HREM, RNAseq and Cell Cycle Analyses Reveal the Role of the G2/M-Regulatory Protein, WEE1, on the Survivability of Chicken Embryos during Diapause

Narayan Pokhrel, Olga Genin, Dalit Sela-Donenfeld*, Yuval Cinnamon*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

4 Scopus citations


Avian blastoderm can enter into diapause when kept at low temperatures and successfully resume development (SRD) when re-incubated in body temperature. These abilities, which are largely affected by the temperature and duration of the diapause, are poorly understood at the cellular and molecular level. To determine how temperature affects embryonic morphology during diapause, high-resolution episcopic microscopy (HREM) analysis was utilized. While blastoderms diapausing at 12C for 28 days presented typical cytoarchitecture, similar to non-diapaused embryos, at 18C, much thicker blastoderms with higher cell number were observed. RNAseq was conducted to discover the genes underlying these phenotypes, revealing differentially expressed cell cycle regulatory genes. Among them, WEE1, a negative regulator of G2/M transition, was highly expressed at 12C compared to 18C. This finding suggested that cells at 12C are arrested at the G2/M phase, as supported by bromodeoxyuridine incorporation (BrdU) assay and phospho-histone H3 (pH 3) immunostaining. Inhibition of WEE1 during diapause at 12C resulted in cell cycle progression beyond the G2/M and augmented tissue volume, resembling the morphology of 18C-diapaused embryos. These findings suggest that diapause at low temperatures leads to WEE1 upregulation, which arrests the cell cycle at the G2/M phase, promoting the perseverance of embryonic cytoarchitecture and future SRD. In contrast, WEE1 is not upregulated during diapause at higher temperature, leading to continuous proliferation and maladaptive morphology associated with poor survivability. Combining HREM-based analysis with RNAseq and molecular manipulations, we present a novel mechanism that regulates the ability of diapaused avian embryos to maintain their cytoarchitecture via cell cycle arrest, which enables their SRD.

Original languageAmerican English
Article number779
Issue number4
StatePublished - Apr 2022

Bibliographical note

Publisher Copyright:
© 2022 by the authors. Licensee MDPI, Basel, Switzerland.


  • G2/M transition
  • RNAseq
  • WEE1
  • cell cycle
  • chicken embryonic blastoderm
  • chicken embryonic diapause
  • high-resolution episcopic microscopy (HREM)
  • mitosis


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