Double UP: A Dual Color, Internally Controlled Platform for in utero Knockdown or Overexpression

Figures

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  FIGURE 1. Construction and characterization of Double UP. Double UP generates equivalent populations of red and green neurons within an individual slice. (A) Schematic of Double UP construct. Representative IUE of 2 μg/μL Double UP without Cre (B) and with 15 ng/μL pCAG-Cre (B’). Embryos were electroporated at E14.5 and allowed 3 days to mature (E14.5+3). Scale bar, 100 μm. (C) Comparison of migration of mNeon-Green- and mScarlet-positive cells, E14.5+3. Each dot represents the mean distance of all cells within one cortical slice to the top of the cortical plate. Connected dots indicate measures from the same slice (n = 61 slices). Large dots and black line indicate mean and SD of all 61 slices. No significant difference was detected between green and red populations (two-way ANOVA). Full data shown in Supplementary Figure S1. (D) Comparison of reliability of controls between section matching and Double UP. 76 comparisons were made for section matching and 61 comparisons were made for Double UP. Only sections that had a perfect match in another brain were included for either analysis. ****p < 0.0001, Kolmogorov–Smirnov t-test, two-tailed. Lines indicate mean and SD. See also Supplementary…

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  FIGURE 2. Automated tracking of cellular location with TRON program. Example of data analysis using the TRON program. (A) Example input. 12, three-color confocal slices from a 100 μm coronal brain slice from an E14.5+3 embryo after IUE. (B) Green and (B’) red colors, processed independently and automatically via: unsharp mask, gaussian blur, conversion to 8-bit, automatic threshold, erode, watershed. (C) Manual drawing of regions of interest (ROIs—red lines at top of cortical plate and ventricle). This is the only manual step in the process. (D,D’) Output of modified 3D Object Counter plugin from Fiji. Center of mass of each cell is identified and mapped; X, Y location and brightness of original is obtained and recorded. (E) Tracings of the top of the cortical plate and ventricle (ROIs). (F) Location of each cell mapped onto the ROIs. Distance from the center of each cell to the nearest point on each ROI is measured and recorded. (G) Graphical representation of distance each cell is from top of the cortical plate. Cells located above the top of the cortical plate are treated as having a “negative” distance. Lines indicate mean and SD. (H) Distance between ROIs can be split into bins,…

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  FIGURE 3. Double UP is not “leaky.” Three tests to determine if green cells express any red protein or associated manipulation. (A) High power image of two adjacent neurons, one green and one red, taken with a 63x/1.4NA oil objective. In the green neuron (solid oval), no signal is visible in the red channel. In the red neuron (dashed oval), a faint signal is visible in the green channel. (B) Number of green and red cells of greater than 1000 units of brightness per slice in Double UP +Cre (three slices, mean = 425 green/320 red cells), Double UP No Cre (four slices, mean = 382 green/0.25 red cells), and Double UP +ERT2-Cre-ERT2 (three slices, mean = 370 green/three red cells). Error bars indicate SEM. All images acquired with identical settings. (C) Quantification of the brightness in the red channel of red neurons from 90 neurons across three brains for: red neurons from Double UP +Cre, as well as green neurons from brains having received Double UP without Cre and Double UP +CreER. Dot above box and whiskers in “Green Cells” column with No Cre indicate an outlier. All other points are contained within the whiskers (Tukey). Boxes are 25/75%, median is also shown. (D) Schematic of Double…

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  FIGURE 4. Double UP provides overexpression with an internal control. Robust replication of previously discovered migration defects utilizing Double UP. (A) Schematic of Double UP Rac1-V12. (B) Schematic of Double UP Rac1-N17. (C) Representative image of Double UP Rac1-V12, E14.5+4. Scale bar, 100 μm. (D) Representative image of Double UP Rac1-N17 E14.5+4. (E) Dot plot of three representative slices from Double UP, Double UP Rac1-V12, and Double UP Rac1-N17. Each dot represents distance from the top of the cortical plate to the center of mass for each neuron. (F) Dot plot of Double UP, Double UP Rac1-V12, and Double UP Rac1-N17 (n = 6, 4, and 5 slices, respectively, each from a different brain). Each dot represents mean distance from the top of the cortical plate to the distance of all cortical neurons in a slice. Connected dots indicate measurements were made in the same brain. ns = not significant, ****p < 0.0001, two-way ANOVA. Error bars not shown for clarity. See also Supplementary Figure S2.

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  FIGURE 5. Double UP combined with pSico provides knockdown with an internal control. Robust replication of previously discovered migration defects, utilizing Double UP and pSico. (A) Representative image of IUE with pSico RapGEF2 (2 μg/μL), Double UP (1 μg/μL), and pCAG-Cre (15 ng/μL). Scale bar: 100 μm. (B) Representative image of IUE with pSuper RapGEF2 (2 μg/μL) and Double UP (1 μg/μL). (C) Dot plot of Double UP and either pSico Scrambled (pSico Scramb), pSico RapGEF2 (pSico RG2), and pSuper RapGEF2 (pSuper RG2) (n = 5, 8, and 5 slices, respectively, each from a different brain). Each dot represents mean distance from the top of the cortical plate to all cortical neurons in a slice. Connected dots indicate measurements were made in the same brain. ns = not significant, ****p < 0.0001. Comparisons within samples (black bars) were two-way ANOVA; comparison between pSuper RapGEF2 and the red cells of pSico RapGEF2 (red bars) were performed with a Student’s t-test. Error bars not shown for clarity. See also Supplementary Figure S3.