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Parker B Antin

Publications and source records attributed to Parker B Antin.

18 recordsLinked to original sources
PubMed2006

Differences in vertebrate microRNA expression.

MicroRNAs (miRNAs) attenuate gene expression by means of translational inhibition and mRNA degradation. They are abundant, highly conserved, and predicted to regulate a large number of transcripts. Several hundred miRNA classes are known, and many are associated with cell proliferation and differentiation. Many exhibit tissue-specific expression, which aids in evaluating their functions, and it has been assumed that their high level of sequence conservation implies a high level of expression conservation. A limited amount of data supports this, although discrepancies do exist. By comparing the expression of approximately 100 miRNAs in medaka and chicken with existing data for zebrafish and mouse, we conclude that the timing and location of miRNA expression is not strictly conserved. In some instances, differences in expression are associated with changes in miRNA copy number, genomic context, or both between species. Variation in miRNA expression is more pronounced the greater the differences in physiology, and it is enticing to speculate that changes in miRNA expression may play a role in shaping the physiological differences produced during animal development.

Animals↗
PubMed2006

Depolymerized hyaluronan induces vascular endothelial growth factor, a negative regulator of developmental epithelial-to-mesenchymal transformation.

Cardiac malformations constitute the most common birth defects, of which heart septal and valve defects are the most frequent forms diagnosed in infancy. These cardiac structures arise from the endocardial cushions through dynamic interactions between cells and the extracellular matrix (cardiac jelly). Targeted deletion of the hyaluronan synthase-2 (Has2) gene in mice results in an absence of hyaluronan (HA), cardiac jelly, and endocardial cushions, a loss of vascular integrity, and death at embryonic day 9.5. Despite the requirements for Has2 and its product, HA, in the developing heart, little is known about the normal processing and removal of HA during development. Cell culture studies show that HA obtains new bioactivity after depolymerization into small oligosaccharides. We previously showed reduction in Has2 expression and diminished presence of HA at later stages of heart development as tissue remodeling formed the leaflets of the cardiac valves. Here we show that small oligosaccharide forms of HA (o-HA) act antagonistically to developmental epithelial-to-mesenchymal transformation (EMT), which is required to generate the progenitor cells that populate the endocardial cushions. We further show that o-HA induces vascular endothelial growth factor (VEGF), which acts as a negative regulator of EMT. This is the first report illustrating a functional link between oligosaccharide HA and VEGF. Collectively, our data indicate that following endocardial cell EMT, native HA is likely processed to o-HA, which stimulates VEGF activity to attenuate cardiac developmental EMT.

Animals↗
PubMed2006

Has2 expression in heart forming regions is independent of BMP signaling.

Heart septation and valve malformations constitute the most common birth defects. These cardiac structures arise from the endocardial cushions through dynamic interactions between cells and the extracellular matrix (cardiac jelly). Targeted deletion of the hyaluronan synthase-2 (Has2) gene in mice results in an absence of cardiac jelly and endocardial cushions, a loss of vascular integrity, and embryonic death at E9.5. Despite the requirements for Has2 and its synthetic product hyaluronan (HA) in the developing cardiovascular system, little is known about the normal expression pattern of Has2 or the factors regulating Has2 gene transcription during development. Bmp signaling is an important regulator of cardiac myogenesis, and is also important for endocardial cushion formation. The current study defines the embryonic expression pattern of Has2 and explores the regulation of Has2 gene expression by Bmp signaling. In situ hybridization studies demonstrate dynamic Has2 expression patterns during myocardial cell development and cardiac tube formation, formation of the cardiac endocardial cushions, and cushion invasion by valve primordial cells. Despite overlapping regional expression of Bmp2 in the late gastrula anterior lateral endoderm and Has2 in the adjacent cardiogenic mesoderm, application of noggin-expressing CHO cells beneath the endoderm failed to perturb normal Has2 expression. Thus, in contrast to many genes expressed in the heart forming region, regulation of Has2 in the cardiogenic mesoderm is independent of Bmp signaling.

Animals↗
PubMed2006

Hot hearts in the Sonoran Desert: the 11th Weinstein Cardiovascular Development Conference in Tucson.

The 11th Annual Weinstein Cardiovascular Development Conference was held May 19-22, 2005 at the Westward Look Resort and Conference Center in Tucson, Arizona. The Westward Look was the site of the 6th Weinstein Meeting in 1999, and this year, 330 basic research scientists and research clinicians returned to Tucson for 3 days of meetings, Mariachis, and margaritas. The meeting was hosted by the cardiovascular research group at the University of Arizona and offered flavors of the desert southwest that included record temperatures, the Skopopelli conference logo modified from the Kokopelli of Native American mythology (Fig. 1), and liberal use of a cattle prod to encourage speaker timeliness.

Animals↗
PubMed2006

MicroRNA expression during chick embryo development.

MicroRNAs (miRNAs) are small, abundant, noncoding RNAs that modulate protein abundance by interfering with target mRNA translation or stability. miRNAs are detected in organisms from all domains and may regulate 30% of transcripts in vertebrates. Understanding miRNA function requires a detailed determination of expression, yet this has not been reported in an amniote species. High-throughput whole mount in situ hybridization was performed on chicken embryos to map expression of 135 miRNA genes including five miRNAs that had not been previously reported in chicken. Eighty-four miRNAs were detected before day 5 of embryogenesis, and 75 miRNAs showed differential expression. Whereas few miRNAs were expressed during formation of the primary germ layers, the number of miRNAs detected increased rapidly during organogenesis. Patterns highlighted cell-type, organ or structure-specific expression, localization within germ layers and their derivatives, and expression in multiple cell and tissue types and within sub-regions of structures and tissues. A novel group of miRNAs was highly expressed in most tissues but much reduced in one or a few organs, including the heart. This study presents the first comprehensive overview of miRNA expression in an amniote organism and provides an important foundation for investigations of miRNA gene regulation and function.

Animals↗
PubMed2006

Latrophilin-2 is a novel component of the epithelial-mesenchymal transition within the atrioventricular canal of the embryonic chicken heart.

Endothelial cells in the atrioventricular canal of the heart undergo an epithelial-mesenchymal transition (EMT) to form heart valves. We surveyed an on-line database (http://www.geisha.arizona.edu/) for clones expressed during gastrulation to identify novel EMT components. One gene, latrophilin-2, was identified as expressed in the heart and appeared to be functional in EMT. This molecule was chosen for further examination. In situ localization showed it to be expressed in both the myocardium and endothelium. Several antisense DNA probes and an siRNA for latrophilin-2 produced a loss of EMT in collagen gel cultures. Latrophilin-2 is a putative G-protein-coupled receptor and we previously identified a pertussis toxin-sensitive G-protein signal transduction pathway. Microarray experiments were performed to examine whether these molecules were related. After treatment with antisense DNA against latrophilin-2, expression of 1,385 genes and ESTs was altered. This represented approximately 12.5% of the microarray elements. In contrast, pertussis toxin altered only 103 (0.9%) elements of the array. There appears to be little overlap between the two signal transduction pathways. Latrophilin-2 is thus a novel component of EMT and provides a new avenue for investigation of this cellular process.

Animals↗
PubMed2005

Disruption in the tropomodulin1 (Tmod1) gene compromises cardiomyocyte development in murine embryonic stem cells by arresting myofibril maturation.

Tropomodulins (Tmods) comprise a family of capping proteins for actin filament pointed ends. To decipher the significance of Tmod1 functions during de novo myofibrillogenesis, we generated Tmod1 null embryonic stem (ES) cells and studied their differentiation into cardiomyocytes. Strikingly, in vitro cardiomyocyte differentiation of wild type (WT) ES cells faithfully recapitulates in vivo cardiomyocyte differentiation, allowing us to evaluate the phenotypes of Tmod1 knockout (KO) myofibrils irrespective of embryonic lethality of Tmod1 KO mice. Immunofluorescence and electron microscopy studies revealed that Tmod1 null cardiac myocytes were round, morphologically immature, and contained underdeveloped myofibrils that were shorter, narrower, and had fewer thin filaments than those in WT cells. Unexpectedly, clear gaps in the staining pattern for F-actin at the H-zone were detected in most KO cells, indicating the presence of filaments at uniform lengths. This indicates that additional mechanisms other than capping proteins are responsible for thin filament length maintenance in cardiac myocytes. Also unexpectedly, approximately 40% of the KO cardiac myocytes exhibited contractile activity. Our data indicate that differentiating ES cells are a powerful system to investigate the functional properties of contractile proteins and that Tmod1 functions are critical for late stages of myofibrillogenesis, and for the maturation of myofibrils.

Animals↗
PubMed2005

Genomic resources for chicken.

The recent sequencing and draft assembly of a chicken genome has provided biologists with an invaluable research tool that complements a growing list of additional avian genomic resources. For many researchers, finding and using these resources is challenging, because information is presented through an increasing number of Web sites and browser navigation frequently requires specific knowledge and expertise. This primer provides an overview of online genomic resources for the chicken, including the Ensembl, UCSC, and NCBI annotated chicken genome browsers; expressed sequence tag and in situ hybridization databases; and sources for microarrays, cDNAs, and bacterial artificial chromosomes (BACs). Several short tutorials oriented toward the biologist with limited bioinformatics skills outline how to retrieve several types of commonly needed information and reagents.

Animals↗
PubMed2004

Hedgehog signaling is essential for endothelial tube formation during vasculogenesis.

During embryonic development, the first blood vessels are formed through the aggregation and subsequent assembly of angioblasts (endothelial precursors) into a network of endothelial tubes, a process known as vasculogenesis. These first vessels generally form in mesoderm that is adjacent to endodermal tissue. Although specification of the angioblast lineage is independent of endoderm interactions, a signal from the endoderm is necessary for angioblasts to assemble into a vascular network and to undergo vascular tube formation. In this study, we show that endodermally derived sonic hedgehog is both necessary and sufficient for vascular tube formation in avian embryos. We also show that Hedgehog signaling is required for vascular tube formation in mouse embryos, and for vascular cord formation in cultured mouse endothelial cells. These results demonstrate a previously uncharacterized role for Hedgehog signaling in vascular development, and identify Hedgehog signaling as an important component of the molecular pathway leading to vascular tube formation.

Animals↗
PubMed2004

Regulation of Hex gene expression and initial stages of avian hepatogenesis by Bmp and Fgf signaling.

The vertebrate liver and heart arise from adjacent cell layers in the anterior lateral (AL) endoderm and mesoderm of late gastrula embryos, and the earliest stages of liver and heart development are interrelated through reciprocal tissue interactions. Although classical embryological studies performed several decades ago in chick and quail defined the timing of hepatogenic induction in birds and the important role for cardiogenic mesoderm in this process, almost nothing is known about the molecular aspects of avian liver development. Here we use in vivo and explantation assays to investigate tissue interactions and signaling pathways regulating Hex, a homeobox gene required for liver development, and the earliest stages of hepatogenesis in the chick embryo. We find that explants of late gastrula anterior lateral endoderm plus mesoderm, which have been used extensively for studies relating to heart development, also produce albumin-expressing hepatoblasts. Expression of Hex, the earliest known molecular marker for the hepatogenic endoderm, and albumin, indicative of early committed hepatoblasts, requires both autocrine Bmp signaling and a specific paracrine signal from the cardiogenic (anterior lateral) mesoderm. Endodermal expression of Fox2a, in contrast, requires the mesoderm but is independent of Bmp signaling. In vivo induction assays show that the ability of BMP2 to activate Hex expression in the endoderm is restricted to a region that is only slightly larger than the endogenous domain of Hex expression. Although Fgfs can substitute for the cardiogenic mesoderm to support the expression of Hex and albumin in the endoderm, several Fgf genes are expressed in the anterior lateral endoderm but an Fgf expressed predominantly in the mesoderm was not identified. Studies also showed that Fgf gene expression in the endoderm does not require a signal from the mesoderm. Mechanisms regulating endodermal signaling pathways activated by Fgfs may therefore be more complex than previously appreciated.

Albumins↗
PubMed2004

GEISHA, a whole-mount in situ hybridization gene expression screen in chicken embryos.

Despite the increasing quality and quantity of genomic sequence that is available to researchers, predicting gene function from sequence information remains a challenge. One method for obtaining rapid insight into potential functional roles of novel genes is through gene expression mapping. We have performed a high throughput whole-mount in situ hybridization (ISH) screen with chick embryos to identify novel, differentially expressed genes. Approximately 1,200 5' expressed sequence tags (ESTs) were generated from cDNA clones of a Hamburger and Hamilton (HH) stage 4-7 (late gastrula) chick embryo endoderm-mesoderm library. After screening to remove ubiquitously expressed cDNAs and internal clustering and after comparison to GenBank sequences, remaining cDNAs (representing both characterized and uncharacterized genes) were screened for expression in HH stage 3-14 embryos by automated high throughput ISH. Of 786 cDNAs for which ISH was successfully performed, approximately 30% showed ubiquitous expression, 40% were negative, and approximately 30% showed a restricted expression pattern. cDNAs were identified that showed restricted expression in every embryonic region, including the primitive streak, somites, developing cardiovascular system and neural tube/neural crest. A relational database was developed to hold all EST sequences, ISH images, and corresponding BLAST report information, and to enable browsing and querying of data. A user interface is freely accessible at http://geisha.biosci.arizona.edu. Results show that high throughput whole-mount ISH provides an effective approach for identifying novel genes that are differentially expressed in the developing chicken embryo.

Animals↗
PubMed2003

The complete mouse nebulin gene sequence and the identification of cardiac nebulin.

Nebulin is a giant (M(r) 750-850kDa), modular sarcomeric protein proposed to regulate the assembly, and to specify the precise lengths of actin (thin) filaments in vertebrate skeletal muscles. Nebulin's potential role as a molecular template is based on its structural and biochemical properties. Its central approximately 700kDa portion associates with actin along the entire length of the thin filament, its N-terminal region extends to thin filament pointed ends, and approximately 80kDa of its C-terminal region integrates within the Z-line lattice. Here, we determined the exon/intron organization of the entire mouse nebulin gene, which contains 165 exons in a 202kb segment. We identified 16 novel exons, 15 of which encode nebulin-repeat motifs (12 from its central region and 3 from its Z-line region). One novel exon shares high sequence homology to the 20 residue repeats of the tight-junction protein, ZO-1. RT-PCR analyses revealed that all 16 novel exons are expressed in mouse skeletal muscle. Surprisingly, we also amplified mRNA transcripts from mouse and human heart cDNA using primers designed along the entire length of nebulin. The expression of cardiac-specific nebulin transcripts was confirmed by in situ hybridization in fetal rat cardiomyocytes and in embryonic Xenopus laevis (frog) heart. On the protein level, antibodies specific for skeletal muscle nebulin's N and C-terminal regions stained isolated rat cardiac myofibrils at the pointed and barbed ends of thin filaments, respectively. These data indicate a conserved molecular layout of the nebulin filament systems in both cardiac and skeletal myofibrils. We propose that thin filament length regulation in cardiac and skeletal muscles may share conserved nebulin-based mechanisms, and that nebulin isoform diversity may contribute to thin filament length differences in cardiac and skeletal muscle.

Amino Acid Sequence↗
PubMed2003

Ephs and ephrins during early stages of chick embryogenesis.

The Eph family of receptor tyrosine kinases and their ligands, the ephrins, are membrane-bound proteins that mediate bidirectional signals between adjacent cells. By modulating cytoskeleton dynamics affecting cell motility and adhesion, Ephs and ephrins orchestrate cell movements during multiple morphogenetic processes, including gastrulation, segmentation, angiogenesis, axonal pathfinding, and neural crest cell migration. The full repertoire of developmental Eph/ephrin functions remains uncertain, however, because coexpression of multiple receptor and ligand family members, and promiscuous interactions between them, can result in functional redundancy. A complete understanding of expression patterns, therefore, is a necessary prerequisite to understanding function. Here, we present a comprehensive expression overview for 10 Eph and ephrin genes during the first 48 hr of chick embryo development. First, dynamic expression domains are described for each gene between Hamburger and Hamilton stages 4 and 12; second, comparative analyses are presented of Eph/ephrin expression patterns in the primitive streak, the somites, the vasculature, and the brain. Complex spatially and temporally dynamic expression patterns are revealed that suggest novel functions for Eph and ephrin family members in both known and previously unrecognized processes. This study will provide a valuable resource for further experimental investigations of Eph and ephrin functions during early embryonic development.

Animals↗
PubMed2003

Tetracycline-inducible system for regulation of skeletal muscle-specific gene expression in transgenic mice.

Tightly regulated control of over-expression is often necessary to study one aspect or time point of gene function and, in transgenesis, may help to avoid lethal effects and complications caused by ubiquitous over-expression. We have utilized the benefits of an optimized tet-on system and a modified muscle creatine kinase (MCK) promoter to generate a skeletal muscle-specific, doxycycline (Dox) controlled over-expression system in transgenic mice. A DNA construct was generated in which the codon optimized reverse tetracycline transactivator (rtTA) was placed under control of a skeletal muscle-specific version of the mouse MCK promoter. Transgenic mice containing this construct expressed rtTA almost exclusively in skeletal muscles. These mice were crossed to a second transgenic line containing a bi-directional promoter centered on a tet responder element driving both a luciferase reporter gene and a tagged gene of interest; in this case the calpain inhibitor calpastatin. Compound hemizygous mice showed high level, Dox dependent muscle-specific luciferase activity often exceeding 10,000-fold over non-muscle tissues of the same mouse. Western and immunocytochemical analysis demonstrated similar Dox dependent muscle-specific induction of the tagged calpastatin protein. These findings demonstrate the effectiveness and flexibility of the tet-on system to provide a tightly regulated over-expression system in adult skeletal muscle. The MCKrtTA transgenic lines can be combined with other transgenic responder lines for skeletal muscle-specific over-expression of any target gene of interest.

Animals↗
PubMed2002

Regulation of Hex gene expression by a Smads-dependent signaling pathway.

The homeobox gene Hex is expressed in multiple cell types during embryogenesis and is required for liver and monocyte development. Hex is expressed in the foregut region of late gastrula avian and mammalian embryos in a pattern that overlaps with expression of bone morphogenetic proteins (BMPs). Here we investigate the relationship between BMP signaling and Hex gene expression. We find that Hex expression in avian anterior lateral endoderm is regulated by autocrine BMP signaling. Characterization of the mouse Hex gene promoter identified a 71-nucleotide BMP-responsive element (BRE) that is required for up-regulation of Hex by an activated BMP signaling pathway. The Hex BRE binds Smad4 and Smad1-Smad4 complexes in vitro, and in transfection assays, it is responsive to Smad1 and Smad4 but not to Smad2 and Smad4 or Smad3 and Smad4. The BRE contains two copies of a GCCGnCGC-like motif that in Drosophila is the binding site for Mad and Madea followed by two CAGAG boxes that are similar to sequences required for transforming growth factor-beta/activin responsiveness of several vertebrate genes. Mutation of the GC elements, but not the two CAGAG boxes, abolishes Smads responsiveness in the intact Hex promoter, whereas mutations in both the GC elements and CAGAG boxes show that they act cooperatively to confer Smads responsiveness to the Hex promoter. The Hex BRE can confer Smads responsiveness to a heterologous promoter, and in this context, both the GC-rich elements and the CAGAG boxes are required for Smads-dependent promoter activity. An element almost identical to the Hex BRE is present within the BMP-responsive Nkx2-5 gene promoter, suggesting that the Hex BRE represents a common response element for genes regulated by BMP signaling in the foregut region of the embryo.

Animals↗
PubMed2002

Precocious expression of cardiac troponin T in early chick embryos is independent of bone morphogenetic protein signaling.

Cardiac troponin T (cTNT) is a component of the troponin complex, which confers calcium sensitivity to contraction in skeletal and cardiac muscle. Although it is thought that most components of the contractile myofibril are expressed exclusively in differentiated muscle cells, we observed that mRNAs coding for cTNT were detectable in explanted late gastrula mesoderm at least 12 hr before cardiac myocyte differentiation. We therefore conducted a detailed analysis of cTNT gene expression in the early chick embryo. Whole-mount in situ hybridization studies showed that by Hamburger and Hamilton stage 5, cTNT mRNAs are detectable in lateral mesoderm and, by stage 6, are observed throughout the lateral embryonic and extraembryonic mesoderm in a distribution that is much broader than the recognized heart field. As myocardial cell differentiation commences, cTNT transcripts become progressively localized to the forming heart and, by stage 14, are completely restricted to heart muscle cells. Western blot analyses demonstrated that cTNT protein expression is under translational control, as cTNT protein is not detectable until stage 9, concomitant with myocardial cell differentiation. Removal of endoderm at stage 5 had no effect on cTNT mRNA levels, and the bone morphogenetic protein (BMP) inhibitor noggin failed to block cTNT expression, even in the heart-forming region and in cases where heart formation was inhibited. Implantation of noggin-expressing CHO cells at the anterior midline of stage 7 embryos resulted in cardia bifida. These findings demonstrate the precocious, BMP-independent expression of a gene coding for a myofibrillar protein and suggest that an additional regulatory pathway exists for activation of some cardiogenic genes.

Animals↗