小鼠四细胞胚胎异质表达基因在胚胎细胞命运决定中的作用及机制研究

It has been reported that heterogeneous expressed genes may initiate the cell-fate decisions in mouse 4-cell embryos. However, the underlying molecular mechanism remains unclear. Previously, we profiled the trancriptome of individual blastomeres of mouse 4-cell embryos. Through bioinformatics analysis, we found that three genes, Tcf23, Id3 and Glis2, which are important for maintaining pluripotency of embryonic stem cells, were highly variable expressed at this stage, highlighting their potential roles in the early cell-fate decision. In this project, we plan to investigate the functions of heterogeneous expressed Tcf23, Id3 and Glis2 in determining preimplantation embryonic cell fates through RNA interference, live cell imaging and identify their downstream genes using single-cell RNA-seq. Furthermore, we will use CRISPR/Cas9 genome-editing, ChIP-seq, NOMe-seq and immunoprecipitation-Mass Spectrometry in mouse embryonic stem cells to reveal the transcriptional regulatory mechanism of these heterogeneous expressed genes. Taken together, these results will give us more insights into the regulatory mechanism of early embryonic cell-fate decision, and may provide the basis for the diagnosis and treatment of cases with repeated embryo development arrest in clinic.

有研究报道，小鼠四细胞胚胎中异质表达基因可能启动了早期细胞命运决定，但具体机制有待阐明。申请人前期检测了小鼠四细胞胚胎各卵裂球的基因表达情况，发现Tcf23、Id3、Glis2在四细胞期呈异质表达，并且这三个基因在维持胚胎干细胞多能性方面具有重要功能，提示这些基因可能参与早期胚胎细胞命运的决定。本课题拟利用小鼠植入前胚胎，通过基因干扰、活细胞成像、单细胞转录组测序等技术研究Tcf23、Id3及Glis2异质表达基因在植入前胚胎第一次细胞命运决定中的作用。进一步在小鼠胚胎干细胞中，通过基因编辑、ChIP-seq、NOMe-seq、蛋白质纯化-质谱等揭示Tcf23、Id3及Glis2调控下游靶基因表达的分子机制。本课题的完成可为揭示植入前胚胎早期细胞命运决定的调控机制提供重要线索，并为临床上发生反复胚胎发育阻滞的病例提供诊疗依据。

小鼠四细胞胚胎中异质表达基因可能参与早期细胞命运决定的启动，但具体机制有待阐明。利用小鼠植入前胚胎，通过基因敲除、细胞成像、单细胞转录组测序等技术，探究了Tcf23、Id3、Glis2的异质性及在早期胚胎发育中的作用机制。结果表明：Tcf23在小鼠四细胞阶段呈异质性表达，通过影响细胞分化等参与调控小鼠囊胚形成。Id3在小鼠早期胚胎发育过程中呈现异质性表达，敲除Id3后通过影响微管、类固醇合成和线粒体磷酸化等影响囊胚功能，最终导致产仔数下降。此外，还探究了不同超排方式、高盐膳食摄入对小鼠卵巢及卵母细胞的影响。本课题的完成为揭示植入前胚胎早期细胞命运决定的调控机制提供重要线索，并为临床上发生反复胚胎发育阻滞的病例提供诊疗依据。