Supplementary Materials

Programmed cell death along the midline axis patterns ipsilaterality in gastrulation

Lisandro Maya-Ramos and Takashi Mikawa

Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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  • Materials and Methods
  • Figs. S1 to S12
  • Table S1
  • Captions for Movies S1 to S10
  • References

Images, Video, and Other Media

Movie S1
Embryo bilaterally electroporated with Flag:2A:H2B-RFP (red) on the left side and Flag: 2A:H2B-GFP (pseudocolored blue) on the right side. Live imaged at 4X with an epifluorescent microscope from stage HH 3 to HH 4+. First panel with cellular tracks and second panel without tracks. Scale bar, 500 μm.
Movie S2
Embryo electroporated with membrane-tethered GFP (pseudocolored white) and live imaged at 20X with a multiphoton microscope. An increased number of cellular blebbing occurs as cells approach the PS midline. Scale bar, 30 μm.
Movie S3
Embryo co-electroporated with Annexin-V (YFP-Sec-A5) reporter (green) and membrane RFP. Live imaged at 20X with an epifluorescent microscope. Midline cell expressing YFP-Sec-A5 reporter and membrane RFP undergoes blebbing, as lateral cells expressing primarily RFP move towards the PS midline. Scale bar, 50 μm.
Movie S4
Embryo apically labeled with Alexa Fluor 488 Annexin V conjugate staining and live imaged at 10X with an epifluorescent microscope. Imaging from stage HH 2 to HH 4+. Tracks were statistically colored to show the start position along the X axis. X axis length = 1400μm. The PS localized between 600-800μm along the X axis, with the midline residing at approximately 700μm. Y axis length = 1050μm. Scale bar, 100 μm.
Movie S5
Embryo electroporated with lissamine oligo (green) at the PS and Flag:2A:H2B-GFP (red) at lateral epiblast.. Live imaged at 4X with an epifluorescent microscope from stage HH 3 to HH 4+. GFP (red) labeled WT cells undergo ipsilateral gastrulation. Scale bar, 300 μm
Movie S6
Embryo electroporated with P35:2A:H2B-GFP at the PS and Flag:2A:H2B-RFP at lateral epiblast. Live imaged at 4X with an epifluorescent microscope from stage HH 3 to HH 4+. RFP labeled WT cells cross the P35 expressing midline to invaded the contralateral side. Scale bar, 500 μm.
Movie S7
Embryo electroporated with lissamine-tagged LAMA1 morpholino at the PS and H2B-GFP at lateral epiblast. Live imaged at 4X with an epifluorescent microscope from stage HH 3 to HH stage 4+. GFP labeled WT cells cross the LAMA1 expressing midline to invade the contralateral side. Scale bar, 400 μm.
Movie S8
Embryo electroporated with hMMP-15:2A:mCherry at the PS and H2B-GFP at lateral epiblast. Live imaged at 4X with an epifluorescent microscope from stage HH 3 to HH 4+. GFP labeled WT cells cross the hMMP-15 expressing midline to invaded the contralateral side. Scale bar, 200 μm.
Movie S9
Embryo electroporated with LAMA1 morpholino at the PS and Flag:2A:H2B-GFP at lateral epiblast. PCD was locally introduced via staurosporine microinjections. Live imaged at 4X then 10X with an epifluorescent microscope from stage HH 3 to HH 4+. Staurosporine induced-PCD suppressed contralateral invasion. While contralateral migration continued in non-injected zones, at ECM-PCD deficient PS regions. Scale bar, 500 μm.
Movie S10
Embryo electroporated with fluorescein-tagged LAMA1 morpholino at the PS and Flag: 2A:H2B-GFP at lateral epiblast. PCD was locally introduced via Hoechst microinjection and focal illumination. Live imaged at 4X from stage HH 3 to HH 4+. Induced-PCD suppressed contralateral invasion. While contralateral migration continued in non-injected zones, at ECMPCD deficient PS regions. Scale bar, 200 μm.