A chemical screen in zebrafish embryonic cells establishes that Akt activation is required for neural crest development

  1. Christie Ciarlo
  2. Charles K Kaufman
  3. Beste Kinikoglu
  4. Jonathan Michael
  5. Song Yang
  6. Christopher D′Amato
  7. Sasja Blokzijl-Franke
  8. Jeroen den Hertog
  9. Thorsten M Schlaeger
  10. Yi Zhou
  11. Eric Liao
  12. Leonard I Zon  Is a corresponding author
  1. Children’s Hospital Boston, Howard Hughes Medical Institute, United States
  2. Harvard Medical School, United States
  3. Washington University School of Medicine, United States
  4. Massachusetts General Hospital, United States
  5. University Medical Center Utrecht, Netherlands
  6. Harvard Stem Cell Institute, United States
7 figures, 5 videos, 2 tables and 1 additional file

Figures

Figure 1 with 1 supplement
Chemical screening in zebrafish embryonic cell cultures identifies inhibitors of neural crest development.

(A) CAPE decreases crestin:EGFP+ cells in culture while leaving ubi:mCherry+ cells unchanged. Scale bar: 100 μm. Characterization of cultured crestin:EGFP+ cells is shown in Figure 1—figure …

https://doi.org/10.7554/eLife.29145.002
Figure 1—source data 1

Ubi:mCherry and crestin:EGFP+ cell numbers in CAPE-treated cultures.

https://doi.org/10.7554/eLife.29145.004
Figure 1—figure supplement 1
Characterization of cultured crestin:EGFP+ cells.

(A) Cell size and morphology visualized by crestin:EGFP. (B) Percentage of crestin:EGFP+ cells determined by FACS. Bars indicate mean and points indicate independent experiments. (C) Crestin:EGFP+ ce…

https://doi.org/10.7554/eLife.29145.003
Figure 1—figure supplement 1—source data 1

Percentage crestin:EGFP+ cells in culture.

https://doi.org/10.7554/eLife.29145.005
Figure 1—figure supplement 1—source data 2

Cell migration speed.

https://doi.org/10.7554/eLife.29145.006
Figure 1—figure supplement 1—source data 3

Fraction EdU+ cells.

https://doi.org/10.7554/eLife.29145.007
Figure 1—figure supplement 1—source data 4

qPCR analysis of neural crest gene expression in cultured crestin:EGFP+ cells.

https://doi.org/10.7554/eLife.29145.008
Figure 2 with 4 supplements
CAPE decreases neural crest gene expression.

(A) CAPE dramatically reduces crestin:EGFP expression at 26 hpf. Figure 2—figure supplement 1 shows the response of a smaller (396 bp) crestin promoter fragment to CAPE and the timing of crestin

https://doi.org/10.7554/eLife.29145.011
Figure 2—source data 1

Expression of neural crest genes by ISH in CAPE-treated embryos.

https://doi.org/10.7554/eLife.29145.016
Figure 2—figure supplement 1
Crestin_296bp:EGFP expression in CAPE-treated embryos and time course of CAPE treatment.

(A) CAPE decreases expression of crestin_296bp:EGFP. Embryos were treated at 2 ss and imaged at 24 hpf. A single clutch is shown. (B) CAPE reduces crestin expression within two hours of treatment. …

https://doi.org/10.7554/eLife.29145.012
Figure 2—figure supplement 1—source data 1

Scoring of crestin_296bp:EGFP expression in CAPE-treated embryos.

https://doi.org/10.7554/eLife.29145.017
Figure 2—figure supplement 2
Neural crest genes not significantly affected by CAPE treatment as determined by ISH.

(A) Representative embryos from ISH. Images are representative of at least two independent experiments. Arrows point to regions with subtle decreases in expression. (B) Scoring of ISH in (A). Images …

https://doi.org/10.7554/eLife.29145.013
Figure 2—figure supplement 2—source data 1

Scoring of neural crest gene expression by ISH in CAPE-treated embryos.

https://doi.org/10.7554/eLife.29145.018
Figure 2—figure supplement 3
Changes in cell number do not explain reduced crestin expression in CAPE-treated embryos.

Embryos were treated continuously starting at 2 ss. (A) CAPE does not affect proliferation of neural crest cells as determined by phospho-histone H3 in the sox10:GFP+ region of embryos. Flat mounts …

https://doi.org/10.7554/eLife.29145.014
Figure 2—figure supplement 3—source data 1

PH3+ cells in sox10:GFP+ region of CAPE-treated embryos.

https://doi.org/10.7554/eLife.29145.019
Figure 2—figure supplement 3—source data 2

TUNEL+ cells in neural crest region of CAPE-treated embryos.

https://doi.org/10.7554/eLife.29145.020
Figure 2—figure supplement 4
Analysis of gene expression changes in sox10:Kaede+ cells by RNA-seq, comparing DMSO- to CAPE-treated embryos.

(A) IPA analysis pointed to increased inflammatory signaling with CAPE treatment. (B) CAPE alters morphogen expression. Red indicates upregulation while green indicates downregulation. Fold change …

https://doi.org/10.7554/eLife.29145.015
Figure 3 with 1 supplement
CAPE reduces Sox10 activity.

(A) ATAC-seq was conducted on sox10:Kaede+ cells from DMSO- or CAPE-treated embryos at two stages. CAPE reduces chromatin accessibility at the mitfa promoter in sox10:Kaede+ cells, and Sox10 binds …

https://doi.org/10.7554/eLife.29145.023
Figure 3—source data 1

Scoring of crestin:EGFP expression in sox10-injected and CAPE-treated embryos.

https://doi.org/10.7554/eLife.29145.025
Figure 3—figure supplement 1
Tfap2c RNA injection (120 pg) increases crestin:EGFP expression in both control and CAPE-treated embryos.

(A) Crestin:EGFP expression in representative embryos. (B) Embryos were scored as in Figure 3E. Sum of three clutches from two independent experiments is shown. (C) Based on flow cytometric analysis …

https://doi.org/10.7554/eLife.29145.024
Figure 3—figure supplement 1—source data 1

Scoring of crestin:EGFP expression in tfap2c-injected and CAPE-treated embryos.

https://doi.org/10.7554/eLife.29145.026
Figure 3—figure supplement 1—source data 2

Percentage sox10:Kaede+ live cells in CAPE-treated embryos.

https://doi.org/10.7554/eLife.29145.027
Figure 4 with 2 supplements
CAPE inhibits neural crest migration and pigment cell differentiation.

Embryos were treated at 2 ss unless otherwise indicated. (A) Sox10:Kaede+ cells in the trunk of zebrafish embryos are more dorsally located at 24 hpf. Dotted line indicates top of yolk sac …

https://doi.org/10.7554/eLife.29145.028
Figure 4—source data 1

Melanocyte numbers in CAPE-treated embryos.

https://doi.org/10.7554/eLife.29145.031
Figure 4—source data 2

Fraction dorsal melanocytes in CAPE-treated embryos.

https://doi.org/10.7554/eLife.29145.032
Figure 4—figure supplement 1
CAPE disrupts iridophore development less dramatically than melanocyte development.

Embryos were treated with the indicated concentration of CAPE at 2 ss either continuously (5 μM) or until 48 hpf (7.5 μM washout) to reduce toxicity. (A) Pigment cell phenotypes at 3 dpf. (B) …

https://doi.org/10.7554/eLife.29145.029
Figure 4—figure supplement 1—source data 1

Number of iridophores in CAPE-treated embryos.

https://doi.org/10.7554/eLife.29145.033
Figure 4—figure supplement 1—source data 2

Fraction dorsal iridophores in CAPE-treated embryos.

https://doi.org/10.7554/eLife.29145.034
Figure 4—figure supplement 2
CAPE disrupts ear development.

Embryos were treated continuously from 2 ss. (A) Otic vesicles of control and CAPE-treated embryos at 24 hpf. (B) Otic vesicles of control and CAPE-treated embryos at 48 hpf. Note lack of …

https://doi.org/10.7554/eLife.29145.030
Figure 5 with 1 supplement
Myr-Akt1 rescues neural crest defects caused by CAPE.

(A) Injection of myr-Akt1 RNA results in high phospho-Akt in heterogeneous neural crest cultures regardless of CAPE treatment. The same result was observed in four independent experiments. (B) …

https://doi.org/10.7554/eLife.29145.037
Figure 5—source data 1

Scoring of crestin expression by ISH in CAPE-treated and myr-Akt1-injected embryos.

https://doi.org/10.7554/eLife.29145.039
Figure 5—source data 2

Melanocyte numbers in CAPE-treated and myr-Akt1-injected embryos.

https://doi.org/10.7554/eLife.29145.040
Figure 5—source data 3

Melanocyte numbers in CAPE-treated and myr-Akt1-injected embryos.

https://doi.org/10.7554/eLife.29145.041
Figure 5—figure supplement 1
Effect of PI3K inhibitors on crestin:EGFP expression in vitro and effect of myr-Akt1 injection on CAPE-induced defects.

(A) PI3K inhibitors selectively reduce crestin:EGFP+ cells compared to ubi:mCherry+ controls representing a random population of cells. Cells were plated with the indicated concentration of …

https://doi.org/10.7554/eLife.29145.038
Figure 5—figure supplement 1—source data 1

Ubi:mCherry+ and crestin:EGFP+ cell numbers in cultures treated with PI-103.

https://doi.org/10.7554/eLife.29145.042
Figure 5—figure supplement 1—source data 2

Ubi:mCherry+ and crestin:EGFP+ cell numbers in cultures treated with GDC0941.

https://doi.org/10.7554/eLife.29145.043
Figure 5—figure supplement 1—source data 3

Developmental defects in CAPE-treated and myr-Akt1-injected embryos.

https://doi.org/10.7554/eLife.29145.044
Figure 6 with 2 supplements
CAPE inhibits FGF-stimulated Akt activation in vitro.

Embryos were plated in neural crest medium and cultured for 2 hr for western blotting. (A) Heterogeneous neural crest cultures after 24 hr in neural crest medium with or without FGF and insulin. …

https://doi.org/10.7554/eLife.29145.045
Figure 6—source data 1

Number of crestin:EGFP+ cells per total ubi:mCherry fluorescence.

https://doi.org/10.7554/eLife.29145.048
Figure 6—source data 2

Quantification of p-Erk/Erk and p-Akt /Akt ratios by western blot and densitometry.

https://doi.org/10.7554/eLife.29145.049
Figure 6—figure supplement 1
CAPE inhibits FGF-stimulated Akt activation.

Whole embryos were cultured for two hours in neural crest medium with the indicated growth factors and inhibitors for all experiments. Unless otherwise noted, cultures contained insulin and FGF. …

https://doi.org/10.7554/eLife.29145.046
Figure 6—figure supplement 1—source data 1

Scoring of crestin expression by ISH in pten mutant embryos.

https://doi.org/10.7554/eLife.29145.050
Figure 6—figure supplement 2
RTK signaling regulates crestin expression.

(A) Treatment of embryos with Chembridge novel kinase inhibitors at 2 ss decreases crestin expression at 15 ss. (B) Quantification of crestin expression in drug-treated embryos based on the scoring …

https://doi.org/10.7554/eLife.29145.047
Figure 6—figure supplement 2—source data 1

Scoring of crestin expression by ISH with kinase inhibitor treatment.

https://doi.org/10.7554/eLife.29145.051
Akt signaling regulates neural crest gene expression in vivo.

(A) PTEN QMA-mCherry (300 pg) reduces phospho-Akt level in whole embryos. (B) Morphology, PTEN QMA-mCherry expression, and crestin:EGFP expression of PTEN QMA-mCherry injected embryos. Scoring …

https://doi.org/10.7554/eLife.29145.052
Figure 7—source data 1

Scoring of crestin:EGFP expression in PTEN QMA-mCherry-injected embryos.

https://doi.org/10.7554/eLife.29145.053
Figure 7—source data 2

Scoring of crestin expression by ISH in PTEN QMA-injected embryos.

https://doi.org/10.7554/eLife.29145.054
Figure 7—source data 3

Scoring of crestin:EGFP expression in LY294002-treated embryos.

https://doi.org/10.7554/eLife.29145.055
Figure 7—source data 4

Scoring of crestin:EGFP in sox10- and PTEN-injected embryos.

https://doi.org/10.7554/eLife.29145.056

Videos

Video 1
Crestin:EGFP time lapse in heterogeneous neural crest cultures.
https://doi.org/10.7554/eLife.29145.009
Video 2
Crestin:EGFP expression in DMSO-treated zebrafish embryos.

Embryos were treated at 2 ss and mounted for imaging at 10 ss. Embryos were imaged for 16.25 hr, and images were collected every 9 min.

https://doi.org/10.7554/eLife.29145.021
Video 3
Crestin:EGFP expression in CAPE-treated (10 μM) zebrafish embryos.

Embryos were treated at 2 ss and mounted for imaging at 10 ss. Embryos were imaged for 16.25 hr, and images were collected every 9 min.

https://doi.org/10.7554/eLife.29145.022
Video 4
Neural crest migration in DMSO-treated zebrafish embryos.

Sox10:GFP transgenic embryos were treated at 2 ss and mounted for imaging at 15 ss. Embryos were imaged for 12 hr, and images were collected every 10 min.

https://doi.org/10.7554/eLife.29145.035
Video 5
Neural crest migration in CAPE-treated (10 μM) zebrafish embryos.

Sox10:GFP transgenic embryos were treated at 2 ss and mounted for imaging at 15 ss. Embryos were imaged for 12 hr, and images were collected every 10 min.

https://doi.org/10.7554/eLife.29145.036

Tables

Table 1
In vitro validated screen hits.

Hits that also validated in vivo are bold.

https://doi.org/10.7554/eLife.29145.010
CompoundLibraryTarget/category
CAPEICCBNFkB/multiple
raloxifeneLOPACEstrogen receptor
mianserinLOPAC5-HT receptor antagonist
GANT61LOPACHedgehog
MnTBAPICCBSOD mimetic
loperamideFDA approvedMu opiod receptor agonist
latanoprostFDA approvedprostaglandin F2a analogue
tetraethylthiuram disulfideLOPACalcohol dehydrogenase
dopamineLOPACdopamine receptor
mycophenolate mofetilLOPACIMPH
genisteinLOPACkinase inhibitor
albendazoleFDA approvedantihelminthic
JFD00244LOPACsirt2 inhibitor
perphenazineFDA approved5-HT receptor
5-NOTFDA approved5-HT agonist
SKF95282LOPAChistamine H2 receptor antagonist
bicalutamideFDA approvedanti-androgen
capsazepineLOPACsodium channels
triflupromazineLOPACmonoamine transporters
flubendazoleFDA approvedantihelminthic
GDC-0941LOPACPI3K
imatinibFDA approvedRTK inhibitor
indatralineLOPACdopamine uptake inhibitor
MBCQICCBPDE5
MDL-28170ICCBcalpain inhibitor
NS8593LOPACpotassium channels
NU6027LOPACATR/CDK2 inhibitor
PD180970LOPACRTK inhibitor
PD173074LOPACsrc inhibitor
PI-103LOPACPI3K
rapamycinLOPACmTOR
SB242084LOPAC5-HT receptor antagonist
triptolideFDA approvedRNA pol II
tyrphostin AG698LOPACtyrosine kinase inhibitor
wiskostatinLOPACactin
PAC-1LOPACproapoptotic zinc chelator
PD407824LOPACchk1 inhibitor
PD173952LOPACsrc inhibitor
sanguinarineLOPACNa/K ATPase
tyrphostin AG835LOPACEGFR
(-)-alpha-methylnorepinephrineLOPACsympathomimetic
chloroquineLOPACantimalarial
M-344LOPACHDAC inhibitor
olmesartan medoxomilFDA approvedangiotensin II receptor antagonist
1,10-phenanthrolineLOPACchelator, MMP
2,3-dimethoxy-1,4-naphthoquinoneLOPACROS
amilorideICCBcalcium channels
fluvastatinFDA approvedHMG co-A reductase
CHM-1LOPACantimitotic
SAHALOPACHDAC inhibitor
nimesulideLOPACCOX-2
mibefradilLOPACcalcium channels
KB-R7493LOPACsodium calcium exchanger
LY165163LOPAC5-HT receptor antagonist
dequaliniumLOPACpotassium channels
AM92016ICCBpotassium channels
2-[4-(1,3-benzodioxol-5-yl)−1H-pyrazol-1-yl]-N-(2-ethyl-2H-1,2,3-triazol-4-yl)acetamideChembridgepredicted adenosine kinase
N-(2-ethyl-2H-1,2,3-triazol-4-yl)−2-{4-[3-(1H-pyrazol-1-yl)phenyl]−1H-pyrazol-1-yl}acetamideChembridgepredicted adenosine kinase
2,2,6,6-tetramethyl-N-(1-methyl-3-phenylpropyl)−4-piperidinamineChembridgepredicted vitamin D receptor
N-[(5-chloro-1H-indol-2-yl)methyl]−2-(3-hydroxyphenyl)acetamideChembridgepredicted TK(FLT3)
5-(1H-indol-2-ylcarbonyl)−4,5,6,7-tetrahydrothieno[3,2 c]pyridineChembridgepredicted TK(FLT3)
1-acetyl-4-{4-[1-(2-fluorophenyl)−1H-pyrazol-4-yl]pyrimidin-2-yl}−1,4-diazepaneChembridgepredicted JNK
4-(4-butyl-1H-1,2,3-triazol-1-yl)−1-{[(1S*,4S*)−3,3-dimethyl-2-methylenebicyclo[2.2.1]hept-1-yl]carbonyl}piperiChembridgepredicted liver X receptor
1-(3-methylbenzyl)−4-thieno[2,3-d]pyrimidin-4-yl-2-piperazinoneChembridgepredicted TK(EGFR, PDGFR, CSFR1); PKC; PKA
1-(2-methoxyphenyl)−2,2-dimethyl-4-(4-methylpentanoyl)piperazineChembridgepredicted androgen receptor
5,6-dimethyl-2-[4-({methyl[(2-methylpyridin-4-yl)methyl]amino}methyl)phenyl]pyrimidin-4(3 hr)-oneChembridgepredicted estrogen receptor
N-[1-(1,5-dimethyl-1H-pyrazol-4-yl)ethyl]thieno[2,3-d]pyrimidin-4-amineChembridgepredicted EGFR
N-(1-cyclohexyl-1H-pyrazol-5-yl)−2-[3-(2-thienyl)−1H-pyrazol-1-yl]acetamideChembridgepredicted VEGFR2, EGF/KDR
2-[1-(3-isobutyl-1,2,4-oxadiazol-5-yl)−2-methylbutyl]−1-isoindolinoneChembridgepredicted RAR(gamma)
1-propyl-N-{1-[4-(1H-pyrazol-1-yl)phenyl]piperidin-4-yl}piperidin-4-amineChembridgepredicted estrogen receptor
5-[5-methyl-4-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl]−4,5,6,7-tetrahydrothieno[3,2 c]pyridineChembridgepredicted TK(VEGFR, KDR, FLK1)
2-[5-(2,6-dimethylphenyl)−1H-indazol-1-yl]-N-(1,3-dimethyl-1H-pyrazol-5-yl)acetamideChembridgepredicted TK(PDGFR, EGFR, FGFR)
Table 2
Primer sequences.
https://doi.org/10.7554/eLife.29145.057
UseGeneForwardReverseReference
qPCRbactin1CGAGCAGGAGATGGGAACCCAACGGAAACGCTCATTGC(McCurley and Callard, 2008)
qPCRsox10ATATCCGCACCTGCACAACGTTCAGCAGTCTCCACAG
qPCRcrestinAGTGCCTGCCAATGTTCACCTGAAAAAGGCCGATGAGTT
qPCRfoxd3CATGCAAAACAAGCCCAAGATGAGGGCGATGTACGAGTAG
qPCRmitfaGGCGGTTTAATATCAATGACAGAGGTGCCTTTATTCCACCTCA
qPCRneurog1CGTGCCATTATCTTCAACACACGATCTCCATTGTTGATAACCTT
qPCRmyf5GCTACAACTTTGACGCACAAAACACGATGCTGGACAAACACT
qPCRrunx1CGTCTTCACAAACCCTCCTCAAGCTTTACTGCTTCATCCGGCT
ISHtfap2aTAATACGACTCACTATAGGGAATCT
TCACAGATGTTAGTGCACAGTTTTTCCGCGAT
AATTAACCCTCACTAAAGGTCAC
TTTCTGTGCTTCTCATCTT
ISHtfap2cTAATACGACTCACTATAGGGACAG
AAACAACATGTTGTGGAAATTAGCAGATAA
AATTAACCCTCACTAAAGGTCA
CTTTCGGTGTTTGTCCATCTT
ISHinka1aAATTAACCCTCACTAAAGGG
GAATCGGGTGACTGTCTGC
TAATACGACTCACTATAGGGATGG
GTGTTCTGCTCCCAG
ISHdlx2aAATTAACCCTCACTAAAGGACAA
CAGCATGAACAGCGTC
TAATACGACTCACTATAGGGACAGGC
GCATGAAACACAT
ISHpax7aAATTAACCCTCACTAAAGGAGAA
CTACCCACGAACCGGA
TAATACGACTCACTATAGGTTGATC
TGTGAAGCGTGCTG
ISHmycaTAATACGACTCACTATAGGGCAAG
TGTCAAAATGCCGGTGAGTGCGAGTTTGGCGT
AATTAACCCTCACTAAAGGTTAATGTG
AACTCCGCAGCTGCTGAA
ISHets1TAATACGACTCACTATAGGGTGTA
CGTTTGAATGCGTGACCATGACGGCAGCTGT
AATTAACCCTCACTAAAGGTCAGGAGC
TCCAACAGGAACTGCCAGA
ISHnr2f2TAATACGACTCACTATAGGGTAGATATGGC
AATGGTAGTGTGGAGAGGCTCCCA
AATTAACCCTCACTAAAGGCTACTGAAT
CGACATATAAGGCCAGTT
ISHmsx1bTAATACGACTCACTATAGGGGATGGTTAA
CGATGAATTCTCCTAAGGGACCCGTT
AATTAACCCTCACTAAAGGTTAAGAC
AAATAATACATCCCATA
ISHdlx5aTAATACGACTCACTATAGGGTTATCCAA
ACTATGACTGGAGTATTCGACAGAAGGA
AATTAACCCTCACTAAAGGTCAGTACAAC
GTTCCTGATCCGAGTGCCAA

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