DOI: 10.5061/dryad.37pvmcvsx
title: Coupling and uncoupling of midline morphogenesis and cell flow in amniote gastrulation

description: 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.
description: 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).
description: # 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](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.