ZFIN Image: Lenard et al., 2013, Fig. S2

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Figure Caption

Fig. S2

Transcellular lumen formation is highly influenced by blood flow. Related to Figure 1 and 3.

A-F Still pictures of a time lapse movie showing development of the PLA in a transgenic embryo Tg(fliep:GFF)^ubs3,(UAS:mRFP),(UAS:VE-cadherinΔC-EGFP)^ubs12 injected with tnnt2 morpholino (sih) blocking the heart beat and hence, blood flow. The formation of PLA sprouts in the sih embryos is normal. As in the wild type, they are led by single tip cells (A, white arrows) attached to the stalk by ring shaped junctions (A, green arrows). All the steps of new contact formation are conserved, including new spot formation (B, white arrow), ring formation (C, white arrow) and cellular rearrangements (D-F, white and green arrows point at borders of junctional rings and new contact formation spots). Movie upon request.

G-H Pdxl2 (apical, green) and ZO-1 (junctional, white) staining of PLA at particular fusion steps in silent heart embryos. Blue bars mark the surface of cell-cell junctional contacts. Pdxl2 staining is only visible within the junctional rings and lines, since no membrane invagination takes place.

I-JStill pictures of a time lapse movie (Movie S7, I – embryo 1; J – embryo 2) showing development of the PLA in transgenic embryos Tg(fliep:GFF)^ubs3,(UAS:mRFP),(UAS:VEcadherinΔC-EGFP)^ubs12 before, during and after acute heart arrest with 4x tricaine. White/black arrows show the new contact length between PLA sprouts. Green arrows show multicellular parts of the sprouts (where continuous cell-cell junctions are visible). Red arrows show luminal membrane invagination/unicellular tube parts. After the heart arrest, lumen collapses and the fusion process continues like it the silent heart embryos. In the younger embryo (I) fusion proceeds up to the VE-cadherin-GFP ring formation. In the slightly older embryo (J) – up to the end of cell rearrangements. When tricaine is removed, lumen inflates again and the fusion continues with unicellular tube formation in the younger embryo (I). In J, lumen is immediately inflated in the already formed multicellular cord. See also Movie S7.

Scale bars: 20 μm.

Acknowledgments

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Reprinted from Developmental Cell, 25(5), Lenard, A., Ellertsdottir, E., Herwig, L., Krudewig, A., Sauteur, L., Belting, H.G., and Affolter, M., In Vivo analysis reveals a highly stereotypic morphogenetic pathway of vascular anastomosis, 492-506, Copyright (2013) with permission from Elsevier. Full text @ Dev. Cell