Lineage-specific CDK activity dynamics characterize early mammalian development

Abstract

Cyclin-dependent kinases (CDKs) regulate proliferation dynamics and cell fate in response to extracellular inputs. It remains largely unknown how CDK activity fluctuates and influences cell commitment during early mammalian development. Here, we generated a mouse model expressing a CDK translocation reporter that enabled quantification of CDK activity in live single cells. By examining pre- and post-implantation mouse embryos at different stages, we observed a progressive decrease in CDK activity in cells from the trophectoderm (TE) prior to implantation. This drop seems to correlate with the available levels of ICM-derived FGF4 as CDK activity downregulation is rescued by exogenous FGF4. Furthermore, we showed that cell fate decisions in the pre-implantation embryo are not determined by the establishment of oscillatory CDK activity or overall changes in CDK activity. Finally, we uncovered the existence of conserved regulatory mechanisms in mammals by revealing lineage-specific regulation of CDK activity in TE-like human cells.

Keywords: CDK; CP: Developmental biology; cyclin-dependent kinase; embryonic stem cells; kinase translocation reporter; pre-implantation development; trophectoderm.

Figure 1.. A mouse model to visualize CDK activity

(A) CDK sensor targeted at the Rosa26 locus. NES, nuclear export signal; NLS, nuclear localization signal. (B) Confocal images of mouse ESC^DHB/H2B treated with CDK1/2i (30 μM). Scale bar, 30 μm. (C) Single-cell CDK activity (green) and nuclear size (blue) traces of representative ESC^DHB/H2B. We tracked the same cell and followed a daughter and granddaughter cell after mitosis (M). (D) Plot showing mean CDK activity with a shading of minimum and maximum values in untreated (ø) or CDK1/2i-treated (30 mM) ESC^DHB/H2B. (E) High-throughput imaging (HTI) quantification of CDK activity in untreated (ø) or CDK1/2i-treated ESC^DHB/H2B cultures at different time points and concentrations (1, 5, and 30 μM). Center lines indicate mean values. All comparisons to untreated cells showed p < 0.0001 from two-tailed unpaired t tests. n.s., non-significant. (F) HTI quantification of CDK activity in untreated (ø) or dTAG-treated CDK1-degron ESC^DHB/H2B combined with PF4091 for 2 h. Center lines indicate mean values. p values are from two-tailed unpaired t tests. ****p < 0.0001. (G) Single-cell CDK activity traces from ROSA26^DHB/H2B-derived MEFs. All tracks were synchronized in silico to mitosis. Criteria to define cells as CDK_inc, CDK_del, and G1 exit are specified in STAR Methods. (H) Confocal images of untreated ROSA26^DHB/H2B-derived MEFs. Scale bar, 50 μm. (I) Confocal images of E7.5 ROSA26^DHB/H2B mice showing the node (dashed lines). Scale bar, 20 μm.

Figure 2.. TE cells show decreased CDK activity upon implantation

(A) Confocal images from representative ROSA26^DHB/H2B embryos. Scale bar, 20 μm. (B) Plot showing a quantification of CDK activity in individual SOX2⁺ or CDX2⁺ cells from ROSA26^DHB/H2B embryos (dots from the same column) staged based on the number of cells. Percentage of cells (in red) below the arbitrary threshold (−0.25) shown for embryos containing 0–100 cells and above 100 cells. N = 54 embryos. (C) Time-lapse microscopy experiment performed in two representative E3.5 ROSA26^DHB/H2B embryos. Note the embryonic (e)-abembryonic (a) axis. Scale bar, 30 μm. (D) Plots showing CDK activity in individual cells obtained from a representative E3.5 ROSA26^DHB/H2B embryo. The embryo was imaged, fixed, and stained for CDX2 and SOX2 (bottom; scale bar, 30 μm) to determine lineage identity at the endpoint. Highlighted tracks are in cyan and magenta. (E) Plot showing a quantification of CDK activity in individual cells obtained from a pool of E4.5 ROSA26^DHB/H2B embryos. Percentage of cells (in red) below the arbitrary threshold (−0.25). p values are from two-tailed unpaired t tests. ****p < 0.0001; N = 5 embryos. (F) Confocal images from E6.5 ROSA26^DHB/H2B embryos. Dashed line surrounds the EPI. Scale bar, 20 μm.

Figure 3.. ICM-derived FGF4 establishes a CDK activity gradient

(A and B) Confocal images (A; scale bar, 100 μm) and single-cell CDK activity traces (B) from TSC^DHB/H2B and ESC^DHB/H2B cultured with or without self-renewal signals for 72 h. In (B), all tracks were synchronized in silico to mitosis. Criteria to define cells as CDK_inc, CDK_del, Endored, G1 exit and G2 exit are specified in STAR Methods. (C) HTI quantification of CDK activity in ESC^DHB/H2B and TSC^DHB/H2B under self-renewal conditions or following self-renewal factor withdrawal for 72 h. Center lines indicate mean values. ****p < 0.0001 from two-tailed unpaired t tests. (D) Confocal images of representative E3.5 untreated or FGF4-treated ROSA26^DHB/H2B embryos cultured for 20 h. Scale bar, 50 μm. Arrows indicate the embryonic (e)-abembryonic (a) axis. (E) Plot showing CDK activity levels in a pool of CDX2⁺ or CDX2⁻ cells from untreated (N = 5 embryos) or FGF4-treated (N = 9 embryos). p value is from a two-tailed unpaired t test. ****p < 0.0001. In red is the percentage of cells below the arbitrary threshold (−0.25). (F) Schematic representation of the development of a E3.5 blastocyst (day 0 for in vitro culture) beyond implantation stages. (G–J) Series of 2DZ-slices obtained from a Z-stack (G and J) or maximum projection (H and I) confocal images of representative ROSA26^DHB/H2B embryos cultured in vitro 3 days after isolation at E3.5. Scale bars, 20 μm (G and H), 50 μm (I), and 30 μm (J).

Figure 4.. Human TE-like cells show nuclear translocation of the CDK sensor

(A) Confocal images of human ESC^DHB/H2B untreated (ø) or CDK1/2i-treated (30 μM) for 1 h. Scale bar, 50 μm. (B) Single-cell CDK activity and nuclear trace of representative human ESC^DHB/H2B. (C) Confocal images of uninduced or BMP4-induced gastruloids. Insets show higher magnification. Dashed lines highlight TE-like cells. Scale bar, 100 μm. (D) Representation of the gastruloid using radial distance (left) or concentric rings (right). (E–G) Plot showing CDK activity levels in 2,000 random cells distributed according to their position (E), 1,000 random CDX2⁺ or CDX2⁻ cells (F), and 600 random CDX2⁺/CDX2⁻ cells according to their radial distance (G). Percentage of cells per ring (in red) below the arbitrary threshold (<−0.25). Data obtained by combining multiple gastruloids (N = 12). p value is from a two-tailed unpaired t test. ****p < 0.0001. (H) Confocal images of representative BMP4-induced gastruloids. Dashed lines highlight TE-like cells. Scale bar, 50 μm.