• A New Functional Zebrafish Gastrointestinal Motility Assay

      Rich, Adam; Hess, Stacey; The College at Brockport (2007-08-01)
      Gastrointestinal (GI) motility is the coordinated contractions of smooth muscles resulting in mixing and propulsion of material through the GI tract. GI motility is influenced by smooth muscle, enteric neurons, and Interstitial cells of Cajal (ICC). New model systems for GI motility are needed because regulation of motility is poorly understood, and the number of drugs which can assist in GI motility disorders is insufficient. The Kit receptor is required for ICC development and maintenance in mammalian model systems and in humans. Mutations in the Kit receptor are associated with GI motility disorders. The objective of these experiments was to determine motility patterns in wildtype zebrafish and compare them to motility patterns in the zebrafish kita null mutant, sparse (spab5). Contraction frequency was measured from 7 minute digital video recordings of anesthetized larvae immobilized in 1.2% agar. Contraction frequency averaged 0.586 ± 0.222 contractions per minute in wildtype larvae at 7 dpf, and 0.329 ± 0.158 contractions per minute in 7 dpf sparse. The apparent contraction intensity was also scored, with a score from 0, no contractions, to 2, complete occlusion of the lumen. Contraction intensity averaged 1.250 ± 0.444 in wildtype larvae and 1.619 ± 0.498 in sparse larvae at 7 dpf. At 11 dpf contraction intensity decreased to 1.000 ± 0.000 and 1.125 ± 0.354, respectively. A functional motility assay was developed to quantify GI motility that contributes to propulsive movement of intestinal contents. Larvae were fed FITC labeled microspheres, washed, and fluorescence intensity of the GI tract was digitally imaged. Larvae were placed in clean system water, and reimaged after 24 hours. Fluorescence intensities were normalized against initial intensity of ingested microspheres at 7 dpf in the intestinal bulb of the anterior GI tract. At 7 dpf the change in fluorescence intensity in the anterior GI tract decreased by 0.371 ± 0.030 and 0.119 ± 0.021 in the posterior GI tract of wildtype larvae. At 11 dpf fluorescence intensity decreased to 0.455 ± 0.056 in the anterior GI tract, and 0.258 ± 0.046 in the posterior GI tract. Peritoneal injection of the ACK-2 anti-Kit antibody results in distention and an increased volume in the GI tract (Figure 12) (Maeda, Yamagata et al. 1992). These effects result from inactivation of Kit function by ACK-2. The size of the GI tract in sparse larvae appeared to be larger compared to wildtype, suggesting that the null kita mutant resulted in a phenotype similar to the ACK-2 effects on mice. We measured the area of a longitudinal cross section of wild type and sparse mutant larvae at 7 dpf. The average area of the GI tract in wildtype larvae was 4491 ± 754 and 5532 ± 880 in sparse larvae. Fluorescence spectroscopy was used to quantify the fluorescence intensity. Total fluorescence emission averaged 62,630 ± 23,780 counts per second immediately after ingestion in 11 dpf larvae. One day later, total fluorescence intensity averaged 53,277 ± 6,333 counts per second, a 15% decrease. 5-hydroxytryptamine (5-HT), a prokinetic agent, was used to validate the assay. One day after loading, the total fluorescence intensity decreased after application of 5-HT to 86,820 ± 21,850. 11 dpf zebrafish larvae ingest fluorescent labeled microspheres, and spectroscopic analysis showed reduced fluorescence intensity one day after loading. Functional studies showed a reduced contraction frequency and an increase in surface area between sparse mutant and wildtype larvae. The decrease in fluorescence intensity in wildtype larvae suggests aboral movement of microspheres, out of the posterior GI tract.
    • Expression Level of Kita, Kitb, Kitla, and Kitlb in Zebrafish Gastrointestional Tract

      Strouse, Jennifer B.; The College at Brockport (2011-06-01)
      Gastrointestinal (GI) motility is the muscular contractions that move intestinal contents in an anterograde (mouth to anus) direction and is necessary for nutrient absorption and elimination of waste. GI motility is highly coordinated and rhythmic contraction patterns. Interstitial cells of Cajal (ICC), enteric neurons, and smooth muscle cells all regulate GI motility. ICC function as pacemaker cells and determine contraction frequency. ICC growth and development is influenced by Kit, a tyrosine kinase receptor located on the plasma membrane of ICC. TMEM16A is a calcium activated chloride channel which contributes to the slow wave in the GI tract. Constipation, delayed gastric emptying, and bloating have been correlated with deficits of ICC in GI tissues. A functional Kit receptor and stimulation of Kit with Kit ligand is necessary for ICC growth and development. However, little is known about ICC development in adults or in developing GI tissue. The objective for this project is to determine the relative and temporal expression levels of Kita, Kitb, Kitla, and Kitlb in the zebrafish model system at several developmental time points. Understanding the temporal and relative expression pattern of these genes is the first step towards a more complete understanding of ICC development and turnover. The zebrafish model system is anatomically similar to the human GI tract and at early time points the zebrafish is transparent. One advantage to this model system is that GI motility may be examined in the intact larvae. RNA was isolated from dissected zebrafish GI tissues and used as template for reverse transcriptase reactions to make eDNA. Relative and temporal expression levels of Kita, Kitb, Kitla, and Kitlb was determined at 5 days post fertilization (dpf), 7 dpf, 11 dpf, 28dpf, and in adult gut tissues using eDNA as template for real time PCR. Kita and Kitla were confirmed as a functional receptor/ligand pair which was first identified in melanocyte migration19. The relative expression data suggests that Kitb and Kitlb are also a functional receptor/ligand pair. Temporal expression data shows high expression of Kitb early in development (5dpf). Besides the early high expression of Kitb, gene expression for all genes of interest peak at 11 dpf. TMEM16A (also called ANOI) was identified as a more accurate marker for gastrointestinal stromal tumors (GIST) than Kit24. RNA isolated from dissected zebrafish GI tract was used to make eDNA which became the template for reverse transcriptase (RT)-PCR and real-tin1e PCR (q-PCR). Anti-ANOI antibodies were used to identify TMEM16A in dissected, fixed zebrafish GI tract. RT-PCR showed that TMEM16A, B, and Care expressed in the zebrafish GI tract. Immunohistochemistry identifies a network of cells in the zebrafish GI tract that is similar in morphology and location to ICC stained by Kit antibodies. Relative and temporal expression was determined using samples isolated at 5, 7, 11, 28dpf, and adult time points. Expression of TMEM16B dominates TMEM16A and B at 28dpf and adult time points.
    • 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 (2009-01-01)
      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.
    • The Role of Kitlb on Development of Coordinated Muscular Contractions in the Zebrafish Gastrointestinal Tract

      Rich, Adam; Heatherington, Brittany A.; The College at Brockport (2012-08-01)
      Gastrointestinal (GI) motility is the spontaneous rhythmic contractions of smooth muscles that mix and propel the contents of the GI tract. Regulation of the complex muscular contractions is controlled by smooth muscles, interstitial cells of the Cajal (ICC) and enteric neurons. ICC act as pacemaker cells in the GI tract and set the frequency of spontaneous contractions. Altering ICC density results in uncoordinated GI muscular contractions. Our lab examines the role of ICC in GI motility and is focused on mechanisms that regulate ICC growth and development. Expression of the Kit receptor tyrosine kinase is used to identify ICC. Kit is stimulated by Kit ligand and stimulation is necessary for the growth and development of ICC. This project specifically examines the role of Kit – Kit ligand signaling on ICC development using the zebrafish model system. The zebrafish has two Kit genes (kita and kith) that are orthologous to human KIT, and two Kit Ligand genes (kit/a and kitlh ). I will examine the role of kitlh on the development and maturation of ICC using morpholino oligonucleotides knockdown in zebrafish. Gene expression was quantified using reverse transcriptase PCR analysis. Digital imaging techniques was used to examine morphology of the GI tract. It is anticipated that continued stimulation of kith by kitlh is necessary for development of the ICC network, and maintenance of the ICC network in adult animals.
    • Three Gastrointestinal Assays

      Rich, Adam; Majtyka, Bailey; State University of New York College at Brockport (2020-09-16)
      Gastrointestinal (GI) functionality relies on the spontaneous, rhythmic and coordinated propagation of muscular contractions in the GI tract, or GI motility. Without these coordinated motor patterns, digestion falters, and results in problems with digestion. Disrupted or un-coordinated motor patterns are associated with altered GI transit times. GI transit is the amount of time necessary for intestinal contents to move through the GI tract. GI transit is measured in patients complaining about abdominal discomfort to determine if discomfort results from a true dysmotility or from idiopathic symptoms. GI transit assays help to determine appropriate treatments but idiopathic symptoms, or pain from an unknown cause, is very common. The zebrafish is an attractive model system for human GI motility because the entire GI tract can be observed in intact zebrafish larva. In current methods, larvae are fed food with a marker substance and movement through the intestine is viewed using a microscope and recorded using a digital camera. However, GI transit time is highly variable. It is possible that this variability is completely normal and results from variable GI physiology. Alternatively, it is possible that the variability is due to the assay. Three distinct GI transit assays have been published. The overall objective for this work is to determine the reliability for each assay and to better understand which assay is most appropriate for future work. The assays will be described and compared, and results comparing the assays will be presented.
    • 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. (2012-01-01)
      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.