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Biomedical subjects

Margaret L Kirby

Publications and source records attributed to Margaret L Kirby.

At least 19 recordsLinked to original sources
PubMed2006

Model systems for the study of heart development and disease. Cardiac neural crest and conotruncal malformations.

Neural crest cells are multipotential cells that delaminate from the dorsal neural tube and migrate widely throughout the body. A subregion of the cranial neural crest originating between the otocyst and somite 3 has been called "cardiac neural crest" because of the importance of these cells in heart development. Much of what we know about the contribution and function of the cardiac neural crest in cardiovascular development has been learned in the chick embryo using quail-chick chimeras to study neural crest migration and derivatives as well as using ablation of premigratory neural crest cells to study their function. These studies show that cardiac neural crest cells are absolutely required to form the aorticopulmonary septum dividing the cardiac arterial pole into systemic and pulmonary circulations. They support the normal development and patterning of derivatives of the caudal pharyngeal arches and pouches, including the great arteries and the thymus, thyroid and parathyroids. Recently, cardiac neural crest cells have been shown to modulate signaling in the pharynx during the lengthening of the outflow tract by the secondary heart field. Most of the genes associated with cardiac neural crest function have been identified using mouse models. These studies show that the neural crest cells may not be the direct cause of abnormal cardiovascular development but they are a major component in the complex tissue interactions in the caudal pharynx and outflow tract. Since, cardiac neural crest cells span from the caudal pharynx into the outflow tract, they are especially susceptible to any perturbation in or by other cells in these regions. Thus, understanding congenital cardiac outflow malformations in human sequences of malformations as represented by the DiGeorge syndrome will necessarily require understanding development of the cardiac neural crest.

Animals↗
PubMed2006

Cardiac neural crest ablation inhibits compaction and electrical function of conduction system bundles.

Retroviral and transgenic lineage-tracing studies have shown that neural crest cells associate with the developing bundles of the ventricular conduction system. Whereas this migration of cells does not provide progenitors for the myocardial cells of the conduction system, the question of whether neural crest affects the differentiation and/or function of cardiac specialized tissues continues to be of interest. Using optical mapping of voltage-sensitive dye, we determined that ventricles from chick embryos in which the cardiac neural crest had been laser ablated did not progress to apex-to-base activation by the expected stage [i.e., Hamburger and Hamilton (HH) 35] but instead maintained basal breakthroughs of epicardial activation consistent with immature function of the conduction system. In direct studies of activation, waves of depolarization originating from the His bundle were found to be uncommon in control hearts from HH34 and HH35 embryos. However, activations propagating from septal base, at or near the His bundle, occurred frequently in hearts from HH34 and HH35 neural crest-ablated embryos. Consistent with His bundle cells maintaining electrical connections with adjacent working myocytes, histological analyses of hearts from neural crest-ablated embryos revealed His bundles that had not differentiated a lamellar organization or undergone a process of compaction and separation from surrounding myocardium observed in controls. Furthermore, measurements on histological sections from optically mapped hearts indicated that, whereas His bundle diameter in control embryos thinned by almost one-half between HH30 and HH34, the His bundle in ablated embryos underwent no such compaction in diameter, maintaining a thickness at HH30, HH32, and HH34 similar to that observed in HH30 controls. We conclude that the cardiac neural crest is required in a novel function involving lamellar compaction and electrical isolation of the basally located His bundle from surrounding myocardium.

Animals↗
PubMed2006

Cardiac arterial pole alignment is sensitive to FGF8 signaling in the pharynx.

Morphogenesis of the cardiac arterial pole is dependent on addition of myocardium and smooth muscle from the secondary heart field and septation by cardiac neural crest cells. Cardiac neural crest ablation results in persistent truncus arteriosus and failure of addition of myocardium from the secondary heart field leading to malalignment of the arterial pole with the ventricles. Previously, we have shown that elevated FGF signaling after neural crest ablation causes depressed Ca2+ transients in the primary heart tube. We hypothesized that neural crest ablation results in elevated FGF8 signaling in the caudal pharynx that disrupts secondary heart field development. In this study, we show that FGF8 signaling is elevated in the caudal pharynx after cardiac neural crest ablation. In addition, treatment of cardiac neural crest-ablated embryos with FGF8b blocking antibody or an FGF receptor blocker rescues secondary heart field myocardial development in a time- and dose-dependent manner. Interestingly, reduction of FGF8 signaling in normal embryos disrupts myocardial secondary heart field development, resulting in arterial pole malalignment. These results indicate that the secondary heart field myocardium is particularly sensitive to FGF8 signaling for normal conotruncal development, and further, that cardiac neural crest cells modulate FGF8 signaling in the caudal pharynx.

Animals↗
PubMed2006

Solving an enigma: arterial pole development in the zebrafish heart.

It is a widely held belief that the arterial pole of the zebrafish heart is unusual among models of comparative cardiogenesis. This is based, in part, on the report that the bulbus arteriosus undergoes a striated-to-smooth muscle phenotypic transition during development. An implication of this is that the zebrafish, a model almost ubiquitously accepted in other fields of comparative biology, may be poorly suited to the study of conotruncal abnormalities in human disease. However, while the use of atrioventricular-specific molecular markers has allowed extensive characterization of the development of the atrium and ventricle, the lack of any bulbus-specific markers has meant that this region of the zebrafish heart is poorly characterized and quite possibly misunderstood. We have discovered that the fluorescent nitric oxide indicator 4,5-diaminofluorescein diacetate (DAF-2DA) specifically labels the bulbus arteriosus throughout development from approximately 48 h post-fertilization. Therefore, using DAF-2DA and an immunohistochemical approach, we attempted to further characterize the development of the bulbus. We have concluded that no such phenotypic transition occurs, that contrary to current thinking, aspects of zebrafish arterial pole development are evolutionarily conserved, and that the bulbus should not be considered a chamber, being more akin to the arterial trunk(s) of higher vertebrates.

Animals↗
PubMed2006

Patterning of coronary arteries in wildtype and connexin43 knockout mice.

Abnormal patterning of coronary arteries (CAs) is a clinically significant problem, and as yet, few animal models have been systematically investigated for coronary patterning defects. Here we characterized coronary artery (CA) insertion and branching patterns of the proximal coronary stems in the hearts of wildtype and heterozygous connexin43 knockout (Cx43alpha1 KO) mice. This study entailed the use of high-resolution micro CT imaging for three-dimensional coronary reconstructions. MicroCT of 17 wildtype mice showed a remarkably consistent pattern of CA deployment in the normal mouse heart. Two main CA stems are inserted from the left and right into the aorta. The right coronary artery then branches immediately into the right main and the septal-conal branch, while the left coronary artery branches further distally into the circumflex and the anterior descending CA. This patterning of CA anatomy was confirmed by histology, and by using a vascular smooth muscle or endothelial cell specific lacZ reporter gene to delineate the CAs. A parallel analysis of 25 heterozygous Cx43alpha1 KO mouse hearts showed 22 had defects in patterning of the CAs. They exhibited a wide variation in CA anatomy, including abnormal origin and course of the main CA stems, multiple accessories, and dual septal-conal branches. Overall, these studies show loss of one Cx43alpha1 allele (haploinsufficiency) leads to a high incidence of coronary patterning defects. These findings suggest CA patterning is sensitive to Cx43alpha1 gene dosage.

Animals↗
PubMed2005

Connexin43 deficiency causes dysregulation of coronary vasculogenesis.

The connexin43 knockout (Cx43alpha1 KO) mouse dies at birth from outflow obstruction associated with infundibular pouches. To elucidate the origin of the infundibular pouches, we used microarray analysis to investigate gene expression changes in the pouch tissue. We found elevated expression of many genes encoding markers for vascular smooth muscle (VSM), endothelial cells, and fibroblasts, cell types that are epicardially derived and essential for coronary vasculogenesis. This was accompanied by increased expression of VEGF and genes in the TGFbeta and VEGF/Notch/Eph cell-signaling pathways known to regulate vasculogenesis/angiogenesis. Using immunohistochemistry and a VSM lacZ reporter gene, we confirmed an abundance of ectopic VSM and endothelial cells in the infundibular pouch and in some regions of the right ventricle forming secondary pouches. This was associated with distinct thinning of the compact myocardium. TUNEL labeling showed increased apoptosis in the pouch tissue, in agreement with the finding of altered expression of many apoptotic genes. Defects in vascular remodeling were indicated by a marked reduction in the branching complexity of the distal coronary arteries. In the near term KO mouse, we also observed a profusion of large coronary vascular plexuses subepicardially. This was associated with elevated epicardial expression of VEGF and abnormal epicardial cell morphology. Together, these observations indicate that dysregulated coronary vasculogenesis plays a pivotal role in formation of the infundibular pouches and suggests an essential role for Cx43alpha1 gap junctions in coronary vasculogenesis and vascular remodeling.

Animals↗
PubMed2005

Ablation of the secondary heart field leads to tetralogy of Fallot and pulmonary atresia.

Recent studies in chick and mouse embryos have identified a previously unrecognized secondary heart field (SHF), located in the ventral midline splanchnic mesenchyme, which provides additional myocardial cells to the outflow tract as the heart tube lengthens during cardiac looping. In order to further delineate the contribution of this secondary myocardium to outflow development, we labeled the right SHF of Hamburger-Hamilton (HH) stage 14 chick embryos via microinjection of DiI/rhodamine and followed the fluorescently labeled cells over a 96-h time period. These experiments confirmed the movement of the SHF into the outflow and its spiraling migration distally, with the right side of the SHF contributing to the left side of the outflow. In contrast, when the right SHF was labeled at HH18, the fluorescence was limited to the caudal wall of the lengthening aortic sac. We then injected a combination of DiI and neutral red dye, and ablated the SHF in HH14 or 18 chick embryos. Embryos were allowed to develop until day 9, and harvested for assessment of outflow alignment. Of the embryos ablated at HH14, 76% demonstrated cardiac defects including overriding aorta and pulmonary atresia, while none of the sham-operated controls were affected. In addition, the more severely affected embryos demonstrated coronary artery anomalies. The embryos ablated at HH18 also manifested coronary artery anomalies but maintained normal outflow alignment. Therefore, the myocardium added to the outflow by the SHF at earlier stages is required for the elongation and appropriate alignment of the outflow tract. However, at later stages, the SHF contributes to the smooth muscle component of the outflow vessels above the pulmonary and aortic valves which is important for the development of the coronary artery stems. This work suggests a role for the SHF in a subset of congenital heart defects that have overriding aorta and coronary artery anomalies, such as tetralogy of Fallot and double outlet right ventricle.

Animals↗
PubMed2005

Cardiac neural crest is necessary for normal addition of the myocardium to the arterial pole from the secondary heart field.

In cardiac neural-crest-ablated embryos, the secondary heart field fails to add myocardial cells to the outflow tract and elongation of the tube is deficient. Since that study, we have shown that the secondary heart field provides both myocardium and smooth muscle to the arterial pole. The present study was undertaken to determine whether addition of both cell types is disrupted after neural crest ablation. Marking experiments confirm that the myocardial component fails to be added to the outflow tract after neural crest ablation. The cells destined to go into the outflow myocardium fail to migrate and are left at the junction of the outflow myocardium with the nascent smooth muscle at the base of the arterial pole. In contrast, the vascular smooth muscle component is added to the arterial pole normally after neural crest ablation. When the myocardium is not added to the outflow tract, the point where the outflow joins the pharynx does not move caudally as it normally should, the aortic sac is smaller and fails to elongate resulting in abnormal connections of the outflow tract with the caudal aortic arch arteries.

Animals↗
PubMed2005

Secondary heart field contributes myocardium and smooth muscle to the arterial pole of the developing heart.

The arterial pole of the heart is the region where the ventricular myocardium continues as the vascular smooth muscle tunics of the aorta and pulmonary trunk. It has been shown that the arterial pole myocardium derives from the secondary heart field and the smooth muscle tunic of the aorta and pulmonary trunk derives from neural crest. However, this neural crest-derived smooth muscle does not extend to the arterial pole myocardium leaving a region at the base of the aorta and pulmonary trunk that is invested by vascular smooth muscle of unknown origin. Using tissue marking and vascular smooth muscle markers, we show that the secondary heart field, in addition to providing myocardium to the cardiac outflow tract, also generates prospective smooth muscle that forms the proximal walls of the aorta and pulmonary trunk. As a result, there are two seams in the arterial pole: first, the myocardial junction with secondary heart field-derived smooth muscle; second, the secondary heart field-derived smooth muscle with the neural crest-derived smooth muscle. Both of these seams are points where aortic dissection frequently occurs in Marfan's and other syndromes.

Animals↗
PubMed2004

Beta-arrestin 2 regulates zebrafish development through the hedgehog signaling pathway.

beta-arrestins are multifunctional proteins that act as scaffolds and transducers of intracellular signals from heptahelical transmembrane-spanning receptors (7TMR). Hedgehog (Hh) signaling, which uses the putative 7TMR, Smoothened, is established as a fundamental pathway in development, and unregulated Hh signaling is associated with certain malignancies. Here, we show that the functional knockdown of beta-arrestin 2 in zebrafish embryos recapitulates the many phenotypes of Hh pathway mutants. Expression of wild-type beta-arrestin 2, or constitutive activation of the Hh pathway downstream of Smoothened, rescues the phenotypes caused by beta-arrestin 2 deficiency. These results suggest that a functional interaction between beta-arrestin 2 and Smoothened may be critical to regulate Hh signaling in zebrafish development.

Animals↗
PubMed2004

ENU induced mutations causing congenital cardiovascular anomalies.

We used non-invasive high frequency ultrasound to screen N-ethyl-N-nitrosourea mutagenized mouse fetuses for congenital cardiovascular anomalies. We ultrasound scanned 7546 mouse fetuses from 262 mutagenized families, and identified 124 families with cardiovascular defects. Represented were most of the major congenital cardiovascular anomalies seen clinically. The ENU-induced mutations in several families were mapped using polymorphic microsatellite DNA markers. One family with forelimb anomalies and ventricular septal defects, phenotypes similar to Holt-Oram syndrome, and one family with transposition of the great arteries and heart situs anomalies were mapped to different regions of mouse chromosome 4. A third mutation causing persistent truncus arteriosus and craniofacial defects, phenotypes reminiscent of DiGeorge syndrome, was mapped to mouse chromosome 2. We note that mouse chromosomes 4 and 2 do not contain Tbx5 or Tbx1, genes previously linked to Holt-Oram and DiGeorge syndromes, respectively. In two other families, the ENU-induced mutation was identified--Sema3CL605P was associated with persistent truncus arteriosus with interrupted aortic arch, and the Gja1W45X connexin43 mutation caused conotruncal malformation and coronary aneurysms. Although our screen was designed as a recessive screen, a number of the mutations showed cardiovascular phenotypes in both heterozygote and homozygote animals. These studies show the efficacy of ENU mutagenesis and high-throughput ultrasound phenotyping in recovering mutations causing a wide spectrum of congenital heart defects. These ENU-induced mutations hold promise in yielding new insights into the genetic basis for human congenital heart disease.

Amino Acid Sequence↗
PubMed2004

Neurotrophin-3 and TrkC are expressed in the outflow tract of the developing chicken heart.

Transcripts encoding trkC and full-length (catalytic) TrkC receptors were detected in the outflow tract of the chicken heart during early development (stage 17; embryonic day [E] 2.5) before the start of septation. Expression of trkC mRNA persisted through early septation (stage 25, E4.5-E5) but was no longer evident by the end of septation (stage 34, E8). Neurotrophin-3 (NT-3) mRNA was also shown to be present in the outflow tract throughout cardiac development. Quail-chick chimeras were used to confirm that cardiac neural crest cells were not present in the outflow tract at stage 17 (E2.5). Our results show that NT-3 interacts with cells in the outflow tract that are not of neural crest origin. This finding indicates that, in addition to effects on neural crest cells, NT-3 may be important for cardiac development due to its interaction with cells in the outflow tract such as those arising from the secondary heart field.

Animals↗
PubMed2004

[Studies on morphogenesis and visualization of the early embryonic heart with regard to the development of conotruncal heart defects].

Most congenital cardiovascular malformations have their origins during early morphogenesis, and some forms of adult-onset cardiovascular disease also arise during embryonic development. Conotruncal heart defects comprise a major category of congenital heart disease and are found in children with a relative high frequency. These defects are associated with a high mortality risk in utero, and after postnatal surgical repair; embryologically they are linked with dextroposed aorta, which is an anomaly of the ventricular outflow tract with malalignment of the great arteries. The etiology and pathogenesis of dextroposed aorta is not known but is thought to be due to abnormal looping and/or incorrect "wedging" of the outflow tract (i.e., wedged positioning of the aorta between the atrioventricular valves) during early heart development. We have studied the morphology and visual development of the embryonic heart in an animal model of dextropsed aorta in a series of experiments to determine possible mechanisms for dextropositioning of the aorta. At this, we have employed besides established methods for analysis of anatomy and pathology (morphological studies, cardiac morphometry, histology, scanning electron microscopy and immunhistochemistry) also new imaging techniques (videocinephotography and time-lapse studies with a digital high-speed video camera, confocal and scanning electron microscopy, optical coherence tomography (OCT) and magnetic resonance microscopy (MRM) for 3D reconstruction of the heart) to achieve a better visualization of normal and pathological changes during heart development. The paper at hand summarizes the results of these studies.

Adult↗
PubMed2003

Hensen's node gives rise to the ventral midline of the foregut: implications for organizing head and heart development.

Patterning of the ventral head has been attributed to various cell populations, including endoderm, mesoderm, and neural crest. Here, we provide evidence that head and heart development may be influenced by a ventral midline endodermal cell population. We show that the ventral midline endoderm of the foregut is generated directly from the extreme rostral portion of Hensen's node, the avian equivalent of the Spemann organizer. The endodermal cells extend caudally in the ventral midline from the prechordal plate during development of the foregut pocket. Thus, the prechordal plate appears as a mesendodermal pivot between the notochord and the ventral foregut midline. The elongating ventral midline endoderm delimits the right and left sides of the ventral foregut endoderm. Cells derived from the midline endoderm are incorporated into the endocardium and myocardium during closure of the foregut pocket and fusion of the bilateral heart primordia. Bilateral ablation of the endoderm flanking the midline at the level of the anterior intestinal portal leads to randomization of heart looping, suggesting that this endoderm is partitioned into right and left domains by the midline endoderm, thus performing a function similar to that of the notochord in maintaining left-right asymmetry. Because of its derivation from the dorsal organizer, its extent from the forebrain through the midline of the developing face and pharynx, and its participation in formation of a single midline heart tube, we propose that the ventral midline endoderm is ideally situated to function as a ventral organizer of the head and heart.

Animals↗
PubMed2003

Neural crest and cardiovascular development: a 20-year perspective.

BACKGROUND: Twenty years ago this year was the first publication describing a region of neural crest cells necessary for normal cardiovascular development. Ablation of this region in chick resulted in persistent truncus arteriosus, mispatterning of the great vessels, outflow malalignments, and hypoplasia or aplasia of the pharyngeal glands. METHODS: We begin with a historical perspective and then review the progress that has been made in the ensuing 20 years in determining the direct and indirect contributions of the neural crest cells, now termed cardiac neural crest cells, in cardiovascular and pharyngeal arch development. Many of the molecular pathways that are now known to influence the specification, migration, patterning and final targeting of the cardiac neural crest cells are also reviewed. RESULTS: Although much knowledge has been gained by using many genetic manipulations to understand the cardiac neural crest cells' role in cardiovascular development, most models fail to explain the phenotypes seen in syndromic and non-syndromic human congenital heart defects, such as the DiGeorge syndrome. CONCLUSIONS: We propose that the cardiac neural crest exists as part of a larger cardiocraniofacial morphogenetic field and describe several human syndromes that result from abnormal development of this field.

Abnormalities, Multiple↗