Extended Data Fig. 12: CellREADR vector targeting of neuron types in human cortical ex vivo tissues.
From: Programmable RNA sensing for cell monitoring and manipulation
a, Schematic of organotypic platform for of human cortical ex vivo tissues. Left panel was adapted from “Brain (lateral view)”, by BioRender.com (2022). Retrieved from https://app.biorender.com/biorender-templates. b, Schematic of a hSyn-eGFP viral construct used to drive widespread neuronal cell labeling. c. AAVrg-hSyn-eGFP labeled cells were distributed across all layers, and exhibited diverse morphologies (c1). Insets from c1 (c2) and c2 (c3, 4) depict numerous cells with pyramidal morphologies, including prominent vertically oriented apical dendrites. d, FOXP2 expression in human neocortex. FOXP2 mRNA expression pattern taken from the Allen Institute human brain-map (specimen# 4312), showing upper and deep layer expression (arrows) (d1). FOXP2 immunostaining in the current study (magenta) also demonstrated both upper and deep labeling (d2, 4). NeuN immunostaining (red) depicting cortical neurons (d3, 4). Dashed lines delineate pia and white matter. e, READRFOXP2(1) labeling in an organotypic slice derived from the same tissue used in d. Overview of bright field and mNeon native fluorescence in the organotypic slice demonstrating highly restricted labeling, as compared to that observed in d. Inset from e2 (e3) depicting morphologies of upper layer pyramidal neurons. f, Schematics of two singular vectors of READRFOXP2. In READRFOXP2(1), the hSyn promoter drives an expression cassette encoding ClipF, sesRNA1, smV5, and tTA2. In READRFOXP2(2), the hSyn promoter drives an expression cassette encoding ClipF, sesRNA2, smFlag, and FlpO. g–h, Seven days after application of READRFOXP2(2) AAV on DIV 1, tissue was fixed and stained with antibodies against FOXP2 and FLAG (g). h, Boxed region from g. FLAG-labeled cells from READRFOXP2(2) exhibited relatively small somata with short apical dendrites (h1–h3, arrowheads). Non-specific background fluorescence signals (e.g. a blood vessel-like profile) are indicated by thin arrows (h2). i, Quantification of CellREADR specificity measured as the percentage of V5+ cells (for READRFOXP2(1)) and FLAG+ cells (for READRFOXP2(2)) labeled by FOXP2 immunostaining, respectively. Each bar in i is the value from one human brain sample performed (n = 1).
