All zebrafish experiments were performed in accordance with IACUC regulations. Injections and in situ hybridizations were performed as previously described (47 (link), 53 (link)). Quantification by flow cytometry using fluorescently labeled transgenic zebrafish embryos was performed as previously described (96 (link), 97 (link)). Morpholinos were purchased from Gene Tools, LLC (Philomath, OR, USA).
Zebrafish embryos at the one-cell stage were injected with Morpholinos (MOs). The sequence of the Morpholinos targeting the exon 4-intron 4 (MO1) and the exon 5-intron 5 (MO2) of D. rerio lat3a (please see “Bioinformatic and statistical analysis” for accession numbers) were as follows: lat3a MO1: 5′-ATAGATCATGTACTCACCTTCTGGT-3′; lat3a MO2: 5′-CATTTTCTGCTGCTCCTTACCGTTA-3′. The MO targeting zebrafish lat1 (NM_001128358) at the exon 1-intron 1 boundary was 5′-AGGTAACAGTTTACTTACGTATACA-3′.
Zebrafish embryos at 19 hpf (20 somites) were chemically dechorionated with pronase (Roche, Basel, Switzerland) and treated with DMSO (vehicle), 50 μM rapamycin, 1 μM torin 1, or 10 μM 4EGI-1 and o-dianisidine stained at 72 hpf (96 (link)).
Zebrafish embryos at the one-cell stage were injected with Morpholinos (MOs). The sequence of the Morpholinos targeting the exon 4-intron 4 (MO1) and the exon 5-intron 5 (MO2) of D. rerio lat3a (please see “Bioinformatic and statistical analysis” for accession numbers) were as follows: lat3a MO1: 5′-ATAGATCATGTACTCACCTTCTGGT-3′; lat3a MO2: 5′-CATTTTCTGCTGCTCCTTACCGTTA-3′. The MO targeting zebrafish lat1 (NM_001128358) at the exon 1-intron 1 boundary was 5′-AGGTAACAGTTTACTTACGTATACA-3′.
Zebrafish embryos at 19 hpf (20 somites) were chemically dechorionated with pronase (Roche, Basel, Switzerland) and treated with DMSO (vehicle), 50 μM rapamycin, 1 μM torin 1, or 10 μM 4EGI-1 and o-dianisidine stained at 72 hpf (96 (link)).
