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. 2022 Dec 7;42(3):e111364. doi: 10.15252/embj.2022111364

Figure EV2. zRbm14 functions in the blastula‐to‐gastrula development through both RNA‐binding and phase separation (related to Figs 4 and 5A–C).

Figure EV2

  • A
    Diagrams showing target sequences of the indicated MOs on zRbm14 mRNAs. Translation initiation codons (AUG) are shown in red. Lowercase letters represent intron sequences. 14a‐MO and 14b‐tMO were designed to respectively repress the translation of both maternal and zygotic mRNAs of zRbm14a and zRbm14b. 14b‐MO was designed to interfere with the splicing of zRbm14b pre‐mRNA and thus only interfered with the zygotic transcript. The efficacies of both 14a‐MO and 14b‐MO were confirmed previously using 24‐hpf morphants (Xiao et al, 2019).
  • B
    Our anti‐zRbm14a antibody only weakly cross‐reacted with zRbm14b. HEK293T cell lysates expressing GFP, GFP‐zRbm14a, or GFP‐zRbm14b were immunoblotted with antibodies against GFP and zRbm14a, respectively.
  • C
    14a‐MO efficiently repressed the translation of zRbm14a mRNA in early embryos. Zebrafish 1‐cell embryos were each microinjected with 4 ng of MO and collected at the indicated time points. Lysates from 5 embryos were loaded in each lane. α‐tubulin served as an internal control.
  • D, E
    14b‐tMO efficiently repressed the translation of zRbm14b mRNA in early embryos. 14b‐200 nt‐GFP, a reporter mRNA in vitro transcribed from a construct containing a 5′ 200‐nucleotide zRbm14b cDNA fragment followed by an in‐frame GFP cDNA (D), was used to assess the efficacy of 14b‐tMO. In total, 100 pg of the reporter mRNA were co‐injected with 4 ng of MO into each 1‐cell embryo. The embryos were imaged at the indicated time points (E). Note that the embryos co‐injected with 14b‐tMO failed to express GFP.
  • F
    A typical set of zebrafish morphants. In total, 8 ng of ctrl‐MO, 14‐tMOs (4 ng 14a‐MO + 4 ng 14b‐tMO), or 14‐MOs (4 ng 14a‐MO + 4 ng 14b‐MO) were co‐injected with 100 pg of in vitro transcribed GFP mRNA (as a tracer) into each 1‐cell embryo to generate control, maternal zRbm14, and zygotic zRbm14 morphants, respectively. GFP‐positive embryos were collected at 2.5 hpf and imaged sequentially at the indicated time points. Representative embryos and quantification results are presented in Fig 4A and B.
  • G
    A typical set of zebrafish embryos in rescue experiments. 800 pg of in vitro transcribed mRNA for one of the indicated proteins were co‐injected with 14‐tMOs (4 ng 14a‐MO + 4 ng 14b‐tMO) into 1‐cell embryos. GFP‐positive morphants were imaged at the indicated time points. Representative embryos (arrows) and quantification results are presented in Fig 5B and C.

Source data are available online for this figure.