• 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 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 kitb) that are orthologous to human KIT, and two Kit Ligand genes (kitla and kitlb). I will examine the role of kitlb 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 kitb by kitlb is necessary for development of the ICC network, and maintenance of the ICC network in adult animals.