• A Fundamental Investigation into the Presence of Interstitial Cells of Cajal within the Gastrointestinal Tract of Dania rerio

      Leddon, Scott; The College at Brockport (2007-07-01)
      Coordinated gastrointestinal (GI) motility results from the complex interactions between interstitial cells of Cajal (ICC), enteric neurons, and smooth muscle cells. Kit positive immunoreactivity has been extensively utilized as a marker for ICC; these cells function to generate rhythmic depolarization in the GI smooth muscle termed the electrical slow wave. Coordinated GI motility is regulated by the electrical slow wave. Directly lesioning ICC populations with neutralizing antibodies results in GI dysmotility and loss of the electrical slow wave. Furthermore, GI dysmotility symptoms and other human pathologies have been correlated with ICC deficiencies within the GI tract. Currently, there are few treatments and therapeutic interventions for GI motility disorders, Irritable Bowl Syndrome alone affects up to 20% of the world population. 1- 5 The high prevalence of GI motility disorders combined with the lack of effective interventions, suggest new treatments are needed. Rational drug development and new treatment design will benefit from the development of new model systems. Greater understanding of the regulation of GI motility, pathologies that affect GI motility, and the development of new therapeutics will all benefit from a new model of GI motility. This thesis provides support to the use of Danio rerio, the zebrafish, as a model of human GI motility. Danio rerio is an excellent model system for several human physiological systems or diseases including macular degeneration, neural degenerative diseases, cardiovascular disease, cholesterol and lipid metabolism, and cardiovascular disease. 6 - 20 The zebrafish model system offers the opportunity for forward genetic analysis; novel genes, genetic elements, and pathologies can be characterized with reverse genetic screens through the evaluation of interesting phenotypes. Zebrafish reach sexual maturity quickly (three months), produce large numbers of offspring (100 per week), and are more cost efficient to house compared to mammalian models. Most seductive of zebrafish traits, optical transparency of larvae, enables direct visualization in-vivo of organ function (including the GI tract) under physiological settings, without perturbing the organism. This thesis critically examines the hypothesis that GI motility in the zebrafish involves ICC. Prior to the data presented within this thesis, there have not been any published reports on the presence of ICC within the zebrafish GI tract. Results from this investigation show Kit-like immunoreactivity within the muscular layers of the zebrafish GI tract. Additionally, orthologs for the human receptor tyrosine kinase Kit, kita and kitb, and orthologs for the human Kit ligand, kitla and kitlb, are shown to be expressed within the zebrafish GI tract. Kit receptor and ligand are necessary for ICC development and maintenance in mammals. Expression of kit receptors and ligands within the zebrafish GI tract are consistent with the hypothesis that ICC support spontaneous rhythmic contractions in the zebrafish-- this suggests that the zebrafish may be a suitable model for GI motility.
    • 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.
    • Gastrointestinal (GI) Motility is Unaltered by Feeding in Zebrafish

      Rich, Adam; Diamond, Amanda Marie; The College at Brockport (2012-08-08)
      Gastrointestinal motility patterns in humans are not constant and respond to the luminal contents and nutrient status (Degen and Phillips, 1996). The zebrafish is a new model system for human GI motility and regulation of motility patterns appears to be controlled by enteric neurons and interstitial cells of Cajal (Huizinga et al., 1995, Farrugia et. al., 2003, Sanders et.al., 2006, Rich et. al., 2007). Although several research laboratories investigate GI motility in zebrafish larvae, no standard protocol for feeding exists and experiments may be performed on fasted or fed larvae. The goal of this study was to examine the effects of feeding on GI motility when ICC and enteric neurons are developed and regulate GI motor patterns in larvae, at 7 days post fertilization ( dpt) (Rich et. al., 2007). Larvae were fasted or fed once daily beginning at 5 dpf. At 7 dpf larvae were fed dry food labeled with FITC-dextran and GI motility was measured using time-lapse imaging and image analysis techniques. Motility was examined in the anterior and the posterior regions of the GI tract. No differences were observed in fish standard length, a developmental marker. The total number and distance of contractions increased in the anterior intestine after feeding. These data suggest that feeding has little influence on GI motility patterns in the posterior intestine. The effects of Cisapride, a prokinetic in humans, was examined and found to increase the contraction number, velocity, and interval. The effects of Niflumic Acid and DIDS were also examined, because anoctamin 1 (ANOl), a chloride-selective channel, has recently been identified as a potential regulator for ICC pacemaker function. Both drugs dramatically reduced the total number of contractions as well as the GI motility index indicating a reduction in coordinated motility patterns. Cisapride, Niflumic Acid, and DIDS have similar effects on GI motility in mice and in the zebrafish, suggesting that similar molecular mechanisms regulate GI motility in zebrafish and mice. The findings contribute to the validation of the zebrafish model system for human GI motility function.
    • McNair Summer Research Journal 2006

      Volpe-van Dijk, Herma; The College at Brockport (The College at Brockport, 2006-01-01)
      2006 McNair Summer Research Journal features abstracts of the summer research and selected papers of the following students: Anna O’Connor, Frances Spezio, Jairo Enrique Bastilla, Jeremiah Kirkland, Jimmie Eddington, Jody Davis, LaToya Janice Evans, Elizabeth Rosa, Peter Gonzalez, Roberta Stanford, Stacy Burke, Alisha Hale, Ashley Ussery, Carl B. Wilson, Farrah Augustin, Lindsey Brown, Michael Anthony Nicholson, and Leia DeVivi.
    • 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.