Zebrafish hhex regulates liver development and digestive organ chirality.
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Animals↗
Biomedical subjects
A J Chin
Publications and source records attributed to A J Chin.
The role of vascular endothelial growth factor (VEGF) in vasculogenesis, angiogenesis, and hematopoiesis in zebrafish development.
Vascular endothelial growth factor (VEGF, VEGF-A), a selective mitogen for endothelial cells is a critical factor for vascular development. Two isoforms that differ in the presence of exons 6 and 7, Vegf(165) and Vegf(121), are the dominant forms expressed in zebrafish embryo. Simultaneous overexpression of both isoforms in the embryo results in increased production of flk1, tie1, scl, and gata1 transcripts, indicating a stimulation of both endothelial and hematopoietic lineages. We also demonstrate that vegf can stimulate hematopoiesis in zebrafish by promoting the formation of terminally differentiated red blood cells. Simultaneous overexpression of both isoforms also causes ectopic vasculature and blood cells in many of the injected embryos as well as pericardial edema in later stage embryos. Overexpression of vegf also resulted in earlier onset of flk1, tie1, scl, and gata1 expression in the embryo, indicating a possible role of vegf in stimulating the differentiation of both vascular and hematopoietic lineages. Co-injection of RNAs for both isoforms results in increased expression of three of these markers over and above that observed when either RNA is singly injected and analysis of vegf expression in the notochord mutants no tail and floating head suggests that the notochord patterns the formation of the dorsal aorta by stimulating adjacent somite cells to express vegf, which in turn functions as a signal in dorsal aorta patterning. Finally, studies of vegf expression in cloche mutant indicate that vegf expression is generally independent of cloche function. These results show that in the zebrafish embryo, vegf can not only stimulate endothelial cell differentiation but also hematopoiesis. Moreover, these effects are most dramatic when both vegf isoforms are co-expressed, indicating a synergistic effect of the expression of the two forms of the VEGF protein.
Animals↗
Evaluation and follow-up of patients with left ventricular apical to aortic conduits with 2D and 3D magnetic resonance imaging and Doppler echocardiography: A new look at an old operation.
BACKGROUND: Although the interposition of left ventricular apical to descending aorta conduits has diminished with the advent of the Ross-Konno operation, it remains a useful option. We reviewed our institutional experience imaging these conduits and tested the hypothesis that the gradient across the native left ventricular outflow tract (LVOT) by echocardiography correlated with the conduit gradient by cardiac catheterization. In a patient with an unobstructed conduit, no gradient should exist across the native LVOT. METHODS: This was a retrospective review of the echocardiography, cardiac catheterization, magnetic resonance imaging (MRI) data, and history of 9 patients with these conduits over an 8-year period. In 7 of 9 patients, 8 conduit obstruction events were assessed by Doppler interrogation of the native LVOT and by cardiac catheterization. Five patients underwent 6 MRI scans. RESULTS: In all cases of obstruction diagnosed by catheterization (56.3 +/- 21.9 mm Hg), Doppler echocardiography demonstrated gradients across the native LVOT (69.3 +/- 21.2 mm Hg, r = 0.67). Because 2D echocardiography could not visualize the entire conduit in any patient, 2- and 3-dimensional MRI was used successfully to evaluate anatomy and identify the site of obstruction. All patients manifested conduit obstruction. Four (44%) of 9 patients died, 3 underwent the Ross operation, 1 continues to live with his original conduit, and 1 was lost to follow-up. CONCLUSIONS: A gradient by Doppler interrogation of the native LVOT is an indirect means of assessing conduit obstruction. MRI is a useful tool to complement anatomic diagnosis by echocardiography. Conduit obstruction is common, and late mortality rates appear to be high.
Adolescent↗
Design and baseline characteristics for the ACE Inhibitor After Anthracycline (AAA) study of cardiac dysfunction in long-term pediatric cancer survivors.
PURPOSE: The ACE Inhibitor After Anthracycline (AAA) study is a randomized, double-blind, controlled clinical trial comparing enalapril with placebo to determine whether treatment can slow the progression of cardiac decline in patients who screen positive for anthracycline cardiotoxicity. METHODS: The primary outcome measure is the rate of decline, over time, in maximal cardiac index (in liters per minute per meters squared) at peak exercise; the secondary outcome measure is the rate of increase in left ventricular end systolic wall stress (in grams per centimeters squared). Patients >2 years off therapy and <4 years from diagnosis, aged 8 years and older, were eligible if they had received anthracyclines and had at least one cardiac abnormality identified at any time after anthracycline exposure. RESULTS: A total of 135 patients were randomized to enalapril or placebo. Baseline characteristics were similar across treatment groups. CONCLUSIONS: The AAA study will provide important information concerning the efficacy of using angiotensin-converting enzyme inhibitors to offset the effects of late anthracycline cardiotoxicity.
Adolescent↗
Analysis of Cx43alpha1 promoter function in the developing zebrafish embryo.
The Cx43alpha1 gap junctions play an important role in cardiovascular development. Studies using transgenic mouse models have indicated that this involves an essential role for Cx43alpha1 in modulating neural crest cell motility. We previously showed that a 6.8 kb mouse genomic sequence containing the promoter and upstream regulatory sequences of the Cx43alpha1 gene can drive lacZ reporter gene expression in all neural crest cell lineages in the mouse embryo. To obtain further insights into the sequence motifs and regulatory pathways involved in targeting Cx43alpha1 gene expression in neural crest cells, we assayed the activity of the mouse Cx43alpha1 promoter in evolutionarily distantly related zebrafish embryos. For these studies, the 6.8kb Cx43alpha1 genomic sequence and various deletion derivatives were used to generate GFP or lacZ expression vectors. The transcriptional activities of these constructs were analyzed in vivo after microinjection into one- or two- cell stage zebrafish embryos. These studies indicated that the mouse Cx43alpha1 promoter can drive lacZ expression in neural crest cells in the zebrafish embryos. Analysis by whole mount in situ hybridization showed that the endogenous zebrafish Cx43alpha1 gene is expressed maternally and zygotically, and expression is observed in regions where neural crest cells are found. To further elucidate the developmental regulation of Cx43alpha1 gene expression, we screened a zebrafish BAC library and identified a clone containing the entire zebrafish Cx43alpha1 gene and flanking upstream and downstream sequences. The upstrean Cx43alpha1 promoter sequences from zebrafish, mouse, and human were analyzed for evolutionarily conserved DNA motifs. Overall these studies suggest that the sequence motifs and transcriptional regulation involved in the targeting Cx43alpha1 expression to neural crest cells are evolutionarily conserved in zebrafish and mouse embryos.
Animals↗
Heart and gut chiralities are controlled independently from initial heart position in the developing zebrafish.
A fundamental problem in developmental biology is how left-right (LR) asymmetry is generated, both on the whole organism level and at the level of an individual organ or structure. To investigate the relationship of organ sidedness to organ chirality, we examined 12 zebrafish mutants for initial heart tube position and later heart looping direction (chirality). Anomalous initial heart position was found in seven mutants, which also demonstrated loss of normal LR asymmetry in lateral plate mesoderm (LPM) antivin/lefty-1 and Pitx2 expression. Those with a relatively normal notochord (cyc(b16), din, and spt) displayed a predictive correlation between initial heart position and heart chirality, whereas initial heart position and heart chirality were independently randomized in those with a defective notochord (flh, boz, ntl, and mom). The predictability of heart chirality in spt, din, and b16 embryos, even in the absence of normal antivin/lefty-1 and Pitx2 expression, strongly suggests that heart chirality is controlled by a process distinct from that which controls appropriate left-sided LPM expression of antivin-Pitx2 signaling pathway molecules. In addition, there was correlation of initial heart position with gut chirality (and also between heart chirality and gut chirality) in the first class of mutants with normal notochord, but not in the second class, which appears to model human heterotaxy syndrome.
Animals↗
Right-sided aortic arch with bilateral ductus: a rare case of nonconfluent pulmonary arteries without associated cardiac anomalies.
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Aorta, Thoracic↗
Maternally controlled (beta)-catenin-mediated signaling is required for organizer formation in the zebrafish.
We have identified and characterized a zebrafish recessive maternal effect mutant, ichabod, that results in severe anterior and dorsal defects during early development. The ichabod mutation is almost completely penetrant, but exhibits variable expressivity. All mutant embryos fail to form a normal embryonic shield; most fail to form a head and notochord and have excessive development of ventral tail fin tissue and blood. Abnormal dorsal patterning can first be observed at 3.5 hpf by the lack of nuclear accumulation of (beta)-catenin in the dorsal yolk syncytial layer, which also fails to express bozozok/dharma/nieuwkoid and znr2/ndr1/squint. At the onset of gastrulation, deficiencies in expression of dorsal markers and expansion of expression of markers of ventral tissues indicate a dramatic alteration of dorsoventral identity. Injection of (beta)-catenin RNA markedly dorsalized ichabod embryos and often completely rescued the phenotype, but no measurable dorsalization was obtained with RNAs encoding upstream Wnt pathway components. In contrast, dorsalization was obtained when RNAs encoding either Bozozok/Dharma/Nieuwkoid or Znr2/Ndr1/Squint were injected. Moreover, injection of (beta)-catenin RNA into ichabod embryos resulted in activation of expression of these two genes, which could also activate each other. RNA injection experiments strongly suggest that the component affected by the ichabod mutation acts on a step affecting (beta)-catenin nuclear localization that is independent of regulation of (beta)-catenin stability. This work demonstrates that a maternal gene controlling localization of (beta)-catenin in dorsal nuclei is necessary for dorsal yolk syncytial layer gene activity and formation of the organizer in the zebrafish.
Active Transport, Cell Nucleus↗
Analysis of two aspects of left-right patterning of the vertebrate heart. Heart tube position and heart tube chirality.
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Animals↗
Escherichia coli cAMP receptor protein-DNA complexes. 1. Energetic contributions of half-sites and flanking sequences in DNA recognition.
In Escherichia coli, the cyclic AMP receptor protein (CRP) serves as a sensor of the intracellular level of cyclic AMP. At increasing concentrations of cyclic AMP, CRP becomes activated upon binding a cyclic AMP molecule. The activated CRP is capable of regulating the expression of more than 20 genes by binding to specific DNA sites. The specific DNA sequences recognized by CRP consist of two-half-sites of the consensus sequence TGTGA......XCAXA. At present, the relative contributions of the two half-site and flanking sequences in the energetics of CRP recognition have not been quantitatively defined. A series of 20 DNA sequences was designed to dissect the contributions of individual half-sites and flanking sequences using the natural gal P1 and lac P1 sequences as the initial targets. The binding of CRP to these DNA sequences was monitored by fluorescence anisotropy. None of the individual half-sites or flanking sequences contributes more to the binding energetics than a random sequence. In the lac P1 sequence, the combination of both half-sites leads to a >100-fold increase in affinity compared to that of an individual half-site in CRP-DNA complex formation. The flanking sequence of lac P1 exhibits a 10- and 0-fold enhancement in affinity for CRP compared to that of a random sequence in the presence and absence of the two half-sites, respectively. The observations of the gal P1 sequence differ from those of the lac P1 sequence. The combination of both half-sites exhibits no significant increase in affinity, but the flanking sequence exhibits a 100-fold enhancement in the presence of the two half-sites. However, there is a disproportionate contribution from the flanking sequence proximal to the conserved TGTGA motif. The total energetics of the gal-CRP complex formation is essentially due to the presence of the conserved half-site and its adjacent flanking sequence. Thus, the relative contributions of the half-site and flanking sequences to the energetics of DNA recognition are operon specific.
Bacterial Proteins↗
Cloning and characterization of vascular endothelial growth factor (VEGF) from zebrafish, Danio rerio.
We have cloned and sequenced a zebrafish (Danio rerio) Vascular Endothelial Growth Factor (vegf) cDNA. It encodes a precursor protein of 188 amino acids with a putative 23 amino acids signal peptide. Sequence comparison analysis indicates that the zebrafish vegf cDNA corresponds to the human VEGF165 isoform and shows about 52% identity to human VEGF165 at the amino acid level. A 2.8 kb vegf message RNA was detected in adult zebrafish by Northern blot analysis. Expression of vegf165 is also detected by RT-PCR in adult fish and throughout the zebrafish embryonic development. Whole mount in situ hybridization of zebrafish embryos indicates strong expression in four areas of the 18-19 h post-fertilization (hpf) embryo: within the anterior central nervous system in the prospective optic stalk, in mesoderm overlapping the bilaterally located merging heart fields, in mesoderm underlying and flanking the hindbrain posterior to rhombomere 4, and in medial regions of the somites. The study of vegf function in zebrafish embryonic vascular development will contribute to our understanding of the mechanisms of vertebrate endothelial cell differentiation and vasculature formation.
Amino Acid Sequence↗
Interfacial communications in recombinant rabbit kidney pyruvate kinase.
Tissue-specific isozymes of pyruvate kinase are particularly attractive systems to elucidate the molecular mechanism(s) of conferring allostery. The muscle- and kidney-type isozymes are coded by the same gene. As a consequence of alternative message RNA splicing, the two primary sequences differ by a small number of residues. However, they exhibit very different regulatory behavior. In an effort to identify the roles of specific residues in conferring allostery, the gene encoding rabbit kidney-type pyruvate kinase was cloned and expressed in Escherichia coli. The primary structure of recombinant rabbit kidney-type pyruvate kinase (rRKPK) and recombinant rabbit muscle-type pyruvate kinase (rRMPK) differ at 22 positions, which are located in a region that forms important intersubunit contacts in the RMPK structure. Velocity sedimentation and analytical gel chromatographic studies show that rRKPK undergoes reversible dimer left and right arrow tetramer assembly with an equilibrium constant of 28 +/- 3 mL/mg. This subunit assembly process provides the opportunity to elucidate the role of this dimer interface in transmission of signal upon binding of substrates and allosteric effectors. The assembly to tetrameric rRKPK is favored by the binding of phosphoenolpyruvate (PEP), one of the two substrates, or fructose 1,6-bisphosphate (FBP), an activator. In contrast, the equilibrium is shifted toward dimeric rRKPK upon binding of adenosine diphosphate (ADP), the other substrate, or l-phenylalanine (Phe), the inhibitor. These observations provide significant new insights to the molecular mechanism of allosteric regulation in the pyruvate kinase system. First, all substrates and effectors communicate through this particular dimer-dimer interface. Second, the thermodynamic signatures of these communications are qualitatively different for the two substrates and between the activator, FBP, and inhibitor, Phe.
Amino Acid Sequence↗
Heart and neural tube defects in transgenic mice overexpressing the Cx43 gap junction gene.
Transgenic mice were generated containing a cytomegaloviral promoter driven construct (CMV43) expressing the gap junction polylpeptide connexin 43. RNA and protein analysis confirmed that the transgene was being expressed. In situ hybridization analysis of embryo sections revealed that transgene expression was targeted to the dorsal neural tube and in subpopulations of neural crest cells. This expression pattern was identical to that seen in transgenic mice harboring other constructs driven by the cytomegaloviral promoter (Kothary, R., Barton, S. C., Franz, T., Norris, M. L., Hettle, S. and Surani, M. A. H. (1991) Mech. Develop. 35, 25-31; Koedood, M., Fitchel, A., Meier, P. and Mitchell, P. (1995) J. Virol. 69, 2194-2207), and corresponded to a subset of the endogenous Cx43 expression domains. Significantly, dye injection studies showed that transgene expression resulted in an increase in gap junctional communication. Though viable and fertile, these transgenic mice exhibited reduced postnatal viability. Examination of embryos at various stages of development revealed developmental perturbations consisting of cranial neural tube defects (NTD) and heart malformations. Interestingly, breeding of the CMV43 transgene into the Cx43 knockout mice extended postnatal viability of mice homozygote for the Cx43 knockout allele, indicating that the CMV43 trangsene may partially complement the Cx43 deletion. Both the Cx43 knockout and the CMV43 transgenic mice exhibit heart defects associated with malformations in the conotruncus, a region of the heart in which neural crest derivatives are known to have important roles during development. Together with our results indicating neural-crest-specific expression of the transgene in our CMV-based constructs, these observations strongly suggest a role for Cx43-mediated gap junctional communication in neural crest development. Furthermore, these observations indicate that the precise level of Cx43 function may be of critical importance in downstream events involving these migratory cell populations. As such, the CMV43 mouse may represent a powerful new model system for examining the role of extracardiac cell populations in cardiac morphogenesis and other developmental processes.
Animals↗
Late ventricular geometry and performance changes of functional single ventricle throughout staged Fontan reconstruction assessed by magnetic resonance imaging.
OBJECTIVES: We sought to test the hypothesis that late ventricular geometry and performance changes occur in functional single ventricles as they progress through staged Fontan reconstruction. BACKGROUND: Indexes of ventricular geometry and performance are important in evaluating the functional state of the heart. Magnetic resonance imaging determines these indexes in complex ventricular shapes with minimal geometric assumptions. Previous studies have shown that 1 week after hemiFontan, the mass/volume ratio markedly increases. METHODS: Multiphase, multislice, spin echo (n = 5) and cine (n = 30) magnetic resonance imaging was performed in 35 patients with a functional single ventricle (1 week to 12 years old) at various stages of Fontan reconstruction (15 in the pre hemiFontan stage, 11 after [6 to 9 months] the hemiFontan procedure and 9 after [1 to 2 years] the Fontan procedure). Volume and mass were calculated at end-systole and end-diastole. Ventricular output was then obtained. Ventricular centroid motion was also calculated. RESULTS: No difference was noted (power > 72%) from the pre hemiFontan stage to 6 to 9 months after the hemiFontan procedure in (mean +/- SD) end-diastolic volume (104 +/- 24 vs. 123 +/- 40 cc/m2), mass (171 +/- 46 vs. 202 +/- 61 g/m2), ventricular output (7.9 +/- 2.2 vs. 6.6 +/- 2.4 liters/min per m2) or centroid motion (6.9 +/- 2.8 vs. 6.7 +/- 2. mm/m2). Patients in the Fontan group demonstrated a marked decrease in all indexes, indicating significant volume unloading and decrease in mass and ventricular performance. Mass/volume ratio was not significantly different among all three groups. CONCLUSIONS: No geometric and performance changes from the volume-loaded stage are noted 6 to 9 months after the hemiFontan procedure; however, major changes occur 1 to 2 years after the Fontan procedure. The dramatic changes in the mass/volume ratio seen early after the hemiFontan procedure were not detected at 6 to 9 months. Furthermore diminution of mass, volume and ventricular performance are present at least 2 years after the Fontan procedure.
Case-Control Studies↗
Interrupted aortic arch. Impact of subaortic stenosis on management and outcome.
Interrupted aortic arch (IAA) is often related developmentally to subaortic obstruction (SAO). When severe, SAO must be addressed in surgical management of IAA. From 1990 to 1993, 25 neonates presented for initial surgical management of IAA complexes. Associated lesions were ventricular septal defect (VSD) with or without atrial septal defect (19 patients), truncus arteriosus (3 patients), tricuspid atresia with transposition of the great arteries (1 patient), aortic atresia with VSD (1 patient), and d-transposition of the great arteries with VSD (1 patient). Overall hospital mortality was 20% (five deaths). One death was related to sepsis and two to sudden hemodynamic decompensation (a 2-kg premature infant after arch repair and VSD closure and a neonate with IAA-truncus arteriosus after arch repair and truncus repair with aortic root replacement). Two deaths were related to low cardiac output in patients with severe subaortic narrowing (< 3 mm by two-dimensional echocardiography), which was not addressed surgically. Of 10 additional patients judged preoperatively to have severe SAO, 1 underwent resection of the infundibular septum together with VSD closure and arch reconstruction, and 9 underwent a modification of Norwood's operation with arch reconstruction and proximal pulmonary artery to aortic anastomosis (7 with systemic to pulmonary artery shunts and 2 with right ventricle to pulmonary artery outflow tract reconstruction). One patient died 2 months after surgery of staphylococcal sepsis. All 9 others were discharged well. Subaortic narrowing is a physiologically important element of IAA complexes. When SAO is severe, satisfactory initial palliation can be achieved by a modification of Norwood's operation.
Aorta, Thoracic↗
Prevalence, relation to spontaneous closure, and association of muscular ventricular septal defects with other cardiac defects.
Previous studies on muscular ventricular septal defect (VSD) have not taken into account the specific defect location in the septum. We retrospectively reviewed all patients with a muscular VSD, with and without associated malformations, diagnosed over 32 months to determine the prevalence and rate of spontaneous closure of single defects in relation to location in the muscular septum. Defects were classified into 4 groups: midmuscular, apical, anterior, and posterior. Two hundred seven patients were identified, of whom 125 had a single defect. The relative prevalence of single muscular VSD was: midmuscular 55 (44%), apical 31 (25%), anterior 33 (26%), and posterior 6 (5%). Thirty patients had signs of spontaneous closure and only 1 underwent surgery. There was no difference in rate of closure with respect to anatomic locations. Patients with multiple muscular VSD were either referred for surgery in the first year of life or had a course similar to patients with a single VSD. Muscular VSD associated with other cardiac malformations was more often encountered in patients with conoventricular VSD and coarctation of the aorta. The distribution of anatomic groups of muscular VSD in association with malformations was similar to the single VSD.
Child, Preschool↗
Subcostal two-dimensional echocardiographic identification of right superior vena cava connecting to left atrium.
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Heart Atria↗
Role of early postoperative surface echocardiography in the pediatric cardiac intensive care unit.
OBJECTIVE: To compare surface echocardiographic data with catheterization and surgical observation as a way of deciding on the need to reoperate to correct hemodynamically important sequelae following pediatric cardiac surgery; to determine the false-negative diagnosis rate of surface echocardiography. DESIGN: Case series. SETTING: Tertiary-care center, pediatric cardiac intensive care unit. PATIENTS: All 39 patients who underwent reoperation because of hemodynamically significant anatomic sequelae following primary or elective secondary surgery in 1 calendar year. INTERVENTIONS: None. MEASUREMENTS: Two-dimensional and color Doppler ultrasound assessment of anatomy and physiology following cardiac surgery. RESULTS: In 85 percent, surface echocardiography provided sufficient information for surgeons to reoperate on the same admission. Detection of important residual shunts or arterial stenoses and identification of anatomic causes of pulmonary undercirculation (or overcirculation) in palliated single ventricle are feasible. CONCLUSION: Early postoperative surface echocardiography is a viable way to decide on the hemodynamic adequacy of cardiac surgery.
Adolescent↗