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ZFIN ID: ZDB-FIG-170306-6
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:
Knockdown Reagents:
Anatomical Terms:
Stage Range: Long-pec to Protruding-mouth
PHENOTYPE:
Fish:
Knockdown Reagents:
Observed In:
Stage Range: Long-pec to Protruding-mouth

Fig. 4

TGFβ-driven LPM morphogenesis requires zeb1a. (A) Knockdown of the transcription factor zeb1a impairs iSMC differentiation. Fluorescent images of Tg(acta2:mCherry)uto5 embryos at 72 hpf after zeb1a morpholino injections. zeb1a knockdown embryos exhibit decreased mCherry and Tagln expression in the gut region (g) compared with controls (arrow). Scale bars: 200 μm. Insets show confocal transverse sections of the posterior gut region (dashed line) in embryos stained for Tagln (green). The number of embryos exhibiting the phenotype is indicated. Nuclei are in blue. Scale bars in insets: 10 μm. n, notochord; h, heart. (B) Knockdown of zeb1a does not alter endoderm morphology and differentiation. Box and whisker plots show the percentage of iSMCs or endodermal cells isolated by fluorescent-activated cell sorting (FACS) experiments from the trunk of double Tg(acta2:mCherry)uto5 (Xia.Eef1a1:GFP)s854 embryos at 72 hpf after zeb1a downregulation. Although the number of iSMCs is severely reduced by zeb1a knockdown, endodermal cells are normal. The boxplots show the maximum, minimum, upper and lower quartiles, and the sample median. Asterisks represent the results of unpaired t-tests of mean difference=0 (***P<0.001; n=6 groups of 10-20 embryos). (C) Knockdown of zeb1a impairs LPM ventral migration. Upper panel: confocal transverse sections of Tg(hand2:EGFP)pd24 stained with phalloidin (red). The number of embryos exhibiting this phenotype is indicated. Scale bars: 15 μm. Lower panel: schematic representation of impaired migration in zeb1a knockdown embryos. (D) zeb1a differentially regulates expression of genes associated with migrating phenotypes in LPM. Histograms show qPCR analyses of defined genes in LPM cells sorted from Tg(hand2:EGFP)pd24 after zeb1a knockdown and the relative controls at 48 hpf. Compared with controls, the knockdown of zeb1a upregulates genes (such as E-cadherin and occludin A) associated with non-migrating epithelial structures (*P<0.05).

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
cdh1 pd24Tg + MO2-zeb1a control Long-pec mesoderm RTPCR
pd24Tg + MO3-zeb1a control Long-pec mesoderm RTPCR
EGFP pd24Tg control Long-pec gut anatomical region IHC
Long-pec mesoderm IHC
pd24Tg + MO2-zeb1a control Long-pec gut anatomical region IHC
Long-pec mesoderm IHC
pd24Tg + MO3-zeb1a control Long-pec gut anatomical region IHC
Long-pec mesoderm IHC
mCherry uto5Tg control Protruding-mouth heart IFL
Protruding-mouth intestine smooth muscle IFL
Protruding-mouth notochord IFL
uto5Tg + MO2-zeb1a control Protruding-mouth heart IFL
Protruding-mouth intestine smooth muscle IFL
Protruding-mouth notochord IFL
uto5Tg + MO3-zeb1a control Protruding-mouth heart IFL
Protruding-mouth intestine smooth muscle IFL
Protruding-mouth notochord IFL
oclna pd24Tg + MO2-zeb1a control Long-pec mesoderm RTPCR
pd24Tg + MO3-zeb1a control Long-pec mesoderm RTPCR
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Anatomy
Ab1-tagln IHC uto5Tg control Protruding-mouth heart
IHC Protruding-mouth intestine smooth muscle
IHC Protruding-mouth notochord
IHC uto5Tg + MO2-zeb1a control Protruding-mouth heart
IHC Protruding-mouth intestine smooth muscle
IHC Protruding-mouth notochord
IHC uto5Tg + MO3-zeb1a control Protruding-mouth heart
IHC Protruding-mouth intestine smooth muscle
IHC Protruding-mouth notochord
Phenotype Details
Fish Conditions Stage Phenotype
pd24Tg + MO2-zeb1a control Long-pec gut anatomical region EGFP expression spatial pattern, abnormal
Long-pec lateral plate mesoderm tissue migration decreased occurrence, abnormal
Long-pec lateral plate mesoderm tissue migration decreased process quality, abnormal
Long-pec mesoderm cdh1 expression decreased amount, abnormal
Long-pec mesoderm oclna expression decreased amount, abnormal
pd24Tg + MO3-zeb1a control Long-pec gut anatomical region EGFP expression spatial pattern, abnormal
Long-pec lateral plate mesoderm tissue migration decreased occurrence, abnormal
Long-pec lateral plate mesoderm tissue migration decreased process quality, abnormal
Long-pec mesoderm cdh1 expression decreased amount, abnormal
Long-pec mesoderm oclna expression decreased amount, abnormal
s854Tg ; uto5Tg + MO2-zeb1a control Protruding-mouth intestine smooth muscle cell decreased amount, abnormal
s854Tg ; uto5Tg + MO3-zeb1a control Protruding-mouth intestine smooth muscle cell decreased amount, abnormal
uto5Tg + MO2-zeb1a control Protruding-mouth intestine smooth muscle ab1-tagln labeling decreased amount, abnormal
Protruding-mouth intestine smooth muscle mCherry expression decreased amount, abnormal
uto5Tg + MO3-zeb1a 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