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. 2022 Aug 5;119(32):e2116956119. doi: 10.1073/pnas.2116956119

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Copyright © 2022 the Author(s). Published by PNAS.

This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

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Fig. 1. — Postmitotic accumulation of H3.3 in cortical neurons and genetic manipulation of H3f3a and H3f3b. (A) snRNA-seq of wild-type E14.5 cortex visualized by Uniform Manifold Approximation and Projection (UMAP). H3f3a and H3f3b mRNA expression was found in each of the 19 identified cell clusters and in 98.0% and 98.2% of cells, respectively. (B) Analysis of H3.3 and pan-H3 protein in cortical development by immunostaining. On E14.5 and E16.5, low levels of H3.3 (green) were present in the VZ and SVZ, composed largely of cycling NPCs. High levels of H3.3 were present in the CP, composed largely of postmitotic neurons. In E16.5 CP and P0 cortex, higher levels of H3.3 were present in the early-born neurons of the SP and deep layers, compared to the late-born neurons of the upper layers (open arrowheads). Pan-H3 (red) was broadly present and showed no preference for early-born neurons; high levels were present in late-born upper-layer neurons (solid arrowhead). (C) Temporal analysis of H3.3 accumulation. Neurons born on E13.5 were labeled by a single pulse of EdU. EdU-labeled neurons (magenta, arrowheads) were analyzed by H3.3 immunostaining (green). (D) Quantitative analysis of immunofluorescence revealed a progressive increase in H3.3 levels over the first postmitotic days (one-way ANOVA with Tukey’s post hoc test, n.s., not significant; *P < 0.01; ***P < 0.0001). (E) A schematic of H3.1/H3.2 and H3.3 accumulation, and genetic manipulation of H3.3-encoding genes H3f3a and H3f3b. (F) Analysis of Cre expression (yellow) by immunostaining confirmed the published specificities of Neurod6^Cre in newly postmitotic neurons and of Emx1^Cre in NPCs at E14.5. (G) Analysis of H3 proteins in P0 control (ctrl) and conditional H3.3 gene double knockouts, Neurod6^Cre;H3f3a^f/f;H3f3b^f/f (dKO-N) and Emx1^Cre;H3f3a^f/f;H3f3b^f/f (dKO-E). In both dKO-N and dKO-E, H3.3 (green) was largely lost from neocortex (Nctx) but preserved in caudate putamen (CPu) and septum (Sep). The levels of pan-H3 (red) were unaffected in dKO-N or dKO-E. CIN, cortical interneuron; CR, Cajal-Retzius cell; CPN, cortical plate neuron; dCP, deep cortical plate; IPC, intermediate progenitor cell; IZ, intermediate zone; Ln, layer n; MZ, marginal zone; RGC, radial glial cell; SPN, subplate neuron; uCP, upper cortical plate.