Incomplete activation of developmentally required genes Alyref1 and Gabpb1 leads to preimplantation arrest in cloned mouse embryos

  1. Shunya Ihashi
  2. Mizuto Hamanaka
  3. Masaya Kaji
  4. Miki Mori
  5. Yuma Imasato
  6. Misaki Nakamura
  7. Masayuki Anzai
  8. Kazuya Matsumoto
  9. Masahito Ikawa
  10. Kei Miyamoto

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Abstract

Differentiated cell nuclei can be reprogrammed after nuclear transfer (NT) to oocytes and the produced NT embryos can give rise to cloned animals. However, development of NT embryos is often hampered by recurrent reprogramming failures, including the incomplete activation of developmental genes, yet specific genes responsible for the arrest of NT embryos are not well understood. Here, we searched for developmentally important genes among the reprogramming-resistant H3K9me3-repressed genes, and identified Alyref and Gabpb1 by siRNA screening. Gene knockout of Alyref and Gabpb1 by the CRISPR/Cas9 system resulted in early developmental arrest in mice. Single embryo RNA-seq revealed that Alyref is needed for the formation of inner cell mass. The supplement of Alyref and Gabpb1 by mRNA injection supported efficient preimplantation development of cloned embryos. Thus, our study shows that the H3K9me3-repressed genes contain developmentally required genes and the incomplete activation of such genes results in preimplantation arrest of cloned embryos.

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    Referee #2

    Evidence, reproducibility and clarity

    Major points:

    1. The authors claim the Alyref and Gabpb1 were repressed by H3K9me3 in SCNT embryos, however, they didn't provide the direct evidence of H3K9me3 modification in Alyref and Gabpb1 promoter or enhancer.
    2. The authors should provide direct evidence that how Alyref and Gabpb1 regulate pluripotency, cell viability and apoptotic related genes which are essential for morula arrest in knock out embryos.

    Minor points:

    1. Figure1A, which developmental stage of SCNT or IVF embryos are used for RNA-seq?
    2. In Figure S2B, FPKM values of Alyref in SCNT embryos with TSA+VC-1 is inconsistent low. More repetitions are recommended.
    3. The samples of single embryo RNA-seq are less. More repetitions are recommended. 4.They lack the data of embryos transfer and offsprings experiment of SCNT embryos by the addition of Alyref and Gabpb1 through mRNA injection.

    Significance

    The manuscript by Ihashi et al aims to find specific genes responsible for the arrest of SCNT embryos. The authors identified Alyref and Gabpb1 by siRNA screening and verified morulae arrest phnotype in Alyref and Gabpb1 KO IVF embryos, and single embryo RNA-seq revealed that Alyref is needed for the formation of inner cell mass. The preimplantation development of cloned embryos was aided by the addition of Alyref and Gabpb1 by mRNA injection. Overall, this is an interesting study that demonstrate incomplete activation of Alyref1 and Gabpb1 will lead to preimplantation arrest of SCNT embryos. However, this is a very preliminary study that some important issues are need to address, thus, this manuscript is far from a publishable form.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The authors picked up candidate genes which might be important for SCNT embryo development based on RNA-seq data (basically genes downregulated in SCNT embryos compared to normal ones) and performed siRNA-knockdown on the candidate 15 genes. Two genes, Alyref and Gabpb1, were required for normal development. KO of each gene resulted in embryonic lethality, confirming the KD data. RNA-seq showed that Alyref KO affected lineage specification while Gabpb1 KO resulted in apoptosis. Finally the authors injected mRNA for Alyref and Gabpb1 into SCNT embryos and observed improved development.

    Major comments:

    1. The data provided are overall convincing and support the authors' conclusion. However, as the authors may understand, the paper lacks mechanistic insights into how Alyref and Gabpb1 work in early embryos. Furthermore, it is still not very clear why Alyref and Gabpb1 are downregulated in SCNT embryos. It seems that the authors speculate that somatic H3K9me3 might regulate the expression of those genes, but it is still possible that H3K9me3 just inhibits the expression of upstream regulator of Alyref and Gabpb1. The paper lacks experiments to address these possibilities.

    2. Fig. 7: Given the current status of the SCNT field, it would be important to show the birth rate of SCNT embryos upon mRNA injection.

    3. Fig. 2C-D: It would be important to know when the differential expression of Alyref or Gabpb1 protein can be seen to understand the relationship between SCNT RNA-seq data and KO embryo phenotype.

    Minor comments:

    1. State clearly in the manuscript which stage of embryos was used for RNA-seq analysis or other assays.

    2. Fig 1A: "DEGs" might be confusing. Do the authors refer to downregulated genes as DEGs?

    3. L113: State clearly what the "transcriptional activators" in this study are. Also, "Repression mix" and "Activation mix" in Fig1C are not easily understandable.

    4. Fig6A: Are the pathways indicated top significant ones? If not, the IPA result should be indicated in an unbiased manner. Also, is it meaningful to show -log10(q-value) = ~1?

    5. S6A: The stage at which the Pou5f1 signal was measured is not clearly indicated; Pou5f1 expression becomes high in blastocysts, so comparison between 72 hpi morula would be appropriate.

    6. L217, Fig 6F: Is this indeed based on unsupervised hierarchical clustering?

    7. "heterozygous mutant mice" should be used rather than "hetero mice".

    Significance

    The study is constructed based on the previous finding that Kdm4d overexpression significantly improved SCNT embryo development. However, it is still important to know why the removal of H3K9me3 can exert such an effect. The authors tackled this question and suggested that two genes (Alyref and Gabpb1) expressed upon H3K9me3 removal play important roles for SCNT embryo development. Unfortunately, while it is now widely accepted that Dux expression is important for SCNT development in the field, the authors did not test nor discuss how Dux is involved in the authors' findings. In addition, there would be many other things to be done in this study (described in major comments) to contribute to the SCNT field.

  5. Version published to 10.1101/2022.04.14.488417v1 on bioRxiv