The early embryonic heart regenerates by compensation of proliferating residual cardiomyocytes after cryoinjury.

The adult mammalian heart is non-regenerative because cardiomyocytes withdraw from the cell cycle shortly after birth. Embryonic mammalian hearts, in which cardiomyocytes are genetically ablated in a salt-and-pepper-like pattern, regenerate due to compensation by residual cardiomyocytes. To date, it remains unknown whether or how transmural ventricular defects at the looped heart stage regenerate after cryoinjury. We established a cryoablation model in stage 16 chick embryonic hearts. In hearts at 5 h post cryoinjury (hpc), cryoinjury-induced defects were approximately 200 µm in width in the primitive ventricle; thereafter, the defect was filled with mesenchymal cells accumulating between the epicardium and endocardium. The defect began to regress at 4 days post cryoinjury (dpc) and disappeared around 9 dpc. Immunohistochemistry showed that there were no isl1-positive cells in either the scar tissue or residual cardiomyocytes. BrdU incorporation into residual cardiomyocytes was transiently downregulated in association with upregulation of p27 (Kip1), suggesting that cell cycle arrest occurred at G1-to-S transition immediately after cryoinjury. Estimated cell cycle length was examined, and the results showed that the shortest cell cycle length was 18 h at stages 19-23; it increased with development due to elongation of the G2-M-G1 phase and 30 h at stages 27-29. The S phase length was constant at 6-8 h. The cell cycle length was elongated immediately after cryoinjury, and it reversed at 1-2 dpc. Cryoablated transmural defects in the early embryonic heart were restored by compensation by residual myocytes.

Msx1 upregulates p27 expression to control cellular proliferation during valvuloseptal endocardial cushion formation in the chick embryonic heart.

Cushion tissues, the primordia of valves and septa of the adult heart, are formed in the atrioventricular (AV) and outflow tract (OFT) regions of the embryonic heart. The cushion tissues are generated by the endothelial-mesenchymal transition (EMT), involving many soluble factors, extracellular matrix, and transcription factors. Moreover, neural crest-derived mesenchymal cells also migrate into the OFT cushion. The transcription factor Msx1 is known to be expressed in the endothelial and mesenchymal cells during cushion tissue formation. However, its exact role in EMT during cushion tissue formation is still unknown. In this study, we investigated the expression patterns of Msx1 mRNA and protein during chick heart development. Msx1 mRNA was localized in endothelial cells of the AV region at Stage 14, and its protein was first detected at Stage 15. Thereafter, Msx1 mRNA and protein were observed in the endothelial and mesenchymal cells of the OFT and AV regions. in vitro assays showed that ectopic Msx1 expression in endothelial cells induced p27, a cell-cycle inhibitor, expression and inhibited fibroblast growth factor 4 (FGF4)-induced cell proliferation. Although the FGF signal reduced the EMT-inducing activities of transforming growth factor ß (TGFß), ectopic Msx1 expression in endothelial cells enhanced TGFß signaling-induced αSMA, an EMT marker, expression. These results suggest that Msx1 may support the transformation of endothelial cells due to a TGFß signal in EMT during cushion tissue formation.

Hypoxia-induced downregulation of Sema3a and CXCL12/CXCR4 regulate the formation of the coronary artery stem at the proper site.

BACKGROUND: During the formation of the coronary artery stem, endothelial strands from the endothelial progenitor pool surrounding the conotruncus penetrate into the aortic wall. Vascular endothelial growth factors (VEGFs) as well as CXCL12/CXCR4 signaling are thought to play a role in the formation of the coronary stem. However, the mechanisms regulating how endothelial strands exclusively invade into the aorta remain unknown. METHODS AND RESULTS: Immunohistochemistry showed that before the formation of endothelial strands, Sema3a was highly expressed in endothelial progenitors surrounding the great arteries. At the onset of/during invasion of endothelial strands into the aorta, Sema3a was downregulated and CXCR4 was upregulated in the endothelial strands. In situ hybridization showed that Cxcl12 was highly expressed in the aortic wall compared with in the pulmonary artery. Using avian embryonic hearts, we established two types of endothelial penetration assay, in which coronary endothelial strands preferentially invaded into the aorta in culture. Sema3a blocking peptide induced an excess number of endothelial strands penetrating into the pulmonary artery, whereas recombinant Sema3a inhibited the formation of endothelial strands. In cultured coronary endothelial progenitors, recombinant VEGF protein induced CXCR4-positive endothelial strands, which were capable of being attracted by CXCL12-impregnated beads. Monoazo rhodamine detected that hypoxia was predominant in aortic/subaortic region in ovo and hypoxic condition downregulated the expression of Sema3a in culture. CONCLUSION: Results suggested that hypoxia in the aortic region downregulates the expression of Sema3a, thereby enhancing VEGF activity to induce the formation of CXCR4-positive endothelial strands, which are subsequently attracted into the Cxcl12-positive aortic wall to connect the aortic lumen.

A novel case of multiple variations in the brachial plexus with the middle trunk originating from the C7 and C8.

The brachial plexus is an important nervous structure from which all major nerves to the upper limb arise. It typically originates from the anterior rami of the C5-T1 spinal nerves. As it passes laterally, the roots are successively organized into three trunks, six divisions, and three cords. The BP is susceptible to injury during the perinatal and postnatal periods, as well as in adulthood. Its structure can show considerable variation, and there is a wealth of literature describing its variations, providing indispensable information to neurosurgeons. Here, we report a novel unilateral variant of the brachial plexus found in an adult Japanese male cadaver. In this case, the middle trunk arose from the C7 and C8 spinal nerves, and the inferior trunk continued from the T1 alone. At the interscalene triangle, the subclavian artery was situated between the C8 and T1 nerves. The posterior cord arose from the posterior divisions of the superior and middle trunks, while the root from the T1 nerve/inferior trunk was absent. The anterior division of the middle trunk gave independent roots to the musculocutaneous and median nerves, without completely establishing the lateral cord. A communicating branch arose from the musculocutaneous nerve to join the median nerve. Some branches from the roots and cords also deviated from typical configurations. This case represents a rare combination of variations in the trunks, divisions, cords, and the median nerve and offers a valuable addition to the literature regarding variations in the brachial plexus.

Retinoic acid signaling in heart development.

Retinoic acid (RA) is a vitamin A metabolite that acts as a morphogen and teratogen. Excess or defective RA signaling causes developmental defects including in the heart. The heart develops from the anterior lateral plate mesoderm. Cardiogenesis involves successive steps, including formation of the primitive heart tube, cardiac looping, septation, chamber development, coronary vascularization, and completion of the four-chambered heart. RA is dispensable for primitive heart tube formation. Before looping, RA is required to define the anterior/posterior boundaries of the heart-forming mesoderm as well as to form the atrium and sinus venosus. In outflow tract elongation and septation, RA signaling is required to maintain/differentiate cardiogenic progenitors in the second heart field at the posterior pharyngeal arches level. Epicardium-secreted insulin-like growth factor, the expression of which is regulated by hepatic mesoderm-derived erythropoietin under the control of RA, promotes myocardial proliferation of the ventricular wall. Epicardium-derived RA induces the expression of angiogenic factors in the myocardium to form the coronary vasculature. In cardiogenic events at different stages, properly controlled RA signaling is required to establish the functional heart.

Inotropic effects of a single intravenous recommended dose of pimobendan in healthy dogs.

We investigated the effects of an injectable pimobendan solution (0.15 mg/kg) on cardiac function in healthy dogs. Fifteen dogs were divided into placebo, intravenous pimobendan injection, and subcutaneous pimobendan injection groups. In the placebo, the heart rate, systolic and end-diastolic left ventricular pressure (LVPs and LVEDP), and peak positive (max dP/dt) and negative (min dP/dt) first derivatives of the left ventricular pressure did not change for 60 min. After the intravenous pimobendan injection, LVEDP decreased significantly within 5 min, while the max dP/dt increased, and the effects continued until 60 min. In comparison, there were no hemodynamic changes after the subcutaneous pimobendan injection. This study demonstrates that injectable pimobendan induced a rapid inotropic effect and decreased the LVEDP in dogs.

Anatomy of the coronary artery and cardiac vein in the quail ventricle: patterns are distinct from those in mouse and human hearts.

Coronary vessel development has been investigated in avian and mouse embryonic hearts. Quail embryos are a useful tool to examine vascular development, particularly because the QH1 antibody and transgenic quail line, Tg (tie1:H2B-eYFP), are useful to trace endothelial cells. However, there are only a few descriptions of the quail coronary vessels. Using ink injection coronary angiography, we examined the course of coronary vessels in the fetal quail heart. The major coronary arteries were the right and left septal arteries, which, respectively, branched off from the right and left coronary stems. The right septal artery ran posteriorly (dorsally) and penetrated the ventricular free wall to distribute to the posterior surface of the ventricles. The left septal artery ran anteriorly (ventrally) and penetrated the ventricular free wall to distribute to the anterior surface of the ventricles. The right and left circumflex arteries were directed posteriorly along the atrioventricular sulci. The cardiac veins consisted of three major tributaries: the middle, great, and anterior cardiac veins. The middle cardiac vein ascended along the posterior interventricular sulcus and emptied into the right atrium. The great cardiac vein ran along the anterior interventricular sulcus, entered the space between the left atrium and conus arteriosus and emptied into the right atrium behind the aortic bulb. The anterior cardiac vein drained the anterior surface of the right ventricle and connected to the anterior base of the right atrium. The course of coronary vessels in the quail heart was basically the same as that observed in chick but was different from those of mouse and human.

Avian coronary endothelium is a mosaic of sinus venosus- and ventricle-derived endothelial cells in a region-specific manner.

The origin of coronary endothelial cells (ECs) has been investigated in avian species, and the results showed that the coronary ECs originate from the proepicardial organ (PEO) and developing epicardium. Genetic approaches in mouse models showed that the major source of coronary ECs is the sinus venosus endothelium or ventricular endocardium. To clarify and reconcile the differences between avian and mouse species, we examined the source of coronary ECs in avian embryonic hearts. Using an enhanced green fluorescent protein-Tol2 system and fluorescent dye labeling, four types of quail-chick chimeras were made and quail-specific endothelial marker (QH1) immunohistochemistry was performed. The developing PEO consisted of at least two cellular populations in origin, one was sinus venosus endothelium-derived inner cells and the other was surface mesothelium-derived cells. The majority of ECs in the coronary stems, ventricular free wall, and dorsal ventricular septum originated from the sinus venosus endothelium. The ventricular endocardium contributed mainly to the septal artery and a few cells to the coronary stems. Surface mesothelial cells of the PEO differentiated mainly into a smooth muscle phenotype, but a few differentiated into ECs. In avian species, the coronary endothelium had a heterogeneous origin in a region-specific manner, and the sources of ECs were basically the same as those observed in mice.

Mechanism responsible for D-transposition of the great arteries: Is this part of the spectrum of right isomerism?

D-transposition of the great arteries (TGA) is one of the most common conotruncal heart defects at birth and is characterized by a discordant ventriculoarterial connection with a concordant atrioventricular connection. The morphological etiology of TGA is an inverted or arrested rotation of the heart outflow tract (OFT, conotruncus), by which the aorta is transposed in the right ventral direction to the pulmonary trunk. The rotational defect of the OFT is thought to be attributed to hypoplasia of the subpulmonic conus, which originates from the left anterior heart field (AHF) residing in the mesodermal core of the first and second pharyngeal arches. AHF, especially on the left, at the early looped heart stage (corresponding to Carnegie stage 10-11 in the human embryo) is one of the regions responsible for the impediment that causes TGA morphology. In human or experimentally produced right isomerism, malposition of the great arteries including D-TGA is frequently associated. Mutations in genes involving left-right (L-R) asymmetry, such as NODAL, ACTRIIB and downstream target FOXH1, have been found in patients with right isomerism as well as in isolated TGA. The downstream pathways of Nodal-Foxh1 play a critical role not only in L-R determination in the lateral plate mesoderm but also in myocardial specification and differentiation in the AHF, suggesting that TGA is a phenotype in heterotaxia as well as the primary developmental defect of the AHF.

Impaired development of left anterior heart field by ectopic retinoic acid causes transposition of the great arteries.

BACKGROUND: Transposition of the great arteries is one of the most commonly diagnosed conotruncal heart defects at birth, but its etiology is largely unknown. The anterior heart field (AHF) that resides in the anterior pharyngeal arches contributes to conotruncal development, during which heart progenitors that originated from the left and right AHF migrate to form distinct conotruncal regions. The aim of this study is to identify abnormal AHF development that causes the morphology of transposition of the great arteries. METHODS AND RESULTS: We placed a retinoic acid-soaked bead on the left or the right or on both sides of the AHF of stage 12 to 14 chick embryos and examined the conotruncal heart defect at stage 34. Transposition of the great arteries was diagnosed at high incidence in embryos for which a retinoic acid-soaked bead had been placed in the left AHF at stage 12. Fluorescent dye tracing showed that AHF exposed to retinoic acid failed to contribute to conotruncus development. FGF8 and Isl1 expression were downregulated in retinoic acid-exposed AHF, and differentiation and expansion of cardiomyocytes were suppressed in cultured AHF in medium supplemented with retinoic acid. CONCLUSIONS: The left AHF at the early looped heart stage, corresponding to Carnegie stages 10 to 11 (28 to 29 days after fertilization) in human embryos, is the region of the impediment that causes the morphology of transposition of the great arteries.

Signaling regulating inner ear development: cell fate determination, patterning, morphogenesis, and defects.

The membranous labyrinth of the inner ear is a highly complex organ that detects sound and balance. Developmental defects in the inner ear cause congenital hearing loss and balance disorders. The membranous labyrinth consists of three semicircular ducts, the utricle, saccule, and endolymphatic ducts, and the cochlear duct. These complex structures develop from the simple otic placode, which is established in the cranial ectoderm adjacent to the neural crest at the level of the hindbrain at the early neurula stage. During development, the otic placode invaginates to form the otic vesicle, which subsequently gives rise to neurons for the vestibulocochlear ganglion, the non-sensory and sensory epithelia of the membranous labyrinth that includes three ampullary crests, two maculae, and the organ of Corti. Combined paracrine and autocrine signals including fibroblast growth factor, Wnt, retinoic acid, hedgehog, and bone morphogenetic protein regulate fate determination, axis formation, and morphogenesis in the developing inner ear. Juxtacrine signals mediated by Notch pathways play a role in establishing the sensory epithelium, which consists of mechanosensory hair cells and supporting cells. The highly differentiated organ of Corti, which consists of uniformly oriented inner/outer hair cells and specific supporting cells, develops during fetal development. Developmental alterations/arrest causes congenital malformations in the inner ear in a spatiotemporal-restricted manner. A clearer understanding of the mechanisms underlying inner ear development is important not only for the management of patients with congenital inner ear malformations, but also for the development of regenerative therapy for impaired function.

Epicardium is required for sarcomeric maturation and cardiomyocyte growth in the ventricular compact layer mediated by transforming growth factor ß and fibroblast growth factor before the onset of coronary circulation.

The epicardium, which is derived from the proepicardial organ (PE) as the third epithelial layer of the developing heart, is crucial for ventricular morphogenesis. An epicardial deficiency leads to a thin compact layer for the developing ventricle; however, the mechanisms leading to the impaired development of the compact layer are not well understood. Using chick embryonic hearts, we produced epicardium-deficient hearts by surgical ablation or blockade of the migration of PE and examined the mechanisms underlying a thin compact myocardium. Sarcomeric maturation (distance between Z-lines) and cardiomyocyte growth (size) were affected in the thin compact myocardium of epicardium-deficient ventricles, in which the amounts of phospho-smad2 and phospho-ERK as well as expression of transforming growth factor (TGF)ß2 and fibroblast growth factor (FGF)2 were reduced. TGFß and FGF were required for the maturation of sarcomeres and growth of cardiomyocytes in cultured ventricles. In ovo co-transfection of dominant negative (dN)-Alk5 (dN-TGFß receptor I) and dN-FGF receptor 1 to ventricles caused a thin compact myocardium. Our results suggest that immature sarcomeres and small cardiomyocytes are the causative architectures of an epicardium-deficient thin compact layer and also that epicardium-dependent signaling mediated by TGFß and FGF plays a role in the development of the ventricular compact layer before the onset of coronary circulation.

Biosynthesis of linoleic acid in Tyrophagus mites (Acarina: Acaridae).

We report here that Tyrophagus similis and Tyrophagus putrescentiae (Astigmata: Acaridae) have the ability to biosynthesize linoleic acid [(9Z, 12Z)-9, 12-octadecadienoic acid] via a Δ12-desaturation step, although animals in general and vertebrates in particular appear to lack this ability. When the mites were fed on dried yeast enriched with d31-hexadecanoic acid (16:0), d27-octadecadienoic acid (18:2), produced from d31-hexadecanoic acid through elongation and desaturation reactions, was identified as a major fatty acid component of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) in the mites. The double bond position of d27-octadecadienoic acid (18:2) of PCs and PEs was determined to be 9 and 12, respectively by dimethyldisulfide (DMDS) derivatization. Furthermore, the GC/MS retention time of methyl 9, 12-octadecadienoate obtained from mite extracts agreed well with those of authentic linoleic acid methyl ester. It is still unclear whether the mites themselves or symbiotic microorganisms are responsible for inserting a double bond into the Δ12 position of octadecanoic acid. However, we present here the unique metabolism of fatty acids in the mites.

New insights into the developmental mechanisms of coronary vessels and epicardium.

During heart development, the epicardium, which originates from the proepicardial organ (PE), is a source of coronary vessels. The PE develops from the posterior visceral mesoderm of the pericardial coelom after stimulation with a combination of weak bone morphogenetic protein and strong fibroblast growth factor (FGF) signaling. PE-derived cells migrate across the heart surface to form the epicardial sheet, which subsequently seeds multipotent subepicardial mesenchymal cells via epithelial-mesenchymal transition, which is regulated by several signaling pathways including retinoic acid, FGF, sonic hedgehog, Wnt, transforming growth factor-ß, and platelet-derived growth factor. Subepicardial endothelial progenitors eventually generate the coronary vascular plexus, which acquires an arterial or venous phenotype, connects with the sinus venosus and aortic sinuses, and then matures through the recruitment of vascular smooth muscle cells under the regulation of complex growth factor signaling pathways. These developmental programs might be activated in the adult heart after injury and play a role in the regeneration/repair of the myocardium.

Ectopic retinoic acid signaling affects outflow tract cushion development through suppression of the myocardial Tbx2-Tgfß2 pathway.

The progress of molecular genetics has enabled us to identify the genes responsible for congenital heart malformations. However, recent studies suggest that congenital heart diseases are induced not only by mutations in certain genes, but also by abnormal maternal factors. A high concentration of maternal retinoic acid (RA), the active derivative of vitamin A, is well known as a teratogenic agent that can cause developmental defects. Our previous studies have shown that the maternal administration of RA to mice within a narrow developmental window induces outflow tract (OFT) septum defects, a condition that closely resembles human transposition of the great arteries (TGA), although the responsible factors and pathogenic mechanisms of the TGA induced by RA remain unknown. We herein demonstrate that the expression of Tbx2 in the OFT myocardium is responsive to RA, and its downregulation is associated with abnormal OFT development. We found that RA could directly downregulate the Tbx2 expression through a functional retinoic acid response element (RARE) in the Tbx2 promoter region, which is also required for the initiation of Tbx2 transcription during OFT development. Tgfb2 expression was also downregulated in the RA-treated OFT region and was upregulated by Tbx2 in a culture system. Moreover, defective epithelial-mesenchymal transition caused by the excess RA was rescued by the addition of Tgfß2 in an organ culture system. These data suggest that RA signaling participates in the Tbx2 transcriptional mechanism during OFT development and that the Tbx2-Tgfß2 cascade is one of the key pathways involved in inducing the TGA phenotype.

Myocardial progenitors in the pharyngeal regions migrate to distinct conotruncal regions.

BACKGROUND: The cardiac progenitor cells for the outflow tract (OFT) reside in the visceral mesoderm and mesodermal core of the pharyngeal region, which are defined as the secondary and anterior heart fields (SHF and AHF), respectively. RESULTS: Using chick embryos, we injected fluorescent-dye into the SHF or AHF at stage 14, and the destinations of the labeled cells were examined at stage 31. Labeled cells from the right SHF were found in the myocardium on the left dorsal side of the OFT, and cells from the left SHF were detected on the right ventral side of the OFT. Labeled cells from the right and left AHF migrated to regions of the ventral wall of the OFT close to the aortic and pulmonary valves, respectively. CONCLUSION: These observations indicate that myocardial progenitors from the SHF and AHF contribute to distinct conotruncal regions and that cells from the SHF migrate rotationally while cells from the AHF migrate in a non-rotational manner.

Expression of the Tgfß2 gene during chick embryogenesis.

We performed a comprehensive analysis of the expression of transforming growth factor (TGF) ß2 during chick embryogenesis from stage 6 to 30 (Hamburger and Hamilton, J Morphol 1951;88:49-92) using in situ hybridization. During cardiogenesis, Tgfß2 was expressed in the endothelial/mesenchymal cells of the valvulo-septal endocardial cushion tissue and in the epicardium until the end of embryogenesis. During the formation of major arteries, Tgfß2 was localized in smooth muscle progenitors but not in the vascular endothelium. During limb development, Tgfß2 was expressed in the mesenchymal cells in the presumptive limb regions at stage 16, and thereafter it was localized in the skeletal muscle progenitors. In addition, strong Tgfß2 expression was seen in the mesenchymal cells in the pharyngeal arches. Tgfß2 mRNA was also detected in other mesoderm-derived tissues, such as the developing bone and pleura. During ectoderm development, Tgfß2 was expressed in the floor plate of the neural tube, lens, optic nerve, and otic vesicle. In addition, Tgfß2 was expressed in the developing gut epithelium. Our results suggest that TGFß2 plays an important role not only in epithelial-mesenchymal interactions but also in cell differentiation and migration and cell death during chick embryogenesis. We also found that chick and mouse Tgfß2 RNA show very similar patterns of expression during embryogenesis. Chick embryos can serve as a useful model to increase our understanding in the roles of TGFß2 in cell-cell interactions, cell differentiation, and proliferation during organogenesis.

Tenascin C may regulate the recruitment of smooth muscle cells during coronary artery development.

Tenascin C (TNC) is an extracellular glycoprotein that is thought to be involved in tissue remodeling during organogenesis and regeneration. Using avian embryonic hearts, we investigated the spatiotemporal expression patterns of TNC during the formation of the proximal coronary artery. Immunohistochemistry showed that TNC was deposited around the developing coronary stem and that TNC colocalized with vascular smooth muscle α-actin. A quail-chick chimera, in which a quail proepicardial organ (PEO) had been transplanted, showed that quail tissue-derived cells contributed to the establishment of the endothelial and mural cells of the proximal coronary artery, and the quail tissue-derived mural cells displayed TNC. Proepicardial cells cultured in TNC showed the myofibroblast/smooth muscle cell phenotype and neutralizing anti-TNC antibody suppressed the expression of smooth muscle markers. These observations suggest that TNC plays a role in the mural smooth muscle development of the nascent proximal coronary artery.

Nodal signal is required for morphogenetic movements of epiblast layer in the pre-streak chick blastoderm.

During axis formation in amniotes, posterior and lateral epiblast cells in the area pellucida undergo a counter-rotating movement along the midline to form primitive streak (Polonaise movements). Using chick blastoderms, we investigated the signaling involved in this cellular movement in epithelial-epiblast. In cultured posterior blastoderm explants from stage X to XI embryos, either Lefty1 or Cerberus-S inhibited initial migration of the explants on chamber slides. In vivo analysis showed that inhibition of Nodal signaling by Lefty1 affected the movement of DiI-marked epiblast cells prior to the formation of primitive streak. In Lefty1-treated embryos without a primitive streak, Brachyury expression showed a patchy distribution. However, SU5402 did not affect the movement of DiI-marked epiblast cells. Multi-cellular rosette, which is thought to be involved in epithelial morphogenesis, was found predominantly in the posterior half of the epiblast, and Lefty1 inhibited the formation of rosettes. Three-dimensional reconstruction showed two types of rosette, one with a protruding cell, the other with a ventral hollow. Our results suggest that Nodal signaling may have a pivotal role in the morphogenetic movements of epithelial epiblast including Polonaise movements and formation of multi-cellular rosette.