ZFIN ID: ZDB-FIG-150320-16
Coxam et al., 2015 - carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (cad) regulates Notch signaling and vascular development in zebrafish. Developmental dynamics : an official publication of the American Association of Anatomists   244(1):1-9 Full text @ Dev. Dyn.
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Fish:
Anatomical Term:
Stage Range: Protruding-mouth to Day 5
PHENOTYPE:
Fish:
Observed In:
Stage: Day 4

Fig. 4 Notch signaling is reduced in cadhu10125 mutants. A–D: The vasculature Tg(flt1:tomato, Tp1-MmHbb:EGFP) in wild-type siblings (n = 43) and cadhu10125 (n = 12) embryos at 4 dpf, (A,B), Tg(flt1:tomato); (C,D), Tg(Tp1-MmHbb:EGFP; flt1:tomato). E–H Computer assisted segregation of vascular specific expression of (Tp1-MmHbb:EGFP) in Tg(flt1:tomato, Tp1-MmHbb:EGFP) wild-type siblings (n = 13 embryos, 40 aISVs analysed) and cadhu10125 (n = 13 embryos, 38 aISVs analysed) at 4 dpf. E,G: aISVs, Tg(flt1:tomato, Tp1-MmHbb:EGFP) positive, in wild-type siblings (E) and cadhu10125 embryos (G) at 4 dpf. F,H: aISVs-restricted expression of Tg(Tp1-MmHbb:EGFP) in wild-type siblings (F) and cadhu10125 embryos (H) at 4 dpf. Fluorescence intensity is displayed as a heat map, with a low to high color-coded from blue to white. I: Quantification of aISV-restricted Notch-reporter intensity based on analysis displayed in (F) and (H) (P = 0.0012). Each point represents the average aISV signal intensity in individual WT sibling embryos (n = 13 embryos, 40 aISVs analysed) or cadhu10125 embryos (n = 13 embryos, 38 aISVs analysed) normalised to average WT sibling signal intensity. J: Western blot detection of GFP expression in 48 hpf pooled embryos from WT and cadhu10125 Notch reporter transgenic embryos. The level of GFP expression is significantly reduced in cadhu10125 embryos (P = 0.0274). The expression of actin was used to monitor protein input and normalise GFP band intensity. K: Quantitative RT-PCR for the Notch target genes efnb2a, hey1, hey2, and nrarpa in embryos treated with 0.1% DMSO (black columns) or 100 µM DAPT (white columns), shows that the expression of these genes is significantly reduced when Notch signaling is inhibited from 24 hpf to 3dpf) (3 biological replicates, e10 embryos each). L,M: Quantitative RT-PCR for the Notch target genes efnb2a, hey1, hey2 and nrarpa in WT (black columns) and cadhu10125 (white columns) embryos at 3 dpf (WT, n = 5; cadhu10125, n = 5 biological replicates, e10 embryos each)(L) and 5 dpf (WT, n = 5; cadhu10125, n = 4 biological replicates, e10 embryos each) (M). efnb2a reduction at 3 dpf, P = 0.0017, 5 dpf P = 0.0082.; hey1 reduction at 5 dpf, P = 0.0437. Other trends nonsignificant in cadhu10125 mutants but significant in DAPT treated embryos.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
ephb2a WT control Protruding-mouth whole organism RTPCR
Day 5 whole organism RTPCR
cadhu10125/hu10125 standard conditions Protruding-mouth whole organism RTPCR
Day 5 whole organism RTPCR
hey1 WT control Protruding-mouth whole organism RTPCR
Day 5 whole organism RTPCR
cadhu10125/hu10125 standard conditions Protruding-mouth whole organism RTPCR
Day 5 whole organism RTPCR
hey2 WT control Protruding-mouth whole organism RTPCR
Day 5 whole organism RTPCR
cadhu10125/hu10125 standard conditions Protruding-mouth whole organism RTPCR
Day 5 whole organism RTPCR
nrarpa WT control Protruding-mouth whole organism RTPCR
Day 5 whole organism RTPCR
cadhu10125/hu10125 standard conditions Protruding-mouth whole organism RTPCR
Day 5 whole organism RTPCR
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
cadhu10125/hu10125; hu5333Tg; um14Tg standard conditions Day 4 Notch signaling pathway decreased intensity, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Developmental dynamics : an official publication of the American Association of Anatomists for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Dev. Dyn.