Aberrant Epigenetic Gene Regulation in GABAergic Interneuron Subpopulations in the Hippocampal Dentate Gyrus of Mouse Offspring Following Developmental Exposure to Hexachlorophene

Abstract

Maternal hexachlorophene (HCP) exposure causes transient disruption of hippocampal neurogenesis in mouse offspring. We examined epigenetically hypermethylated and downregulated genes related to this HCP-induced disrupted neurogenesis. Mated female mice were dietary exposed to 0 or 100 ppm HCP from gestational day 6 to postnatal day (PND) 21 on weaning. The hippocampal dentate gyrus of male offspring was subjected to methyl-capture sequencing and real-time reverse transcription-polymerase chain reaction analyses on PND 21. Validation analyses on methylation identified three genes, Dlx4, Dmrt1, and Plcb4, showing promoter-region hypermethylation. Immunohistochemically, DLX4+, DMRT1+, and PLCB4+ cells in the dentate hilus co-expressed GAD67, a γ-aminobutyric acid (GABA)ergic neuron marker. HCP decreased all of three subpopulations as well as GAD67+ cells on PND 21. PLCB4+ cells also co-expressed the metabotropic glutamate receptor, GRM1. HCP also decreased transcript level of synaptic plasticity-related genes in the dentate gyrus and immunoreactive granule cells for synaptic plasticity-related ARC. On PND 77, all immunohistochemical cellular density changes were reversed, whereas the transcript expression of the synaptic plasticity-related genes fluctuated. Thus, HCP-exposed offspring transiently reduced the number of GABAergic interneurons. Among them, subpopulations expressing DLX4, DMRT1, or PLCB4 were transiently reduced in number through an epigenetic mechanism. Considering the role of the Dlx gene family in GABAergic interneuron migration and differentiation, the decreased number of DLX4+ cells may be responsible for reducing those GABAergic interneurons regulating neurogenesis. The effect on granule cell synaptic plasticity was sustained until the adult stage, and reduced GABAergic interneurons active in GRM1-PLCB4 signaling may be responsible for the suppression on weaning.

Figure 1

Experimental design for the developmental exposure study of hexachlorophene (HCP) using mated female mice. Eight offspring were preserved in each dam after culling on postnatal day (PND) 3, and offspring were subjected to molecular and immunohistochemical analyses in the hippocampal dentate gyrus. One offspring per dam was used in each analysis.

Figure 2

Transcript expression results obtained from MethylCap-seq analysis of hypermethylated genes in the hippocampal dentate gyrus of mice on postnatal day (PND) 21 and PND 77. A, PND 21. B, PND 77. Values are normalized to Hprt (left) or Gapdh (right) and expressed as the mean + SD; n = 6/group. *p < .05, **p < .01, significantly different from 0 ppm controls by Student’s or Aspin-Welch’s t test.

Figure 3

Quantitative methylation-specific PCR data of selected genes in the hippocampal dentate gyrus on postnatal day (PND) 21. Values are expressed as the mean + SD; n = 5/group. *p < .05, **p < .01, significantly different from 0 ppm controls by Student’s or Aspin-Welch’s t test.

Figure 4

Pyrosequencing results for Dlx4 and Dmrt1 in the hippocampal dentate gyrus on postnatal day (PND) 21. A, Dlx4. B, Dmrt1. All cytosine bases within CpG site are numbered as 1–16 or 1–5. White columns, 0 ppm controls; black columns, 100 ppm hexachlorophene (HCP). Values are expressed as the mean + SD.; n = 4/group. *p < .05, significantly different from 0 ppm controls by Student’s or Aspin-Welch’s t test.

Figure 5

Pyrosequencing results for Plcb4 and Reps1 in the hippocampal dentate gyrus on postnatal day (PND) 21. A, Plcb4. B, Reps1. All cytosine bases within CpG site are numbered as 1–6 or 1–7. White columns, 0 ppm controls; black columns, 100 ppm hexachlorophene (HCP). Values are expressed as the mean + SD; n = 4/group. *p < .05, significantly different from 0 ppm controls by Student’s or Aspin-Welch’s t test.

Figure 6

Density of cells immunoreactive for DLX4, DMRT1, and PLCB4 in the hippocampal dentate hilus of male offspring on postnatal day (PND) 21 and PND 77 after maternal exposure to hexachlorophene (HCP) from gestational day 6 to PND 21. A, DLX4. B, DMRT1. C, PLCB4. Representative images from mice exposed to 0 ppm controls (left) and 100 ppm HCP (right) on PND 21. Arrowheads indicate immunoreactive cells. Magnification, ×400; scale bar, 50 µm. Graphs show the density of cells immunoreactive for the indicated molecule in the dentate hilus. n = 10/group. *p < .05, significantly different from 0 ppm controls by Student’s or Aspin-Welch’s t test.

Figure 7

Density of cells immunoreactive for DLX4, DMRT1, and PLCB4 in the dentate hilus in relation to GAD67⁺ cells. A–C, Cellular identity of DLX4, DMRT1, and PLCB4 with GAD67⁺ cells analyzed using serial mirror sections. A, DLX4 vs. GAD67. B, DMRT1 vs. GAD67. C, PLCB4 vs. GAD67. Arrowheads indicate cells positive for both GAD67 and the indicated molecule. Magnification, ×400; scale bar, 50 µm. D, Density of GAD67⁺ cells in the dentate hilus of male offspring on postnatal day (PND) 21 and PND 77 after maternal exposure to hexachlorophene (HCP) from gestational day 6 to PND 21. Representative images from mice exposed to 0 ppm controls (left) and 100 ppm HCP (right) on PND 21. Arrowheads indicate GAD67⁺ cells. Magnification, ×400; scale bar, 50 µm. Graphs show the density of GAD67⁺ cells in the dentate hilus. n = 10/group. **p < .01, significantly different from 0 ppm controls by Student’s or Aspin–Welch’s t test.

Figure 8

Distribution of GRM1⁺ cells in the dentate hilus in relation to PLCB4⁺ cells, and alteration in the number of ARC⁺ granule cells after maternal exposure to hexachlorophene (HCP). A, Identity of PLCB4⁺ cells as GRM1⁺ cells analyzed using serial mirror sections. Arrowheads indicate cells positive for both PLCB4 and GRM1. Magnification, ×400; scale bar, 50 µm. B, Number of ARC⁺ granule cells in male offspring on postnatal day (PND) 21 and PND 77 after maternal exposure to HCP from gestational day 6 to PND 21. Representative images from mice exposed to 0 ppm controls (left) and 100 ppm HCP (right) on PND 21. Magnification, ×400; scale bar, 50 µm. Graphs show the number of ARC⁺ cells in the granule cell layer. n = 8–10/group. *p < .05, significantly different from 0 ppm controls by Student’s or Aspin–Welch’s t test.