• Ultrastructure of Zebra Fish Dorsal Aortic Cells

      Miano, Joseph A.; Georger, Mary A.; Rich, Adam; De Mesy Bentley, Karen L.; The College at Brockport; University of Rochester (1/1/2006)
      Expression of vascular smooth muscle cell (VSMC) markers such as serum response factor (SRF) is complicated in zebrafish because of the ill-defined histology of the dorsal aorta and the presence of perivascular pigment. We report the ultrastructure of aortic cells in 7-day, 1-month, and 3-month-old zebrafish and provide clear evidence for the presence of perivascular melanocytes harboring an abundance of melanin. In 7-day-old larvae, endothelial cells (EC) and synthetic mural cells that display little evidence of VSMC differentiation comprise the dorsal aorta. The latter mural cells appear to fully differentiate into VSMC by 1 month of age. In 3-month-old adult zebrafish, EC exhibit greater differentiation as evidenced by the accumulation of electron-dense bodies having a diameter of approximately 200 nm. Adult zebrafish aortae also exhibit at least one clear layer of VSMC with the characteristic array of membrane-associated dense plaques, myofilament bundles, and a basement membrane. Subjacent to VSMC are collagen-producing adventitial fibroblasts and melanocytes. These studies indicate that fully differentiated VSMC occur only after day 7 in zebrafish and that such cells are arranged in at least one lamellar unit circumscribing the endothelium. These findings provide new data about the timing and accumulation of VSMC around the zebrafish aorta, which will be useful in phenotyping mutant zebrafish that exhibit defects in blood circulation.
    • The ? 1H Ca2+ channel subunit is expressed in mouse jejunal interstitial cells of Cajal and myocytes

      Gibbons, Simon J.; Strege, Peter R.; Lei, Sha; Roeder, Jaime L.; Mazzone, Amelia; Ou, Yijun; Rich, Adam; Farrugia, Gianrico; Mayo Medical School; The College at Brockport (1/1/2009)
      T-type Ca2+ currents have been detected in cells from the external muscular layers of gastrointestinal smooth muscles and appear to contribute to the generation of pacemaker potentials in interstitial cells of Cajal from those tissues. However, the Ca2+ channel subunit responsible for these currents has not been determined. We established that the ? subunit of the ?1H Ca2+ channel is expressed in single myocytes and interstitial cells of Cajal using reverse transcription and polymerase chain reaction from whole tissue, laser capture microdissected tissue and single cells isolated from the mouse jejunum. Whole-cell voltage clamp recordings demonstrated that a nifedipine and Cd2+ resistant, mibefradil-sensitive current is present in myocytes dissociated from the jejunum. Electrical recordings from the circular muscle layer demonstrated that mibefradil reduced the frequency and initial rate of rise of the electrical slow wave. Gene targeted knockout of both alleles of the cacna1h gene, which encodes the ? 1H Ca2+ channel subunit, resulted in embryonic lethality because of death of the homozygous knockouts prior to E13.5 days in utero. We conclude that a channel with the pharmacological and molecular characteristics of the ? 1H Ca2+ channel subunit is expressed in interstitial cells of Cajal and myocytes from the mouse jejunum, and that ionic conductances through the ? 1H Ca2+ channel contribute to the upstroke of the pacemaker potential. Furthermore, the survival of mice that do not express the ? 1H Ca2+ channel protein is dependent on the genetic background and targeting approach used to generate the knockout mice.
    • Ultra-Structural Identification of the Interstitial Cells of Cajal in the Zebrafish Danio rerio

      Ball, Evan R.; Matsuda, Miho M.; Dye, Louis; Hoffmann, Victoria; Zerfas, Patricia M.; Szarek, Eva; Rich, Adam; Chitnis, Ajay; Stratakis, Constantine A.; National Institute of Child Health and Human Development; et al. (1/1/2012)
      The interstitial cells of Cajal (ICCs) are important mediators of gastrointestinal motility due to their role as pacemakers in the GI tract. In addition to their function, ICCs are also structurally distinct cells most easily identified by their ultra-structural features and expression of the tyrosine kinase receptor c-KIT. ICCs have been described in mammals, rodents, birds, reptiles and amphibians ; there are no reports at the ultra-structural level of ICC’s within the GI tract of an organism from the teleost lineage. This report describes the presence of cells in the muscularis of the zebrafish intestine with similar features to ICCs in other vertebrates. ICC-like cells were associated with the muscularis, were more electron dense than surrounding smooth muscle cells, possessed long cytoplasmic processes and mitochondria, and were situated opposing to enteric nervous structures. In addition, immunofluorescent and immunoelectron microscopic studies using antibodies targeting the zebrafish ortholog of a putative ICC marker, c-KIT (kita), demonstrated c-kit immunoreactivity in zebrafish ICCs. Taken together, these data represent the first ultra-structural characterization of cells in the muscularis of the zebrafish Danio rerio and suggest ICC differentiation in vertebrate evolution may date back to the teleost lineage.
    • Drosophila Enhancer of Rudimentary Homolog, ERH, Is a Binding Partner of RPS3, RPL19, and DDIT4, Suggesting a Mechanism for the Nuclear Localization of ERH

      Tsubota, Stuart I.; Phillips, Anthony C.; Saint Louis University; The College at Brockport (1/1/2016)
      The protein enhancer of rudimentary homolog, ERH, is a small, highly conserved protein that has been found in animals, plants, and protists. Genetic and biochemical interactions have implicated ERH in the regulation of pyrimidine biosynthesis, DNA replication, transcription, mRNA splicing, cellular proliferation, tumorigenesis, and the Notch signaling pathway. In vertebrates and insects, ERH is nuclearly localized; however, an examination of the ERH amino-acid sequence does not reveal any nuclear localization signals. In this paper we show that the first 24 amino acids contain sequences necessary and sufficient for nuclear localization. Through yeast two-hybrid screens, three new binding partners of ERH, RPS3, RPL19, andDDIT4,were identified. RPS3 was isolated from both human and Drosophila screens. These interactions suggest functions of ERH in cell growth, cancer, and DNA repair. The ERH sequences necessary for the interactions between ERH and RPS3 and RPL19 are mapped onto the same 24-amino-acid region in ERH which are necessary for nuclear localization, suggesting that ERH is localizing to the nucleus through binding to one of its DNA-binding partners, such as RPS3 or RPL19.
    • Expression and Comparative Genomics of Two Serum Response Factor Genes in Zebrafish

      Davis, Jody L.; Long, Xiaochun; Georger, Mary A.; Scott, Ian C.; Rich, Adam; Miano, Joseph A.; The College at Brockport; The Hospital for Sick Children; University of Rochester (2/1/2008)
      Serum response factor (SRF) is a single copy, highly conserved transcription factor that governs the expression of hundreds of genes involved with actin cytoskeletal organization, cellular growth and signaling, neuronal circuitry and muscle differentiation. Zebrafish have emerged as a facile and inexpensive vertebrate model to delineate gene expression, regulation, and function, and yet the study of SRF in this animal has been virtually unexplored. Here, we report the existence of two srf genes in zebrafish, with partially overlapping patterns of expression in 3 and 7 day old developing animals. The mammalian ortholog (srf1) encodes for a 520 amino acid protein expressed in adult vascular and visceral smooth muscle cells, cardiac and skeletal muscle, as well as neuronal cells. The second zebrafish srf gene (srf2), encoding for a presumptive protein of only 314 amino acids, is transcribed at lower levels and appears to be less widely expressed across adult tissues. Both srf genes are induced by the SRF coactivator myocardin and attenuated with a short hairpin RNA to mammalian SRF. Promoter studies with srf1 reveal conserved CArG boxes that are the targets of SRF-myocardin in embryonic zebrafish cells. These results reveal that SRF was duplicated in the zebrafish genome and that its protein expression in all three muscle cell types is highly conserved across vertebrate animals suggesting an ancient code for transcriptional regulation of genes unique to muscle cell lineages.
    • The Human and Drosophila ERH are Functionally Equivalent: Evidence from Transgenic Studies

      Tsubota, Stuart; Ryan, Theodore; Rizzo, Nicholas; Hing, Huey; The College at Brockport (9/1/2016)
      The enhancer of rudimentary, e(r), gene encodes a small highly conserved protein, enhancer of rudimentary homolog (ERH), which has been shown to have a regulatory function in cell division, Notch signaling, and cancer progression. Human and Drosophila ERH, both 104 amino acids in length, are 76% identical and 84% similar. The high sequence identity translates into nearly identical tertiary structures. Previous studies on the expression of the human and Drosophila e(r) genes reveal that the two genes are similarly regulated. Data in the present study using an e(r)-eGFP reporter gene confirm these results, showing a high expression of the reporter in the ovaries, testes, and brain. The high structural and regulatory conservation of e(r) and ERH argue that human and Drosophila ERH may be biochemically and functionally equivalent. To test this hypothesis, a chimeric transgene containing the Drosophila e(r) non-coding regions and the human e(r) coding region was constructed and used to establish transgenic Drosophila stocks. This transgene can rescue all of the mutant phenotypes of an e(r) deletion, and Drosophila stocks in which the fly ERH has been replaced with the human ERH are fully healthy and viable. These studies demonstrate that the human and Drosophila ERH are functionally equivalent, suggesting that studies on the activity of the human ERH can be done in Drosophila, where a multitude of genetic and developmental tools are available.