Harnessing diversity in the CC and DO populations for

the study of behavior

Elissa J. Chesler, PhD

The importance of behavioral genetics

2

Behavioral variation and ability to engage in behavioral change pervades health and disease

Many toxicological effects are manifest in behavioral phenomena

Psychotropic drug toxicity is a problem for patients and environments

Mouse genetics has been a versatile means to discover the mechanisms of behavioral heterogeneity among individuals and among traits.

Depression and Alcoholism

Hyperactivity

Anxiety and Depression Gene 2

Environment

Environment

Gene 1 Gene

Gene

GXGXE

ExE

Biological Processes

Gene-Environment Interactions

Natural perturbations Gene-trait

associations Trait-trait Associations

Chesler EJ and Langston, MA RECOMB Systems Biology and Regulatory Genomics 2005: 150-165

Systems genetic analysis holistically connects biology and behavior

3

Data integration in recombinant inbred mice: A genetic reference population

GENOTYPES GENE EXPRESSION CLASSICAL PHENOTYPES

GENO

TYPE

SGE

NE E

XPRE

SSIO

NCL

ASSI

CAL P

HENO

TYPE

S

Linkage Map Construction

CO-EXPRESSION NETWORKS

GENETIC CORRELATION OF

COMPLEX PHENOTYPES

QTL MAPPING Expression QTLs

QTL MAPPING Classical QTLs

GENE EXPRESSION RELATIONSHIP TO

COMPLEX PHENOTYPES

Chesler EJ, et al. Neuroinformatics. 2003;1(4):343-57. Chesler EJ, Williams RW. Int Rev Neurobiol. 2004;60:59-95. 4

5

Finding relations among behaviors using multidimensional genetics

Chesler and Bubier, Neurotherapeutics, 2012

Finding disorders and genes related to dimensions of behavior

Rea

ctiv

ity F

acto

r

Preference for alcohol vs. water QTL mapping to find genes that influence behavior

Philip VM, et al. Genes Brain Behav. 2010 Mar 1;9(2):129-59. 6

Gene expression analysis in early populations shows massive co-expression networks

Chesler et al, Nature Genetics, 2005

Baldwin et al. J Biomed Biotechnol. 2005 Jun 30;2005(2):172-80.

Getting behavior out of the bottleneck Although many discoveries have been made in behavioral genetics using the laboratory mouse, precision and variation have been limited

Conventional mice were selected for behavior and subject to bottlenecks

o Affects phenotypic variation o Affects phenotypic co-variation o Affects mapping precision

The CC and DO populations increase diversity by randomly segregating wild-derived and common alleles.

o Heterozygosity o Multi-locus combinations

Advanced Mouse Populations

9

Churchill, 2004; Chesler et al, Mammalian Genome, 2008; Philip et al, Genome Research 2012

Characterization of the ORNL Collaborative Cross breeding colony

Darla R. Miller Philip VM, et al. Genome Res. 2011 Aug;21(8):1223-38.

Wildness in the Collaborative Cross

Frequency of extreme high and low responses declines, beginning with outcrosses

More mice have moderate to high scores

Docility is polygenic Philip VM, et al. Genome Res. 2011 Aug;21(8):1223-38.

Increased phenotypic diversity for other behavioral traits

BXD ☐ CC

Philip VM, Genome Res. 2011 Aug;21(8):1223-38.

High QTL mapping precision in the Collaborative Cross

Philip VM, et al Genome Res. 2011 Aug;21(8):1223-38.

Summary of Significant QTLs in CC Mice Phenotype 1.5 LOD CI (Mbp) # Positional

Candidates Effect Size

Peak activity after sleep deprivation (*) .5 2 7.77

Periosteal circumference (*) 1 5 11.6

Hot plate latency 15.5 6 6.7

Open field locomotion in the first 3 minutes 9.3 16 8.23

Average percent sleep at night 6 39 7.3

Integrated distance from center 3.6 45 9.54

Body length 5.6 70 6.93

Red cell distribution width 7 221 25.99

Philip VM, et al. Genome Res. 2011 Aug;21(8):1223-38.

300 DO mice

4 Tissues isolated for RNAseq

Day 1 Open Field

Day 3 Light-Dark Box

Day 4 Visual

Cliff

Day 5 Hot plate

Days 9-11 Tail Suspension

Logan RW, Robledo et al, Genes Brain Behavior 2013. Recla et al, Mammalian Genome, 2013.

y o

RaRobled

Systems genetics of behavior in the DO

PWK decreasor NOD and CAST decreasors

Activity and anxiety like behavior in the Open Field

Logan RW, Robledo et al, Genes Brain Behavior, 2013

Positional Candidates

Traitsa Interval Width

(Mb) Protein Coding Pseudo-gene miRNA Total Habituation to center of

open-field 1.61 25 7 0 32

Immobility in open-field 7 12 21 2 35 1.87 12 3 0 15 1.74 14 5 2 21

Time spent in light of light-dark box 2.89 82 11 3 96

Habituation to light of light-dark box 1.53 33 5 3 41

Climbing frequency during tail-suspension test 1.12 3 2 0 5

Activity in bottom area of visual-cliff 1.63 13 1 0 14

Logan RW, Robledo et al, Genes, Brain Behavior 2013.

Improved precision of mapping populations for representative behavioral traits

Expanded BXD

BXD

CC

F2

DO

CSS

` Mb

Bubier JA, Chesler EJ. Neurotherapeutics. 2012 Apr;9(2):338-48 18

Quantification of gene expression in DO founders

Building an individualized transcriptome

F F D D D D D C C A A A A A A A G G E E E E E E H H H H

G G G G B B B B B A A H H H H F F F F F F E E C C C D D

Pseudo-phased Chromosomes

Two Haploid Chromosomes

Genotype DNA @ 8-80k informative SNPs

FG FG DG DG DB DB DB CB CB AA AA AH AH AH AH AF GF GF EF EF EF EE EE EC HC HC HD HD

Infer 36-state

genotypes

Munger et al Genetics 2014

Align

A proximal QTL for expression of Per3

Improved precision of eQTLs in DO

Narayanan Raghupathy, Al Simon, Matt Hibbs, Gene Expression Service, Computational Science Service

Chesler et al, Nature Genetics 2005

BXD DO

The AP-1 Binding transcriptional network

Improved resolution in systems genetics network analyses

Behaviors mRNA

DNA Behaviors mRNA

DNA

Historic Populations CC/DO Chesler EJ. Mammalian Genome 2013

24

The Consequences of Limited Variation: “Mice don’t do that”

25

Poor response to selection Poor correlation to drug intake

Diversity Matters: Sign Tracking & Goal Tracking A

AJ B6 129 NOD CAST

Sig

n-tra

ckin

g (s

/tria

l)

0.0

0.5

1.0

1.5

2.0

2.5PairedUnpaired

* ** *

B

AJ B6 129 NOD CAST

Goa

l-tra

ckin

g (s

/tria

l)

0

2

4

6

8

10PairedUnpaired

****

Dickson et al, in review

Pavlovian conditioned response Do subjects attend to a sign (lighted lever paired with food reward), or do they attend to the goal (food delivery area)? Sign tracking is associated with drug intake in rats. C57BL/6J do not show robust sign tracking.

27

Sex differences in sign- and goal-tracking in collaborative cross founder strains

Males

CAST12

9 A/J B6NOD

GT

Dur

atio

n (s

)

0

1

2

3

4A

A/J 129 B6

NODCAST

ST D

urat

ion

(s)

0

1

2

3

4B

Males

A/J 129 B6

NODCAST

ST:G

T R

atio

0

1

2

3

4C

Males

Females

NOD A/JCAST

129 B6

GT

Dur

atio

n (s

)

0

1

2

3

4D

129 B6 A/J

CASTNOD

ST D

urat

ion

(s)

0

1

2

3

4E

Females

129 B6

CAST A/JNOD

ST:G

T R

atio

0

1

2

3

4F

Females

Dickson et al., in review

Cocaine self-administration in DO mice

Session0 1 2 3 4 5 6 7 8 9 10Pe

rcen

tage

of r

espo

nses

on th

e ac

tive

leve

r

060

70

80

90

100A Cocaine IVSA

Final Day of Acquisition

Leve

r pre

sses

0

10

20

30

40

50

60

*

B

*

Active Inactive

Dickson et al, Psychopharmacology, 2014

Canonical Correlation of drug seeking and novelty reference traits.

novelty-related: distance traveled in the open field, novelty preference, hole board exploration, time in the center of the open field. addiction-related: infusions on the FR1 schedule, breakpoint, IVSA acquisition (i.e., did or did not reach criterion during cocaine IVSA testing), sex

Dickson et al, Psychopharmacology, 2014

The quest for consilience

“…the evidence in favour of our induction is of a much higher and more forcible character when it enables us to explain and determine cases of a kind different from those which were contemplated in the formation of our hypothesis…

No accident could give rise to such extraordinary coincidence.”

-W. Whewell, 1847

Cross-species and cross-population integration in GeneWeaver

Drug Related Gene (DRG) Database

User submitted & published gene sets from GWAS, QTL, transcriptomics, text mining, gene co-expression networks, expert lists, curated annotations, etc.

http://geneweaver.org

~60,279 Gene Sets 9 supported species

Gene Weaver A system for the integration of Functional genomics experiments.

Baker EJ, Jay JJ, Bubier JA, Langston MA, Chesler EJ. Nucleic Acids Res. 2012 31

Comparative Toxicogenomics Database

A single positional candidate supported by high convergent functional evidence

32

Bubier et al, Genetics 2014

Convergent evidence across populations and traits enables causal SNP identification

Bubier et al, Genetics 2014

Conclusions Precise mouse genetic systems can enable refined gene-trait and trait-trait associations.

High diversity in advanced mouse populations enable study of many behaviors

High precision facilitates genetic mapping and discovery of genomic networks underlying behavioral variation.

Cross-population data integration enables discovery of functional and genetic mechanisms of behavioral heterogeneity.

34

Acknowledgements

35

Dr. Vivek Philip Dr. Jill Recla Dr. Joel H. Graber Dr. Ryan W. Logan Dr. Dan Gatti Dr. Erich J. Baker Dr. Ray F. Robledo Dr. Narayanan Raghupathy Dr. Michael A. Langston Jeremy Jay Dr. Matthew A. Hibbs Dr. Judith Blake Dr. Jason A. Bubier Dr. Carol Bult Dr. Yun Zhang Troy Wilcox Dr. Andrew Holmes Dr. John C. Crabbe James Clark Dr. Gary A. Churchill Pamela Metten

Laura Anderson Darla Miller Dr. David Threadgill

Dr. Juliet Ndukum Dr. Ken Manly Dr. Price Dickson Dr. Brynn Voy

Dr. Bem Culiat Dr. Bruce O’Hara

P50 GM76468 Center for Genome Dynamics

P30 AG38070 Nathan Shock Center on Aging at JAX

The Ellison Medical Foundation

R01 AA18776 R01 DA37927

Department of Energy Office of Science

The Jackson Laboratory