Notch signaling restricts FGF pathway activation in parapineal cells to promote their collective migration
- Université de Toulouse, CNRS (UMR 5547), France
Figures
Figure 1 with 3 supplements
Increased activation of FGF signaling in mindbomb mutants correlates with defects in migration and specification of parapineal cells.
(A–D) Confocal sections showing the expression of the Tg(dusp6:d2EGFP) transgene (Green, (A–B’) or sox1a (red, (C–D) in the epithalamia of 32 hpf in control (A, A’, n = 36 and C, n = 30) and in mib-/…
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Figure 1—source data 1
Source files for data used to generate dot plots in Figure 1.
- https://doi.org/10.7554/eLife.46275.007
Figure 1—figure supplement 1
The number but not the mean intensity of Tg(dusp6:d2EGFP) expressing cells is increased in mib-/- mutants.
(A–B) Dot plots showing the mean GFP fluorescence intensity (A, B) of Tg(dusp6 :d2EGFP) expressing parapineal cells in the first (A) and second experiment (B) in controls (blue dots; A, n = 21; B, n …
Figure 1—figure supplement 2
rbpja/b morphants phenocopy mindbomb mutants.
(A–B’) Confocal sections showing the expression of gfi1ab (red) at 48 hpf in control embryos (A, n = 33) and in two representative embryos injected with 4 ng of rbpja/b morpholinos (MO) (B, B’); …
Figure 1—figure supplement 3
Components of the Notch pathway are mosaically expressed in the parapineal.
(A–B) Confocal sections showing the expression of Tg(Tp1bglob:EGFP) transgene (green, A, B), Topro-3 nuclear staining (gray, A’–B’) and merge (A’’–B’’) in the epithalamia of two embryos at 30 hpf (n …
Figure 2 with 3 supplements
Uncoupled roles for the Notch pathway in the specification, laterality and migration of parapineal cells.
(A–B) Confocal sections showing the expression of gfi1ab (red) in embryos treated with DMSO (A; n = 24) or 100 µM LY411575 (B; n = 32) from 22 to 32 hpf and fixed at 48 hpf, merged with nuclear …
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Figure 2—source data 1
Source files for data used to generate dot plots in Figure 2.
- https://doi.org/10.7554/eLife.46275.012
Figure 2—figure supplement 1
Effect of treatment with high dose of γ-secretase inhibitor from 22 to 32 hpf on the specification and migration of parapineal cells.
(A, B) Upper panel shows a schematic of LY411575 treatment timeline: wild-type (+/+) or mib+/- embryos were treated from 22 to 32 hpf with 200 µM of LY411575. Dot plots show the number (A) and the …
Figure 2—figure supplement 2
LY411575 treatment after 32 hpf does not affect the migration or the specification of parapineal cells.
(A–B) Upper panels show a schematic of the LY411575 treatment timeline, from 32 to 36 hfp for dot plots (A, C) or from 22 to 48 hpf (B, D). Dot plots show, for each embryo, the mean position (A–B) …
Figure 2—figure supplement 3
Early Notch loss-of-function results in bilateral Nodal pathway activation in the epithalamus.
(A–C) Confocal sections showing Left (A), Bilateral (B) or Absent (C) pitx2c expression in the epithalamus at 32 hpf (red). Embryo view is dorsal, anterior is up; scale bar = 10 µm. (D–H) Histogram …
Figure 3 with 1 supplement
Ectopic Notch signaling triggers decreased FGF activation and defects in migration and specification of parapineal cells.
(A–F) Confocal sections showing the expression of Tg(dusp6 :d2EGFP) (A-B, green), sox1a (C-D, red) or tbx2b (E-F, red) at 36 hpf, in control embryos (A, n = 16; C, n = 12; E, n = 5) or in Tg(hsp70l:G…
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Figure 3—source data 1
Source files for data used to generate dot plots in Figure 3.
- https://doi.org/10.7554/eLife.46275.015
Figure 3—figure supplement 1
Effect of activation of the Notch pathway at 22, 24, 28 and 32 hpf on the migration and specification of parapineal cells.
(A–H) Confocal sections showing the expression of gfi1ab (red) at 48 hpf in control embryos (A, n = 27; C, n = 17; E, n = 28; G, n = 8) or in Tg(hsp70l:Gal4);Tg(UAS:myc-Notch1a-intra) double …
Figure 4
Decreasing or increasing FGF signaling rescues or aggravates the parapineal migration defect in Notch loss-of-function.
(A–D) Confocal sections showing the expression of gfi1ab (red) merged with nuclei staining (gray) at 48 hpf in representative control sibling (A–B) or mib-/- mutant embryos (C–D) treated from 24 to …
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Figure 4—source data 1
Source files for data used to generate dot plots in Figure 4.
- https://doi.org/10.7554/eLife.46275.017
Author response image 1
Expression of others FGF target genes in the epithalamus.
Confocal sections of the head of Tg(dusp6:d2EGFP) embryos (A-C’’’) at 36 hpf after a whole-mount in situ hybridization against erm (red, A-A’’’), pea3 (red, B-B’’’), etv5a (red, C-C’’’) or of Tg(spro…
Author response image 2
The number but not the mean intensity of Tg(dusp6:d2EGFP) expressing cells is increased in mib-/- mutants.
(A-B) Dot plots showing the mean GFP fluorescence intensity (A, B) of Tg(dusp6:d2EGFP) expressing parapineal cells in the first (A) and second experiment (B) in controls (blue dots; A, n=21; B, …
Author response image 3
Notch target genes her6 and her9 are mosaïcally expressed in parapineal cells.
(A-D) Confocal sections showing the expression of her6 (A, B) and her9 (C, D) (red) at 32 hpf in control embryos (A, C) and in mib-/- mutant embryos (B, D) merged with a cell nuclei staining (grey). …
Author response image 4
Late LY41 treatment does not affect parapineal migration.
(A-B) Upper panels show a schematic of the LY411575 treatment timeline, from 32 to 36 hfp (A) or 22 to 48 hpf (B) for dot plots. Dot plots showing, for each embryo, the mean position of gfi1ab …
Author response image 5
Notch ligand deltaB is mosaically expressed in the parapineal.
(A-B’’) Confocal sections showing the expression of Tg(dusp6:d2EGFP) transgene (green, A, B) or deltaB (red, A’-B’) and merge (A’’-B’’) in the epithalamia of two representative embryos at 32 hpf. del…
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (D. rerio) | mibta52b | Itoh et al., 2003 | RRID:ZFIN_ZDB-GENO-071218-1 | |
| Genetic reagent (D. rerio) | fgf8ti282a (ace) | Reifers et al., 1998 | RRID:ZFIN_ZDB-GENO-980202-822 | |
| Genetic reagent (D. rerio) | Tg(hsp70:Gal4)kca4 | Scheer et al., 2001 | RRID:ZFIN_ZDB-ALT-020918-6 | |
| Genetic reagent (D. rerio) | Tg(UAS:myc-Notch1a-intra)kca3 | Scheer and Campos-Ortega, 1999 | RRID:ZFIN_ZDB-ALT-020918-8 | |
| Genetic reagent (D. rerio) | Tg(hsp70:ca-fgfr1; cryaa:DsRed)pd3 | Marques et al., 2008 Neilson and Friesel, 1996 | RRID:ZFIN_ZDB-GENO-090127-1 | |
| Genetic reagent (D. rerio) | Tg(dusp6:d2EGFP)pt6 | Molina et al., 2007 | RRID:ZFIN_ZDB-GENO-071017-5 | |
| Genetic reagent (D. rerio) | Tg(Tp1bglob:EGFP)ia12 | Corallo et al., 2013 | RRID:ZFIN_ZDB-ALT-130115-3 | |
| Sequence-based reagent | rbpja/su(H)1; rbpjb/su(H)2 | Echeverri and Oates, 2007 | ZFIN ID : ZDB-FISH-150901–13686 | |
| Antibody | Rabbit anti-GFP | Torrey Pines Biolabs | TP-401 RRID:AB_10013661 | 1/1000 |
| Antibody | Goat anti-rabbit IgG Alexa 488-conjugated | Molecular probe | A11034 | 1/1000 |
| Chemical compound, drug | LY411575 | MedChem Express | HY-50752 | Rothenaigner et al., 2011 |
| Chemical compound, drug | SU5402 | Calbiochem | 572630 | Mohammadi et al., 1997 |
| Other | Fast Red | Sigma Aldrich | F46-48 | |
| Other | ToPro-3 | Molecular probe | T3605 | 1/1000 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.46275.018
