Page 71 - Q. Neuroscience

Study on molecular mechanisms of gait switching in C. elegans

Kyeong Min Moon, Jihye Cho, Jimin Kim, and Kyuhyung Kim

Department of Brain and Cognitive sciences, DGIST, Daegu, 42988, Republic of Korea

Abstract
Gait is the movement pattern of the animals. Most animals exhibit multiple forms of gaits and switch gaits depending upon external and/or internal
conditions. However, molecular mechanisms underlying gait switching are not fully understood. The nematode Caenorhabditis elegans is a good model
system that allows us to study gait switching, because animals exhibit well-defined locomotive behaviors or gaits which are flexible. For example, animals
crawl on solid surfaces with low frequency and short wavelength (Karbowski J, et al., 2006), and swim under liquid with high frequency and long-wavelength
(Korta et al., 2007; Pierce-Shimomura et al., 2008). To identify the molecular mechanisms of gait switching in C. elegans, we performed EMS mutagenesis
and isolated ten mutants (lsk56-68), which are defective in crawl-to-swim switching or swimming. We classified these mutants into two groups depending
on their behavior phenotypes. The first group of five mutants (lsk56,57,59,67,68) exhibited delayed crawl-to-swim switching and severely decreased
swimming frequency. The second group of two mutant (lsk58, 66) exhibited normal crawl-to-swim switching, but decreased swimming frequency. Currently,
we are performing chromosomal mapping to clone newly found swimming defective mutants.

Results EMS mutagenesis for finding new genes Swimming onset time is not coupled to Head muscle contraction may be involved in
involved in gait transition swimming frequency gait transition
C. elegans switches gait in response to change
of environmental condition Ethyl methanesulfonate Slow onset lsk56,57,59,66,67,68
Slow swimming
x SMBs
Random Pick swimming (DL, DR, VL, VR)
mutation defective mutant Fast onset
Slow swimming lsk58
Crawling Swimming Swimming behavior test
Molecular mechanism of gait transition is 100 Kim et al., (2015)
still unclear *** ***
80 - The SMB neurons innervate neck muscle
- lim-4 gene is expressed in the SMB neurons
Onset time (sec) 40 100
? 60 *** ** - The SMB neurons are not functional in lim-4 mutants
Serotonin Dopamine 20 In C. elegans gait transition, transition time may 80 ***
?
affect swimming speed and motion
Crawling Swimming 0 lsk56 lsk57 lsk58 lsk59 lsk66 lsk67 lsk68 Chromosome mapping for identifying genetic 60
? ? Wild-type Onset time (sec) 40
Sensory neuron mutation site
Interneuron 6 GFP
Swimming frequency (Hz) *** mutant GFP marker worm
Motorneuron ** 20
T.Pierce-Shimomura et al.,(2008), Vidal-Gadea et al.,(2011) 4 *** *** P 0 EMS Chromosome 0 Wild-type lim-4(ky403)
(male)
Crawl-to-Swim assays 2 *** *** F1 6
0 Assay in chromosomal 4 ***
Wild-type lsk56 lsk57 lsk58 lsk59 lsk66 lsk67 lsk68 F2 marker strains Swimming frequency (Hz)
Free moving on a NGM Drop M9 buffer Recording
plate for 1 min *Swim onset Crawling behavior test Non-GFP GFP GFP 2
*Swim onset = The time between touching the liquid and initiating a full Wave width I lsk58,59
swimming movement. 0
II Wild-type lim-4(ky403)
Candidate mutants Screen III lsk56,57
100 Wave length IV Questions
500
80 450 *** * V ?
Onset time (sec) 60 ** Wave length (µm) 400 ** X
40
350
during swimming
20 300 lsk59 mutants display abnormal head movement
0 250
Wild-type lon-2 lon-2 mec-7 mec-2 tph-1 lgc-40 Wild-type lsk56 lsk57 lsk66 lsk67 lsk68 Still, we want to know how they detect
250 *** environmental change and mechanisms
6 * * 200 when worm swims, its head moves first and then underlying gait switching
Swimming frequency (Hz) 4 2 Wave width (µm) 150 * x 1. Investigate mechanisms by which animals
the body moves
Future works
100
50
0 0 lsk56 lsk57 lsk66 lsk67 lsk68 lsk59 mutants do not move their head detect change in environmental conditions
2. Investigate neural circuit of gait transition
Wild-type lon-2 lon-2 mec-7 mec-2 tph-1 lgc-40 Wild-type during swimming.
Gene name(Allele) Description References
lon-2(e678) Glypican family, growth factor binding activity 〮 T.Pierce-Shimomura et al., (2008) “Genetic analysis of crawling and swimming locomotory patterns in
C. elegans”, PNAS
mec-7(e1506) Beta-tubulin, GTP binding activity K. Kim lab
〮 Wallece et al., (1959) “The moment of eelworms in water films”, Ann Appl Biol
mec-2(e75) Stomatin homolog, cholesterol binding activity
〮 Vidal-Gadea et al., (2011) “Caenorhabditis elegans selects distinct crawling and swimming gaits via
tph-1(n4622) Tryptophan hydroxylase, serotonin biosynthesis dopamine and serotonin”, PNAS CGC
NBRP
lgc-40(n4545) Glycine receptor homolog, low-affinity 〮 Kim et al., (2015) “The evolutionarily conserved LIM homeodomain protein LIM-4_LHX6 specifies the
serotonin receptor
terminal identity of a cholinergic and peptidergic C. elegans sensory/inter/motor neuron-type”, Plos Genetics