Fluorescein labeled dextran

For the evaluation of tumor vessel leakiness, mice were given 1 mg of 70-kDa lysine fixable fluorescein labeled dextran (Thermo Fisher). After 10 min, the mouse was subjected to whole-animal perfusion. The tumors were carefully excised and cut into two halves along the maximum diameter. Both halves were fixed with 4% formaldehyde overnight at 4 °C, and then frozen. The FITC/CD31 double-positive vessels were identified as perfused vessels and counted, and the fluorescence intensity of the FITC-only positive area (extravasated FITC-dextran) was standardized for the perfused vessel counts.

A pXT7 construct containing Pitxc partial 5′UTR (207-bp), full-length coding sequence, and partial 3′UTR (217-bp) was conserved in a previous study [24 (link)]. Pitxc mRNAs was synthesized from the construct with T7 mMESSAGE mMACHINE kit (Ambion) following the kit manual. The mRNAs (300 ng/μL), together with 3 mg/ml fluorescein-labeled dextran (10,000 MW, Thermo Fisher Scientific) and 12.5% glycerol, were injected into amphioxus unfertilized eggs or one blastomere of 2-cell embryos following steps described in previous reports [72 (link)].

Regulatory sequences of Lhx3 (a 500-bp fragment upstream of its transcriptional start) and Hex (a 554-bp fragment upstream of the transcriptional start) genes were determined according to the ATAC-seq data of B. lanceolatum [79 ]. They were respectively inserted upstream of the firefly luciferase gene in pGL3 (Promega) and modified by PCR to generate mutant versions (Additional file 2: Table S3). Pitx binding sites in the regulatory sequences of Lhx3 and Hex were predicted using JASPAR profiles at http://jaspar.genereg.net/. Injection solutions were prepared to contain 60 ng/μL of Lhx3 or Hex constructs, or Renilla luciferase vector pRL-SV40 (control), and 3 mg/ml fluorescein-labeled dextran (10,000 MW, Thermo Fisher Scientific), 12.5% glycerol, with or without 100 ng/μL Pitxc mRNA. Embryos were injected at unfertilized egg stage [72 (link)] and assayed at N4 stage (eight somites, 60 embryos for each experiment); uninjected embryos from the same batch were used as the negative control. Firefly luciferase expression levels from pGL3 and Renilla luciferase from pRL-SV40 were detected with the Dual Luciferase Kit (Promega) using a GloMax luminometer with an integration of 15 s, and the level of firefly luciferase was normalized to Renilla luciferase activity for each experiment.

Cas9 mRNA/sgRNA injection solution was prepared with 5 mg/mL fluorescein-labeled dextrans (10,000 MW, Thermo Fisher Scientific), 20% glycerol, 376.7 μg/μL Cas9 mRNA and 178.6 μg/μL sgRNA. Cas9 nuclease/sgRNA injection solution was prepared to contain 3 mg/mL fluorescein-labeled dextrans, 12.5% glycerol (not including the glycerol from Cas9 nuclease) and the desired concentration of Cas9 protein and sgRNA. Three Cas9 nucleases which were purchased from New England BioLabs (20 μM, equal to around 3 μg/μL), Thermo Fisher Scientific (1 μg/μL) and TaKaRa (3 μg/μL) were tested in the study. sgRNA was used directly or predenatured as previously described [12 (link)]. Before injection, the Cas9 nuclease/sgRNA injection solution was incubated at 37 °C for 5 min in a thermal cycler to facilitate Cas9/sgRNA RNP complex formation. Microinjection of unfertilized eggs was carried out as previously described [13 (link)], while single blastomere injection at the two-cell stage was conducted as recently described [14 (link)]. Around 100–200 unfertilized eggs or 50 two-cell embryos were injected for each experiment.