alomone labs agc  (Alomone Labs)

Journal: bioRxiv

Article Title: Human neuron subtype programming through combinatorial patterning with scSeq readouts

doi: 10.1101/2023.12.12.571318

Figure Lengend Snippet: (A) Experimental scheme and timeline of the NGN2-iN combinatorial patterning screen with single-cell transcriptomics readout. NGN2-iNs exposed to each of the 192 morphogen combinations were individually analyzed with snRNA-seq using split-pool combinatorial barcoding (Parse Biosciences). (B) UMAP embedding of 184,431 cells in the dataset, colored by cluster identity and the source of AP- or DV-morphogens. (C) Feature plots of representative marker genes. (D) Heatmap of cluster markers, transcription factors and ion channel expression in each patterned NGN2-iN cell cluster. (E) UMAP embeddings colored based on the annotations transferred from primary neuron reference atlases, including division, region, neuron type and corresponding mapping score. CNS, central nervous system; PNS, peripheral nervous system; SYM, sympathetic nervous system; ENS, enteric nervous system; TG, trigeminal ganglia; DRG, dorsal root ganglia; GLUT: glutamatergic neuron; CHO, cholinergic neuron; NOR, noradrenergic neuron; NBL, neuroblast-like cells. (F) Heatmap of representative cell type markers in each patterned NGN2-iN cluster with transferred annotations as side bars. (G) Immunofluorescent staining of week 6 patterned NGN2-iNs with MAP2 (top row) and SLC17A6 (bottom row) (yellow). DAPI is shown in cyan. Scale bar: 20 μm. (H-I) Feature plots (H) and immunofluorescent staining (I) of neuron subtype-specific markers, including LHX9, SLC5A7, NTRK1 and TRPM8. Scale bar: 20 μm. (J) Example spike-triggered electrical footprints of NGN2-iNs recorded on high-density microelectrode arrays (HD-MEAs) for five patterning conditions. Colors indicate the latencies of action potentials propagating along the various neurites (from blue to red). Scale bar: 200 μm. (K) Clustering of iN patternings could be clustered based on the electrophysiological features obtained from HD-MEA recordings (each dot represents one neuronal network). (L) CNQX application significantly reduced the spontaneous electrical activity of iNs across all patterning conditions; coloring as in (K).

Article Snippet: Antibodies against the following proteins were purchased from the indicated vendors: MAP2 (EMD Millipore AB5622); SLC17A6 (Alomone Labs AGC-036-GP); SLC5A7 (Invitrogen PA5-117124); LHX9 (Sigma-Aldrich HPA009695); TRPM8 (Alomone Labs ACC-049), NTRK1 (FabGennix International TRKA-101Y).

Techniques: Single-cell Transcriptomics, Marker, Expressing, Staining, Activity Assay

Journal: bioRxiv

Article Title: Human neuron subtype programming through combinatorial patterning with scSeq readouts

doi: 10.1101/2023.12.12.571318

Figure Lengend Snippet: (A) Experimental scheme and timeline for the pre- and post-patterning screen coupled with ASCL1/DLX2 induction. (B) UMAP embedding of pre-and post-patterned cells after ASCL1/DLX2 induction. ASCL1/DLX2-iNs are mostly neuronal (MAP2+) and GABAergic (GAD1+), with no or low expression of excitatory markers (SLC17A6-). (C) UMAP embedding of integrated atlas of all neuronal cells (n=430,936 cells) showing sample distribution and concentration gradients. (D) Scatterplots showing mean distance to control cells (normalized to 1) in relation to morphogen concentrations. Error bars represent SEM. (E) Diversity of clusters generated in all datasets, shown by dendrogram based on transcriptomic distance (cosine, TF marker genes). Annotation heatmap shows concentrations for the condition which has the highest relative composition of the cluster. Purity shown in percentage as barplots. GO heatmap shows enrichment of disease genes in marker genes of clusters. Expression heatmap shows row-normalized expression of key neurotransmitter genes, highlighting the diversity. (F) Comparison of regulons inferred from post-patterned NGN2-iNs and ASCL1/DLX2-iNs and their correlation with BMP4 and RA. Regulons inferred separately from NGN2- and ASCL1/DLX2 datasets represented by different shapes were scored for activity correlation to indicated morphogen gradients in both datasets. Regulons are colored with the regional identity where maximal activity is registered, while the size of the regulons was represented as the size of the dots. (G) Heatmap showing importances of morphogens for a Random Forest regressor to predict expression of neurotransmitter and neuropeptide genes. Importances were multiplied with the sign of the Pearson correlation to indicate directionality. (H) Boxplots showing examples of neurotransmitter/neuropeptides correlated with morphogen concentrations. The box plots show the median (center line), upper and lower quartiles (box limits) and 1.5×interquartile range (whiskers). Outliers shown.

Article Snippet: Antibodies against the following proteins were purchased from the indicated vendors: MAP2 (EMD Millipore AB5622); SLC17A6 (Alomone Labs AGC-036-GP); SLC5A7 (Invitrogen PA5-117124); LHX9 (Sigma-Aldrich HPA009695); TRPM8 (Alomone Labs ACC-049), NTRK1 (FabGennix International TRKA-101Y).

Techniques: Expressing, Concentration Assay, Control, Generated, Marker, Comparison, Activity Assay

Journal: eLife

Article Title: Sparse genetically defined neurons refine the canonical role of periaqueductal gray columnar organization

doi: 10.7554/eLife.77115

Figure Lengend Snippet: ( A ) Example histology images showing immunostaining of pan-neuronal marker NeuN (top row), viral-mediated expression of GFP in CCK-expressing cells (middle row), and overlay of NeuN and CCK-GFP (bottom row) in the dorsolateral (left column), lateral (middle column), and ventrolateral (right column) PAG. Scale bars, 10 μm. ( B ) Raw counts of CCK-GFP+ and NeuN+ cells in the dorsolateral PAG (dlPAG), lPAG, and vlPAG. ( C ) Fraction of NeuN-labeled cells that are also GFP-labeled in the dlPAG and l/vlPAG. CCK-expressing cells comprise ~5% of l/vlPAG neurons and constitute significantly more of l/vlPAG neurons than dlPAG neurons (n = 4; paired t-test, ** P =.0032). ( D ) Immunostaining of glutamatergic marker vGlut2 in CCK cells. Example histology images showing vGlut2 (top), CCK-GFP (middle) and vGlut2/GFP overlay (bottom). White arrow indicates vGlut2+/GFP+ cell. Dashed outline indicates vGlut2+/GFP- cell. Scale bar, 10 μm. ( E ) 9.6% of vGlut2-labeled cells in the l/vlPAG are also GFP-labeled (n = 4; 302 vGlut2/GFP+ of 3115 vGlut2+ cells). ( F ) A majority (94.8%) of GFP-labeled cells in the l/vlPAG are also vGlut2-labeled (n = 4; 302 vGlut2+/GFP+ of 317 GFP+ cells). Errorbars: mean ± SEM.

Article Snippet: Sections were then incubated at 4°C for 16 hr with polyclonal anti-VGLUT2 antibody (#AGC-036, Alomone Labs) made in rabbit (1:500 dilution) in blocking solution.

Techniques: Immunostaining, Marker, Expressing, Labeling

Journal: eLife

Article Title: Sparse genetically defined neurons refine the canonical role of periaqueductal gray columnar organization

doi: 10.7554/eLife.77115

Figure Lengend Snippet: ( A ) In situ hybridization labeling of l/vlPAG neurons showing mRNAs for CCK (green) and VGLUT2 (red). Nuclei stained by DAPI are shown in blue. Example image (×40 objective) of vglut2-labeled cells (red rectangle) and double-labeled vglut2/CCK-labeled (green rectangle) in l/vlPAG. Scale bar, 20 µm. ( B ) Zoomed-in images of subregions in ( A ). Scale bar, 20 µm. ( C ) 8.58% of vglut2-labeled cells are also CCK-labeled (15 of 183 total cells). Errorbars: mean ± SEM.

Article Snippet: Sections were then incubated at 4°C for 16 hr with polyclonal anti-VGLUT2 antibody (#AGC-036, Alomone Labs) made in rabbit (1:500 dilution) in blocking solution.

Techniques: In Situ Hybridization, Labeling, Staining