating regions with VSX2 and FOXG1 (magenta). Scale bars, 200 mm (whole organoid) and 50 mm (inset). Cell line Crx-iPS. n = 14 organoids from at least 4 batches

Article Snippet: Primary antibodies: Source: Cat.No. raised against: Dilution: Alpha A/ alpha B Crystallin Enzo Life Sciences ADI-SPA-224-D rabbit 1:50 Alpha-PKC Sigma Aldrich P4334 rabbit 1:1000 Anti-CRX R&D systems #O43186 sheep 1:100 Anti-Ctip2 (25B6) Abcam ab18465 rat 1:500 Anti-Doublecortin (DCX) Synaptic Systems 326003 rabbit 1:400 Anti-FoxG1 abcam ab18259 rabbit 1:400 Anti-Keratin K3/K76 (clone AE5) Millipore #CBL218-I rabbit 1:1000 Arl13B Proteintech 17711-1-AP rabbit 1:200 Brn-3 (A-4) Santa Cruz Sc-390780 mouse 1:50 NRL R&D systems AF2945 goat 1:200 NRL Proteintech 13780-1-AP rabbit 1:200 Myelin Basic Protein (D8X4Q) Cell Signaling #78896 rabbit 1:50 Nestin (4D11) Novus biologicals NBP1-92717 mouse 1:400 OTX2 GeneTex GTX133210 rabbit 1:200 OTX2 R&D systems AF1979 goat 1:200 ONECUT2 R&D Systems AF6294 sheep 1:200 PAX6 DSHB PAX6 mouse 1:50 PCP4 Proteintech 14705-1-AP rabbit 1:200 Phalloidin-TRITC Sigma Aldrich P1951 - 0.1 mg/ml PHF-1 kind gift from Peter Davis Tau phos Ser396/Ser404 (PHF-1) mouse 1:200 Rax abcam ab23340 rabbit 1:50 Recoverin Millipore AB5585 rabbit 1:1000 Rhodopsin Novus Biologicals NBP2-25159SS mouse 1:1000 Synapsin-1 Cell Signaling #5297 rabbit 1:200 VSX2 Santa Cruz Sc365519 goat 1:50 VSX2 Santa Cruz Sc21692 mouse 1:50 VSX2 Proteintech 25825-1-AP rabbit 1:50 Secondary antibodies: Source: Cat.No.

Techniques:

Journal: eLife

Article Title: A cell atlas of the chick retina based on single-cell transcriptomics

doi: 10.7554/eLife.63907

Figure Lengend Snippet:

Article Snippet: Antibodies used in this study were: rabbit polyclonal antibodies to GFP (Millipore, AB3080P); mouse monoclonal anti-Brn3a (clone, 5A3.2, Millipore, MAB1585); rabbit anti-calretinin (Millipore, AB5054); rabbit anti-calbindin (Swant, CB38); rabbit anti-protein kinase C- α (PKCα) (Sigma, P4334); rabbit anti-VSX2 (Chx10, GeneTex, GTX114143); rat monoclonal anti-HA tag (Roche, 3F10); rabbit anti-Satb1(Abcam, ab109122); mouse monoclonal anti-Satb2 (Abcam, ab51502); rabbit anti-Met-enkephalin (ImmunoStar, 20065); rabbit anti-neuropeptide Y (Abcam ab10980).

Techniques: In Situ Hybridization, CRISPR, Recombinant, Purification, Magnetic Beads, Electroporation, Extraction, Software