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Rho kinases play an obligatory role in vertebrate embryonic organogenesis

Lei Wei^1,2,4,*, Wilmer Roberts^1,*, Lu Wang^2,4, Miho Yamada¹, Shuxing Zhang¹, Zhiyong Zhao⁵, Scott A. Rivkees⁵, Robert J. Schwartz^1,2,3,4, and Kyoko Imanaka-Yoshida⁶

¹ Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA
² Cardiovascular Sciences Section, Baylor College of Medicine, Houston, TX 77030, USA
³ Center for Cardiovascular Development, Baylor College of Medicine, Houston, TX 77030, USA
⁴ Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA
⁵ Yale Child Health Research Center, Yale University School of Medicine, New Haven, CN, USA
⁶ Department of Pathology, Mie University, School of Medicine, Tsu, Mie, Japan
^* These authors contributed equally to this work

View larger version (92K): [in a new window]   Fig. 1. Transcripts of Rho kinases are enriched in regions of early organogenesis. (A-H) Whole-mount in situ hybridization analysis of p160ROCK (A-C) and ROK (E-G) expression in chick embryos at Hamburger and Hamilton (HH) stages 6 to 8 (indicated in the bottom left-hand corner of each photograph). Ventral views of stage 6 (A,E), stage 7 (B,F) and stage 8 (C,G) chick embryos. The position of the first pair of somites is indicated by the arrows. (D,H) Transverse sections of stage 8 embryo at the levels indicated in C and G. (I-N) Whole-mount in situ hybridization analysis of p160ROCK (I-L) and ROK (M,N) expression in mouse embryos at E7.5 to E9.0. (I,M) Ventral views of E7.5 embryos. (J) Transverse section of an E8.5 embryo. (K) Sagittal section of an E9.0 embryo. (L) Enlargement of the box areas in K. (N) Lateral view of an E9.0 embryo. The following embryonic tissues are marked: cc, cardiac crescent; ec, endocardium; en, endoderm; et, extra-embryonic tissue; h, head; hf, head fold; hn, Hensen’s node; ht, heart tube; lp, lateral plate mesoderm; mc, myocardium; nf, neural fold; ng, neural groove; np, neural plate; ps, primitive streak.

View larger version (54K): [in a new window]   Fig. 2. Y27632, a specific Rho kinase inhibitor, caused severe cardiac and neural developmental defects. (A,B) Ventral views of chick embryos cultured with or without Y27632 (indicated in the bottom left-hand corner of each photograph) from stages 4-6. Whole-mount in situ hybridization was performed with cardiac -actin. (C,D) Transverse sections at levels indicated in A,B. (E-H) Mouse embryos cultured with (F,H) or without (E,G) Y27632 from E7.5 (E,F) or E8.0 (G,H). Embryos are shown in lateral (E,G) or dorsal view (F,H). Whole-mount in situ hybridization was performed with Nkx2.5. In the drug-treated mouse embryos from stage E8.0 (H), the head formation and the turning process were disrupted so that the head was under the heart tube. (I,J) Immunostaining for -actinin of transverse sections of mouse embryos cultured with (J) or without (I) Y27632. No lumen was formed in the heart of drug-treated embryos. The following embryonic tissues are marked as a, atrial; ec, endocardium; h, head; ht, heart tube; mc, myocardium; nc, notochord; np, neural plate; nt, neural tube; ph, pharynx; v, ventricle.

View larger version (80K): [in a new window]   Fig. 3. Y27632 prevented the regression of Hensen’s node and induced a laterality defect. Chick embryos of stage 5 were treated with or without Y27632 (indicated in the bottom left-hand corner of each photograph) and continued to grow to stage 12 at evaluation for Shh expression (A) or to stage 9 for Nodal expression (B) by whole-mount in situ hybridization. Shh was expressed in the notochord anterior to Hensen’s node (indicated by arrows), thus showing the position of Hensen’s node. Nodal expression (indicated by arrow) was detected on the left-hand side of the lateral plate in the control embryo, but on both sides of the drug-treated embryo. All embryos are shown in ventral view.

View larger version (109K): [in a new window]   Fig. 4. Y27632 did not inhibit expression of GATA5, an endoderm differentiation marker. (A-D) Whole-mount in situ hybridization of GATA5 expression in chick embryos that were treated with or without Y27632 (indicated as Y or C, respectively, in the bottom left-hand corner of each photograph) from stages 5-6 and allowed to develop to stages 9-12 as indicated. (E,F) Transverse sections at levels indicated in C,D. GATA5 expression was abundant in cardiac mesoderm, the underlying endoderm and gut endoderm. The following embryonic tissues are marked: ec, endocardium; en, endoderm; mc, myocardium; ff, ventral floor of the foregut.

View larger version (82K): [in a new window]   Fig. 5. Y27632 induced precocious expression of cardiac -actin, expanded the paired heart fields at early stages and partially retained cardiac anteroposterior polarity. (A-D) Whole-mount in situ hybridization analysis of cardiac -actin (A), GATA4 (B), SRF (C) and Nkx2.5 (D) expression in chick embryos which were treated with (Y) or without Y27632 (C in corner) from stages 5-6 and continued to grow to stages 8-12 as indicated. The position of the first pair of somites in each embryo is indicated by an arrow. Abnormal somite formation in drug-treated embryos is indicated by an arrowhead in C. (E) The timecourse of expression of cardiac -actin, GATA4, SRF or Nkx2.5 was analyzed by semi-quantitative RT-PCR with the pool of three embryos for each sample. For each gene, two or three cycle numbers were tested to be certain that PCR product accumulates within a linear range. Their expression was normalized to GAPDH and is shown for Y27632-treated embryos relative to control embryos. Data represent averages of three to five PCR reactions + s.e.m. (vertical bars). (F) Whole-mount in situ hybridization analysis of Irx4 and AMHC1 compared with that of cardiac -actin. A bar separates the ventricular and atrial regions in the control embryos. A bar separates the anterior and posterior regions of bilateral heart tubes in the embryos treated with Y27632. All embryos are shown in ventral view.

View larger version (18K): [in a new window]   Fig. 6. Rho kinase antisense oligonucleotides reduced Rho kinase transcripts, reproducing Y27632-induced embryonic defects. (A) Semi-quantitative RT-PCR analysis of chick embryos after treatment with p160ROCK antisense oligonucleotide (p160-AS) or ROK antisense oligonucleotide (ROK-AS) or randomized control oligonucleotides (control oligo 1 and control oligo 2) from stage 5 for 4 hours. Three identically treated embryos were pooled. For each gene, two or three cycle numbers were tested to be certain that PCR product accumulates within a linear range. Their expression was normalized to GAPDH as described in Materials and Methods and is shown for oligonucleotide-treated embryos relative to control embryos treated with DMSO. (B) Incidence of heart anomalies induced by antisense oligonucleotide treatment. In addition to cardia bifida, other heart anomalies include reversed and/or abnormal looping and poor contractility. (C) Ventral view of chick embryos treated with control or antisense oligonucleotides and the deformities produced. Whole-mount in situ hybridization was performed with cardiac -actin.