Planarians are non-parasitic flatworms that usually live in fresh water, one of many flatworms in the class Turbellaria, phylum Platyhelminthes (Baguñá, 2012). Planarians are bilaterally symmetric metazoans and triploblastic, their bodies comprised of three germ layers (ectoderm, mesoderm, and endoderm) (Orii et al., 2002). Planarians are thought to have been amongst the first animals to develop a central nervous system (CNS), the nervous system of the flatworm is comprised of two ventral longitudinal nerve chords that span the length of the body with the brain comprising of two cephalic ganglia connected to the nerve chords (Agata et al., 1998). They also lack a circulatory system and due to their small size, their method of obtaining oxygen and removing carbon dioxide is through diffusion across the body cells. (Lund, 1921). Locomotion is achieved by cilia located on the outside of epithelial cells on the body (Stevenson and Beane, 2010). As planarians do not have a coelom they use sensory structures found in the anterior to carry out tasks; photoreceptors and chemoreceptors (Coward and Johannes, 1969; Salo et al., 2009; Cross et al., 2015). The photoreceptors along with pigment cells form two simple eye structures in the head region of the planarian (Agata and Umesono, 2008). For feeding and ingestion of food, the planarian will extrude its pharynx which is found in the middle section of its body (Wulzen, 1917). The pharynx is also the mechanism of removing waste due to lack of an anus. (Forsthoefel et al., 2015). Planarians contain at least two distinct types of musculature, DjMHC-A is expressed in the pharynx and intestine to aid in peristalsis, whereas DjMHC-B is expressed in the body wall in which aids in direction of movement (Kobayashi et al., 1998). They have amazing regenerative capacity due to their population of adult pluripotent stem cells referred to as neoblasts (Agata and Watanabe, 1999). Around 25-30% of all planarian cells are neoblasts (Baguñá et al., 1989) In order for an organism to undergo successful wound healing they must produce a mass of cell at the site of the wound, these cells need to be undifferentiated initially and then differentiate in order to reform the lost tissues/organs. This mass of cells is referred to as a blastema. (Tasaki et al., 2011). Tasaki et al. (2011) also suggests that ERK signalling controls the cell differentiation within the blastema during regeneration.
Mitogen-activated protein kinases (MAPKs) are a group of highly conserved serine/threonine protein kinases that are essential for many fundamental processes that occur within a cell. They also play a role in the transduction of extracellular signals into cellular responses. MAPKs are part of protein kinase cascades, MAPK kinase kinase (MAPKKK) phosphorylates MAPK kinase (MAPKK) which in turn phosphorylates MAPK (Widmann et al.,1999).
The pathway begins with an extracellular ligand binding to a receptor tyrosine kinase (RTK) which causes the RTK to autophosphorylate (Figure 1). The SH2 domain of Grb2, an adapter protein, now interacts with the phosphotyrosine residue on the RTK (Pawson, 1997). Sos interacts with the SH3 domain of Grb2, linking the proline-rich region of Sos. Sos acts of a guanine nucleotide exchange factor (GEF) which aids in the conversion of GDP-bound Ras (inactive) to GTP bound Ras (active). Activated Ras can now interact with Raf, a serine/threonine kinase, a MAPKKK (Jelinek et al., 1996). MEK1/2 is then phosphorylated, a MAPKK. The cascade ends with ERK1/2 being phosphorylated. ERK1/2 can then go on to phosphorylate transcription factors or to interact with other kinases (Kyriakis et al., 1992).
Tasaki et al. (2011) proposes that in order for planarian blastemas to switch from a state of proliferation to differentiation that ERK signalling activation must occur. MAPK/ERK is found to be active in planarian wound sites. The Dugesia japonica MAPK phosphatase gene, DjmkpA, is induced at wound sites as well which forms a negative feedback loop with ERK in order to control blastema cell differentiation (Tasaki et al., 2011). ERK signalling is also shown to be involved in maintaining the anterior-posterior axis during replication. ERK signalling is present in the anterior whereas in the posterior ERK signalling is negatively modulated by b-catenin activity (Umesono et al., 2013)
Aims of the Project
The aims of this project are to understand more about the mechanism of planarian regeneration in relation to the MAPK/ERK pathway. Utilising the MEK inhibitor U0126 to inhibit the MAPK/ERK pathway to determine if this pathway is involved in the regeneration of wound closure. In doing so this could lead to a greater understanding of how the MAPK/ERK pathway is involved in regeneration mechanisms of higher mammals. This could then be further studied to understand if the same processes could be applied to regeneration in humans. It could lead to novel clinical treatments for human injuries.
Materials and Methods
Dugesia planarians were collected from the pond on the grounds of Kingston University and placed in a tank containing water from the same pond. The water was gradually replaced with filtered water using a Metal Reduction water filter (Silverline, Devon, UK). This process was done gradually to ensure the planarians were able to adjust to the conditions. Feeding occurred once a week with small amounts of chicken liver over a period of one hour, planarians were transferred to a separate tank for feeding to avoid contamination of main tank water. Dugesia were starved for 2-3 days before experiments. Planarians were transferred using plastic Pasteur pipettes and soft paint brushes to avoid damage to the body.
Preliminary tests were carried out to determine the best method of wounding. The first method was to remove the head of the planarian using a scalpel so the area and depth of the wound of the head fragment could be determined. Another method was to puncture a hole in the middle of the body using a WellTech Rapid–Core tissue hole-puncturing device to measure the area of the hole closure. This method was not selected due to the difficulty in getting repeatable wounding in the same area of each planarian, there was also difficulty in not puncturing the pharynx, so the first method was chosen. Immobilisation using a weak ethanol concentration to de-ciliate the planarians was considered but ultimately not undertaken due to immobilising result lasting less than the time of the experiment and the coverslip kept most planarians relatively still (Stevenson and Beane, 2010).
Control and inhibitor treatment
Planarians were placed in individual wells in a 12 well plate using a Pasteur pipette. For controls, the planarians head was removed and placed on a microscope slide with a coverslip on top to limit movement and to stop the planarian drying out. Each planarian head was then imaged using a microscope at 30-minute intervals for 3 hours. DMSO controls were also carried out in the same fashion. DMSO was made up to 0.1% concentration, the same concentration as used for the inhibitor, and the planarian was incubated for 1 hour before dissection.
The U0126 inhibitor was made up to 10mM in 100% DMSO as per manufacturer instructions (Cell Signalling Technology). This stock was then frozen in 2µL aliquots. When needed, the stock aliquots were defrosted and diluted and made up to concentrations of 10µM and 5µM using water and the appropriate volumes of DMSO.
Planarians of similar size were selected and incubated for 1 hour with either 5µM or 10µM inhibitor. After incubation, the head of the planarian was removed with a scalpel and placed under a coverslip with appropriate inhibitor solution added periodically to stop the planarian drying out and being crushed by the coverslip. Planarians were then photographed at 30-minute intervals for 3 hours using a microscope. 3 photos were taken at each time point to allow for muscle contraction to be taken into account. Towards the end of the second hour, muscle contractions were less apparent.
Image analysis was performed using ImageJ. The area of the wound was measured horizontally from each side of the wound then using the freehand tool around the depth of the wound. The depth of the wound was also measured from the centre of the horizontal line down to the lowest point in the wound.
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