Mutations in Bcl9 and Pygo genes cause congenital heart defects by tissue-specific perturbation of Wnt/ß-catenin signaling.

Bcl9 and Pygopus (Pygo) are obligate Wnt/ß-catenin cofactors in Drosophila, yet their contribution to Wnt signaling during vertebrate development remains unresolved. Combining zebrafish and mouse genetics, we document a conserved, ß-catenin-associated function for BCL9 and Pygo proteins during vertebrate heart development. Disrupting the ß-catenin-BCL9-Pygo complex results in a broadly maintained canonical Wnt response yet perturbs heart development and proper expression of key cardiac regulators. Our work highlights BCL9 and Pygo as selective ß-catenin cofactors in a subset of canonical Wnt responses during vertebrate development. Moreover, our results implicate alterations in BCL9 and BCL9L in human congenital heart defects.

Effects of in utero exposure to Δ-9-tetrahydrocannabinol on cardiac extracellular matrix expression and vascular transcriptome in rhesus macaques.

Delta-9-tetrahydrocannabinol (THC), the psychoactive component of cannabis, remains a schedule I substance, thus safety data regarding the effects on the cardiovascular and prenatal health are limited. Importantly, there is evidence showing prenatal cannabis exposure can negatively impact fetal organ development, including the cardiovascular system. THC can cross the placenta and bind to cannabinoid receptors expressed in the developing fetus, including on endothelial cells. To understand the impact of prenatal THC exposure on the fetal cardiovascular system, we used our rhesus macaque model of prenatal daily edible THC consumption. Before conception, animals were acclimated to THC (2.5 mg/7 kg/day, equivalent to a heavy medical cannabis dose) and maintained on this dose daily throughout pregnancy. Fetal tissue samples were collected at gestational day 155 (full term is 168 days). Our model showed that in utero THC exposure was associated with a decreased heart weight-to-body weight ratio in offspring, warranting further mechanistic investigation. Histological examination of the fetal cardiac and vascular tissues did not reveal any significant effect of THC exposure on the maturity of collagen within the fetal heart or the aorta. Total collagen III expression and elastin production and organization were unchanged. However, bulk RNA-sequencing of vascular cells in the umbilical vein, umbilical artery, and fetal aorta demonstrated that THC alters the fetal vascular transcriptome and is associated with upregulated expression of genes involved in carbohydrate metabolism and inflammation. The long-term consequences of these findings are unknown but suggest that prenatal THC exposure may affect cardiovascular development in offspring.NEW & NOTEWORTHY Prenatal cannabis use is increasing and despite the public health relevance, there is limited safety data regarding its impact on offspring cardiovascular health outcomes. We used a translational, nonhuman primate model of daily edible Δ-9-tetrahydrocannabinol (THC) consumption during pregnancy to assess its effects on the fetal cardiovascular system. THC-exposed fetal vascular tissues displayed upregulation of genes involved in cellular metabolism and inflammation, suggesting that prenatal THC exposure may impact fetal vascular tissues.

Sertraline-Induced 5-HT Dysregulation in Mouse Cardiomyocytes and the Impact on Calcium Handling.

Selective serotonin reuptake inhibitors (SSRIs) are prescribed in 15% of pregnancies in the United States for depression. Maternal use of SSRIs has been linked to an increased risk of congenital heart defects, but the exact mechanism of pathogenesis is unknown. SSRIs, including sertraline, are permeable to the placenta and can produce direct fetal exposure. Previously, we have shown decreased cardiomyocyte proliferation, left ventricle size, and cardiac expression of the serotonin receptor 5-HT2B in offspring of mice exposed to the SSRI sertraline relative to offspring of saline-exposed mice. Using a mouse model of in utero plus neonatal sertraline exposure, we observed lengthened peak-to-peak time of calcium oscillation (saline 784 ±76 ms; sertraline 1121 ± 130 ms, p<0.001) and decreased expression of critical genes in calcium regulation. We also observed significant up-regulation of specific miRNAs that modulate serotonin signaling in neonatal cardiac tissues (Slc6a4: miR-223-5p, miR-92a-2-5p, miR-182-5p; Htr2a: miR-34b-5p, miR-182-5p; Htr2b: miR-223-5p, miR-92a-2-5p, miR-337-5p) (p<0.05) with corresponding levels of the target mRNAs down-regulated (Slc6a4 0.73 ± 0.05; Htr2a 0.67 ± 0.04; Htr2b 0.72 ± 0.03; all p< 0.01), resulting in decreased production of the cognate proteins. Adult mice at 10 weeks showed altered cardiac parameters including decreased heart rates in males (saline 683 ± 8 vs sertraline 666 ± 6 beats per minute, p< 0.05) and ejection fraction in females (saline 83.9 ± 0.6% vs sertraline 80.6 ± 1.1%, p<0.05). These findings raise the question if sertraline exposure during development may increase the potential risk for cardiac disease when subjected to stress.

Molecular regulators of defective placental and cardiovascular development in fetal growth restriction.

Placental insufficiency is one of the major causes of fetal growth restriction (FGR), a significant pregnancy disorder in which the fetus fails to achieve its full growth potential in utero. As well as the acute consequences of being born too small, affected offspring are at increased risk of cardiovascular disease, diabetes and other chronic diseases in later life. The placenta and heart develop concurrently, therefore placental maldevelopment and function in FGR may have profound effect on the growth and differentiation of many organ systems, including the heart. Hence, understanding the key molecular players that are synergistically linked in the development of the placenta and heart is critical. This review highlights the key growth factors, angiogenic molecules and transcription factors that are common causes of defective placental and cardiovascular development.

Nile Tilapia (Oreochromis niloticus) Patched1 Mutations Disrupt Cardiovascular Development and Vascular Integrity through Smoothened Signaling.

Hedgehog (Hh) signaling is crucial in cardiovascular development and maintenance. However, the biological role of Patched1 (Ptch1), an inhibitory receptor of the Hh signaling pathway, remains elusive. In this study, a Ptch1 ortholog was characterized in Nile tilapia (Oreochromis niloticus), and its function was investigated through CRISPR/Cas9 gene knockout. When one-cell embryos were injected with CRISPR/Cas9 targeting ptch1, the mutation efficiency exceeded 70%. During 0-3 days post fertilization (dpf), no significant differences were observed between the ptch1 mutant group and the control group; at 4 dpf (0 day after hatching), about 10% of the larvae showed an angiogenesis defect and absence of blood flow; from 5 dpf, most larvae exhibited an elongated heart, large pericardial cavity, and blood leakage and coagulation, ultimately dying during the 6-8 dpf period due to the lack of blood circulation. Consistently, multiple differentially expressed genes related to angiogenesis, blood coagulation, and heart development were enriched in the ptch1 mutants. Furthermore, Smoothened (Smo) antagonist (cyclopamine) treatment of the ptch1 mutants greatly rescued the cardiovascular disorders. Collectively, our study suggests that Ptch1 is required for cardiovascular development and vascular integrity via Smo signaling, and excessive Hh signaling is detrimental to cardiovascular development.

Tek (Tie2) is not required for cardiovascular development in zebrafish.

Angiopoietin/TIE signalling plays a major role in blood and lymphatic vessel development. In mouse, Tek (previously known as Tie2) mutants die prenatally due to a severely underdeveloped cardiovascular system. In contrast, in zebrafish, previous studies have reported that although embryos injected with tek morpholinos (MOs) exhibit severe vascular defects, tek mutants display no obvious vascular malformations. To further investigate the function of zebrafish Tek, we generated a panel of loss-of-function tek mutants, including RNA-less alleles, an allele lacking the MO-binding site, an in-frame deletion allele and a premature termination codon-containing allele. Our data show that all these mutants survive to adulthood with no obvious cardiovascular defects. MO injections into tek mutants lacking the MO-binding site or the entire tek locus cause similar vascular defects to those observed in MO-injected +/+ siblings, indicating off-target effects of the MOs. Surprisingly, comprehensive phylogenetic profiling and synteny analyses reveal that Tek was lost in the largest teleost clade, suggesting a lineage-specific shift in the function of TEK during vertebrate evolution. Altogether, these data show that Tek is dispensable for zebrafish development, and probably dispensable in most teleost species.

CHD7 regulates cardiovascular development through ATP-dependent and -independent activities.

CHD7 encodes an ATP-dependent chromatin remodeling factor. Mutation of this gene causes multiple developmental disorders, including CHARGE (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth/development, Genital abnormalities, and Ear anomalies) syndrome, in which conotruncal anomalies are the most prevalent form of heart defects. How CHD7 regulates conotruncal development remains unclear. In this study, we establish that deletion of Chd7 in neural crest cells (NCCs) causes severe conotruncal defects and perinatal lethality, thus providing mouse genetic evidence demonstrating that CHD7 cell-autonomously regulates cardiac NCC development, thereby clarifying a long-standing controversy in the literature. Using transcriptomic analyses, we show that CHD7 fine-tunes the expression of a gene network that is critical for cardiac NCC development. To gain further molecular insights into gene regulation by CHD7, we performed a protein-protein interaction screen by incubating recombinant CHD7 on a protein array. We find that CHD7 directly interacts with several developmental disorder-mutated proteins including WDR5, a core component of H3K4 methyltransferase complexes. This direct interaction suggested that CHD7 may recruit histone-modifying enzymes to target loci independently of its remodeling functions. We therefore generated a mouse model that harbors an ATPase-deficient allele and demonstrates that mutant CHD7 retains the ability to recruit H3K4 methyltransferase activity to its targets. Thus, our data uncover that CHD7 regulates cardiovascular development through ATP-dependent and -independent activities, shedding light on the etiology of CHD7-related congenital disorders. Importantly, our data also imply that patients carrying a premature stop codon versus missense mutations will likely display different molecular alterations; these patients might therefore require personalized therapeutic interventions.

Adaptive Growth of the Ductus Arteriosus and Aortic Isthmus in Various Ductus-Dependent Complex Congenital Heart Diseases.

BACKGROUND: The ductus arteriosus (DA) is critical in maintaining postnatal circulation in neonates with obstructed systemic circulation (OSC) and pulmonary circulation (OPC). We hypothesized that the size of the DA and aortic isthmus (AoI) undergoes adaptive growth in utero to counteract the hemodynamic challenges in these congenital heart diseases (CHD). METHODS: Postnatal echocardiograms of neonates diagnosed prenatally with ductal-dependent CHD who were started on prostaglandins within 24 h of birth were reviewed. We assessed the cross-sectional area of the aortic valve opening, pulmonary valve opening, AoI, and DA by calculating (diameter)2/body surface area. Neonates were classified into OSC or OPC then subgrouped depending upon the patency of semilunar valves: OSC with and without aortic atresia (OSC-AA and OSC-nAA, respectively) and OPC with and without pulmonary atresia (OPC-PA and OPC-nPA, respectively). RESULTS: Ninety-four cases were studied. The DA in OSC was significantly larger than OPC, and the DA in OSC-AA was significantly larger than OSC-nAA. The size of the AoI was significantly larger in OPC than OSC and larger in OSC-AA than OSC-nAA. Within the OSC-nAA group, there was no significant difference in the size of the DA, AoI, or pulmonary valve opening between those with retrograde flow (RF) at the AoI and without (nRF) except the aortic valve opening was significantly larger in nRF. All groups had comparable cross-sectional areas of systemic output. CONCLUSIONS: Our findings suggest that DA and AoI show compensatory growth to maintain critical blood flow to vital organs against primary anatomical abnormalities in ductus-dependent CHD. (249 words).

The advantages of naming rather than numbering the arteries of the pharyngeal arches.

Controversies continue as to how many pharyngeal arches, with their contained arteries, are to be found in the developing human. Resolving these controversies is of significance to paediatric cardiologists since many investigating abnormalities of the extrapericardial arterial pathways interpret their findings on the basis of persistence of a fifth set of such arteries within an overall complement of six sets. The evidence supporting such an interpretation is open to question. In this review, we present the history of the existence of six such arteries, emphasising that the initial accounts of human development had provided evidence for the existence of only five sets. We summarise the current evidence that substantiates these initial findings. We then show that the lesions interpreted on the basis of persistence of the non-existing fifth arch arteries are well described on the basis of the persistence of collateral channels, known to exist during normal development, or alternatively due to remodelling of the aortic sac.

Local fluid shear stress operates a molecular switch to drive fetal semilunar valve extension.

BACKGROUND: While much is known about the genetic regulation of early valvular morphogenesis, mechanisms governing fetal valvular growth and remodeling remain unclear. Hemodynamic forces strongly influence morphogenesis, but it is unknown whether or how they interact with valvulogenic signaling programs. Side-specific activity of valvulogenic programs motivates the hypothesis that shear stress pattern-specific endocardial signaling controls the elongation of leaflets. RESULTS: We determined that extension of the semilunar valve occurs via fibrosa sided endocardial proliferation. Low OSS was necessary and sufficient to induce canonical Wnt/ß-catenin activation in fetal valve endothelium, which in turn drives BMP receptor/ligand expression, and pSmad1/5 activity essential for endocardial proliferation. In contrast, ventricularis endocardial cells expressed active Notch1 but minimal pSmad1/5. Endocardial monolayers exposed to LSS attenuate Wnt signaling in a Notch1 dependent manner. CONCLUSIONS: Low OSS is transduced by endocardial cells into canonical Wnt signaling programs that regulate BMP signaling and endocardial proliferation. In contrast, high LSS induces Notch signaling in endocardial cells, inhibiting Wnt signaling and thereby restricting growth on the ventricular surface. Our results identify a novel mechanically regulated molecular switch, whereby fluid shear stress drives the growth of valve endothelium, orchestrating the extension of the valve in the direction of blood flow.

Diverse contribution of amniogenic somatopleural cells to cardiovascular development: With special reference to thyroid vasculature.

BACKGROUND: The somatopleure serves as the primordium of the amnion, an extraembryonic membrane surrounding the embryo. Recently, we have reported that amniogenic somatopleural cells (ASCs) not only form the amnion but also migrate into the embryo and differentiate into cardiomyocytes and vascular endothelial cells. However, detailed differentiation processes and final distributions of these intra-embryonic ASCs (hereafter referred to as iASCs) remain largely unknown. RESULTS: By quail-chick chimera analysis, we here show that iASCs differentiate into various cell types including cardiomyocytes, smooth muscle cells, cardiac interstitial cells, and vascular endothelial cells. In the pharyngeal region, they distribute selectively into the thyroid gland and differentiate into vascular endothelial cells to form intra-thyroid vasculature. Explant culture experiments indicated sequential requirement of fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) signaling for endothelial differentiation of iASCs. Single-cell transcriptome analysis further revealed heterogeneity and the presence of hemangioblast-like cell population within ASCs, with a switch from FGF to VEGF receptor gene expression. CONCLUSION: The present study demonstrates novel roles of ASCss especially in heart and thyroid development. It will provide a novel clue for understanding the cardiovascular development of amniotes from embryological and evolutionary perspectives.

Emerging role of Piezo ion channels in cardiovascular development.

Mechanical cues are crucial for vascular development and the proper differentiation of various cell types. Piezo1 and Piezo2 are mechanically activated cationic channels expressed in various cell types, especially in vascular smooth muscle and endothelial cells. It is present as a transmembrane homotrimeric complex, regulating calcium influx. Local blood flow associated shear stress, in addition to blood pressure associated cell membrane stretching are key Piezo channel activators. There is rising proof, showcasing Piezo channels significance in myocytes, cardiac fibroblast, vascular tone maintenance, atherosclerosis, hypertension, NO generation, and baroreceptor reflex. Here, we review the role of Piezo channels in cardiovascular development and its associated clinical disorders. Also, emphasizing on Piezo channel modulators which might lead to novel therapies for cardiovascular diseases.

PAX Genes in Cardiovascular Development.

The mammalian heart is a four-chambered organ with systemic and pulmonary circulations to deliver oxygenated blood to the body, and a tightly regulated genetic network exists to shape normal development of the heart and its associated major arteries. A key process during cardiovascular morphogenesis is the septation of the outflow tract which initially forms as a single vessel before separating into the aorta and pulmonary trunk. The outflow tract connects to the aortic arch arteries which are derived from the pharyngeal arch arteries. Congenital heart defects are a major cause of death and morbidity and are frequently associated with a failure to deliver oxygenated blood to the body. The Pax transcription factor family is characterised through their highly conserved paired box and DNA binding domains and are crucial in organogenesis, regulating the development of a wide range of cells, organs and tissues including the cardiovascular system. Studies altering the expression of these genes in murine models, notably Pax3 and Pax9, have found a range of cardiovascular patterning abnormalities such as interruption of the aortic arch and common arterial trunk. This suggests that these Pax genes play a crucial role in the regulatory networks governing cardiovascular development.

Transcriptional regulation of transcriptional Mediator complexes in cardiovascular development and disease.

During the development of the mammalian cardiovascular system, the formation of a mature and fully functional cardiovascular system needs the fine coordination of the morphogenesis of various molecules, cells, tissues, and organs. Abnormalities in these processes usually lead to serious congenital heart defects. The determination and maintenance of cell fate in multicellular organisms depend to a large extent on the precise timing and control of RNA polymerase II (Pol II) transcription, and the transcription Mediator complex plays an irreplaceable role in the Pol II transcription process. Mediator is an evolutionarily conserved multi-subunit protein complex, including four parts: head, middle, tail, and kinase. It is a functional bridge between transcription factors and basic transcription machines. In recent years, due to the key role of Mediator in the transcriptional regulation of gene expression, many of human heart diseases have been confirmed to be related to specific Mediator gene mutations, such as heart valve defects, translocation of the great arteries, DiGeorge syndrome and some cardiovascular diseases related to energy homeostasis. In this review, we summarize the role of Mediator in cardiovascular development and disease, focusing on the role of Mediator in the development of cardiovascular disease, and provides a broad idea for the research on Mediator-related cardiovascular system development and diseases.

Methyltransferase SET domain family and its relationship with cardiovascular development and diseases.

Abnormal epigenetic modification is closely related to the occurrence and development of cardiovascular diseases. The SET domain (SETD) family is an important epigenetic modifying enzyme containing SETD. They mainly affect gene expression by methylating H3K4, H3K9, H3K36 and H4K20. Additionally, the SETD family catalyzes the methylation of non-histone proteins, thereby affects the signal transduction of signal transduction and activator of transcription (STAT) 1, Wnt/ß-catenin, hypoxia-inducible factor (HIF)-1α and Hippo/YAP pathways. The SETD family has the following regulatory effects on cardiovascular development and diseases: regulating coronary artery formation and cardiac development; protecting cardiac tissue from ischemia reperfusion injury; regulating inflammation, oxidative stress and apoptosis in cardiovascular complications of diabetes; participating in the formation of pulmonary hypertension; regulating thrombosis, cardiac hypertrophy and arrhythmia. This article summarizes the basic structures, expression regulation mechanisms and the role of existing SETD family members in cardiovascular development and diseases, in order to provide a basis for understanding the molecular mechanism of cardiovascular disease and exploring the therapeutic targets.

Novel role of sex-determining region Y-box 7 (SOX7) in tumor biology and cardiovascular developmental biology.

The sex-determining region Y-box 7 (Sox7) is an important member of the SOX F family, which is characterized by a high-mobility-group DNA-binding domain. Previous studies have demonstrated the role of SOX7 in cardiovascular development. SOX7 expression could be detected in normal adult tissues. Furthermore, the expression levels of SOX7 were different in different tumors. Most studies showed the downregulation of SOX7 in tumors, while some studies reported its upregulation in tumors. In this review, we first summarized the upstream regulators (including transcription factors, microRNAs (miRNAs), long noncoding RNAs (lncRNAs) and some exogenous regulators) and downstream molecules (including factors in the Wnt/ß-catenin signaling pathway and some other signaling pathways) of SOX7. Then, the roles of SOX7 in multiple tumors were presented. Finally, the significance of divergent SOX7 expression during cardiovascular development was briefly discussed. The information compiled in this study characterized SOX7 during tumorigenesis and cardiovascular development, which should facilitate the design of future research and promote SOX7 as a therapeutic target.

FRS2α-dependent cell fate transition during endocardial cushion morphogenesis.

Atrioventricular valve development requires endothelial-to-mesenchymal transition (EndMT) that induces cushion endocardial cells to give rise to mesenchymal cells crucial to valve formation. In the adult endothelium, deletion of the docking protein FRS2α induces EndMT by activating TGFß signaling in a miRNA let-7-dependent manner. To study the role of endothelial FRS2α during embryonic development, we generated mice with an inducible endothelial-specific deletion of Frs2α (FRS2αiECKO). Analysis of the FRS2αiECKO embryos uncovered a combination of impaired EndMT in AV cushions and defective maturation of AV valves leading to development of thickened, abnormal valves when Frs2α was deleted early (E7.5) in development. At the same time, no AV valve developmental abnormalities were observed after late (E10.5) deletion. These observations identify FRS2α as a pivotal controller of cell fate transition during both EndMT and post-EndMT valvulogenesis.

Msx1 haploinsufficiency modifies the Pax9-deficient cardiovascular phenotype.

BACKGROUND: Successful embryogenesis relies on the coordinated interaction between genes and tissues. The transcription factors Pax9 and Msx1 genetically interact during mouse craniofacial morphogenesis, and mice deficient for either gene display abnormal tooth and palate development. Pax9 is expressed specifically in the pharyngeal endoderm at mid-embryogenesis, and mice deficient for Pax9 on a C57Bl/6 genetic background also have cardiovascular defects affecting the outflow tract and aortic arch arteries giving double-outlet right ventricle, absent common carotid arteries and interruption of the aortic arch. RESULTS: In this study we have investigated both the effect of a different genetic background and Msx1 haploinsufficiency on the presentation of the Pax9-deficient cardiovascular phenotype. Compared to mice on a C57Bl/6 background, congenic CD1-Pax9-/- mice displayed a significantly reduced incidence of outflow tract defects but aortic arch defects were unchanged. Pax9-/- mice with Msx1 haploinsufficiency, however, have a reduced incidence of interrupted aortic arch, but more cases with cervical origins of the right subclavian artery and aortic arch, than seen in Pax9-/- mice. This alteration in arch artery defects was accompanied by a rescue in third pharyngeal arch neural crest cell migration and smooth muscle cell coverage of the third pharyngeal arch arteries. Although this change in phenotype could theoretically be compatible with post-natal survival, using tissue-specific inactivation of Pax9 to maintain correct palate development whilst inducing the cardiovascular defects was unable to prevent postnatal death in the mutant mice. Hyoid bone and thyroid cartilage formation were abnormal in Pax9-/- mice. CONCLUSIONS: Msx1 haploinsufficiency mitigates the arch artery defects in Pax9-/- mice, potentially by maintaining the survival of the 3rd arch artery through unimpaired migration of neural crest cells to the third pharyngeal arches. With the neural crest cell derived hyoid bone and thyroid cartilage also being defective in Pax9-/- mice, we speculate that the pharyngeal endoderm is a key signalling centre that impacts on neural crest cell behaviour highlighting the ability of cells in different tissues to act synergistically or antagonistically during embryo development.

Trans-ovo permethrin exposure affects growth, brain morphology and cardiac development in quail.

Permethrin is a commonly used, highly effective pesticide in poultry agriculture, and has recently been trialed in conservation efforts to protect Galápagos finch hatchlings from an invasive ectoparasite. Although permethrin is considered safe for adults, pesticides can have health consequences when animals are exposed during early life stages. The few studies that have examined permethrin's effects in embryonic chicks and rats have shown hydrocephaly, anencephaly, reduced cellular energy conversion, and disruption of developing heart muscle. To test whether trans-ovo exposure of permethrin affects early development in birds, we exposed Japanese quail (Coturnix japonica) eggs to cotton treated with 1% permethrin that was incorporated into nests in two amounts (0.2, 0.8 g), each with a paired untreated cotton control group. When measured on incubation Day 15, we found permethrin-treated developing birds were smaller and showed signs of microcephaly, although mortality rates were the same. Despite no difference in heart mass, ventricular tissue was less compact, cardiac arteries were reduced and heart rates were slower in permethrin-treated birds. Differences in heart development were also observed at 5 days of incubation, indicating that abnormalities are present from early in cardiac development. Future studies are needed to examine permethrin's effects on developmental pathways and to determine if these effects persist after hatching to affect offspring health. This study provides evidence that permethrin can cross the eggshell to cause non-lethal but adverse effects on embryonic development, and studies should look beyond hatching when monitoring the efficacy of permethrin on wild bird populations.

Generation and evaluation of a transgenic zebrafish for tissue-specific expression of a dominant-negative Rho-associated protein kinase-2.

The Ras homologous (Rho) proteins are a family of small GTPases, which regulate the cytoskeleton and are related to stress fibers and focal adhesion. The Rho-associated protein kinases (ROCK) constitute part of the Rho effectors that regulate cell shape and movement via phosphorylation of the myosin light chain and actin depolymerizing factor/cofilin. ROCK members are widely expressed and play roles in various cell types during vertebrate development and morphogenesis; therefore, ROCK-knockout animals exhibit multiple defects mostly initiated at the embryonic stage. Analyzing the distinct roles of ROCK in cell shape and movement during the embryonic stages using live mammalian models is difficult. Here, we inhibited the Rho/ROCK pathway in zebrafish, which is a small fish that can be conveniently used as a developmental animal model in place of mammals. To inhibit the Rho/ROCK pathway, we designed a dominant-negative ROCK-2 (dnROCK-2) that lacked the kinase domain and was under the control of an upstream activation sequence (UAS). To evaluate the effects of expression of dnROCK-2, transgenic zebrafish lines were generated by mating strains expressing the construct with counterpart strains expressing the Gal4 activator in target tissues. In this study, we crossed the dnROCK-2-expressing line with two such Gal4-expressing lines; (1) SAGFF(LF)73A for expression in the whole body, and (2) Tg(fli1a: Gal4FF)ubs4 for endothelial cell-specific expression. The phenotypes of the fish obtained were observed by fluorescent stereomicroscopy or confocal microscopy. Overexpression of dnROCK-2 in the whole body resulted in an inhibition of development, notably in cephalic formation, at 1-day post-fertilization (dpf). Confocal microscopy revealed that Hensen's zone became unclear in the trunk muscle fibers expressing dnROCK-2. Endothelial cell-specific expression of dnROCK-2 caused abnormalities in cardiovascular formation at 2-dpf. These results suggest that dnROCK-2 can act as a dominant negative construct of the Rho/ROCK pathway to affect regulation of the cytoskeleton. This construct could be a convenient tool to investigate the function of ROCK members in other vertebrate cell types.