*2.5. Zebrafish*

Experiments were conducted in accordance with Dutch guidelines for the care and use of laboratory animals, with the approval of the Animal Experimentation Committee (DEC) of the Royal Netherlands Academy of Arts and Sciences (KNAW). Mutant *zebrafish* possessed the previously described vhlhu2117 mutation [12]. For RNA collection, *zebrafish* embryos were collected at 5 dpf based on these phenotypes and RNA was collected using Trizol reagen<sup>t</sup> (Ambion) as per manufacturer's instructions.

#### *2.6. Injections and Visualizations*

Wild-type and mutant *Zebrafish* embryos were injected at the 1–2-cell stage with 1 nL of 5 uM of the same miRNA mimics and antagonists used for the cell culture experiments described above. Mimics and antagonists were diluted in pure water with 0.1% phenol red for visualization and injected using a nanoject2000 microinjector (World Precision Instruments). *Zebrafish* were selected without bias at 5dpf and then imaged using an LSM700 microscope (Zeiss). DNA was then collected from theses embryos using lysis bu ffer, and then embryos were genotyped using a KASP ™ genotyping system (LGC Genomics, Teddington, Middlesex, England) kit designed against the *vhlhu2117* mutation or by sanger sequencing using the following primers: Fw: 5'-TAA GGG CTT AGC GCA TGT TC-3' and Rv: 5'-CGA GTT AAA CGC GTA GAT AG-3'.

#### *2.7. Statistical Analysis*

Data was analyzed with Graphpad Prism 6 and comparisons were performed with student t-test or paired *t*-test between two groups and with ANOVA for more than two groups. Data are presented as mean ± SEM. *p*-values are indicated as follows: \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; *p* < 0.05 is considered significant.
