AhR-mediated gene expression in the developing mouse telencephalon

Abstract

We hypothesize that TCDD-induced developmental neurotoxicity is modulated through an AhR-dependent interaction with key regulatory neuronal differentiation pathways during telencephalon development. To test this hypothesis we examined global gene expression in both dorsal and ventral telencephalon tissues in E13.5 AhR-/- and wildtype mice exposed to TCDD or vehicle. Consistent with previous biochemical, pathological and behavioral studies, our results suggest TCDD initiated changes in gene expression in the developing telencephalon are primarily AhR-dependent, as no statistically significant gene expression changes are evident after TCDD exposure in AhR-/- mice. Based on a gene regulatory network for neuronal specification in the developing telencephalon, the present analysis suggests differentiation of GABAergic neurons in the ventral telencephalon is compromised in TCDD exposed and AhR-/- mice. In addition, our analysis suggests Sox11 may be directly regulated by AhR based on gene expression and comparative genomics analyses. In conclusion, this analysis supports the hypothesis that AhR has a specific role in the normal development of the telencephalon and provides a mechanistic framework for neurodevelopmental toxicity of chemicals that perturb AhR signaling.

Figure 1. Frozen sections of developing telencephalon in AhR wild-type (A. and B.) and knockout (C. and D.) E13.5 mouse exposed to vehicle (A. and C.) or TCDD (B. and D.)

A.-C. show sections cut at 6 μm and stained with HemaCen® (American Master Tech Sci Inc., Lodi, CA) for tissue mapping. D. shows a section cut at 8 μm and stained with 1% aqueous Cresyl Violet acetate (Sigma-Aldrich, St. Louis, MO) and highlights the dorsal and ventral regions removed using laser capture microdissection for further gene expression analysis.

Figure 2. Venn diagram of global gene expression analysis results in dorsal (A.) and ventral (B.) telencephalon

KO refers to AhR -/-, WT refers to wildtype (AhR +/+), TR refers to treatment of TCDD on GD 11.5, and C refers to vehicle control group. Each circle reports the number of differentially expressed genes (p value < 0.001 and fold change greater than 1.2) within each comparison and the overlap between each comparison.

Figure 3. Impact of TCDD exposure and AhR status on a Gene Regulatory Network (GRN) of neurogenesis in the developing telencephalon

GRN is based on robust experimental evidence as detailed in [48]. Connection strengths were tested using a network quantification algorithm described in detail previously [49]. Increased thickness of an arrow identifies a significant increase (p< 0.10) in connection strengths when the algorithm is run using TCDD-exposed or AhR-/- datasets. A dotted arrow identifies a significant decrease (p< 0.10) in connection strength when the algorithm is run using TCDD-exposed or AhR -/- datasets.

Figure 4. Comparative genomics analysis of the Sox11 gene

Using the ECRbrowser, Mulan, and MultiTF tools on the www.dcode.org site [70], an extended AhR/ARNT binding site (GCGCTGGCATGCAAACTCT) as described in [51] was identified in the 5' untranslated region (UTR) 400 bp upstream of the transcription start site (TSS). This site is conserved across Sox 11 genes in human (hg 18 chr2:5749959-5758967), monkey (rheMac2 chr13:5770499-5779504), dog (canFam2 chr17:6556513-6563934), opossum (monDom4 chr1:535907235-535917096), and chicken (galGal3 chr3:97427132-97434301).