TOXICOLOGICAL SCIENCES 103(1), 125–136 (2008)
Advance Access publication January 27, 2008
2,3,7,8-Tetracholorodibenzo-p-Dioxin Exposure Disrupts Granule
Neuron Precursor Maturation in the Developing Mouse Cerebellum
Loretta L. Collins, Mary A. Williamson,1Bryan D. Thompson, Daniel P. Dever, Thomas A. Gasiewicz, and Lisa A. Opanashuk2
Department of Environmental Medicine, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642
Received September 29, 2007; accepted January 21, 2008
The widespread environmental contaminant 2,3,7,8-tetrachlor-
odibenzo-p-dioxin (TCDD) has been linked to developmental
neurotoxicity associated with abnormal cerebellar maturation in
both humans and rodents. TCDD mediates toxicity via binding to
the aryl hydrocarbon receptor (AhR), a transcription factor that
regulates the expression of xenobiotic metabolizing enzymes and
growth regulatory molecules. Our previous studies demonstrated
that cerebellar granule neuron precursor cells (GNPs) express
transcriptionally active AhR during critical developmental
periods. TCDD exposure also impaired GNP proliferation and
survival in vitro. Therefore, this study tested the hypothesis that
TCDD exposure disrupts cerebellar development by interfering
with GNP differentiation. In vivo experiments indicated that
TCDD exposure on postnatal day (PND) 6 resulted in increased
expression of a mitotic marker and increased thickness of the
external granule layer (EGL) on PND10. Expression of the early
differentiation marker TAG-1 was also more pronounced in
postmitotic, premigratory granule neurons of the EGL, and
increased apoptosis of GNPs was observed. On PND21, expression
of the late GNP differentiation marker GABAAa6 receptor
(GABARAa6) and total estimated cell numbers were both reduced
following exposure on PND6. Studies in unexposed adult AhR2/2
mice revealed lower GABARAa6levels and DNA content. In vitro
studies showed elevated expression of the early differentiation
marker p27/Kip1 and the GABARAa6in GNPs following TCDD
exposure, and the expression patterns of proteins related to
granule cell neurite outgrowth, bIII-tubulin and polysialic acid
neural cell adhesion molecule, were consistent with enhanced
neuroblast differentiation. Together, our data suggest that TCDD
disrupts a normal physiological role of AhR, resulting in
compromised GNP maturation and neuroblast survival, which
impacts final cell number in the cerebellum.
Key Words: neurogenesis; neurotoxicity; neural progenitor;
differentiation; TCDD; cell cycle.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous
and persistent environmental contaminant that exerts develop-
mental toxicity via binding to the aryl hydrocarbon receptor
(AhR) (Hankinson, 1995), a ligand-activated transcription
factor. AhR is a member of the basic helix–loop–helix/
Per–Arnt–Sim superfamily (Gu et al., 2000) whose family
members are known to play important roles in a variety of
cellular processes, including neuronal development, cell fate
determination, and differentiation (Gu et al., 2000; Hahn, 2002;
Lee, 1997). Ligand-activated AhR has also been found to
regulate the expression of genes known to participate in cell
cycle regulation and apoptosis, such as p21CIP1, p27KIP1, and
Bax (Barnes-Ellerbe et al., 2004; Kolluri et al., 1999;
Matikainen et al., 2001). We previously determined that AhR
is robustly expressed and transcriptionally active in cerebellar
granule neuron precursors (GNPs) (Williamson et al., 2005)
during a critical period of neurogenesis. These observations
lead to the hypothesis that TCDD exposure interferes with the
spatiotemporal expression of cell cycle and differentiation
factors that participate in granule neuroblast maturation in the
Several observations are consistent with the possibility
that TCDD exposure disrupts cerebellar development. Children
exposed to mixtures containing polychlorinated biphenyls and
TCDD have been reported to suffer from neurobehavioral
deficits associated with abnormal cerebellar maturation, such as
delayed motor development, higher incidences of hypotonia,
and increased activity levels (Neuberger et al., 1999; Rogan
and Gladen, 1992). Moreover, rodents prenatally exposed to
TCDD exhibit delayed development of the righting reflex and
impaired rotarod performance, which are behaviors normally
dependent upon proper cerebellar development and function
(Thiel et al., 1994). Gestational TCDD exposure has been
shown to alter the developmental expression profile of Sp1,
a zinc finger transcription factor critical for neuronal growth
and differentiation in the cerebellar and cerebral cortices
(Nayyar et al., 2002). Our laboratory has previously reported
that TCDD significantly reduced [3H]-thymidine incorporation
in granule neuroblasts, which was accompanied by a modest
decrease in cell viability, suggesting abnormal cell cycle
activity and cell loss (Williamson et al., 2005). These
1Present address: Department of Hospital Laboratories, University of
Massachusetts Memorial Hospital, Worcester, MA 01605.
2To whom correspondence should be addressed at Box EHSC Department
of Environmental Medicine, University of Rochester Medical Center,
Rochester, NY 14642. Fax: (585) 276-0453. E-mail: Lisa_Opanashuk@urmc.
Loretta L. Collins and Mary A. Williamson contributed equally to this work.
? The Author 2008. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
For Permissions, please email: email@example.com
by guest on June 4, 2013
observations raised the hypothesis that TCDD disrupts the
process of GNP differentiation during cerebellar development.
Numerous dynamic cellular processes including, prolifera-
tion, differentiation, migration, and apoptosis are essential for
proper cerebellar development (Altman and Bayer, 1997;
Encha-Razavi and Sonigo, 2003; White and Barone, 2001).
Following proliferation in the superficial external granular
layer (EGL), which peaks between postnatal days (PND) 5–7,
(Miyazawa et al., 2000), become postmitotic, and migrate into
the deep zone of the EGL. Postmitotic, premigratory, and
migrating GNPs elaborate neurites and express the neuron-
specific cytoskeletal protein, bIII-tubulin (Tuj1) (Przyborski
and Cambray-Deakin, 1997), as well as TAG-1, a cell surface
glycoprotein (Furley et al., 1990). Neuroblasts then migrate
inward to form the internal granule cell layer (IGL) of the
cerebellar cortex by PND21 (Hatten, 1999). In the IGL, granule
neuroblasts terminally differentiate and undergo synaptogene-
sis, which is characterized by polysialic acid neural cell
adhesion molecule (PSA-NCAM) and GABAAa6 receptor
(GABARAa6) expression (Rutishauser and Landmesser, 1996;
Zheng et al., 1993). Therefore, the adult cerebellar cortex has
three highly defined layers, the molecular layer (ML), Purkinje
layer, and IGL (Altman and Bayer, 1997). Disruption at any
stage of this tightly regulated developmental process may alter
subsequent cellular events and result in permanent deficits.
The proper timing and balance between cell proliferation and
cell death is crucial in regulating the number and types of cells
present during central nervous system development (Encha-
Razavi and Sonigo, 2003). Two discrete waves of apoptosis
occur during development of the rodent cerebellum. In the rat,
the first occurs within the EGL and peaks at PND10–12,
coinciding with the peak period of neuroblast migration from
the EGL to the IGL (White and Barone, 2001). The second
wave of apoptosis reportedly occurs in the IGL around PND21
(White and Barone, 2001), which is also a time of extensive
synaptogenesis. Developmental apoptosis is essential for the
establishment of final cell numbers and proper neuroanatomical
connections in the mature brain.
This study tested the hypothesis that TCDD exposure
interferes with cerebellar development by disrupting granule
neuroblast maturation. We examined early and late GNP
differentiation in mice following TCDD exposure during
a critical period of neurogenesis that coincides with peak
AhR expression. Cerebellar DNA content, bromodeoxyuridine
(BrdU) incorporation, and terminal transferase deoxyuridine
triphosphate nick-end labeling (TUNEL) were measured as
indications of changes in cell proliferation/death and cell fate in
the cerebellum following oral exposure to TCDD on PND6.
Apoptosis was also estimated by examining expression of the
proapoptotic Bax protein (Krajewska et al., 2002). Differen-
tiation was investigated by monitoring neurite outgrowth and
the temporally expressed developmental markers TAG-1, p27,
bIII-tubulin, PSA-NCAM, and GABARAa6. Our results
suggest that TCDD interferes with granule neuroblast matura-
tion. The dysregulation of GNP differentiation associated with
TCDD exposure during neurogenesis appeared to reduce final
cell number. Interestingly, AhR?/?mice also exhibited an
abnormal cerebellar phenotype in the absence of TCDD
exposure. Therefore, this study raises interesting questions
regarding the molecular mechanism of TCDD neurotoxicity as
well as the endogenous function of AhR in the cerebellum.
MATERIALS AND METHODS
Reagents. TCDD was obtained from Cambridge Isotopes (Cambridge,
MA) and solubilized in dimethyl sulfoxide (DMSO). Triton X-100, bovine
serum albumin (BSA), and DMSO were purchased from Sigma (St Louis,
MO). Dulbecco’s phosphate buffered saline (DPBS) was purchased from Gibco
(Grand Island, NY).
129-Ahrtm1Bra) were obtained from Jackson Laboratories (Bar Harbor, ME).
Mice were maintained on a 12-h light/dark cycle with food and water provided
ad libitum and kept in accordance with the guidelines set by the University of
Rochester University Committee on Animal Resources and the American
Association for Laboratory Animal Science. Both male and female mice were
analyzed in the following experiments, with no sex-specific differences in the
effects of TCDD on cerebellar development observed.
animals. C57BL/6J and AhR?/?
In vivo TCDD exposure. Male and female mice were gavaged with 1.0
lg/kg TCDD dissolved in olive oil (vehicle) or with vehicle alone on PND6.
Animals from at least three separate litters were subjected to each exposure
condition. For all experiments, one male and one female pup per litter were
analyzed for each exposure condition. Differences between males and females
were not observed. A subset of animals was perfused via cardiac puncture, at
the appropriate exposure endpoint, with saline followed by 4% paraformalde-
hyde. The tissue was then processed for immunohistochemical analysis.
Separate animals were anesthetized with CO2on PND7, 8, 10, or 21, and
cerebella were quickly removed and frozen at ?80?C until they were processed
for immunoblot analysis or DNA quantification.
Quantification of DNA content. DNA levels were measured using a dye-
binding method (Labarca and Paigen, 1980) modified as follows. Tissues were
homogenized briefly in 50mM sodium phosphate, 2M NaCl, 2mM ethyl-
enediaminetetraacetic acid (pH 7.4) (Polytron PT 1200C, Brinkmann Instru-
ments, Westbury, NY). Hoechst 33258 (Sigma) was added to samples at a final
concentration of 1 lg/ml and samples were then read in a spectrofluorometer
(Molecular Devices, Sunnyvale, CA) using an excitation wavelength of 356 nm
and an emission wavelength of 458 nm. The amount of DNA in each sample
was extrapolated using linear regression with purified calf thymus DNA
(Sigma) as a standard.
Western blot analysis. Cerebellar tissue was homogenized in PBS
containing 0.1% Triton X-100 and antiprotease cocktail (Roche Molecular
Biochemical, Manheim, Germany). Protein concentrations were determined by
the microBCA assay (Pierce, Rockford, IL). Proteins (20–75 lg) were
fractionated on 7% acrylamide gels and transferred to Immun-Blot PVDF
membranes (BioRad, Hercules, CA). Membranes were blocked with 5%
powdered milk containing 0.2% Tween-20 and probed with antibodies specific
for p27 (1:2500; Santa Cruz, Plymouth Meeting, PA), GABARAa6(1:250;
Chemicon, Temecula, CA), or Bax (1:5000; Chemicon, Temecula, CA),
overnight at 4?C. Membranes were then probed with the appropriate
horseradish peroxidase-conjugated secondary antibodies (1:5000; Jackson
Immunoresearch, Westgrove, PA) for 2 h, at room temperature. Proteins were
visualized using a chemiluminescent substrate (Amersham Biosciences,
Piscataway, NJ). All immunoblots were stripped and reprobed with anti-b-
actin (1:5000; Sigma) to confirm equal protein loading. GABARAa6, p27, and
COLLINS ET AL.
by guest on June 4, 2013
of GNPs in the IGL (Gault and Siegel, 1997) demonstrated that
GABARAa6levels were elevated following TCDD exposure.
This observed increase in GABARAa6protein may appear to
contradict our in vivo results of reduced expression at PND21.
There are possible explanations to reconcile these differences.
The culture system includes KCl to mimic excitatory input that
is required for terminal differentiation of precursors into
functional granule neurons, but it does not preserve cellular
interactions within the cerebellum. Moreover, the TCDD insult
in culture does not precisely mimic the temporal exposure
paradigm in our animal studies, which target precursors at
earlier stages. A more logical reason for this discrepancy in
GABARAa6expression is that the impact of TCDD on GNP
differentiation in the EGL could prevent cells from achieving
the proper spatiotemporal context necessary for survival.
Therefore, fewer mature granule cells would exist and/or
express GABARAa6 protein at later stages of development
in vivo. Alternatively, if mossy fiber input to the cerebellar cortex
is perturbed, then terminal neuronal differentiation into
a functional granule cell could be compromised. Future
investigations are necessary to ascertain the impact of TCDD
on terminal neuronal differentiation in the IGL. Nevertheless,
our data support the concept that TCDD promotes GNP
differentiation at various developmental stages, which could
adversely impact the final cellular profiles in the mature
The central hypothesis of our research is that AhR
participates in granule cell neurogenesis, which is disrupted
by TCDD. This study suggests that an initial TCDD insult
during the GNP expansion phase produces long-term alter-
ations in cerebellar cell composition. Reduced cerebellar cell
number, inferred from DNA content analysis, along with
decreased expression of the late-stage differentiation marker
GABARAa6on PND21, indicates that GNPs are lost earlier in
development. Alternatively, lower GABARAa6 expression
could reflect compromised functional differentiation, which
may also involve a TCDD-mediated impairment of critical
signaling from Purkinje cells or mossy fibers that serve to
integrate granule neurons into the circuitry. Interestingly,
analysis of cerebella from adult AhR?/?mice revealed
a reduction in DNA content and GABARAa6 expression.
These observations suggest that an AhR deficiency leads to
abnormal cerebellar cytoarchitecture and compromised granule
neuron maturation or maintenance. Moreover, they raise
interesting possibilities regarding physiological roles for AhR
in neurogenesis and the manner in which TCDD may interfere
with this process.
We postulate that TCDD exposure disrupts the intrinsic
cellular program that controls granule cell neurogenesis
through the inappropriate activation or suppression of AhR
function. Our data suggest that early developmental insult leads
to later-stage defects that may have long-term effects on
formation of the cerebellum and ultimately on function. Based
on defects observed in AhR?/?mice, we propose that AhR
signaling is involved in guiding neuronal maturation or
survival in the cerebellum. Future studies are necessary to
gain mechanistic insight into the adverse impact of TCDD on
GNP maturation by investigating whether TCDD exposure
leads to improper modulation of AhR-mediated signaling
during critical developmental periods.
National Institutes of Health grants (ES013512, ES09430,
P30 ES01247, and T32 ES07026).
We would like to thank Dr. Emmanuel DiCicco-Bloom
(UMDNJ) for his very helpful discussions and suggestions
regarding this study. We also appreciate the generosity of Drs.
Robert Gross and Michael O’Reilly for the use of their
microscopy facilities. Jason Walrath is acknowledged for
maintenance of the AhR?/?mouse colony.
Akahoshi, E., Yoshimura, S., and Ishihara-Sugano, M. (2006). Over-expression
of AhR (aryl hydrocarbon receptor) induces neural differentiation of
Neuro2a cells: neurotoxicology study. Environ. Health 5, 24.
Altman, J., and Bayer, S. A. (1997). Development of the Cerebellar System
in Relation to its Evolution, Structure, and Functions. CRC Press, Boca
Barnes-Ellerbe, S., Knudsen, K. E., and Puga, A. (2004). 2,3,7,8-Tetrachlor-
odibenzo-p-dioxin blocks androgen-dependent cell proliferation of LNCaP
cells through modulation of pRB phosphorylation. Mol. Pharmacol. 66,
Burgoyne, R. D., Cambray-Deakin, M. A., Lewis, S. A., Sarkar, S., and
Cowan, N. J. (1988). Differential distribution of beta-tubulin isotypes in
cerebellum. EMBO J. 7, 2311–2319.
Cheng, Y., Black, I. B., and DiCicco-Bloom, E. (2002). Hippocampal granule
neuron production and population size are regulated by levels of bFGF. Eur.
J. Neurosci. 15, 3–12.
Dong, W., Teraoka, H., Yamazaki, K., Tsukiyama, S., Imani, S., Imagawa, T.,
Stegeman, J. J., Peterson, R. E., and Hiraga, T. (2002). 2,3,7,8-
Tetrachlorodibenzo-p-dioxin toxicity in the zebrafish embryo: Local
circulation failure in the dorsal midbrain is associated with increased
apoptosis. Toxicol. Sci. 69, 191–201.
Durbec, P., and Cremer, H. (2001). Revisiting the function of PSA-NCAM in
the nervous system. Mol. Neurobiol. 24, 53–64.
Encha-Razavi, F., and Sonigo, P. (2003). Features of the developing brain.
Childs Nerv. Syst. 19, 426–428.
Furley, A. J., Morton, S. B., Manalo, D., Karagogeos, D., Dodd, J., and
Jessell, T. M. (1990). The axonal glycoprotein TAG-1 is an immunoglobulin
superfamily member with neurite outgrowth-promoting activity. Cell 61,
Gao, W. O., Heintz, N., and Hatten, M. E. (1991). Cerebellar granule cell
neurogenesis is regulated by cell-cell interactions in vitro. Neuron 6,
DIOXIN DISRUPTS GRANULE CELL NEUROGENESIS
by guest on June 4, 2013
Gault, L. M., and Siegel, R. E. (1997). Expression of the GABAA receptor
delta subunit is selectively modulated by depolarization in cultured rat
cerebellar granule neurons. J. Neurosci. 17, 2391–2399.
Gu, Y. Z., Hogenesch, J. B., and Bradfield, C. A. (2000). The PAS superfamily:
Sensors of environmental and developmental signals. Annu. Rev. Pharmacol.
Toxicol. 40, 519–561.
Hahn, M. E. (2002). Aryl hydrocarbon receptors: Diversity and evolution.
Chem. Biol. Interact. 141, 131–160.
Hankinson, O. (1995). The aryl hydrocarbon receptor complex. Annu. Rev.
Pharmacol. Toxicol. 35, 307–340.
Hanlon, P. R., Ganem, L. G., Cho, Y. C., Yamamoto, M., and Jefcoate, C. R.
(2003). AhR- and ERK-dependent pathways function synergistically to
mediate 2,3,7,8-tetrachlorodibenzo-p-dioxin suppression of peroxisome
proliferator-activated receptor-gamma1 expression and subsequent adipocyte
differentiation. Toxicol. Appl. Pharmacol. 189, 11–27.
Hatten, M. E. (1999). Central nervous system neuronal migration. Annu. Rev.
Neurosci. 22, 511–539.
p-dioxin (TCDD) inhibition of coronary development is preceded by
a decrease in myocyte proliferation and an increase in cardiac apoptosis.
Teratology 64, 201–212.
Juan, G., Traganos, F., James, W. M., Ray, J. M., Roberge, M., Sauve, D. M.,
Anderson, H., and Darzynkiewicz, Z. (1998). Histone H3 phosphorylation
and expression of cyclins A and B1 measured in individual cells during their
progression through G2 and mitosis. Cytometry 32, 71–77.
Kolluri, S. K., Weiss, C., Koff, A., and Gottlicher, M. (1999). p27(Kip1)
induction and inhibition of proliferation by the intracellular Ah receptor in
developing thymus and hepatoma cells. Genes Dev. 13, 1742–1753.
Krajewska, M., Mai, J. K., Zapata, J. M., Ashwell, K. W., Schendel, S. L.,
Reed, J. C., and Krajewski, S. (2002). Dynamics of expression of apoptosis-
regulatory proteins Bid, Bcl-2, Bcl-X, Bax and Bak during development of
murine nervous system. Cell Death Differ. 9, 145–157.
Labarca, C., and Paigen, K. (1980). A simple, rapid, and sensitive DNA assay
procedure. Anal. Biochem. 102, 344–352.
Lee, J. E. (1997). Basic helix-loop-helix genes in neural development. Curr.
Opin. Neurobiol. 7, 13–20.
Matikainen, T., Perez, G. I., Jurisicova, A., Pru, J. K., Schlezinger, J. J.,
Ryu, H. Y., Laine, J., Sakai, T., Korsmeyer, S. J., Casper, R. F., et al. (2001).
Aromatic hydrocarbon receptor-driven Bax gene expression is required for
premature ovarian failure caused by biohazardous environmental chemicals.
Nat. Genet. 28, 355– 360. [see comment].
Miyazawa, K., Himi, T., Garcia, V., Yamagishi, H., Sato, S., and Ishizaki, Y.
(2000). A role for p27/Kip1 in the control of cerebellar granule cell precursor
proliferation. J. Neurosci. 20, 5756–5763.
Mooney, S. M., and Miller, M. W. (2001). Effects of prenatal exposure to
ethanol on the expression of bcl-2, bax and caspase 3 in the developing rat
cerebral cortex and thalamus. Brain Res. 911, 71–81.
Nayyar, T., Zawia, N. H., and Hood, D. B. (2002). Transplacental effects of
2,3,7,8-tetrachlorodibenzo-p-dioxin on the temporal modulation of Sp1 DNA
binding in the developing cerebral cortex and cerebellum. Exp. Toxicol.
Pathol. 53, 461–468.
Neuberger, M., Rappe, C., Bergek, S., Cai, H., Hansson, M., Ja ¨ger, R.,
Kundi, M., Lim, C. K., Wingfors, H., and Smith, A. G. (1999). Persistent
health effects of dioxin contamination in herbicide production. Environ. Res.
Nowak, S. J., and Corces, V. G. (2004). Phosphorylation of histone H3: A
balancing act between chromosome condensation and transcriptional
activation. Trends Genet. 20, 214–220.
Opanashuk, L. A., Pauly, J. R., and Hauser, K. F. (2001). Effect of nicotine
on cerebellar granule neuron development. Eur. J. Neurosci. 13, 48–56.
Przyborski, S. A., and Cambray-Deakin, M. A. (1997). Profile of glutamylated
tubulin expression during cerebellar granule cell development in vitro. Brain
Res. Dev. Brain Res. 100, 133–138.
Strobek,M., andKnudsen,E.S. (2002).Roleofthe arylhydrocarbonreceptor
in cell cycle regulation. Toxicology 181-182, 171–177.
Ray, S. S., and Swanson, H. I. (2003). Alteration of keratinocyte differentiation
and senescence by the tumor promoter dioxin. Toxicol. Appl. Pharmacol.
Rogan, W. J., and Gladen, B. C. (1992). Neurotoxicology of PCBs and related
compounds. Neurotoxicology 13, 27–35.
Rutishauser, U., and Landmesser, L. (1996). Polysialic acid in the vertebrate
nervous system: A promoter of plasticity in cell-cell interactions. Trends
Neurosci. 19, 422–427.
Thiel, R., Koch, E., Ulbrich, B., and Chahoud, I. (1994). Peri- and postnatal
exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin: Effects on physiological
development, reflexes, locomotor activity and learning behaviour in Wistar
rats. Arch. Toxicol. 69, 79–86.
Vogel, M. W. (2002). Cell death, Bcl-2, Bax, and the cerebellum. Cerebellum
White, L. D., and Barone, S., Jr. (2001). Qualitative and quantitative estimates
of apoptosis from birth to senescence in the rat brain. Cell Death Differ. 8,
Williamson, M. A., Gasiewicz, T. A., and Opanashuk, L. A. (2005). Aryl
hydrocarbon receptor expression and activity in cerebellar granule neuro-
blasts: Implications for development and dioxin neurotoxicity. Toxicol. Sci.
Yamamoto, M., Hassinger, L., and Crandall, J. E. (1990). Ultrastructural
localization of stage-specific neurite-associated proteins in the developing rat
cerebral and cerebellar cortices. J. Neurocytol. 19, 619–627.
Zheng, T., Santi, M. R., Bovolin, P., Marlier, L. N., and Grayson, D. R. (1993).
Developmental expression of the alpha 6 GABAA receptor subunit mRNA
occurs only after cerebellar granule cell migration. Brain Res. Dev. Brain
Res. 75, 91–103.
COLLINS ET AL.
by guest on June 4, 2013