Biomedical subjects

Takashi Mikawa

Publications and source records attributed to Takashi Mikawa.

At least 19 recordsLinked to original sources

PubMed2006

Bone marrow Oct3/4+ cells differentiate into cardiac myocytes via age-dependent paracrine mechanisms.

The mechanisms that govern the capacity of the bone marrow stem cells to generate cardiac myocytes are still unknown. Herein we demonstrate that the cardiomyogenic potential of bone marrow-derived Oct3/4(+)/cKit(+/-)/CXCR4(+/-)/CD34(-)/Sca1(-) cells is governed by age-dependent paracrine/juxtacrine platelet-derived growth factor (PDGF) pathways. Specifically, bone marrow cell cultures from both 3- and 18-month-old mice formed aggregates of Oct3/4(+) cells circumscribed by PDGFRalpha(+)/Oct3/4(-)/Sca1(+) cells. In young (3-month) bone marrow cell cultures, induction of PDGF-AB preceded the induction of cardiac genes and was required for the generation of cardiomyogenesis. Indeed, in old (18-month) cultures, diminished PDGF-B induction was associated with impaired cardiomyogenic potential, despite having Oct3/4 levels similar to those in the young cells. Importantly, supplementation with PDGF-AB specifically restored the cardiac differentiation capacity of the old bone marrow cells. Together these results demonstrate that, regardless of age, the bone marrow niche contains Oct3/4 stem cells that are capable of differentiating into cardiac myocytes. Moreover, this differentiation is governed by age-dependent PDGF-AB-mediated paracrine/juxtacrine pathways that may be essential in the translation of bone marrow cell-mediated cardiomyogenesis.

Animals↗

PubMed2006

TrkB/BDNF signaling regulates photoreceptor progenitor cell fate decisions.

Neurotrophins, via activation of Trk receptor tyrosine kinases, serve as mitogens, survival factors and regulators of arborization during retinal development. Brain-derived neurotrophic factor (BDNF) and TrkB regulate neuronal arborization and survival in late retinal development. However, TrkB is expressed during early retinal development where its functions are unclear. To assess TrkB/BDNF actions in the early chick retina, replication-incompetent retroviruses were utilized to over-express a dominant negative truncated form of TrkB (trunc TrkB), or BDNF and effects were assessed at E15. Clones expressing trunc TrkB were smaller than controls, and proliferation and apoptosis assays suggest that decreased clone size correlated with increased cell death when BDNF/TrkB signaling was impaired. Analysis of clonal composition revealed that trunc TrkB over-expression decreased photoreceptor numbers (41%) and increased cell numbers in the middle third of the inner nuclear layer (INL) (23%). Conversely, BDNF over-expression increased photoreceptor numbers (25%) and decreased INL numbers (17%). Photoreceptors over-expressing trunc TrkB demonstrated no increase in apoptosis nor abnormalities in lamination suggesting that TrkB activation is not required for photoreceptor cell survival or migration. These studies suggest that TrkB signaling regulates commitment to and/or differentiation of photoreceptor cells from retinal progenitor cells, identifying a novel role for TrkB/BDNF in regulating cell fate decisions.

Animals↗

PubMed2006

VEGF family members regulate myocardial tubulogenesis and coronary artery formation in the embryo.

This study tested the hypothesis that coronary tubulogenesis and coronary artery formation require VEGF family members. Quail embryos were injected with soluble vascular endothelial growth factor (VEGF) receptors R1 (Flt-1), R2 (Flk-1), R3 (Flt-4), VEGF-Trap (a chimera of R1 and R2), or neutralizing antibodies to VEGF-A, VEGF-B, or fibroblast growth factor (FGF)-2. Our data document that tubulogenesis is temporally dependent on multiple VEGF family members, because the early stage of tubulogenesis was markedly inhibited by VEGF-Trap and to a lesser extent by soluble VEGFR-1. Some inhibition of tubulogenesis was documented when anti-FGF-2, but not anti-VEGF-A, antibodies were injected at embryonic day 6 (E6). Most importantly, we found that VEGF-Trap injected at either E6 or E7 prevented the formation of coronary arteries. Soluble VEGFR-1 and soluble VEGFR-2 modified the formation of coronary arteries, whereas soluble VEGFR-3 was without effect. Antibodies to VEGF-B, but not VEGF-A, had a strong inhibitory effect on coronary artery development. The absence of coronary artery stems, and thus a functional coronary circulation, in the embryos injected with VEGF-Trap caused an accumulation of erythrocytes in the subepicardium and muscular interventricular septum. Using retroviral cell tagging, we showed that some of the erythrocytes in blood islands and small vascular tubes were progeny of the proepicardium. Thus, another salient finding of this study is the first definitive documentation of proepicardially derived hemangioblasts, which can differentiate into erythrocytes.

Animals↗

PubMed2006

Enhanced sensitivity and stability in two-color in situ hybridization by means of a novel chromagenic substrate combination.

Double in situ hybridization analysis is a fundamental technique for studying the expression of two genes with high temporal and spatial resolution. However, due to the lack of sensitivity in current detection methods, this approach is powerful only when at least one transcript is abundantly expressed. Here, we report a new enzyme/chromagenic substrate combination that provides sufficient sensitivity for detecting two less abundant transcripts and stability for subsequent paraffin sectioning.

Animals↗

PubMed2005

Normal patterning of the coronary capillary plexus is dependent on the correct transmural gradient of FGF expression in the myocardium.

The formation of the coronary vessel system is vital for heart development, an essential step of which is the establishment of a capillary plexus that displays a density gradient across the myocardial wall, being higher on the epicardial than the endocardial side. This gradient in capillary plexus formation develops concurrently with transmural gradients of myocardium-derived growth factors, including FGFs. To test the role of the FGF expression gradient in patterning the nascent capillary plexus, an ectopic FGF-over-expressing site was created in the ventricular myocardial wall in the quail embryo via retroviral infection from E2-2.5, thus abolishing the transmural gradient of FGFs. In FGF virus-infected regions of the ventricular myocardium, the capillary density across the transmural axis shifted away from that in control hearts at E7. This FGF-induced change in vessel patterning was more profound at E12, with the middle zone becoming the most vascularized. An up-regulation of FGFR-1 and VEGFR-2 in epicardial and subepicardial cells adjacent to FGF virus-infected myocardium was also detected, indicating a paracrine effect on induction of vascular signaling components in coronary precursors. These results suggest that correct transmural patterning of coronary vessels requires the correct transmural expression of FGF and, therefore, FGF may act as a template for coronary vessel patterning.

Animals↗

PubMed2005

Somatic transgenesis in the avian model system.

The chick embryo is a versatile model system, in which classical embryology can be combined with modern molecular approaches. In the last two decades, several efficient methods have been developed to introduce exogenous genes into the chick embryo. These techniques allow alteration of gene expression levels in a spatially and temporally restricted manner, thereby circumventing embryonic lethality and/or eliminating secondary effects in other tissues. Here, we present the current status of avian somatic transgenic techniques, focusing on electroporation and retrovirus-mediated gene transfer. Electroporation allows quick and efficient gain-of-function studies based on transient misexpression of genes. Retroviral vectors, which are capable of integrating exogenous genes into the host chromosome, permit analysis of long-term effects of gene misexpression. The variety of methods available for somatic transgenesis, along with the recent completion of the chicken genome, are transforming the chick embryo into one of the most attractive model systems to examine function of genes that are important for embryonic development.

Animals↗

PubMed2004

A role for Tbx5 in proepicardial cell migration during cardiogenesis.

Transcriptional regulatory cascades during epicardial and coronary vascular development from proepicardial progenitor cells remain to be defined. We have used immunohistochemistry of human embryonic tissues to demonstrate that the TBX5 transcription factor is expressed not only in the myocardium, but also throughout the embryonic epicardium and coronary vasculature. TBX5 is not expressed in other human fetal vascular beds. Furthermore, immunohistochemical analyses of human embryonic tissues reveals that unlike their epicardial counterparts, delaminating epicardial-derived cells do not express TBX5 as they migrate through the subepicardium before undergoing epithelial-mesenchymal transformation required for coronary vasculogenesis. In the chick, Tbx5 is expressed in the embryonic proepicardial organ (PEO), which is composed of the epicardial and coronary vascular progenitor cells. Retrovirus-mediated overexpression of human TBX5 inhibits cell incorporation of infected proepicardial cells into the nascent chick epicardium and coronary vasculature. TBX5 overexpression as well as antisense-mediated knockdown of chick Tbx5 produce a cell-autonomous defect in the PEO that prevents proepicardial cell migration. Thus, both increasing and decreasing Tbx5 dosage impairs development of the proepicardium. Culture of explanted PEOs demonstrates that untreated chick proepicardial cells downregulate Tbx5 expression during cell migration. Therefore, we propose that Tbx5 participates in regulation of proepicardial cell migration, a critical event in the establishment of the epicardium and coronary vasculature.

Animals↗

PubMed2004

Platelet-derived growth factor-AB promotes the generation of adult bone marrow-derived cardiac myocytes.

The directed generation of cardiac myocytes from endogenous stem cells offers the potential for novel therapies for cardiovascular disease. To facilitate the development of such approaches, we sought to identify and exploit the pathways directing the generation of cardiac myocytes from adult rodent bone marrow cells (BMCs). In vitro cultures supporting the spontaneous generation of functional cardiac myocytes from murine BMCs demonstrated induced expression of platelet-derived growth factor (PDGF)-A and -B isoforms with alpha- and beta-myosin heavy chains as well as connexin43. Supplementation of PDGF-AB speeded the kinetics of myocyte development in culture by 2-fold. In a rat heart, myocardial infarction pretreatment model PDGF-AB also promoted the derivation of cardiac myocytes from BMCs, resulting in a significantly greater number of islands of cardiac myocyte bundles within the myocardial infarction scar compared with other treatment groups. However, gap junctions were detected only between the cardiac myocytes receiving BMCs alone, but not BMCs injected with PDGF-AB. Echocardiography and exercise testing revealed that the functional improvement of hearts treated with the combination of BMCs and PDGF-AB was no greater than with injections of BMCs or PDGF-AB alone. These studies demonstrated that PDGF-AB enhances the generation of BMC-derived cardiac myocytes in rodent hearts, but suggest that alterations in cellular patterning may limit the functional benefit from the combined injection of PDGF-AB and BMCs. Strategies based on the synergistic interactions of PDGF-AB and endogenous stem cells will need to maintain cellular patterning in order to promote the restoration of cardiac function after acute coronary occlusion.

Animals↗

PubMed2004

Hemodynamic-dependent patterning of endothelin converting enzyme 1 expression and differentiation of impulse-conducting Purkinje fibers in the embryonic heart.

Impulse-conducting Purkinje fibers differentiate from myocytes during embryogenesis. The conversion of contractile myocytes into conduction cells is induced by the stretch/pressure-induced factor, endothelin (ET). Active ET is produced via proteolytic processing from its precursor by ET-converting enzyme 1 (ECE1) and triggers signaling by binding to its receptors. In the embryonic chick heart, ET receptors are expressed by all myocytes, but ECE1 is predominantly expressed in endothelial cells of coronary arteries and endocardium along which Purkinje fiber recruitment from myocytes takes place. Furthermore, co-expression of exogenous ECE1 and ET-precursor in the embryonic heart is sufficient to ectopically convert cardiomyocytes into Purkinje fibers. Thus, localized expression of ECE1 defines the site of Purkinje fiber recruitment in embryonic myocardium. However, it is not known how ECE1 expression is regulated in the embryonic heart. The unique expression pattern of ECE1 in the embryonic heart suggests that blood flow-induced stress/stretch may play a role in patterning ECE1 expression and subsequent induction of Purkinje fiber differentiation. We show that gadolinium, an antagonist for stretch-activated cation channels, downregulates the expression of ECE1 and a conduction cell marker, Cx40, in ventricular chambers, concurrently with delayed maturation of a ventricular conduction pathway. Conversely, pressure-overload in the ventricle by conotruncal banding results in a significant expansion of endocardial ECE1 expression and Cx40-positive putative Purkinje fibers. Coincident with this, an excitation pattern typical of the mature heart is precociously established. These in vivo data suggest that biomechanical forces acting on, and created by, the cardiovascular system during embryogenesis play a crucial role in Purkinje fiber induction and patterning.

Animals↗

PubMed2004

Developmental transitions in electrical activation patterns in chick embryonic heart.

The specialized conduction tissue network mediates coordinated propagation of electrical activity through the adult vertebrate heart. Following activation of the atria, the activation wave is slowed down in the atrioventricular canal or node, then spreads rapidly into the left and right ventricles via the His-Purkinje system (HPS). This results in the ventricle being activated from the apex toward the base and is thought to represent HPS function. The development of mature HPS function in embryogenesis follows significant phases of cardiac morphogenesis. Initially, cardiac impulse propagates in a slow, linear, and isotropic fashion from the sinus venosus at the most caudal portion of the tubular heart. Although the speed of impulse propagation gradually increases, ventricular activation in the looped heart still follows the direction of blood flow. Eventually, the immature base-to-apex sequence of ventricular activation undergoes an apparent reversal, maturing to apex-to-base pattern. The embryonic chick heart has been studied intensively by both electrophysiological and morphological techniques, and the morphology of its conduction system (which is similar to mammals) is well characterized. One interesting but seldom studied feature is the anterior septal branch (ASB), which came sharply to focus (together with the rest of the ventricular conduction system) in our birthdating studies. Using an optical mapping approach, we show that ASB serves to activate ventricular surface between stages 16 and 25, predating the functionality of the His bundle/bundle branches. Heart morphogenesis and conduction system formation are thus linked, and studying the abnormal activation patterns could further our understanding of pathogenesis of congenital heart disease.

Action Potentials↗

PubMed2004

Induction and patterning of the primitive streak, an organizing center of gastrulation in the amniote.

The primitive streak is the organizing center for amniote gastrulation. It defines the future embryonic midline and serves as a conduit of cell migration for germ layer formation. The migration patterns of endodermal and mesodermal precursors through the streak have been studied in great detail. Additional new breakthroughs recently have revealed the cell biological and molecular mechanisms that govern streak induction and patterning. These findings include (1) identification of the ontogeny and inductive signals of streak precursors, (2) the potential cellular mechanism of streak extension, and (3) the molecular and functional diversification along the anterior-posterior and mediolateral axes within the primitive streak. These findings indicate that amniote embryos initiate gastrulation by using both evolutionarily conserved and divergent mechanisms. The data also provide a foundation for understanding how the midline axis is defined and maintained during gastrulation of the amniotes.

Amniotic Fluid↗

PubMed2004

Folding of the tectal cortex by local remodeling of neural differentiation.

The folding pattern of the brain cortex is a precisely regulated process, but the mechanism involved during development remains unclear. A proposed theory predicts that the initiation of cortical folding depends, at least partly, on nonuniform distribution of neuronal differentiation and neurite growth. We tested this theory experimentally, by remodeling the normal pattern of neuronal cell differentiation within the embryonic optic tectum. Multiple foci of activated fibroblast growth factor signaling were created in the tectal cortex to locally change the neural differentiation and axonal growth patterns. At these foci, tectal cells remained undifferentiated and their radial and tangential migration was suppressed. These local changes in the neuronal cell differentiation resulted in a conversion of the tectal cortex from smoothly extended into precociously folded. The results provide in vivo experimental evidence that microscopic changes in the neuronal cell differentiation pattern can induce or remodel the folding pattern of the brain cortex.

Animals↗

PubMed2004

Somatic transgenesis using retroviral vectors in the chicken embryo.

The avian embryo is an excellent model system for experimental studies because of its accessibility and ease of microsurgical manipulations. While the complete chicken genome sequence will soon be determined, a comprehensive germ cell transmission-based genetic approach is not available for this animal model. Several techniques of somatic cell transgenesis have been developed in the past decade. Of these, the retroviral shuttle vector system provides both (1) stable integration of exogenous genes into the host cell genome, and (2) constant expression levels in a target cell population over the course of development. This review summarizes retroviral vectors available for the avian model and outlines the uses of retroviral-mediated gene transfer for cell lineage analysis as well as functional studies of genes and proteins in the chick embryo.

Animals↗

PubMed2004

Negative regulation of midline vascular development by the notochord.

The negative regulation of vascular patterning is one of the least understood processes in vascular biology. In amniotes, blood vessels develop throughout the embryonic disc, except for a midline region surrounding the notochord. Here we show that the notochord is the primary signaling center for the inhibition of vessel formation along the embryonic midline. Notochord ablation in quail embryos results in vascular plexus formation at midline. Implantation of the notochord into paraxial and lateral mesoderm inhibits vessel formation locally. The notochord-expressed BMP antagonists Chordin and Noggin inhibit endothelial cell migration in vitro, and their ectopic expression in vivo results in a local disruption of vessel formation. Conversely, BMP-4 activates endothelial cell migration in vitro, and its ectopic expression along the notochord induces vascular plexus formation at midline. These data indicate an inhibitory role of the notochord in defining an avascular zone at the embryonic midline, in part via BMP antagonism.

Animals↗

PubMed2003

Optic cup morphogenesis requires pre-lens ectoderm but not lens differentiation.

The formation of the vertebrate optic cup is a morphogenetic event initiated after the optic vesicle contacts the overlying surface/pre-lens ectoderm. Placodes form in both the optic neuroepithelium and lens ectoderm. Subsequently, both placodes invaginate to form the definitive optic cup and lens, respectively. We examined the role of the lens tissue in inducing and/or maintaining optic cup invagination in ovo. Lens tissue was surgically removed at various stages of development, from pre-lens ectoderm stages to invaginating lens placode. Removal of the pre-lens ectoderm resulted in persistent optic vesicles that initiated neural retinal differentiation but failed to invaginate. In striking contrast, ablation of the lens placode gave rise to optic vesicles that underwent invagination and formed the optic cup. The results suggest that: (1) the optic vesicle neuroepithelium requires a temporally specific association with pre-lens ectoderm in order to undergo optic cup morphogenesis; and (2) the optic cup can form in the absence of lens formation. If ectopic BMP is added, a neural retina does not develop and optic cup morphogenesis fails, although lens formation appears normal. FGF-induced neural retina differentiation in the absence of the pre-lens ectoderm is not sufficient to create an optic cup. We hypothesize the presence of a signal coming from the pre-lens ectoderm that induces the optic vesicle to form an optic cup.

Animals↗

PubMed2003

Fibroblast growth factor (FGF)-4 can induce proliferation of cardiac cushion mesenchymal cells during early valve leaflet formation.

While much has been learned about how endothelial cells transform to mesenchyme during cardiac cushion formation, there remain fundamental questions about the developmental fate of cushions. In the present work, we focus on the growth and development of cushion mesenchyme. We hypothesize that proliferative expansion and distal elongation of cushion mesenchyme mediated by growth factors are the basis of early valve leaflet formation. As a first step to test this hypothesis, we have localized fibroblast growth factor (FGF)-4 protein in cushion mesenchymal cells at the onset of prevalve leaflet formation in chick embryos (Hamburger and Hamilton stage 20-25). Ligand distribution was correlated with FGF receptor (FGFR) expression. In situ hybridization data indicated that FGFR3 mRNA was confined to the endocardial rim of the atrioventricular (AV) cushion pads, whereas FGFR2 was expressed exclusively in cushion mesenchymal cells. FGFR1 expression was detected in both endocardium and cushion mesenchyme as well as in myocardium. To determine whether the FGF pathways play regulatory roles in cushion mesenchymal cell proliferation and elongation into prevalvular structure, FGF-4 protein was added to the cushion mesenchymal cells explanted from stage 24-25 chick embryos. A significant increase in proliferative ability was strongly suggested in FGF-4-treated mesenchymal cells as judged by the incorporation of 5'-bromodeoxyuridine (BrdU). To determine whether cushion cells responded similarly in vivo, a replication-defective retrovirus encoding FGF-4 with the reporter, bacterial beta-galactosidase was microinjected into stage 18 chick cardiac cushion mesenchyme along the inner curvature where AV and outflow cushions converge. As compared with vector controls, overexpression of FGF-4 clearly induced expansion of cushion mesenchyme toward the lumen. To further test the proliferative effect of FGF-4 in cardiac cushion expansion in vivo (ovo), FGF-4 protein was microinjected into stage 18 chick inner curvature. An assay for BrdU incorporation indicated a significant increase in proliferative ability in FGF-4 microinjected cardiac cushion mesenchyme as compared with BSA-microinjected controls. Together, these results suggest a role of FGF-4 for cardiac valve leaflet formation through proliferative expansion of cushion mesenchyme.

Animals↗

PubMed2003

Evidence for an extracellular matrix bridge guiding proepicardial cell migration to the myocardium of chick embryos.

During heart development, the proepicardium (PE) gives rise to cells of the epicardial epithelium, connective tissue of the subepicardium and the myocardium, and smooth muscle, endothelium, and connective tissue of the coronary arteries. The PE arises as an outgrowth of the pericardial serosa at embryonic day 2 (Hamburger and Hamilton stage [HH] 14) of chick development. Between stages HH14 and HH17, multicellular villous projections extend from the PE toward the dorsal aspect of the lesser curvature of the myocardium. On reaching the atrioventricular (AV) junction, the cells spread over the myocardium, eventually enveloping the complete heart surface as a simple squamous epithelium. Although the lineage of the PE cells is well established, it remains uncertain how cells of the PE reach the myocardial surface and specifically target the AV junction. By using a combination of serial section reconstructions, immunofluorescence, and electron microscopy, we have identified an extracellular matrix bridge (ECMB) spanning the coelomic cavity between the PE and the myocardium. The ECMB is first detectable at HH14 and persists until the PE contacts the bare myocardial surface. This ECMB stains intensely with ruthenium red and Alcian blue, contains heparan sulfate and fibronectin, and exhibits both fibrillar and globular ultrastructure, reminiscent of proteoglycans. After PE attachment to the myocardium (HH16-HH17), the subepicardium exhibited strong staining for heparan sulfate. Heparinase injection into the pericardial coelom at HH15 resulted in aberrant development of the primordial epicardium. On the basis of these studies, we suggest that the ECMB may participate in migration and targeting of the PE to the myocardium.

Animals↗

PubMed2003

Epicardium is required for the full rate of myocyte proliferation and levels of expression of myocyte mitogenic factors FGF2 and its receptor, FGFR-1, but not for transmural myocardial patterning in the embryonic chick heart.

Proper heart development requires patterning across the myocardial wall. Early myocardial patterning is characterized by a transmural subdivision of the myocardium into an outer, highly mitotic, compact zone and an inner, trabecular zone with lower mitotic activity. We have shown previously that fibroblast growth factor receptor (FGFR) -mediated signaling is central to myocyte proliferation in the developing heart. Consistent with this, FGFR-1 and FGF2 are more highly expressed in myocytes of the compact zone. However, the mechanism that regulates the transmural pattern of myocyte proliferation and expression of these mitogenic factors is unknown. The present study examined whether this transmural patterning occurs in a myocardium-autonomous manner or by signals from the epicardium. Microsurgical inhibition of epicardium formation in the embryonic chick gives rise to a decrease in myocyte proliferation, accounting for a thinner compact myocardium. We show that the transmural pattern of myocyte mitotic activity is maintained in these hearts. Consistent with this, the expression patterns of FGF1, FGF2, and FGFR-1 across the myocardium persist in the absence of the epicardium. However, FGF2 and FGFR-1 mRNA levels are reduced in proportion to the depletion of epicardium. The results suggest that epicardium-derived signals are essential for maintenance of the correct amount of myocyte proliferation in the compact myocardium, by means of levels of mitogen expression in the myocardium. However, initiation and maintenance of transmural patterning of the myocardium occurs largely independently of the epicardium.

Animals↗