Transcript Hurem S. et al - laboratory of radiation biology

Effects of low dose rate gamma
irradiation during early life stages
in the zebrafish model
Selma Huremab, Hans Christian Teienac, Ole Christian Lindac, Dag Anders Bredeac, Turid
Hertel-Aasac, Lene Sørlie Heierac, Vidar Bergab, Leonardo Martinab, Deborah Oughtonac, Brit
Salbuac, Peter Alestrømab, Jan Ludvig Lycheab
aCentre
for Environmental Radioactivity (CERAD CoE), Norway
University of Life Sciences, Vetbio, Campus Oslo, Norway
cNorwegian University of Life Sciences, Department of Environmental Sciences, Campus Ås, Norway
bNorwegian
[email protected]
Meeting at St. Petersburg, Russia, 2-6 June 2015
________________________________________________
IV International conference
MODERN PROBLEMS OF GENETICS, RADIOBIOLOGY,
RADIOECOLOGY, AND EVOLUTION
CERAD CoE – Centre for environmental
radioactivity
UMB 3 Radiosensitivity
M. edulis
C. elegans
A. thaliiana
E. fetida
S. salar
D. magna
C. reinhardtii
L. minor
D. rerio
M. musculus
UMB 5 Transgenerational hereditary,
reproductive and epigenetic effects
The Zebrafish model (Danio rerio)
Large quantity of embryo production
External embryogenesis
Optical transparency
Extensive information network
Various HTS methods available
Relatively short generation interval
Shares 70% of the genes with humans
Howe et al. Nature 2013
The Zebrafish model (Danio rerio)
Sensitive life stages
Embryogenesis
ZF: < 72 hours
Mammals: weeks
Gametogenesis
ZF: 2-3 weeks
Mammals: 18 weeks
embryology.med.unsw.edu.au
The two main stages in the life cycle when complete de-methylation
followed by gradual re-methylation occur
How does the zebrafish embryo program its
developmental gene expression pattern?
ZGA
Kimmel et al. Dev. Dynamics 1995
• 3 hours of development until zygotic genome activation (ZGA)
• Essentially no transcription during this period
• Also a great opportunity to test the hypothesis of an epigenetic prepatterning of developmental gene expression
The Zebrafish model (Danio rerio)
Transgenerational studies example
The goal of transgenerational epigenetic studies is to correlate
epigenetic profiles at specific developmental stages to patterns of gene
expression across stages
•
This might enable us to find state-to-state epigenetic patterns that are predictive of
biological process (Gene Ontology) and anatomically-specific (ZFIN) expression
and further…
Allow us to create an atlas of zebrafish developmental epigenetics
with prediction value
And apply it to other species or fields
Marking of the genome by histone modifications
before, during and after onset of ZGA
Non-expressed genes
RNA-seq
(Aanes et al., 2011. Genome Res.)
Maternal-zygotic genes
Zygotic genes
Epigenetic patterning of zebrafish developmental gene
expression before ZGA onset
Pre-MBT
MBT
Post-MBT
Pre-patterning
Priming
Activation
H3K4me3
H3K36me3
H3K27me3
H3K9me3
Hypomethylation
RNAPII
TSS
 H3K4me3, H3K9me3 and H3K27me3 mark the zebrafish genome before ZGA
 The marked genes are transcriptionally silent and developmentally regulated
 H3K4me3 marking is linked to propensity for transcriptional activation after
ZGA
 The data suggest a developmental instructive function of epigenetic marking
before onset of ZGA
Lindeman, Andersen et al., 2011. Dev Cell
Epigenetic patterning of zebrafish developmental gene
expression before ZGA onset
Pre-MBT
MBT
Post-MBT
Pre-patterning
Priming
Activation
H3K4me3
H3K36me3
H3K27me3
H3K9me3
Hypomethylation
RNAPII
TSS
Where do chromatin modifications come from?
?
Data currently supports 2 models:
• transgenerational
inheritance of chromatin marks?
• post-fertilization removal & reconstitution of epigenetic marks based on
DNA sequence rather than through a copy of histone modifications?
 Both models are compatible with a de novo epigenetic marking of genes post-fertilization
early life stages
trans-generational effects:
F0, F1, F2, F3
•RNA •RNA
•ChIP •ChIP
•DNA
damage
•oncogen
markers
pathology
•liver
•gonads
•gut
•RNA •RNA
•ChIP •ChIP
•DNA
damage
•oncogen
markers
6 MONTHS
F1
F1
PUBERTY
6 MONTHS
F0
PUBERTY
F0
MBT
zebrafish
PUBERTY
MBT
zEmbryo
96 w
MBT
γrad
F2
pathology
•liver
•gonads
•gut
We are in F2 in our
transgenerational study
Study 1: Dose-response
Aim:
Assess relevant gamma doses for a multigenerational experiment.
Subtask:
Assess potential phenotypic effects and changes in gene expression
at the midblastula transition/ early gastrula stage of development
(exposed 2.5-5.5 hpf).
From previously we know that:
• It takes 3 hours of development (hpf) until zygotic genome activation
(ZGA) in zebrafish
• Essentially no transcription during this period
Methods:
Zebrafish embryo toxicity test (ZFET), OECD
zFET: OECD Test Guideline (TG) 236, Fish Embryo Acute Toxicity (FET) Test, 26 July 2013, http://www.oecdilibrary.org/environment/test-no-236-fish-embryo-acute-toxicity-fet-test_9789264203709-en
RNA-seq
Gamma irradiation
IR start: 2.5 hours post fertilization (hpf)
IR end: 5 days post fertilization (dpf)
Dose-rates: 0.5, 1, 5, 25, 40 mGy/h
Dosimetry: Landauer nanoDot™ OSLD
Analysis: day 2 and 5
Sampling: RNA-seq at 5.5 hpf
AB wild type
Gamma irradiation - FIGARO
1030 cm
1 mGy/h
40 mGy/h
0.5 mGy/h
10 mGy/h
25 mGy/h
Co60
5 mGy/h
332 cm
Control 1
Control 2
Demographic endpoints
1. Mortality
2. Hatching success
3. Deformities
Exposure
group mGy/h
0
0.5
1
5
25
40
N
embryo Analyzed day 2 Analyzed day 5
384
191
193
390
188
202
377
190
187
379
189
190
403
214
189
343
145
198
Mortality
Mortality at 5 dpf
25%
Indicators:
1.
Coagulation of
fertilized eggs
2.
Lack of somite
formation
3.
Lack or detachment
of the tail bud from
the yolk sack
4.
Lack of heart beat
20%
15%
10%
5%
0%
8.6 %
12.6 %
18.6 %
12.4 %
11.2 %
16.9 %
0
0.5
1
5
25
40
IR dose-rate (mGy/h)
Hatching
Deformities
Most frequently
observed:
1.
Short tail
2.
Lack of pigment
3.
Retardation in
development
4.
Unhatched after
73hpf added to
deformities
RNA-seq samples
Sampling was performed using TRIzol Reagent
(invitrogen). At 5.5hpf, 20 embryos per sample would
have sufficient yield.
Total RNA isolation/ 120 samples
Qiagen kit/ Modified Invitrogen protocol
>200 ng/ul;
260/280 >1.8;
260/230 >2
Agilent 2100 Bioanalyzer RIN (all samples)>8.5;
28S/18S>1
Select samples based on relevance for sequencing and
quality
Samples sent to BGI for RNA-sequencing
Gene expression changes in early
embryos exposed to gamma
Numbers of genes differentially
expressed compared to controls
Dose-rate
Total
0.5 mGy/h 19
Up/down
17/2
5 mGy/h 158
132/26
10 mGy/h 795
589/206
Number of overlapping genes
differentially expressed in exposed
compared to controls
Comparison of 10 and 5 mGy/h
The mechanistic target of rapamycin,
also known as mammalian target of
rapamycin (mTOR) is a protein that in
humans is encoded by
the MTOR gene. Regulates cell
growth, cell proliferation, cell motility,
cell survival, protein synthesis,
and transcription.
Brown et al. Nature 2010
Eukaryotic initiation factors (eIFs) are
proteins involved in the initiation phase
of eukaryotic translation.
No diseases directly observed, but many
illnesses caused by down-regulation of
eIF2 through its upstream kinases. It is
associated with neurodegenerative
diseases.
Jackson et al. Nature 2010
Physiological pathways affected by 5 and 10 mGy/h expsure
Key regulator genes affected by 5 and 10 mGy/h expsure
Analysis and intepretation are still in the initial phase.
Data is compared with data from human studies.
Common responses are suggested between the species.
Some further comparisons…
The mechanistic target of rapamicin (mTOR)
pathway is implicated in the premature
senescence of primary human endothelial
cells exposed to chronic gamma radiation.
Yentrapalli et al., 2013
HNF4A: Hepatic nuclear factor 4 alpha ccurs in stem cells. Associated with
diabetes type 2 and colorectal cancer etc. It may play a role in the development
of the liver kidney and intestines. The ZF ortholog hnf4a.
In B-CLL patients, gene networks around
MYC, HNF1A and HNF4A, YWHAG, NFκB1 and SP1, CEBPA, YWHAG, SATB1 and
RB1 are regulated. Savli et al. Exp Oncol. 2012
Global gene expression responses to low- or
high-dose radiation in a human three-dimensional
tissue model. 175:677-688. Mezentsev et al. 2011.
Radiat Res.
Transgenerational study result comparison
ZF research: Hnf4a is a key regulator in the maintenance of hepatocyte
differentiation and the control of lipid homeostasis. Dongling Zheng et al. BMC
Genomics 2010
In mice, recent in vitro experiments suggest that HNF4a is
indispensable for hepatocyte differentiation.
HNF4alpha-null mice die during embryogenesis.
In our multigenerational study, we found a 100% mortality occuring at the
gastrulation stage in all offspring of parental fish irradiated to a dose-rate of
40 mGy/h during 27 days.
ZF F1-40 offspring, 5,5 - 8hpf. Cytation 3 Imaging
kinetic
MYCN: Excess MYCN associated
with various tumors
CEBPA: Dysregulation associated with
hematopoietic
abnormalities including myeloid leukemia.
This intronless gene encodes a transcription factor
that contains a basic leucine zipper (bZIP) domain.
VetBio
Campus Oslo
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Jan Ludvig Lyche
Peter Aleström
Leonardo M. Martin
Håvard Aanes
Leif Lindeman
Vidar Berg
NRPA
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Terje Christensen
Campus Ås
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Hans-Christian Teien
Ole Christian Lind
Dag Anders Brede
Deborah H. Oughton
Brit Salbu
Turid Hertel-Aas
Lene Sørlie Heier
Thank you for your attention.