Mmessage mmachine t3 kit

pT3Ts-nCas9n plasmid was linearized with XbaI (NEB), treated with RNAsecure (Thermo Fisher Scientific) and purified using the QIAquick Gel Extraction Kit (Qiagen). In vitro Cas9 mRNA transcription was performed using the mMESSAGE mMACHINE T3 Kit (Thermo Fisher Scientific) followed by A-tailing using a Poly(A) Tailing Kit (Thermo Fisher Scientific). These transcripts were purified with the RNeasy Mini Kit (Qiagen), following manufacturer’s instructions.

The ORF of MYMK cDNA was tagged by Flag sequence at 3’ end and subcloned into pGEM^®-T Easy Vector (Promega), and the mRNA was generated using a MAXIscript^TM T7 transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) and then poly-A tailed (Thermo Fisher Scientific). The ORF of GFP cDNA was amplified with the T3 promoter region included at the 5ʹ-end, and the mRNA was generated using a mMESSAGE mMACHINE^® T3 kit (Thermo Fisher Scientific) and then poly-A tailed. The mRNAs were transfected using Lipofectamine^® MessengerMAX^TM Reagent (Thermo Fisher Scientific). Delivery efficiency was almost 90% about GFP, and 60-70% about MYMK, which was confirmed by the immunocytochemistry of GFP and Flag. The representative picture was shown in Supplementary Fig. 3.

The zebrafish reck^ulb3 allele was engineered by targeted genome editing using the CRISPR/Cas9 system, as described previously (Martin et al., 2022 (link); America et al., 2022 (link)). sgRNA target site was designed using the CRISPR Design website (http://crispr.mit.edu). The oligos (5′ – taggCCTGACAGTACTCACGAC – 3′ and 5′ – aaacGTCGTGAGTACTGTCAGG – 3′) were annealed and ligated into the pT7-gRNA vector (#46759, Addgene) (Jao et al., 2013 (link)) after digesting the plasmid with BsmBI (NEB). The sgRNA was transcribed from the BamHI linearized pT7-gRNA vector using the MEGAshortscript T7 kit (#AM1354, Thermo Fisher Scientific). The synthetic Cas9 mRNA was transcribed from the XbaI linearized pT3TS-nls-zCas9-nls vector (#46757, Addgene) using the mMESSAGE mMACHINE T3 Kit (#AM1348, Thermo Fisher Scientific). sgRNA (30 pg) and nls-zCas9-nls mRNA (150 pg) were injected into one-cell stage zebrafish embryos. The reck^ulb3 mutant allele harbors a 12 base pair deletion in the exon that encodes part of the third cysteine knot motif. Please refer to Figure 1—figure supplement 2 for more information. Genotyping of reck^ulb3 mutant fish was performed by HRMA of PCR products using the following primers:

Constructs encoding Cdx2, dn aPKC, and Gap43 tagged with RFP or GFP were cloned into a pBluescript RN3P vector and mRNA was synthesized from T3 promotor using mMessage mMachine T3 kit (Ambion), as established previously (Zernicka-Goetz et al., 1997 (link)). Construct encoding Histone 2B tagged with RFP was cloned into pGEMHE vector and synthesized from T7 promotor using mMessage mMachine T7 kit (Ambion). mRNAs (0.05 μg/μl for Cdx2, 0.23 μg/μl for dn aPKC, 0.34 μg/μl for Gap43-RFP and Gap43-GFP, 0.05 μg/μl for H2B-RFP) were injected into one 2-cell embryo blastomere and injected embryos were cultured in KSOM until late 4- or 8-cell stage. In some experiments 8-cell embryos׳ cells were injected with either rabbit pan-kinesin antibody (Cytoskeleton, 250 µg/ml), or control rabbit IgG (250 µg/ml). In both cases rhodamine dextran was used as a cell lineage tracer. Embryos were transferred to M2 medium and imaged in 12–15 planes (5–7 µm apart) every 15 min for 12 h over the 8–16-cell transition. Imaging was performed on Leica scanning confocal microscope, Zeiss spinning-disc confocal microscope or Deltavision fluorescence microscope, equipped with 37.5 °C chambers.