#2005-10-12355C Horikawa et. al.,_ Supplementary Methods
Supplementary Methods
Cell-transplantation
Cell-transplantation at the blastula stage was performed as described previouslyS17.
Briefly, zebrafish embryos were labeled with 1% Rhodamine-/FITC-dextran
(MW-10,000: Molecular Probes) and 1% biotin-dextran (MW-10,000, lysine fixable:
Molecular Probes) at the one-cell stage. A few donor cells (5-25 cells) were then
transplanted into the marginal zone of the normal host embryos at the 2,000-4,000-cell
stage.
Direct cell-transplantation was carried out as follows:
To isolate fluorescently
labeled PSM cells, donor embryos at the 8-somite stage were briefly treated with 50
mg/ml of Dispase (Gibco: 17105-041) and pieces of PSM were collected in the L-15
medium.
Host embryos were embedded in 1.2% ultra-LMP-agarose gel (Sigma:
A-9045) and their enveloping layer and ectoderm were locally pierced by mechanical
force using micropipettes filled with 1mg/ml of fluorescently labeled Dispase. The
pressure was balanced so as not to cause leakage of the enzyme into the embryo.
Donor cells (10-20 cells) were gently transferred into the PSM of the host embryo using
a glass capillary connected to the oil-assisted micro-manipulator (eppendorf:
5176-000-033), under a fluorescent-stereomicroscope (Leica: MZFLIII).
Biotin-labeled donor cells were detected by either TSA-647 or ELF97 kits (Molecular
Probes: E-6605) at the final step of the ISH.
Morpholino anti-sense oligonucleotides and cRNA probes
Morpholino anti-sense oligonucleotides against both her1/her7S7 and deltaCS6 were
described elsewhere. Exonic-probes for her1, deltaC and myoD were transcribed from
full-length cDNAs that had been cloned into pBSII. Probes for the third intron of her1
(1.2 kb) were generated as described previouslyS18.
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#2005-10-12355C Horikawa et. al.,_ Supplementary Methods
High resolution ISH
To preserve the cellular localization of mRNAs, embryos were fixed with 4% PFA for 2
hours at room temperature. During the course of the study, we found that ice-cold
fixative never produced nuclear signals.
DIG- or FITC-labeled cRNA probes were
detected with TSA-Alexa488 (Molecular Probes: T-20932) or TSA-Alexa647
(Molecular Probes: T-20936), according to the manufacturer’s instructions and ref S19.
Detailed procedure is also described in ref S4.
Nuclei were stained with 0.3 mg/ml of
propidium iodide (Molecular Probes: P3566) for several days at 4ºC. Flat-mounted
embryos were optically scanned with a confocal microscope (Olympus: FV500).
Z-stack images of 5 µm thickness (5-slices with a 1 µm interval) were acquired with
UApo40W (Olympus: NA. 1.15).
DAPT treatment
A 10 mM stock of DAPT in DMSO was diluted in L15 medium (Gibco:21083-027)
containing 0.0001% Triton-X 100 and 2% DMSO. Manually dechorionated embryos
were treated with 100 M DAPT from the 6-somite to 10-somite stage on a rocking
shaker at 28.5ºCS20.
Image analysis
For the graphical analysis of her1 expression, the distance of the her1-positive cells
(detected by BCIP/NBT) from the 8th segment was digitally scored NIH image
software. The relative positions in the PSM were normalized by the lengths between
the 8th segment and posterior end of the axial mesoderm. To determine the numbers
of nuclear-her1-cells, whole PSMs of flat-mounted embryos were optically scanned at
1-µm intervals (Olympus: FV500). PSM cells located at the levels between the 5th
and 15th adaxial cells from the posterior end were subjected to this analysis.
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#2005-10-12355C Horikawa et. al.,_ Supplementary Methods
distinct z-positions in each PSM were sterically analyzed using Volocity (Improvision).
The values from each assessment were analyzed graphically using Microsoft Excel.
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