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Figures
- Fig. 1.
ZIKV causes postnatal microcephaly and growth restriction. (A) Confocal imaging of infected E17.5 cerebral cortex stained with antibodies against Sox2 (labeling NPCs; magenta) and Flavivirus group antigen (ZIKV; green). Hoechst stains nuclei (blue). Scale bar: 50 μm. (B) Viral titers in P3 pup brains were determined using the TCID50 assay. A significantly higher titer of ZIKV (1×109.5 TCID50/ml) was detected in P3 pup brains compared with mock controls. Error bars indicate s.e.m. of three independent measurements with one mock and one ZIKV-infected brain in each measurement (***P<0.0001, Student's t-test). (C) Viral titers were determined in brains at different stages using the TCID50 assay. Error bars indicate the s.e.m. of three independent measurements with one mock and one ZIKV-infected brain in each measurement. Analysis of variance (ANOVA) detects a significant increase in viral titer as development proceeds. (D) Dorsal views of P3 pups. ZIKV (∼1 μl 1.7×106 TCID50/ml) was injected into cerebral ventricles of E14.5 brains followed by analyses at P3. Scale bar: 0.5 cm. (E) ZIKV-infected brains are smaller than controls at P3. Scale bar: 2 mm. (F,G) Dorsal views of P3 hearts and lungs after ZIKV intracerebral inoculation of E14.5 mouse brains. Scale bar: 1 mm. (H) Relative weights of different organs from control or ZIKV-infected pups at P3. Error bars indicate the s.e.m. of six independent experiments with one mock and one ZIKV-infected brain in each experiment (*P<0.01, Student's t-test).
- Fig. 2.
ZIKV infection results in neuronal loss and cortical thinning. (A) Coronal sections of P3 cerebral cortex stained with H&E. Scale bars: 0.2 mm (left panels); 50 μm (right panels). Black brackets indicate the measurement of cortical radial thickness. Black boxed areas are enlarged in insets and show a substantial number of dead cells indicated by dark staining in ZIKV-infected brains. (B) Quantification of cortical radial thickness from the experiment shown in A. Error bars indicate s.e.m. of nine sections from three independent experiments. *P<0.05 (Student's t-test). (C) Confocal imaging of P3 cerebral cortex stained with antibodies against NeuN (green). Hoechst stains nuclei (blue). Right panels are enlargements of the regions outlined by red boxes in left panels. Scale bars: 200 μm (left panels); 100 μm (right panels). (D) Quantification of NeuN-positive cells per mm2 in a 3.385×105 μm2 boxed area of P3 cerebral cortex from the experiment shown in C. Error bars indicate s.e.m. of nine sections from three independent experiments. *P<0.05 (Student's t-test). (E) Confocal microscope images of coronal sections from P3 cortex stained with antibodies against Tbr1 (green), Ctip2 (magenta) and Cux1 (green). Hoechst stains nuclei (blue). Right panels are enlargements of the regions outlined by red boxes in left panels. Scale bars: 200 μm (left panels); 100 μm (right panels). (F) Quantification of percentage of Tbr1-, Ctip2- and Cux1-positive cells per mm2 in a 5.274×105 μm2 boxed area of P3 cerebral cortex from the experiment shown in E. Error bars indicate s.e.m. of nine sections from three independent experiments (*P<0.05, Student's t-test).
- Fig. 3.
ZIKV infection leads to massive neuronal death and axonal rarefaction. (A) Confocal imaging of P3 cerebral cortex stained with antibodies against cleaved caspase 3 (red) and Flavivirus group antigen (ZIKV, green). Hoechst stains nuclei (blue). Right panels are enlargements of the regions outlined by red boxes in left panels. White arrowhead indicates the ventricular zone (VZ) with fewer viruses detected. Scale bars: 200 μm (left panels); 100 μm (right panels). (B) TUNEL staining (green) on coronal sections of P3 cortex reveals extensive apoptotic cell labeling in the ZIKV-infected cortex compared with controls. Hoechst stains nuclei (blue). Right panels are enlargements of the regions outlined by red boxes in left panels. White arrowhead indicates the ventricular zone (VZ) with less viruses detected. Scale bars: 200 μm (left panels); 100 μm (right panels). (C) Quantification of percentage of caspase-3- and TUNEL-positive cells out of total cells in a 5.625×105 μm2 boxed area in the experiments shown in A and B. Error bars indicate s.e.m. of nine sections from three independent experiments. ***P<0.001 (Student's t-test). (D) Confocal microscope images of coronal sections from P3 cortex stained with antibodies against L1 (magenta; labels axons), NF (neurofilament; red; labels mature neurons), Map2 (green; labels dendrites) and TuJ1 (red; labels newly generated neurons). Hoechst stains nuclei (blue). Right panels are enlargements of the regions outlined by red boxes in left panels. Note the drastically reduced L1 and NF staining indicated by white arrowhead in ZIKV-infected cerebral cortex. Scale bars: 200 μm (left panels); 100 μm (right panels). (E) Quantification of relative signal intensities from L1, NF, Map2 and TuJ1 staining using ImageJ. Error bars indicate s.e.m. of nine sections from three independent experiments (*P<0.05, Student's t-test).
- Fig. 4.
Abnormal vasculature and leaky BBB in ZIKV-infected brains. (A,C) Confocal micrographs of P3 (A) or P0 (C) coronal cortical sections stained with antibodies against PECAM-1 (green) after viral infection at E14.5. Hoechst stains nuclei (blue). Right panels are enlargements of the white boxed areas. Scale bars: 50 μm (left panels in A); 20 μm (right panels in A), 100 μm (left panels in C) and 10 μm (right panels in C). (B,D) Quantification of relative vessel density and diameter from the experiments shown in A and C. Measurements of vessel density and diameter in ZIKV-infected brain sections were normalized to that in controls. Error bars indicate s.e.m. of nine sections from three independent experiments. *P<0.05 (Student's t-test). (E,F) Confocal micrographs of coronal sections of P0 brains stained with antibodies against PECAM-1 (green) together with 10 kDa dextran tracer (red). Hoechst stains nuclei (blue). Scale bars: 50 μm. Dextran tracer (10 kDa) revealed a leaky BBB (white arrowheads) in P0 brains after ZIKV infection at E14.5.
- Fig. 5.
Extensive microglial activation and astrogliosis in ZIKV-infected brain. (A,B) Confocal micrographs of coronal sections of E17.5 (A) and P3 (B) brains stained with antibodies against Iba1 (green) or GFAP (red). Hoechst stains nuclei (blue). Right panels are enlargements of the regions outlined by white boxes in left panels. Scale bars: 50 μm (left panels in A and B); 10 μm (right panels in A) and 20 μm (right panels in B). (C) Quantification of Iba1-positive cells per mm2 in a 1.314×105 μm2 boxed area of E17.5 cerebral cortex from the experiment shown in A, and a 1.314×105 μm2 boxed area of P3 cerebral cortex from the experiment shown in B. Error bars indicate s.e.m. of nine sections from three independent experiments. *P<0.05, ***P<0.001 (Student's t-test for comparison of mock- versus ZIKV-infected brains). Two-way ANOVA analysis detected a significant difference in the increase of Iba1-positive cells between E17.5 and P3 (*P<0.05). (D) Quantification of GFAP-positive cells in a 1.314×105 μm2 boxed area of P3 cerebral cortex from the experiment shown in B. Error bars indicate s.e.m. of nine sections from three independent experiments. ***P<0.001 (Student's t-test).
- Fig. 6.
ZIKV induces dysregulation of genes involved in the immune response. (A) RT-PCR analysis of expression of IL-1β and TNF-α in NPCs. NPCs isolated from E14.5 developing brains were infected with ZIKV (multiplicity of infection: 5) followed by RNA isolation at 48 h post-infection. Error bars indicate s.e.m. of three independent experiments. **P<0.01 (Student's t-test). (B) RNA-Seq analyses of E17.5 brains after ZIKV infection at E14.5. The dysregulated genes in ZIKV-infected brains compared with controls were subjected to GO analysis. (C) Gene set enrichment analysis (GSEA) reveals dysregulated genes in immune response in ZIKV-infected brains. x-axis represents the gene ranks in ordered immune response dataset. (D) RNA expression analyses of RNA-Seq data (Table S1) show that the top ten most significantly upregulated genes in infected brains are involved in immune response. ***P<0.001 (Student's t-test). (E) A diagram showing that ZIKV causes neuronal death, abnormal vasculature and BBB leakage, and cell cycle arrest and apoptosis of NPCs, resulting in postnatal microcephaly with brain damage.
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