Sample Preparation for Computed Tomography-based Three-dimensional Visualization of Murine Hind-limb Vessels.

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ function. Clarifying the mechanism of blood vessel formation is therefore extremely useful for elucidating developmental processes and pathological mechanisms. Murine hind-limb vessels are often used as a model for physiological and pathological angiogenesis. Evaluation is mainly performed via a two-dimensional method using tissue sections. However, methods for evaluating three-dimensional (3D) vascular morphology are particularly limited. This paper introduces a method for visualizing murine hind-limbs using computed tomography (CT). Radiation-opaque resin is injected through the descending aorta, and whole vessels are filled with dye. By adjusting the time of dye injection, arterial-specific filling is also possible, and samples can be obtained with any micro-X-ray CT device. This contrast method provides a basic technique for the 3D evaluation of murine blood vessels in the lower extremities. Furthermore, this method can be used to visualize all blood vessels below the diaphragm and evaluate blood vessels in the abdominal organs.

A role of Hey2 transcription factor for right ventricle development through regulation of Tbx2-Mycn pathway during cardiac morphogenesis.

A basic helix-loop-helix transcription factor Hey2 is expressed in the ventricular myocardium and endocardium of mouse embryos, and Hey2 null mice die perinatally showing ventricular septal defect, dysplastic tricuspid valve and hypoplastic right ventricle. In order to understand region-specific roles of Hey2 during cardiac morphogenesis, we generated Hey2 conditional knockout (cKO) mice using Mef2c-AHF-Cre, which was active in the anterior part of the second heart field and the right ventricle and outflow tract of the heart. Hey2 cKO neonates reproduced three anomalies commonly observed in Hey2 null mice. An earliest morphological defect was the lack of right ventricular extension along the apico-basal axis at midgestational stages. Underdevelopment of the right ventricle was present in all cKO neonates including those without apparent atresia of right-sided atrioventricular connection. RNA sequencing analysis of cKO embryos identified that the gene expression of a non-chamber T-box factor Tbx2 was ectopically induced in the chamber myocardium of the right ventricle. Consistently, mRNA expression of the Mycn transcription factor, which was a cell cycle regulator transcriptionally repressed by Tbx2, was down regulated, and the number of S-phase cells was significantly decreased in the right ventricle of cKO heart. These results suggest that Hey2 plays an important role in right ventricle development during cardiac morphogenesis, at least in part, through mitigating Tbx2-dependent inhibition of Mycn expression.

Expression of Hey2 transcription factor in the early embryonic ventricles is controlled through a distal enhancer by Tbx20 and Gata transcription factors.

Development of multi-chambered heart is associated with spatio-temporal regulation of gene expression. A basic helix-loop-helix transcription factor Hey2 is specifically expressed in the embryonic mouse ventricles and is indispensable for ventricular myocyte differentiation, compartment identity and morphogenesis of the heart. However, how Hey2 transcription is precisely regulated in the heart remains unclear. In this study, we identified a distal Hey2 enhancer conserved in the mouse and human to possess specific transcriptional activity in ventricular free wall myocytes at the looping stage of cardiac development. Deletion of the enhancer significantly decreased endogenous Hey2 expression in the ventricular myocardium but not in other tissues of mouse embryos. Mutation/deletion of the conserved binding sites for T-box and Gata proteins, but not NK-2 proteins, abolished the enhancer activity, and Tbx20 null mice completely lost the enhancer activity in the embryonic ventricles. Luciferase reporter analysis suggested that the ventricular enhancer activity was controlled by Tbx20 through its DNA binding and cooperative function with cardiac Gata proteins. These results delineate a regulatory mechanism of ventricular Hey2 expression and help fully understand molecular cascades in myocardial cell differentiation and cardiac morphogenesis during embryonic development.

Importance of endothelial Hey1 expression for thoracic great vessel development and its distal enhancer for Notch-dependent endothelial transcription.

Thoracic great vessels such as the aorta and subclavian arteries are formed through dynamic remodeling of embryonic pharyngeal arch arteries (PAAs). Previous work has shown that loss of a basic helix-loop-helix transcription factor Hey1 in mice causes abnormal fourth PAA development and lethal great vessel anomalies resembling congenital malformations in humans. However, how Hey1 mediates vascular formation remains unclear. In this study, we revealed that Hey1 in vascular endothelial cells, but not in smooth muscle cells, played essential roles for PAA development and great vessel morphogenesis in mouse embryos. Tek-Cre-mediated Hey1 deletion in endothelial cells affected endothelial tube formation and smooth muscle differentiation in embryonic fourth PAAs and resulted in interruption of the aortic arch and other great vessel malformations. Cell specificity and signal responsiveness of Hey1 expression were controlled through multiple cis-regulatory regions. We found two distal genomic regions that had enhancer activity in endothelial cells and in the pharyngeal epithelium and somites, respectively. The novel endothelial enhancer was conserved across species and was specific to large-caliber arteries. Its transcriptional activity was regulated by Notch signaling in vitro and in vivo, but not by ALK1 signaling and other transcription factors implicated in endothelial cell specificity. The distal endothelial enhancer was not essential for basal Hey1 expression in mouse embryos but may likely serve for Notch-dependent transcriptional control in endothelial cells together with the proximal regulatory region. These findings help in understanding the significance and regulation of endothelial Hey1 as a mediator of multiple signaling pathways in embryonic vascular formation.

Amniogenic somatopleure: a novel origin of multiple cell lineages contributing to the cardiovascular system.

The somatopleure is the amniotic primordium in amniote development, but its boundary to the embryonic body at early embryonic stages and the fate of cells constituting this structure are not well characterized. It also remains unclear how cells behave during the demarcation between intra- and extra-embryonic tissues. Here we identify cellular alignments, which indicate two streams towards the sites of dorsal amniotic closure and ventral thoracic wall formation. A subpopulation of mesodermal cells moving ventrally from the somatopleural region adjacent to the base of the head fold enter the body of the embryo and distribute to the thoracic wall, pharyngeal arches and heart. These cells are induced to differentiate into vascular endothelial cells and cardiomyocytes possibly by FGF and BMP signaling, respectively. These results indicate that the somatopleure acting as the amniotic primordium also serves as a source of embryonic cells, which may contribute to cardiovascular development.

2.56 Tbit/s/ch (640 Gbaud) polarization-multiplexed DQPSK non-coherent Nyquist pulse transmission over 525 km.

We demonstrate a single-channel 2.56 Tbit/s polarization-multiplexed DQPSK transmission using 640 Gbaud non-coherent optical Nyquist pulses. By virtue of a large tolerance to polarization-mode dispersion, the detrimental depolarization-induced crosstalk was reduced by 3.8 dB compared with RZ pulses. As a result, the transmission distance was substantially extended to 525 km compared with the distance of 300 km obtained with a Gaussian pulse.

Ultrafast Nyquist OTDM demultiplexing using optical Nyquist pulse sampling in an all-optical nonlinear switch.

We propose the ultrahigh-speed demultiplexing of Nyquist OTDM signals using an optical Nyquist pulse as both a signal and a sampling pulse in an all-optical nonlinear switch. The narrow spectral width of the Nyquist pulses means that the spectral overlap between data and control pulses is greatly reduced, and the control pulse itself can be made more tolerant to dispersion and nonlinear distortions inside the nonlinear switch. We apply the Nyquist control pulse to the 640 to 40 Gbaud demultiplexing of DPSK and DQPSK signals using a nonlinear optical loop mirror (NOLM), and demonstrate a large performance improvement compared with conventional Gaussian control pulses. We also show that the optimum spectral profile of the Nyquist control pulse depends on the walk-off property of the NOLM.

640 Gbaud (1.28 Tbit/s/ch) optical Nyquist pulse transmission over 525 km with substantial PMD tolerance.

We report a substantial increase in PMD tolerance in a single-channel ultrahigh-speed transmission using optical Nyquist pulses. We demonstrate both analytically and experimentally a large reduction in depolarization-induced crosstalk with optical Nyquist pulses, which is one of the major obstacles facing polarization-multiplexed ultrashort pulse transmission. By taking advantage of the high PMD tolerance, a low-penalty 1.28 Tbit/s/ch optical Nyquist TDM transmission at 640 Gbaud was achieved over 525 km.

Fatty acid composition in fetal, neonatal, and cultured cardiomyocytes in rats.

Reconstructed myocardial tissue still does not have enough pulsatile contraction. It is well known that fetal and mature neonatal cardiomyocytes utilize glucose and lipid, respectively, as their energy substrates, and that cultured ones mainly use glucose in spite of their age comparable to neonate ones, probably due to insufficient supply of lipids from culture medium. In the present study, we compared 7 saturated, 6 monounsaturated, and 11 polyunsaturated fatty acid contents in cultured cardiomyocytes (Cul group) with those in fetal (Fet group, approximately 17 d after impregnation) and neonatal (Neo group, 9 d old) rats, where the age of the Cul cells were set nearly equal to the Neo ones. Saturated fatty acid contents in the Cul group were generally lower than those in the Fet group and were close to those in the Neo group, except for C12:0 of which content was highest in the Neo group. Monounsaturated fatty acid contents in the Cul group were generally lower than those in the Fet group but similar to or higher than those in the Neo group, except for C24:1n-9 of which content was again highest in the Neo group. In contrast, most of polyunsaturated fatty acid (PUFA) contents in the Cul group appeared lower than those in both the Fet and Neo groups, and differences in 5 of 10 detected PUFAs were significant between the Cul and Neo groups. The results suggest that PUFA contents in cultured cardiomyocytes might be insufficient to exert enough contractile ability. In conclusion, it could be necessary for cultured cardiomyocytes to uptake more lipid; PUFAs in particular.

Preotic neural crest cells contribute to coronary artery smooth muscle involving endothelin signalling.

Neural crest cells constitute a multipotent cell population that gives rise to diverse cell lineages. The neural crest arising from the postotic hindbrain is known as the 'cardiac' neural crest, and contributes to the great vessels and outflow tract endocardial cushions, but the neural crest contribution to structures within the heart remains largely controversial. Here we demonstrate that neural crest cells from the preotic region migrate into the heart and differentiate into coronary artery smooth muscle cells. Preotic neural crest cells preferentially distribute to the conotruncal region and interventricular septum. Ablation of the preotic neural crest causes abnormalities in coronary septal branch and orifice formation. Mice and chicks lacking endothelin signalling show similar abnormalities in the coronary artery, indicating its involvement in neural crest-dependent coronary artery formation. This is the first report that reveals the preotic neural crest contribution to heart development and smooth muscle heterogeneity within a coronary artery.

Viscoelastic characteristics of contracted collagen gels populated with rat fibroblasts or cardiomyocytes.

The viscoelastic characteristics of contracted collagen gels populated with rat fibroblasts or cardiomyocytes were investigated by uniaxial tensile testing. Rat type I collagen-Dulbecco's modified Eagle's medium solution (each 2 ml in volume, 0.5 mg/ml collagen concentration) containing 2.0 million rat fibroblasts or cardiomyocytes were cast in a circular shape. After gelation and culture for 10 days the contracted gels were first stretched to a tensile strain of approximately 0.20 at 4.6 × 10(-3)/s strain rate, and then the strain was kept unchanged for 3 min. The tensile stress in the gels was recorded. The results were regressed against the equations of the Kelvin viscoelastic model. It was found that the two elastic coefficients in the model were 6.5 ± 1.7 and 10.2 ± 3.2 kPa, respectively, for gels with cardiomyocytes and 5.1 ± 1.6 and 4.5 ± 0.9 kPa for those with fibroblasts; the values for gels with cardiomyocytes were significantly higher than those for gels with fibroblasts. The viscous coefficient was 169.6 ± 60.7 kPa s for the cardiomyocytes and 143.6 ± 44.7 kPa s for the fibroblasts. The relaxation time constant for gels with cardiomyocytes was 19.6 ± 10.6 s, significantly smaller than for gels with fibroblasts (36.4 ± 13.3 s). This study is the first to obtain viscoelastic data for living cell-contracted collagen gels. These data show that the viscous effect has a vital effect on the mechanical behavior of the gels and cannot be neglected in the culture and function of artificial substitutes based on contracted collagen gels. Furthermore, the data may imply that viscous coefficient of the gels might be closely related to collagen density rather than to cross linking among collagen fibrils.