Rho-associated kinase activity is required for proper morphogenesis of the inner cell mass in the mouse blastocyst

Abstract

The blastocyst consists of the outer layer of trophectoderm and pluripotent inner cell mass (ICM), the precursor of the placenta and fetus, respectively. During blastocyst expansion, the ICM adopts a compact, ovoidal shape, whose proper morphology is crucial for normal embryogenesis. Rho-associated kinase (ROCK), an effector of small GTPase RHO signaling, mediates the diverse cellular processes of morphogenesis, but its role in ICM morphogenesis is unclear. Here, we demonstrate that ROCK is required for cohesion of ICM cells and formation of segregated tissues called primitive endoderm (PrE) and epiblast (Epi) in the ICM of the mouse blastocyst. Blastocyst treatment with ROCK inhibitors Y-27632 and Fasudil caused widening or spreading of the ICM, and intermingling of PrE and Epi. Widening of ICM was independent of trophectoderm because isolated ICMs as well as colonies of mouse embryonic stem cells (mESC) also spread upon Y-27632 treatment. PrE, Epi, and trophectoderm cell numbers were similar between control and treated blastocysts, suggesting that ROCK inhibition affected ICM morphology but not lineage differentiation. Rock1 and Rock2 knockdown via RNA interference in mESC also induced spreading, supporting the conclusion that morphological defects caused by the pharmacological inhibitors were due to ROCK inactivation. When blastocysts were transferred into surrogates, implantation efficiencies were unaffected by ROCK inhibition, but treated blastocysts yielded greater fetal loss. These results show that proper ICM morphology is dependent on ROCK activity and is crucial for fetal development. Our studies have wider implication for improving efficiencies of human assisted reproductive technologies that diminish pregnancy loss and promote successful births.

Keywords: assisted reproductive technologies (ART); cell lineage; developmental biology; early development; embryo; fetal loss; implantation; preimplantation.

FIG. 1

Inhibition of ROCK activity during blastocyst cavity expansion disrupts the ICM morphology but not cell proliferation and differentiation. Blastocysts were cultured in the absence (control) or presence of 10 μM Y-27632 from E3.5 to E4.5 and immunostained for cell lineage-specific transcription factors. A) GATA4-positive PrE (red) and NANOG-positive Epi (green) cells are less tightly packed together and appear scattered in the inhibitor-treated blastocyst than in the control blastocyst. Confocal images are z-series projections. B) Three-dimensional reconstructions of confocal microscopic images of the ICM of embryos in A. Reconstructed three-dimensional images are rotated to show views from the PrE and Epi side of the ICM. C) Comparison of ICM size based on the widest length of ICM cell dispersion. Circles represent the ICM size in individual embryos, and horizontal bars represent the mean value for each group. The ICM is significantly wider (Student t-test) in inhibitor-treated embryos (n = 22) compared to the control embryos (n = 23). D) Comparison of total cell numbers. Graph shows the mean and standard error. There is no statistically significant difference (P = 0.49; Student t-test) between control (n = 24) and inhibitor-treated (n = 25) embryos. E) Comparison of the number of PrE (red bar) and Epi (green bar) cells shows no statistically significant differences (P[PrE] = 0.61, P[Epi] = 0.07; Student t-test) between control (n = 24) and inhibitor-treated (n = 25) embryos. Graph shows the mean and standard error. Data presented in C, D, and E are compilations of two replicates of the experiment that were performed on different occasions. F) The treated blastocyst shows no differences to the control blastocyst with respect to CDX2 (red) protein immunostaining of the TE cells. The ICM is immunostained for pluripotency marker POU5F1 (green) protein. Confocal images are z-series projections. Blue, DAPI. Bars = 20 μm (A, B, F).

FIG. 2

Fasudil treatment phenocopies the Y-27632-induced disruption of the ICM morphology. Blastocysts were cultured in the absence (control) or presence of 5 μM Fasudil from E3.5 to E4.5 and immunostained for cell lineage-specific transcription factors. A) GATA4-positive PrE (red) and NANOG-positive Epi (green) cells are less tightly packed together and are more spread out in the Fasudil-treated embryo than in the control embryo. Confocal images are z-series projections. B) Three-dimensional reconstructions of confocal microscopic images of the ICM of embryos in A. Reconstructed three-dimensional images are rotated to show views from the PrE and Epi side of the ICM. C) Comparison of ICM size based on the widest length of ICM cell dispersion. Circles represent the ICM size in individual embryos, and horizontal bars represent the mean value for each group. The ICM is significantly wider (Student t-test) in inhibitor-treated embryos (n = 12) than in the control embryos (n = 14). D) Comparison of total cell numbers. Graph shows the mean and standard error. There is no statistically significant difference (P = 0.87; Student t-test) between control (n = 14) and inhibitor-treated (n = 14) embryos. E) Comparison of the number of PrE (red bar) and Epi (green bar) cells shows no statistically significant differences (P[PrE] = 0.16, P[Epi] = 0.29; Student t-test) between control (n = 14) and inhibitor-treated (n = 14) embryos. Graph shows the mean and standard error. Data presented in C, D, and E are compilations of two replicates of the experiment that were performed on different occasions. Blue, DAPI. Bars = 20 μm (A, B).

FIG. 3

Transient reduction of the blastocyst cavity does not interfere with ICM morphogenesis. A) Time-lapse recordings show the changes in the size of the blastocyst cavity between E3.5 and E4.5. Top panels show still images from time-lapse movies (Supplemental Movies), specifically of the start and end points of recordings. In the graphs, each colored line represents the cavity size of individual blastocysts. Y-27632-treated blastocysts (n = 15) tend to show more drastic reduction in cavity size than control blastocysts (n = 16). B) Blastocyst cavity is transiently reduced by brief cytochalasin D (CD, 0.5 μg/ml) treatment. Nonetheless, the E4.5 blastocyst shows segregated GATA4-positive PrE (red) and NANOG-positive Epi (green) tissue layers, similar to that in the control blastocyst. Confocal images are z-series projections. C) Comparison of ICM size based on the widest length of ICM cell dispersion. Circles represent the ICM size in individual embryos, and horizontal bars represent the mean value for each group. There is no statistically significant difference (P = 0.50; Student t-test) in the ICM size between CD-treated (n = 12) and control (n = 8) embryos. Data presented in C are compilations of two replicates of the experiment that were performed on different occasions. Blue, DAPI. Bar = 20 μm (B).

FIG. 4

ROCK inhibition by Y-27632 treatment induces spreading of isolated ICMs. A) Representative examples of morphology of control and treated ICMs. B) Comparison of surface area in control and treated ICMs. Circles represent surface area in individual ICMs, and horizontal bars represent the mean value for each group. Red circles represent ICMs that formed a cavity during the course of the experiment. Treated ICMs (n = 29) have significantly increased surface area (Student t-test) than control ICMs (n = 29). C) Fluorescence microscopic images of control and treated ICMs immunostained for the cell adhesion molecule CDH1 and cytoskeletal components, namely filamentous actin (F-actin) and microtubules. Data presented in B are compilations of two replicates of the experiment that were performed on different occasions. Nuclei were stained with DAPI. Bars = 20 μm (A, C).

FIG. 5

Disruption of the ICM morphology by Y-27632 (10 μM) treatment is not reversible. A) GATA4-positive PrE (red) and NANOG-positive Epi (green) cells appear more widely spread apart in the treated/washed blastocyst (E4.5+1day) than in the control blastocyst. Confocal images are z-series projections. B) Comparison of ICM size of blastocysts (E4.5+1day) based on the widest length of ICM cell dispersion. Circles represent the ICM size in individual embryos, and horizontal bars represent the mean value for each group. The ICM is significantly wider (Student t-test) in treated/washed embryos (n = 16) than in control embryos (n = 23). C) Comparison of the number of PrE (red bar) and Epi (green bar) cells shows no statistically significant differences (P[PrE] = 0.83, P[Epi] = 0.22; Student t-test) between control (n = 24) and treated/washed (n = 21) embryos. Graph shows the mean and standard error. Data presented in B and C are compilations of two replicates of the experiment that were performed on different occasions. D) Fluorescence microscopic images of blastocysts (E4.5+1day) immunostained for pluripotency markers. Top row shows POU5F1-positive ICM (red) cells and a subset of NANOG-positive Epi (green) cells in the control blastocyst. Second and third rows show an ectopic clump of NANOG/POU5F1-positive cells (arrowhead) at different planes of focus in the treated/washed blastocyst. E) Fluorescence microscopic images of blastocysts (E4.5+1day). The treated/washed blastocyst shows no differences to the control blastocyst with respect to CDX2 (red) protein immunostaining of the TE nuclei. The ICM is immunostained for POU5F1 (green) protein. Note that ICM cells are scattered in the Y-27632-treated blastocyst. Blue, DAPI (for nuclear staining [+Nuc]). Bars = 20 μm (A, D, E).

FIG. 6

Loss-of-function of ROCK induces spreading of mESC colonies. A) Quantitative RT-PCR analysis of Rock1 and Rock2 transcript levels in P19 cells. P19 cells have been transfected with control, Rock1-specific, or Rock2-specific shRNA plasmid. Expressions of Rock1 and Rock2 are normalized with a house-keeping gene Gapdh, and expression levels are presented relative to the control. White and gray bars represent independent set of experiments performed on different occasions. B) Quantitative RT-PCR analysis of Rock1, Rock2, and Rhoa transcript levels at E3.5 and E4.5 in mouse embryos that have been injected with control or a mixture of Rock1-specific and Rock2-specific shRNA plasmids at E0.5. Expressions are normalized with respect to Gapdh and presented relative to the control at each developmental stage. Graphs show average values and standard deviation of triplicates using a set of cDNAs that have been synthesized from pooled RNA samples of 15–25 embryos. Note that the levels of Rhoa, encoding a component of RHO/ROCK signaling, are not affected by Rock1 and Rock2 shRNA plasmids. C) Representative examples of morphology of control and Y-27632-treated mESC colony. D) Representative examples of morphology of mESC colony that have been transfected with the control shRNA plasmid or a mixture of Rock1 and Rock2 shRNA plasmids. Bars = 50 μm (C, D).