Targeted disruption of the mouse rho-associated kinase 2 gene results in intrauterine growth retardation and fetal death

Abstract

Rho-associated kinase (ROCK), including the ROCK-I and ROCK-II isoforms, is a protein kinase involved in signaling from Rho to actin cytoskeleton. However, in vivo functions of each ROCK isoform remain largely unknown. We generated mice deficient in ROCK-II by gene targeting. ROCK-II(-/-) embryos were found at the expected Mendelian frequency until 13.5 days postcoitum, but approximately 90% died thereafter in utero. ROCK-II(-/-) mice of both genders that survived were born runts, subsequently developed without gross abnormality, and were fertile. Whole-mount staining for a knocked-in lacZ reporter gene revealed that ROCK-II was highly expressed in the labyrinth layer of the placenta. Disruption of architecture and extensive thrombus formation were found in the labyrinth layer of ROCK-II(-/-) mice. While no obvious alteration in actin filament structures was found in the labyrinth layer of ROCK-II(-/-) placenta and stress fibers were formed in cultured ROCK-II(-/-) trophoblasts, elevated expression of plasminogen activator inhibitor 1 was found in ROCK-II(-/-) placenta. These results suggest that ROCK-II is essential in inhibiting blood coagulation and maintaining blood flow in the endothelium-free labyrinth layer and that loss of ROCK-II leads to thrombus formation, placental dysfunction, intrauterine growth retardation, and fetal death.

FIG. 1.

Generation of ROCK-II-deficient mice. (A) Targeting strategy. Schematic representations of the domain structure of ROCK-II, the wild-type ROCK-II allele, targeting vector, and the targeted allele are shown. The positions of β-galactosidase (β-gal), neomycin resistance (Neo), and thymidine kinase (TK) genes, restriction sites KpnI (K), HindIII (H), PstI (P), EcoRV (E), BamHI (B), ClaI (C), and NotI (N), and exons 2 (ex2) and 3 (ex3) are shown. The positions of primers for PCR analysis are indicated by the arrowheads. The external probe is a unique 3′ genomic probe that distinguishes the wild-type 9.7-kb PstI fragment from a 5-kb PstI fragment generated by the targeted alleles. (B) Southern blot analysis of genomic DNA obtained from visceral yolk sacs at 13.5 dpc. (C) Genotyping by PCR on genomic DNA obtained from visceral yolk sacs at 13.5 dpc.

FIG. 2.

Western blot analysis. Wild-type and mutant dpc 13.5 embryo lysates or wild-type and mutant whole adult brain lysates of ROCK-II^+/+, ROCK-II^+/−, and ROCK-II^−/− mice were probed with antibodies specific for ROCK-II (A), ROCK-I (B), and β-tubulin (C), respectively. The positions of molecular mass markers (in kilodaltons) are shown to the right of the blots.

FIG. 3.

Growth retardation of ROCK-II^−/− mice. Wild-type (ROCK-II^+/+) and ROCK-II^−/− dpc 18.5 embryos (A) and neonates (B) are shown. Note that the ROCK-II^−/− mouse was a runt. Hematomas in the hind limbs of the ROCK-II^−/− mouse are indicated by arrows.

FIG. 4.

ROCK-II expression in 13.5-dpc embryo and placenta as revealed by X-Gal staining of the lacZ reporter gene. (A) Expression in the embryo at 13.5 dpc. X-Gal staining is detected in umbilical blood vessels, heart, and dorsal root ganglion. (B and C) Expression in the placenta. Strong X-Gal staining is found in the labyrinth layer of ROCK-II^−/− placenta (B), which is rich in vasculature as shown in ROCK-II^+/+ placenta (C). (D) Uniform X-Gal staining in trophoblasts in the labyrinth layer. (E) Expression of the ROCK-II-lacZ reporter gene in a blood vessel in the chorionic plate. Parallel staining for actin (data not shown) indicates that the vessel stained with X-Gal (below) is the umbilical artery, while the vessel devoid of the X-Gal staining (above) is a vein. Bars, 100 μm.

FIG. 5.

Morphology and histology of wild-type (ROCK-II^+/+) and ROCK-II^−/− embryos and placentas. (A and B) Embryos isolated together with their placentas. Blood clots found in the ROCK-II^−/− placenta (arrows) and hemorrhage in the hind limb of the ROCK-II^−/− embryo (arrowhead) are indicated. (C to F) Hematoxylin and eosin staining of a 12.5-dpc wild-type placenta (C), a 12.5-dpc ROCK-II^−/− placenta (D), and a 13.5-dpc ROCK-II^−/− placenta (E). Panel F shows a higher-magnification view of the labyrinth layer shown in panel E. (G) Immunohistochemical staining of the labyrinth layer of a ROCK-II^−/− placenta with an anti-PECAM antibody. The arrow indicates a blood clot found in this section. The absence of nucleated blood cells and PECAM1-positive endothelial cells around the clot indicates that the clot had a maternal origin. Bars, 500 μm (C, D, and E), 300 μm (F), and 200 μm (G).

FIG. 6.

Architecture of actin fibers in the labyrinth layer and cultured trophoblasts from the placentas of wild-type (ROCK-II^+/+) and ROCK-II^−/− mice. (A) Cytoarchitecture of actin fibers in the labyrinth layer of wild-type and ROCK-II^−/− placentas. (B and C) Actin structures in clusters of cultured trophoblasts from wild-type (B) and ROCK-II^−/− (C) placentas. Cells were cultured in the absence or presence of 10 μM Y-27632. Note that the structure of actin bundles is not different in the wild-type and ROCK-II^−/− cells, and the structures of the actin bundles in the wild-type and ROCK-II^−/− cells treated with Y-27632 were similar. Bars, 20 μm (A) and 50 μm (B and C).

FIG. 7.

Elevated expression of PAI-1 in ROCK-II^−/− placenta. (A) RT-PCR analysis of PAI-1 expression in ROCK-II^+/− and ROCK-II^−/− 13.5-dpc placentas. (B) Northern blot analysis of PAI-1 mRNA expression in mROCK-II^+/− and mROCK-II^−/− 13.5-dpc placentas.

FIG. 8.

Hemorrhage in the hind limb of a ROCK-II^−/− mouse. (A) Hemorrhage in a 13.5 dpc ROCK-II^−/− embryo. Hemorrhages in both hind limbs and at the tip of the tail are indicated by the arrows. (B) Deformity of the hind limb of a ROCK-II^−/− mouse. (C to E) Hematoxylin-and-eosin-stained sections of the hind limb bud from a 12.5-dpc wild-type (ROCK-II^+/+) embryo (C) and 12.5-dpc (D) and 13.5-dpc (E) ROCK-II^−/− homozygous embryos. Small capillaries in the hind limbs of wild-type and knockout mice are indicated by the arrows. In panels C, D, and E, lower-magnification (left) and higher-magnification (right) views are shown to the right. Bars, 300 μm (left micrographs of panels C, D, and E) and 100 μm (right micrographs of panels C, D, and E).