HREM, RNAseq and cell-cycle analyses reveal the role of the G2/M-regulatory protein, Wee1, on the survivability of chicken embryos during diapause.

Avian blastoderm can enter into diapause when kept at low temperatures, and successfully re-sume 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 12oC for 28 days presented typical cytoarchitecture, similar to non-diapaused embryos, at 18oC much thicker blastoderms with higher cell-number were ob-served. RNAseq was conducted to discover the genes underlying these phenotypes, revealing differentially-expressed cell-cycle regulatory genes. Amongst them, Wee1, a negative-regulator of G2/M transition, was highly expressed at 12oC compared to 18oC. This finding suggested that cells at 12oC are arrested at the G2/M phase, as supported by bromodeoxyuridine incorporation (BrdU) assay and phosho-histone-H3 (pH3) immuno-staining. Inhibition of Wee1 during dia-pause at 12oC resulted in cell-cycle progression beyond the G2/M and augmented tissue volume, resembling the morphology of 18oC-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 up-regulated 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.