Journal: Journal of Clinical Investigation

Article Title: A systems biology approach identifies the role of dysregulated PRDM6 in the development of hypertension

doi: 10.1172/jci160036

Figure Lengend Snippet: Figure 9. Fate mapping of neural crest PRDM6. (A) ZsGreen1 and renin-expressing cells in the kidneys of Prdm6fl/fl Wnt1-Cre and WT littermates. The plot on the right shows the quantification of ZsGreen1 (NCC) and renin-positive cells. (B) Augmented part of panel A demonstrates the close proximity of ZsGreen1 and renin-expressing cells on the vascular wall of in Prdm6fl/fl Wnt1-Cre kidneys marked by arrowheads. Arrows show renin. (C) The localization of Sox6 in relationship to αSMA and ZsGreen1positive cells (NCC) in Prdm6fl/fl Wnt1-Cre kidneys and WT littermates. Scale bars: 20 μm. Unpaired 2-tailed t test; *P < 0.05. n = 6 mice per group in A–C.

Article Snippet: Cg-E2f1Tg(Wnt1-cre)2Sor/J) and ZsGreen1 (Rosa-CAG-LSL-ZsGreen1-WPRE, Jax 007906) were purchased from The Jackson Laboratory.

Techniques: Expressing

Journal: Neuroscience

Article Title: A Transgenic Mouse Model to Selectively Identify α 3 Na,K-ATPase Expressing Cells in the Nervous System

doi: 10.1016/j.neuroscience.2018.07.018

Figure Lengend Snippet: (A) Representative florescent image of a frozen brain section (60 μm) obtained from a Atp1a3-ZsGreen1 male mouse (approximate Allen’s Mouse Brain Reference Atlas (AMB) level 75). Brain regions of interests (ROI) have been outlined using the Fiji Image J ROI Manager tool and are based on the AMB. (B)Fluorescent signal remaining after subtraction of the background intensity as calculated by the rolling ball method (see Experimental Procedures for more details). (C) Intensity of labeling of ROIs shown in panels A and B (sorted by brain region and then by intensity). Abbreviations (order as in Panel C): White matter tracts: fi - fimbria, st - stria terminalis, cpd - cerebellar peduncle, fr - fasciculus retroflexus; Isocortex: AUD- auditory cortex, TEa-ECT-PERI - temporal association area and perirhinal and entorhinal cortical areas, SSp - primary somato-sensory cortex; Olfactory areas: PIR - piriform area, PAA - piriform amygdalar area, COA - cortical amygdalar area, posterior part; Retrohippocampal region and hippocampus: ENTI - entorhinal cortical area, lateral part, CA - Ammon’s horn; DG - dentate gyrus; Cortical subplate - CTXsp; Cerebral nuclei: CP - caudate putamen, MEApv - medial amygdalar nucleus, posteroventral part, MEApd - medial amygdalar nucleus, posterodorsal part; Thalamus and Epithalamus: PO - posterior complex of the thalamus, LGd - lateral geniculate complex, dorsal part, VP - ventral posterior complex of the thalamus, LP - lateral posterior nucleus, RT - reticular nucleus; LGv - lateral geniculate complex, ventral part, MT - medial thalamic nuclei, LH - lateral habenula; VM- ventral medial nucleus, PF - parafascicular nucleus; Hypothalamus: STN - subthalamic nucleus; HY- hypothalamus major proper; Zl- zona inserta

Article Snippet: Atpla3-ZsGreen1 plasmid construction A portion of the mouse Atp1a3 gene (promoter and entire 5’-untranslated region) linked to the expression cassette from pZsGreen1–1 (Clontech, Palo Alto, CA, USA) was synthesized and cloned into the EcoRV site of pUC57 by GenScript (Piscataway, NJ, USA) to generate the Atp1a3-ZsGreen1 plasmid.

Techniques: Labeling

Journal: Neuroscience

Article Title: A Transgenic Mouse Model to Selectively Identify α 3 Na,K-ATPase Expressing Cells in the Nervous System

doi: 10.1016/j.neuroscience.2018.07.018

Figure Lengend Snippet: (A)Gel picture of RT-PCR results from Tg and WT mice at the indicated postnatal days (P) of age. Each lane represents a unique animal except for the no template (H2O) control. The 18S rRNA product was used as a reference for normalization. Both the Atp1a3-ZsGreen1 transgene and endogenous Atp1a3 gene were expressed well in brain, but not in liver. (B) Plot of the mean level of expression for Atp1 a3- ZsGreen1 and Atp1a3 relative to 18S rRNA in brain during development as determined by measuring the band intensities in Panel A. Numbers next to Atp1a3 data points indicate sample size (i.e. number of animals analyzed). Both the transgene and endogenous Atp1a3 gene demonstrate a similar pattern of temporal expression in brain. (C) Plot of the band intensities between Atp1a3-ZsGreen1 and Atp1a3 signals for each animal in Panel A. Shown is a linear regression (solid line) of the data obtained from transgenic mice (filled circles) flanked by the 95% confidence limits (dotted lines). Open circles indicate data obtained with non-transgenic (WT) mice.

Article Snippet: Atpla3-ZsGreen1 plasmid construction A portion of the mouse Atp1a3 gene (promoter and entire 5’-untranslated region) linked to the expression cassette from pZsGreen1–1 (Clontech, Palo Alto, CA, USA) was synthesized and cloned into the EcoRV site of pUC57 by GenScript (Piscataway, NJ, USA) to generate the Atp1a3-ZsGreen1 plasmid.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transgenic Assay

Journal: Neuroscience

Article Title: A Transgenic Mouse Model to Selectively Identify α 3 Na,K-ATPase Expressing Cells in the Nervous System

doi: 10.1016/j.neuroscience.2018.07.018

Figure Lengend Snippet: (A, B) Image captured at 5× magnification of frozen sections (30 μm) from liver of an Atp1a3-ZsGreen1 Tg mouse. Panel A represents the original image and Panel B is the same image after performing rolling ball background subtraction. (C, D) Image of the cerebellum from the same Tg mouse as in Panels A and B. Panel C represents the original image, while the image in Panel D is after rolling ball background subtraction. (E-H) Images of cerebellum captured at 2.5× magnification of a brain slice (60 μm) obtained from an Atp1a3-ZsGreen1 Tg mouse (E, F) and a non-transgenic littermate (G, H) cut at approximately the same sagittal level. Panels E and G are original images and Panels F and H represent the corresponding images after rolling ball background subtraction. Labels: DCN - deep cerebellar nucleus, PJ - Purkinje cell layer, wt - white matter tracts. The scale bar in Panel A represents 2 mm and is applicable for Panels A-D. The scale bar in Panel E represents 1 mm and is applicable for Panels E-H.

Article Snippet: Atpla3-ZsGreen1 plasmid construction A portion of the mouse Atp1a3 gene (promoter and entire 5’-untranslated region) linked to the expression cassette from pZsGreen1–1 (Clontech, Palo Alto, CA, USA) was synthesized and cloned into the EcoRV site of pUC57 by GenScript (Piscataway, NJ, USA) to generate the Atp1a3-ZsGreen1 plasmid.

Techniques: Slice Preparation, Transgenic Assay

Journal: Neuroscience

Article Title: A Transgenic Mouse Model to Selectively Identify α 3 Na,K-ATPase Expressing Cells in the Nervous System

doi: 10.1016/j.neuroscience.2018.07.018

Figure Lengend Snippet: The image was taken at 20× magnification and does not show any co-localization of ZsGreen1 fluorescence (green) with immunostaining for GFAP (red) in the dentate gyrus (A), deep cerebellar nucleus (B), or parabigeminal nucleus (C). The scale bar (100 μm) is indicated in Panel C, which is the same for all images. DAPI staining (blue) in Panel A specifies cell nuclei.

Article Snippet: Atpla3-ZsGreen1 plasmid construction A portion of the mouse Atp1a3 gene (promoter and entire 5’-untranslated region) linked to the expression cassette from pZsGreen1–1 (Clontech, Palo Alto, CA, USA) was synthesized and cloned into the EcoRV site of pUC57 by GenScript (Piscataway, NJ, USA) to generate the Atp1a3-ZsGreen1 plasmid.

Techniques: Fluorescence, Immunostaining, Staining

Journal: Neuroscience

Article Title: A Transgenic Mouse Model to Selectively Identify α 3 Na,K-ATPase Expressing Cells in the Nervous System

doi: 10.1016/j.neuroscience.2018.07.018

Figure Lengend Snippet: (A) Schematic illustration of coronal frozen sections (60 μm) isolated from the brains of Atp1a3- ZsGreen1 mice at 3 to 3.5 months of age (n = 3 per sex). Arrows indicate the relative location of the brain slices. At least three sections per mouse were analyzed for fluorescence in subcortical structures and major brain regions. (B) ZsGreen1 fluorescence in subcortical areas (gray shaded areas in the illustration on the left side). Mean pixel intensity indicates the average amount after rolling ball background subtraction of the combined area(s) as noted. Brain regions according to the AMB atlas: cerebral nuclei (CN), hypothalamus (HY), medulla (MY), midbrain (MB), pons (P), thalamus (TH). (C) ZsGreen1 fluorescence in major brain sub-regions (gray shaded areas depicted in the illustration to the left) according to the procedure used in Panel B, except mean pixel intensity is plotted against a particular brain sub-region. The level of the AMB atlas selected was based on being most representative for each brain region (e.g. AMB atlas coronal layers 85–93 for MB and 61–69 for TH). Additional abbreviations: cerebellum (CB), cortical subplate (CTXsp), hippocampal formation (HIF), isocortex (ISO), midbrain (MB), olfactory-related cortical area (OLF), pallidum (PAL), striatum (STR).

Article Snippet: Atpla3-ZsGreen1 plasmid construction A portion of the mouse Atp1a3 gene (promoter and entire 5’-untranslated region) linked to the expression cassette from pZsGreen1–1 (Clontech, Palo Alto, CA, USA) was synthesized and cloned into the EcoRV site of pUC57 by GenScript (Piscataway, NJ, USA) to generate the Atp1a3-ZsGreen1 plasmid.

Techniques: Isolation, Fluorescence

Journal: Neuroscience

Article Title: A Transgenic Mouse Model to Selectively Identify α 3 Na,K-ATPase Expressing Cells in the Nervous System

doi: 10.1016/j.neuroscience.2018.07.018

Figure Lengend Snippet: Effect of rostro-caudal subcortical region position on the intensity of ZsGreen1 labeling *

Article Snippet: Atpla3-ZsGreen1 plasmid construction A portion of the mouse Atp1a3 gene (promoter and entire 5’-untranslated region) linked to the expression cassette from pZsGreen1–1 (Clontech, Palo Alto, CA, USA) was synthesized and cloned into the EcoRV site of pUC57 by GenScript (Piscataway, NJ, USA) to generate the Atp1a3-ZsGreen1 plasmid.

Techniques: Labeling

Journal: Neuroscience

Article Title: A Transgenic Mouse Model to Selectively Identify α 3 Na,K-ATPase Expressing Cells in the Nervous System

doi: 10.1016/j.neuroscience.2018.07.018

Figure Lengend Snippet: Effect of brain region on the intensity of ZsGreen1 labeling *

Article Snippet: Atpla3-ZsGreen1 plasmid construction A portion of the mouse Atp1a3 gene (promoter and entire 5’-untranslated region) linked to the expression cassette from pZsGreen1–1 (Clontech, Palo Alto, CA, USA) was synthesized and cloned into the EcoRV site of pUC57 by GenScript (Piscataway, NJ, USA) to generate the Atp1a3-ZsGreen1 plasmid.

Techniques: Labeling

Journal: Neuroscience

Article Title: A Transgenic Mouse Model to Selectively Identify α 3 Na,K-ATPase Expressing Cells in the Nervous System

doi: 10.1016/j.neuroscience.2018.07.018

Figure Lengend Snippet: (A) pallidum/striatum, (B) thalamus and epithalamus, (C) hypothalamus and cerebellum, (D) midbrain, (E) pons, (F) medulla. Each column represents the mean level of fluorescence per specified sub-region studied in sections from 4 to 6 animals. Sub-regions were based on the presence of at least one section per brain in which the borders of the nuclei could be reliably determined. Nuclei whose borders could not be established due to its relative small size were not studied or were studied as a part of a larger CNS subdivision. For example, most hypothalamic nuclei except for zona inserta (Zl), supramammillary (SUM), medial mammillary (MM) and subthalamic (STN) nuclei were studied by grouping them into a single hypothalamic (HY) region of interest. Results are presented as mean pixel intensity (± SD) from coronal sections (60 μm) according to the procedure described in Fig. 1. Horizontal dashed lines represent mean + 3SD pixel intensity value (4.027 shades of gray) measured in 18 randomly selected white matter regions (such as corpus callosum, fimbria or cerebellar peduncle, see Fig. 1; 3 ROI per brain) having only background levels of fluorescence. Thus, brain nuclei with a mean intensity exceeding this value are considered to have specific fluorescence due to expression of ZsGreen1 protein with more than 99% certainty. See Table 1 for a complete list of abbreviations for brain sub-regions.

Article Snippet: Atpla3-ZsGreen1 plasmid construction A portion of the mouse Atp1a3 gene (promoter and entire 5’-untranslated region) linked to the expression cassette from pZsGreen1–1 (Clontech, Palo Alto, CA, USA) was synthesized and cloned into the EcoRV site of pUC57 by GenScript (Piscataway, NJ, USA) to generate the Atp1a3-ZsGreen1 plasmid.

Techniques: Fluorescence, Expressing

Journal: Neuroscience

Article Title: A Transgenic Mouse Model to Selectively Identify α 3 Na,K-ATPase Expressing Cells in the Nervous System

doi: 10.1016/j.neuroscience.2018.07.018

Figure Lengend Snippet: (A) Low power image of a frozen sagittal section (60 μm) at AMB atlas sagittal level 10. The rectangle specifies the hippocampal region, which is shown at higher (5× magnification in Panel B. Designated structures: interposed nucleus (IP), parabigeminal nucleus (PBG), substantia nigra (SNR), zona incerta (Zl), globus paiidus external part (GPe) and substantia innominate (SI). (B) Enlarged boxed area from Panel A containing the hippocampal region. Designated structures: molecular (mo) and polymorphic (po) layers of the dentate gyrus; hippocampal lacunosum-moleculare (slm), radiatum (sr) and oriens (so) layers; subiculum (SUB) and postsubiculum (POST). Arrows point to three ZsCreen1-positive cells in the pyramidal layer region which are shown at higher (10x) magnification in Panel C. (C) The majority of the neurons within the hippocampal pyramidal layer are ZsGreen1 -negative, with a few exceptions (arrows indicate ZsGreen1 fluorescent cells).

Article Snippet: Atpla3-ZsGreen1 plasmid construction A portion of the mouse Atp1a3 gene (promoter and entire 5’-untranslated region) linked to the expression cassette from pZsGreen1–1 (Clontech, Palo Alto, CA, USA) was synthesized and cloned into the EcoRV site of pUC57 by GenScript (Piscataway, NJ, USA) to generate the Atp1a3-ZsGreen1 plasmid.

Techniques:

Journal: Neuroscience

Article Title: A Transgenic Mouse Model to Selectively Identify α 3 Na,K-ATPase Expressing Cells in the Nervous System

doi: 10.1016/j.neuroscience.2018.07.018

Figure Lengend Snippet: (A) Low power image of a frozen section (60 μm; scale bar 0.5 mm) of cerebellum (AMB atlas sagittal layer 20–21) stained with antibodies against calbindin to identify Purkinje (PJ) neurons. Roman numerals indicate the different cerebellar lobules. (B) Image of lobule II at higher (20×) magnification (scale bar for images B-D is 100 μm, as depicted in Panel D). The granular (gr) and molecular (mo) layers of cerebellum are indicated. A semi-transparent selection line has been drawn over a portion of the PJ cell layer towards the right side of Panel B to illustrate how the area was traced in order to determine the average pixel intensity and pixel intensity profile within a given lobule. (C) Overlay of images for lobule II. Green indicates ZsGreen1-positive cells, while red indicates cells that express calbindin. Cells expressing both ZsGreen1 and calbindin are yellow. (D) Image from Panel C after Sharpen and Find Edges pre-processing and rolling ball background subtraction protocols in Image J. Arrows in Panels C and D indicate ZsGreen1 -negative PJ neurons.(E) Linear ZsGreen1 labeling intensity profile in the PJ cell layer for all of lobule II. Horizontal dashed line indicates arbitrary cut-off value (≥10 pixel intensity units) used to denote strongly labeled PJ neurons. (F) Mean pixel intensity for the PJ cell layer within different cerebellar lobules (n = 3 animals). (G) Counts (number per PJ cell layer length in mm) of strongly labeled PJ neurons in the different cerebellar lobules. (H) Percentage of ZsGreen1-negative neurons within the PJ cell layer of a given lobule. Asterisk indicates a significant difference (p<0.05) between the amount of ZsGreen1 -negative cells in lobule VII compared with other lobules.

Article Snippet: Atpla3-ZsGreen1 plasmid construction A portion of the mouse Atp1a3 gene (promoter and entire 5’-untranslated region) linked to the expression cassette from pZsGreen1–1 (Clontech, Palo Alto, CA, USA) was synthesized and cloned into the EcoRV site of pUC57 by GenScript (Piscataway, NJ, USA) to generate the Atp1a3-ZsGreen1 plasmid.

Techniques: Staining, Selection, Expressing, Labeling

Journal: Neuroscience

Article Title: A Transgenic Mouse Model to Selectively Identify α 3 Na,K-ATPase Expressing Cells in the Nervous System

doi: 10.1016/j.neuroscience.2018.07.018

Figure Lengend Snippet: (A) ZsGreen1-positive neuron and its response to a depolarizing current ramp. (B) ZsGreen1-negative interneuron from the same animal, and its response to a depolarizing current ramp. Scale bars for all images are 25 μm. Scale bars for current command and voltage response (vertical bars) traces are 30 pA and 20 mV, respectively. Time scale (horizontal bars) is 200 ms.

Article Snippet: Atpla3-ZsGreen1 plasmid construction A portion of the mouse Atp1a3 gene (promoter and entire 5’-untranslated region) linked to the expression cassette from pZsGreen1–1 (Clontech, Palo Alto, CA, USA) was synthesized and cloned into the EcoRV site of pUC57 by GenScript (Piscataway, NJ, USA) to generate the Atp1a3-ZsGreen1 plasmid.

Techniques: