1. Ecology
  2. Genetics and Genomics
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Novel adverse outcome pathways revealed by chemical genetics in a developing marine fish

  1. Elin Sørhus  Is a corresponding author
  2. John P Incardona
  3. Tomasz Furmanek
  4. Giles W Goetz
  5. Nathaniel L Scholz
  6. Sonnich Meier
  7. Rolf B Edvardsen
  8. Sissel Jentoft
  1. Institute of Marine Research, Norway
  2. University of Oslo, Norway
  3. Northwest Fisheries Science Center, National Marine Fisheries Service, United States
  4. University of Agder, Norway
Research Article
Cite this article as: eLife 2017;6:e20707 doi: 10.7554/eLife.20707
8 figures, 3 tables and 1 additional file

Figures

Figure 1 with 1 supplement
Terminal phenotypes after high dose exposure.

Control (A) and exposed (B) three days post hatch (dph) larvae (6 days post embryonic exposure). Open arrowheads in (A) indicate the marginal finfold surrounding the larvae and the white asterisk indicate the location of the connection between the dorsal space and the ventral yolk sac in the vicinity of the pectoral fin. In (B) the black arrowhead indicates severely reduced craniofacial outgrowth, while the black arrow indicates yolk sac edema. The ventricle and atrium in control (C) and embryonically exposed (D) animals are indicated by black and white arrows, respectively. (E) Normal craniofacial structure in control, and (F) moderate and (G) severe craniofacial defects in exposed animals. (H) Normal marginal finfold in control, (I) exposed animals with severe reduction of anterior marginal finfold (open arrowheads). Yolk mass (*) in control (J) and embryonically exposed larvae (K). (L) Control and (M) exposed 18 dph larvae. Open arrowheads indicate increased anterior marginal finfold, black arrowhead indicates reduced upper jaw outgrowth, and black arrow indicates edema formation in the peritoneal cavity in oil-exposed larvae (M). Scale bar: 0.2 mm (C,D; EG; HK) and 1 mm (A,B and L,M).

https://doi.org/10.7554/eLife.20707.003
Figure 1—figure supplement 1
Normal development of liver and lateral line in the severe phenotypes.

Normally developed livers and neuromast cells are indicated by black arrows and arrowheads in control (A, C) and severely affected hunchback phenotypes (B, D), respectively. Scale bar 0.2 mm.

https://doi.org/10.7554/eLife.20707.004
Appearance of phenotypes over time.

In each panel control and high-dose-exposed embryos are shown on the left and right, respectively. (AC) Lateral overview of whole embryos showing accumulation of edema (anterior to the left). (A) 6 dpf/E2 sampling point. (B) 8 dpf (between E2 and E3 sampling points). Heart (h) and liver bud (l) are indicated. White arrowheads indicate outer margins of the yolk sac membranes; asterisk indicates small pocket of edema. Black arrowheads indicate the hindbrain ventricle. (C) 10 dpf/E3 sampling point. Arrowheads same as (B); asterisks indicate expanded yolk sac edema. (DE) High-magnification ventral views of the heart (anterior at top). (D) 6 dpf/E2. Dashed turquoise lines indicate outer border and lumen of midline cardiac cone. (E) 9 dpf (between E2 and E3). Arrows indicate the atrium (a), ventricle (v) and bulbus arteriosus (ba). (F) 0 dph (E5 sampling point). Chambers indicated as in (E). (GI) Lateral views of the developing head (anterior to the left). (G) 8 dpf (between E2 and E3). (H) 0 dph (E5). Arrow indicates abnormal lower jaw cartilages in oil-exposed larva. (I) 3 dph (E6 sampling point). Red bars indicate difference in eye diameter between control and exposed larvae.

https://doi.org/10.7554/eLife.20707.005
Figure 3 with 2 supplements
Exposure regimes and differentially expressed genes (DEGs) during embryonic development.

(A) Embryos were exposed to a continuous high dose (black line; 6.7 ± 0.2 μg/L TPAH), a pulsed dose (red line; 0.09 ± 0.02–6.8 ± 1.0 μg/L TPAH) and a continuous low dose (blue line; 0.58 ± 0.05 μg/L TPAH) of crude oil. Photos indicate normal developmental progress at each of six sampling time points (E1–E6). Venn diagrams show shared and exclusive DEGs for each of the three oil exposures at E1–E6. (B) Venn diagrams illustrating the number of shared and exclusive DEGs at each stage in development up to hatching for the three exposure regimes.

https://doi.org/10.7554/eLife.20707.006
Figure 3—figure supplement 1
Most regulated KEGG pathways.

(A) Pathways (Total) with highest number of DEGs ≥2 FC during and after embryonic exposure. (B) Pathways with the largest fraction of DEGs ≥2 FC/ Total number of genes in pathway (Normalized) during and after embryonic exposure.

https://doi.org/10.7554/eLife.20707.007
Figure 3—figure supplement 2
Comparison of mRNA read count data with real-time qPCR for selected genes during and after embryonic exposure.

Genes include cp1a (cytochrome p450 1 a), wnt11 (wingless-type MMTV integration site family member 11), kcnh2 (potassium voltage-gated channel subfamily H member 2), cac1c (voltage-dependent L-type calcium channel), nac1 (sodium/calcium exchanger 1), at2a2 (sarcoplamsic-endoplasmic reticulum calcium ATPase). (A) Real-time qPCR. (B) Read count data from RNA sequencing. Data were normalized as described in Materials and methods.

https://doi.org/10.7554/eLife.20707.008
Figure 4 with 2 supplements
Exposure regimes and differentially expressed genes (DEGs) during larval development.

(A) Larvae were exposed to a continuous high dose (black line; 7.6 ± 0.7 μg/L TPAH), a pulsed dose (red line; 0.3 ± 0.3–6.1 ± 0.5 μg/L TPAH), and a continuous low dose (blue line; 0.65 ± 0.08 μg/L TPAH) of crude oil. Photos indicate normal developmental progress at each of five sampling time points (L1–L5). Venn diagrams show shared and exclusive DEGs for each of the three oil exposures at L1–5. (B) Venn diagrams illustrating the number of shared and exclusive DEGs at each larval stage for the three exposure regimes.

https://doi.org/10.7554/eLife.20707.010
Figure 4—figure supplement 1
Most regulated KEGG pathways.

(A) Pathways (Total) with highest number of DEGs ≥2 FC during larval exposure. (B) Pathways with the largest fraction of DEGs ≥2 FC/ Total number of genes in pathway (Normalized) during and after embryonic exposure.

https://doi.org/10.7554/eLife.20707.011
Figure 4—figure supplement 2
Comparison of mRNA read count data with real time qPCR for selected genes during larval exposure.

Genes include cp1a (cytochrome p450 1 a), wnt11, kcnh2 (potassium voltage-gated channel subfamily H member 2), cac1c (voltage-dependent L-type calcium channel), nac1 (sodium/calcium exchanger 1), and at2a2 (sarcoplamsic-endoplasmic reticulum calcium ATPase). (A) Real-time qPCR. (B) Read count data from RNA sequencing. Data were normalized as described in Materials and methods.

https://doi.org/10.7554/eLife.20707.012
DEGs involved in cardiogenesis.

Regulation of genes involved in cardiogenesis during and after embryonic exposure. Purple: increased expression, red: decreased expression in exposed group.

https://doi.org/10.7554/eLife.20707.015
DEGs involved in E–C coupling.

Embryonic developmental samples (E1–6) were collected during (black lettering) and after (blue lettering) crude oil exposure. Oil exposure was continuous across the larval sampling points (L1–5).

https://doi.org/10.7554/eLife.20707.016
DEGs involved in craniofacial development.

(a) Regulation of genes involved in craniofacial development during and after embryonic exposure. (b) Regulation of myosin heavy chain genes. Purple: increased expression, red: decreased expression in exposed group.

https://doi.org/10.7554/eLife.20707.017
DEGs involved in osmoregulation.

E1–E6: Embryonic exposure, L1–L5: Larval exposure. Black letters: during exposure, blue letters: after exposure.

https://doi.org/10.7554/eLife.20707.018

Tables

Table 1

Genes expressed at all stages during and after embryonic exposure (E2–E6) in high dose group. SP, swissprot; GB, genebank; IE; increased expression; DE; decreased expression.

https://doi.org/10.7554/eLife.20707.009

Cod ID

Swissprot annotation

SP ID

GB ID

Category

Regulation

ENSGMOG00000018302

Fumarylacetoacetase

faaa

fah

Tyrosine metabolism

IE

ENSGMOG00000000318

Cytochrome P450 1A1

cp1a1

cyp1a1

xenobiotic metabolism and stress

IE

ENSGMOG00000012518

Glutathione S-transferase P

gstp1

gstp1

xenobiotic metabolism and stress

IE

ENSGMOG00000016016

Glutathione S-transferase omega-1

gsto1

gsto1

xenobiotic metabolism and stress

IE

ENSGMOG00000018752

3-hydroxyanthranilate 3,4-dioxygenase

3hao

haao

xenobiotic metabolism and stress

IE

ENSGMOG00000006796

3-beta-hydroxysteroid-Delta(8),Delta(7)-isomerase

ebp

ebp

xenobiotic metabolism and stress

IE

ENSGMOG00000007636

Glutamine synthetase

glna

glul

xenobiotic metabolism and stress

IE

ENSGMOG00000015234

Heat shock protein HSP 90-alpha

h90a1

hsp90a.1

xenobiotic metabolism and stress

IE

ENSGMOG00000012029

Peptidyl-prolyl cis-trans isomerase

ppia

-

xenobiotic metabolism and stress

IE

ENSGMOG00000000218

Ammonium transporter Rh type A OS=Mus

rhag

rhag

xenobiotic metabolism and stress

Mainly IE

ENSGMOG00000003353

Ferritin, middle subunit

frim

-

xenobiotic metabolism and stress

IE

ENSGMOG00000018206

Filamin-C

flnc

Flnc

myofibrillar establishment and repair

IE

ENSGMOG00000001317

Iron-sulfur cluster assembly enzyme ISCU, mitochondrial

iscu

Iscu

cardiac defects

IE

ENSGMOG00000010446

Fatty acid-binding protein, heart

fabph

fabp3

cardiac defects and repair

IE

ENSGMOG00000007115

Lanosterol 14-alpha demethylase

cp51a

cyp51a1

Cholesterol syntheis

IE

ENSGMOG00000005565

Squalene monooxygenase

erg1

Sqle

Cholesterol syntheis

IE

ENSGMOG00000018991

Farnesyl pyrophosphate synthase

fpps

fdps

Cholesterol syntheis

IE

ENSGMOG00000005774

3-hydroxy-3-methylglutaryl-coenzyme A reductase

hmdh

hmgcr

Cholesterol syntheis

IE

ENSGMOG00000015657

Epididymal secretory protein

npc2

npc2

Cholesterol syntheis

IE

ENSGMOG00000001249

Putative adenosylhomocysteinase

sahh3

ahcyl2

cardiac defects

DE

ENSGMOG00000013374

Peptide Y OS=Dicentrarchus

py

-

CNS function and development

IE

ENSGMOG00000014820

Complement C1q-like protein

c1ql2

c1ql2

CNS function and development

IE

ENSGMOG00000017148

Augurin-A OS=Danio rerio

augna

zgc:112443

CNS function and development

IE

ENSGMOG00000001072

C-4 methylsterol oxidase

erg25

sc4mol

CNS function and development

IE

ENSGMOG00000013980

Fatty acid-binding protein, brain

fabp7

fabp7

CNS function and development

IE

ENSGMOG00000014938

Maltase-glucoamylase, intestinal

mga

mgam

ATP metabolism

IE

ENSGMOG00000003530

ADP/ATP translocase

adt3

slc25a6

ATP metabolism

DE

ENSGMOG00000006172

IEF0762 protein C6orf58 homolog

cf058

-

not known

IE

Table 2

Genes expressed at all stages during larval exposure (L1–L5) in high-dose group. SP, swissprot; GB, genebank; IE; increased expression; DE; decreased expression.

https://doi.org/10.7554/eLife.20707.013

Cod ID

Swissprot annotation

SP ID

GB ID

Category

Regulation

ENSGMOG00000009114

Aryl hydrocarbon receptor repressor

ahrr

ahrr

Xenobiotic metabolism

IE

ENSGMOG00000020141

Cytochrome P450 1B1

cp1b1

cyp1b1

Xenobiotic metabolism

IE

ENSGMOG00000006842

Cytochrome P450 1B1

cp1b1

cyp1b1

Xenobiotic metabolism

IE

ENSGMOG00000019790

Cytochrome P450 1B1

cp1b1

cyp1b1

Xenobiotic metabolism

IE

ENSGMOG00000000318

Cytochrome P450 1A1

cp1a1

cyp1a1

Xenobiotic metabolism

IE

ENSGMOG00000014967

Keratinocyte growth factor

fgf7

fgf7

Myocardial development and tissue repair

IE

ENSGMOG00000020500

Forkhead box protein Q1

foxq1

foxq1

Transcription factor

IE

ENSGMOG00000000218

Ammonium transporter Rh type A

rhag

rhag

Gas transport

IE

Table 3

Time course of pathway enrichment relating to affected and unaffected developmental and functional phenotypes.

https://doi.org/10.7554/eLife.20707.014

Phenotype

Development stage*

3 dpf/E1

6 dpf/E2

10 dpf/E3

11 dpf/E4

0 Dph/E5

three Dph/E6

Cardiovascular

0 (0/8)

22.4 (11/49)

5.7 (4/70)

7.0 (4/57)

4.7 (2/43)

2.1 (1/48)

Craniofacial

0 (0/8)

12.2 (6/49)

10 (7/70)

5.3 (3/57)

7.0 (3/43)

2.1 (1/48)

Liver

12.5 (1/8)

0 (0/49)

5.7 (4/70)

8.8 (5/57)

0 (0/43)

0 (0/48)

Eye

0 (0/8)

4.1 (2/49)

20 (14/70)

48.6 (17/35)

51.2 (22/43)

50.0 (24/48)

Osmoregulation

--

43.3 (13/30)

29.3 (12/41)

15.0 (3/20)

16 (4/25)

--

Cholesterol

0/30

0 (0/27)

27.1 (13/48)

31.3 (10/32)

25.5 (12/47)

--

Lipid

0/30

40.7 (11/27)

35.4 (17/48)

50.0 (16/32)

48.9 (23/47)

--

  1. *Percentage of total enriched pathways (absolute values).

  2. Numbers of affected pathways representing Cardiovascular, Craniofacial, Liver and Eye were extracted from the combined Development category in IPA results; numbers of pathways representing osmoregulation/ion transport were extracted from the Molecular Transport category; numbers of pathways affecting Cholesterol/sterol metabolism and other non-cholesterol lipids (Lipid) were extracted from the Lipid Metabolism category.

Additional files

Supplementary file 1

Tables.

(A) Read count data for a selection of genes expressed in distinct tissues. (B) Ten most regulated Ingenuity Pathway Analysis (Categories: Development, Lipid metabolism, Molecular transport) in high dose at stages E1-E6. #, number of molecules (C) Ingenuity Pathway Analysis. Top five pathways in low, pulse and high dose at all time points during and after embryonic exposure in the categories: Top canonical pathways, Molecular and cellular functions, Physiological system development and function and Cardiotoxicity. #, number of molecules (D) Ten most up- and down-regulated genes. Genes that are represented among the ten most at more than one stage are collapsed into one row. SP, swissprot; E1-E6, embryonic exposure; L1-L5, larval exposure; PMID, PubMed identification. (E) Regulation of differentially expressed genes involved in cardiogenesis SP, swissprot; GB, genebank; FC, fold change; E1-E6, embryonic exposure; C, control; H, high dose. (F) Manually curated list of genes involved in cardiac development and function and craniofacial development and bone and cartilage maintenance. SP, swissprot; GB, genebank; Ref, references; PMID: National Center for Biotechnology Information (NCBI) PubMed identification. (G) List of excitation contraction coupling genes examined. SP, swissprot; GB, genebank. (H) Regulation of differentially expressed genes involved in excitation contraction coupling in exposed haddock. SP, swissprot; GB, genebank; FC, fold change; E1-E6, embryonic exposure; C, control; H, high dose; L1-L5, larval exposure. (I) Regulation of differentially expressed genes involved in craniofacial development. SP, swissprot; GB, genebank; FC, fold change; E1-E6, embryonic exposure; C, control; H, high dose. (J) Regulation of differentially expressed myosin heavy chain genes. SP, swissprot; GB, genebank; FC, fold change; E1-E6, embryonic exposure C, control; H, high dose. (K) Regulation of differentially expressed key genes involved in osmoregulation. SP, swissprot; GB, genebank; FC, fold change; E1-E6, embryonic exposure; C, control; H, high dose; L1-L5, larval exposure. (L) Differentially expressed genes involved in osmoregulation. SP, swissprot; GB, genebank; IE, increased expression; DE, decreased expression. (M) Genes involved in liver and lateral line development. SP, swissprot; GB, genebank. (N) Primers and probes for real time qPCR. SP, swissprot.

https://doi.org/10.7554/eLife.20707.019

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