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Imidacloprid exposure suppresses neural crest cells generation during early chick embryo development Chao-jie Wang, Guang Wang, Xiao-yu Wang, Meng Liu, Manli Chuai, Kenneth Ka-ho Lee, Xiao-Song He, Da-xiang Lu, and xuesong Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01478 • Publication Date (Web): 19 May 2016 Downloaded from http://pubs.acs.org on May 20, 2016
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Imidacloprid exposure inhibits cranial osteogenesis of chick embryos. 829x761mm (150 x 150 DPI)
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Imidacloprid exposure retards the development of gastrulating chick embryos. 822x790mm (150 x 150 DPI)
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Imidacloprid exposure represses production of cranial NCCs. 792x780mm (150 x 150 DPI)
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Imidacloprid exposure suppresses the apoptosis of cranial NCCs 796x773mm (150 x 150 DPI)
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Imidacloprid inhibits Msx1 expression in the cranial neural tubes of chick embryos. 799x787mm (150 x 150 DPI)
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Imidacloprid represses BMP4 expression in the cranial neural tubes of chick embryos. 799x799mm (150 x 150 DPI)
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Imidacloprid exposure up-regulates Cad6B expression 799x799mm (150 x 150 DPI)
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Imidacloprid exposure represses NCCs production in cranial neural tubes explants 799x799mm (150 x 150 DPI)
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A proposed model depicting how imidacloprid exposure could lead to defective cranial NCCs generation. 799x799mm (150 x 150 DPI)
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Primer sets used in the RT- PCR analysis. 799x811mm (150 x 150 DPI)
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Imidacloprid exposure suppresses neural crest cells generation during early
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chick embryo development
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Chao-jie Wang†∥, Guang Wang†∥, Xiao-yu Wang†, Meng Liu†, Manli Chuai§, Kenneth
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Ka Ho Lee#, Xiao-Song He‡, Da-xiang Lu⊥, Xuesong Yang*†
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†
Division of Histology & Embryology, Key Laboratory for Regenerative Medicine of
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the Ministry of Education, Medical College, Jinan University, Guangzhou 510632,
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China §
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Division of Cell and Developmental Biology, University of Dundee, Dundee, DD1 5EH, UK
10 #
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Key Laboratory for Regenerative Medicine of the Ministry of Education, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin, Hong Kong
12 ‡
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Research Academy of Environmental Sciences, Beijing 100012, China
14
15
State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese
⊥
Division of pathophysiology, Medical College, Jinan University, Guangzhou 510632, China
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17 ∥
contribute to the work equally
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*
The corresponding author: Xuesong Yang (
[email protected])
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Abstract
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Imidacloprid is a neonicotinoid pesticide that is widely used in the control pests
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found on crops and fleas on pets. However, it is still unclear whether imidacloprid
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exposure could affect early embryo development - despite some studies having been
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conducted on the gametes. In this study, we demonstrated that imidacloprid exposure
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could lead to abnormal craniofacial osteogenesis in the developing chick embryo.
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Cranial neural crest cells (NCCs) are the progenitor cells of the chick cranial skull.
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We found that the imidacloprid exposure retards the development of gastrulating
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chick embryos. HNK-1, PAX7 and Ap-2ɑ immunohistological stainings indicated that
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cranial NCCs generation was inhibited after imidacloprid exposure. Double
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immunofluorescent staining (Ap-2ɑ and PHIS3 or PAX7 and c-Caspase3) revealed
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that imidacloprid exposure inhibited both NCC proliferation and apoptosis. In
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addition, it inhibited NCCs production by repressing Msx1 and BMP4 expression in
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the developing neural tube and by altering expression of EMT-related adhesion
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molecules (Cad6B, E-Cadherin and N-cadherin) in the developing neural crests. We
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also determined that imidacloprid exposure suppressed cranial NCCs migration and
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their ability to differentiate. In sum, we have provided experimental evidence that
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imidacloprid exposure during embryogenesis disrupts NCCs development which in
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turn causes defective cranial bone development.
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Key words: imidacloprid, cranial neural crest cells, osteogenesis, cell proliferation,
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apoptosis.
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Introduction
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Imidacloprid is one of two types of neonicotinoid pesticides that have a toxic
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effect on insects by mimicking nicotine. Nicotine exists naturally in many plants such
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as tobacco and it is toxic to insects. Presently, imidacloprid is widely used to control
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sucking insects, termites and fleas (1, 2). It is available in liquid and granule forms and
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has been on sale in US since 1994. As a systemic insecticide, it is usually sprayed on 2
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soil, water
and leaves which then disperse throughout the plant stem, leaves, fruit
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and flowers
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nervous system. Human are also exposed to imidacloprid which get through their eyes
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and skin or ingested when handling the pesticide or their treated pets. If imidacloprid
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was ingested via tainted vegetables and fruits, it would disrupt nerve signal
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transduction
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imidacloprid on the early developing embryo despite reports that it reduces
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reproductive ability in human (7-9).
(4, 5)
. Once the insects eat the treated plants, imidacloprid damages their
(6)
. However, we still do not know the potential toxic effect of
Neural tube defects (NTDs) is a common severe congenital abnormality that
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(10)
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affects 0.5–2/1000 confirmed pregnancies worldwide
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developed neural tubes, where the neural tube remains open at birth, which may occur
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in the brain or spinal cord. Both genetics and the external environment can cause
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NTDs
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NTDs in chick embryo (unpublished data). NTDs can also occur in the company of
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other birth defects, especially malformations associated with the neural crest cells
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(NCCs)(12). NCCs have remarkable ability to migrate extensively within the
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developing embryo and differentiate like pluripotent cells into many tissue derivatives
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(13)
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the fourth embryonic germ layer. NCCs derive from the borders of the neural plate
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and undergo the process of neural induction, delamination, epithelial-mesenchymal
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transition (EMT) and migration to give rise to most tissues in the embryo - including
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the peripheral nervous system
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craniofacial skeleton, cerebral ganglions, enteric nervous system and schwann cells (15,
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16)
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of transcriptional and epigenetic genes (13) and abnormal development of NCCs could
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results in neural tube defects, atrioventricular septal defect, patent ductus arteriosus,
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and Waardenburg's syndrome
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concentrations of neonicotinoid pesticides could significant affect early zebrafish
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development (20).
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. NTDs refer to improperly
(10, 11)
. In our previously study, we found exposing imidacloprid could lead to
. It is because of these remarkable abilities that NCCs are sometimes referred to as
(14)
. In the cranial region, NCCs form all of the
. It has been reported that NCC generation is spatiotemporally regulated by a series
(17-19)
. In zebrafish, it has been demonstrated that high
Scientists are now more aware of the toxic side-effects of pesticides on the 3
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developing embryo, especially dysplasia caused by abnormal NCCs development.
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The NCCs are especially sensitive and vulnerable to external insults because these
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cells migrate and differentiation extensively within the embryo during development. It
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has been reported that hyperglycemia exposure impaired the ability of NCCs to
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differentiate into cranial bone
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embryo model
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cranial NCCs and elucidate the cellular and molecular mechanism involved.
(22, 23)
(21)
. In this study, we have used the classical chick
to investigated the toxic effects of imidacloprid exposure on the
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Materials and Methods
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Chick embryos
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Fertilized leghorn eggs were acquired from the Avian Farm of South China
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Agriculture
University
(Guangzhou,
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Hamburger-Hamilton (HH) stage 0
China).
The
fertilized
eggs,
at
(24)
, were incubated in the presence of DMSO
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(control) or 500µM imidacloprid ( Santa cruz ) inside a humidified incubator (Yiheng
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Instrument, Shanghai, China) set at 38°C and 70% humidity. The embryos from these
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incubated eggs were harvested for analysis when they reached the desired
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developmental (HH)(25) stage .
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Alizarin red staining of whole embryos
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The craniofacial skeleton was visualized in 12-day-old chick embryos by staining
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with alizarin red dyes (Solarbio, Beijing, China), as previously described . Briefly, the
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embryos were fixed in 95% ethanol for 3 days, and then the skin and viscera were
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carefully removed before post-fixation for an additional 1 day. The embryos were then
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pretreated in 0.5% KOH (Jinan University, Guangzhou, China) for 48 hours before
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they were stained with the alizarin red dye suspended in 0.5% KOH for 3 days.
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Finally, the embryos were cleared in a graded series of glycerol. The craniofacial
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skeleton was photographed using a stereomicroscope (Olympus MVX10, Japan).
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Immunofluorescent staining
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Chick embryos were harvested after incubation and fixed in 4% PFA ( Newprobe
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Bioscience Technology Co.,Ltd, Beijing, China) overnight at 4oC. Whole-mount
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embryo immunofluorescent staining or primary explant was performed using the
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following antibodies: phospho-Histone3 (pHIS3; 1:400, Santa Cruz, USA), Pax7
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(1:300, DSHB, USA), AP-2α (1:100, DSHB, USA), HNK-1 (1:200, Sigma, USA),
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c-Caspase3 (1:200, Cell Signaling Technology, USA) and Laminin (1:100, Sigma,
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USA). Briefly, the fixed chick embryos were incubated with the primary antibodies at
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4oC overnight on a shaker. After extensive rinsing in PBST (0.1% Tween-20), the
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embryos were treated with the corresponding Alexa Fluor® 555 or 488 labelled
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secondary antibody (1:1000, Invitrogen, USA) at 4oC overnight on a shaker. For
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double immunofluorescent staining, the antibodies were incubated one after the other.
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All the embryos were later counterstained with DAPI (1:1000, Invitrogen) at room
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temperature for 1 hour. Subsequently, the stained embryos were sectioned at 10µm
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using a cryostat (Leica CM1900).
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In situ hybridization Whole-mount in situ hybridization of chick embryos was performed according to (26)
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standard in situ hybridization protocol
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synthesized to detect Msx1, BMP4 and Cad6B mRNAs. [Msx1: forward:
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GGATGAAGGGAGCGGAGAAGTC reverse: ATTAACCCTCACTAAAGGGCGA
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GGAGGAGAGCGACAAAC. BMP4: forward: TTATAAAAGCTTGCGGCCGCAG
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AATATATGTTTGGCTGCGAAGG
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AAAGGGCTGGTTGGTGGAGTTGAG. Cad6B: forward: CAAGTGGAA GATGTA
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GATGAACCCC
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TCTGCG (GEISHA ISH Analysis)]. Whole-mount stained embryos were
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photographed and then frozen sections at thickness of 16 µm were prepared from
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these embryos for histological analysis.
reverse:
reverse:
. Digoxigenin-labeled probes were
GCTCTAGAAATTAACCCTCACT
ATTAACCCTCACTAAAGGTTCTGGCACAATGTC
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RNA isolation and qPCR analysis 5
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Total RNA was isolated from HH10 chick embryos using a Trizol kit (Invitrogen,
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USA) according to the manufacturer's instructions. First-strand cDNA synthesis and
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SYBR® Green qPCR assay were performed using a PrimeScriptTM RT reagent kit
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(Takara, Japan). All specific primers used are described in Supplementary Figure S1
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(27, 28)
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S1000TM (Bio-Rad, USA) and ABI 7000 thermal cyclers, respectively. The
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housekeeping gene GAPDH was run in parallel to confirm that equal amounts of
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RNA were used in each reaction. The ratio between intensity of the fluorescently
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stained bands corresponding to genes and GAPDH was calculated to quantify the
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level of the transcripts for those genes mRNAs. The RT-PCR result was representative
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of three independent experiments.
. Reverse transcription and amplification reactions were performed in Bio-Rad
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Primary NCC cultures
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NCCs were prepared from cranial neural tubes isolated from chick embryos,
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according to the methods previously described (29). Briefly, fertilized chick eggs were
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incubated until the 7-9 somite stage (HH9). The neural tube was then dissected from
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the head region of the embryo and explanted into 3.5mm dishes containing DMEM
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and 10% FBS for 6 hours at 37oC and 5% CO2, to allow the explants adhere. The
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explants were incubated until a few NCCs were observed migrating out of the neural
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tubes and then 1ml of culture medium containing DMSO (control) or 500µM
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imidacloprid (dissolved in DMSO,
