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Early Environmental Regulation of Gene
Expression: How Early Experience Exerts a
Sustained Influence on Neuronal Function
[email protected]
Michael J Meaney
Douglas Mental Health University Institute
McGill University
and
Singapore Institute for Clinical Sciences
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Summary
The experience of the child is “biologically
embedded” and serves to influence health and
capacity over the lifespan.
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Summary
The experience of the child is “biologically embedded”
and serves to influence health and capacity over the
lifespan.
This effect is apparent even at the level of the
DNA of the individual; the activity of genes implicated
in brain development and function
are directly regulated by the social environment.
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Summary
The experience of the child is “biologically embedded”
and serves to influence health and capacity over the
lifespan.
This effect is apparent even at the level of the
DNA of the individual; the activity of genes implicated
in brain development and function
are directly regulated by the social environment.
This effect is potentially stable over time; the
imprint of of childhood adversity on the genome
is apparent at later ages, providing a biological basis
for an enduring effect on health and capacity.
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The development of an individual is an active
process of adaptation that occurs within a
social and economic context:
• To resource (food, shelter, safety)
availability.
• To social interactions (e.g., parental
signals).
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The development of an individual is an active
process of adaptation that occurs within a
social and economic context:
• To resource (food, shelter, safety)
availability.
• To social interactions.
This influence is apparent in the epigenetic mechanisms
that regulate genomic structure and function
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Early experience
Abuse
Family strife
Emotional neglect
Harsh discipline
Health Risks
Depression
Drug abuse
Anxiety
Diabetes
Heart disease
Obesity
Developmental Origins of Adult Disease
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Early experience
Abuse
Family strife
Emotional neglect
Harsh discipline
Health Risks
Depression
Drug abuse
Anxiety
Diabetes
Heart disease
Obesity
Individual differences
in neural and endocrine
responses to stress (defensive responses)
How does family life influence health over the life span?
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Early experience
Abuse
Family strife
Emotional neglect
Harsh discipline
Health Risks
Depression
Drug abuse
Anxiety
Diabetes
Heart disease
Obesity
Individual differences
in neural and endocrine
responses to stress
Poverty
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Nutrient Supply
Mate Quality
Violence
Infection
Population density
Parental
investment
Defensive Strategies
Foraging/Metabolism
Reproductive Strategies
Environmental Parental Developmental
condition signal outcome
Robert Hinde: Evolution has shaped the young to use parental
signals as a ‘forecast’ of the quality of the environment
into which they have been born. For most species, there is
no single, optimal phenotype.
Evolutionary biology - Maternal effects
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Health and
Human Capacity
Early experience
Abuse
Family strife
Emotional neglect
Harsh discipline
Individual differences
in neural and endocrine
responses to stress
Adversity
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Environmental regulation of phenotypic variation
What is the biological basis for ‘programming’ effects
whereby environmental signals acting over perinatal
development associate with stable changes in transcription
and complex phenotypes (physiology, behaviour)?
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Summary
• Parental care affects the activity of
genes in the brain that regulate stress
responses, neural development and
reproduction.
• This parental effect involves a form
a “plasticity” at the level of the DNA.
Epigenetics: Any functional change in the genome that
does not involve an alteration of DNA sequence.
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If they ask you anything you don’t know,
just say its due to epigenetics.
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Nucleosome core particle: ribbon traces for the 146-bp DNA phosphodiester backbones
(brown and turquoise) and eight histone protein chains (Luger et al. Nature 1997).
+ -
Prevents TF
binding to DNA
TF binding involves
alteration of
chromatin structure
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Nucleosome core particle: ribbon traces for the 146-bp DNA phosphodiester backbones
(brown and turquoise) and eight histone protein chains (Luger et al. Nature 1997).
+ -
Prevents TF
binding to DNA
TF binding involves
alteration of
chromatin structure
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Acetyl group
B. Turner. Chromatin structure and gene regulation. 2001.
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Nucleosome core particle: ribbon traces for the 146-bp DNA phosphodiester backbones
(brown and turquoise) and eight histone protein chains (Luger et al. Nature 1997).
+ -
Prevents TF
binding to DNA
TF binding involves
alteration of
chromatin structure
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C T A C G T A C T C G G A A T C T C G CH
3 CH
3 CH
3
C T A C G T A C T C G G A A T C T C G
Genetic code is defined by the sequence of four
nucleotides that produce proteins.
RNAs, proteins
Epigenetic effects refer to modifications of the DNA
that alter the activity of the gene, but not its function.
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SIN
SIN3a
CH 3
CH 3
MeCP2/
MBD2
HDAC MeCP2/
MBD2
DNA Methylation can inhibit gene expression
by blocking transcription factor binding
HDAC HDAC: Histone deacetylase
Methylated DNA
binding protein CH 3
CH 3
CH 3
CH 3
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HDAC
B. Turner. Chromatin structure and gene regulation. 2001.
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Nucleosome core particle: ribbon traces for the 146-bp DNA phosphodiester backbones
(brown and turquoise) and eight histone protein chains (Luger et al. Nature 1997).
+ -
Prevents TF
binding to DNA
TF binding involves
alteration of
chromatin structure
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Every cell in your body has the same nuclear genes,
but…?
Multiple phenotypes from a common genotype
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Maternal licking/grooming
Environmental epigenetics hypothesis: Environmental
events activate intracellular signals that remodel
the epigenome, leading to sustained alterations in
the structure and function of the genome, and thus
stable effects on gene transcription.
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Parental care Phenotype
Gene
expression
Epigenetic
marks
Parental signals as a source of phenotypic plasticity?
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Parental care Phenotype
Gene
expression Epigenetic
marks
Parental signals as a source of phenotypic plasticity?
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Maternal licking/grooming
Source of tactile stimulation/nurturance: Enhances
activity of endocrine systems (e.g., GH/IGF) that
promote somatic growth, suppresses those
(glucocorticoids) that inhibit growth
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Variations in maternal care
6 11 1 6 0
50
100
150
200
Fre
qu
en
cy c
ou
nt
% Licking/grooming
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Stress responses
Neural development
Learning & memory
Metabolism
Reproduction (females)
Broad range of parental effects
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Parental care Phenotype
Gene
expression Epigenetic
marks
Parental signals as a source of phenotypic plasticity?
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Stress responses
Neural development
Learning & memory
Metabolism
Reproduction (females)
Broad range of parental effects
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Glucocorticoid Receptor
Hypothalamus
Pituitary
Adrenals
Hippocampus
Glucocorticoids
Stress
(-)
CRF/AVP
ACTH
CRF: corticotropin releasing factor. ACTH: adrenocorticotropin
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Hypothalamus
Pituitary
Adrenals
High LG offspring
Glucocorticoids
(-)
CRF
ACTH
(-)
Hypothalamus
Pituitary
Adrenals
Low LG offspring
Glucocorticoids
(-)
CRF
ACTH
(-)
Individual differences in glucocorticoid receptor
levels lead to altered pituitary-adrenal responses
to stress
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High LG offspring show more modest HPA stress responses
Pre S10 S20 P20 P40 P60 P120 0
200
400
600
800
1000 High LG Low LG
Pla
sm
a A
CT
H (
pg
/ml)
Pre 10 20 40 60 80 100 0
10
20
30
40
50
60
70
80 High LG Low LG
Co
rtic
oste
ron
e (
µg
/dl)
Disruption of hippocampal GR signaling eliminates this
maternal effect
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Hypothalamus
Pituitary
Adrenals
High LG offspring
Glucocorticoids
(-)
CRF
ACTH
(-)
Hypothalamus
Pituitary
Adrenals
Low LG offspring
Glucocorticoids
(-)
CRF
ACTH
(-)
Cross-fostering reveals evidence for direct, postnatal
effects of maternal care
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Hippocampal GR(17) mRNA
1 2 3 4 5 6 7 8 9 1011 12 2 3 4 5 67 8 9
5’ 3’
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DG CA1 CA2 CA3 0.00
0.05
0.10
0.15
High LG
Low LG
Re
lati
ve
O
DU
Hippocampal GR(17) mRNA
*
* * *
1 2 3 4 5 6 7 8 9 1011 12 2 3 4 5 67 8 9
5’ 3’
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1681 ccc
1741 ctctgctagt gtgacacact t1cg2cgcaact c3cgcagttgg 4cggg5cg6cgga ccacccctg7c
1801 ggctctgc8cg gctggctgtc accct9cgggg gctctggctg c10cgaccca11cg ggg12cgggct
1861 c13cgag14cggtt ccaagcct15cg gagtggg16cg gggg17cgggag ggagcctggg agaa
DNA sites that regulate glucocorticoid receptor gene
NGFI-A
GR Promoter 17 Sequence
1 2 3 4 5 6 7 8 910 1112 2 3 4 5 6 78 9
Variable exon 1 region Constant region
5’ 3’
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NGFI-A GR gene
GR mRNA
NGFI-A regulates GR gene expression
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5’ 3’ 0.0
0.2
0.4
0.6
0.8
1.0
Low LG
High LG
c-M
eth
yla
tio
n
*
NGFI-A site
5’…tgcgggggcgggg…3’
NGFI-A
CH 3 CH
3
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Low/Low High/High Low/High High/Low
0.0
0.2
0.4
0.6
0.8
c-m
eth
yla
tio
n
5' CpG region of NGFI-A/RE
5’…tgcgggggcgggg…3’
NGFI-A
CH 3
CH 3
CH 3
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SIN
SIN
CH 3
CH 3
CH 3
CH 3
MeCP2/
MBD2
HDAC CH 3
CH 3
MeCP2/
MBD2
DNA Methylation can inhibit gene expression
by blocking transcription factors binding
HDAC HDAC: Histone deacetylase
Methylated DNA
binding protein
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HDAC
B. Turner. Chromatin structure and gene regulation. 2001.
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High Low
0.0
0.2
0.4
0.6
0.8
1.0
*
0.0
0.2
0.4
0.6
0.8
1.0
An
tib
od
y/I
np
ut
*
Histone
Acetylation
NGFI-A
Association
Increased methylation of the exon 17 GR promoter associates with
decreased H3K9ac, reduced NGFI-A binding and GR expression
0
0.1
0.2
0.3
0.4
GR
IR
(R
OD
-IO
D)
*
GR
Expression
High Low High Low
Effects are reversed with intra-hippocampal infusion of HDAC inhibitor
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Low/Veh Low/TSA High/Veh High/TSA
Basal Stress Stress + 40 Stress + 60 Stress + 90 0
10
20
30
40
50
Co
rtic
oste
ron
e (
µ
g/d
l)
Time (min)
Low/Veh
Low/TSA
High/Veh
High/TSA
Glucocorticoid stress response
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GGAGCCTGGGAGAACCAAGCCTCGGAGTGGGCGGGGGCGGGAG CH
3 CH
3
GGAGCCTGGGAGAACCAAGCCTCGGAGTGGGCGGGGGCGGGAG CH
3
Offspring of Low LG mothers
Offspring of High LG mothers
Lower levels of GR in hippocampus -
Increased stress response
High levels of GR in hippocampus -
Modest stress response
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cAMP
5-HT7 receptor
5-HT Tactile stimulation
(maternal LG)
PKA CBP
Summary of in vivo and in vitro studies
NGFI-A (egr-1, zif-268, krox-24)
(T4 - T3)
SP1
CBP NGFI-A SP1
CpGme Exon 17 promoter
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C T A C G T A C T C G G A A T C T C G
CH 3
CH 3
CH 3
cAMP
NGFI-A
5-HT7 rec
5-HT
GR gene
PKA CBP
SP1
CBP SP1
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Do comparable processes occur in humans?
• Post-mortem studies of hippocampus.
• Samples from suicide victims/controls.
• QSBB (Gustavo Turecki) – forensic phenotyping.
• Human exon 1F promoter (Turner & Muller,
2005)
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Human glucocorticoid receptor gene
A B CD E F G H I J 2 3 4 5 67 8 9
Variable exon 1 region Constant region
5’ 3’
Exon 1F is the human ortholog of the rat exon 17 GR promoter
(70% homology) and contains an NGFI-A consensus sequence.
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Human glucocorticoid receptor gene
A B C DE F G H I J 2 3 4 5 67 8 9
Variable exon 1 region Constant region
5’ 3’
Control Suicide 0.0
0.5
1.0
1.5 GRtotal
GR
mR
NA
*
Control Suicide 0.0
0.3
0.6
0.9
1.2 GR1F
GR
1-F
vari
an
ts
*
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Suicide vs abuse - GR expression
GRtotal
GR
mR
NA
/GA
PD
H (
log
co
nc.)
Control Suicide Suicide
- Abuse + Abuse
0.0
0.4
0.8
1.2
*
GR
-1F
mR
NA
/GA
PD
H (
log
co
nc.)
Control Suicide Suicide
- Abuse + Abuse
0.0
0.5
1.0
1.5
*
GR1F
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GR
1-F
CpG
Meth
yla
tion (
%)
Control Suicide Suicide
- Abuse + Abuse
0
20
40
60 *
Suicide vs abuse - CpG methylation
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Hypothalamus
Pituitary
Adrenals
Controls
Glucocorticoids
(-)
CRF
ACTH
(-)
Hypothalamus
Pituitary
Adrenals
Maltreatment
Glucocorticoids
(-)
CRF
ACTH
(-)
Childhood maltreatment associates with increased central
CRF levels and greater HPA and autonomic responses to stress
(DeBellis et al 1994; Heim et al 2000; Lee et al 2005)
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Childhood adversity and NR3C1 promoter methylation in DNA
from periperhal samples
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Early experience
Abuse
Family strife
Emotional neglect
Harsh discipline
Health Risks
Depression
Drug abuse
Anxiety
Diabetes
Heart disease
Obesity
Individual differences
in neural and endocrine
responses to stress (defensive responses)
How does family life influence health over the life span?
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Childhood adversity and NR3C1 promoter methylation in DNA
from periperhal samples
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345 genes
Secure:
TBCD (23.89%: Body)
CEBPE (19.70%: TSS200)
Attachment at 18 months (Secure Vs Insecure)
associates with epigenetic variation (6-8 yrs)
612 probes
≥. 5% (p<0.05)
Insecure:
ABR (21.35%: Body)
SHANK2 (9.4%: 1st Exon)
DRD4 (5.76%: Body)
Microtubule
formation LTP
Synaptic
density
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Reference 1: ADHDgene Database (I)
64
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213 Genes in the ADHDgene database (24
Genes are hot genes)
36 Probes (25 Genes) of ADHD database
genes are found in the 2907 DMPs list.
Reference 1: ADHDgene Database (II)
ADRA1A FGF10
ANK3 GNAO1
ARRB2 HK1
ATXN1 HLA-DRB1
BAIAP2 ITGA11
BAIAP2 MEIS2
BAIAP2 MEIS2
BAIAP2 NCKAP5
BAIAP2 PTPRG
BDNF PTPRG
CACNA1C SH3BP5
CDH13 SH3BP5
CHRNA4 SLC6A3
CHRNA4 UNC5B
COMT UNC5B
DBH ZNF423
DRD4 ZNF423
EMP2 ZNF423
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Child attention problems and co-methylation
Child attention problems (SDQ) associated with
differential methylation of 1747 probes (957
genes).
30 probes (22 genes) associated with child
attention problems (-0.3 < r >0.3) show co-
methylation (-0.6 < r >0.6).
rs= -0.36, p=0.02 rs= 0.36, p=0.02 rs= -0.64, p<0.001
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7 Genes(GO) + 94 Genes(Pathway)
--123 Probes--
Hierarchical clustering of pair-wise correlation
DMPs
DM
Ps
Pair-wise Correlation
20 40 60 80 100 120
20
40
60
80
100
120-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
The Most
highly
correlated
cluster (30
probes, Avg
Corr=0.8)
67 Co-methylation Clusters
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The ‘partial’ mediating role of maternal
psychopathology
Maternal
depression
(36 months)
Maternal
emotional
neglect
Maternal
depression
(36 months)
Child negative
emotionality (36
moths) p = 0.01
p = 0.008 p = 0.001
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The ‘partial’ mediating role of maternal
psychopathology
Maternal
depression
(36 months)
Maternal
emotional
neglect
Maternal
depression
(36 months)
Child negative
emotionality (36
moths) p = 0.01
p = 0.008 p = 0.001
(Parenting)
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Do effective treatment programs that target
mothers affect DNA methylation profiles
in the children?
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90 Unique Samples
Infinium 450K Array
Blood Samples
27 years old
Variables: methy_grp (0= control/no treatment ;1=intervention)
ETXGROUP: 1 and 2 = different forms of no treatment versus 3 and 4 which represent prenatal intervention and combined pre and postnatal intervention, respectively
CHILDGENDER 1=male 2=female
Child Abuse at age 4 and age 15
Child abuse is physical, sexual or emotional mistreatment or neglect
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Principal Component Analysis
Component Fstat p
Gender (3.0%) 7.32 .003 x 10-8
Abuse/4 yrs (3%) 5.80 .002 x 10-2
Abuse/15yrs 1.67 ns
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Principal Component Analysis
Component Fstat p
Gender (3%) 7.32 .003 x 10-8
Abuse/4 yrs (3%) 5.80 .002 x 10-2
Abuse/15yrs 1.67 ns
Treatment (8%) 3.07 .002 x 10-4
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Conclusions
• The function of the genome is regulated by
epigenetic signals that are subject to
environmental regulation.
• These epigenetic signals reflect the quality of the
dearly environment, and guide the development
and function of the brain.
• These effects are potentially stable, but are
subject to modification, potentially over the
lifespan.
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Family
Parent-
child
Family
Parent-
child
Transgenerational
influences
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Family
Parent-
child
Family
Parent-
child
Transgenerational
influences
Page 77
Family
Parent-
child
Family
Parent-
child
Transgenerational
influences
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Control Perfume Snake 0
1
2
3
4
Response to snake odours
To
ng
ue-F
lick
Offspring
are significantly
larger and with
longer tails
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Defense to snake predation in skink lizards
Most frequent prey
• smaller
• shorter tails
• less reactive to
snake cues
If mother has been exposed to the scent of a
predatory snake then offspring are larger, with
longer tails and ….
Page 80
Nutrient Supply
Mate Quality
Violence
Infection
Population density
Parental
investment
Defensive Strategies
Foraging/Metabolism
Reproductive Strategies
Environmental Parental Developmental
condition signal outcome
Evolutionary biology - Maternal effects