Protein profiling of zebrafish embryos unmasks regulatory layers during early embryogenesis.

The maternal-to-zygotic transition is crucial in embryonic development, marked by the degradation of maternally provided mRNAs and initiation of zygotic gene expression. However, the changes occurring at the protein level during this transition remain unclear. Here, we conducted protein profiling throughout zebrafish embryogenesis using quantitative mass spectrometry, integrating transcriptomics and translatomics datasets. Our data shows that unlike RNA changes, protein changes are less dynamic. Further, increases in protein levels correlate with mRNA translation, whereas declines in protein levels do not, suggesting active protein degradation processes. Interestingly, proteins from pure zygotic genes are present at fertilization, challenging existing mRNA-based gene classifications. As a proof of concept, we utilized CRISPR-Cas13d to target znf281b mRNA, a gene whose protein significantly accumulates within the first two hours post-fertilization, demonstrating its crucial role in development. Consequently, our protein profiling, coupled with CRISPR-Cas13d, offers a new approach to unravel maternal mRNAs function during embryonic development. Overall design: Two biological replicate samples containing 50 zebrafish oocytes per stage were collected by pairing females in natural matings to ''purge'' mature eggs and used to establish an oogenesis time-line. Fish were euthanized and ovaries harvested within 1–11 days post-purging (dpp). Stage I and II oocytes were collected at 1–2 dpp, stage III oocytes (germinal vesicle in central position) at 4–7 dpp, and stage IV oocytes (germinal vesicle asymmetrically located) at 8–10 dpp. Oocyte isolations were based on73, and conducted in isolation medium Leibovitz L-15 (Sigma-Aldrich #L5520) plus Collagenase I and II depending on the stages as follows. Stages I and II were isolated with 3 mg/mL Collagenase I (Sigma-Aldrich C0130) and 3 mg/mL Collagenase II (Gibco 17101015). Stage III was isolated with 3 mg/mL Collagenase I (Sigma-Aldrich C0130). Stage IV was isolated by mechanical stripping with forceps and needle without the use of Collagenases. Isolated oocytes were snap-frozen and RNA was extracted with TRIzol (Invitrogen #15596026) following manufacturer's protocols. Three biological replicate samples containing 20 dechorionated zebrafish embryos were snap-frozen in TRIzol (Invitrogen #15596026). Four time points of embryonic development were included: 1-cell stage (0-hpf, hours post fertilization), 2-hpf, 4-hpf, and 6-hpf. RNA was extracted with Direct-zol RNA MicroPrep kit (Zymo Research #R2062) following manufacturer's protocols.