Journal: bioRxiv
Article Title: R-loop landscapes in the developing human brain are linked to neural differentiation and cell-type specific transcription
doi: 10.1101/2023.07.18.549494
Figure Lengend Snippet: Expression of the RNASEH1 ΔMLS transgene, delivered via in utero electroporation into neural precursor cells of the ventricular zone in E15 mouse embryos, affects dendrite and spine morphology of cortical projection neurons. A) Expression plasmids of GFP combined with tdTomato are used as controls, while GFP combined with RNASEH1 ΔMLS are used to destroy neural R-loops in mice during prenatal development. B) Overview of the in utero electroporation technique. TdTomato or RH1ΔMLS along with GFP reporters are transfected and electroporated unilaterally in E15 mouse ventricular zone. In adult mouse, P28, strong unilateral GFP expression is observed. Scale bar = 1 mm. C) In utero electroporation experimental timeline. D) Expression of exogenous (human) RNASEH1 in RH1ΔMLS mice was confirmed in FAC-sorted GFP+ neurons, whereas no difference was observed for endogenous expression of (mouse) Rnaseh1 between groups via qPCR (Normalized to Gapdh expression). Student’s two-tailed t test; n.s. = not significant. E) Expression of human RH1ΔMLS reduces S9.6 immunoreactivity in the nucleus of mouse neural cells at P0. F) FlSH images showing RNA expression of RNASEH1 (red) in the P28 cortex of tdTomato controls and RH1ΔMLS mice, and colocalization of GFP (green) and RNASEH1 in RH1ΔMLS cells. Scale bar = 100 mm. G) Representative images of (left) control and (right) RH1ΔMLS-expressing frontal pyramidal neuron dendritic trees at P28. H) Sholl analysis, showing reduced dendritic complexity in (left) control and (right) RH1ΔMLS-expressing pyramidal neurons at P28 (tdTomato control n = 3, RH1ΔMLS n = 4). I) Representative images of (left) control and (right) RH1ΔMLS frontal pyramidal neuron dendritic spines at P28. J) Apical and basal spine densities, assessed by Imaris tracing + FOCM (see text), are significantly decreased in RH1ΔMLS-expressing prefrontal pyramidal neurons. N = 3/group. *P < 0.05, **P < 0.01 determined by Two-Tailed Student’s t-test.
Article Snippet: To analyze the complexity of each neuron, Sholl analysis was performed via the Filament Sholl Analysis XTension for Imaris with Sholl sphere radius set to 20 μm with a maximum of 30 spheres.
Techniques: Expressing, In Utero, Electroporation, Transfection, Two Tailed Test, RNA Expression, Control
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