Lineage-specific CDK activity dynamics characterize early mammalian development

Background:  In mammals, the pre-implantation embryo takes all the big decisions on its own. Cell-cycle is the force driving it towards becoming a completely autonomous entity, with two distinct cell-types – the trophoectoderm (TE) and the inner cell mass (ICM). The cell-cycle is different between the embryo and the somatic cells, even though cyclins and cyclin-dependent kinases (CDKs) are involved in both. In this preprint, the authors turned into cinematographers, capturing the real time dynamics of CDKs using a very sensitive sensor.

Key findings of the study:

Generating the mouse model – Generating genetically modified mouse lines is a difficult task. A good reporter for studying cell-cycle dynamics in the pre-implantation embryo should capture  – low levels of protein, the dynamics of protein at single-cell level and rapid changes in the subcellular localisation of the protein.

The authors achieved all of these by stitching five DNA elements together –

1. A portion of DNA human helicase B (DHB) containing four CDK phosphorylation sites
2. Fluorescent protein mClover3
3. Self-cleaving T2A peptide sequence
4. Histone 2B (H2B) tag
5. Fluorescent protein mRuby3

The CDK activity increases from G0/G1-S-G2/M phase and the mClover reporter translocate between the nucleus (low activity) and the cytoplasm (high activity) in a CDK-dependent manner, providing a quantifiable readout of CDK activity at a single-cell level.

Developmental stage specific CDK activity – The signal from the CDK sensor was apparent in the cytoplasm during the morula and mid-blastocyst stages suggesting high CDK activity. The late blastocyst has three major cell types – outer trophoblast layer (CDX2+), the epiblast (NANOG+), and the primitive endoderm layer (GATA6+). The authors made several fascinating observations giving us a peek into molecular events that are otherwise hard to probe. CDK activity decreased in the outer trophoblast as the embryo made preparations for  implanting itself in the uterus, however CDK activity remained high in the epiblast and primitive endoderm cells.

The molecule that causes the dip in CDK activity in the trophoblast – Interestingly, the three cell-types present in the blastocyst can be maintained in-vitro as self-renewing stem cells. To achieve this, trophoblast stem cells (TSCs) rely on fibroblast growth factor 4 (FGF4) whereas embryonic stem cells (ESCs) require leukaemia inhibitory factor (LIF) to self-renew. The authors performed FGF4 or LIF withdrawal experiments on TSCs and ESCs respectively and found that TSC were intrinsically more prone than ESC to downregulate CDK activity upon changes in the levels of self-renewing signals. This simple experiment offers a fundamental finding which the field lacked for a long-time.

Conservation of lineage-specific regulation of CDK activity in human TE-like cells –  The authors, leveraging their experimental strengths, used a 2D system to mimic human gastrulation to study CDK dynamics in human TE-like cells. The human ESCs were cultured in the circular micropatterned chip and stimulated with BMP4 to induce differentiation. The outermost ring of TE-like cells (CDX2+) showed nuclear enrichment of the CDK reporter, consistent with the observations in mouse embryo.

What I like about the preprint:

This preprint describes a beautiful tool that can be used to understand cell-cycle dynamics in early embryogenesis. Recently, there has been an explosion of papers that describe self-organising properties of stem cells into embryo-like structures. But how exactly do cells self-organise? The tool  described here can potentially be used to address some of the fundamental processes underlying self-organisation, and the autonomous developmental potential that stem cells carry. It is an elegantly designed system which can be used very easily and efficiently. The authors have used it to reveal the differences in cell cycle dynamics between ESCs and TSCs, which in itself is intriguing.

 

doi: https://doi.org/10.1242/prelights.38013

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