DEV003244 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. A splice-blocking morpholino targeted to dll4. (a) Schematic representation of the region spanning exon 4 through exon 7 of dll4. Target sites for MODll4 and the PCR primers used for RT-PCR are indicated. (b) RT-PCR analysis of dll4 splicing upon injection with 5 or 10 ng MODll4. In embryos injected with 10 ng MODll4, the normal transcript is undetectable and replaced by a predominant splice variant lacking exon 6 and with a frame-shift introducing a stop codon in exon 7. A total of 10 ng of the morpholino was used for all subsequent experiments. EF-1α was used as a loading control. The sequence for MODll4 is 5′-TAGGGTTTAGTCTTACCTTGGTCAC-3′. PCR was performed in two rounds using heminested primers. The first reaction was performed using the primer 5′-CATTGATTATTGAGGCCTGG-3′ (located 5′ of the region shown in a) and PCR-rev (5′-CTGTCACACTCTCGCACCTG-3′). A small amount of the resulting product was then amplified using PCR-for (5′-GTTTCGGCCACTACACCTGC-3′) and PCR-rev.

• Movie 1 -

  Movie 1. Time-lapse movie showing DLAV and ISVs in a normal embryo. Endothelial cells in a normal embryo at 2.5 dpf are quiescent and display few filopodia.

• Movie 2 -

  Movie 2. Time-lapse movie showing DLAV and ISVs in an MODll4-injected embryo. Similar region of an embryo injected with 10 ng MODll4. Endothelial cells continue to extend and retract filopodia. Notice the formation of an ectopic sprout into the space between two ISVs.

• Movie 3 -

  Movie 3. Time-lapse movie showing DLAV and ISVs in a control embryo treated with DMSO starting at 33 hpf. Control embryo at 54 hpf treated with DMSO overnight starting at 33 hpf.

• Movie 4 -

  Movie 4. Time-lapse movie showing DLAV and ISVs in an embryo treated with DAPT starting at 33 hpf. Similar region of a sibling treated with DAPT. Endothelial cells actively extend and retract filopodia and ectopic sprouts can be seen invading the space between ISVs.