Abstract
Cul4b-null (Cul4bΔ/Y) mice undergo growth arrest and degeneration during the early embryonic stages and die at E9.5. The pathogenic causes of this lethality remain incompletely characterized. However, it has been hypothesized that the loss of Cul4b function in extraembryonic tissues plays a key role. In this study, we investigated possible causes of death for Cul4b-null embryos, particularly in regard to the role of embryonic Cul4b. First, we show that the loss of embryonic Cul4b affects the growth of the inner cell mass in vitro and delays epiblast development during the gastrulation period at E6.5~E7.5 in vivo, as highlighted by the absence of the epiblastic transcription factor Brachyury from E6.5~E7.5. Additionally, at E7.5, strong and laterally expanded expression of Eomes and Fgf8 signaling was detected. Sectioning of these embryos showed disorganized primitive streak layer cells. Second, we observed that Mash2-expressing cells were present in the extraembryonic tissues of Cul4b-deficient embryos at E6.5 but were absent at E7.5. In addition, the loss of Cul4b resulted in decreased expression of cyclin proteins, which are required for the cell cycle transition from G1 to S. Taken together, these observations suggest that the embryonic expression of Cul4b is important for epiblast growth during E6.5~E7.5, and the loss of Cul4b results in either delayed growth of the epiblast or defective localization of primitive streak layer cells. As a result, the signaling activity mediated by the epiblast for subsequent ectoplacental cone development is affected, with the potential to induce growth retardation and lethality in Cul4bΔ/Y embryos.
Highlights
During embryogenesis, pluripotent stem cells, under precise control of DNA replication and repair, can give rise to an entire organism
Studies have previously demonstrated that Cul4b heterozygous female mice (Cul4bΔ/+) generated from Cul4bflox/+ females carrying the Cul4b-null X chromosome from their mother crossed with EIIa-Cre or CAG-Cre male mice can survive up to E13.5 [20, 21]
To achieve different tissue specificities to clarify the timing of the lethality of Cul4b-null mice and to determine whether Cre activity could influence the survival of Cul4bΔ/+ female mice, we set up different mating strategies to produce Cul4bΔ/+ female mice either using Cre activity (Zp3-Cre oocyte-specific knockout mice bred with wild type male, S1B Fig) or not (Cul4b+/Δ female mice bred with wild type male, S1A Fig)
Summary
Pluripotent stem cells (e.g., embryonic stem cells), under precise control of DNA replication and repair, can give rise to an entire organism. At the blastocyst stage of mouse embryos, typically embryonic day 3.5 (E3.5), two distinct cell lineages contribute to early embryonic and extraembryonic tissue development [1]. These cell lineages include the inner cell mass (ICM) and the trophectoderm, respectively. Epiblast cells emerging from the primitive streak between the embryonic ectoderm and overlying visceral endoderm give rise to the mesoderm [2,3,4]. Further development of the primitive streak gives rise to three embryonic germ layers, namely, the ectoderm, mesoderm, and endoderm. The primitive endoderm layer is eventually incorporated into the extraembryonic ectoderm to become part of the placenta
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