Notch signalling maintains Hedgehog responsiveness via a Gli-dependent mechanism during spinal cord patterning in zebrafish
- Alberta Children’s Hospital Research Institute, University of Calgary, Canada
Figures
Figure 1 with 2 supplements
Characterization of the her12:Kaede reporter.
(A) Schematic drawing of the her12:Kaede BAC reporter. A BAC containing the her12 locus and surrounding regulatory elements was modified to replace the first exon of her12 with a cassette containing …
Figure 1—figure supplement 1
Co-expression of her12 and other Notch target genes in the spinal cord.
Whole-mount double fluorescent in situ hybridisation was performed in wild-type embryos at 24 hpf for her2 and her12 (A), her4 and her12 (B) and hes5 and her12 (C). Brackets in lateral views and …
Figure 1—figure supplement 2
LY-411575 and cyclopamine are efficient inhibitors of Notch and Hh signalling, respectively.
Wild-type embryos were treated with LY-411575 or cyclopamine for 0, 1, 2, 4, 6, 8, or 10 hr, and fixed at 24 hpf. Whole mount in situ hybridisation was then performed for her12 in LY-411575 treated …
Figure 2 with 3 supplements
PHRESH analysis of the temporal dynamics of Notch and Hh signalling.
(A) Schematic representation of the experimental design. A section of the spinal cord above the yolk extension was photoconverted by the UV light at t0 and the fluorescent profile was analysed after …
Figure 2—figure supplement 1
PHRESH signalling profiles show similar trends across multiple embryos.
ptc2:Kaede and her12:Kaede embryos were photoconverted at 24 hpf (A), 48 hpf (B) and 72 hpf (C) and imaged 6 hr post-conversion. Each line (grey) represents Kaedegreen fluorescent intensity along …
Figure 2—figure supplement 2
Notch and Hh response profiles after the ‘signalling termination’ phase.
Continuation of the time course described in Figure 2, where her12:Kaede and ptc2:Kaede embryos were photoconverted at 84 hpf or 90 hpf, and imaged 6 hr post-conversion. Lateral views of confocal …
Figure 2—figure supplement 3
her12 and ptc2 are expressed in the same domain corresponding to sox2+ neural progenitors.
Whole-mount double fluorescent in situ hybridisation was performed in wild-type embryos for her12 and ptc2 (A), sox2 and her12 (B), and sox2 and ptc2 (C) at either 24, 48, or 72 hpf. Dotted lines …
Figure 3 with 1 supplement
Inhibition of Notch signalling abolishes Hh response in the spinal cord.
(A) ptc2:Kaede and her12:Kaede embryos were incubated with DMSO, cyclopamine (Cyc) or LY-411575 from 20 to 30 hpf, photoconverted at 24 hpf and imaged at 30 hpf. Lateral views of confocal …
Figure 3—figure supplement 1
Inhibition of Notch signalling results in loss of Hh response in the spinal cord.
(A) Whole-mount in situ hybridisation was performed in mindbomb mutants or their sibling controls for ptc2 expression at 30 hpf. n = 15 embryos per staining. (B) Embryos were injected with the …
Figure 4
Ectopic Notch activation results in increased and expanded Hh response.
hsp:rSmoM2-tRFP embryos and wild-type controls (A), or hsp:Gal4; UAS:NICD embryos and wild-type controls (B) were heat shocked at 11 hpf and stained for the expression of ptc2 and her12 at 24 hpf. …
Figure 5
Inhibition of Notch signalling results in loss of Hh response followed by loss of neural progenitor identity.
(A) Wild-type embryos were incubated in LY-411575 from 20 hpf for 1, 2 or 3 hr and DMSO for the entire duration. Embryos were stained for the expression of ptc2 or sox2. Brackets in lateral views …
Figure 6
Activation of Hh signalling by rSmoM2 cannot rescue Notchoff spinal cords.
(A) Schematic representation of the manipulation to the Hh pathway caused by ectopic expression of rSmoM2-tRFP. The point of manipulation is highlighted in green with an asterisk. (B) Experimental …
Figure 7
Notch signalling regulates Hh response independent of primary cilia.
(A) Schematic representation of the manipulation to the Hh pathway caused by the loss of primary cilia in iguana mutants. The point of manipulation is highlighted in green with an asterisk. (B) …
Figure 8 with 1 supplement
Notch signalling regulates Hh response at the Gli level.
(A) Whole-mount in situ hybridisation for gli1 was performed on wild-type embryos treated with DMSO, cyclopamine, or LY-411575 from 20 to 30 hpf. (B) Schematic representation of the manipulation of …
Figure 8—figure supplement 1
Notch signalling regulates the expression of all Gli family members in the spinal cord.
Wild-type embryos were treated with DMSO, cyclopamine, or LY-411575 from 20 to 30 hpf, and stained with gli2a, gli2b or gli3 probes. Brackets in lateral views and dotted lines in transverse views …
Author response image 1
Notch signalling regulates Hh response at the Gli level.
(A) Experimental design. Embryos were heat shocked (HS) for 30 minutes in the solution containing the drug at 1 hour after the drug treatment. (B) hsp:EGFP-Gli1 and wild type control embryos were …
Videos
Video 1
The re-emergence of ptc2:Kaede expression after photoconversion.
A ptc2:Kaede embryo was photoconverted at 28 hpf and then underwent time-lapse imaging for 18 hr. The vertical line indicates the boundary between photoconverted and unconverted regions at the start …
Video 2
The ptc2:Kaede response profile along the anterior-posterior axis.
ptc2:Kaede embryos were photoconverted at 48 hpf and imaged 6 hr after. Individual transverse sections generated by 3D reconstruction were prepared into a video. The first frame is the most anterior …
Video 3
The her12:Kaede response profile along the anterior-posterior axis.
her12:Kaede embryos were photoconverted at 48 hpf and imaged 6 hr after. Individual transverse sections generated by 3D reconstruction were prepared into a video. The first frame is the most …
Tables
Key resources table
| Resource type | Designation | Source/Reference | Identifier |
|---|---|---|---|
| Zebrafish strain (Danio rerio) | hsp:Gal4 | Scheer and Campos-Ortega, 1999, PMID: 10072782 | RRID:ZFIN_ZDB-ALT-020918-6 |
| Zebrafish strain (Danio rerio) | UAS:NICD | Scheer and Campos-Ortega, 1999, PMID: 10072782 | RRID:ZFIN_ZDB-ALT-020918-8 |
| Zebrafish strain (Danio rerio) | hsp:rSmoM2-tRFP | This paper. | NA |
| Zebrafish strain (Danio rerio) | hsp:EGFP-Gli1 | Huang and Schier, 2009 PMID: 19700616 | RRID:ZFIN_ZDB-ALT-110207-11 |
| Zebrafish strain (Danio rerio) | iguts294 (iguana) | Sekimizu et al., 2004; Wolff et al., 2004 PMIDs: 15115751; 15198976 | RRID:ZFIN_ZDB-ALT-980203-1553 |
| Zebrafish strain (Danio rerio) | mib1ta52b (mindbomb) | Itoh et al., 2003 PMID: 12530964 | RRID:ZFIN_ZDB-ALT-980203-1374 |
| Zebrafish strain (Danio rerio) | her12:Kaede | This paper: Generated using BAC clone zK5I17 (DanioKey) | NA |
| Zebrafish strain (Danio rerio) | ptc2:Kaede | Huang et al., 2012 PMID: 22685423 | RRID:ZFIN_ZDB-ALT-120810-2 |
| Morpholino oligonucleotide | rbpja/bMO (Previously Su(H)1+2 MO) | Gene Tools, LLC | ZFIN ID: ZDB- MRPHLNO-070410–11 |
| Antibody | Rabbit polyclonal anti-Kaede | MBL International | Cat# PM012, RRID:AB_592060 |
| Antibody | Goat anti-rabbit IgG, Alexa Fluor 555 | Thermo Fisher Scientific | Cat# A-21428, RRID:AB_2535849 |
| DNA dye | Draq5 | Biostatus | Cat# DR50050, RRID:AB_2314341 |
| Small molecule inhibitor | Cyclopamine | Toronto Chemical | Cat# C988400 |
| Small molecule inhibitor | LY-411575 | Millipore Sigma | Cat# 209984-57-6 |
| Software package | Fiji-ImageJ | Schindelin et al., 2012 PMID: 22743772 https://fiji.sc | RRID:SCR_002285 |
| Software package | Graphpad Prism | https://www.graphpad.com/scientific-software/prism/ | RRID:SCR_002798 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.49252.020
