1. Genetic approaches to understand variability in FASD facial and neural phenotypes
Specific Aim 1: Screen community resources to identify ethanol-sensitive loci.
Swartz et al. ACER 2014
Lovely et al. Alcohol 2014
McCarthy and Eberhart Cell Mol Life Science 2014
McCarthy et al. Dev Dynamics in press
McCarthy et al. Dev Bio in revision
Lovely et al. Development in revision
Eberhart and Parnell ACER in press
Lovely et al. Zebrafish in revision
Lovely et al. SDB Wire in preparation
kif3a
pdgfra
vangl2

2. Ethanol disrupts convergent extension.
p-value = 7.564e-11 ***
p-value = 9.211e-09 ***
Tukey’s for shh
Control wt vs. etoh wt – p = 0.009
Control het vs. etoh het – p = 0.008
Control mut vs. etoh mut – p = (not sig)
Control wt vs. control het – p = 0.02
Control het vs. control mut – p = 0.07 (not sig)
Etoh wt vs. etoh het – p = (not sig)
Etoh het vs. etoh mut – p = 0.005
Tukey’s for pax2
Control wt vs. etoh wt – p = 0.02
Control het vs. etoh het – p = 0.003
Control mut vs. etoh mut – p = 0.046
Control wt vs. control het – p = (not sig)
Control het vs. control mut – p = (not sig)
Etoh wt vs. etoh het – p = (not sig)
Etoh het vs. etoh mut – p = (not sig)

3. Pilot RNA-seq experimental design
Gastrulation
2 h
2 h
6 hpf
8 hpf
10 hpf
5 control individuals
5 control +
5 ethanol
individuals
5 control +
5 ethanol
individuals
1% ethanol initiated at 6 hpf for all treatments
25 total samples
1 embryo/sample
Nextseq 500 PE 2x75
660 million reads total
~26.4 million reads/sample

4. Subteratogenic levels of ethanol increase transcript abundance noise
8 hpf
10 hpf
FDR adjusted p-value of 0.1Significant genes:
rin3
dpp4
si:ch c3.2
cystatin 14a, tandem duplicate 2

5. Some embryos fail to undergo regulative development

6. Specific Aim 2: Characterize mutants from our forward genetic screen for ethanol-sensitive loci regulating facial development.
-R01AA023426
recruited Desirè Buckley and Yohaan Fernandes
-K99/R00 Ben Lovely
-lrp13b Tim Kuka

7. Mean distance to stimulus (cm)
Preliminary data shows no effect of genotype on sensitivity to ethanol-induced behavioral deficits 1 5 20 10 23
+/-
+/+
Mean distance to stimulus (cm) 50 30 40
One minute intervals
Exposure to 0.5% EtOh @ 16 hpf

8. Skeletal defects are present in ethanol-treated aus67 embryos by 72 hpf
fli1:EGFP
Jaw support
Jaw
Jaw support
Jaw

9. The penetrance of defects in ethanol-treated aus67 embryos may be genetically regulated

10. Interactions with other projects
McCarthy et al., Development, co-authored publication with Tatiana Foroud.
Eberhart and Parnell, ACER.
Tina Chambers, Rajesh Miranda-genetic/epigenetic underpinnings of FASD

11. Possible Aims for CIFASD IV
High throughput analysis of candidate FASD genes
Genes identified via RNA-seq
Genes identified in humans
F1 screens of CRISPR-Cas9
Face, brain, behavior
Multiplexed injections
Tracking sensitive cell populations
Transgenics to label/isolate individual cells
Knock-down genes in specific tissues
Identify suppressors of ethanol sensitivity
Genetic & chemical screens
exon

12. Priority areas in FASD research
Early identification
Biomarkers, etc.
Targeted interventions
Nutritional, environmental, etc therapies to improve outcomes in FASD
Multifactorial causes of FASD
Adult health issues in FASD

14. aus67 embryos are sensitive to ethanol from 24-48 hpf
To determine the most crucial time window in development in terms of lower jaw development in the prescence of ethanol. Embryos were exposed from or 72 hpf /72, and hpf. You can see the exposure times beginning at 6 hpf a continuous increase in the number of jaw defects corresponds with the length of exposure to ethanol. The latter time points, those beginning at 24hpf suggest that later time points may be more important has very low number of jaw defects, while an equal length of time at a later time point causes a larger number of defects.