The Development of the Epicardium in the Sturgeon Acipenser naccarii

This article reports on the development of the epicardium in alevins of the sturgeon Acipenser naccarii, aged 4–25 days post‐hatching (dph). Epicardial development starts at 4 dph with formation of the proepicardium (PE) that arises as a bilateral structure at the boundary between the sinus venosus and the duct of Cuvier. The PE later becomes a midline organ arising from the wall of the sinus venosus and ending at the junction between the liver, the sinus venosus and the transverse septum. This relative displacement appears related to venous reorganization at the caudal pole of the heart. The mode and time of epicardium formation is different in the various heart chambers. The conus epicardium develops through migration of a cohesive epithelium from the PE villi, and is completed through bleb‐like aggregates detached from the PE. The ventricular epicardium develops a little later, and mostly through bleb‐like aggregates. The bulbus epicardium appears to derive from the mesothelium located at the junction between the outflow tract and the pericardial cavity. Strikingly, formation of the epicardium of the atrium and the sinus venosus is a very late event occurring after the third month of development. Associated to the PE, a sino‐ventricular ligament develops as a permanent connection. This ligament contains venous vessels that communicate the subepicardial coronary plexus and the sinus venosus, and carries part of the heart innervation. The development of the sturgeon epicardium shares many features with that of other vertebrate groups. This speaks in favour of conservative mechanisms across the evolutionary scale. Anat Rec, 2009. © 2009 Wiley‐Liss, Inc.     

Insights Into Coronary Artery Development in Model of Maternal Protein Restriction in Mice

Programming of fetal development is considered to be an important risk factor for noncommunicable diseases of adulthood, including coronary heart disease (CHD). Aiming to investigate the association between maternal nutrition and the development of the coronary arteries (CAs) in staged mice embryos, C57BL/6 mice embryos from Stages 16 to 23 were taken from mothers fed a normal protein (NP) or low protein (LP) diet, and the CA were studied. Although the LP embryos had lower masses, they had faster heart growth rates when compared with the NP embryos. The subepicardial plexuses were observed earlier in the NP embryos (Stage 20) than in the LP ones (Stage 22; P < 0.01). Apoptotic nuclei were seen around the aortic peritruncal ring beginning at Stage 18 in the NP and LP embryos. FLK1⁺ (fetal liver kinase 1 = VEGF‐r2 or vascular endothelial growth factor receptor 2) cells had a homogeneous distribution in the NP embryos as early as Stage 18, whereas a similar distribution in the LP embryos was only seen at Stages 22 and 23. Maternal protein restriction in mice leads to a delay in the growth of the heart in the embryonic period modifying the development of the subepicardial peritruncal plexus and the apoptosis in the future coronary orifice region. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

In normal development pulmonary veins are connected to the sinus venosus segment in the left atrium

Background: Classic theories descibe that the common pulmonary vein develops as an outgrowth from either the sinus venosus or atrial segment. Recent studies show that the pulmonary veins are connected to the sinu-atrial region before its differentiation into a sinus venosus and atrial segment. Methods: The development of the sinu-atrial region with regard to the developing common pulmonary vein and the growth of the atrial septum was investigated in avian embryos, using both scanning electron microscopy and immunohistochemistry. Embryos ranging between stage HH12 and HH28 were incubated with QH-1 that recognizes quail endothelial cells and precursors, HNK-1, that appears in this study to detect the myocardium of the sinus venosus, or with HHF-35, being specific for muscle actins. Also vascular casts of the heart were produced by injecting prepolymerized Mercox into the vascular system. Results: In preseptation stages the common pulmonary vein drains into the left part of the sinus venosus, that is clearly demarcated by the sinuatrial fold and HNK-1 expression. During atrial septation the left part of the sinus venosus, in contrast to the right part, loses its HNK-1 antigen from stage HH23 onwards, while at the same time the sinu-atrial fold in the left atrial dorsal wall flattens and disappears. From stage HH25 onwards HNK-1 expression is restricted to the right part of the sinus venosus, which contributes to the right atrium. The myocardial atrial septum never expresses the HNK-1 antigen, suggesting that the septum is of atrial origin. Discussion: It appeared that the sinus venosus does not only contribute to the sinus venarum of the right atrium, but also to the left atrium. © 1995 Wiley-Liss, Inc.     

Development of the pulmonary vein in the American alligator (Alligator mississippiensis)

The origin of the embryonic common pulmonary vein in terrestrial vertebrates is still uncertain. Most earlier studies in nonhuman embryos describe the vein as entering the sinus venosus. The currently prevailing view, however, based largely on the study of human material, is that the embryonic common pulmonary vein is associated with the left atrium from its inception. We recently observed the pulmonary vein entering the sinus venous part of the right atrium in several normal dog embryos of a stage comparable to horizon XIV in man (Streeter: Contrib. Embryol. Carnegie Inst. Wash., 31:53, 1945). In slightly older specimens the vein entered the left atrium just to the left of septum primum. This observation, and the fact that some atrial septal and pulmonary venous anomalies in man still await a plausible pathogenetic explanation, stimulated a restudy of the origin of the vein. The alligator was used because we already had prepared a large number of closely graded serially sectioned embryos for other purposes. Wax plate reconstructions clearly showed that the common pulmonary vein entered the left side of the sinus venosus. With the formation of the atrial septum, this part of the sinus venosus is "pinched off" and becomes incorporated into the left atrium, thus transferring the pulmonary venous ostium to that atrium.     

The identification of different endothelial cell populations within the mouse proepicardium

The proepicardium is a transient embryonic structure that is a source of precursors of the epicardium, coronary smooth muscle cells, and may be a source of coronary endothelial cells (EC). To better understand proepicardium development a systematic analysis of EC appearance was performed. Multiple marker analysis showed that EC are present in the mouse proepicardium at embryonic day (E) 9.0 through E9.75. Distinct populations of EC were found that were associated with the liver bud, and the sinus venosus, as well as a population that do not appear to be associated with either of these structures. There was a temporal increase in the number of EC and temporal changes in the distribution of EC within the different populations during PE development. These findings indicate that EC exist in the proepicardium before coronary vasculogenesis, and support a model in which there is a heterogeneous origin for EC in the proepicardium.     

Platelet-derived growth factors in the developing avian heart and maturating coronary vasculature.

Platelet-derived growth factors (PDGFs) are important in embryonic development. To elucidate their role in avian heart and coronary development, we investigated protein expression patterns of PDGF-A, PDGF-B, and the receptors PDGFR-alpha and PDGFR-beta using immunohistochemistry on sections of pro-epicardial quail-chicken chimeras of Hamburger and Hamilton (HH) 28-HH35. PDGF-A and PDGFR-alpha were expressed in the atrial septum, sinus venosus, and throughout the myocardium, with PDGFR-alpha retreating to the trabeculae at later stages. Additionally, PDGF-A and PDGFR-alpha were present in outflow tract cushion mesenchyme and myocardium, respectively. Small cardiac nerves and (sub)epicardial cells expressed PDGF-B and PDGFR-beta. Furthermore, endothelial cells expressed PDGF-B, while vascular smooth muscle cells and interstitial epicardium-derived cells expressed PDGFR-beta, indicating a role in coronary maturation. PDGF-B is also present in ventricular septal development, in the absence of any PDGFR. Epicardium-derived cells in the atrioventricular cushions expressed PDGFR-beta. We conclude that all four proteins are involved in myocardial development, whereas PDGF-B and PDGFR-beta are specifically important in coronary maturation.     

Formation and remodeling of the coronary vascular bed in the embryonic avian heart.

To study the formation of the coronary vessels in the developing avian heart, we stained developmentally staged quail hearts with the endothelial specific antibody QH-1. QH-1 reacted with individual cells in the proepicardial organ in Hamburger and Hamilton stage (HH) 17 embryos only after it had contacted the heart. In HH18-26 hearts, individual QH-1+ cells accumulated over the surface of the atria and ventricles. The first endothelial vessels appeared in the dorsal atrioventricular groove in HH23 hearts. CD45+ hematopoietic precursors accumulated on the heart surface, demonstrating the close temporal relationship of hematopoiesis with vasculogenesis during heart development. However, CD45 expression preceded association of these cells with the vasculature, suggesting hematopoietic commitment precedes formation of blood islands in the coronary vasculature. Endothelial tubules first appeared on the dorsal and then the ventral aspects of the heart, coalescing into large sinusoids. These sinusoids remodeled into compact muscularized vessels by HH35. Smooth muscle cell markers were first expressed at HH27 and only in association with developing vasculature. We did not observe markers of smooth muscle differentiation in the proepicardium, but it remains uncertain whether cells in the proepicardium are committed to this cell fate. Our data support a strictly vasculogenic mechanism for the formation of the coronary vessels and blood islands.     

人胚心静脉窦和传导系统的早期发育

目的 探讨早期人胚心静脉窦及传导系的发生发育机制. 方法 用抗α-平滑肌肌动蛋白(α-SMA)、抗α-横纹肌肌动蛋白(α-SCA)和抗结蛋白(DES)抗体对29例C10～C16期人胚心连续切片行免疫组织化学染色. 结果 人胚发育C12～C13期,系统静脉汇集形成的静脉窦出现于心包腔尾端原始横膈间充质中,静脉窦壁间充质细胞逐渐分化为α-SCA阳性的静脉窦心肌细胞.C14期,心包腔的扩张使静脉窦进入心包腔内,参与了右心房的形成.DES阳性传导系心肌的分化始于C10期心房室管右侧壁,随发育逐渐向室间沟心肌扩展,发育为房室传导系的希氏束、左右束支及心室腔面的小梁心肌.在心房,DES表达首先出现于C11期心房背侧壁,在C13期,可见静脉窦左背侧壁α-SCA、α-SMA、DES阳性心肌带与左心房底部、房室管背侧壁相延续,这条心肌带可能参与了人胚心静脉窦至房室管传导系的发育.C14～C16期,DES强阳性染色从窦房结经左、右静脉瓣及心房的背、腹侧壁延伸至房室管右侧壁,可能是原始的心房传导通路. 结论 心包腔尾端原始横膈间充质是人胚静脉窦心肌发生区,原始横膈间充质细胞逐渐分化为心肌细胞,添加到人胚心管静脉端,形成心静脉窦心肌.人胚心传导系心肌的分化始于房室管,随心管发育逐渐向动、静脉端扩展,在C16期,已分化为形态清晰可辨的DES阳性胚胎心传导系.     

MAP kinase activation in avian cardiovascular development.

Signaling pathways mediated by receptor tyrosine kinases (RTK) and mitogen-activated protein kinase (MAPK) activation have multiple functions in the developing cardiovascular system. The localization of diphosphorylated extracellular signal regulated kinase (dp-ERK) was monitored as an indicator of MAPK activation in the forming heart and vasculature of avian embryos. Sustained dp-ERK expression was observed in vascular endothelial cells of embryonic and extraembryonic origins. Although dp-ERK was not detected during early cardiac lineage induction, MAPK activation was observed in the epicardial, endocardial, and myocardial compartments during heart chamber formation. Endocardial expression of dp-ERK in the valve primordia and heart chambers may reflect differential cell growth associated with RTK signaling in the heart. dp-ERK localization in the epicardium, subepicardial fibroblasts, myocardial fibroblasts, and coronary vessels is consistent with MAPK activation in epicardial-derived cell lineages. The complex temporal-spatial regulation of dp-ERK in the heart supports diverse regulatory functions for RTK signaling in different cell populations, including the endocardium, myocardium, and epicardial-derived cells during cardiac organogenesis.     

Patterning of embryonic blood vessels.

Morphometric methods were developed to characterize the geometry of vascular patterns in avian and murine embryos. By using these methods, we found that networks of blood vessels formed during vasculogenesis share similar geometric properties (i.e., mean blood vessel diameters and avascular space diameters) regardless of developmental stage, location, or species in which they form. We also found that endothelial cell density within a unit area of an embryonic vasculature could be used to accurately distinguish between a small diameter, capillary-like vascular network (low endothelial cell density) and a large diameter, presinusoidal network (high endothelial cell density). Furthermore, we show that endothelial cell size remains constant in small and large diameter vessels, indicating that increased endothelial cell size is not the basis for diversity in vessel diameter. These observations serve as a foundation for future studies seeking to evaluate the effects of agents or genetic mutations on aspects of vasculogenesis.     

Tissue‐specific venous expression of the EPH family receptor EphB1 in the skin vasculature

Background : The major arteries and veins are formed early during development. The molecular tools to identify arterial and venous endothelial cells improve our understanding of arterial–venous differentiation and branching morphogenesis. Compared with arterial differentiation, relatively little is known about what controls venous development, due to lack of definitive molecular markers for venous endothelial cells. Results: Here we report that the antibody against EphB1, an EphB class receptor, makes it possible to establish a reliable whole‐mount immunohistochemical analysis of venous identity with greater resolution than previously possible in embryonic and adult skin vasculature models. EphB1 expression is restricted to the entire venous vasculature throughout embryonic development to adulthood, whereas the previously established venous marker EphB4 is also detectable in lymphatic vasculature. This venous‐restricted expression of EphB1 is established after the vascular remodeling of the primary capillary plexus has occurred. Compared with its venous‐specific expression in the skin, however, EphB1 is not restricted to the venous vasculature in yolk sac, trunk and lung. Conclusions: These studies introduce EphB1 as a new venous‐restricted marker in a tissue‐specific and time‐dependent manner. Developmental Dynamics 242:976–988, 2013. © 2013 Wiley Periodicals, Inc.     

斑马鱼血管系统的发育遗传学研究进展

斑马鱼血管系统在原肠胚形成后不久便开始发育，血管系统的发育过程可分为血管发生和血管生成这两个不同的阶段，其过程受到多种信号通路的的调控，这些信号协同作用，以确保血管发育的正常进行。文中综述了主要以模式生物斑马鱼来研究的血管发育遗传的过程，并介绍调节血管发育进程的一些关键的调控。以斑马鱼为模式生物来研究血管系统的发育遗传学，为理解人类血管的发育和再生，为缺血性疾病和肿瘤等疾病的治疗提供了新的途径。     

Developmental Progression of the Coronary Vasculature in Human Embryos and Fetuses

Although considerable advances in our understanding of mammalian and avian embryonic coronary development have occurred during the last decade, our current knowledge of this topic in humans is limited. Accordingly, the aim of this study was to determine if the development of the human coronary vasculature in humans is like that of other mammals and avians. The data document a progression of events involving mesenchymal cell‐containing villi from the proepicardium, establishment of blood islands and a capillary network. The major finding of the study is direct evidence that the capillary plexus associated with spindle cells and erythroblasts invades the base of the aorta to form coronary ostia. A role for the dorsal mesocardium is also indicated by the finding that cells from this region are continuous with the aorta and pulmonary artery. The development of the tunica media of the coronary arteries follows the same base‐apex progression as in other species, with the development of branches occurring late in the embryonic period. The fetal period is characterized by 1) growth and a numerical increase in the smallest arterial branches, veins, and venules, 2) innervation of arteries, and 3) inclusion of elastic fibers in the tunica media of the coronary arteries and development of the tunica adventitia. In conclusion, the data demonstrate that the development of the coronary system in humans is similar to that of other mammalian and avian species, and for the first time documents that the formation of the ostia and coronary stems in humans occurs by ingrowth of a vascular plexus and associated cells from the epicardium. Anat Rec, 299:25–41, 2016. © 2015 Wiley Periodicals, Inc.     

Apoptosis during coronary artery orifice development in the chick embryo

Previous studies regarding the development of proximal segments of the coronary arteries in the chick have demonstrated that these vessels do not develop as angiogenic outgrowths from the aorta. Rather, the proximal segments of the coronary arteries arise from a peritruncal capillary plexus in the epicardium that coalesces around the aortic and pulmonary outflow tracts. Vessels from the peritruncal plexus grow toward and attach to the aorta at about Hamburger and Hamilton (HH) Stage 32 to establish the definitive coronary circulation. Currently, little is known about the process by which patent connections are established between these peritruncal vessels and the aorta. The hypothesis that apoptosis is involved in the formation of the coronary artery orifices was tested in the present study. Aortic and periaortic tissue from HH 29-35 chick embryos was examined using routine light and electron microscopy and TUNEL assays. Apoptotic cells were observed in close spatial and temporal association with the invasion of peritruncal vessels into the aorta (HH 29-31), the initial formation of coronary orifices (HH 32-33), and the further development of the definitive coronary arteries and orifices (HH 34-35). Whereas the origin of these apoptotic cells and the specific factors regulating their death remain unknown, the results of the present study strongly correlate apoptosis with the formation of proximal coronary arteries and their orifices. Our findings suggest avenues for further research and indicate that factors involved in regulating apoptosis should be included in future models of coronary artery development.     

Development of the coronary blood supply: changing concepts and current ideas

Earlier views of the development of the coronary vasculature included angiogenic budding and growth of arteries from the aortic sinuses and veins from the coronary sinus. The current concept begins with the establishment of the epicardium from the proepicardial organ, an outgrowth of the dorsal wall of the pericardial cavity. Capillaries form in a subepicardial mesenchymal population, extending as a plexus toward the truncus arteriosus and the atria. Multiple vessels grow from a peritruncal ring of capillaries, preferentially invading the newly formed aorta. In a process involving apoptotic changes of both the aortic wall and the invading capillaries, orifices open at the level of the aortic sinuses. Smooth muscle cells and pericytes, recruited from the surrounding mesenchyme, contribute to the vessel walls, and the definitive coronary artery pattern is established. Similar events are occurring on the venous side of the coronary circulation, following a slightly earlier time course. Multiple factors govern this process, including VEGF and FGF-1 stimulating vasculogenesis and angiogenesis, and the angiopoietins and their tyrosine kinase receptors modulating interactions between endothelial cells and the mural components. As remodeling of the capillary plexus and the coronary orifices progresses, TGF beta released by apoptotic cells or from other sources likely modulates VEGF and FGF-1, and also contributes to further apoptotic changes. A better appreciation of the controls of the mechanisms of coronary vessel development may direct further research in the prevention of arteriosclerosis and ischemic tissue injuries.     

Development of the origin of the coronary arteries, a matter of ingrowth or outgrowth?

Summary Inconsistencies still exist with regard to the exact mode of development of proximal coronary arteries and coronary orifices. In this regard 15 quail embryos were investigated using a monoclonal anti-endothelium antibody, enabling a detailed study of the development of endothelium-lined vasculature. Coronary orifices emerged at 7–9 days of incubation (Zacchei stages 24–26) and were invariably present at 10 days of incubation (Zacchei stage 27).We never observed more than 2 coronary orifices; these were always single in either of the facing sinuses of the aorta. A coronary orifice was always observed being connected to an already developed proximal coronary artery, which belonged to a peritruncal ring of coronary arterial vasculature. We did not find any coronary orifice without a connection to a proximal coronary artery. Moreover, at 7–9 days of incubation (Zacchei stages 24–26) we observed coronary arteries from the peritruncal ring penetrating the aortic media. In 2 specimen this coronary artery, with a lumen, was in contact with the still intact endothelial lining of the aorta.We conclude that coronary arteries do not grow out of the aorta, but grow into the aorta from the peritruncal ring of coronary arterial vasculature. This throws new light on normal and abnormal development of proximal coronary arteries and coronary orifices.     

Rare coronary anastomoses between the aorta,pulmonary trunk,left coronary artery,and subclavian artery

We report a rare case of coronary anastomoses in an 83‐year‐old male cadaveric heart. Anomalous vessels arose from the right sinus of the aorta, left main coronary artery, left anterior descending artery, left anterior medial atrial artery, and left subclavian artery. These vessels bifurcated and anastomosed, and finally connected to the pulmonary trunk. The present case is categorized as a multilateral coronary artery fistula in cardiology. Clin. Anat. 25:969–972, 2012. © 2012 Wiley Periodicals, Inc.     

The orphan nuclear receptor COUP-TFII is required for angiogenesis and heart development

The embryonic expression of COUP-TFII, an orphan nuclear receptor, suggests that it may participate in mesenchymal-epithelial interactions required for organogenesis. Targeted deletion of the COUP-TFII gene results in embryonic lethality with defects in angiogenesis and heart development. COUP-TFII mutants are defective in remodeling the primitive capillary plexus into large and small microcapillaries. In the COUP-TFII mutant heart, the atria and sinus venosus fail to develop past the primitive tube stage. Reciprocal interactions between the endothelium and the mesenchyme in the vascular system and heart are essential for normal development of these systems. In fact, the expression of Angiopoietin-1, a proangiogenic soluble factor thought to mediate the mesenchymal-endothelial interactions during heart development and vascular remodeling, is down-regulated in COUP-TFII mutants. This down-regulation suggests that COUP-TFII may be required for bidirectional signaling between the endothelial and mesenchymal compartments essential for proper angiogenesis and heart development.