Endogenous Circadian Clock Machinery in Cortical NG2-Glia Regulates Cellular Proliferation

Terry Dean; Aissia Victoria Koffi; Evan Goldstein; Javid Ghaemmaghami; Vittorio Gallo

doi:10.1523/ENEURO.0110-22.2022

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Figure 1.
Circadian clock gene expression, including Bmal1, oscillate in NG2-glia. A, Bar graph depicting normalized read counts of circadian clock gene transcripts isolated via TRAP-seq from Pdgfra-TRAP mice. Samples were collected at P18, P22, P30, and P45. Each dot is representative of one animal. (mean ± SD). B, Graphs depicting relative expression, via qPCR, of circadian clock gene transcripts (Bmal1, left; Per1, middle; Nr1d2, right) isolated from Pdgfra-TRAP mice throughout several times of day (CT 0200, 0600, 1000, 1400, 1800, and 2200). Each dot is representative of one animal (n = 21 over six groups). The p-values from RAIN^a. R² is from goodness of fit to depicted sine wave for illustrative purpose^b. C, Representative images of RNA ISH of Bmal1 (magenta) and Cspg4 (green) with nuclear staining (DAPI, blue) at CT 0000 and 1200 in somatosensory cortex. Cspg4⁺ cells are denoted with solid arrowheads (▴). Scale bar, 10 μm. D, Graph depicting quantification of Bmal1 RNA ISH in cortical Cspg4⁺ cells at CT 0000 and CT 1200 in somatosensory cortex. N = 3 animals/condition (68 and 77 cells, respectively). The p-value is from the Kolmogorov–Smirnov test^c. E, Graph depicting quantification of Per2 RNA ISH in cortical Cspg4⁺ cells at CT 0000 and CT 1200 in somatosensory cortex. N = 3 animals/condition (134 and 81 cells, respectively). The p-value is from the Kolmogorov–Smirnov test^c. F, Representative images of immunofluorescence for nuclear BMAL1 (gray) in OLIG2⁺ (magenta) PDGFRA⁺ (green) NG2-glia in somatosensory cortex. Nuclei are stained with DAPI (red). NG2-glia nuclei are indicated with solid arrowheads (▴). Scale bar, 10 μm. G, Graph depicting quantification of peak intensity of nuclear BMAL1 immunofluorescence in Pdgfra-expressing NG2-glia from somatosensory cortex at several times of day (CT 0200, 0600, 1000, 1400, 1800, and 2200). N = 2–3 animals/time point (73 nuclei total). Each dot is representative of one nucleus. The p-value is from RAIN^d. R² is from the goodness-of-fit to depicted sine wave for illustrative purpose^e.
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Figure 2.
Circadian oscillation of Bmal1 regulates NG2-glia proliferation in naive cortex. A, Representative images of immunofluorescence for cortical NG2-glia from somatosensory cortex at CT 0000 and CT 1200. NG2-glia from Pdgfra-TRAP mice identified as ramified NG2⁺ GFP⁺ cells, with proliferative cells indicated by Ki67 staining (magenta). Proliferative single NG2-glia are indicated by a solid arrowhead (▴), and doublets are indicated by a caret (^). Scale bar, 50 μm. B, Graph depicting quantification of OLIG2⁺ Ki67⁺ density in somatosensory cortex of C57BL/6 mice at several times of day (CT 0200, 0600, 1000, 1400, 1800, and 2200). N = 4–8 animals/time point (31 total). Each dot is representative of one animal. The p-value is from RAIN^f. R² is from the goodness of fit to depicted sine wave for illustrative purpose^g. C, Mating scheme and experimental timeline for Bmal1 CKO experiments. D, Representative images of immunofluorescence for BMAL1 in CKO mice (Pdgfra-creERT:Bmal1^fl/fl:R26R-EYFP), indicated by colabeling for EYFP reporter (magenta), DAPI (blue), and BMAL1 (green). Nuclei from recombined NG2-glia with EYFP reporter, but lacking BMAL1 staining, are indicated by a solid arrowhead (▴). Scale bar, 10 μm. E, Representative images of immunofluorescence for NG2-glia, indicated by colabeling for NG2 (magenta) and OLIG2 (green) from cortex of Pdgfra-creERT:Bmal1^fl/fl CKO and Ctl Pdgfra-creERT:Bmal1^+/+ mice 3 weeks following tamoxifen injection. Samples collected at CT 0000. Scale bar, 20 μm. F, Graph depicting quantification of NG2-glia (NG2⁺ and OLIG2⁺) from cortices of CKO and Ctl mice 3 weeks following tamoxifen injection. Samples were collected at CT 0000. Each dot is representative of one animal (mean ± SD). The p-value is from Mann-Whitney test^k. G, Graph depicting quantification of proliferative NG2⁺ Ki67⁺ cell density from cortices of CKO and Ctl mice 3 weeks following tamoxifen injection. Samples collected at CT 0000. Each dot is representative of one animal (mean ± SD). The p-value is from Mann-Whitney test^l.
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Figure 3.
Endogenous Bmal1 expression regulates NG2-glia proliferation after TB1. A, Graph depicting quantification of Bmal1 RNA ISH in Ng2⁺ Pdgfra^TRAP-GFP cells from cortex adjacent (Ipsi) and contralateral (Contra) to the injury. Samples collected at CT 1200. N = 4 animals. The p-value from Kolmogorov–Smirnov test^c. B, Mating scheme and experimental timeline for Bmal1 CKO TBI experiments. C, Representative images of immunofluorescence for proliferative NG2-glia in cortical penumbra at 48 h after TBI from Pdgfra^creERTBmal1^fl/fl CKO and pooled Ctl animals. Proliferative NG2-glia [NG2⁺ (gray) OLIG2⁺ (green) Ki67⁺ (magenta)] are indicated with solid arrowheads (▴), while nonproliferative NG2-glia (NG2⁺ OLIG2⁺ Ki67^–) are indicated by caret (^). DAPI nuclear staining in blue. Scale bar, 10 μm. D, Graph depicting quantification of NG2-glia (NG2⁺ OLIG2⁺) in cortical penumbra at 48 h after TBI from CKO and Ctl mice. Each dot is representative of one animal (mean ± SD). The p-value is from Mann-Whitney testⁿ. E, Graph depicting quantification of proliferative NG2-glia (NG2⁺ OLIG2⁺ Ki67⁺) in cortical penumbra at 48 h after TBI from CKO and Ctl mice. N = 5 and 4, respectively. Each dot is representative of one animal (mean ± SD). The p-value is from Mann-Whitney test^o.

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Table 1

Statistical table

	Data Structure	Type of test	Power (Conf. Int)
a	Non-normal distribution	Rank test for umbrella alternatives (i.e., RAIN)	n/a
b	Nonlinear regression (sine)	Goodness of fit	n/a
c	Non-normal distribution	Kolmogorov–Smirnov	n/a
d	Non-normal distribution	RAIN	n/a
e	Nonlinear regression (sine)	Goodness of fit	n/a
f	Non-normal distribution	RAIN	n/a
g	Nonlinear regression (sine)	Goodness of fit	n/a
h	Non-normal distribution	Mann–Whitney	Rank sum: 36 vs 19, U = 4
i	Non-normal distribution	Mann–Whitney	Rank sum: 11 vs 10, U = 4
j	Non-normal distribution	Mann–Whitney	Rank sum: 8 vs 13, U = 2
k	Non-normal distribution	Mann–Whitney	Rank sum: 44 vs 11, U = 1
l	Non-normal distribution	Mann–Whitney	Rank sum: 43 vs 12, U = 2
m	Non-normal distribution	Mann–Whitney	Rank sum: 35 vs 10, U = 0
n	Non-normal distribution	Mann–Whitney	Rank sum: 30 vs 15, U = 0
o	Non-normal distribution	Mann–Whitney	Rank sum: 30 vs 15, U = 0

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Figure 1-1
Circadian variation in total nuclear BMAL1 expression. Graph depicting quantification of total intensity of nuclear BMAL1 immunofluorescence in Pdgfra-expressing NG2-glia at several times of day (CT 0200, 0600, 1000, 1400, 1800, and 2200). N = 2–3 animals/time point (68 nuclei total). Each dot is representative of one nucleus. The p-value is from RAIN^d. R² is from the goodness of fit to the depicted sine wave for illustrative purpose^e. Graph depicting quantification of total intensity of nuclear BMAL1 immunofluorescence in Pdgfra-expressing NG2-glia at several times of day (CT 0200, 0600, 1000, 1400, 1800, and 2200). n/a, Not applicable. Download Figure 1-1, file.