Coupling and uncoupling of midline morphogenesis and cell flow in amniote gastrulation
Data files
Apr 08, 2024 version files 18.10 GB
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Aphidicolin-pMyosin-ZO1-staining.nd2.zip
6.86 GB
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Control-pMyosin-ZO1-staining.nd2.zip
11.24 GB
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README.md
3.43 KB
Abstract
Large-scale cell flow characterizes gastrulation in animal development. In amniote gastrulation, particularly in avian gastrula, a bilateral vortex-like counter-rotating cell flow, called ‘polonaise movements’, appears along the midline. Here, through experimental manipulations, we addressed relationships between the polonaise movements and morphogenesis of the primitive streak, the earliest midline structure in amniotes. Suppression of the Wnt/planar cell polarity (PCP) signaling pathway maintains the polonaise movements along a deformed primitive streak. Mitotic arrest leads to diminished extension and development of the primitive streak and maintains the early phase of the polonaise movements. Ectopically induced Vg1, an axis-inducing morphogen, generates the polonaise movements, aligned to the induced midline, but disturbs the stereotypical cell flow pattern at the authentic midline. Despite the altered cell flow, induction and extension of the primitive streak are preserved along both authentic and induced midlines. Finally, we show that ectopic axis-inducing morphogen, Vg1, is capable of initiating the polonaise movements without concomitant PS extension under mitotic arrest conditions. These results are consistent with a model wherein primitive streak morphogenesis is required for the maintenance of the polonaise movements, but the polonaise movements are not necessarily responsible for primitive streak morphogenesis. Our data describe a previously undefined relationship between the large-scale cell flow and midline morphogenesis in gastrulation.
README: Immunofluorescent staining images of control- and aphidicolin-treated embryos at Hamburger Hamilton stage 3 (HH3) with ZO-1 and pMyosin.
Data description:
We have submitted our raw data of phospho-myosin light chain II (pMyosin) and ZO-1 immunofluorescent staining images in the control (Control-pMyosin-ZO1-staining.nd2.zip) and aphidicolin-treated (aphidicolin is a DNA polymerase inhibitor; Aphidicolin-pMyosin-ZO1-staining.nd2.zip) embryos. ZO-1 is a tight junction protein, and pMyosin is related to cell migration and tissue contraction.
Both the control and aphidicolin-treated embryos exhibited a large-scale ring-like distribution pattern of the pMyosin cables in the epiblast.
These datasets are related to Figure 2, figure 2–figure supplement 2, figure 2–figure supplement 3, and Video 2. Further, the nd2 files can be opened by imaging analysis softwares, such as Fiji, NIS-elements, and Imaris. We processed these images by using Imaris for figure 2–figure supplement 3.
[Access this dataset on Dryad]
https://doi.org/10.5061/dryad.37pvmcvsx
Control-pMyosin-ZO1-staining.nd2
Whole-mount immunofluorescent staining image of a control chick embryo with anti-pMyosin (RFP channel) and anti-ZO-1 (Cy5 channel) antibody immunofluorescent staining. ZO-1 was localized at a tight junction in the epiblast. pMyosin cables distributed in the epiblast cell layer. Experimental procedure is described below.
Aphidicolin-pMyosin-ZO1-staining.nd2
Whole-mount immunofluorescent staining image of an aphidicolin-treated chick embryo with anti-pMyosin (RFP channel) and anti-ZO-1 (Cy5 channel) antibody immunofluorescent staining. ZO-1 was localized at a tight junction in the epiblast. Despite of aphidicolin-treatment, the large-scale ring-like pattern of the pMyosin cables remained in the epiblast. Experimental procedure is described below.
Experimental Procedure:
1) Fertilized eggs of White Leghorn (Gallus Gallus domesticus) were obtained from Petaluma Farms (Petaluma, CA).
2)Embryos were isolated, and soaked in Tyrode’s solution with either 0.3% DMSO (control), or 100uM of aphidicolin for 15 minutes at 37°C. Embryos were then cultured using the New culture method at 37°C until HH3.
3) After culture, the embryos were fixed in 4% PFA/PBS for 30 minutes at RT and washed in PBS 3X for 30 minutes each.
4) Embryos were then incubated in blocking reagent [1% bovine serum albumin (BSA) and 0.1% triton in PBS] for 1 hour at RT.
5) Embryos were then incubated with primary antibodies [anti-phospho-myosin light chain 2 antibody (1:300, Cell Signaling Technologies, 3674), anti-ZO1 antibody (1:300, Thermo Fisher Scientific, 33-9100)] at 4°C O/N.
6) After washing in PBS 3 X for 30 minutes each, embryos were then incubated with secondary antibodies [anti-rabbit IgG (H+L) Alexa Fluor 568 antibodies (1:500, Thermo Fisher Scientific, A10042), Anti-mouse-IgG (H+L) Alexa Fluor 647 antibody (1:500, Thermo Fisher Scientific, A-31571)]for 2 hours at RT.
7) Embryos were then washed in PBS 2X for 30 minutes each.
8) Embryos were incubated with DAPI for 1 hour, washed for 30 minutes, then mounted between cover slips (VWR, 48366249), and slide glasses (Thermo Fisher Scientific, 12-544-7) with Aqua-PolyMount (Polysciences, Inc., #18606-20).
9) The samples were imaged by Crest LFOV Spinning Disk with Nikon Ti2-E.
Methods
- Fertilized eggs of White Leghorn (Gallus Gallus domesticus) were obtained from Petaluma Farms (Petaluma, CA).
- Embryos were isolated, and soaked in Tyrode’s solution with either 0.3% DMSO (control), or 100uM of aphidicolin (Aphidicolin) for 15 minutes at 37°C. Embryos were then cultured using the New culture method at 37°C until HH3.
- After culture, the embryos were fixed in 4% PFA/PBS for 30 minutes at RT and washed in PBS 3X for 30 minutes each.
- Embryos were then incubated in blocking reagent [1% bovine serum albumin (BSA) and 0.1% triton in PBS] for 1 hour at RT.
- Embryos were then incubated with primary antibodies [anti-phospho-myosin light chain 2 antibody (1:300, Cell Signaling Technologies, 3674), anti-ZO1 antibody (1:300, Thermo Fisher Scientific, 33-9100)] at 4°C O/N.
- After washing in PBS 3 X for 30 minutes each, embryos were then incubated with secondary antibodies [anti-mouse IgG Alexa Fluor 594 antibody (1:1000, Thermo Fisher Scientific, A21203), anti-rabbit IgG Alexa Fluor 647(A31573)] for 2 hours at RT.
- Embryos were then washed in PBS 2X for 30 minutes each.
- Embryos were incubated with DAPI for 1 hour, washed for 30 minutes, then mounted between cover slips (VWR, 48366249), and slide glasses (Thermo Fisher Scientific, 12-544-7) with Aqua-PolyMount (Polysciences, Inc., #18606-20).