Parker Antin

Parker Antin, PhD is focused on understanding the molecular regulation of early developmental processes in vertebrate embryos. By primarily using the chicken embryo as a model organism, he approaches research questions from the dual perspective of how individual molecules function and how their functions can be integrated into network models. One present research emphasis is concerned with understanding epithelial to mesenchymal transition (EMT) during avian gastrulation. Microarray studies have shown that more than 1800 genes are upregulated in the epiblast adjacent to the primitive streak. Many of these genes are regulated by FGF signaling, including members of several other signaling pathways and at least thirty differentially expressed transcription factors.  Fgf signaling therefore appears to be a key upstream regulator of EMT.  Studies are investigating the intracellular signaling pathways downstream of Fgf receptor activation, including the MAPK, PI3K and AKT pathways.  The MAPK pathway in particular directly regulates downstream gene transcription via activation of several transcription factors, including members of the Ets and T Box families.  Studies are investigating downstream transcriptional targets of these factors.    Another long standing research interest in the Antin lab is the mechanisms controlling early stages of cardiac myogenesis, from the emergence of premyocardial cells during gastrulation to formation of the primitive heart tube.  Bmp and Fgf signaling are well known activators of genes in the cardiogenic pathway, however relatively few direct transcriptional targets of these signaling pathways have been identified.  By combining classical experimental embryological approaches with genome wide microarray analyses, we are working to generate a large-scale model of cardiac myogenesis.
The Antin lab also hosts the GEISHA in situ hybridization database and website. The GEISHA project (gallus expression in situ hybridization analysis) began in 1998 to investigate using high throughput whole mount in situ hybridization to identify novel, differentially expressed genes in chicken embryos. An initial expression screen of approximately 900 genes demonstrated feasibility of the approach, and also highlighted the need for a centralized repository of in situ hybridization expression data. Funding was eventually obtained for this purpose. The goals of the GEISHA project are to obtain whole mount in situ hybridization expression information for all differentially expressed genes in the chicken embryo between HH stages 1-25, to integrate expression data with the chicken genome browsers, and to offer this information through a user-friendly graphical user interface.
Dr. Antin is also a member of the Molecular Cardiovascular Research Program.
 
			
Olfactomedin 1 Activity Identifies A Cell Invasion Checkpoint During Epithelial Mesenchymal Transition In The Embryonic Heart. Source: Disease Models & Mechanisms
December 20th, 2012 PMID: 23264563 Parker Antin Raymond Runyan
Endothelia in the atrioventricular (AV) canal of the developing heart undergo a prototypical epithelial mesenchymal transition (EMT) to begin heart valve formation. Using an in vitro invasion assay, an extracellular matrix protein found in the heart, Olfactomedin-1 (OLFM1), increases mesenchymal cell numbers. Both anti-OLFM1 antibody and OLFM1 siRNA treatment inhibit mesenchymal cell formation. OLFM1 does not alter cell proliferation, migration or apoptosis. Dispersion, but lack of invasion in the presence of inhibiting antibody, identifies a specific role for OLFM1 in cell invasion during EMT. This role is conserved in other epithelia, as OLFM1 similarly enhances invasion by MDCK epithelial cells in a trans-well assay. OLFM-1 activity is cooperative with TGFβ, as synergy is observed when TGFβ2 and OLFM1 are added to MDCK cell cultures. Inhibition of both OLFM1 and TGFβ in heart invasion assays shows a similar cooperative role during development. To explore OLFM1 activity during EMT, representative EMT markers were examined. Effects of OLFM1 protein and anti-OLFM1 on transcripts of cell-cell adhesion molecules and the transcription factors, Snail-1, Snail-2, Twist1, and Sox-9, argue that OLFM1 does not initiate EMT. Rather, regulation of transcripts of Zeb1 and Zeb2, secreted proteases and mesenchymal cell markers by both OLFM1 and anti-OLFM1 is consistent with regulation of the cell invasion step of EMT. We conclude that OLFM1 is present and necessary during EMT in the embryonic heart. Its role in cell invasion and mesenchymal cell gene regulation suggests an invasion checkpoint in EMT where OLFM1 acts to promote cell invasion into the three-dimensional matrix.<br /><br />
Fibroblast Growth Factor (Fgf) Signaling During Gastrulation Negatively Modulates The Abundance Of Micro Rn As That Regulate Proteins Required For Cell Migration And Embryo Patterning. Source: The Journal Of Biological Chemistry
September 20th, 2012 PMID: 22995917 Parker Antin
FGF signaling plays a pivotal role in regulating cell movements and lineage induction during gastrulation. Here we identify 44 microRNAs that are expressed in the primitive streak region of gastrula stage chicken embryos. We show that the primary effect of FGF signaling on microRNA abundance is to negatively regulate the levels of miR-let-7b, -9, -19b, -107, -130b, and -218. LIN28B inhibits microRNA processing and is positively regulated by FGF signaling. Gain- and loss-of-function experiments show that LIN28B negatively regulates the expression of miR-19b, -130b, and let-7b, whereas negative modulation of miR-9, -107, and -218 appears to be independent of LIN28B function. Predicted mRNA targets of the FGF-regulated microRNAs are over-represented in serine/threonine and tyrosine kinase receptors, including ACVR1, ACVR2B, PDGFRA, TGFBR1, and TGFBR3. Luciferase assays show that these and other candidates are targeted by FGF-regulated microRNAs. PDGFRA, a receptor whose activity is required for cell migration through the primitive streak, is a target of miR-130b and -218 in vivo. These results identify a novel mechanism by which FGF signaling regulates gene expression by negatively modulating microRNA abundance through both LIN28B-dependent and LIN28B-independent pathways.<br /><br />
Micro Rna 195 And 451 Regulate The Lkb1/Ampk Signaling Axis By Targeting Mo25. Source: Plo S One
July 23rd, 2012 PMID: 22844503 Parker Antin
BACKGROUND:<br />Recently, MicroRNAs (miR) and AMP-kinase (AMPK) have emerged as prominent players in the development of cardiac hypertrophy and heart failure. We hypothesized that components of the adenosine monophosphate-activated kinase (AMPK) pathway are targeted by miRs and alter AMPK signaling during pathological cardiac stress.<br /><br />METHODOLOGY/PRINCIPAL FINDINGS:<br />Using a mouse model of hypertrophic cardiomyopathy (HCM), we demonstrated early elevation of miR-195 and miR-451 in HCM hearts, which targets MO25, a central component of the MO25/STRAD/LKB1 complex that acts as an upstream kinase for AMPK. We show functional targeting of MO25 by miR-195 and -451. Further in vitro interrogation of MO25 as a functional target validated this hypothesis where over-expression of miR-195 in C2C12 cells knocked down MO25 expression levels and downstream AMPK signaling (phosphorylation of Acetyl CoA carboxylase [ACC] and AMPK activity assay), similar to MO25 knockdown in C2C12 cells by siRNA. Parallel changes were measured in 60 day R403Q HCM male hearts that were rescued by short-term administration of AICAR, an AMPK agonist.<br /><br />CONCLUSIONS/SIGNIFICANCE:<br />Elevated miR-195 targets the LKB1/AMPK signaling axis in HCM progression and implicates a functional role in HCM disease progression. MiR-195 may serve as potential therapeutics or therapeutic targets for heart disease.<br /><br />
Fgf Signalling Through Ras/Mapk And Pi3 K Pathways Regulates Cell Movement And Gene Expression In The Chicken Primitive Streak Without Affecting E Cadherin Expression. Source: Bmc Developmental Biology
March 21st, 2011 PMID: 21418646 Parker Antin
BACKGROUND:<br />FGF signalling regulates numerous aspects of early embryo development. During gastrulation in amniotes, epiblast cells undergo an epithelial to mesenchymal transition (EMT) in the primitive streak to form the mesoderm and endoderm. In mice lacking FGFR1, epiblast cells in the primitive streak fail to downregulate E-cadherin and undergo EMT, and cell migration is inhibited. This study investigated how FGF signalling regulates cell movement and gene expression in the primitive streak of chicken embryos.<br /><br />RESULTS:<br />We find that pharmacological inhibition of FGFR activity blocks migration of cells through the primitive streak of chicken embryos without apparent alterations in the level or intracellular localization of E-cadherin. E-cadherin protein is localized to the periphery of epiblast, primitive streak and some mesodermal cells. FGFR inhibition leads to downregulation of a large number of regulatory genes in the preingression epiblast adjacent to the primitive streak, the primitive streak and the newly formed mesoderm. This includes members of the FGF, NOTCH, EPH, PDGF, and canonical and non-canonical WNT pathways, negative modulators of these pathways, and a large number of transcriptional regulatory genes. SNAI2 expression in the primitive streak and mesoderm is not altered by FGFR inhibition, but is downregulated only in the preingression epiblast region with no significant effect on E-cadherin. Furthermore, over expression of SNAIL has no discernable effect on E-cadherin protein levels or localization in epiblast, primitive streak or mesodermal cells. FGFR activity modulates distinct downstream pathways including RAS/MAPK and PI3K/AKT. Pharmacological inhibition of MEK or AKT indicate that these downstream effectors control discrete and overlapping groups of genes during gastrulation. FGFR activity regulates components of several pathways known to be required for cell migration through the streak or in the mesoderm, including RHOA, the non-canonical WNT pathway, PDGF signalling and the cell adhesion protein N-cadherin.<br /><br />CONCLUSIONS:<br />In chicken embryos, FGF signalling regulates cell movement through the primitive streak by mechanisms that appear to be independent of changes in E-cadherin expression or protein localization. The positive and negative effects on large groups of genes by pharmacological inhibition of FGF signalling, including major signalling pathways and transcription factor families, indicates that the FGF pathway is a focal point of regulation during gastrulation in chicken.<br /><br />
Leiomodin 2 Is An Antagonist Of Tropomodulin 1 At The Pointed End Of The Thin Filaments In Cardiac Muscle. Source: Journal Of Cell Science
August 24th, 2010 PMID: 20736303 Parker Antin
Regulation of actin filament assembly is essential for efficient contractile activity in striated muscle. Leiomodin is an actin-binding protein and homolog of the pointed-end capping protein, tropomodulin. These proteins are structurally similar, sharing a common domain organization that includes two actin-binding sites. Leiomodin also contains a unique C-terminal extension that has a third actin-binding WH2 domain. Recently, the striated-muscle-specific isoform of leiomodin (Lmod2) was reported to be an actin nucleator in cardiomyocytes. Here, we have identified a function of Lmod2 in the regulation of thin filament lengths. We show that Lmod2 localizes to the pointed ends of thin filaments, where it competes for binding with tropomodulin-1 (Tmod1). Overexpression of Lmod2 results in loss of Tmod1 assembly and elongation of the thin filaments from their pointed ends. The Lmod2 WH2 domain is required for lengthening because its removal results in a molecule that caps the pointed ends similarly to Tmod1. Furthermore, Lmod2 transcripts are first detected in the heart after it has begun to beat, suggesting that the primary function of Lmod2 is to maintain thin filament lengths in the mature heart. Thus, Lmod2 antagonizes the function of Tmod1, and together, these molecules might fine-tune thin filament lengths.<br /><br />
Embryonic Expression Of The Chicken Krüppel Like (Klf) Transcription Factor Gene Family. Source: Developmental Dynamics : An Official Publication Of The American Association Of Anatomists
May 26th, 2010 PMID: 20503383 Parker Antin
The Krüppel-like transcription factors (KLF) are zinc finger proteins that activate and suppress target gene transcription. Although KLF factors have been implicated in regulating many developmental processes, a comprehensive gene expression analysis has not been reported. Here we present the chicken KLF gene family and expression during the first five days of embryonic development. Fourteen chicken KLF genes or expressed sequences have been previously identified. Through synteny analysis and cDNA mapping, we have identified the KLF9 gene and determined that the gene presently named KLF1 is the true ortholog of KLF17 in other species. In situ hybridization expression analyses show that in general KLFs are broadly expressed in multiple cell and tissue types. Expression of KLFs 3, 7, 8, and 9, is widespread at all stages examined. KLFs 2, 4, 5, 6, 10, 11, 15, and 17 show more restricted patterns that suggest multiple functions during early stages of embryonic development.<br /><br />
Arsenic Exposure Perturbs Epithelial Mesenchymal Cell Transition And Gene Expression In A Collagen Gel Assay. Source: Toxicological Sciences : An Official Journal Of The Society Of Toxicology
March 22nd, 2010 PMID: 20308225 Raymond Runyan Parker Antin
Arsenic is a naturally occurring metalloid and environmental contaminant. Arsenic exposure in drinking water is reported to cause cancer of the liver, kidneys, lung, bladder, and skin as well as birth defects, including neural tube, facial, and vasculogenic defects. The early embryonic period most sensitive to arsenic includes a variety of cellular processes. One key cellular process is epithelial-mesenchymal transition (EMT) where epithelial sheets develop into three-dimensional structures. An embryonic prototype of EMT is found in the atrioventricular (AV) canal of the developing heart, where endothelia differentiate to form heart valves. Effects of arsenic on this cellular process were examined by collagen gel invasion assay (EMT assay) using explanted AV canals from chicken embryo hearts. AV canals treated with 12.5-500 ppb arsenic showed a loss of mesenchyme at 12.5 ppb, and mesenchyme formation was completely inhibited at 500 ppb. Altered gene expression in arsenic-treated explants was investigated by microarray analysis. Genes whose expression was altered consistently at exposure levels of 10, 25, and 100 ppb were identified, and results showed that 25 ppb in vitro was particularly effective. Three hundred and eighty two genes were significantly altered at this exposure level. Cytoscape analysis of the microarray data using the chicken interactome identified four clusters of altered genes based on published relationships and pathways. This analysis identified cytoskeleton and cell adhesion-related genes whose disruption is consistent with an altered ability to undergo EMT. These studies show that EMT is sensitive to arsenic and that an interactome-based approach can be useful in identifying targets.<br><br>
Whole Mount In Situ Hybridization Detection Of M Rn As Using Short Lna Containing Dna Oligonucleotide Probes. Source: Rna (New York, N.Y.)
January 19th, 2010 PMID: 20086052 Parker Antin
In situ hybridization is widely used to visualize transcribed sequences in embryos, tissues, and cells. For whole mount detection of mRNAs in embryos, hybridization with an antisense RNA probe is followed by visual or fluorescence detection of target mRNAs. A limitation of this approach is that a cDNA template of the target RNA must be obtained in order to generate the antisense RNA probe. Here we investigate the use of short (12-24 nucleotides) locked nucleic acid (LNA) containing DNA probes for whole mount in situ hybridization detection of mRNAs. Following extensive protocol optimization, we show that LNA probes can be used to localize several mRNAs of varying abundances in chicken embryos. LNA probes also detected alternatively spliced exons that are processed in a tissue specific manner. The use of LNA probes for whole mount in situ detection of mRNAs will enable in silico design and chemical synthesis and will expand the general use of in situ hybridization for studies of transcriptional regulation and alternative splicing.<br /><br />
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