Maternal Immune Activation Alters Fetal Brain Development and Enhances Proliferation of Neural Precursor Cells in Rats

Abstract

Maternal immune activation (MIA) caused by exposure to pathogens or inflammation during critical periods of neurodevelopment is a major risk factor for behavioral deficits and psychiatric illness in offspring. A spectrum of behavioral abnormalities can be recapitulated in rodents by inducing MIA using the viral mimetic, PolyI:C. Many studies have focused on long-term changes in brain structure and behavioral outcomes in offspring following maternal PolyI:C exposure, but acute changes in prenatal development are not well-characterized. Using RNA-Sequencing, we profiled acute transcriptomic changes in rat conceptuses (decidua along with nascent embryo and placenta) after maternal PolyI:C exposure during early gestation, which enabled us to capture gene expression changes provoked by MIA inclusive to the embryonic milieu. We identified a robust increase in expression of genes related to antiviral inflammation following maternal PolyI:C exposure, and a corresponding decrease in transcripts associated with nervous system development. At mid-gestation, regions of the developing cortex were thicker in fetuses prenatally challenged with PolyI:C, with females displaying a thicker ventricular zone and males a thicker cortical mantle. Along these lines, neural precursor cells (NPCs) isolated from fetal brains prenatally challenged with PolyI:C exhibited a higher rate of self-renewal. Expression of Notch1 and the Notch ligand, delta-like ligand 1, which are both highly implicated in maintenance of NPCs and nervous system development, was increased following PolyI:C exposure. These results suggest that MIA elicits rapid gene expression changes within the conceptus, including repression of neurodevelopmental pathways, resulting in profound alterations in fetal brain development.

Keywords: PolyI:C; fetal brain development; maternal immune activation; neural precursor cells; pregnancy.

Figure 1

Gene expression changes in the conceptus following maternal exposure to PolyI:C. (A) Volcano plot depicting number of unique transcripts expressed in the conceptus following PolyI:C exposure relative to conceptuses exposed to saline. The x-axis represents magnitude of fold-change (log₂), and the y-axis shows P-value (log₁₀). Transcripts altered 2-fold or more (false discovery rate P < 0.01) following PolyI:C are shown in red. (B) Pie chart showing number of transcripts upregulated and downregulated in conceptuses exposed to PolyI:C compared to those exposed to saline. (C) Heat map showing RPKM values (log₂) of select transcripts in conceptuses following maternal exposure to saline or PolyI:C. (D) Quantitative RT-PCR validation of select transcripts in relation to values obtained using RNA-Seq. Data are normalized to values obtained from saline-exposed conceptuses. All data are based on lysates from three conceptuses pooled from each of three dams (nine conceptuses total) per treatment.

Figure 2

Pathway analysis of unique gene signatures upregulated and downregulated in the conceptus following maternal exposure to PolyI:C. (A) Top 20 gene pathways upregulated in the conceptus following administration of PolyI:C to pregnant dams. (B) Top 20 gene pathways downregulated in the conceptus following administration of PolyI:C to pregnant dams. Pathway analysis was conducted by inputting genes changed more than 2-fold compared to conceptuses collected following maternal exposure to saline, and with a false discovery rate < 0.01.

Figure 3

Prenatal exposure to PolyI:C prompts changes in cortical architecture. Dams were injected with either saline or PolyI:C on E8.5, and whole fetal brains were collected 1 week later. (A) Schematic depiction of the layers of the rat fetal cortex at E15.5. (B) Coronal sections of the cortex at E15.5 were stained for β3T (red) and Sox2 (green) to delineate the CM and VZ, respectively. (C) Measurements showing total cortical thickness and thickness of the CM and VZ in male and female fetuses. Graphs represent means ± SEM. A star () denotes the ventricle. Statistical analyses were performed using two-way analysis of variance and Sidak's multiple comparison. Data are represented as mean ± SEM and asterisks denote statistical significance (*P < 0.05; N ≥ 10 fetuses per sex from at least three dams per group). Scale bar = 100 μm.

Figure 4

Maternal exposure to PolyI:C increases proliferation potential of neurospheres prepared from fetal cortices. Pregnant rats were administered saline or PolyI:C on E8.5, and NPCs were isolated from E15.5 fetal cortices for neurosphere culture. (A) Schematic depiction of experimental protocol. (B) The two left panels show neurospheres produced from cortical NPCs imaged using brightfield. The middle two panels show EdU incorporation in cryosectioned neurospheres. The right two panels show immunofluorescence for phospho-histone H3 (P-HH3). In the middle and right panels, Hoechst is used to counterstain nuclei. Scale bar = 100 μm. (C) Percent EdU positive cells in each neurosphere were quantified from each treatment group. (D) Western blot analysis of P-HH3 expression in neurospheres following 24, 48, and 72 h culture. β-actin was used as a loading control. Statistical analyses were performed using Student's t-test. Data are represented as mean ± SEM. Data significantly different (P < 0.05) from controls are indicated by an asterisk (*5–6 fetuses from each of 3 dams per treatment were used for neurosphere generation).

Figure 5

NPCs isolated from fetal cortices challenged prenatally with PolyI:C exhibit increased proliferation potential. Pregnant rats were administered saline or PolyI:C on E8.5, and NPCs were isolated from E15.5 cortices for neurosphere culture. Neurospheres from male or female embryos were pooled, and then mechanically dissociated to form monolayers. Monolayers were subsequently cultured in media to promote stem or differentiated states. (A) Representative images of NPC monolayers cultured in proliferation conditions stained for Pax6 (green) and Nestin (red), or cultured in differentiation conditions stained for GFAP (green) and NeuN (red). Hoechst (blue) was used to counterstain nuclei. (B) Representative images of EdU incorporation in NPCs prepared in proliferation conditions from male and female cortices challenged prenatally with saline or PolyI:C 1 week prior. (C) Percentage of male and female NPCs that incorporated EdU during culture in proliferation conditions. (D) Percentage of male and female NPCs maintained in differentiation media that incorporated EdU. (E) Percentage of male and female NPCs maintained in differentiation media immunoreactive for NeuN indicating their differentiation capacity. Statistical analyses were performed using Student's t-test. Data are represented as mean ± SEM. Data significantly different (P < 0.05) from controls are indicated by an asterisk (*2–3 fetuses per sex collected from at least 3 dams). Scale bar = 50 μm.

Figure 6

Maternal exposure to PolyI:C alters expression of Notch signaling components in the conceptus. (A) Transcript levels of Notch receptors (Notch1-4) and ligands (Dll1, Dll3, Dll4, Jag1, Jag2) in conceptuses 6 h following maternal exposure to PolyI:C. Transcript levels were normalized to levels in conceptuses from saline-exposed dams. NE, Not expressed (below threshold of detection). (B) Immunofluorescence showing Notch1 expression in E15.5 fetal cortices 1-week following maternal exposure to saline or PolyI:C. β3T was used to delineate the CM. Hoechst was used to stain nuclei. Note that Notch1 is expressed throughout the cortices, but qualitatively increased expression and nuclear accumulation of Notch1 is evident following prenatal challenge with PolyI:C. Scale bar = 10 μm. (C) Transcript levels of Notch1 expression in neurospheres collected from cortices of E15.5 fetuses 6 h following maternal exposure to PolyI:C or saline. (D) Cytoplasmic and nuclear levels of Notch1 in NPCs collected from fetal cortices 1 week after maternal exposure to saline or PolyI:C. β-actin was used as a loading control for cytoplasmic lysates; histone H3 was used as a loading control for nuclear lysates. Statistical analyses were performed using Student's t-test. Data are represented as mean ± SEM. Data significantly different (P < 0.05) from controls are indicated by an asterisk (*3 fetuses collected from each of 3 dams per treatment).

Figure 7

Schematic depicting increased NPC proliferation mediated by Notch1 signaling following MIA. MIA elicits increased expression of Notch1 and its ligand Dll1 in NPCs, which may contribute to altered NPC proliferation and differentiation dynamics leading to changes in cortical structure. NICD, Notch Intracellular Domain.