Parker B Antin
- Professor Emeritus
- (520) 621-5242
- AHSC, Rm. 4205
- TUCSON, AZ 85724-5044
- pba@arizona.edu
Biography
Parker Antin is Professor of Cellular and Molecular Medicine in the College of Medicine, Associate Vice President for Research for the Division of Agriculture, Life and Veterinary Medicine, and Cooperative Extension, and Associate Dean for Research in the College of Agriculture and Life Sciences. In his positions of Associate Vice President and Associate Dean, he is responsible for developing and implementing the research vision for the Colleges of Agriculture and Life Sciences and the College of Veterinary Medicine, with total research expenditures of approximately $65M per year. His responsibilities include oversight of research strategy and portfolio investment, grants and contracts pre award services, research intensive faculty hires and retentions, research communication and marketing, research facilities, and research compliance services. In collaboration with Division and College leadership teams, he has shared responsibilities for philanthropy, budgets and information technology.
Dr. Antin is a vertebrate developmental biologist whose research is concerned with the molecular mechanisms of embryonic development. His research has been supported by NIH, NSF, NASA, USDA, and the DOE, as well as several private foundations including the American Heart Association and the Muscular Dystrophy Association, He is the Principal Investigator of CyVerse, a $115M NSF funded cyberinfrastructure project whose mission is to design, deploy and expand a national cyberinfrastructure for life sciences research, and train scientists in its use (http://cyverse.org). With 65,000 users worldwide, CyVerse enables scientists to manage and store data and experiments, access high-performance computing, and share data and results with colleagues and the public.
Dr. Antin is also active nationally in the areas of science policy and funding for science. He is a past President of the Federation of Societies for Experimental Biology (FASEB), an umbrella science policy and advocacy organization representing 32 scientific societies and 135,000 scientists. His continued work with FASEB, along with his duties as Associate Vice President and Associate Dean for Research, and CyVerse PI, brings him frequently to Washington, DC, where he advocates for support of science and science policy positions that enhance the scientific enterprise.
Degrees
- Ph.D. Anatomy
- University of Pennsylvania, Philadelphia, Pennsylvania, United States
- B.S. Biology
- Union College, Schenectady, New York, United States
Work Experience
- University of Arizona, Tucson, Arizona (2014 - Ongoing)
- University of Arizona, Tucson, Arizona (2009 - Ongoing)
- University of Arizona, Tucson, Arizona (2004 - 2009)
- University of Arizona, Tucson, Arizona (2000 - 2004)
- University of Arizona, Tucson, Arizona (1998 - 2000)
- University of Arizona, Tucson, Arizona (1992 - 1997)
- University of California San Francisco, San Francisco, California (1986 - 1991)
- Cornell University Medical College (1984 - 1986)
- University of Pennsylvania, Philadelphia, Pennsylvania (1982 - 1983)
Awards
- Flinn Young Investigator Award
- American Heart Association, Spring 1994
- Postdoctoral Fellowship Award
- National Institutes of Health, Spring 1986
- Revson Winston Postdoctoral Fellowship
- Revson Winston Foundation, Spring 1985
- College of Medicine Faculty Mentoring Award
- UA College of Medicine, Spring 2015
- Editor in Chief
- Developmental Dynamics, Spring 2013
- Merck Visiting Scholar
- Union College, Spring 2010
- Fellow
- American Association of Anatomists, Spring 2009
Interests
No activities entered.
Courses
No activities entered.
Scholarly Contributions
Journals/Publications
- Malatesta, M., Mori, G., Acquotti, D., Campanini, B., Peracchi, A., Antin, P. B., & Percudani, R. (2020). Birth of a pathway for sulfur metabolism in early amniote evolution. Nature ecology & evolution, 4(9), 1239-1246.More infoAmong amniotes, reptiles and mammals are differently adapted to terrestrial life. It is generally appreciated that terrestrialization required adaptive changes of vertebrate metabolism, particularly in the mode of nitrogen excretion. However, the current paradigm is that metabolic adaptation to life on land did not involve synthesis of enzymatic pathways de novo, but rather repurposing of existing ones. Here, by comparing the inventory of pyridoxal 5'-phosphate-dependent enzymes in different amniotes, we identify in silico a pathway for sulfur metabolism present in chick embryos but not in mammals. Cysteine lyase contains haem and pyridoxal 5'-phosphate co-factors and converts cysteine and sulfite into cysteic acid and hydrogen sulfide, respectively. A specific cysteic acid decarboxylase produces taurine, while hydrogen sulfide is recycled into cysteine by cystathionine beta-synthase. This reaction sequence enables the formation of sulfonated amino acids during embryo development in the egg at no cost of reduced sulfur. The pathway originated around 300 million years ago in a proto-reptile by cystathionine beta-synthase duplication, cysteine lyase neofunctionalization and cysteic acid decarboxylase co-option. Our findings indicate that adaptation to terrestrial life involved innovations in metabolic pathways, and reveal the molecular mechanisms by which such innovations arose in amniote evolution.
- Yang, J., Antin, P. B., Berx, G., Blanpain, C., Brabletz, T., Bronner, M., Campbell, K., Cano, A., Casanova, J., Christofgori, G., Dedhar, S., Derynk, R., Ford, H. L., Fluxe, J., Garcia de Herreros, A., Goodall, G. J., Hadjantonakis, A., Huang, R. J., Kalcheim, C., , Kalluri, R., et al. (2020). Definitions and Guidelines for Research on Epithelial-Mesenchymal Transition. Nature Reviews Molecular Cell Biology.More infoA consensus of major researchers the field of EMT with alphabetical order on authors except for 1st and last. I wrote the 1st draft of the Introduction and History sections and made the 1st figure and the two tables in addition to a few editorial comments on the other sections.
- O'Hare, E. A., Antin, P. B., & Delany, M. E. (2019). Two Proximally Close Priority Candidate Genes for diplopodia-1, an Autosomal Inherited Craniofacial-Limb Syndrome in the Chicken: MRE11 and GPR83. The Journal of heredity, 110(2), 194-210.More infoNext-generation sequencing (NGS) and expression technologies were utilized to investigate the genes and sequence elements in a 586 kb region of chicken chromosome 1 associated with the autosomal recessive diplopodia-1 (dp-1) mutation. This mutation shows a syndromic phenotype similar to known human developmental abnormalities (e.g., cleft palate, polydactyly, omphalocele [exposed viscera]). Toward our goal to ascertain the variant responsible, the entire 586 kb region was sequenced following utilization of a specifically designed capture array and to confirm/validate fine-mapping results. Bioinformatic analyses identified a total of 6142 sequence variants, which included SNPs, indels, and gaps. Of these, 778 SNPs, 146 micro-indels, and 581 gaps were unique to the UCD-Dp-1.003 inbred congenic line; those found within exons and splice sites were studied for contribution to the mutant phenotype. Upon further validation with additional mutant samples, a smaller subset (of variants [51]) remains linked to the mutation. Additionally, utilization of specific samples in the NGS technology was advantageous in that fine-mapping methodologies eliminated an additional 326 kb of sequence information on chromosome 1. Predicted and confirmed protein-coding genes within the smaller 260 kb region were assessed for their developmental expression patterns over several stages of early embryogenesis in regions/tissues of interest (e.g., digits, craniofacial region). Based on these results and known function in other vertebrates, 2 genes within 5 kb of each other, MRE11 and GPR83, are proposed as high-priority candidates for the dp-1 mutation.
- Antin, P. B. (2017). Transitions. Developmental dynamics : an official publication of the American Association of Anatomists, 246(12), 969.
- Antin, P. B. (2016). A conversation with rudolf jaenisch. Developmental dynamics : an official publication of the American Association of Anatomists, 245(7), 698-701.
- Khan, P., Cooley, J. R., Zeltzer, S. L., Yatskievych, T. A., & Antin, P. B. (2016). Gastrulation EMT is Independent of Epithelial Cadherin Downregulation. PLoS One, 11(4). doi:10.1371/journal.pone.0153591
- Merchant, N., Lyons, E., Goff, S., Matt, V., Doreen, W., & Antin, P. B. (2016). The iPlant Collaborative: Cyberinfrastructure for Enabling Data to Discovery for the Life Sciences. PLoS Biology, 10(1371).
- Moly, P. K., Cooley, J. R., Zeltzer, S. L., Yatskievych, T. A., & Antin, P. B. (2016). Gastrulation EMT Is Independent of P-Cadherin Downregulation. PloS one, 11(4), e0153591.More infoEpithelial-mesenchymal transition (EMT) is an evolutionarily conserved process during which cells lose epithelial characteristics and gain a migratory phenotype. Although downregulation of epithelial cadherins by Snail and other transcriptional repressors is generally considered a prerequisite for EMT, recent studies have challenged this view. Here we investigate the relationship between E-cadherin and P-cadherin expression and localization, Snail function and EMT during gastrulation in chicken embryos. Expression analyses show that while E-cadherin transcripts are detected in the epiblast but not in the primitive streak or mesoderm, P-cadherin mRNA and protein are present in the epiblast, primitive and mesoderm. Antibodies that specifically recognize E-cadherin are not presently available. During EMT, P-cadherin relocalizes from the lateral surfaces of epithelial epiblast cells to a circumferential distribution in emerging mesodermal cells. Cells electroporated with an E-cadherin expression construct undergo EMT and migrate into the mesoderm. An examination of Snail function showed that reduction of Slug (SNAI2) protein levels using a morpholino fails to inhibit EMT, and expression of human or chicken Snail in epiblast cells fails to induce EMT. In contrast, cells expressing the Rho inhibitor peptide C3 rapidly exit the epiblast without activating Slug or the mesoderm marker N-cadherin. Together, these experiments show that epiblast cells undergo EMT while retaining P-cadherin, and raise questions about the mechanisms of EMT regulation during avian gastrulation.
- Ward, A., Baldwin, T. O., & Antin, P. B. (2016). Research data: Silver lining to irreproducibility. Nature, 532(7598), 177.
- Schmid, M., Smith, J., Burt, D. W., Aken, B. L., Antin, P. B., Archibald, A. L., Ashwell, C., Blackshear, P. J., Boschiero, C., Brown, C. T., Burgess, S. C., Cheng, H. H., Chow, W., Coble, D. J., Cooksey, A., Crooijmans, R. P., Damas, J., Davis, R. V., de Koning, D., , Delany, M. E., et al. (2015). Third Report on Chicken Genes and Chromosomes 2015. Cytogenetic and genome research, 145(2), 78-179.
- Antin, P. B., Yatskievych, T. A., Davey, S., & Darnell, D. K. (2014). GEISHA: An evolving gene expression resource for the chicken embryo. Nucleic Acids Research, 42(D1), D933-D937.More infoAbstract: GEISHA (Gallus Expression In Situ Hybridization Analysis; http://geisha.arizona.edu) is an in situ hybridization gene expression and genomic resource for the chicken embryo. This update describes modifications that enhance its utility to users. During the past 5 years, GEISHA has undertaken a significant restructuring to more closely conform to the data organization and formatting of Model Organism Databases in other species. This has involved migrating from an entry-centric format to one that is gene-centered. Database restructuring has enabled the inclusion of data pertaining to chicken genes and proteins and their orthologs in other species. This new information is presented through an updated user interface. In situ hybridization data in mouse, frog, zebrafish and fruitfly are integrated with chicken genomic and expression information. A resource has also been developed that integrates the GEISHA interface information with the Online Mendelian Inheritance in Man human disease gene database. Finally, the Chicken Gene Nomenclature Committee database and the GEISHA database have been integrated so that they draw from the same data resources. © 2013 The Author(s). Published by Oxford University Press.
- Antin, P. B., Yatskievych, T. A., Davey, S., & Darnell, D. K. (2014). GEISHA: an evolving gene expression resource for the chicken embryo. Nucleic acids research, 42(Database issue), D933-7.More infoGEISHA (Gallus Expression In Situ Hybridization Analysis; http://geisha.arizona.edu) is an in situ hybridization gene expression and genomic resource for the chicken embryo. This update describes modifications that enhance its utility to users. During the past 5 years, GEISHA has undertaken a significant restructuring to more closely conform to the data organization and formatting of Model Organism Databases in other species. This has involved migrating from an entry-centric format to one that is gene-centered. Database restructuring has enabled the inclusion of data pertaining to chicken genes and proteins and their orthologs in other species. This new information is presented through an updated user interface. In situ hybridization data in mouse, frog, zebrafish and fruitfly are integrated with chicken genomic and expression information. A resource has also been developed that integrates the GEISHA interface information with the Online Mendelian Inheritance in Man human disease gene database. Finally, the Chicken Gene Nomenclature Committee database and the GEISHA database have been integrated so that they draw from the same data resources.
- Cooley, J. R., Yatskievych, T. A., & Antin, P. B. (2014). Embryonic expression of the transforming growth factor beta ligand and receptor genes in chicken. Developmental Dynamics, 243(3), 497-508.More infoAbstract: Background: Transforming growth factor-beta (TGFβ) signaling regulates a myriad of biological processes during embryogenesis, in the adult, and during the manifestation of disease. TGFβ signaling is propagated through one of three TGFβ ligands interacting with Type I and Type II receptors, and Type III co-receptors. Although TGFβ signaling is regulated partly by the combinatorial expression patterns of TGFβ receptors and ligands, a comprehensive gene expression analysis has not been published. Results: Here we report the embryonic mRNA expression patterns in chicken embryos of the canonical TGFβ ligands (TGFB1, TGFB2, and TGFB3) and receptors (TGFBR1, TGFBR2, TGFBR3), plus the Activin A receptor, type 1 (ACVR1) and co receptor Endoglin (ENG) that also transduce TGFβ signaling. Conclusions: TGFB ligands and receptors show dynamic and frequently overlapping expression patterns in numerous embryonic cell layers and structures. Integrating expression information identifies combinations of ligands and receptors that are involved in specific developmental processes including somitogenesis, cardiogenesis and vasculogenesis. Developmental Dynamics 243:497-508, 2014. © 2013 Wiley Periodicals, Inc.
- Cooley, J. R., Yatskievych, T. A., & Antin, P. B. (2014). Embryonic expression of the transforming growth factor beta ligand and receptor genes in chicken. Developmental dynamics : an official publication of the American Association of Anatomists, 243(3), 497-508.More infoTransforming growth factor-beta (TGFβ) signaling regulates a myriad of biological processes during embryogenesis, in the adult, and during the manifestation of disease. TGFβ signaling is propagated through one of three TGFβ ligands interacting with Type I and Type II receptors, and Type III co-receptors. Although TGFβ signaling is regulated partly by the combinatorial expression patterns of TGFβ receptors and ligands, a comprehensive gene expression analysis has not been published.
- Darnell, D. K., & Antin, P. B. (2014). LNA-based in situ hybridization detection of mRNAs in embryos. Methods in molecular biology (Clifton, N.J.), 1211, 69-76.More infoIn situ hybridization (ISH) in embryos allows the visualization of specific RNAs as a readout of gene expression during normal development or after experimental manipulations. ISH using short DNA probes containing locked nucleic acid nucleotides (LNAs) holds the additional advantage of allowing the detection of specific RNA splice variants or of closely related family members that differ in only short regions, creating new diagnostic and detection opportunities. Here we describe methods for using short (14-24 nt) DNA probes containing LNA nucleotides to detect moderately to highly expressed RNAs in whole chick embryos during the first 5 days of embryonic development. The protocol is easily adaptable for use with embryos of other vertebrate species.
- Antin, P. (2013). A digital upgrade as 113 years of print publication comes to an end. Developmental Dynamics, 242(12), 1347-1347.More infoPMID: 24259481;
- Antin, P. (2013). The interesting times of science. Developmental Dynamics, 242(1), 1-.More infoPMID: 23192952;
- Antin, P. B. (2013). A digital upgrade as 113 years of print publication comes to an end. Developmental dynamics : an official publication of the American Association of Anatomists, 242(12), 1347.
- Lencinas, A., Chhun, D. C., Dan, K. P., Ross, K. D., Hoover, E. A., Antin, P. B., & Runyan, R. B. (2013). Olfactomedin-1 activity identifies a cell invasion checkpoint during epithelial-mesenchymal transition in the chick embryonic heart. DMM Disease Models and Mechanisms, 6(3), 632-642.More infoPMID: 23264563;PMCID: PMC3634647;Abstract: 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, Olfactomedin-1 (OLFM1), was found to increase mesenchymal cell numbers in AV canals from embryonic chick hearts. Treatment with both anti-OLFM1 antibody and siRNA targeting OLFM1 inhibits 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 transwell assay. Synergy is observed when TGFβ2 and OLFM1 are added to MDCK cell cultures, indicating that OLFM-1 activity is cooperative with TGFβ. 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 chick 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. © 2013. Published by The Company of Biologists Ltd.
- Lencinas, A., Chhun, D. C., Dan, K. P., Ross, K. D., Hoover, E. A., Antin, P. B., & Runyan, R. B. (2013). Olfactomedin-1 activity identifies a cell invasion checkpoint during epithelial-mesenchymal transition in the chick embryonic heart. Disease models & mechanisms, 6(3), 632-42.More infoEndothelia 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, Olfactomedin-1 (OLFM1), was found to increase mesenchymal cell numbers in AV canals from embryonic chick hearts. Treatment with both anti-OLFM1 antibody and siRNA targeting OLFM1 inhibits 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 transwell assay. Synergy is observed when TGFβ2 and OLFM1 are added to MDCK cell cultures, indicating that OLFM-1 activity is cooperative with TGFβ. 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 chick 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.
- Robb, E. A., Antin, P. B., & Delany, M. E. (2013). Defining the Sequence Elements and Candidate Genes for the Coloboma Mutation. PLoS ONE, 8(4).More infoAbstract: The chicken coloboma mutation exhibits features similar to human congenital developmental malformations such as ocular coloboma, cleft-palate, dwarfism, and polydactyly. The coloboma-associated region and encoded genes were investigated using advanced genomic, genetic, and gene expression technologies. Initially, the mutation was linked to a 990 kb region encoding 11 genes; the application of the genetic and genomic tools led to a reduction of the linked region to 176 kb and the elimination of 7 genes. Furthermore, bioinformatics analyses of capture array-next generation sequence data identified genetic elements including SNPs, insertions, deletions, gaps, chromosomal rearrangements, and miRNA binding sites within the introgressed causative region relative to the reference genome sequence. Coloboma-specific variants within exons, UTRs, and splice sites were studied for their contribution to the mutant phenotype. Our compiled results suggest three genes for future studies. The three candidate genes, SLC30A5 (a zinc transporter), CENPH (a centromere protein), and CDK7 (a cyclin-dependent kinase), are differentially expressed (compared to normal embryos) at stages and in tissues affected by the coloboma mutation. Of these genes, two (SLC30A5 and CENPH) are considered high-priority candidate based upon studies in other vertebrate model systems. © 2013 Robb et al.
- Antin, P., Bobbs, A. S., Saarela, A. V., Yatskievych, T. A., & Antin, P. B. (2012). Fibroblast growth factor (FGF) signaling during gastrulation negatively modulates the abundance of microRNAs that regulate proteins required for cell migration and embryo patterning. The Journal of biological chemistry, 287(46).More infoFGF 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.
- Bobbs, A. S., Saarela, A. V., Yatskievych, T. A., & Antin, P. B. (2012). Fibroblast Growth Factor (FGF) signaling during gastrulation negatively modulates the abundance of microRNAs that regulate proteins required for cell migration and embryo patterning. Journal of Biological Chemistry, 287(46), 38505-38514.More infoPMID: 22995917;PMCID: PMC3493895;Abstract: 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. PredictedmRNAtargets 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. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
- Chen, H., Untiveros, G. M., A., L., Perez, J., Jing, L. i., Antin, P. B., & Konhilas, J. P. (2012). Micro-RNA-195 and -451 regulate the LKB1/AMPK signaling axis by targeting MO25. PLoS ONE, 7(7).More infoPMID: 22844503;PMCID: PMC3402395;Abstract: Background: 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. Methodology/Principal Findings: 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. Conclusions/Significance: 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. © 2012 Chen et al.
- Chen, H., Untiveros, G. M., McKee, L. A., Perez, J., Li, J., Antin, P. B., & Konhilas, J. P. (2012). Micro-RNA-195 and -451 regulate the LKB1/AMPK signaling axis by targeting MO25. PloS one, 7(7), e41574.More infoRecently, 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.
- Menendez, L., Yatskievych, T. A., Antin, P. B., & Dalton, S. (2012). Wnt signaling and a Smad pathway blockade direct the differentiation of human pluripotent stem cells to multipotent neural crest cells (Proceedings of the National Academy of Sciences (2011) 108, 48 (19240-19245) DOI: 10.1073/pnas.1113746108). Proceedings of the National Academy of Sciences of the United States of America, 109(23), 9220-.
- Antin, P., Hardy, K. M., Yatskievych, T. A., Konieczka, J., Bobbs, A. S., & Antin, P. B. (2011). FGF signalling through RAS/MAPK and PI3K pathways regulates cell movement and gene expression in the chicken primitive streak without affecting E-cadherin expression. BMC developmental biology, 11.More infoFGF 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.
- Hardy, K. M., Yatskievych, T. A., Konieczka, J., Bobbs, A. S., & Antin, P. B. (2011). FGF signalling through RAS/MAPK and PI3K pathways regulates cell movement and gene expression in the chicken primitive streak without affecting E-cadherin expression. BMC Developmental Biology, 11.More infoPMID: 21418646;PMCID: PMC3071786;Abstract: Background: 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. Results: 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. Conclusions: 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. © 2011 Hardy et al; licensee BioMed Central Ltd.
- Menendez, L., Yatskievych, T. A., Antin, P. B., & Dalton, S. (2011). Wnt signaling and a Smad pathway blockade direct the differentiation of human pluripotent stem cells to multipotent neural crest cells. Proceedings of the National Academy of Sciences of the United States of America, 108(48), 19240-19245.More infoPMID: 22084120;PMCID: PMC3228464;Abstract: Neural crest stem cells can be isolated from differentiated cultures of human pluripotent stem cells, but the process is inefficient and requires cell sorting to obtain a highly enriched population. No specific method for directed differentiation of human pluripotent cells toward neural crest stem cells has yet been reported. This severely restricts the utility of these cells as a model for disease and development and for more applied purposes such as cell therapy and tissue engineering. In this report, we use small-molecule compounds in a single-step method for the efficient generation of self-renewing neural crest-like stem cells in chemically defined media. This approach is accomplished directly from human pluripotent cells without the need for coculture on feeder layers or cell sorting to obtain a highly enriched population. Critical to this approach is the activation of canonical Wnt signaling and concurrent suppression of the Activin A/Nodal pathway. Over 12-14 d, pluripotent cells are efficiently specified along the neuroectodermlineage toward p75 + Hnk1 + Ap2 + neural crest-like cells with little or no contamination by Pax6 + neural progenitors. This cell population can be clonally amplified and maintained for >25 passages (>100 d) while retaining the capacity to differentiate into peripheral neurons, smooth muscle cells, and mesenchymal precursor cells. Neural crest-like stem cell-derived mesenchymal precursors have the capacity for differentiation into osteocytes, chondrocytes, and adipocytes. In sum, we have developed methods for the efficient generation of self-renewing neural crest stem cells that greatly enhance their potential utility in disease modeling and regenerative medicine.
- Menendez, L., Yatskievych, T. A., Antin, P. B., & Dalton, S. (2011). Wnt signaling and a Smad pathway blockade direct the differentiation of human pluripotent stem cells to multipotent neural crest cells. Proceedings of the National Academy of Sciences of the United States of America, 108(48), 19240-5.More infoNeural crest stem cells can be isolated from differentiated cultures of human pluripotent stem cells, but the process is inefficient and requires cell sorting to obtain a highly enriched population. No specific method for directed differentiation of human pluripotent cells toward neural crest stem cells has yet been reported. This severely restricts the utility of these cells as a model for disease and development and for more applied purposes such as cell therapy and tissue engineering. In this report, we use small-molecule compounds in a single-step method for the efficient generation of self-renewing neural crest-like stem cells in chemically defined media. This approach is accomplished directly from human pluripotent cells without the need for coculture on feeder layers or cell sorting to obtain a highly enriched population. Critical to this approach is the activation of canonical Wnt signaling and concurrent suppression of the Activin A/Nodal pathway. Over 12-14 d, pluripotent cells are efficiently specified along the neuroectoderm lineage toward p75(+) Hnk1(+) Ap2(+) neural crest-like cells with little or no contamination by Pax6(+) neural progenitors. This cell population can be clonally amplified and maintained for >25 passages (>100 d) while retaining the capacity to differentiate into peripheral neurons, smooth muscle cells, and mesenchymal precursor cells. Neural crest-like stem cell-derived mesenchymal precursors have the capacity for differentiation into osteocytes, chondrocytes, and adipocytes. In sum, we have developed methods for the efficient generation of self-renewing neural crest stem cells that greatly enhance their potential utility in disease modeling and regenerative medicine.
- Yaklichkin, S. Y., Darnell, D. K., Pier, M. V., Antin, P. B., & Hannenhalli, S. (2011). Accelerated evolution of 3'avian FOXE1 genes, and thyroid and feather specific expression of chicken FoxE1. BMC Evolutionary Biology, 11(1).More infoPMID: 21999483;PMCID: PMC3207924;Abstract: Background: The forkhead transcription factor gene E1 (FOXE1) plays an important role in regulation of thyroid development, palate formation and hair morphogenesis in mammals. However, avian FOXE1 genes have not been characterized and as such, codon evolution of FOXE1 orthologs in a broader evolutionary context of mammals and birds is not known. Results: In this study we identified the avian FOXE1 gene in chicken, turkey and zebra finch, all of which consist of a single exon. Chicken and zebra finch FOXE1 are uniquely located on the sex-determining Z chromosome. In situ hybridization shows that chicken FOXE1 is specifically expressed in the developing thyroid. Its expression is initiated at the placode stage and is maintained during the stages of vesicle formation and follicle primordia. Based on this expression pattern, we propose that avian FOXE1 may be involved in regulating the evagination and morphogenesis of thyroid. Chicken FOXE1 is also expressed in growing feathers. Sequence analysis identified two microdeletions in the avian FOXE1 genes, corresponding to the loss of a transferable repression domain and an engrailed homology motif 1 (Eh1) C-terminal to the forkhead domain. The avian FOXE1 proteins exhibit a significant sequence divergence of the C-terminus compared to those of amphibian and mammalian FOXE1. The codon evolution analysis (dN/dS) of FOXE1 shows a significantly increased dN/dS ratio in the avian lineages, consistent with either a relaxed purifying selection or positive selection on a few residues in avian FOXE1 evolution. Further site specific analysis indicates that while relaxed purifying selection is likely to be a predominant cause of accelerated evolution at the 3'-region of avian FOXE1, a few residues might have evolved under positive selection. Conclusions: We have identified three avian FOXE1 genes based on synteny and sequence similarity as well as characterized the expression pattern of the chicken FOXE1 gene during development. Our evolutionary analyses suggest that while a relaxed purifying selection is likely to be the dominant force driving accelerated evolution of avian FOXE1 genes, a few residues may have evolved adaptively. This study provides a basis for future genetic and comparative biochemical studies of FOXE1. © 2011 Yaklichkin et al.; licensee BioMed Central Ltd.
- Yaklichkin, S. Y., Darnell, D. K., Pier, M. V., Antin, P. B., & Hannenhalli, S. (2011). Accelerated evolution of 3'avian FOXE1 genes, and thyroid and feather specific expression of chicken FoxE1. BMC evolutionary biology, 11, 302.More infoThe forkhead transcription factor gene E1 (FOXE1) plays an important role in regulation of thyroid development, palate formation and hair morphogenesis in mammals. However, avian FOXE1 genes have not been characterized and as such, codon evolution of FOXE1 orthologs in a broader evolutionary context of mammals and birds is not known.
- Antin, P. B., Pier, M., Sesepasara, T., Yatskievych, T. A., & Darnell, D. K. (2010). Embryonic expression of the chicken Krüppel-like (KLF) transcription factor gene family. Developmental Dynamics, 239(6), 1879-1887.More infoPMID: 20503383;PMCID: PMC2925473;Abstract: 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. © 2010 Wiley-Liss, Inc.
- Antin, P. B., Pier, M., Sesepasara, T., Yatskievych, T. A., & Darnell, D. K. (2010). Embryonic expression of the chicken Krüppel-like (KLF) transcription factor gene family. Developmental dynamics : an official publication of the American Association of Anatomists, 239(6), 1879-87.More infoThe 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.
- Antin, P., Darnell, D. K., Stanislaw, S., Kaur, S., & Antin, P. B. (2010). Whole mount in situ hybridization detection of mRNAs using short LNA containing DNA oligonucleotide probes. RNA (New York, N.Y.), 16(3).More infoIn 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.
- Darnell, D. K., Stanislaw, S., Kaur, S., & Antin, P. B. (2010). Whole mount in situ hybridization detection of mRNAs using short LNA containing DNA oligonucleotide probes. RNA, 16(3), 632-637.More infoPMID: 20086052;PMCID: PMC2822927;Abstract: 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. Published by Cold Spring Harbor Laboratory Press. Copyright © 2010 RNA Society.
- Lencinas, A., Broka, D. M., Konieczka, J. H., Klewer, S. E., Antin, P. B., Camenisch, T. D., & Runyan, R. B. (2010). Arsenic exposure perturbs epithelial-mesenchymal cell transition and gene expression in a collagen gel assay. Toxicological Sciences, 116(1), 273-285.More infoPMID: 20308225;PMCID: PMC2886855;Abstract: 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. © The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org.
- Lencinas, A., Broka, D. M., Konieczka, J. H., Klewer, S. E., Antin, P. B., Camenisch, T. D., & Runyan, R. B. (2010). Arsenic exposure perturbs epithelial-mesenchymal cell transition and gene expression in a collagen gel assay. Toxicological sciences : an official journal of the Society of Toxicology, 116(1), 273-85.More infoArsenic 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.
- Lopes, L., Konieczka, J., Foulk, V., & Antin, P. (2010). Network Elucidation Template: A framework for human-guided network inference. Computers and Industrial Engineering, 58(4), 680-690.More infoAbstract: Network elucidation is the problem of inferring all parameters of a network from a subset of those parameters. We introduce the Network Elucidation Template (NET), which provides a framework upon which algorithms for such problems can be built. NET algorithms take advantage of novel methods for collaboration between human operators and computers. They use visualizations of the peculiar structures that appear in optimal solutions to aid the parameter search. By design, NET is at a high enough level of abstraction to describe a class of algorithms, as opposed to a single algorithm. Given a problem, and the structure of that problem, an effective instantiation of the template into an algorithm can be created. We describe one such instantiation: using a network flow framework to implement a NET algorithm for uncovering smuggling networks; as well as the general template. © 2010 Elsevier Ltd. All rights reserved.
- Tsukada, T., Pappas, C. T., Moroz, N., Antin, P. B., Kostyukova, A. S., & Gregorio, C. C. (2010). Leiomodin-2 is an antagonist of tropomodulin-1 at the pointed end of the thin filaments in cardiac muscle. Journal of Cell Science, 123(18), 3136-3145.More infoPMID: 20736303;PMCID: PMC2931607;Abstract: 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.
- Tsukada, T., Pappas, C. T., Moroz, N., Antin, P. B., Kostyukova, A. S., & Gregorio, C. C. (2010). Leiomodin-2 is an antagonist of tropomodulin-1 at the pointed end of the thin filaments in cardiac muscle. Journal of cell science, 123(Pt 18), 3136-45.More infoRegulation 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.
- Antin, P., Konieczka, J. H., Drew, K., Pine, A., Belasco, K., Davey, S., Yatskievych, T. A., Bonneau, R., & Antin, P. B. (2009). BioNetBuilder2.0: bringing systems biology to chicken and other model organisms. BMC genomics, 10 Suppl 2.More infoSystems Biology research tools, such as Cytoscape, have greatly extended the reach of genomic research. By providing platforms to integrate data with molecular interaction networks, researchers can more rapidly begin interpretation of large data sets collected for a system of interest. BioNetBuilder is an open-source client-server Cytoscape plugin that automatically integrates molecular interactions from all major public interaction databases and serves them directly to the user's Cytoscape environment. Until recently however, chicken and other eukaryotic model systems had little interaction data available.
- Burt, D. W., Carrë, W., Fell, M., Law, A. S., Antin, P. B., Maglott, D. R., Weber, J. A., Schmidt, C. J., Burgess, S. C., & McCarthy, F. M. (2009). The Chicken Gene Nomenclature Committee report. BMC genomics, 10 Suppl 2, S5.More infoComparative genomics is an essential component of the post-genomic era. The chicken genome is the first avian genome to be sequenced and it will serve as a model for other avian species. Moreover, due to its unique evolutionary niche, the chicken genome can be used to understand evolution of functional elements and gene regulation in mammalian species. However comparative biology both within avian species and within amniotes is hampered due to the difficulty of recognising functional orthologs. This problem is compounded as different databases and sequence repositories proliferate and the names they assign to functional elements proliferate along with them. Currently, genes can be published under more than one name and one name sometimes refers to unrelated genes. Standardized gene nomenclature is necessary to facilitate communication between scientists and genomic resources. Moreover, it is important that this nomenclature be based on existing nomenclature efforts where possible to truly facilitate studies between different species. We report here the formation of the Chicken Gene Nomenclature Committee (CGNC), an international and centralized effort to provide standardized nomenclature for chicken genes. The CGNC works in conjunction with public resources such as NCBI and Ensembl and in consultation with existing nomenclature committees for human and mouse. The CGNC will develop standardized nomenclature in consultation with the research community and relies on the support of the research community to ensure that the nomenclature facilitates comparative and genomic studies.
- Burt, D. W., Carrë, W., Fell, M., Law, A. S., Antin, P. B., Maglott, D. R., Weber, J. A., Schmidt, C. J., Burgess, S. C., & McCarthy, F. M. (2009). The chicken gene nomenclature committee report. BMC Genomics, 10(SUPPL. 2).More infoPMID: 19607656;PMCID: PMC2966335;Abstract: Comparative genomics is an essential component of the post-genomic era. The chicken genome is the first avian genome to be sequenced and it will serve as a model for other avian species. Moreover, due to its unique evolutionary niche, the chicken genome can be used to understand evolution of functional elements and gene regulation in mammalian species. However comparative biology both within avian species and within amniotes is hampered due to the difficulty of recognising functional orthologs. This problem is compounded as different databases and sequence repositories proliferate and the names they assign to functional elements proliferate along with them. Currently, genes can be published under more than one name and one name sometimes refers to unrelated genes. Standardized gene nomenclature is necessary to facilitate communication between scientists and genomic resources. Moreover, it is important that this nomenclature be based on existing nomenclature efforts where possible to truly facilitate studies between different species. We report here the formation of the Chicken Gene Nomenclature Committee (CGNC), an international and centralized effort to provide standardized nomenclature for chicken genes. The CGNC works in conjunction with public resources such as NCBI and Ensembl and in consultation with existing nomenclature committees for human and mouse. The CGNC will develop standardized nomenclature in consultation with the research community and relies on the support of the research community to ensure that the nomenclature facilitates comparative and genomic studies. © 2009 Burt et al; licensee BioMed Central Ltd.
- Cole, L., Anderson, M., Antin, P. B., & Limesand, S. W. (2009). One process for pancreatic beta-cell coalescence into islets involves an epithelial-mesenchymal transition. The Journal of endocrinology, 203(1), 19-31.More infoIslet replacement is a promising therapy for treating diabetes mellitus, but the supply of donor tissue for transplantation is limited. To overcome this limitation, endocrine tissue can be expanded, but this requires an understanding of normal developmental processes that regulate islet formation. In this study, we compare pancreas development in sheep and human, and provide evidence that an epithelial-mesenchymal transition (EMT) is involved in beta-cell differentiation and islet formation. Transcription factors know to regulate pancreas formation, pancreatic duodenal homeobox factor 1, neurogenin 3, NKX2-2, and NKX6-1, which were expressed in the appropriate spatial and temporal pattern to coordinate pancreatic bud outgrowth and direct endocrine cell specification in sheep. Immunofluorescence staining of the developing pancreas was used to co-localize insulin and epithelial proteins (cytokeratin, E-cadherin, and beta-catenin) or insulin and a mesenchymal protein (vimentin). In sheep, individual beta-cells become insulin-positive in the progenitor epithelium, then lose epithelial characteristics, and migrate out of the epithelial layer to form islets. As beta-cells exit the epithelial progenitor cell layer, they acquire mesenchymal characteristics, shown by their acquisition of vimentin. In situ hybridization expression analysis of the SNAIL family members of transcriptional repressors (SNAIL1, -2, and -3; listed as SNAI1, -2, -3 in the HUGO Database) showed that each of the SNAIL genes was expressed in the ductal epithelium during development, and SNAIL-1 and -2 were co-expressed with insulin. Our findings provide strong evidence that the movement of beta-cells from the pancreatic ductal epithelium involves an EMT.
- Cole, L., Anderson, M., Antin, P. B., & Limesand, S. W. (2009). One process for pancreatic β-cell coalescence into islets involves an epithelial-mesenchymal transition. Journal of Endocrinology, 203(1), 19-31.More infoPMID: 19608613;PMCID: PMC3071757;Abstract: Islet replacement is a promising therapy for treating diabetes mellitus, but the supply of donor tissue for transplantation is limited. To overcome this limitation, endocrine tissue can be expanded, but this requires an understanding of normal developmental processes that regulate islet formation. In this study, we compare pancreas development in sheep and human, and provide evidence that an epithelial-mesenchymal transition (EMT) is involved in β-cell differentiation and islet formation. Transcription factors know to regulate pancreas formation, pancreatic duodenal homeobox factor 1, neurogenin 3, NKX2-2, and NKX6-1, which were expressed in the appropriate spatial and temporal pattern to coordinate pancreatic bud outgrowth and direct endocrine cell specifi-cation in sheep. Immunofluorescence staining of the developing pancreas was used to co-localize insulin and epithelial proteins (cytokeratin, E-cadherin, and β-catenin) or insulin and a mesenchymal protein (vimentin). In sheep, individual β-cells become insulin-positive in the progenitor epithelium, then lose epithelial characteristics, and migrate out of the epithelial layer to form islets. As β-cells exit the epithelial progenitor cell layer, they acquire mesenchymal characteristics, shown by their acquisition of vimentin. In situ hybridization expression analysis of the SNAIL family members of transcriptional repressors (SNAIL1, -2, and -3; listed as SNAI1, -2, -3 in the HUGO Database) showed that each of the SNAIL genes was expressed in the ductal epithelium during development, and SNAIL-1 and -2 were co-expressed with insulin. Our findings provide strong evidence that the movement of β-cells from the pancreatic ductal epithelium involves an EMT. © 2009 Society for Endocrinology.
- Konieczka, J. H., Drew, K., Pine, A., Belasco, K., Davey, S., Yatskievych, T. A., Bonneau, R., & Antin, P. B. (2009). BioNetBuilder2.0: Bringing systems biology to chicken and other model organisms. BMC Genomics, 10(SUPPL. 2).More infoPMID: 19607657;PMCID: PMC2966329;Abstract: Background: Systems Biology research tools, such as Cytoscape, have greatly extended the reach of genomic research. By providing platforms to integrate data with molecular interaction networks, researchers can more rapidly begin interpretation of large data sets collected for a system of interest. BioNetBuilder is an open-source client-server Cytoscape plugin that automatically integrates molecular interactions from all major public interaction databases and serves them directly to the user's Cytoscape environment. Until recently however, chicken and other eukaryotic model systems had little interaction data available. Results: Version 2.0 of BioNetBuilder includes a redesigned synonyms resolution engine that enables transfer and integration of interactions across species; this engine translates between alternate gene names as well as between orthologs in multiple species. Additionally, BioNetBuilder is now implemented to be part of the Gaggle, thereby allowing seamless communication of interaction data to any software implementing the widely used Gaggle software. Using BioNetBuilder, we constructed a chicken interactome possessing 72,000 interactions among 8,140 genes directly in the Cytoscape environment. In this paper, we present a tutorial on how to do so and analysis of a specific use case. Conclusion: BioNetBuilder 2.0 provides numerous user-friendly systems biology tools that were otherwise inaccessible to researchers in chicken genomics, as well as other model systems. We provide a detailed tutorial spanning all required steps in the analysis. BioNetBuilder 2.0, the tools for maintaining its data bases, standard operating procedures for creating local copies of its back-end data bases, as well as all of the Gaggle and Cytoscape codes required, are open-source and freely available at http://err.bio.nyu.edu/cytoscape/bionetbuilder/. © 2009 Konieczka et al; licensee BioMed Central Ltd.
- Schoenwolf, G. C., Antin, P. B., Mikawa, T., & Solnica-Krezel, L. (2009). In a world with many development journals, why choose to publish in developmental dynamics?. Developmental Dynamics, 238(1), 1-.
- Albertine, K. H., Antin, P. B., Padhye, S., & Pendleton, A. (2008). Open Access: AR is Fully Compliant with Mandates from NIH and Other Funding Agencies. Anatomical Record, 291(12), 1573-.
- Antin, P., Warkman, A. S., Yatskievych, T. A., Hardy, K. M., Krieg, P. A., & Antin, P. B. (2008). Myocardin expression during avian embryonic heart development requires the endoderm but is independent of BMP signaling. Developmental dynamics : an official publication of the American Association of Anatomists, 237(1).More infoMyocardin, a serum response factor cofactor, plays an important role in regulating heart and smooth muscle development. To investigate myocardin function during early stages of heart development, we isolated the chicken orthologue of myocardin and characterized its expression between Hamburger and Hamilton stages 3 and 15. At stage 4, myocardin transcripts are detected in the lateral and extraembryonic mesoderm, become progressively localized to the precardiac mesoderm and the differentiated myocardium and are also seen in smooth muscle cells of the developing vascular plexus. Surprisingly, myocardin expression within the developing chicken embryo precedes that of the homeodomain transcription factor Nkx2.5. Embryonic dissection studies demonstrate that signals from the endoderm are required for myocardin expression within the precardiac mesoderm. However, unlike Nkx2.5, myocardin expression is not regulated by bone morphogenetic protein (BMP) signaling. These results suggest that initial expression of myocardin in the precardiac mesoderm is regulated by a signaling pathway that is parallel to, and independent of, Nkx2.5 expression.
- Hardy, K. M., Garriock, R. J., Yatskievych, T. A., D'Agostino, S. L., Antin, P. B., & Krieg, P. A. (2008). Non-canonical Wnt signaling through Wnt5a/b and a novel Wnt11 gene, Wnt11b, regulates cell migration during avian gastrulation. Developmental Biology, 320(2), 391-401.More infoPMID: 18602094;PMCID: PMC2539108;Abstract: Knowledge of the molecular mechanisms regulating cell ingression, epithelial-mesenchymal transition and migration movements during amniote gastrulation is steadily improving. In the frog and fish embryo, Wnt5 and Wnt11 ligands are expressed around the blastopore and play an important role in regulating cell movements associated with gastrulation. In the chicken embryo, although Wnt5a and Wnt5b are expressed in the primitive streak, the known Wnt11 gene is expressed in paraxial and intermediate mesoderm, and in differentiated myocardial cells, but not in the streak. Here, we identify a previously uncharacterized chicken Wnt11 gene, Wnt11b, that is orthologous to the frog Wnt11 and zebrafish Wnt11 (silberblick) genes. Chicken Wnt11b is expressed in the primitive streak in a pattern similar to chicken Wnt5a and Wnt5b. When non-canonical Wnt signaling is blocked using a Dishevelled dominant-negative protein, gastrulation movements are inhibited and cells accumulate in the primitive streak. Furthermore, disruption of non-canonical Wnt signaling by overexpression of full-length or dominant-negative Wnt11b or Wnt5a constructions abrogates normal cell migration through the primitive streak. We conclude that non-canonical Wnt signaling, mediated in part by Wnt11b, is important for regulation of gastrulation cell movements in the avian embryo. © 2008 Elsevier Inc. All rights reserved.
- Hardy, K. M., Garriock, R. J., Yatskievych, T. A., D'Agostino, S. L., Antin, P. B., & Krieg, P. A. (2008). Non-canonical Wnt signaling through Wnt5a/b and a novel Wnt11 gene, Wnt11b, regulates cell migration during avian gastrulation. Developmental biology, 320(2), 391-401.More infoKnowledge of the molecular mechanisms regulating cell ingression, epithelial-mesenchymal transition and migration movements during amniote gastrulation is steadily improving. In the frog and fish embryo, Wnt5 and Wnt11 ligands are expressed around the blastopore and play an important role in regulating cell movements associated with gastrulation. In the chicken embryo, although Wnt5a and Wnt5b are expressed in the primitive streak, the known Wnt11 gene is expressed in paraxial and intermediate mesoderm, and in differentiated myocardial cells, but not in the streak. Here, we identify a previously uncharacterized chicken Wnt11 gene, Wnt11b, that is orthologous to the frog Wnt11 and zebrafish Wnt11 (silberblick) genes. Chicken Wnt11b is expressed in the primitive streak in a pattern similar to chicken Wnt5a and Wnt5b. When non-canonical Wnt signaling is blocked using a Dishevelled dominant-negative protein, gastrulation movements are inhibited and cells accumulate in the primitive streak. Furthermore, disruption of non-canonical Wnt signaling by overexpression of full-length or dominant-negative Wnt11b or Wnt5a constructions abrogates normal cell migration through the primitive streak. We conclude that non-canonical Wnt signaling, mediated in part by Wnt11b, is important for regulation of gastrulation cell movements in the avian embryo.
- Schoenwolf, G., Antin, P., Padhye, S., & Pendleton, A. (2008). DD is fully compliant (and then some) with NIH and other funding agencies. Developmental Dynamics, 237(9), 2283-.
- Warkman, A. S., Yatskievych, T. A., Hardy, K. M., Krieg, P. A., & Antin, P. B. (2008). Myocardin expression during avian embryonic heart development requires the endoderm but is independent of BMP signaling. Developmental Dynamics, 237(1), 216-221.More infoPMID: 18069699;Abstract: Myocardin, a serum response factor cofactor, plays an important role in regulating heart and smooth muscle development. To investigate myocardin function during early stages of heart development, we isolated the chicken orthologue of myocardin and characterized its expression between Hamburger and Hamilton stages 3 and 15. At stage 4, myocardin transcripts are detected in the lateral and extraembryonic mesoderm, become progressively localized to the precardiac mesoderm and the differentiated myocardium and are also seen in smooth muscle cells of the developing vascular plexus. Surprisingly, myocardin expression within the developing chicken embryo precedes that of the homeodomain transcription factor Nkx2.5. Embryonic dissection studies demonstrate that signals from the endoderm are required for myocardin expression within the precardiac mesoderm. However, unlike Nkx2.5, myocardin expression is not regulated by bone morphogenetic protein (BMP) signaling. These results suggest that initial expression of myocardin in the precardiac mesoderm is regulated by a signaling pathway that is parallel to, and independent of, Nkx2.5 expression. © 2007 Wiley-Liss, Inc.
- Antin, P. B., Kaur, S., Stanislaw, S., Davey, S., Konieczka, J. H., Yatskievych, T. A., & Darnell, D. K. (2007). Gallus expression in situ hybridization analysis: A chicken embryo gene expression database. Poultry Science, 86(7), 1472-1477.More infoPMID: 17575198;Abstract: With sequencing of the chicken genome largely completed, significant effort is focusing on gene annotation, including acquiring information about the patterns of gene expression. The chicken embryo is ideally suited to provide detailed temporal and spatial expression information through in situ hybridization gene expression analysis in vivo. We have developed the Gallus expression in situ hybridization analysis (GEISHA) database and user interface (http://geisha.arizona.edu) to serve as a centralized repository of in situ hybridization photos and metadata from chicken embryos. This report describes the design and implementation the GEISHA database and Web site and illustrates its usefulness for researchers in the biomedical and poultry science communities. Results from a recent comprehensive expression analysis of microRNA expression in chicken embryos are also presented. ©2007 Poultry Science Association Inc.
- Antin, P. B., Kaur, S., Stanislaw, S., Davey, S., Konieczka, J. H., Yatskievych, T. A., & Darnell, D. K. (2007). Gallus expression in situ hybridization analysis: a chicken embryo gene expression database. Poultry science, 86(7), 1472-7.More infoWith sequencing of the chicken genome largely completed, significant effort is focusing on gene annotation, including acquiring information about the patterns of gene expression. The chicken embryo is ideally suited to provide detailed temporal and spatial expression information through in situ hybridization gene expression analysis in vivo. We have developed the Gallus expression in situ hybridization analysis (GEISHA) database and user interface (http://geisha.arizona.edu) to serve as a centralized repository of in situ hybridization photos and metadata from chicken embryos. This report describes the design and implementation the GEISHA database and Web site and illustrates its usefulness for researchers in the biomedical and poultry science communities. Results from a recent comprehensive expression analysis of microRNA expression in chicken embryos are also presented.
- Darnell, D. K., Kaur, S., Stanislaw, S., Davey, S., Konieczka, J. H., Yatskievych, T. A., & Antin, P. B. (2007). GEISHA: An in situ hybridization gene expression resource for the chicken embryo. Cytogenetic and Genome Research, 117(1-4), 30-35.More infoPMID: 17675842;Abstract: An important and ongoing focus of biomedical and agricultural avian research is to understand gene function, which for a significant fraction of genes remains unknown. A first step is to determine when and where genes are expressed during development and in the adult. Whole mount in situ hybridization gives precise spatial and temporal resolution of gene expression throughout an embryo, and a comprehensive analysis and centralized repository of in situ hybridization information would provide a valuable research tool. The GEISHA project (gallus expression in situ hybridization analysis) was initiated to explore the utility of using high-throughput in situ hybridization as a means for gene discovery and annotation in chicken embryos, and to provide a unified repository for in situ hybridization information. This report describes the design and implementation of a new GEISHA database and user interface (www.geisha.arizona.edu), and illustrates its utility for researchers in the biomedical and poultry science communities. Results obtained from a high throughput screen of microRNA expression in chicken embryos are also presented. Copyright © 2007 S. Karger AG.
- Darnell, D. K., Kaur, S., Stanislaw, S., Davey, S., Konieczka, J. H., Yatskievych, T. A., & Antin, P. B. (2007). GEISHA: an in situ hybridization gene expression resource for the chicken embryo. Cytogenetic and genome research, 117(1-4), 30-5.More infoAn important and ongoing focus of biomedical and agricultural avian research is to understand gene function, which for a significant fraction of genes remains unknown. A first step is to determine when and where genes are expressed during development and in the adult. Whole mount in situ hybridization gives precise spatial and temporal resolution of gene expression throughout an embryo, and a comprehensive analysis and centralized repository of in situ hybridization information would provide a valuable research tool. The GEISHA project (gallus expression in situ hybridization analysis) was initiated to explore the utility of using high-throughput in situ hybridization as a means for gene discovery and annotation in chicken embryos, and to provide a unified repository for in situ hybridization information. This report describes the design and implementation of a new GEISHA database and user interface (www.geisha.arizona.edu), and illustrates its utility for researchers in the biomedical and poultry science communities. Results obtained from a high throughput screen of microRNA expression in chicken embryos are also presented.
- Darnell, D. K., Kaur, S., Stanislaw, S., Konieczka, J. H., Yatskievych, T. A., & Antin, P. B. (2007). Erratum: MicroRNA expression during chick embryo development (Developmental Dynamics 235 (3156-3165)). Developmental Dynamics, 236(1), 333-.
- Antin, P., Darnell, D. K., Kaur, S., Stanislaw, S., Konieczka, J. H., Konieczka, J. K., Yatskievych, T. A., & Antin, P. B. (2006). MicroRNA expression during chick embryo development. Developmental dynamics : an official publication of the American Association of Anatomists, 235(11).More infoMicroRNAs (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.
- Antin, P., Hardy, K. M., Mjaatvedt, C. H., & Antin, P. B. (2006). Hot hearts in the Sonoran Desert: the 11th Weinstein Cardiovascular Development Conference in Tucson. Developmental dynamics : an official publication of the American Association of Anatomists, 235(1).More infoThe 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.
- Ason, B., Darnell, D. K., Wittbrodt, B., Berezikov, E., Kloosterman, W. P., Wittbrodt, J., Antin, P. B., & H., R. (2006). Differences in vertebrate microRNA expression. Proceedings of the National Academy of Sciences of the United States of America, 103(39), 14385-14389.More infoPMID: 16983084;PMCID: PMC1599972;Abstract: 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 ≈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. © 2006 by The National Academy of Sciences of the USA.
- Darnell, D. K., Kaur, S., Stanislaw, S., Konieczka, J. K., Yatskievych, T. A., & Antin, P. B. (2006). MicroRNA expression during chick embryo development. Developmental Dynamics, 235(11), 3156-3165.More infoPMID: 17013880;Abstract: 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. © 2006 Wiley-Liss, Inc.
- Doyle, S. E., Scholz, M. J., Greer, K. A., Hubbard, A. D., Darnell, D. K., Antin, P. B., Klewer, S. E., & Runyan, R. B. (2006). Latrophilin-2 is a novel component of the epithelial-mesenchymal transition within the atrioventricular canal of the embryonic chicken heart. Developmental Dynamics, 235(12), 3213-3221.More infoPMID: 17016846;Abstract: 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. © 2006 Wiley-Liss, Inc.
- Hardy, K. M., Mjaatvedt, C. H., & Antin, P. B. (2006). Hot hearts in the sonoran desert: The 11th Weinstein Cardiovascular Development Conference in Tucson. Developmental Dynamics, 235(1), 170-175.More infoPMID: 16273525;Abstract: 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. © 2005 Wiley-Liss, Inc.
- Klewer, S. E., Yatskievych, T., Pogreba, K., Stevens, M. V., Antin, P. B., & Camenisch, T. D. (2006). Has2 expression in heart forming regions is independent of BMP signaling. Gene Expression Patterns, 6(5), 462-470.More infoPMID: 16458617;Abstract: 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. © 2005 Elsevier B.V. All rights reserved.
- Rodgers, L. S., Lalani, S., Hardy, K. M., Xiang, X., Broka, D., Antin, P. B., & Camenisch, T. D. (2006). Depolymerized hyaluronan induces vascular endothelial growth factor, a negative regulator of developmental epithelial-to-mesenchymal transformation. Circulation Research, 99(6), 583-589.More infoPMID: 16931798;Abstract: 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. © 2006 American Heart Association, Inc.
- Antin, P. B., & Konieczka, J. H. (2005). Genomic resources for chicken. Developmental Dynamics, 232(4), 877-882.More infoPMID: 15739221;Abstract: 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. © 2005 Wiley-Liss, Inc.
- Ono, Y., Schwach, C., Antin, P. B., & Gregorio, C. C. (2005). Disruption in the tropomodulin1 (Tmod1) gene compromises cardiomyocyte development in murine embryonic stem cells by arresting myofibril maturation. Developmental Biology, 282(2), 336-348.More infoPMID: 15950601;Abstract: 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, ∼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. © 2005 Elsevier Inc. All rights reserved.
- Antin, P. B., Fallon, J. F., & Schoenwolf, G. C. (2004). The Chick Embryo Rules (Still)!. Developmental Dynamics, 229(3), 413-.More infoPMID: 14991695;
- Antin, P. B., Schoenwolf, G. C., & Olsen, N. A. (2004). A bigger bang for your buck: Enhanced access to your chick data. Developmental Dynamics, 230(3), 391-.More infoPMID: 15188424;
- Antin, P., Bell, G. W., Yatskievych, T. A., & Antin, P. B. (2004). GEISHA, a whole-mount in situ hybridization gene expression screen in chicken embryos. Developmental dynamics : an official publication of the American Association of Anatomists, 229(3).More infoDespite 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.
- Antin, P., Zhang, W., Yatskievych, T. A., Baker, R. K., & Antin, P. B. (2004). Regulation of Hex gene expression and initial stages of avian hepatogenesis by Bmp and Fgf signaling. Developmental biology, 268(2).More infoThe 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.
- Bell, G. W., Yatskievych, T. A., & Antin, P. B. (2004). GEISHA, a Whole-Mount in Situ Hybridization Gene Expression Screen in Chicken Embryos. Developmental Dynamics, 229(3), 677-687.More infoPMID: 14991723;Abstract: 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. © 2004 Wiley-Liss, Inc.
- Vokes, S. A., Yatskievych, T. A., Heimark, R. L., McMahon, J., McMahon, A. P., Antin, P. B., & Krieg, P. A. (2004). Hedgehog signaling is essential for endothelial tube formation during vasculogenesis. Development, 131(17), 4371-4380.More infoPMID: 15294868;Abstract: 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.
- Zhang, W., Yatskievych, T. A., Baker, R. K., & Antin, P. B. (2004). Regulation of Hex gene expression and initial stages of avian hepatogenesis by Bmp and Fgf signaling. Developmental Biology, 268(2), 312-326.More infoPMID: 15063170;Abstract: 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. © 2004 Elsevier Inc. All rights reserved.
- Antin, P., Baker, R. K., & Antin, P. B. (2003). Ephs and ephrins during early stages of chick embryogenesis. Developmental dynamics : an official publication of the American Association of Anatomists, 228(1).More infoThe 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.
- Antin, P., Grill, M. A., Bales, M. A., Fought, A. N., Rosburg, K. C., Munger, S. J., & Antin, P. B. (2003). Tetracycline-inducible system for regulation of skeletal muscle-specific gene expression in transgenic mice. Transgenic research, 12(1).More infoTightly 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.
- Baker, R. K., & Antin, P. B. (2003). Ephs and ephrins during early stages of chick embryogenesis. Developmental Dynamics, 228(1), 128-142.More infoPMID: 12950087;Abstract: 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. © 2003 Wiley-Liss, Inc.
- Grill, M. A., Bales, M. A., Fought, A. N., Rosburg, K. C., Munger, S. J., & Antin, P. B. (2003). Tetracycline-inducible system for regulation of skeletal muscle-specific gene expression in transgenic mice. Transgenic Research, 12(1), 33-43.More infoPMID: 12650523;Abstract: 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.
- Kazmierski, S. T., Antin, P. B., Witt, C. C., Huebner, N., McElhinny, A. S., Labeit, S., & Gregorio, C. C. (2003). The complete mouse nebulin gene sequence and the identification of cardiac nebulin. Journal of Molecular Biology, 328(4), 835-846.More infoPMID: 12729758;Abstract: Nebulin is a giant (Mr 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 ∼700kDa portion associates with actin along the entire length of the thin filament, its N-terminal region extends to thin filament pointed ends, and ∼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. © 2003 Elsevier Science Ltd. All rights reserved.
- Antin, P. B., Bates, M. A., Zhang, W., Garriock, R. J., & Yatskievych, T. A. (2002). Precocious expression of cardiac troponin T in early chick embryos is independent of bone morphogenetic protein signaling. Developmental Dynamics, 225(2), 135-141.More infoPMID: 12242713;Abstract: 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. © 2002 Wiley-Liss, Inc.
- Antin, P. B., Zhang, W., Bales, M. A., Garriock, R. J., & Yatskievych, T. A. (2002). Erratum: Precocious expression of cardiac troponin T in early chick embryos is independent of bone morphogenetic protein signaling (Developmental Dynamics). Developmental Dynamics, 225(3), 376-.
- Antin, P., Zhang, W., Yatskievych, T. A., Cao, X., & Antin, P. B. (2002). Regulation of Hex gene expression by a Smads-dependent signaling pathway. The Journal of biological chemistry, 277(47).More infoThe 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.
- Zhang, W., Yatskievych, T. A., Cao, X., & Antin, P. B. (2002). Regulation of Hex gene expression by a Smads-dependent signaling pathway. Journal of Biological Chemistry, 277(47), 45435-45441.More infoPMID: 12270938;Abstract: 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-β/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.
- Baker, R. K., Vanderboom, A. K., Bell, G. W., & Antin, P. B. (2001). Expression of the receptor tyrosine kinase gene EphB3 during early stages of chick embryo development. Mechanisms of Development, 104(1-2), 129-132.More infoPMID: 11404090;Abstract: The expression pattern of the receptor tyrosine kinase gene EphB3 was examined during the early stages of chick embryogenesis, and is described in this report. In the gastrula, EphB3 is expressed in epiblast cells adjacent to and entering the anterior portion of the primitive streak; expression is extinguished once cells have ingressed. At headfold stages, EphB3 is strongly transcribed in the floor of the foregut and in anterior lateral endoderm, and is expressed in the subjacent cardiogenic mesoderm. EphB3 is transiently expressed in the lateral ectoderm, neural tube, and neural crest during these stages. Later neural expression is localized to the mesencephalon. In the somitic mesoderm, EphB3 is initially expressed in the sclerotome, but later is expressed predominantly in the dermatome. Prominent expression is also detected in the developing heart, liver, posterior ventral limb bud mesenchyme, pharyngeal arches, and head mesenchyme. Copyright © 2001 Elsevier Science Ireland Ltd.
- Rudy, D. E., Yatskievych, T. A., Antin, P. B., & Gregorio, C. C. (2001). Assembly of thick, thin, and titin filaments in chick precardiac explants. Developmental Dynamics, 221(1), 61-71.More infoPMID: 11357194;Abstract: De novo cardiac myofibril assembly has been difficult to study due to the lack of available cell culture models that clearly and accurately reflect heart muscle development in vivo. However, within precardiac chick embryo ex-plants, premyocardial cells differentiate and commence beating in a temporal pattern that corresponds closely with myocyte differentiation in the embryo. Immunofluorescence staining of explants followed by confocal microscopy revealed that distinct stages of cardiac myofibril assembly, ranging from the earliest detection of sarcomeric proteins to the late appearance of mature myofibrils, were consistently recognized in precardiac cultures. Assembly events involved in the early formation of sarcomeres were clearly visualized and accurately reflected observations described by others during chick heart muscle development. Specifically, the early colocalization of α-actinin and titin dots was observed near the cell periphery representing I-Z -I-like complex formation. Myosin-containing thick filaments assembled independently of actin-containing thin filaments and appeared centered within sarcomeres when titin was also linearly aligned at or near cell borders. An N-terminal epitope of titin was detected earlier than a C-terminal epitope; however, both epitopes were observed to alternate near the cell periphery concomitant with the earliest formation of myofibrils. Although vascular actin was detected within cells during early assembly stages, cardiac actin predominated as the major actin isoform in mature thin filaments. Well-aligned thin filaments were also observed in the absence of organized staining for tropomodulin at thin filament pointed ends, suggesting that tropomodulin is not required to define thin filament lengths. Based on these findings, we conclude that the use of the avian precardiac explant system accurately allows for direct investigation of the mechanisms regulating de novo cardiac myofibrillogenesis. © 2001 Wiley-Liss, Inc.
- Gregorio, C. C., & Antin, P. B. (2000). To the heart of myofibril assembly. Trends in Cell Biology, 10(9), 355-362.More infoPMID: 10932092;Abstract: One of the most fascinating examples of cytoskeletal assembly is the myofibril, the contractile structure of striated (i.e. skeletal and cardiac) muscle. Myofibrils are composed of repeating contractile units known as sarcomeres, perhaps the most highly ordered macromolecular structures in eukaryotic cells. When skeletal and cardiac muscle cells differentiate, thousands of structural and regulatory molecules assemble into the semicrystalline sarcomeric contractile units. As a consequence of this precise assembly, many different classes of proteins function together to convert the molecular interactions of actin and myosin efficiently into the macroscopic movements of contractile activity. Copyright (C) 2000 Elsevier Science Ltd.
- Gerber, W. V., Yatskievych, T. A., Antin, P. B., Correia, K. M., Conlon, R. A., & Krieg, P. A. (1999). The RNA-binding protein gene, hermes, is expressed at high levels in the developing heart. Mechanisms of Development, 80(1), 77-86.More infoPMID: 10096065;Abstract: In a screen for novel sequences expressed during embryonic heart development we have isolated a gene which encodes a putative RNA-binding protein. This protein is a member of one of the largest families of RNA- binding proteins, the RRM (RNA Recognition Motif) family. The gene has been named hermes (for HEart, RRM Expressed Sequence). The hermes protein is 197- amino acids long and contains a single RRM domain. In situ hybridization analysis indicates that hermes is expressed at highest levels in the myocardium of the heart and to a lesser extent in the ganglion layer of the retina, the pronephros and the epiphysis. Expression of hermes in each of these tissues begins at approximately the time of differentiation and is maintained throughout development analysis of the RNA expression of the hermes orthologues from chicken and mouse reveals that, like Xenopus, the most prominent tissue of expression is the developing heart. The sequence and expression pattern of hermes suggests a role in post-transcriptional regulation of heart development.
- Yatskievych, T. A., Pascoe, S., & Antin, P. B. (1999). Expression of the homeobox gene Hex during early stages of chick embryo development. Mechanisms of Development, 80(1), 107-109.More infoPMID: 10096068;Abstract: Whole mount in situ hybridization studies were performed to investigate the expression pattern of the homeobox gene Hex (also known as Prh) during early stages of chick embryogenesis. At the time of laying, cHex transcripts are detected in Koller's sickle and the forming hypoblast. During gastrulation (HH stage 4), chex is expressed in anteriorly-displaced hypoblast cells. At stage 6, cHex transcripts are observed within endoderm in an anterior arc that overlaps the cardiogenic region. Later chex expression is observed within pharyngeal endoderm immediately adjacent to the forming myocardium, in the endocardium and in the liver and thyroid gland primordia. cHex transcripts are also detected within blood islands beginning at stage 4, and in extraembryonic and intraembryonic vascular endothelial cells as vessels form.
- Cong, M., Goll, D. E., & Antin, P. B. (1998). cAMP responsiveness of the bovine calpastatin gene promoter. Biochimica et Biophysica Acta - Gene Structure and Expression, 1443(1-2), 186-192.More infoPMID: 9838106;Abstract: Previous studies have shown that transcription of the gene encoding bovine calpastatin, an inhibitor of the calcium-activated cysteine protease calpain, is upregulated following activation of cAMP-dependent signaling pathways. In this study, deletion and site-directed mutagenesis experiments were performed to identify cis elements conferring cAMP responsiveness. Heterologous promoter assays demonstrated that all cAMP-responsive cis elements were' located within -102 nucleotides (nt) of transcription initiation. Deletion of an element (GTCA) at nt +13 that is identical to half of the palindromic cAMP-responsive element (TGACGTCA) identified in other cAMP-responsive gene promoters had no effect on the response of the calpastatin promoter to dibutyryl-cAMP, although a 67% reduction in basal promoter activity was observed. In contrast, two point mutations in a cis element at nt -76 (GTCA to aTCt) abolished cAMP responsiveness. These results demonstrate that the calpastatin promoter sequence between nt -1653 and +130 contains a single cAMP-responsive element (GTCA) located at nt -76, and suggest a direct molecular pathway by which activation of cAMP signaling could lead to increased calpastatin gene transcription and reduction in calpain-mediated proteolysis.
- Cong, M., Thompson, V. F., Goll, D. E., & Antin, P. B. (1998). The bovine calpastatin gene promoter and a new N-terminal region of the protein are targets for cAMP-dependent protein kinase activity. Journal of Biological Chemistry, 273(1), 660-666.More infoPMID: 9417129;Abstract: To investigate the regulation of calpastatin gene expression, we isolated bovine heart calpastatin cDNAs and 5'-regions of the calpastatin gene. Analysis of 5'-cDNA sequence identified a new translation initiation site that is in frame and 204 nucleotides upstream of the previously designated start site. Conceptual translation from this upstream AUG produces a protein containing 68 additional N-terminal amino acids. This 'XL' region contains three potential PKA phosphorylation sites but shares no homology with other regions of calpastatin or with any known protein. Immunoblot studies demonstrated that heart and liver contain a calpastatin protein of 145 kDa on SDS-polyacrylamide gel electrophoresis that comigrates with full- length bacterially expressed calpastatin and calpastatin produced by coupled in vitro transcription-translation from the upstream AUG. An antibody raised against the XL region recognized the 145-kDa band, demonstrating that the upstream AUG is utilized and that the 145-kDa band represents full-length calpastatin in vivo. Transient transfection assays demonstrated that sequence within 272 nucleotides upstream of transcription initiation of the calpastatin gene is sufficient to direct moderate level transcription. Promoter sequences further upstream act to inhibit or stimulate transcriptional activity. Exposure of transfected cells to dibutyryl cAMP resulted in a 7-20-fold increase in promoter activity for constructs containing at least 272 nucleotides of upstream promoter sequence. Deletion analysis indicates that at least one cAMP-responsive element resides within 102 nucleotides of transcription initiation.
- Ladd, A. N., Yatskievych, T. A., & Antin, P. B. (1998). Regulation of avian cardiac myogenesis by activin/TGFβ and bone morphogenetic proteins. Developmental Biology, 204(2), 407-419.More infoPMID: 9882479;Abstract: Previous studies have identified two signaling interactions regulating cardiac myogenesis in avians, a hypoblast-derived signal acting on epiblast and mediated by activin or a related molecule and an endoderm-derived signal acting on mesoderm and involving BMP-2. In this study, experiments were designed to investigate the temporal relationship between these signaling events and the potential role of other TGFβ superfamily members in regulating early steps of heart muscle development. We find that while activin or TGFβ can potently induce cardiac myogenesis in pregastrula epiblast, they show no capacity to convert noncardiogenic mesoderm toward a myocardial phenotype. Conversely, BMP-2 or BMP-4, in combination with FGF-4, can readily induce cardiac myocyte formation in posterior mesoderm, but shows no capacity to induce cardiac myogenesis in epiblast cells. Activin/TGFβ and BMP-2/BMP-4 therefore have distinct and reciprocal cardiac-inducing capacities that mimic the tissues in which they are expressed, the pregastrula hypoblast and anterior lateral endoderm, respectively. Experiments with noggin and follistatin provide additional evidence indicating that BMP signaling lies downstream of an activin/TGFβ signal in the cardiac myogenesis pathway. In contrast to the cardiogenic-inducing capacities of BMP-2/BMP-4 in mesoderm, however, we find that BMP-2 or BMP-4 inhibits cardiac myogenesis prior to stage 3, demonstrating multiple roles for BMPs in mesoderm induction. These and other published studies suggest a signaling cascade in which a hypoblast-derived activin/TGFβ signal is required prior to and during early stages of gastrulation, regulated both spatially and temporally by an interplay between BMPs and their antagonists. Later cardiogenic signals arising from endoderm, and perhaps transiently from ectoderm, and mediated in part by BMPs, act on emerging mesoderm within cardiogenic regions to activate or enhance expression of cardiogenic genes such as GATA and cNkx family members, leading to cardiac myocyte differentiation.
- Yatskievych, T. A., Ladd, A. N., & Antin, P. B. (1997). Induction of cardiac myogenesis in avian pregastrula epiblast: The role of the hypoblast and activin. Development, 124(13), 2561-2570.More infoPMID: 9216998;Abstract: An in vitro assay has been developed to investigate tissue interactions regulating myocardial cell specification in birds. Explants from the posterior region of stage XI-XIV blastulas were found to form heart muscle at high frequency with a timing that corresponded to onset of cardiac myocyte differentiation in vivo. Isolation and recombination experiments demonstrated that a signal from the hypoblast was required to induce cardiac myogenesis in the epiblast, and regional differences in epiblast responsiveness and hypoblast inductiveness restrict appearance of cardiac myocytes to the posterior region. Explantation studies provided evidence that myocardial cell specification is underway by stage 3, indicating that the hypoblast-derived signal occurs shortly before specification is detected. Recombinations were also performed to compare cardiac-inducing capacities of pregastrula hypoblast and stage 5 anterior lateral endoderm. The hypoblast possessed broad capacity to induce heart muscle cells in pregastrula and mid-gastrula epiblast, and modest ability to induce cardiac myogenesis in stage 4 posterior primitive streak. Stage 5 anterior lateral endoderm, in contrast, showed no ability to induce heart development in epiblast cells but was a potent inducer of cardiac myogenesis in cells from stage 4 posterior primitive streak. These findings suggest that the hypoblast-derived signal likely acts upstream of proposed heart-inducing signals provided by anterior lateral endoderm. Experiments were also performed to investigate whether activin, or an activin-like molecule, is involved in regulating cardiac myogenesis. Follistatin blocked cardiac myogenesis in stage XI-XIV posterior region explants and activin induced cardiac myogenesis in a dose-dependent fashion in posterior epiblast. These findings indicate that activin, or an activin-like molecule, is required for and is sufficient to stimulate cardiac myogenesis in posterior region pregastrula epiblast. Three models are presented to explain these results.
- Antin, P. B., Yatskievych, T., Dominguez, J. L., & Chieffi, P. (1996). Regulation of avian precardiac mesoderm development by insulin and insulin-like growth factors. Journal of Cellular Physiology, 168(1), 42-50.More infoPMID: 8647921;Abstract: Endoderm within the heart forming regions of vertebrate embryos has pronounced effects on myocardial cell development. Previous studies have suggested that these effects are mediated by soluble growth factors, in particular fibroblast growth factor 2 (FGF-2) and activin-A. Since both insulin and insulin-like growth factors (IGFs) are present in developing avian embryos at the time of heart formation, we have investigated the potential role of these molecules in promoting development of premyocardial cells in quail. Culture of precardiac mesoderm explants from stage 5 quail embryos in medium containing insulin, IGF-I, or IGF-II increased proliferation of premyocardial cells, with maximal stimulation observed at approximately 25 nM for each ligand. A direct comparison of the proliferative response of precardiac mesoderm to endoderm, fetal calf serum, insulin, IGF-I, IGF-II, activin-A, and FGF-2 showed that FGF-2 and activin-A increased proliferation of premyocardial cells approximately 2-fold, while insulin, IGF-I, and IGF-II stimulated proliferation approximately 3-fold. Insulin and IGF-I enhanced the rate of myocyte differentiation, similar to previously reported effects of endoderm. In contrast, exposure of precardiac mesoderm explants to transforming growth factor beta (TGFβ) reduced proliferation of premyocardial cells and moderated the proliferative effects of IGF-I. TGFβ did not block the differentiation of stage 5 premyocardial cells. Reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that mRNAs encoding insulin, IGF-II, insulin receptor, and IGF-I receptor were present in both precardiac mesoderm and endoderm, as well as in the forming heart at stage 8. Since premyocardial cells can survive and differentiate in a defined medium lacking these factors precardiac mesoderm may produce IGF-II and insulin at levels that are sufficient to stimulate myocyte development. Taken together, these results suggest that insulin and/or IGF-II may promote cardiac development in vivo by both autocrine and paracrine mechanisms. Cardiogenesis may therefore be promoted by the combined action of several classes of growth factors.
- Niu, S., Antin, P. B., & Morkin, E. (1996). Cloning and sequencing of a developmentally regulated avian mRNA containing the LEA motif found in plant seed proteins. Gene, 175(1-2), 187-191.More infoPMID: 8917097;Abstract: We report the cloning of a bromodeoxyuridine (BrdU)-sensitive transcript of 918 bp from an immortalized quail heart cell line containing an open reading frame (ORF) of 215 amino acids (aa) (≃ 23 kDa). Analysis of the secondary structure predicts two amphipathic α-helices with oppositely oriented amphipathic surfaces at the C-terminus of the protein. Each of the helices contains an LEA (late embryogenesis abundant) consensus sequence (A/TAEKAK/RETKD) which has been previously described only in a group of plant seed-specific proteins. Temporal and spatial distribution patterns of the transcript during chick embryo development were examined by whole-mount in situ hybridization and Northern blot analysis. At H and H (Hamburger and Hamilton, 1951) stages 11-14, the message was expressed strongly in blood islands in the area opaca. At day 5, strong signals were found in the liver primordia, mesonephrons, and nephric duct. Frozen sections of whole mount-stained embryonic liver demonstrated that the message was restricted to developing blood cells. The expression pattern of this transcript suggests that its protein product may be involved in hematopoiesis during avian development.
- Niu, S., Antin, P. B., Akimoto, K., & Morkin, E. (1996). Expression of avian glypican is developmentally regulated. Developmental Dynamics, 207(1), 25-34.More infoPMID: 8875073;Abstract: An avian cDNA homologue of human and rat glypicans has been cloned from a stage 17 chicken heart cDNA library and used to analyze the distribution of this proteoglycan during development by Northern analysis and whole mount in situ hybridization. At stages 7-12, strong signals were detected in the cephalic region of the neural folds, rostral portion of paraxial mesoderm, and newly formed epithelial somites. At stages 2025, strong expression was observed in the mantle zone of the telencephalon, the apical epidermal ridge and proximal region of developing limb. Transcripts also were found in the truncus arteriosus and arteriovenous-canal region of the heart, but not in the myocardium. This distribution pattern suggests that the avian glypican may be involved in the morphogenesis of limb, somite, heart, and brain. The expression of glypican also overlaps FGFs in limb bud, FGF receptors in heart and somite, and NGF receptors in forebrain. The affinity of heparan sulfate proteoglycans for growth factors and the distribution of the avian glypican are consistent with a role for this molecule in growth factor-mediated signals.
- Antin, P. B., Taylor, R. G., & Yatskievych, T. (1994). Precardiac mesoderm is specified during gastrulation in quail. Developmental Dynamics, 200(2), 144-154.More infoPMID: 7919500;Abstract: The establishment of precardiac mesoderm and the role of anterolateral endoderm and ectoderm in regulating heart muscle cell development have been studied in quail using explant cultures. Mesoderm from precardiac regions of stage 4+-6 embryos was explanted alone or in combination with adjacent endoderm or ectoderm, cultured for 12 to 72 hr in several types of culture media, and then assayed by morphological and immunocytochemical criteria for the presence of differentiated cardiac myocytes. Results show that mesoderm from heart forming regions is capable of differentiating into beating cardiac myocytes in a defined medium lacking potential signaling molecules by stage 4+, the earliest time at which we could isolate mesoderm from adjacent cell layers. Although an interaction with anterolateral endoderm from stage 4+ onward is therefore not required for the specification of precardiac mesoderm in quail, explants consisting of mesoderm plus endoderm show an enhanced rate of myocyte differentiation and a shortened delay between expression of myosin heavy chain and the onset of beating. Endoderm also plays a central role in early heart morphogenesis since beating heart tubes form only in explants that contain both mesoderm and endoderm. In contrast, ectoderm from stage 4+-5+ embryos does not support development of precardiac mesoderm. These results suggest that early heart muscle cell development involves an initial specification step that occurs prior to or during gastrulation and which leads to the appearance of myocardial precursor cells, and a subsequent differentiation step during which endoderm plays a central role in enhancing the rate of myocyte differentiation and the degree of heart tube morphogenesis.
- Zhang, Z., Lin, Z., Youyong, L. u., Meng, S., Han, Y., & Antin, P. B. (1994). Expression of gap junction protein Cx43 in cultured human normal and malignant lung cells. Chinese Journal of Cancer Research, 6(2), 95-101.More infoAbstract: Gap junctional intercellular communication-exchange of small molecules and ions between contiguous cells through membranous gap junctional channels-is essential for growth control and tissue homeostasis. This work concerns the functional expression of gap junction protein connexin 43 (Cx43) in normal human lung cells and the changes in lung carcinoma cells. By using Northern blot hybridization analysis and Cx43 immunocytochemical methods, it was observed that cultured normal human embryonic lung cells expressed a high level of Cx43 in both mRNA and protein levels. The Cx43 immunofluorescence was localized at cell membrane regions corresponding to the location of gap junctions. These normal lung cells were competent of intercellular communication function as detected by Lucifer yellow dye transfer. In contrast to normal cells, Cx43 mRNA and protein was not detectable in the carcinoma PG cell line. These tumor cells were defective of intercellular communication function. These results demonstrate that Cx43 is expressed in normal cultured human embryonic lung cells but not in lung tumor cells. The lack of intercellular communication in the lung tumor cell line correlates with dysfunctional intercellular communication. The suggestive role of Cx as a tumor suppersor gene is discussed. © 1994 Chinese Journal of Cancer Research.
- Antin, P. B., & Ordahl, C. P. (1991). Isolation and characterization of an avian myogenic cell line. Developmental Biology, 143(1), 111-121.More infoPMID: 1985013;Abstract: Myogenic cell lines have proven extremely valuable for studying myogenesis in vitro. Although a number of mammalian muscle cell lines have been isolated, attempts to produce cell lines from other classes of animals have met with only limited success. We report here the isolation and characterization of seven avian myogenic cell lines (QM1-4 and QM6-8), derived from the quail fibrosarcoma cell line QT6. A differentiation incompetent QM cell derivative was also isolated (QM5DI). The major features of QM cell differentiation in vitro closely resemble those of their mammalian counterparts. Mononucleated QM cells replicate in medium containing high concentrations of serum components. Upon switching to medium containing low serum components, cells withdraw from the cell cycle and fuse to form elongated multinucleated myotubes. Cultures typically obtain fusion indices of 43-49%. Northern blot and immunoblot analyses demonstrate that each differentiated QM cell line expresses a wide variety of genes encoding muscle specific proteins: desmin, cardiac troponin T, skeletal troponin T, cardiac troponin C, skeletal troponin I, α-tropomyosin, muscle creatine kinase, myosin light chain 2, and a ventricular isoform of myosin heavy chain. While all QM lines analyzed to date express at least some myosin light chain 2, only one line, QM7, expresses this gene at high levels. Surprisingly, none of the QM lines reported here express any known form of α-actin. The absence of sarcomeric actin expression may explain the absence of myofibrils in QM myotubes. These novel features of muscle gene expression in QM cells may prove useful for studying the role of specific muscle proteins during myogenesis. More importantly, however, the isolation of QM cell lines indicates that it may be feasible to isolate other avian myogenic cells lines with general utility for the study of muscle development. © 1991 Academic Press, Inc.
- Antin, P. B., Karp, G. C., & Ordahl, C. P. (1991). Transgene expression in the QM myogenic cell line. Developmental Biology, 143(1), 122-129.More infoPMID: 1985014;Abstract: We have isolated an avian muscle cell line (QM) which has the essential features of established mammalian muscle cell lines. The experiments reported here were undertaken to determine the suitability of QM cells for the introduction and analysis of cloned transgenes. The promoter of the cardiac troponin T (cTNT) gene has been previously shown to contain sequence elements which govern muscle-specific expression of the chloramphenicol acetyltransferase (CAT) gene in transiently transfected primary cell cultures. We show here that QM cells stably harboring cTNT promoter-CAT fusion genes up-regulate CAT expression in concert with myogenic differentiation, and that as few as 110 upstream nucleotides are sufficient for such differentiation-dependent regulation. In addition, both transient and stable transfection experiments demonstrate that differentiated QM cells possess trans-acting factors necessary for the expression of the skeletal α-actin promoter, despite the absence of mRNA or protein product from the endogenous sarcomeric actin genes in these cells. Finally, to follow the developmental potential of QM cells in vivo, we created a clone, QM2ADH, which constitutively expresses the histochemical marker transgene encoding Drosophila alcohol dehydrogenase. When surgically inserted into the limb buds of developing chick embryos, QM2ADH cells are incorporated into endogenous developing muscles, indicating that QM cells are capable of recognizing and responding to host cues governing muscle morphogenesis. Thus, QM cells are versatile as recipients of transgenes for the in vitro and in vivo analysis of molecular events in muscle development. © 1991 Academic Press, Inc.
- Antin, P. B., Mar, J. H., & Ordahl, C. P. (1988). Single cell analysis of transfected gene expression in primary heart cultures containing multiple cell types. BioTechniques, 6(7), 640-649.More infoPMID: 3078720;
- Mar, J. H., Antin, P. B., Cooper, T. A., & Ordahl, C. P. (1988). Analysis of the upstream regions governing expression of the chicken cardiac troponin T gene in embryonic cardiac and skeletal muscle cells. Journal of Cell Biology, 107(2), 573-585.More infoPMID: 3047142;PMCID: PMC2115209;Abstract: The chicken gene encoding cardiac troponin T (cTNT) is expressed in both cardiac and skeletal muscle during early embryonic development, but is specifically repressed in skeletal muscle during fetal development. To determine if the cis-acting sequences governing transcription of a single gene in these two related cell types are the same, we have transfected promoter/upstream segments of the cTNT gene coupled to the bacterial chloramphenicol acetyltransferase gene into primary cultures of early embryonic cardiac and skeletal muscle cells. Using this assay system, chloramphenicol acetyltransferase activity directed by the cTNT promoter/upstream region was between two and three orders of magnitude higher in cardiac or skeletal muscle cells than in fibroblast cells, indicating that cis elements responsible for cell-specific expression reside in this region of the cTNT gene. Deletion experiments showed that a 67-nucleotide DNA segment residing between 268 and 201 nucleotides upstream of cTNT transcription initiation site is required for the cTNT promoter activity in embryonic cardiac cells. This region is not required in embryonic skeletal muscle cells because promoter construction containing only 129 upstream nucleotides is transcriptionally active in these cells. These results demonstrate that different cis-acting sequences are required for cTNT expression in early embryonic cardiac and skeletal muscle cells. Nonessential regions residing farther upstream, on the other hand, affected the level of expression of these minimum regions in a similar manner in both cell types. The data from these experiments indicate, therefore, that transcription of the cTNT promoter in early embryonic cardiac and skeletal muscle cells is governed both by common and divergent regulatory elements in cis and in trans.
- Antin, P. B., Tokunaka, S., Nachmias, V. T., & Holtzer, H. (1986). Role of stress fiber-like structures in assembling nascent myofibrils in myosheets recovering from exposure to ethyl methanesulfonate. Journal of Cell Biology, 102(4), 1464-1479.More infoPMID: 3958057;PMCID: PMC2114158;Abstract: When day 1 cultures of chick myogenic cells were exposed to the mutagenic alkylating agent ethyl methanesulfonate (EMS) for 3 d, 80% of the replicating cells were killed, but postmitotic myoblasts survived. The myoblasts fused to form unusual multinucleated 'myosheets': extraordinarily wide, flattened structures that were devoid of myofibrils but displayed extensive, submembraneous stress fiber-like structures (SFLS). Immunoblots of the myosheets indicated that the carcinogen blocked the synthesis and accumulation of the myofibrillar myosin isoforms but not that of the cytoplasmic myosin isoform. When removed from EMS, widely spaced nascent myofibrils gradually emerged in the myosheets after 3 d. Striking co-localization of fluorescent reagents that stained SFLS and those that specifically stained myofibrils was observed for the next 2 d. By both immunofluorescence and electron microscopy, individual nascent myofibrils appeared to be part of, or juxtaposed to, preexisting individual SFLS. By day 6, all SFLS had disappeared, and the definitive myofibrils were displaced from their submembranous site into the interior of the myosheet. Immunoblots from recovering myosheets demonstrated a temporal correlation between the appearance of the myofibrillar myosin isoforms and the assembly of thick filaments. The assembly of definitive myofibrils did not appear to involve desmin intermediate filaments, but a striking aggregation of sarcoplasmic retriculum elements was seen at the level of each I-Z-band. Our findings suggest that SFLS in the EMS myosheets function as early, transitory assembly sites for nascent myofibrils.
- Holtzer, H., Forry-Schaudies, S., & Antin, P. (1986). Effects of a phorbol ester (TPA) and a carcinogen (EMS) on myofibrillogenesis. Toxicology and Industrial Health, 2(2), 121-.
- Holtzer, H., Sasse, J., Horwitz, A., Antin, P., & Pacifici, M. (1986). Myogenic lineages and myofibrillogenesis.. Bibliotheca anatomica, 109-125.More infoPMID: 3524557;
- Holtzer, H., Forry-Schaudies, S., Antin, P., Dubyak, G., & Nachmias, V. (1985). Induction of incoordinate synthesis of muscle proteins by the tumor promoter TPA and the carcinogen EMS.. Advances in Experimental Medicine and Biology, 182, 179-192.More infoPMID: 4003157;
- Holtzer, H., Forry-Schaudies, S., Dlugosz, A., Antin, P., & Dubyak, G. (1985). Interactions between IFs, microtubules, and myofibrils in fibrogenic and myogenic cells.. Annals of the New York Academy of Sciences, 455, 106-125.More infoPMID: 2867727;
- Dlugosz, A. A., Antin, P. B., Nachmias, V. T., & Holtzer, H. (1984). The relationship between stress fiber-like structures and nascent myofibrils in cultured cardiac myocytes. Journal of Cell Biology, 99(6), 2268-2278.More infoPMID: 6438115;PMCID: PMC2113583;Abstract: The topographic relationship between stress fiber-like structures (SFLS) and nascent myofibrils was examined in cultured chick cardiac myocytes by immunofluorescence microscopy. Antibodies against muscle-specific light meromyosin (anti-LMM) and desmin were used to distinguish cardiac myocytes from fibroblastic cells. By various combinations of staining with rhodamine-labeled phalloidin, anti-LMM, and antibodies against chick brain myosin and smooth muscle α-actinin, we observed the following relationships between transitory SFLS and nascent and mature myofibrils: (a) a more SFLS were present im immature than mature myocytes; (b) in immature myocytes a single fluorescent fiber would stain as a SFLS distally and as a striated myofibril proximally, towards the center of the cell; (c) in regions of a myocyte not yet penetrated by the elongating myofibrils, SFLS were abundant; and (d) in regions of a myocyte with numerous mature myofibrils, SFLS had totally disappeared. Spontaneously contracting striated myofibrils with definitive Z-band regions were present long before antidesmin localized in the I-Z-band region and long before morphologically recognizable structures periodically link Z-bands to the sarcolemma. These results suggest a transient one-on-one relationship between individual SFLS and newly emerging individual nascent myofibrils. Based on these and other relevant data, a complex, multistage molecular model is presented for myofibrillar assembly and maturation. Lastly, it is of considerable theoretical interest to note that mature cardiac myocytes, like mature skeletal myotubes, lack readily detectable stress fibers.
- Holtzer, H., Biehl, J., Antin, P., Tokunaka, S., Sasse, J., Pacifici, M., & Holtzer, S. (1983). Quantal and proliferative cell cycles: how lineages generate cell diversity and maintain fidelity.. Progress in clinical and biological research, 134, 213-227.More infoPMID: 6664999;Abstract: There are no known differences between the mechanisms that generate diverse differentiation programs in a mosaic embryo such as Caenorhabdites elegans or in a regulative embryo such as a chick. Transit through an invariant sequence of compartments in a lineage is obligatory for a given precursor cell 1) to inherit its differentiation program from its mother, and 2) to transmit to its daughters, by way of a predetermined binary decision, a new differentiation program. The inheritability of a differentiation program must be encoded in a structural molecule. We postulate that during an S period of a quantal cell cycle, chromosomal structures are so altered that a network of genes that could not be transcribed in the mother becomes available for transcription in the daughters. We do not view as a likely possibility the traditional notion that cell-cell or cell-matrix interactions instruct or commit blank, naive cells to transform into cells with unique differentiation programs. From this perspective, we have initiated experiments to determine the minimal rounds of DNA synthesis, following fertilization, that are required to generate founder cells for several major lineages in the chick. Somewhere between the 15th and 18th generations after fertilization erythrogenic hematocytoblasts that are cytokeratin-positive and vimentin- and hemoglobin-negative undergo a quantal cell cycle. Their daughters are cytokeratin-negative and vimentin- and hemoglobin-positive. DNA synthesis, but not cytokinesis, is an obligatory requirement for this switch in differentiation programs. Essentially similar findings are presented for cells in the cardiogenic, neurogenic, melanogenic, and endothelial lineages. There is no evidence that cell-cell or cell-matrix interactions are required for this diversification. Such interactions, however, may be required for the large number of proliferative cell cycles within particular compartments of particular lineages that are characteristic of all growing or expanding systems. With respect to classical "CFU cells" it is of interest that definitive white blood cells have not yet been identified in these cultures. Lastly, the high ratio of primitive red blood cells to non-red blood cells in the first 40 hours of culture is consistent with the notion that the majority of all cells present in the blastodisc at these early stages are in fact already committed to a unipotent erythrogenic lineage [5, 18, 23, 44, 45]. The issue of changing ratios of cells within compartments of a lineage, as well as of cells in different lineages, is much neglected in consideration of (a) normal embryogenesis, (b) cell-renewal in mature organisms and, particularly,
- Antin, P. B., Forry-Schaudies, S., & Friedman, T. M. (1981). The effects of taxol on postmitotic myoblasts in culture. Journal of Cell Biology, 91(2 II), No. 20033.
- Antin, P. B., Forry-Schaudies, S., Friedman, T. M., Tapscott, S. J., & Holtzer, H. (1981). Taxol induces postmitotic myoblasts to assemble interdigitating microtubule-myosin arrays that exclude actin filaments. Journal of Cell Biology, 90(2), 300-308.More infoPMID: 6116716;PMCID: PMC2111880;
- Bennett, G. S., Tapscott, S. J., Kleinbart, F. A., Antin, P. B., & Holtzer, H. (1981). Different proteins associated with 10-nanometer filaments in cultured chick neurons and nonneuronal cells. Science, 212(4494), 567-569.More infoPMID: 6163217;
- Bennett, G. S., Tapscott, S. J., Kleinbart, F. A., Antin, P. B., & Holtzer, H. (1981). Different proteins associated with 10-nanometer filaments in cultured chick neurons and nonneuronal cells. Science, 212(4494), 569-571.More infoAbstract: A protein of molecular size 180 kilodaltons is associated with 10-nanometer filaments in neurons and is immunologically distinct from smaller putative neurofilament subunits and from 10-nanometer filament proteins in non-neuronal cells, such as myotubes and fibroblasts. Neurons do not contain vimentin, the major filament protein in many other cells, including the nonneuronal cells in cultures of neural tissue. Copyright © 1981 AAAS.