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ZFIN ID: ZDB-FIG-170306-4
Gays et al., 2017 - An exclusive cellular and molecular network governs intestinal smooth muscle cell differentiation in vertebrates. Development (Cambridge, England)   144(3):464-478 Full text @ Development
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Antibody:
Fish:
Condition:
Knockdown Reagent:
Anatomical Terms:
Stage Range: Long-pec to Protruding-mouth
PHENOTYPE:
Fish:
Condition:
Knockdown Reagent:
Observed In:
Stage Range: Long-pec to Protruding-mouth

Fig. 3

LPM migration is guided by TGFβ signaling. (A) Pharmacological and genetic TGFβ signaling blockade impairs iSMC differentiation. Fluorescent images of Tg(acta2:mCherry)uto5 embryos treated with SB431542 (a TGFβ type I receptors inhibitor) or ltbp3 knockdown (encoding a protein that regulates the bioavailability of TGFβ ligands) exhibit no or few iSMCs, as evaluated by mCherry and Tagln expression in the gut region (g) (arrow) at 72 hpf. Scale bars: 200 μm. n, notochord; h, heart. Insets show confocal transverse sections of posterior gut regions (dashed vertical line) of SB431542-treated embryos and embryos injected with ltbp3 morpholino and stained for Tagln (green). The numbers of embryos showing the phenotype are indicated. Blue indicates nuclei. Scale bars in insets: 10 μm. (B) Alk5 blockade does not affect endoderm development and differentiation. Box and whisker plots show the percentage of iSMCs or endodermal cells isolated by fluorescent-activated cell sorting (FACS) experiments from the trunks spanning from somite 1 to 13 of the double Tg(acta2:mCherry)uto5 (Xia.Eef1a1:GFP)s854 embryos at 72 hpf after chemical (SB431542) or genetic (ltbp3 KD) Alk5 signaling blockade. The boxplots show the maximum, minimum, upper and lower quartiles, and the sample median. Asterisks represent the results of one-way ANOVA-Dunnett's post-hoc test (**P<0.01, ****P<0.0001; from left to right: n=8, n=3, n=5, n=5, n=5 and n=5 groups of 10-20 embryos). (C) Blockade of TGFβ signaling impairs LPM migration. Upper panel: confocal transverse sections of Tg(hand2:EGFP)pd24 stained with phalloidin (red) and Hoechst (blue) showing a reduced LPM migration at 48 hpf (arrows) after TGFβ inhibition. Scale bars: 15 μm. The number of embryos exhibiting this phenotype is indicated. Lower panel: schematic representation of impaired LPM migration (black arrows) observed with the lack of TGFβ signaling in zebrafish embryos. Green, LPM; pink, endoderm. (D) Alk5 blockade does not affect LPM. Box and whisker plots show the percentage of LPM cells isolated by fluorescent-activated cell sorting (FACS) experiments from the trunks of Tg(hand2:EGFP)pd24 embryos at 48 hpf after chemical (SB431542) or genetic (ltbp3 KD) Alk5 signaling inhibition. The boxplots show the maximum, minimum, upper and lower quartiles, and the sample median. From left to right: n=13, n=18 and n=10 groups of 10-20 embryos.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
EGFP pd24Tg control Long-pec mesoderm IHC
pd24Tg chemical treatment by environment: SB 431542 Long-pec mesoderm IHC
pd24Tg + MO2-ltbp3 control Long-pec mesoderm IHC
mCherry uto5Tg control Protruding-mouth heart IFL
Protruding-mouth intestine smooth muscle IFL
Protruding-mouth notochord IFL
uto5Tg chemical treatment by environment: SB 431542 Protruding-mouth heart IFL
Protruding-mouth intestine smooth muscle IFL
Protruding-mouth notochord IFL
uto5Tg + MO2-ltbp3 control Protruding-mouth heart IFL
Protruding-mouth intestine smooth muscle IFL
Protruding-mouth notochord IFL
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Anatomy
Ab1-tagln IHC uto5Tg control Protruding-mouth intestine smooth muscle
IHC uto5Tg chemical treatment by environment: SB 431542 Protruding-mouth intestine smooth muscle
IHC uto5Tg + MO2-ltbp3 control Protruding-mouth intestine smooth muscle
Phenotype Details
Fish Conditions Stage Phenotype
pd24Tg chemical treatment by environment: SB 431542 Long-pec lateral plate mesoderm tissue migration decreased occurrence, abnormal
Long-pec lateral plate mesoderm tissue migration decreased process quality, abnormal
pd24Tg + MO2-ltbp3 control Long-pec lateral plate mesoderm tissue migration decreased occurrence, abnormal
Long-pec lateral plate mesoderm tissue migration decreased process quality, abnormal
s854Tg ; uto5Tg chemical treatment by environment: SB 431542 Protruding-mouth intestine smooth muscle cell decreased amount, abnormal
s854Tg ; uto5Tg + MO2-ltbp3 control Protruding-mouth intestine smooth muscle cell decreased amount, abnormal
uto5Tg chemical treatment by environment: SB 431542 Protruding-mouth intestine smooth muscle ab1-tagln labeling decreased amount, abnormal
Protruding-mouth intestine smooth muscle mCherry expression decreased amount, abnormal
uto5Tg + MO2-ltbp3 control Protruding-mouth intestine smooth muscle ab1-tagln labeling decreased amount, abnormal
Protruding-mouth intestine smooth muscle mCherry expression decreased amount, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Development (Cambridge, England) for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Development