header logo image header logo text
Downloads Login
Research
General Information
ZIRC
ZFIN ID: ZDB-FIG-070314-26
Ma et al., 2007 - Jagged2a-notch signaling mediates cell fate choice in the zebrafish pronephric duct. PLoS Genetics   3(1):e18 Full text @ PLoS Genet.
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Fish:
Knockdown Reagents:
Anatomical Term:
Stage Range: Prim-5 to Prim-25
PHENOTYPE:
Fish:
Knockdown Reagents:
Observed In:
Stage: Prim-5

Fig. 4 Multi-Cilia Cell Hyperplasia Is Due to Mib-Mediated Jagged2a Signaling Pathway via Notch1a and Notch3 Receptors(A) Effectiveness of splicing jagged2a-sp MO. RT-PCR of control embryos generates a 230-bp jagged2a fragment, bridging parts of exon 1 and exon 2 at 24 hpf (lane 1) and 48 hpf (lane 5). jagged2a-sp MO-injected embryos analyzed with the same primers at the same timepoints (lanes 3 and 7) show a larger amplicon of 708 bp caused by a nonsplicing intron 1, which encodes a premature stop codon. Lane 9 shows the amplicon from genomic DNA, and lane 10 shows the amplicon from jagged2a cDNA. No fragment can be amplified in the RT-PCR without reverse transcriptase in 24-hpf (lane 2) or 48-hpf (lane 6) wt embryos or in 24-hpf (lane 4) or 48-hpf (lane 8) jagged2a-sp MO-injected embryos. Lane L: 100-bp ladder. (B) Pronephric duct (arrow) integrity is not affected in jagged2a morphants. Panels C–L focus on the duct between somite 10 and 13. (C–H) Multi-cilia cell number is increased in (D and F) jagged2a-atg morphants compared to (C and E) wt embryos as shown by (C and D) rfx2 and (E and F) centrin2 expression at 24 hpf, but principal cell number is decreased in (H) jagged2a-atg morphants compared to (G) wt embryos as revealed by Na+, K+ ATPase β1a expression at 24 hpf. (I–L) Multi-cilia cell number is increased in (J) mibta52b embryos compared to (I) wt embryos as shown by rfx2 expression at 24 hpf, but principal cell number is decreased in (L) mibta52b embryos compared to (K) wt embryos as revealed by Na+, K+ ATPase α1a2 expression at 24 hpf. Panels M–R focus on the duct around somite 11 to 13. (M–O) Fluorescent double in situ hybridization of rfx2 (green) and Na+, K+ ATPase β1a (red) in 36-hpf (M) wt embryos, (N) jagged2a-sp morphants, and (O) mibta52b mutants shows multi-cilia cell hyperplasia in jagged2a morphants and mibta52b mutants. Arrows point to the rfx2-expressing cells in the duct of (M) wt embryos; arrowheads point to the Na+, K+ ATPase β1a-expressing cells in the pronephric duct of (N) jagged2a-sp morphants. (P–R) Double immunohistochemistry of α6F (green) and Pcm1 (red) in 36-hpf (P) wt embryos, (Q) jagged2a-sp morphants, and (R) mibta52b mutants shows multi-cilia cell hyperplasia in jagged2a morphants and mibta52b mutants. Arrows point to the Pcm1 staining in the pronephric duct of (P) wt embryos; arrowheads point to α6F staining in the pronephric duct of (Q) jagged2a-sp morphants. (S) Immunoprecipitation of Myc-Jagged2a and Myc-Jagged2aicd by Flag-Mibta52b. IP, immunoprecipitation; IB, immunoblotting. (T–U) Expression of Myc-Jagged2a (T) and cotransfection of Myc-Jagged2a and Flag-Mib (U) in COS7 cells. (V–Y) Compared to (V) wt embryos, mild cilia cell hyperplasia is observed in (W) notch1a (desth35b) mutants and (X) notch3-utr morphants, while severe cilia cell hyperplasia is observed in (Y) notch3-utr MO-injected notch1a (desth35b) mutants as shown by rfx2 expression at 24 hpf. All embryos, anterior to the left. Bar scale: 100 μm (B), 75 μm (C–L and V–Y), 50 μm (M–R), and 30 μm (T and U).

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
atp1a1a.2 WT standard conditions Prim-5 pronephric duct ISH
mib1ta52b/ta52b standard conditions Prim-5 pronephric duct ISH
atp1b1a WT standard conditions Prim-5 pronephric duct ISH
Prim-25 pronephric duct ISH
WT + MO5-jag2b standard conditions Prim-5 pronephric duct ISH
WT + MO7-jag2b standard conditions Prim-25 pronephric duct ISH
mib1ta52b/ta52b standard conditions Prim-25 pronephric duct ISH
cetn4 WT standard conditions Prim-5 pronephric duct ISH
WT + MO5-jag2b standard conditions Prim-5 pronephric duct ISH
rfx2 WT standard conditions Prim-5 pronephric duct ISH
Prim-25 pronephric duct ISH
WT + MO5-jag2b standard conditions Prim-5 pronephric duct ISH
WT + MO5-notch3 standard conditions Prim-5 pronephric duct ISH
WT + MO7-jag2b standard conditions Prim-25 pronephric duct ISH
mib1ta52b/ta52b standard conditions Prim-5 pronephric duct ISH
Prim-25 pronephric duct ISH
notch1ath35b/th35b standard conditions Prim-5 pronephric duct ISH
notch1ath35b/th35b + MO5-notch3 standard conditions Prim-5 pronephric duct ISH
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
WT + MO5-jag2b standard conditions Prim-5 pronephric duct morphology, normal
WT + MO6-jag2b standard conditions Prim-5 pronephric duct morphology, normal
WT + MO7-jag2b standard conditions Prim-5 pronephric duct morphology, normal
Acknowledgments:
ZFIN wishes to thank the journal PLoS Genetics for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ PLoS Genet.