Sp6 message machine kit

Translation (rdh10a MO1–5’-GATGTTCATCACCATGTTTAATGCC) and splice-blocking (rdh10a MO2–5’-TAAAAAGAGGCTCACCCAGAAGTGC) MOs were used to target rdh10a (S1 Fig). To knockdown Rdh10a, a cocktail of both MOs was used at 2.5 ng MO1 and 0.7 ng MO2. Sequences for pax2a and pax2b were reported previously. 9 ng pax2a MO and 7.5 ng pax2b MO were used for injections. The dose of pax2a MO used produced a phenotype indistinguishable from the noi mutants [53 (link), 54 (link)]. For all injection experiments, 2 ng of p53 MO was used to help suppress non-specific MO-induced cell death [69 (link)]. Full-length rdh10a was cloned into pCS2+ and capped mRNA was made using a Sp6 Message Machine Kit (Ambion). 200 and 300 pg of rdh10a mRNA were used for experiments, as indicated in the Results. For the rdh10a-E2 reporter, 20 pg of DNA was co-injected with 25 pg Tol2 mRNA.

Morpholinos were purchased from Gene Tools (Philomath, Oregon, USA). The following Morpholinos were used in this study:
amotl2a MO 5′-CTGATGATTCCTCTGCCGTTCTCAT-3′^{14 , 15 (link)}amotl2b MO 5′-TGAGTATTTATGATCTGAGCTGAAC-3′^{14 , 15 (link)}par3 MO1 5′-TCAAAGGCTCCCGTGCTCTGGTGTC-3′^{17 (link)},
par3 MO3 5′-TCCCGTGCTCTGGTGTCAAGATCAT-3′^{17 (link)}control 5′-CCTCTTACCTCAGTTACAATTTATA-3′
The amotl2a morpholino was injected at 1.5 ng per embryo and the amotl2b morpholino was injected at 3 ng per embryo. Morpholino-injected zebrafish embryos were maintained at 28 °C in standard E3 water supplemented with 0.003% phenyl-2-thiourea.
mRNA encoding wildtype or mutant human AMOTL2 or PAR3 were synthesized using the SP6 Message Machine kit (Ambion, Austin, TX, USA), and 50 pg per embryos were co-injected with Morpholinos for rescue experiments. Embryos were fixed in 4% PFA at 28hpf for cell area analysis and at 34 hpf quantification of cell corners. For visualization of Par3-GFP^{18 (link)}, living embryos were sedated with tricaine and mounted into 1% low-melt agarose on a coverslip. All experiments were performed in accordance with relevant guidelines and regulations. All experimental protocols were approved by the regional ethics board (Jordbruksverket.se).

The human wild-type^{67 (link)} mRNA of RHEB (NM_005614) was cloned into the pCS2+ vector and transcribed in vitro using the SP6 Message Machine kit (Ambion). The variants identified in RHEB in our patient cohort (RHEBp.P37L, RHEBp.S68P) were introduced using Phusion high-fidelity DNA polymerase (New England Biolabs) and custom-designed primers. We injected 50 pg of WT or mutant RNA into wild-type zebrafish embryos at the 1- to 4-cell stage. For the experiments with rapamycin treatment we added 2.7 nM of ready-made rapamycin solution in DMSO (R8781, Sigma-Aldrich) in each of the injection cocktails. For the headsize assay, the injected larvae were grown to 5 dpf and imaged live on dorsal view. The area of the head was traced excluding the eyes from the measurements and statistical significance was calculated using Student’s t-test. All experiments were repeated three times and scored blind to injection cocktail.