Gene-environment interactions

(Lovely, et al., 2016)

The beautiful fish face

4 dpf

4 dpf 4 dpf

4 dpfMarySwartz

Gene-ethanol interactions generate distinct phenotypes

untr

eate

det

hano

l

pdgfra+/- vangl2-/- bmp2b+/-

Loss of pdgfra sensitizes embryos to ethanol teratogenesis

(McCarthy, et al., 2013)

pdgfra-ethanol interaction causes crest apoptosis

24 hpf 30 hpf

2

6

10

14

+/+ +/- -/- -/-+/-+/+

ethanoluntreated

apop

totic

cel

ls

Ethanol treatmentfrom 10-24 hpf

(McCarthy, et al., 2013)

Elevating mTOR signaling rescues pdgfra mutants

control 1% ethanol

pten mo L-leucine

PI3K AKT mTOR CellSurvival

Pten L-leucine

(McCarthy, et al., 2013)

Gene-ethanol interactions can be synergistic

PI3K

mTOR

eIF4b

ethanol

Growth factor signaling

PI3K

mTOR

eIF4b

Growth factor signaling

PI3K

mTOR

eIF4b

Growth factor signaling

ethanol

normal development

normal development FASD

Combined loss of pdgfra and pdgfrb resembles ethanol-treated pdgfra mutants

pdgfra+/+ pdgfra-/-

pdgf

rb+/

+pd

gfrb

-/-

(McCarthy, et al., Dev Bio 2016)

There are many inputs into the mTOR pathway

(Huang & Manning, Biochem J, 2008)

Reduction in Tsc1a function protects pdgfraheterozygotes from ethanol teratogenesis

Combined ethanol exposure and Gsk3 inhibition mimics the ethanol-pdgfra interaction

8 hpf

5 control individuals

5 control + 5 ethanolindividuals

5 control + 5 ethanolindividuals

Gastrulation

6 hpf 10 hpf

mTOR insights from transcriptomic profiling of single embryos

2 h 2 h

(Alfire Sidik)

Ethanol exposure contributes to transcriptional differences between embryos

PC1:27% variance PC1:27% variance

PC2:

11%

var

ianc

e

PC2:

12%

var

ianc

e

EthanolControl

2 hrs ethanol 4 hrs ethanol

The mTOR pathway is enriched in the transcriptomic data

Conclusions

• Synergy: pdgfra-ethanol• Convergence: pdgfra-ethanol vs pdgfra;pdgfrb• How are the different functions of Pdgfra

modulated, migration vs survival?• Is the mTOR pathway a common target of ethanol?• There are multiple mechanisms of gene-ethanol

interactions

Desirè BuckleyYohaan FernandesRanjeet KarTim KukaBen Lovely

NIH/NIDCR-R00DE018088-R01DE020884NIH/NIAAA-U24AA014811-R01AA023426-R21AA024043ABMRF

University of HawaiiMichelle Talquist

IUPUITatiana ForoudLeah Wetherill

Angie MartinezPatrick McGurkAlfire SidikMary SwartzTaylor Yamakawa

Neural crest cells fail to condense in pdgfra;pdgfrbdouble mutants

pdgfra-/- pdgfra-/-;pdgfrb-/-

(McCarthy, et al., Dev Bio 2016)

Three gene clusters associate with ethanol exposure

Rosy Brown:•patched 2•intersectin 1 (SH3 domain protein) •eukaryotic translation initiation factor 2B, subunit 3 gamma•minichromosome maintenance complex component 3 associated protein

Mint Cream:•proteasome 26S subunit, non-ATPase 1•tetratricopeptide repeat domain 14•CAMP responsive element binding protein 5b•dedicator of cytokinesis 4•surfactant protein Ba

Pale Green: •fat3a•solute carrier family 18, subfamily B, member 1

Conclusions: Gene-ethanol interactions

(Eberhart & Parnell, 2016)

-Synergy-Variability (by genotype and by individual)-No single mechanism-A minority of genes appear to interact with ethanol-Ethanol-sensitive genes do not fall into a single functional class

Unraveling the impact of gene-environment interacts is difficult

Genetic screens for ethanol-sensitive mutants

Gene-ethanol interactionpdgfra vangl2 bmp2bfgf8a foxi1 hinfp mars4 forward genetic mutants

armc10lrp13b

Lovely et al., Alcohol 2014Swartz et al., ACER 2014McCarthy et al., Development 2013

Strong interaction between vangl2 and ethanol

(Alfire Sidik)

wt vangl2+/- vangl2-/-

cont

rol

Loss of vangl2 sensitizes embryos to ethanol during early embryogenesis.

+/+

+/-

-/-

(Alfire Sidik)

Ethanol disrupts convergent extension in vangl2 mutants and heterozygotes

Ethanol disrupts convergent extension

Eye field separation is defective in ethanol-treated vangl2 mutants

Ethanol disrupts forebrain development in vangl2 mutants

Vangl2-/- WTco

ntro

let

hano

l

(DesireBuckley)

Subteratogenic levels of ethanol increase transcript abundance noise

8 hpf 10 hpf

(Alfire Sidik)

Gene-ethanol interactions can be synergisticun

trea

ted

etha

nol

pdgfra+/- vangl2-/- bmp2b+/-

The endoderm generates serially-reiterated pharyngeal arches

Endoderm is responsive to Bmp during pharyngeal pouch morphogenesis

14 hpf 16 hpf 18 hpf 26 hpf

BRE:

GFP

endo

derm

(Lovely et al., in revision)

Bmp signaling is necessary for endoderm morphogenesis

Endoderm; Neural crest cells (Lovely et al., in revision)

Disrupted arch morphology at 26 hpf

Endoderm; Neural crest cells (Lovely et al., in revision)

Blocking Bmp signaling from 10-18 hpf disrupts craniofacial morphology

(Lovely et al., in revision)

Wild-type endoderm rescues DNBmpR1 embryos

sox32 mRNA;sox17:DsRed

wt into wt wt into DN-BmpR1 mistargeted

(Lovely et al., in revision)

Bmp signaling is necessary for proper Fgf signal reception

Control DM

Endoderm; Fgf response (Lovely et al., in revision)

The Bmp and Fgf pathways genetically interactsmad5 wt smad5-/-fg

f8a

wt

fgf8

a-/-

(Lovely et al., in revision)

Forward genetics effectively identifies ethanol-sensitive loci

Screened 100 F2 families

Isolated 10 mutants

Jaw support

Jaw

fli1:EGFP

Jaw support

Jaw

(Lovely et al., in revision)

0%

5%

10%

15%

20%

25%

30%

Full Pen. Weak Pen. Full x Weak

Full Pen.Weak Pen.Full x Weak

Signaling interactions in endoderm morphogenesis

Bmp

Fgf

Shh

aus67

Gene-environment interactions

Signaling pathways

Genetic background effects

Development is contextual-Multifactorial interactions-Synergy-ConvergenceA minority of genes interact with ethanol-but generate unique phenotypes

Ethanol sensitive mutants isolated in a forward genetic screen.

50% have no defects in the absence of ethanol

Other growth factor pathways interact with ethanol

ethanol

untreated untreated

fgf8a-/-

fgf8a-/- fgf8a-/-

rapamycin

wt

Neil McCarthy

Eberhart labDelany DowdKim HoBen LovelyNeil McCarthyPatrick McGurkAnna PercyJenna RozackyAlfire SidikMary Swartz

AlumniVan NguyenKelly Sheehan-RooneyBen Wells

Mik Di U M h t

NIH/NIDCR-R00DE018088-R01DE020884NIH/NIAAA-U24AA014811ABMRFUT Austin

Alanine (Ancestral) Valine

SNP/ethanol interactions in humans

(Alfire Sidik)

124

126

128

130

132

134

136

138

140

CC CT TT

low

er fa

cial

dep

th

genotype (minor allele = T)

FAT4 missense SNP rs1039808

alcohol

no alcohol

PDGFRA-ethanol interaction

110

115

120

125

130

135

AA AC CC

alc exposed no alc exposure

Mid

faci

alde

pth

(mm

)

genotype

Pdgfra SNP rs4358459

Causes of variation in craniofacial defects

Hypothesis: Gene/environment interactions generate variability-zebrafish model of Fetal Alcohol Spectrum Disorders.

Eberhart labBen LovelyNeil McCarthyPatrick McGurkAnna PercyAlfire SidikMary Swartz

IUPUITatiana ForoudLeah Wetherill

UCLPeter HammondMike Suttie

NIH/NIDCR-R00DE018088-R01DE020884NIH/NIAAA-U24AA014811ABMRF

• Eberhart lab• Delany Dowd• Melissa Griffin• Kim Ho• Luana Kohnke• Ben Lovely• Neil McCarthy• Patrick McGurk• Brandon Pekarek• Jenna Rozacky• Mary Swartz• Ben Wells

• Charles Kimmel UO• Tatiana Foroud IUPUI NIH/NIDCR, NIH/NIAAA, ABMRF

vangl2 mutants and heterozygotes are sensitive to 1% ethanol during early embryogenesis.

+/+

+/-

-/-