Article Figures & Data
Figures
- Figure 1.
Mice deficient for inflammasome pathway genes exhibit behavior abnormalities. This figure is supported by Extended Data Figure 1-1. A, General activity of male WT (black circles, N = 11, from 5 litters) and Casp1−/− (red triangles, N = 12, from 5 litters) mice was determined by open field assay and is shown as total distance moved (centimeters); p = 0.0049, df = 21, t = 3.142. B, Anxiety levels of male WT and Casp1−/− mice were determined using the elevated plus maze assay and are shown as time spent in open arms (seconds); p = 0.0029, df = 21, t = 3.371. C, D, General movement (C) and anxiety levels (D) of male WT (black circles, N = 13, from 4 litters), Il-1r−/− (blue squares, N = 10, from 3 litters), and Gsdmd−/− (green triangles, N = 11, from 4 litters) mice were determined as in A, B, respectively. C, p = 0.0159, df = 33, F = 4.748. Tukey’s multiple comparison test: WT versus Il-1r−/− p = 0.0147, WT versus Gsdmd−/− p = 0.1270, Il-1r−/− versus Gsdmd−/− p = 0.5866. D, p = 0.0050, df = 33, F = 6.306. Tukey’s multiple comparison test: WT versus Il-1r−/− p = 0.1809, WT versus Gsdmd−/− p = 0.0036, Il-1r−/− versus Gsdmd−/− p = 0.2727. E, F, General movement (E) and anxiety (F) of male WT (black circles, N = 10, from 3 litters) and Nlrp3−/− mice (orange diamonds, N = 9, from 2 litters) were determined as in A, B, respectively. E, p = 0.0220, df = 17, t = 2.520. F, p = 0.0130, df = 17, t = 2.776. Data shown are individual mice and error bars indicate standard error of the mean (SEM); *p < 0.05, **p < 0.01.
- Figure 2.
Casp1 re-expression in CX3CR1+ cells restores normal behavior. This figure is supported by Extended Data Figure 2-1. A, Experimental design is shown. Three mouse lines (Casp1−/−, iCasp1, and Cx3cr1-Cre) were crossed to generate Casp1−/−; iCasp1; Cx3cr1-Cre positive mice (experimental) and Casp1−/−; iCasp1; Cre negative littermates (control). Mice expressing Cre had induced expression of Casp1 in CX3CR1+ cells, but not in CX3CR1– cells. B, General movement of male WT (black circles, N = 7, from 3 litters), littermate control (blue triangles, N = 6, from 4 litters), and experimental (red squares, N = 12, from 8 litters) mice was determined by open field assay and is shown as total distance moved (centimeters); p = 0.0581, df = 24, F = 3.247. Tukey’s multiple comparison test: WT versus control p = 0.0997, WT versus experimental p = 0.5502, control versus experimental p = 0.3072. C, Anxiety levels of male WT (black circles, N = 7, from 3 litters), control (blue triangles, N = 10, from 6 litters), and experimental (red squares, N = 12, from 8 litters) mice were determined by elevated plus maze assay and are shown as time spent in open arms (seconds); p < 0.0001, df = 28, F = 15.97. Tukey’s multiple comparison test: WT versus control p < 0.0001, WT versus experimental p = 0.0760, control versus experimental p = 0.0023. Data indicates individual animals and error bars are shown as SEM; ****p < 0.0001; n.s., not significant.
- Figure 3.
Lytic cell death occurs in a spatiotemporal manner in the fetal brain. This figure is supported by Extended Data Figure 3-1 and Movies 1, 2. A, Experimental scheme is shown. PI was injected into pregnant female mice at specific gestational stages. Ten to 30 min after PI injection, total fetal bodies were recovered, cleared, and imaged by LSFM. B, Representative sagittal and coronal LSFM images of the LGE and primitive iTh of a WT E12.5 fetal brain (male, N = 6, from 2 litters) are shown. Arrows indicate the cluster. Green, GFP; purple, PI. Scale bars: 500 μm. C, Representative images of a WT E14.5 fetal brain (male, N = 6, from 3 litters) are shown. Arrows indicate the cluster. Green, GFP; purple, PI. Scale bars: 500 μm. D, Representative coronal LSFM images of a Casp1−/− brain (male, N = 5, from 3 litters) at E14.5 are shown. Scale bars: 500 μm. E, Numbers of mice that exhibited clusters in the LGE and iTh at E14.5 are shown; *p < 0.05, **p < 0.01; n.s., not significant.
- Figure 4.
Numbers of neurons but not microglia are increased in the TRN in adult Casp1−/− mice. This figure is supported by Extended Data Figure 4-1. A, Enlarged image of the GFP+PI+ cluster shown in Figure 3. Mice were injected with PI as shown in Figure 3A, and fetal brain slices were stained with anti-Pax6 antibody. Images of Cx3cr1-GFP (top left), PI (top right), anti-Pax6 (bottom left), and merged signals are shown. Scale bar: 50 μm. B, Representative images of anti-NeuN staining signals from male Casp1−/− (N = 3, right, from 2 litters) and sex/age-matched WT (N = 3, left, from 2 litters) adult brains are shown. TRN regions are outlined in yellow. Scale bar: 200 μm. C, Average anti-NeuN signals from Casp1−/− (red triangles) and WT (black circles) mice are shown as cell number/mm2. Dots indicate individual animals and are the average cell numbers from a total of six serial sections stained as shown in B; p = 0.0209, df = 4, t = 3.694. Error bars indicate SEM; *p < 0.05. D, Representative images of anti-Iba1 antibody staining in the TRNs (regions outlined in yellow) of male WT (N = 3, left, from 2 litters) and Casp1−/− (N = 3, right, 2 litters) mice are shown. Scale bar: 200 μm. E, Average number of Iba1+ cells in Casp1−/− (red triangles) and WT (black circles) mice are shown as mean cell number/mm2. Dots indicate individual animals and are the average of cell numbers from a total of six serial sections stained as shown in D; p = 0.7533, df = 4, t = 0.336. Error bars indicate SEM; n.s., not significant.
- Figure 5.
Fetal exposure to inflammasome inhibitors leads to aberrant behaviors. This figure is supported by Extended Data Figure 5-1. A, Experimental scheme is shown. Pregnant WT female mice were injected with inflammasome inhibitors (VX-765 or MCC950 to inhibit CASP1 or NLRP3, respectively) or control (Veh: vehicle) at E12.5, E13.5, and E14.5. Behaviors of the male offspring of injected mice were tested at eight weeks old. B, General activity of the male offspring of control-injected (vehicle; black circles, N = 11, from 3 litters), VX-765-injected (blue diamonds, N = 10, from 3 litters), and MCC950-injected mothers (pink triangles, N = 8, from 3 litters) was determined by open field assay and is shown as the total distance moved (centimeters); p = 0.0412, df = 28, F = 3.614. Tukey’s multiple comparison test: Veh versus VX-765 p = 0.0963, Veh versus MCC950 p = 0.0591, VX-765 versus MCC950 p = 0.9250. Dots indicate individual animals, and error bars indicate SEM. C, Anxiety levels in the indicated offspring were determined by elevated plus maze assay and are shown as time spent in open arms (seconds); p = 0.0325, df = 28, F = 3.92. Tukey’s multiple comparison test: Veh versus VX-765 p = 0.1736, Veh versus MCC950 p = 0.0307, VX-765 versus MCC950 p = 0.6151. Data are shown as individual animals, and error bars indicate SEM; *p < 0.05; n.s., not significant. D, Working model is shown. Activation of the NLRP3-CASP1-GSDMD/IL-1β cascade in fetal microglia is required for normal brain development. Genetic or pharmacological disruption of this pathway results in aberrant behaviors.
Tables
Behavior Assay Hyperactivity Open field assay (total distance they moved) Anxiety Elevated plus maze assay Attention 5-CSRTT assay The behavior assays used in this study are shown.
Name Vendor Catalog # Dilution NeuN Millipore Sigma ABN78 1:300 Parvalbumin Millipore Sigma P3088 1:1000 Iba1 Thermo Fisher PA5-18039 1:250 Pax6 Eurogentec PRB-278P-100 1:100 The information on primary antibodies used in this study is shown.
Figure t value df p value Fig. 1A 3.142 21 0.0049 Fig. 1B 3.371 21 0.0029 Fig. 1E 2.520 17 0.0220 Fig. 1F 2.776 17 0.0130 Fig. 4C 3.694 4 0.0209 Fig. 4E 0.336 4 0.7533 Extended Data Fig. 1-1B 3.371 21 0.0029 Extended Data Fig. 1-1C 4.425 21 0.0002 Extended Data Fig. 1-1D 2.432 15 0.0280 Extended Data Fig. 1-1E 0.704 5 0.5129 Extended Data Fig. 1-1F 0.022 7 0.9829 Extended Data Fig. 1-1I 2.776 17 0.0130 Extended Data Fig. 1-1J 3.392 17 0.0035 Extended Data Fig. 2-1A 688.100 2 <0.0001 Extended Data Fig. 2-1B 1319.000 2 <0.0001 Extended Data Fig. 2-1C 598.900 2 <0.0001 Extended Data Fig. 2-1D 220.900 2 <0.0001 Extended Data Fig. 3-1D 3.935 30 0.0005 Extended Data Fig. 4-1, cerebrum 0.191 4 0.8580 Extended Data Fig. 4-1, thalamus 0.691 4 0.5274 Extended Data Fig. 4-1, striatum 0.084 4 0.9372 For the Student’s t tests used in our analyses, the t values, df, and p values are summarized.
SS df MS F(dfn,dfd) p Fig. 1C Between columns 43308629 2 21654315 F(2,31) = 4.748 p = 0.0159 Within columns 141389895 31 4560964 Total 184698524 33 Fig. 1D Between columns 17029 2 8514 F(2,31) = 6.306 p = 0.0050 Within columns 41856 31 1350 Total 58885 33 Fig. 2B Between columns 18214401 2 9107200 F(2,22) = 3.247 p = 0.0581 Within columns 61708704 22 2804941 Total 79923105 24 Fig. 2C Between columns 21480 2 10740 F(2,26) = 15.97 p < 0.0001 Within columns 17489 26 672.7 Total 38969 28 Fig. 5B Between columns 2577676 2 1288838 F(2,26) = 3.614 p = 0.0412 Within columns 9272962 26 356652 Total 11850639 28 Fig. 5C Between columns 14905 2 7453 F(2,26) = 3.92 p = 0.0325 Within columns 49424 26 1901 Total 64329 28 Extended Data Fig. 1-1G Between columns 473 2 236.5 F(2,31) = 6.304 p = 0.0050 Within columns 1163 31 37.51 Total 1636 33 Extended Data Fig. 1-1H Between columns 136.1 2 68.07 F(2,31) = 2.005 p = 0.1518 Within columns 1053 31 33.96 Total 1189 33 Extended Data Fig. 2-1E Between columns 11.75 2 5.876 F(2,8) = 16.19 p = 0.0015 Within columns 2.904 8 0.363 Total 14.66 10 Extended Data Fig. 2-1F Between columns 597.6 2 298.8 F(2,26) = 15.92 p < 0.0001 Within columns 488 26 18.77 Total 1086 28 Extended Data Fig. 2-1G Between columns 230.5 2 115.3 F(2,26) = 3.902 p = 0.0330 Within columns 768 26 29.54 Total 998.5 28 Extended Data Fig. 5-1B Between columns 414 2 207 F(2,26) = 3.921 p = 0.0325 Within columns 1373 26 52.8 Total 1787 28 Extended Data Fig. 5-1C Between columns 78.67 2 39.34 F(2,26) = 1.724 p = 0.1981 Within columns 593.2 26 22.82 Total 671.9 28 For our one-way ANOVA analyses, the SS, df, the MS, F ratios, and p values used to assess the variation between columns, within columns, and total are summarized.
Movies
- Movie 1.
Representative LSFM imaging of male E14.5 WT fetal brain. Embryos had one copy of Cx3cr1-GFP and were injected with PI 10–30 min before tissue harvest and visualization. Movies show sequential z-planes of a typical acquisition followed by 3D maximum intensity projection. Purple, PI; green, Cx3cr1-GFP; white, merged.
- Movie 2.
Representative LSFM imaging of male E14.5 Casp1−/− fetal brain. Embryos had one copy of Cx3cr1-GFP and were injected with PI 10–30 min before tissue harvest and visualization. Movies show sequential z-planes of a typical acquisition followed by 3D maximum intensity projection. Purple, PI; green, Cx3cr1-GFP; white, merged.
Extended Data Figure 1-1
Inflammasome cascade and attention behavior of Casp1−/− mice. A, Diagram of the inflammasome cascade. NLRP3, an inflammasome protein, is activated and provides a platform to cleave pro-CASP1 to generate CASP1. CASP1 cleaves pro-GSDMD to GSDMD, which assembles and generates pores in the plasma membrane and induces lytic cell death. CASP1 also cleaves pro-IL-1β to generate mature IL-1β cytokine. B, G, I, Elevated plus maze assay results from the indicated male mice to support Figure 1B,D,F are shown as the time spent in open arms compared to the total time spent on the apparatus (%). C, H, J, Elevated plus maze assay results from the indicated male mice to support Figure 1B,D,F are shown as the number of entries into open arms compared to the total number of entries (%). D, Attention behavior of male WT (N = 7, black circles, from 3 litters) and Casp1−/− (N = 10, red triangles, from 4 litters) mice was determined by the 5-CSRTT and is shown as the accuracy of their responses (%). E, General activity of female WT (black circles, N = 4, from 2 litters) and Casp1−/− (red squares, N = 3, from 2 litters) mice was determined by open field assay and is shown as total distance moved (centimeters). F, Anxiety levels of female WT (black circles, N = 5) and Casp1−/− (red squares, N = 4) mice were determined using the elevated plus maze assay and are shown as the time spent in open arms (seconds). Dots indicate individual animals and error bars show SEM. B, p = 0.0029, df = 21, t = 3.371. C, p = 0.0002, df = 21, t = 4.425. D, p = 0.028, df = 15, t = 2.432. E, p = 0.5129, df = 5, t = 0.704. F, p = 0.9829, df = 7, t = 0.022. G, p = 0.0050, df = 33, F = 6.304. Tukey’s multiple comparison test: WT versus Il-1r−/− p = 0.1809, WT versus Gsdmd−/− p = 0.0036, Il-1r−/− versus Gsdmd−/− p = 0.2729. H, p = 0.1518, df = 33, F = 2.005. Tukey’s multiple comparison test: WT versus Il-1r−/− p = 0.2964, WT versus Gsdmd−/− p = 0.1717, Il-1r−/− versus Gsdmd−/− p = 0.9610. I, p = 0.0130, df = 17, t = 2.776. J, p = 0.0035, df = 17, t = 3.392; *p < 0.05, **p < 0.01; n.s., not significant. Download Figure 1-1, TIF file.
Extended Data Figure 3-1
Quantification and qualification of clusters in WT and Casp1 re-expression mice. A, Numbers of WT mice (male, N = 6) that exhibited clusters in the LGE and iTh at E12.5 are shown; n.s., not significant. B, Cell numbers in the clusters from the LGE and iTh at E14.5 are shown. C, A representative brightfield image of the cluster including swollen microglial cells. Scale bar: 100 μm. D, Diameters of GFP+ and GFP+PI+ cells in the clusters are shown; p = 0.0005, df = 30, t = 3.935. Diameters of individual cells and the averages are shown. Error bars indicate SEM; ***p < 0.001. E, Casp1−/−; iCasp1; Cx3cr1-Cre mice express GFP upon Cre-mediated recombination. Mice were injected with PI as shown in Figure 3A. Representative brightfield (top left), Cx3cr1-GFP staining signal (top right), PI signal (bottom left), and merged (bottom right) images are shown. Scale bar in the bright field image indicates 230 μm and the others indicate 130 μm. Download Figure 3-1, TIF file.
Extended Data Figure 2-1
Casp1 is expressed in microglia from fetal brains and adult Casp1−/−; iCasp1; Cx3cr1-Cre mice. A–D, mRNA expressions of Casp1 (A), Nlrp3 (B), Cd200 (C), and Celf4 (D) in Cx3cr1-GFP+ and Cx3cr1-GFP- cells from male fetal brain at E14.5, as determined by RT-qPCR. A, p < 0.0001, df = 2, t = 688.100. B, p < 0.0001, df = 2, t = 1319.000. C, p < 0.0001, df = 2, t = 598.900. D, p < 0.0001, df = 2, t = 220.900. Data are shown as the average of duplicates, and error bars indicate SEM. E, Microglial cells were isolated from the brains of adult male WT (black circles), control (blue rectangles), and experimental (red rectangles) mice and the mRNA expressions of Casp1 were determined by RT-qPCR. F, Elevated plus maze assay results supporting Figure 2C are shown as the time spent in open arms compared to the total time spent on the apparatus (%). G, Elevated plus maze assay results supporting Figure 2C are shown as the number of entries into open arms compared to the total number of entries (%). E, p = 0.0015, df = 10, F = 16.19. Tukey’s multiple comparison test: WT versus control p = 0.2260, WT versus experimental p = 0.0230, control versus experimental p = 0.0013. F, p < 0.0001, df = 28, F = 15.92. Tukey’s multiple comparison test: WT versus control p < 0.0001, WT versus experimental p = 0.0759, control versus experimental p = 0.0024. G, p = 0.0330, df = 28, F = 3.902. Tukey’s multiple comparison test: WT versus control p = 0.0743, WT versus experimental p = 0.0340, control versus experimental p = 0.9469. Data indicates individual mice and averages are shown. Error bars indicate SEM; **p < 0.01, ****p < 0.0001. Download Figure 2-1, TIF file.
Extended Data Figure 4-1
Brain volume does not change in Casp1−/− mice. Volumes of the cerebrum, thalamus, and striatum of adult male WT (black circles) and Casp1−/− (red triangles) mouse brains were determined by Nissl staining and stereology. Cerebrum p = 0.8580, df = 4, t = 0.191. Thalamus p = 0.5247, df = 4, t = 0.691. Striatum p = 0.9372, df = 4, t = 0.084. Data are shown as volumes (μm3) of regions from individual mice and their averages. Error bars indicate SEM; n.s., not significant. Download Figure 4-1, TIF file.
Extended Data Figure 5-1
Evaluation of inhibitor-exposed offspring in the elevated plus maze and our working model of the role of lytic microglial death in NPC development. A, Elevated plus maze assay results supporting Figure 5C are shown as the time spent in open arms compared to the total time spent on the apparatus (%). B, Elevated plus maze assay results supporting Figure 5C are shown as the number of entries into open arms compared to the total number of entries (%). A, p = 0.0325, df = 28, F = 3.921. Tukey’s multiple comparison test: Veh versus VX-765 p = 0.1736, Veh versus MCC950, p = 0.0307, VX-765 versus MCC950 p = 0.6151. B, p = 0.1981, df = 28, F = 1.724. Tukey’s multiple comparison test: Veh versus VX-765 p = 0.4081, Veh versus MCC950 p = 0.2001, VX-765 versus MCC950 p = 0.8550; *p < 0.05; n.s., not significant. C, Cartoon describing our working model of the fetal brain iTh at E14.5. Damage-associated molecular patterns (DAMPs), such as ATP, are released from dead or dying neural cells to activate microglial NLRP3 and initiate the NLRP3-CASP1-GSDMD/IL-1β cascade depicted in Figure 5D. As a result, sterile inflammation (perhaps mediated in part by IL-1β) influences the development of NPCs. We propose that proinflammatory cytokines are required to promote the death of TRN precursor cells in the iTh, which is why we observe increased numbers of neurons in the TRN region of adult Casp1−/− brains. Download Figure 5-1, TIF file.
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