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. 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.

母源到合子转换（maternal-to-zygotic transition）在胚胎发育过程中至关重要，其标志性事件为母源提供的mRNA降解以及合子基因表达的启动。然而，该转换过程中蛋白质层面的变化仍有待阐明。本研究利用定量质谱技术对斑马鱼胚胎发生的全过程进行蛋白质谱分析，并整合了转录组学（transcriptomics）与翻译组学（translatomics）数据集。研究数据显示，与RNA层面的变化不同，蛋白质层面的变化动态性更低。进一步分析发现，蛋白质水平的升高与mRNA翻译效率正相关，而蛋白质水平的下降则无此关联，这提示该过程存在活跃的蛋白质降解机制。值得注意的是，合子特异性基因编码的蛋白质在受精时即已存在，这一发现挑战了现有的基于mRNA的基因分类体系。作为概念验证，我们利用CRISPR-Cas13d靶向znf281b mRNA——该基因编码的蛋白质在受精后前两小时内显著积累——实验证实其在胚胎发育中发挥关键作用。综上，本研究的蛋白质谱分析结合CRISPR-Cas13d技术，为解析胚胎发育过程中母源mRNA的功能提供了全新的研究策略。本研究通过将雌鱼进行自然交配以排空体内成熟卵子，随后收集每个发育阶段含50枚斑马鱼卵母细胞的两份生物学重复样本，以此构建卵发生的时间线。在排空成熟卵子后的1至11天（dpp）内，对鱼类实施安乐死并采集卵巢。其中，I期与II期卵母细胞于1-2 dpp收集，III期卵母细胞（生发泡（germinal vesicle）位于中央）于4-7 dpp收集，IV期卵母细胞（生发泡位置不对称）于8-10 dpp收集。卵母细胞的分离参照文献73的方法，分离培养基采用Leibovitz L-15（Sigma-Aldrich #L5520），并根据不同阶段添加不同的胶原酶，具体如下：I期与II期卵母细胞使用3 mg/mL胶原酶I（Sigma-Aldrich C0130）与3 mg/mL胶原酶II（Gibco 17101015）；III期卵母细胞使用3 mg/mL胶原酶I（Sigma-Aldrich C0130）；IV期卵母细胞则采用镊子与针头进行机械剥离，无需使用胶原酶。分离得到的卵母细胞经快速冷冻后，按照制造商的操作流程使用TRIzol（Invitrogen #15596026）提取RNA。本研究同时收集了三份含20枚去壳斑马鱼胚胎的生物学重复样本，将其在TRIzol（Invitrogen #15596026）中快速冷冻。选取的四个胚胎发育时间点为：1细胞期（0 hpf，即受精后小时数）、2 hpf、4 hpf及6 hpf。按照制造商的操作流程，使用Direct-zol RNA MicroPrep试剂盒（Zymo Research #R2062）提取总RNA。