(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 …
Ubi:mCherry and crestin:EGFP+ cell numbers in CAPE-treated cultures.
(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…
Percentage crestin:EGFP+ cells in culture.
Cell migration speed.
Fraction EdU+ cells.
qPCR analysis of neural crest gene expression in cultured crestin:EGFP+ cells.
(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 …
Expression of neural crest genes by ISH in CAPE-treated embryos.
(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. …
Scoring of crestin_296bp:EGFP expression in CAPE-treated embryos.
(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 …
Scoring of neural crest gene expression by ISH 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 …
PH3+ cells in sox10:GFP+ region of CAPE-treated embryos.
TUNEL+ cells in neural crest region of 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 …
(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 …
Scoring of crestin:EGFP expression in sox10-injected 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 …
Scoring of crestin:EGFP expression in tfap2c-injected and CAPE-treated embryos.
Percentage sox10:Kaede+ live cells in CAPE-treated embryos.
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 …
Melanocyte numbers in CAPE-treated embryos.
Fraction dorsal melanocytes in CAPE-treated embryos.
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) …
Number of iridophores in CAPE-treated embryos.
Fraction dorsal iridophores in CAPE-treated embryos.
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 …
(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) …
Scoring of crestin expression by ISH in CAPE-treated and myr-Akt1-injected embryos.
Melanocyte numbers in CAPE-treated and myr-Akt1-injected embryos.
Melanocyte numbers in CAPE-treated and myr-Akt1-injected embryos.
(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 …
Ubi:mCherry+ and crestin:EGFP+ cell numbers in cultures treated with PI-103.
Ubi:mCherry+ and crestin:EGFP+ cell numbers in cultures treated with GDC0941.
Developmental defects in CAPE-treated and myr-Akt1-injected embryos.
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. …
Number of crestin:EGFP+ cells per total ubi:mCherry fluorescence.
Quantification of p-Erk/Erk and p-Akt /Akt ratios by western blot and densitometry.
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. …
Scoring of crestin expression by ISH in pten mutant embryos.
(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 …
Scoring of crestin expression by ISH with kinase inhibitor treatment.
(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 …
Scoring of crestin:EGFP expression in PTEN QMA-mCherry-injected embryos.
Scoring of crestin expression by ISH in PTEN QMA-injected embryos.
Scoring of crestin:EGFP expression in LY294002-treated embryos.
Scoring of crestin:EGFP in sox10- and PTEN-injected 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.
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.
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.
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.
Hits that also validated in vivo are bold.
Compound | Library | Target/category |
---|---|---|
CAPE | ICCB | NFkB/multiple |
raloxifene | LOPAC | Estrogen receptor |
mianserin | LOPAC | 5-HT receptor antagonist |
GANT61 | LOPAC | Hedgehog |
MnTBAP | ICCB | SOD mimetic |
loperamide | FDA approved | Mu opiod receptor agonist |
latanoprost | FDA approved | prostaglandin F2a analogue |
tetraethylthiuram disulfide | LOPAC | alcohol dehydrogenase |
dopamine | LOPAC | dopamine receptor |
mycophenolate mofetil | LOPAC | IMPH |
genistein | LOPAC | kinase inhibitor |
albendazole | FDA approved | antihelminthic |
JFD00244 | LOPAC | sirt2 inhibitor |
perphenazine | FDA approved | 5-HT receptor |
5-NOT | FDA approved | 5-HT agonist |
SKF95282 | LOPAC | histamine H2 receptor antagonist |
bicalutamide | FDA approved | anti-androgen |
capsazepine | LOPAC | sodium channels |
triflupromazine | LOPAC | monoamine transporters |
flubendazole | FDA approved | antihelminthic |
GDC-0941 | LOPAC | PI3K |
imatinib | FDA approved | RTK inhibitor |
indatraline | LOPAC | dopamine uptake inhibitor |
MBCQ | ICCB | PDE5 |
MDL-28170 | ICCB | calpain inhibitor |
NS8593 | LOPAC | potassium channels |
NU6027 | LOPAC | ATR/CDK2 inhibitor |
PD180970 | LOPAC | RTK inhibitor |
PD173074 | LOPAC | src inhibitor |
PI-103 | LOPAC | PI3K |
rapamycin | LOPAC | mTOR |
SB242084 | LOPAC | 5-HT receptor antagonist |
triptolide | FDA approved | RNA pol II |
tyrphostin AG698 | LOPAC | tyrosine kinase inhibitor |
wiskostatin | LOPAC | actin |
PAC-1 | LOPAC | proapoptotic zinc chelator |
PD407824 | LOPAC | chk1 inhibitor |
PD173952 | LOPAC | src inhibitor |
sanguinarine | LOPAC | Na/K ATPase |
tyrphostin AG835 | LOPAC | EGFR |
(-)-alpha-methylnorepinephrine | LOPAC | sympathomimetic |
chloroquine | LOPAC | antimalarial |
M-344 | LOPAC | HDAC inhibitor |
olmesartan medoxomil | FDA approved | angiotensin II receptor antagonist |
1,10-phenanthroline | LOPAC | chelator, MMP |
2,3-dimethoxy-1,4-naphthoquinone | LOPAC | ROS |
amiloride | ICCB | calcium channels |
fluvastatin | FDA approved | HMG co-A reductase |
CHM-1 | LOPAC | antimitotic |
SAHA | LOPAC | HDAC inhibitor |
nimesulide | LOPAC | COX-2 |
mibefradil | LOPAC | calcium channels |
KB-R7493 | LOPAC | sodium calcium exchanger |
LY165163 | LOPAC | 5-HT receptor antagonist |
dequalinium | LOPAC | potassium channels |
AM92016 | ICCB | potassium channels |
2-[4-(1,3-benzodioxol-5-yl)−1H-pyrazol-1-yl]-N-(2-ethyl-2H-1,2,3-triazol-4-yl)acetamide | Chembridge | predicted adenosine kinase |
N-(2-ethyl-2H-1,2,3-triazol-4-yl)−2-{4-[3-(1H-pyrazol-1-yl)phenyl]−1H-pyrazol-1-yl}acetamide | Chembridge | predicted adenosine kinase |
2,2,6,6-tetramethyl-N-(1-methyl-3-phenylpropyl)−4-piperidinamine | Chembridge | predicted vitamin D receptor |
N-[(5-chloro-1H-indol-2-yl)methyl]−2-(3-hydroxyphenyl)acetamide | Chembridge | predicted TK(FLT3) |
5-(1H-indol-2-ylcarbonyl)−4,5,6,7-tetrahydrothieno[3,2 c]pyridine | Chembridge | predicted TK(FLT3) |
1-acetyl-4-{4-[1-(2-fluorophenyl)−1H-pyrazol-4-yl]pyrimidin-2-yl}−1,4-diazepane | Chembridge | predicted 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}piperi | Chembridge | predicted liver X receptor |
1-(3-methylbenzyl)−4-thieno[2,3-d]pyrimidin-4-yl-2-piperazinone | Chembridge | predicted TK(EGFR, PDGFR, CSFR1); PKC; PKA |
1-(2-methoxyphenyl)−2,2-dimethyl-4-(4-methylpentanoyl)piperazine | Chembridge | predicted androgen receptor |
5,6-dimethyl-2-[4-({methyl[(2-methylpyridin-4-yl)methyl]amino}methyl)phenyl]pyrimidin-4(3 hr)-one | Chembridge | predicted estrogen receptor |
N-[1-(1,5-dimethyl-1H-pyrazol-4-yl)ethyl]thieno[2,3-d]pyrimidin-4-amine | Chembridge | predicted EGFR |
N-(1-cyclohexyl-1H-pyrazol-5-yl)−2-[3-(2-thienyl)−1H-pyrazol-1-yl]acetamide | Chembridge | predicted VEGFR2, EGF/KDR |
2-[1-(3-isobutyl-1,2,4-oxadiazol-5-yl)−2-methylbutyl]−1-isoindolinone | Chembridge | predicted RAR(gamma) |
1-propyl-N-{1-[4-(1H-pyrazol-1-yl)phenyl]piperidin-4-yl}piperidin-4-amine | Chembridge | predicted estrogen receptor |
5-[5-methyl-4-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl]−4,5,6,7-tetrahydrothieno[3,2 c]pyridine | Chembridge | predicted TK(VEGFR, KDR, FLK1) |
2-[5-(2,6-dimethylphenyl)−1H-indazol-1-yl]-N-(1,3-dimethyl-1H-pyrazol-5-yl)acetamide | Chembridge | predicted TK(PDGFR, EGFR, FGFR) |
Use | Gene | Forward | Reverse | Reference |
---|---|---|---|---|
qPCR | bactin1 | CGAGCAGGAGATGGGAACC | CAACGGAAACGCTCATTGC | (McCurley and Callard, 2008) |
qPCR | sox10 | ATATCCGCACCTGCACAA | CGTTCAGCAGTCTCCACAG | |
qPCR | crestin | AGTGCCTGCCAATGTTCAC | CTGAAAAAGGCCGATGAGTT | |
qPCR | foxd3 | CATGCAAAACAAGCCCAAG | ATGAGGGCGATGTACGAGTAG | |
qPCR | mitfa | GGCGGTTTAATATCAATGACAGA | GGTGCCTTTATTCCACCTCA | |
qPCR | neurog1 | CGTGCCATTATCTTCAACACA | CGATCTCCATTGTTGATAACCTT | |
qPCR | myf5 | GCTACAACTTTGACGCACAAAA | CACGATGCTGGACAAACACT | |
qPCR | runx1 | CGTCTTCACAAACCCTCCTCAA | GCTTTACTGCTTCATCCGGCT | |
ISH | tfap2a | TAATACGACTCACTATAGGGAATCT TCACAGATGTTAGTGCACAGTTTTTCCGCGAT | AATTAACCCTCACTAAAGGTCAC TTTCTGTGCTTCTCATCTT | |
ISH | tfap2c | TAATACGACTCACTATAGGGACAG AAACAACATGTTGTGGAAATTAGCAGATAA | AATTAACCCTCACTAAAGGTCA CTTTCGGTGTTTGTCCATCTT | |
ISH | inka1a | AATTAACCCTCACTAAAGGG GAATCGGGTGACTGTCTGC | TAATACGACTCACTATAGGGATGG GTGTTCTGCTCCCAG | |
ISH | dlx2a | AATTAACCCTCACTAAAGGACAA CAGCATGAACAGCGTC | TAATACGACTCACTATAGGGACAGGC GCATGAAACACAT | |
ISH | pax7a | AATTAACCCTCACTAAAGGAGAA CTACCCACGAACCGGA | TAATACGACTCACTATAGGTTGATC TGTGAAGCGTGCTG | |
ISH | myca | TAATACGACTCACTATAGGGCAAG TGTCAAAATGCCGGTGAGTGCGAGTTTGGCGT | AATTAACCCTCACTAAAGGTTAATGTG AACTCCGCAGCTGCTGAA | |
ISH | ets1 | TAATACGACTCACTATAGGGTGTA CGTTTGAATGCGTGACCATGACGGCAGCTGT | AATTAACCCTCACTAAAGGTCAGGAGC TCCAACAGGAACTGCCAGA | |
ISH | nr2f2 | TAATACGACTCACTATAGGGTAGATATGGC AATGGTAGTGTGGAGAGGCTCCCA | AATTAACCCTCACTAAAGGCTACTGAAT CGACATATAAGGCCAGTT | |
ISH | msx1b | TAATACGACTCACTATAGGGGATGGTTAA CGATGAATTCTCCTAAGGGACCCGTT | AATTAACCCTCACTAAAGGTTAAGAC AAATAATACATCCCATA | |
ISH | dlx5a | TAATACGACTCACTATAGGGTTATCCAA ACTATGACTGGAGTATTCGACAGAAGGA | AATTAACCCTCACTAAAGGTCAGTACAAC GTTCCTGATCCGAGTGCCAA |