An independent project for Developmental Biology Lab (BIOL340), testing the effects of octanol exposure on the regeneration of Schmidtea mediterranea planaria. We tested to see whether or not actin was involved in the gap junction communication of migrating neoblasts during regeneration. We performed RT-PCR and Western Blot assays to detect any changes in actin levels from RNA and protein. Our results show that there seems to be little correlation between actin and gap junctions. We would like to conduct future studies on myosin, F-actin, and G-actin. The poster for this project was presented as part of the Spring 2016 Investigative Biology Labs Poster Session at the University of Maryland, Baltimore County (UMBC). Including BIOL340L, the session also features research projects from students in the Molecular and General Genetics Lab, and the Phage Hunters: Genome Analysis Lab. The session serves to inform the public about the upper-level laboratories offered by the Biological Sciences department at UMBC.

1. The Effects of Octanol on Gap Junctions and Actin of Neoblasts during
Smed Planarian Regeneration
Ian Crosby, Marianne Gadiano, Shana Gregory, & Nkemdilim Ndubuizu
BIOL 340L Developmental Biology Laboratory, Spring 2016
University of Maryland, Baltimore County
Abstract
Planaria have an amazing ability to regenerate into entirely new
beings from 1/279th
of a piece. Their ability to regenerate is due to
their stem cells called neoblasts being able to proliferate and move to
the site of damage before differentiating. Cell movement is mediated
by cytoskeletal rearrangement, specifically, actin and myosin
dynamics. This detailed process must be coordinated by signaling
between cells. One way that cells can relay messages for coordinated
movements is through gap junctions. However, if the signal is not
relayed between cells or relayed improperly, cell migration is
abnormal. We believe that by inhibiting the function of gap junctions
using octanol, we will impair the signal for cell movement between
cells, therefore altering the levels of actin and myosin in planarian
neoblasts. Our data suggests that there is little correlation between
gap junction inhibition and levels of actin within regenerating planaria.
Understanding the signaling mechanism in planarian regeneration is
important because it can be applied to the field of regenerative
medicine.
Introduction
❖ The remarkable regenerative abilities of the Schmidtea
mediterranea planarian have captured the attention of scientists
who hope to apply this ability to humans, such as the regeneration
of insulin cells for those with diabetes and the regeneration of
nerve cells for those with spinal cord injuries.
❖ Following injury, the planarian cells begin the process of
regeneration by contracting around the injury site, reducing the
wound size. After 24 hours, local cell death peaks and neoblasts
proliferate around the site [2]. Within 6-8 hours following injury,
mitotic activity of neoblasts is at its peak. This peak is more
sustained three days after injury. At these timepoints, it is thought
that progenitors which were generated during wounding migrate
to the injury site and form a regeneration blastema [4]. Within a
week, the planarian would have regenerated to its full size.
❖ The process of regeneration would not be possible without
sufficient cell communication. We believe that gap junctions, a
specialized intercellular connection used to diffuse certain
molecules directly between the cytoplasm of two different cells,
and actin, the motility protein that is involved with localization of
cells, may play a role in neoblast migration during planarian
regeneration [3][1].
Experimental Design
Data/Results
References
[1] A Mogilner et al(1996). Call motility driven by actin polymerization.
Biophys J. 71(6): 3030–3045.
[2] Harshani, P. T., Hoyer, K. K., & Oviedo, N. J. (2014). Innate immune
system and tissue regeneration in planarians: An area ripe for exploration.
Seminars in Immunology, 26(4), 295-302.
[3] N.J. Oviedo et al (2010) Long-range neural and gap junction protein-
mediated cues control polarity during planarian regeneration. Dev. Biol.,
339 , pp. 188–199
[4] Rink, J. (n.d). Stem cell systems and regeneration in planaria.
Development Genes And Evolution, 223(1-2), 67-84.
Discussion
❖ Under the microscope, it seems that regeneration and
movement was not hindered with the increasing concentrations
of octanol for all three timepoints. (Figs. 2-4)
❖ Looking at the data from the western blots, it appears that
the levels of actin remained fairly constant. (Figs. 5-7) However,
this would need to be determined using a loading control.
❖ Actin band was vivid in the 18-hr 0% octanol planaria heads
(Figure 8).
❖ Most actin bands were too faint to tell whether actin levels
have changed while the planaria were exposed to octanol. (Figs.
8-10) This may be due to sample evaporation during PCR or
insufficient loading of samples onto the gels.
❖ Based on how far the actin bands travelled, it seems that
actin is consistent at all concentrations and timepoints.
❖ It seems that actin and gap junctions have little correlation.
More experiments will need to be conducted.
Future Directions
❖ Gap junctions might regulate the certain secondary
structures of actin which could lead to the janus heads
observed when planaria are treated with octanol, and so we
would like to repeat the experiment focusing on these
structures.
❖ Examine presence of filamentous actin through the use of
immuno fluorescence.
Figure 1: Well plate were set-up with different octanol concentrations
and timepoints; H = heads, T = tails, 0 = planaria water (negative
control), 25 = 25% concentration (63.5 uM), 50 = 50% concentration
(127 uM), 75 = 75% concentration (190.5 uM).
Figures 2-4 (above, from left to right): 18 hour, 4 day, and 1 week planaria (respectively) in well-plate before dispensing in
RNAlater.
KEY
Lane:1,10 Ladder
Lane:2,6 Negative Control
Lane:3,7 25%
Lane:4,8 50%
Lane:5,9 75%
Figure 8 (Top Left): 18 hour PCR gel
Figure 9 (Bottom): 4 day PCR gel
Figure 10 (Top Right): 1 week PCR gel
Acknowledgements
❖ Dr. Cynthia Wagner for facilitating the class
❖ BIOL 340L Grad TA’s Jesse Fox and Jong Park for general lab
help
❖ BIOL 340L Classmates
α-JLA 20
43kDa
Figure 5 (Top Left): 18 hour western blot gel
Figure 6 (Bottom): 4 day western blot gel
Figure 7 (Top Right): 1 week western blot gel
KEY
Lane 1: 100bp DNA Ladder
Lane 2: 0% Octanol Heads (OH)
Lane 3: 0% Octanol Tails (OT)
Lanes 4-9: 25H, 25T, 50H,
50T, 75H, 75T
A: Actb (Beta-actin)
C: HSP60 (control)
Actb
Actb
Actb
Lanes: 1 2 3 4 5 6 7 8 9
Heads Tails
HSP601,622 bp
1,162 bp