54
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.
+/+
+/-
-/-
