Journal: Nature Neuroscience

Article Title: Unique functional responses differentially map onto genetic subtypes of dopamine neurons

doi: 10.1038/s41593-023-01401-9

Figure Lengend Snippet: ( a ) Schematic showing the distribution of somas and axons across the SNc and striatum for three previously described subtypes (See Poulin et al. 2018 for in depth characterization of each of these subtypes). ( b ) Representative distribution of somas for different subtypes within SNc. Scale bar 100 um. Thresholds for intensity scaling and gamma changes were set for each individual channel to maximize visibility of stained cells. ( c ) Representative projection patterns of different subtypes in striatum. Scale bar 500 um. Thresholds for intensity scaling and gamma changes were set for each individual channel to maximize visibility of stained axons. ( d ) Example recordings for each subtype studied (two from Aldh1a1 with different functional signaling patterns, Type 1 and Type 2), showing fluorescence traces (ΔF/F), velocity, acceleration, licking, and reward delivery times. Isosbestic control shown in blue. Large accelerations = ▲, large decelerations = ▽. ( e ) Cross-correlation between ΔF/F traces and acceleration for traces shown in D. Isosbestic control shown in blue. ( f ) Average cross-correlation between ΔF/F traces and acceleration for all recordings of each subtype and DAT (subtypes indiscriminately labeled). Isosbestic control shown in blue. Shaded regions denote mean ± s.e.m. Heatmap shows cross-correlations for each recording, sorted by the integral of the cross-correlation at positive lags. Vglut2 mice = 12, n = 42 recordings; Calb1 mice = 6, n = 22; Aldh1a1 mice = 14, n = 75 DAT mice = 14, n = 74. ( g ) ΔF/F triggered averages on reward delivery times for all recordings of each subtype and DAT. Isosbestic control shown in light blue, same scale as ΔF/F average. Acceleration shown in gray in the background (scale bar = 0.2 m/s 2 ). Shaded regions denote mean ± s.e.m. Heatmap shows triggered average for each recording, sorted by size of reward response. Vglut2 mice = 11, n = 28 recordings; Calb1 mice = 8, n = 17; Aldh1a1 mice = 8, n = 30; DAT mice = 11, n = 63. ( h ) Distribution of locomotion response (integral of the cross-correlation at positive lags) along the dorso-ventral axis of the striatum for all recordings of all subtypes and DAT, showing how in Aldh1a1 dorsal recordings show acceleration correlation (Type 1) while more ventral recordings show deceleration correlation (Type 2). Black line represents moving average (0.5 mm bins). ( i ) Relationship between reward response and locomotion response for each recording of each subtype, showing how in Aldh1a1 larger reward responses correspond with deceleration correlation (Type 2), while small or negative reward responses correspond with acceleration correlation (Type 1).

Article Snippet: Sections were incubated with primary antibodies against Aldh1a1 (goat, R&D Systems, AF5869, RRID:AB_2044597, 1:500 dilution), TH (mouse, Sigma-Aldrich, T2928, RRID:AB_477569; Pel-Freez Biologicals, P40101-0, RRID:AB_461064, 1:1,000 dilution) and mCherry (rat, Thermo Fisher Scientific, M11217, RRID:AB_2536611, 1:2,000 dilution) in blocking buffer for 24 h, followed by four washes in PBS-Tween 20 and incubation with secondary antibodies diluted 1:250 (donkey anti-goat Alexa Fluor 488 (Molecular Probes, A-11055, RRID:AB_2534102), donkey anti-mouse Alexa Fluor 647 (Thermo Fisher Scientific, A-31571, RRID:AB_162542), donkey anti-rabbit Alexa Fluor 647 (Thermo Fisher Scientific, A-31573, RRID:AB_2536183), donkey anti-rat Cy3 (Jackson ImmunoResearch, 712-165-153, RRID:AB_2340667) and DAPI (Thermo Fisher Scientific, 62248)) for 2 h at room temperature.

Techniques: Staining, Functional Assay, Fluorescence, Control, Labeling

Journal: Nature Neuroscience

Article Title: Unique functional responses differentially map onto genetic subtypes of dopamine neurons

doi: 10.1038/s41593-023-01401-9

Figure Lengend Snippet: ( a ) Resulting clusters from integrating datasets. ( b ) Expression patterns of Anxa1 and Aldh1a1 , the top defining markers for cluster 1. Expression of Anxa1 appears to be limited to a subset of Aldh1a1 -expressing neurons. ( c ) Violin plots of number of genes and RNA counts from each source dataset, which were used to determine cutoffs for quality control filtering. ( d ) LIGER clustering of the meta-dataset, revealing one cluster that was more distantly related to all other DA neurons and came solely from the Tiklova et al. dataset. This cluster was subsequently removed. ( e ) Cells colored by cluster (left) or source dataset (right), which reveals that all clusters were represented by each dataset. ( f ) Violin plots of the top 2 defining marker genes for each cluster.

Article Snippet: Sections were incubated with primary antibodies against Aldh1a1 (goat, R&D Systems, AF5869, RRID:AB_2044597, 1:500 dilution), TH (mouse, Sigma-Aldrich, T2928, RRID:AB_477569; Pel-Freez Biologicals, P40101-0, RRID:AB_461064, 1:1,000 dilution) and mCherry (rat, Thermo Fisher Scientific, M11217, RRID:AB_2536611, 1:2,000 dilution) in blocking buffer for 24 h, followed by four washes in PBS-Tween 20 and incubation with secondary antibodies diluted 1:250 (donkey anti-goat Alexa Fluor 488 (Molecular Probes, A-11055, RRID:AB_2534102), donkey anti-mouse Alexa Fluor 647 (Thermo Fisher Scientific, A-31571, RRID:AB_162542), donkey anti-rabbit Alexa Fluor 647 (Thermo Fisher Scientific, A-31573, RRID:AB_2536183), donkey anti-rat Cy3 (Jackson ImmunoResearch, 712-165-153, RRID:AB_2340667) and DAPI (Thermo Fisher Scientific, 62248)) for 2 h at room temperature.

Techniques: Expressing, Control, Marker

Journal: Nature Neuroscience

Article Title: Unique functional responses differentially map onto genetic subtypes of dopamine neurons

doi: 10.1038/s41593-023-01401-9

Figure Lengend Snippet: a , Schematic of snRNA-seq experimental pipeline. b , UMAP reduction of resulting clusters. In total, 15 clusters were found. Notably, four clusters (12, 13, 14 and 15) had weak dopaminergic characteristics (see Extended Data Fig. for details). c , Expression of Aldh1a1 and Anxa1 , the latter of which is expressed only within a subset of Aldh1a1 -expressing neurons. d , Expression patterns of the additional markers used for genetic access in experiments here, as well as Otx2 , a classical marker of most VTA neurons, enriched in clusters 5, 6 and 7. e , Immunofluorescence images of Aldh1a1 and Anxa1 protein expression in SNc ( n = 4 mice). Anxa1 expression is limited to a ventral subset of Aldh1a1 + neurons. Thresholds for intensity scaling and gamma changes were set for each individual channel to maximize visibility of stained cells. f , Zoomed-in crops of section shown in e . Anxa1 expression was ventrally biased within SNc neurons. g , Right, projection patterns of Anxa1 + SNc axons based on viral labeling ( n = 4 mice), which appear highly restricted to dorsolateral striatum and patches. Left, projection patterns of Aldh1a1 + SNc axons using the same virus ( n = 4 mice); projections extend more ventrally relative to Anxa1 + . Maximum thresholds for image intensity scaling were set to the highest detected pixel intensity in each section to better enable direct comparisons across brains.

Article Snippet: Sections were incubated with primary antibodies against Aldh1a1 (goat, R&D Systems, AF5869, RRID:AB_2044597, 1:500 dilution), TH (mouse, Sigma-Aldrich, T2928, RRID:AB_477569; Pel-Freez Biologicals, P40101-0, RRID:AB_461064, 1:1,000 dilution) and mCherry (rat, Thermo Fisher Scientific, M11217, RRID:AB_2536611, 1:2,000 dilution) in blocking buffer for 24 h, followed by four washes in PBS-Tween 20 and incubation with secondary antibodies diluted 1:250 (donkey anti-goat Alexa Fluor 488 (Molecular Probes, A-11055, RRID:AB_2534102), donkey anti-mouse Alexa Fluor 647 (Thermo Fisher Scientific, A-31571, RRID:AB_162542), donkey anti-rabbit Alexa Fluor 647 (Thermo Fisher Scientific, A-31573, RRID:AB_2536183), donkey anti-rat Cy3 (Jackson ImmunoResearch, 712-165-153, RRID:AB_2340667) and DAPI (Thermo Fisher Scientific, 62248)) for 2 h at room temperature.

Techniques: Expressing, Marker, Immunofluorescence, Staining, Labeling, Virus

Journal: Nature Neuroscience

Article Title: Unique functional responses differentially map onto genetic subtypes of dopamine neurons

doi: 10.1038/s41593-023-01401-9

Figure Lengend Snippet: ( a ) Quantification of stability (via normalized Jaccard similarity index) of all 15 clusters from n = 100 iterations of stability calculations (simulated randomly down-sampled datasets, see ). Lower stability measurements imply the possibility of further subdivisions within the cluster, or additional subpopulations that may have been split across adjacent clusters. Center represents median, upper and lower box bounds represent 75 th and 25 th percentiles respectively, whiskers represent maxima and minima excluding outliers (data points more than 1.5 times the IQR outside the box bounds). ( b ) Mapping clusters across progressively higher resolutions reveals potential subdivisions either within clusters (for example, the splitting of Cluster 8 into two stable clusters) or across adjacent clusters (for example a novel cluster emerging at the intersection of clusters 3 and 10 as resolution increases). Four clusters with lower stability, as shown in panel A, are colored to highlight the potential source of their instabilities. ( c ) Scatter plots comparing the average expression for all genes across two clusters. Several examples of distinguishing genes with notably enriched expression patterns are highlighted. Top: Clusters 4 ( Anxa1 +/ Aldh1a1 +) vs. 1 ( Anxa1 -/ Aldh1a1 +). Bottom: Clusters 11 ( Calb1 + SNc) vs. 9 ( Vglut2 + SNc/SNL). Transcriptomic similarity of cluster pairs can be approximated by the correlation coefficient of their average gene expressions. ( d ) In situ hybridization images from the Allen Mouse Brain Atlas of ventral tier marker genes. Note that Hs6st3 , which is highly enriched in our Anxa1 + cluster, appears limited to ventral-most SNc and highly resembles the expression of Anxa1 (black arrows). Images available from Allen Mouse Brain Atlas, mouse.brain-map.org ( e ) Additional ISH images showing expression of two marker genes that distinguish Cluster 9 ( Vglut2 +) from Cluster 11 ( Calb1 +), further corroborating the distinct identities of these populations. DAT expression is shown for reference to highlight the localization of these markers to SN pars lateralis, matching the previously described location of Vglut2 + SN DA neurons and thus supporting Cluster 9 as the Vglut2 + neurons investigated in the GCaMP activity recordings in this study. Source images available from Allen Mouse Brain Atlas, mouse.brain-map.org.

Article Snippet: Sections were incubated with primary antibodies against Aldh1a1 (goat, R&D Systems, AF5869, RRID:AB_2044597, 1:500 dilution), TH (mouse, Sigma-Aldrich, T2928, RRID:AB_477569; Pel-Freez Biologicals, P40101-0, RRID:AB_461064, 1:1,000 dilution) and mCherry (rat, Thermo Fisher Scientific, M11217, RRID:AB_2536611, 1:2,000 dilution) in blocking buffer for 24 h, followed by four washes in PBS-Tween 20 and incubation with secondary antibodies diluted 1:250 (donkey anti-goat Alexa Fluor 488 (Molecular Probes, A-11055, RRID:AB_2534102), donkey anti-mouse Alexa Fluor 647 (Thermo Fisher Scientific, A-31571, RRID:AB_162542), donkey anti-rabbit Alexa Fluor 647 (Thermo Fisher Scientific, A-31573, RRID:AB_2536183), donkey anti-rat Cy3 (Jackson ImmunoResearch, 712-165-153, RRID:AB_2340667) and DAPI (Thermo Fisher Scientific, 62248)) for 2 h at room temperature.

Techniques: Expressing, In Situ Hybridization, Marker, Activity Assay

Journal: Nature Neuroscience

Article Title: Unique functional responses differentially map onto genetic subtypes of dopamine neurons

doi: 10.1038/s41593-023-01401-9

Figure Lengend Snippet: ( a ) Schematic representation of Aldh1a1-iCre transgenic line. Endogenous Aldh1a1 gene was targeted for insertion of a P2A peptide and iCre immediately following the peptide encoded by Exon 13. ( b ) Ratio of mCherry virally labelled cells co-staining for Aldh1a1 (n = 4 mice). ( c ) Substantia nigra pars compacta immunofluorescence staining from Aldh1a1-iCre mice injected with an AAV5-DIO-mCherry virus (n = 4 mice). Co-staining shows excellent efficiency and fidelity of iCre recombination, which is notably limited to TH+ cells in this region. White arrows: examples of mCherry and Aldh1a1 co-stained cells. Orange arrows: mCherry-expressing cells with undetectable Aldh1a1 staining, which were primarily localized to the dorsal and lateral SNc. Thresholds for intensity scaling and gamma changes were set for each individual channel to maximize visibility of stained cells. ( d ) Schematic representation of Anxa1-iCre transgenic line. ( e ) Ratios of virally labelled cells co-staining for Anxa1 protein (n = 4 mice), showing high fidelity of Cre recombination. ( f ) High magnification of immunofluorescence staining from Anxa1-iCre mice injected with an AAV1-CAG-FLEX-GCaMP6f virus (n = 4 mice) shows that recombination occurs in cells with both high Anxa1 (white arrow) and low Anxa1 (orange arrow), with ~10% of labelled cells showing undetectable Anxa1 protein (red arrows). Thresholds for intensity scaling and gamma changes were set for each individual channel to maximize visibility of stained cells. ( g ) High magnification of immunofluorescence staining from Anxa1-iCre mice injected with an AAV5-DIO-mCherry virus (n = 4 mice) confirms that recombination occurs in cells with both high Anxa1 protein staining (orange arrows) as well as low Anxa1 protein (white arrows), making it difficult to assess specificity using protein staining alone. Thresholds for intensity scaling and gamma changes were set for each individual channel to maximize visibility of stained cells. ( h ) IF staining of GFP and Aldh1a1 in Anxa1-iCre, TH-Flpo, RC::FrePe mice (n = 2 mice). Recombination by iCre and Flpo leads to GFP expression in Anxa1+ DA neurons. Co-staining with Aldh1a1 corroborates that Anxa1-iCre recombination is less broad than Aldh1a1 expression and confirms that viral labelling results were not due to insufficient viral delivery / diffusion (example cells with Aldh1a1 staining but no recombination shown with white arrows). Thresholds for intensity scaling and gamma changes were set for each individual channel to maximize visibility of stained cells.

Article Snippet: Sections were incubated with primary antibodies against Aldh1a1 (goat, R&D Systems, AF5869, RRID:AB_2044597, 1:500 dilution), TH (mouse, Sigma-Aldrich, T2928, RRID:AB_477569; Pel-Freez Biologicals, P40101-0, RRID:AB_461064, 1:1,000 dilution) and mCherry (rat, Thermo Fisher Scientific, M11217, RRID:AB_2536611, 1:2,000 dilution) in blocking buffer for 24 h, followed by four washes in PBS-Tween 20 and incubation with secondary antibodies diluted 1:250 (donkey anti-goat Alexa Fluor 488 (Molecular Probes, A-11055, RRID:AB_2534102), donkey anti-mouse Alexa Fluor 647 (Thermo Fisher Scientific, A-31571, RRID:AB_162542), donkey anti-rabbit Alexa Fluor 647 (Thermo Fisher Scientific, A-31573, RRID:AB_2536183), donkey anti-rat Cy3 (Jackson ImmunoResearch, 712-165-153, RRID:AB_2340667) and DAPI (Thermo Fisher Scientific, 62248)) for 2 h at room temperature.

Techniques: Transgenic Assay, Staining, Immunofluorescence, Injection, Virus, Expressing, Diffusion-based Assay

Journal: Nature Neuroscience

Article Title: Unique functional responses differentially map onto genetic subtypes of dopamine neurons

doi: 10.1038/s41593-023-01401-9

Figure Lengend Snippet: ( a ) Locomotion response (PC1/PC2) mapped onto recording location for each subtype and DAT. Same as Fig. but without collapsing slices for compactness and without random mediolateral shifting of recording locations to reduce overlap. ( b ) Average ΔF/F triggered on large accelerations (left, ▲) and large decelerations (right, ▽) for Aldh1a1 recordings (as Fig. ). Isosbestic control shown in light blue, same scale as ΔF/F average but shifted. Acceleration shown in grey in the background (scale bar = 0.2 m/s 2 ). Shaded regions denote mean ± s.e.m. Heatmap shows triggered average for each recording, sorted by PC1/PC2 angle (see Fig. ). Aldh1a1 mice = 14, n = 75 recordings. ( c ) Average cross-correlation between ΔF/F traces and acceleration for Aldh1a1 recordings (as Fig. ). Isosbestic control shown in blue. Shaded regions denote mean ± s.e.m. Heatmap shows cross-correlation for each recording, sorted as in B. ( d ) Average acceleration triggered on large transients for Aldh1a1 recordings (as Fig. ). ΔF/F average and isosbestic control shown in the background (scale bar = 5% Norm ΔF/F.) Shaded regions denote mean ± s.e.m. Heatmap shows triggered average for each recording, sorted as in B. ( e ) Principal component scores for each recording along PC1 and PC2 for Aldh1a1 (same as Fig. ) but with each Aldh1a1 recording color-coded by depth within striatum, showing that Aldh1a1 axons deeper in striatum show similar locomotion signaling to Calb1. ( f ) Timing of the calcium transient peak in triggered averages on decelerations (Fig. , right) for each recording from Calb1 and Vglut2. Means Vglut2 = 0.35, Calb1 = 0.23; p-value for comparison between subtypes = 0.01 (two-sided Wilcoxon rank-sum test). Vglut2 mice = 12, n = 42 recordings; Calb1 mice = 6, n = 22 (as in Fig. ). Error bars denote mean ± s.e.m. ( g ) Timing of the deceleration peak in triggered averages on ΔF/F transient peaks (Fig. ) for each recording from Calb1 and Vglut2. Means Vglut2 = 0.47, Calb1 = 0.34; p-value for comparison between subtypes = 0.005 (two-sided Wilcoxon rank-sum test). Same n as F. Error bars denote mean ± s.e.m. ( h ) The locomotion signaling observed in DAT mice across depths (H) can be explained by mixtures of the Anxa1 and Calb1 subtypes in varying ratios matching the relative abundance of each subtypes’ axons in that depth (H’). ( i ) Average cross-correlation between ΔF/F traces and acceleration for all recordings of each functionally homogeneous subtype (as Fig. ) but averaged per mouse. Shaded regions denote mean ± s.e.m. Heatmap shows cross-correlation average for each mouse, sorted by PC1/PC2 angle (see Fig. ). ( j ) Difference in locomotion signaling (measured as the difference in PC1/2 angle, as shown in Fig. ) between pairs of recordings made at difference distances from each other, for pairs of recordings from the same subtype (Vglut2, Calb1, and Anxa1, in colors), from DAT mice (mixture of subtypes, in grey), or from mismatched subtypes (Vglut2-Calb1, Vglut2-Anxa1, and Calb1-Anxa1). P-values for comparison between pairs within same subtype vs mismatch subtypes: Vglut2 = 3 × 10 −05 , 6 × 10 −23 , 2 × 10 −25 , 5 × 10 −25 , 1 × 10 −08 , 0.03, 9 × 10 −04 , 1, 0.9; Calb1 = 2 × 10 −04 , 5 × 10 −22 , 4 × 10 −10 , 1 × 10 −07 ; Anxa1 = 8 × 10 −08 , 6 × 10 −35 , 1 × 10 −21 , 5 × 10 −09 , 0.06, 0.02, 1, 1 (two-sided Mann-Whitney U test with Bonferroni correction). Number of pairs of recordings per distance bin (from 0 in steps of 0.3 mm): Vglut2 = [24, 107, 108, 91, 58, 29, 28, 14, 6, 0, 0], Calb1 = [37, 83, 29, 18, 1, 3, 0, 0, 0, 0, 0], Anxa1 = [252, 410, 250, 52, 49, 30, 28, 10, 0, 0, 0], Mismatch = [47, 245, 438, 525, 461, 661, 542, 615, 367, 189, 49]. Error bars denote mean ± s.e.m.

Article Snippet: Sections were incubated with primary antibodies against Aldh1a1 (goat, R&D Systems, AF5869, RRID:AB_2044597, 1:500 dilution), TH (mouse, Sigma-Aldrich, T2928, RRID:AB_477569; Pel-Freez Biologicals, P40101-0, RRID:AB_461064, 1:1,000 dilution) and mCherry (rat, Thermo Fisher Scientific, M11217, RRID:AB_2536611, 1:2,000 dilution) in blocking buffer for 24 h, followed by four washes in PBS-Tween 20 and incubation with secondary antibodies diluted 1:250 (donkey anti-goat Alexa Fluor 488 (Molecular Probes, A-11055, RRID:AB_2534102), donkey anti-mouse Alexa Fluor 647 (Thermo Fisher Scientific, A-31571, RRID:AB_162542), donkey anti-rabbit Alexa Fluor 647 (Thermo Fisher Scientific, A-31573, RRID:AB_2536183), donkey anti-rat Cy3 (Jackson ImmunoResearch, 712-165-153, RRID:AB_2340667) and DAPI (Thermo Fisher Scientific, 62248)) for 2 h at room temperature.

Techniques: Control, Comparison, MANN-WHITNEY

Journal: Nature Neuroscience

Article Title: Unique functional responses differentially map onto genetic subtypes of dopamine neurons

doi: 10.1038/s41593-023-01401-9

Figure Lengend Snippet: ( a ) ΔF/F average triggered on reward delivery times for all recordings from Aldh1a1 (as Fig. ). Isosbestic control shown in light blue, same scale as ΔF/F average. Acceleration shown in gray in the background (scale bar = 0.2 m/s 2 ). Shaded regions denote mean ± s.e.m. Heatmap shows triggered average for each recording, sorted by size of reward response. Aldh1a1 mice = 8, n = 30 recordings. ( b ) Licking average triggered on reward delivery times (same as A) for all recording from Aldh1a1 (as Fig. ). Shaded regions denote mean ± s.e.m. Heatmap shows triggered average for each recording, sorted as in A. ( c ) ΔF/F average triggered on air puff delivery times for all recordings from Aldh1a1 (as Fig. ). Isosbestic control shown in light blue, same scale as ΔF/F average. Acceleration shown in gray in the background (scale bar = 0.2 m/s 2 ). Shaded regions denote mean ± s.e.m. Heatmap shows triggered average for each recording, sorted by reward size as in A, B. Aldh1a1 mice = 8, n = 30 recordings. ( d ) Reward vs air puff responses for Aldh1a1 (as shown in Fig. for other subtypes). X shows mean. ( e ) ΔF/F averages triggered on rewards delivered during rest for all recordings of each subtype and DAT. Isosbestic control shown in light blue, same scale as ΔF/F average. Acceleration shown in gray in the background (scale bar = 0.2 m/s 2 ). Shaded regions denote mean ± s.e.m. Heatmaps show triggered average for each recording, sorted by size of the reward response. Vglut2 mice = 6, n = 8 recordings; Calb1 mice = 6, n = 10; Anxa1 mice = 8, n = 42; DAT mice = 10, n = 42. ( f ) Comparison between response to rewards at rest (E) vs response to rewards not at rest for all recordings of each subtype and DAT. Diagonal dotted line represents identity line (same response to rewards at rest vs all rewards). p-values: Vglut2 = 1 (ns), Calb1 = 1 (ns), Anxa1 = 0.4 (ns), DAT = 0.2 (ns), two-sided paired Wilcoxon signed-rank test with Bonferroni correction. ( g ) Subtypes can still be distinguished by their air puff and reward responses after min-max scaling the responses. ( H ) ΔF/F average triggered on reward delivery times for all recordings of each functionally homogeneous subtype (as Fig. ) but averaged per mouse. Shaded regions denote mean ± s.e.m. Heatmap shows triggered average for each recording, randomly sorted. ( i ) Same as H but for air puffs. ( j ) Reward response mapped onto recording locations for each subtype and DAT. Same as Fig. but without collapsing slices for compactness and without random mediolateral shifting of recording locations to reduce overlap. ( k ) Same as J but for air puff response (matching Fig. but without collapsing slices or random shifting).

Article Snippet: Sections were incubated with primary antibodies against Aldh1a1 (goat, R&D Systems, AF5869, RRID:AB_2044597, 1:500 dilution), TH (mouse, Sigma-Aldrich, T2928, RRID:AB_477569; Pel-Freez Biologicals, P40101-0, RRID:AB_461064, 1:1,000 dilution) and mCherry (rat, Thermo Fisher Scientific, M11217, RRID:AB_2536611, 1:2,000 dilution) in blocking buffer for 24 h, followed by four washes in PBS-Tween 20 and incubation with secondary antibodies diluted 1:250 (donkey anti-goat Alexa Fluor 488 (Molecular Probes, A-11055, RRID:AB_2534102), donkey anti-mouse Alexa Fluor 647 (Thermo Fisher Scientific, A-31571, RRID:AB_162542), donkey anti-rabbit Alexa Fluor 647 (Thermo Fisher Scientific, A-31573, RRID:AB_2536183), donkey anti-rat Cy3 (Jackson ImmunoResearch, 712-165-153, RRID:AB_2340667) and DAPI (Thermo Fisher Scientific, 62248)) for 2 h at room temperature.

Techniques: Control, Comparison

Journal: Nature Neuroscience

Article Title: Unique functional responses differentially map onto genetic subtypes of dopamine neurons

doi: 10.1038/s41593-023-01401-9

Figure Lengend Snippet: ( a - e ) Same as Fig. but for recordings made in SNc. (a) ΔF/F averages triggered on reward delivery times for all recordings of each subtype and DAT. Isosbestic control shown in light blue, same scale as ΔF/F average. Acceleration shown in gray in the background (scale bar = 0.2 m/s 2). Shaded regions denote mean ± s.e.m. Heatmap shows triggered average for each recording, sorted by size of reward response. Vglut2 mice = 9, n = 25 recordings; Calb1 mice = 5, n = 10; Anxa1 mice = 5, n = 23; Aldh1a1 mice = 11, n = 40; DAT mice = 7, n = 39. (b) ΔF/F averages triggered on air puff delivery times for all recordings of each subtype and DAT. Isosbestic control shown in light blue, same scale as ΔF/F average. Acceleration shown in gray in the background (scale bar = 0.2 m/s 2). Shaded regions denote mean ± s.e.m. Heatmap shows triggered average for each recording, sorted by reward size as in A. Vglut2 mice = 9, n = 25 recordings; Calb1 mice = 5, n = 10; Anxa1 mice = 5, n = 25; Aldh1a1 mice = 11, n = 41; DAT mice = 8, n = 47. (c) Average reward and air puff responses for each subtype. Error bars denote ± s.e.m. p-values for reward: Vglut2 = 5 × 10 −05 , Calb1 = 0.007, Anxa1 = 1 (not significant), DAT = 2 × 10 −07 . p-values for air puff: VGlut2 = 5 × 10 −05 , Calb1 = 0.008, Anxa1 = 1 (not significant), DAT = 3 × 10 −05 . Two-sided Wilcoxon sign-rank test with Bonferroni correction. Same recordings and n as A,B. (d) Reward vs air puff responses for all recordings of each subtype and DAT. X shows mean for each subtype. Shaded regions are areas representing greater air puff than reward response (for Vglut2) or greater reward vs air puff response (for Calb1). (e) Comparison of responses to small vs large rewards for each subtype. Error bars denote mean ± s.e.m. p-values: Vglut2 = 0.05 (not significant), Calb1 = 0.03, Anxa1 = 1 (not significant). Two-sided paired Wilcoxon Signed Rank test with Bonferroni correction. Vglut2 mice = 9, n = 25 recordings; Calb1 mice = 5, n = 10; Anxa1 mice = 5, n = 23. (F) 3D plot showing locomotion (PC1/PC2 angle), reward and air puff responses for each recording and each subtype, comparing striatal recordings (same as Fig. ) and SNc recordings. (G) 2D plots for each pair of variables shown in the 3D plot in F. ( h - k ) Same as Fig. but for recordings made in SNc. ( h ) Average cross-correlation between ΔF/F traces and acceleration for all recordings of each subtype. Isosbestic control shown in blue. Shaded regions denote mean ± s.e.m. Heatmap shows cross-correlation for each recording, sorted by PC1/PC2 angle (see Fig. ). Vglut2 mice = 11, n = 28 recordings; Calb1 mice = 3, n = 6; Anxa1 mice = 8, n = 34; Aldh1a1 mice = 13, n = 42; DAT mice = 8, n = 31. ( I ) ΔF/F averages triggered on large accelerations (left, ▲) and large decelerations (right, ▽) for all recordings of each subtype. Isosbestic control shown in light blue, same scale as ΔF/F average but shifted. Acceleration shown in gray in the background (scale bar = 0.2 m/s ). Shaded regions denote mean ± s.e.m. Heatmap shows triggered average for each recording, sorted as in H. ( J ) Acceleration averages triggered on large transients for all recordings of each subtype. ΔF/F average and isosbestic control shown in the background (scale bar = 5% Norm ΔF/F.) Shaded regions denote mean ± s.e.m. Heatmap shows triggered average for each recording, sorted as in H. ( K ) Principal component scores for each recording of each subtype along PC1 and PC2 (same PCs obtained from the striatal recordings, as shown in Fig. ). X shows mean for each subtype. Striatal PCs explain 77.6% of SNc variance (52.4% PC1, 25.2% PC2).

Article Snippet: Sections were incubated with primary antibodies against Aldh1a1 (goat, R&D Systems, AF5869, RRID:AB_2044597, 1:500 dilution), TH (mouse, Sigma-Aldrich, T2928, RRID:AB_477569; Pel-Freez Biologicals, P40101-0, RRID:AB_461064, 1:1,000 dilution) and mCherry (rat, Thermo Fisher Scientific, M11217, RRID:AB_2536611, 1:2,000 dilution) in blocking buffer for 24 h, followed by four washes in PBS-Tween 20 and incubation with secondary antibodies diluted 1:250 (donkey anti-goat Alexa Fluor 488 (Molecular Probes, A-11055, RRID:AB_2534102), donkey anti-mouse Alexa Fluor 647 (Thermo Fisher Scientific, A-31571, RRID:AB_162542), donkey anti-rabbit Alexa Fluor 647 (Thermo Fisher Scientific, A-31573, RRID:AB_2536183), donkey anti-rat Cy3 (Jackson ImmunoResearch, 712-165-153, RRID:AB_2340667) and DAPI (Thermo Fisher Scientific, 62248)) for 2 h at room temperature.

Techniques: Control, Comparison

Journal: Nature Neuroscience

Article Title: Unique functional responses differentially map onto genetic subtypes of dopamine neurons

doi: 10.1038/s41593-023-01401-9

Figure Lengend Snippet: ( a ) Example recording for Aldh1a1 showing simultaneous fluorescence traces (ΔF/F) from SNc and striatum. Isosbestic control shown in blue. ▼= Example transients present in SNc and in striatum. ( b ) Cross-correlation between ΔF/F traces from striatum and SNc shown in A. Isosbestic control shown in blue. ( c ) Average cross-correlation between ΔF/F traces from striatum and SNc for all recordings of Aldh1a1 (as Fig. ). Isosbestic control shown in blue. Shaded regions denote mean ± s.e.m. Heatmap shows cross correlations for each paired recording sorted by peak magnitude. Aldh1a1 mice = 8, n = 29 recordings. ( d ) Distribution of peak cross correlations between SNc and striatum for recordings of Aldh1a1 and DAT shown in C (as Fig. ). P-value for comparison DAT-Aldh1a1 = 0.03 (two-sided Mann-Whitney U test with Bonferroni correction). ( e ) Peak cross correlations between dorsal striatum recordings (from Aldh1a1 or DAT) vs different relative depths in SNc, showing that for Aldh1a1 dorsal striatum signaling is best correlated to ventral SNc.

Article Snippet: Sections were incubated with primary antibodies against Aldh1a1 (goat, R&D Systems, AF5869, RRID:AB_2044597, 1:500 dilution), TH (mouse, Sigma-Aldrich, T2928, RRID:AB_477569; Pel-Freez Biologicals, P40101-0, RRID:AB_461064, 1:1,000 dilution) and mCherry (rat, Thermo Fisher Scientific, M11217, RRID:AB_2536611, 1:2,000 dilution) in blocking buffer for 24 h, followed by four washes in PBS-Tween 20 and incubation with secondary antibodies diluted 1:250 (donkey anti-goat Alexa Fluor 488 (Molecular Probes, A-11055, RRID:AB_2534102), donkey anti-mouse Alexa Fluor 647 (Thermo Fisher Scientific, A-31571, RRID:AB_162542), donkey anti-rabbit Alexa Fluor 647 (Thermo Fisher Scientific, A-31573, RRID:AB_2536183), donkey anti-rat Cy3 (Jackson ImmunoResearch, 712-165-153, RRID:AB_2340667) and DAPI (Thermo Fisher Scientific, 62248)) for 2 h at room temperature.

Techniques: Fluorescence, Control, Comparison, MANN-WHITNEY

Journal: Nature

Article Title: Lymphoangiocrine signals promote cardiac growth and repair

doi: 10.1038/s41586-020-2998-x

Figure Lengend Snippet: a , Wild type mouse cardiac lymphatic vasculature development as depicted by anti-Lyve1 whole mount immunostaining. Yellow arrowheads indicate cardiac lymphatics at E14.5. b-c, Bright field images of E17.5 control and Prox1 ΔLEC/ΔLEC embryos and hearts. White arrow indicates edema in Prox1 ΔLEC/ΔLEC embryos. d-i, Whole mount immunostaining shows that E17.5 Prox1 ΔLEC/ΔLEC hearts lack Lyve1+ cardiac lymphatics and have normal major coronary arteries and veins, as indicated by α-SMA and endomucin (EMCN) staining. Arrowheads indicate developing lymphatics in control hearts. j, Quantification of organ weight relative to body length (BL) shows reduced heart size and normal liver and kidney sizes in E17.5 Prox1 ΔLEC/ΔLEC embryos ( N =13 controls and N =10 Prox1 ΔLEC/ΔLEC embryos; 3 different litters). Data is presented as mean ± S.E.M. *** p =3.19062E-06 by unpaired two-tailed Student’s t test. n.s, not significant. Control embryos are TAM treated Cre- and Cre + ; Prox1 +/+ littermates. HW, heart weight; LW, liver weight; KW, kidney weight. N = 3 embryos/genotype (a, d-i). Scale bars, 500 μm (a, c-i), 2 mm (b).

Article Snippet: Primary antibodies were used as follows: α-Actinin (Mouse, Sigma, A7811, 1:500), cardiac Troponin C (Mouse, Abcam, ab8295, 1:1000), Ki67 (Rabbit, Invitrogen, SP6, MA5-14520, 1:200), active Caspase-3 (Rabbit, BD Pharmingen, C92- 605, 559565, 1:200), Lyve1 (Goat, R&D, AF2125, 1:200), Reelin (Goat, R&D, AF3820, 1:50), Lyve1 (Rabbit, AngioBio, 11-034, 1:500), Prox1 (Rabbit, AngioBio, 11002, 1:500), Prox1 (Goat, R&D, AF2727, 1:100) and Mef2c (Rabbit, LSBio, LSC356188, 1:1000).

Techniques: Immunostaining, Staining, Two Tailed Test

Journal: Nature

Article Title: Lymphoangiocrine signals promote cardiac growth and repair

doi: 10.1038/s41586-020-2998-x

Figure Lengend Snippet: a. EdU labeling shows an overall reduction in the number of EdU+ cells in sections of E17.5 Prox1 ΔLEC/ΔLEC hearts. Dashed boxes indicate the corresponding areas of the heart that are shown at higher magnification in panels b-e. b-e, Immunostaining results show the presence of Prox1+Lyve1+ cardiac lymphatics (white arrows) in sections of control hearts ( b, c ), and lack of lymphatics in Prox1 ΔLEC/ΔLEC hearts ( d, e ). Yellow arrows indicate Lyve1+ Prox1- macrophages. N = 3 embryos/genotype from 3 separate litters. f , CM proliferation is reduced in the myocardium of the left ventricle area (LV), the right ventricle area (RV) and the septum. N = 4 embryos/genotype from 3 separate litters. At least 3 images/region and 3 separate regions/heart were quantified. Data are presented as mean ± S.E.M. * p = 0.01, ** p =0.003, 0.006 and * p =0.02 (upper panel); *** p = 0.0001, ** p= 0.004, 0.002 and 0.005 (middle panel); ** p = 0.001, 0.001, * p= 0.01 and ** p =0.002 (bottom panel) as calculated by unpaired two-tailed Student’s t test. g, Immunostaining with antibodies against Vimentin (fibroblasts), PECAM1 (blood endothelial cells), CD68 (macrophages), Six2 (nephron progenitors) or Hnf4α (hepatocytes) together with EdU labeling (white arrows) shows no differences in proliferation in those cell types between E17.5 Prox1 ΔLEC/ΔLEC and control hearts (TAM injected at E13.5 and E14.5). n.s. no significant differences by unpaired two-tailed Student’s t test. N = 3 embryos/genotype from 3 separate litters. Control are TAM treated Cre - embryos and Cre + ; Prox1 +/+ littermates. Data are presented as mean ± S.E.M. Scale bars, 200 μm (a), 100 μm (b-e), 25μm (g).

Article Snippet: Primary antibodies were used as follows: α-Actinin (Mouse, Sigma, A7811, 1:500), cardiac Troponin C (Mouse, Abcam, ab8295, 1:1000), Ki67 (Rabbit, Invitrogen, SP6, MA5-14520, 1:200), active Caspase-3 (Rabbit, BD Pharmingen, C92- 605, 559565, 1:200), Lyve1 (Goat, R&D, AF2125, 1:200), Reelin (Goat, R&D, AF3820, 1:50), Lyve1 (Rabbit, AngioBio, 11-034, 1:500), Prox1 (Rabbit, AngioBio, 11002, 1:500), Prox1 (Goat, R&D, AF2727, 1:100) and Mef2c (Rabbit, LSBio, LSC356188, 1:1000).

Techniques: Labeling, Immunostaining, Two Tailed Test, Injection

Journal: Nature

Article Title: Lymphoangiocrine signals promote cardiac growth and repair

doi: 10.1038/s41586-020-2998-x

Figure Lengend Snippet: a, Bright field images of whole E17.5 Vegfr3 kd/kd and WT embryos and hearts. Quantification of organ weight (heart, liver and kidney) relative to body length indicates that the heart is smaller and the liver and kidney have comparable sizes between Vegfr3 kd/kd and control embryos. N =10 (WT) and N =8 ( Vegfr3 kd/kd ). Embryos are from 3 different litters. * p =0.019. b, Lyve1 whole mount immunostaining shows that ventral and dorsal sides of the heart are devoid of lymphatics in Vegfr3 kd/kd embryos. N = 3/genotype. c-f, Co-immunostaining using antibodies against cell proliferation markers (EdU, pH3, Ki67 and AuroraB) and antibodies against CM markers (cardiac Troponin C [cTnC], Prox1, αActinin and/or Mef2c) shows reduced CM proliferation in Vegfr3 kd/kd hearts compared to wild-type hearts at E17.5. Arrows indicate representative proliferating CMs. g, Quantification shows significantly reduced percentage of EdU+ and Ki67+ CMs and significantly reduce number of pH3+ and AuroraB+ CMs in Vegfr3 kd/kd hearts compared to controls. N = 4 embryos/genotype from 3 separate litters. ** p =0.005 (EdU), 0.001(Ki67, pH3), * p =0.02 (AuroraB). h, Active Caspase-3 immunostaining shows increased CM apoptosis (white arrows) in Vegfr3 kd/kd hearts compared to wild-type hearts at E17.5. Right panel is the quantitative data showing significantly increased percentage of active caspase-3+ CMs (Prox1+) in Vegfr3 kd/kd hearts compared to wild-types. N = 4 embryos/genotype from 3 separate litters. * p =0.032. i, Co-immunostaining with antibodies against Vimentin, PECAM1, CD68, Six2 and Hnf4α, together with EdU labeling shows comparable proliferation of cardiac fibroblasts, blood endothelial cells and macrophages, and of nephron progenitors and hepatocytes between wild-type and Vegfr3 kd/kd embryos at E17.5. White arrows indicate EdU+ proliferating cells. Quantification of the proliferation for each of those cell types is shown on the right panels. n.s. not significant. N = 3 embryos/genotype from 3 separate litters. Data are presented as mean ± S.E.M. p values were calculated by unpaired two-tailed Student’s t test. Scale bars, 1 mm (a), 500 μm (b), 25 μm (c-f, h), 25μm (i). Lower magnification images for panels c-e and h are included in .

Article Snippet: Primary antibodies were used as follows: α-Actinin (Mouse, Sigma, A7811, 1:500), cardiac Troponin C (Mouse, Abcam, ab8295, 1:1000), Ki67 (Rabbit, Invitrogen, SP6, MA5-14520, 1:200), active Caspase-3 (Rabbit, BD Pharmingen, C92- 605, 559565, 1:200), Lyve1 (Goat, R&D, AF2125, 1:200), Reelin (Goat, R&D, AF3820, 1:50), Lyve1 (Rabbit, AngioBio, 11-034, 1:500), Prox1 (Rabbit, AngioBio, 11002, 1:500), Prox1 (Goat, R&D, AF2727, 1:100) and Mef2c (Rabbit, LSBio, LSC356188, 1:1000).

Techniques: Immunostaining, Labeling, Two Tailed Test

Journal: Nature

Article Title: Lymphoangiocrine signals promote cardiac growth and repair

doi: 10.1038/s41586-020-2998-x

Figure Lengend Snippet: a, qPCR analysis shows reduced Reln expression in E17.5 Prox1 ΔLEC/ΔLEC hearts (TAM injected at E13.5 and E14.5). N = 3 embryos/genotype from the same litter. Control embryos are TAM treated Cre - embryos and Cre + ; Prox1 +/+ littermates. * p =0.014. b, qPCR analysis validates the expression of candidates from the LECs secretome ( SERPINE1, FN1, RELN, HSPG2, MMRN1, LAMA4, FSTL1 and THBS1 ). Experiments were repeated 3 times using different batches of LECs. Gene expression is normalized as a fold change relative to 100x Gapdh. c, Reelin protein can be detected in 3 different batches of LEC conditioned media and the relative Reelin level is quantified by ELISA according to the OD intensity. d-e, Immunostaining of sections of E17.5 WT hearts shows Reelin is highly expressed in cardiac lymphatics of the epicardium and myocardium. Some blood vessels in the heart express low levels of Reelin ( e , arrows). N = 3 WT embryos. f, Immunostaining of E17.5 control and Prox1 ΔLEC/ΔLEC heart sections with antibodies against Reelin and Lyve1 shows that cardiac lymphatics and Reelin are absent in Prox1 ΔLEC/ΔLEC hearts (TAM injected at E13.5 and E14.5). N = 3 embryos/genotype from the same litter. Control embryos are TAM treated littermate Cre - and Cre + ; Prox1 +/+ embryos. g, Representative bright field images show smaller hearts in E17.5 Reln −/− embryos. h , Quantifications of organ weight (heart, liver and kidney) relative to body length indicate that hearts are smaller in E17.5 Reln −/− embryos compared to controls. N = 7 (WT) and N =6 ( Reln −/− ) embryos from 3 separate litters. * p =0.03. i, Whole mount immunostaining shows that cardiac lymphatic development is normal in Reln −/− embryos. N = 3 embryos/genotype from 2 separate litters. Data are presented as mean ± S.E.M. p values were calculated by unpaired two-tailed Student’s t test. n.s, not significant. Scale bar, Scale bar, 25 μm (d, e, f), 1mm (g), 500 μm (i).

Article Snippet: Primary antibodies were used as follows: α-Actinin (Mouse, Sigma, A7811, 1:500), cardiac Troponin C (Mouse, Abcam, ab8295, 1:1000), Ki67 (Rabbit, Invitrogen, SP6, MA5-14520, 1:200), active Caspase-3 (Rabbit, BD Pharmingen, C92- 605, 559565, 1:200), Lyve1 (Goat, R&D, AF2125, 1:200), Reelin (Goat, R&D, AF3820, 1:50), Lyve1 (Rabbit, AngioBio, 11-034, 1:500), Prox1 (Rabbit, AngioBio, 11002, 1:500), Prox1 (Goat, R&D, AF2727, 1:100) and Mef2c (Rabbit, LSBio, LSC356188, 1:1000).

Techniques: Expressing, Injection, Enzyme-linked Immunosorbent Assay, Immunostaining, Two Tailed Test

Journal: Nature

Article Title: Lymphoangiocrine signals promote cardiac growth and repair

doi: 10.1038/s41586-020-2998-x

Figure Lengend Snippet: a , Immunostaining of E17.5 control and Reln ΔLEC/ΔLEC heart sections with antibodies against Reelin and Lyve1 confirms that Reelin is deleted from cardiac lymphatics in Reln ΔLEC/ΔLEC hearts (TAM injected at E13.5 and E14.5). N = 3 embryos/genotype from 2 separate litters. Control embryos are TAM treated Cre - embryos and Cre + ; Reln +/+ embryos. b, Co-immunostaining with antibodies against Vimentin, PECAM1, CD68, Six2 and Hnf4α, together with EdU labeling shows comparable proliferation of cardiac fibroblasts, blood endothelial cells and macrophages, and of nephron progenitors and hepatocytes between controls and E17.5 Reln ΔLEC/ΔLEC hearts (TAM injected at E13.5 and E14.5). White arrows indicate EdU+ proliferating cells. Quantification of the proliferation for each of those cell types is shown on the right panels. N = 3 embryos/genotype from 2 separate litters. Control embryos are TAM treated Cre - and Cre + ; Reln +/+ littermates. Data are presented as mean ± S.E.M. n.s. not significant difference by unpaired two-tailed Student’s t test. Scale bar, 25 μm.

Article Snippet: Primary antibodies were used as follows: α-Actinin (Mouse, Sigma, A7811, 1:500), cardiac Troponin C (Mouse, Abcam, ab8295, 1:1000), Ki67 (Rabbit, Invitrogen, SP6, MA5-14520, 1:200), active Caspase-3 (Rabbit, BD Pharmingen, C92- 605, 559565, 1:200), Lyve1 (Goat, R&D, AF2125, 1:200), Reelin (Goat, R&D, AF3820, 1:50), Lyve1 (Rabbit, AngioBio, 11-034, 1:500), Prox1 (Rabbit, AngioBio, 11002, 1:500), Prox1 (Goat, R&D, AF2727, 1:100) and Mef2c (Rabbit, LSBio, LSC356188, 1:1000).

Techniques: Immunostaining, Injection, Labeling, Two Tailed Test

Journal: Nature

Article Title: Lymphoangiocrine signals promote cardiac growth and repair

doi: 10.1038/s41586-020-2998-x

Figure Lengend Snippet: a, qPCR analysis shows efficient Reln knockdown in LECs after siRNA treatment. N =3. Mean ± S.E.M. * p < 0.05 by unpaired two-tailed Student’s t test. b, Representative Western blot of primary CMs cultured with DMEM, siCtrl and siReln treated conditioned media, or with conditioned media + Integrinβ1 blocking antibody o/n. Addition of the LEC conditioned media ( siCtrl group) to primary CMs increases Dab1, FAK, AKT and ERK activities. These activities are reduced when cultured CMs are treated with Reelin deficient LECs conditioned media or with LECs conditioned media with β1 blocking antibody. Experiments were repeated 3 times. Data are presented as mean ± S.E.M. * p < 0.05; ** p <0.01; *** p< 0.001 by two-way ANOVA followed by Bonferroni test. c , Ki67 quantification of immunostained cultured cells (similar to ) shows that addition of the LEC conditioned media ( siCtrl group) to cultured primary CMs improves CM proliferation and this effect is partially abolished in CMs treated with Reln (siReln) deficient LECs conditioned media or with LECs conditioned media containing β1 blocking antibody. Percentage of CM proliferation was quantified by the number of Ki67+ Prox1+ CMs relative to total numbers of Prox1+CMs. N =3. Mean ± S.E.M. ** p < 0.01 by two-way ANOVA followed by Bonferroni test. d, Quantification of active Caspase 3 immunostained cultured CMs shows that addition of the LEC conditioned media ( siCtrl group) to primary CMs protect them from apoptosis and this effect is partially abolished in CMs treated with Reln deficient LECs conditioned media or with LECs conditioned media with β1 blocking antibodies. Percentage of apoptotic CMs was quantified by the number of active Caspase 3+ CMs relative to Prox1+ CMs. N =3. Mean ± S.E.M. * p < 0.05; ** p <0.01 by two-way ANOVA followed by Bonferroni test. e, Representative Western blot of primary CMs after treatment with Reelin conditioned media from Reelin transfected cells (Reelin), or conditioned media from mock-transfected cells (control) or Reelin conditioned media with Integrin β1 blocking antibody (Reelin + β1 blocking ab) shows that Reelin treatment increases Dab1, FAK, AKT and ERK activities in primary CMs, and these activities are reduced by adding the Integrinβ1 blocking antibody. N =3. Data are presented as mean ± S.E.M. * p < 0.05; ** p <0.01 by one-way ANOVA followed by Tukey’s test. f , Bright field images show no difference in embryo size at E17.5 among control, Reln +/− , β1 ΔCM/+ and β1 ΔCM/+ ; Reln +/− embryos. Quantification of organ weight (heart, liver and kidney) relative to body length indicates that hearts are smaller in E17.5 β1 ΔCM/+ ; Reln +/− embryos. N =9 (control), N =7 ( Reln +/− ) , N= 6 ( β1 ΔCM/+ ) and N =6 ( β1 ΔCM/+ ; Reln +/− ) embryos from 3 separate litters. Data are presented as mean ± S.E.M. * p =0.015 by one-way ANOVA followed by Tukey’s test. n.s, not significant. g , Whole mount immunostaining using Lyve1 antibodies shows normal cardiac lymphatic development in control, β1 ΔCM/+ , β1 ΔCM/+ ; Reln +/− and Reln +/− embryos. N = 3 embryos/genotype from 3 separate litters. Scale bars, 1 mm (f), 500 μm (g). For western blot source data, see and . Exact p values included in Source Data.

Article Snippet: Primary antibodies were used as follows: α-Actinin (Mouse, Sigma, A7811, 1:500), cardiac Troponin C (Mouse, Abcam, ab8295, 1:1000), Ki67 (Rabbit, Invitrogen, SP6, MA5-14520, 1:200), active Caspase-3 (Rabbit, BD Pharmingen, C92- 605, 559565, 1:200), Lyve1 (Goat, R&D, AF2125, 1:200), Reelin (Goat, R&D, AF3820, 1:50), Lyve1 (Rabbit, AngioBio, 11-034, 1:500), Prox1 (Rabbit, AngioBio, 11002, 1:500), Prox1 (Goat, R&D, AF2727, 1:100) and Mef2c (Rabbit, LSBio, LSC356188, 1:1000).

Techniques: Two Tailed Test, Western Blot, Cell Culture, Blocking Assay, Transfection, Immunostaining

Journal: Nature

Article Title: Lymphoangiocrine signals promote cardiac growth and repair

doi: 10.1038/s41586-020-2998-x

Figure Lengend Snippet: a. Immunostaining with Reelin, Prox1 and PECAM shows Reelin is highly expressed in cardiac lymphatics in the epicardium and myocardium nearby the base of the heart at E17.5. Reelin expression level is gradually downregulated during development from P2 to P14. N = 3 hearts/stage. Arrows indicate Prox1+ cardiac lymphatics. b , qPCR analysis using sorted cardiac lymphatics shows Reln levels are drastically downregulated in cardiac LECs during development. N =3. Reln relative level from each experiment is presented as fold changes relative to E17.5. Data are presented as mean ± S.E.M. ** p =0.009 (P2 vs E17.5), 0.004 (P7 vs E17.5), 0.001 (P14 vs E17.5) by one-way ANOVA followed by Tukey’s test. c, Immunostaining shows Reelin expression is highly upregulated in the newly formed cardiac lymphatics in WT P7 pups (myocardial infarction was performed at P2). Notably, the pre-existing cardiac lymphatics in the non-infarcted area express low levels of Reelin. Reln −/− hearts are completely devoid of Reelin expression in both, newly formed cardiac lymphatics and pre-existing lymphatics. Arrows indicate cardiac lymphatics. N = 3 hearts/group. d , Immunostaining against the pan-endothelial marker PECAM1 and the lymphatic marker Lyve1 shows normal lymphangiogenesis in WT and Reln −/− hearts 21 days after MI (MI performed at P2). N = 3 hearts/group. Data are presented as mean ± S.E.M. n.s. not significant difference by unpaired two-tailed Student’s t test. e , EdU labeling shows no differences in LECs proliferation in WT and Reln −/− hearts 21 days after MI (MI performed at P2). N = 3 hearts/group. Data are presented as mean ± S.E.M. n.s. not significant difference by unpaired two-tailed Student’s t test. Arrow indicates EdU+ LECs. Scale bars, 100μm (d), 25μm (a,c,e).

Article Snippet: Primary antibodies were used as follows: α-Actinin (Mouse, Sigma, A7811, 1:500), cardiac Troponin C (Mouse, Abcam, ab8295, 1:1000), Ki67 (Rabbit, Invitrogen, SP6, MA5-14520, 1:200), active Caspase-3 (Rabbit, BD Pharmingen, C92- 605, 559565, 1:200), Lyve1 (Goat, R&D, AF2125, 1:200), Reelin (Goat, R&D, AF3820, 1:50), Lyve1 (Rabbit, AngioBio, 11-034, 1:500), Prox1 (Rabbit, AngioBio, 11002, 1:500), Prox1 (Goat, R&D, AF2727, 1:100) and Mef2c (Rabbit, LSBio, LSC356188, 1:1000).

Techniques: Immunostaining, Expressing, Marker, Two Tailed Test, Labeling