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inx-1 is a negative regulator of the expulsion step of the defecation motor program in C. elegans

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inx-1 is a negative regulator of the expulsion step of the defecation motor program in C. elegans

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inx-1 is a Negative Regulator of the Expulsion Step
of the Defecation Motor Program in C. elegans

by
Ukjin Choi

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
August 2016

Copyright 2016 Ukjin Choi
i

Acknowledgements
My thesis work would not have been possible without my mentor Dr. Derek Sieburth.
I would like to thank him for his guidance, encouragement and support during my master’s
program. I very much appreciate his patience and trust when the project did not go well.
Also, I appreciate him for providing me the materials whenever I needed to set up new
experiments for my project.
I would also like to thank the current members from Dr. Sieburth’s lab, Dr. Sungjin
Kim, Dr. Mingxi Hu, and Qi Jia for their help. They really helped a lot by giving precious
advice to my project. I also want to thank Dr. Han Wang, a former member of Dr. Sieburth’s
lab and currently a postdoc at California Institute of Technology. I could not have
accomplished this much without his previous work. Also, I want to thank Dr. Karen Chang
and her lab members for their advice and help.
I am thankful to Dr. Zoltan Tokes, who is the chair of my committee. He has been
inspiring and encouraging throughout the two years of my master’s program, and helped me
to pursue academic achievement. I would also like to thank my committee member, Dr.
Pragna Patel for her guidance and support.
Finally, I want to thank my parents in Korea for their support and trust. It would not
have been possible without their full support. Also, I want to thank my friends here for
helping me whenever I needed support. Especially, I want to thank Karen Ra and Sean
Chung for not only supporting me and giving me advice with my academic work, but also
encouraging me and helping me with my life here.

ii

Table of Contents
Acknowledgements .................................................................................................................. i
List of figures .......................................................................................................................... iii
Abbreviation ........................................................................................................................... iv
Abstract .................................................................................................................................... v
Chapter 1: Introduction ......................................................................................................... 1
1.1 Defecation motor program .............................................................................................. 1
1.2 Expulsion step ................................................................................................................. 2
1.3 Innexin ............................................................................................................................. 4
Chapter 2: Materials and Methods ....................................................................................... 6
2.1 C. elegans Strains ............................................................................................................ 6
2.2 Molecular Biology........................................................................................................... 6
2.2 Transgenic Strains ........................................................................................................... 7
2.3 Behavioral assay .............................................................................................................. 8
2.4 Fluorescence imaging ...................................................................................................... 8
2.5 In vivo calcium imaging .................................................................................................. 9
Chapter 3: Results................................................................................................................. 10
3.1 inx-1 mutations restore Exp defect of nlp-40 mutants .................................................. 10
3.2 inx-1 functions in the GABAergic neurons to negatively regulate the Exp step .......... 13
3.3 inx-1 expresses in DVB ................................................................................................. 15
3.4 inx-1 acts downstream of PKA and upstream of calcium channels .............................. 17
3.5 INX-1 localizes to synaptic regions .............................................................................. 18
3.6 inx-1 mutation disrupts rhythmic calcium influx in the GABAergic neurons .............. 22
3.7 inx-1 requires expression in both AVL and DVB neurons to function ......................... 25
3.8 AVL neuron negatively regulates DVB neuron through INX-1 ................................... 28
3.9 inx-1 positively regulates potassium channels .............................................................. 30
Chapter 4: Discussion ........................................................................................................... 32
References .............................................................................................................................. 36

iii

List of figures
Figure 1. Schematic diagram of the defecation motor program in C. elegans
Figure 2. Schematic view of signal pathway regulating the Exp step
Figure 3. inx-1 mutants restore Exp defects
Figure 4. inx-1 functions in GABAergic neurons
Figure 5. inx-1 functions post-developmentally
Figure 6. CRISPR-Cas9 mediated GFP knock-in at endogenous inx-1 loci
Figure 7. inx-1 functions downstream of PKA and upstream of calcium channels
Figure 8. Functional inx-1 fusion protein and expression in GABAergic neurons
Figure 9. Searching for the functional motif within inx-1
Figure 10. inx-1 mutation causes ectopic calcium influx in DVB neuron
Figure 11. inx-1 requires both AVL and DVB expression
Figure 12. Phenotype of other innexins in the Exp step
Figure 13. AVL negatively regulates DVB through INX-1
Figure 14. inx-1 mutation restores Exp defect of gain of function potassium channels
iv

Abbreviation
aBoc anterior body wall muscle contraction
ACY adenylyl cyclase
cAMP cyclic adenosine monophosphate
Exp expulsion or enteric muscle contraction
GABA gamma-aminobutyric acid
GCaMP a genetically-encoded calcium indicator
GPCR G protein-coupled receptor
pBoc posterior body wall muscle contraction
PKA cAMP-dependent protein kinase or protein kinase A
PKA[CA] constitutively active PKA
PKA[DN] dominant negative PKA
VGCC voltage-gated calcium channel

v

Abstract
Gap junctions are specialized connections between cells that regulate the intercellular
transfer of chemical and electrical signals. Gap junctions play important roles during
development and in the functions of mature tissues such as neurons and muscles. Gap
junctions are composed of two hemi-channels, each made up of hexamers of connexin
subunits (or innexin subunits in invertebrates). There are over two dozen connexin (and over
one dozen innexin) family members that can combine to form either homo- or hetero hemi-
channels, and the composition of the hemichannels is thought to contribute to the structural
and functional diversity of gap junctions. Here, we identify a novel role for a putative gap
junction protein, inx-1/innexin in negatively regulating the contraction of enteric muscles
during a pacemaker-controlled motor program in C. elegans. inx-1 mutants restore enteric
muscle contraction and presynaptic calcium influx to animals with defects in pacemaker-
induced activation of the enteric muscle neuromuscular junction. Our genetic analysis reveals
that that inx-1 functions in the motor neurons that innervate the enteric muscles and inx-1
regulates calcium influx at presynaptic terminals possibly by regulating the activity of
presynaptic potassium channels. We find that the INX-1 protein is concentrated at
presynaptic terminals where it may form gap junctions that connect two motor neurons that
control the enteric muscles contraction. We propose that INX-1 functions to negatively
regulate motor neuron activation by inhibiting the generation of rhythmic calcium spikes in
motor axons at this synapse.
1

Chapter 1
Introduction
1.1 Defecation motor program
The defecation motor program is a rhythmic behavior that is accurately and rigidly
regulated by an endogenous biological clock that occurs about every 50 seconds at 20°C in
the presence of food in C. elegans. The motor program consists of three sequential steps
involving contraction of the posterior body-wall muscles (pBoc), anterior body-wall muscles
(aBoc), and enteric muscles (Exp). First, the posterior body-wall muscles contract (pBoc),
pushes the intestinal contents to the anterior region. After 3 seconds, the anterior body-wall
muscles contract (aBoc) to push the gut contents to the posterior intestine (the pre-anal
region). Immediately after aBoc, contraction of enteric muscles occurs which leads to
expulsion (Exp) of the gut contents (Figure 1). These events can be all observed by dissecting
microscopy, allowing the use of the defection motor program as an assay for identifying
genes that are involved in this process. The defecation motor program is controlled by the
intestine, GABAergic neurons (AVL and DVB), anterior body-wall muscles, posterior body-
wall muscles, and enteric muscles (Branicky & Hekimi, 2006).

2

Figure 1. Schematic diagram of the defecation motor program in C. elegans
The diagram is modified from (Beg, Ernstrom, Nix, Davis, & Jorgensen, 2008). The
defecation motor program occurs every 50 seconds. First, the posterior body wall muscles
contract (pBoc), and after 3 seconds, the anterior body wall muscles contract (aBoc),
immediately followed by enteric muscle contraction, which leads to expulsion (Exp).

1.2 Expulsion step
In the defecation motor program, the expulsion (Exp) step directly controls the
release of the gut contents from the anus. The circuit of the expulsion consists of the
intestine, the GABAergic neurons (AVL and DVB), and the enteric muscles. Expulsion
defects manifest as lack of enteric muscle contraction and result in constipation, revealing an
expansion of the intestine under the dissecting microscope due to accumulation of the gut
contents. Studies using mutants which show these phenotypes have revealed the underlying
mechanism of the signaling pathway of the Exp step (Branicky & Hekimi, 2006). The Exp
step starts with propagation of a calcium wave through the intestine which is the pacemaker
(Peters, Teramoto, White, Iwasaki, & Jorgensen, 2007). Then, the calcium wave triggers the
release of the neuropeptide-like protein, NLP-40 from dense core vesicles (DCVs) via a the
DCV calcium sensor SNT-2/synaptotagmin. The released NLP-40 binds to its receptor,
3

AEX-2/GPCR which is on the membrane of the GABAergic neurons and activates the
heterotrimeric G protein s (G s) and adenylate cyclase (ACY) to generate cAMP (Mahoney
et al., 2008; Wang et al., 2013). cAMP activates PKA which leads to calcium influx through
calcium channels. The calcium influx drives GABA release from the GABAergic neurons
(Wang & Sieburth, 2013). GABA released from the two neurons binds to EXP-1 which is an
excitatory GABA receptor, resulting in enteric muscle contractions (Beg & Jorgensen, 2003)
(Figure 2). Enteric muscle contraction which leads to expulsion of the intestinal contents is
stimulated by two GABAergic motor neurons, AVL and DVB (McLntire, Jorgensen, Kaplan,
& Horvitz, 1993).

4

Figure 2. Schematic view of signal pathway regulating the Exp step
The diagram is modified from (Wang et al., 2013). The Exp step is initiated by a calcium
spike in the intestine. SNT-2/synaptotagmin on DVCs senses the calcium oscillation and
release of NLP-40 is promoted from the intestine. Secreted NLP-40 activates AEX-2/GPCR
which leads to activation of G s adenylyl cyclase (ACY) to generate cAMP. Then, cAMP
activates PKA which triggers calcium influx in GABAergic neurons (AVL and DVB).
Calcium influx promotes release GABA from the neurons and the GABA binds to the
excitatory GABA receptor, EXP-1 to trigger enteric muscle contractions (Exp).

1.3 Innexin
Gap junctions form specialized connections between adjacent cells in eukaryotes that
are important for intracellular communication (Sohl, Maxeiner, & Willecke, 2005). Gap
junctions are composed of two hemichannels called innexons and these hemichannels consist
of six innnexin subunits. Hemichannels can also function as channels on the plasma
membrane. Gap junctions allow small molecules (<1.5kDa) including ions, metabolites,
5

neurotransmitters, and signaling molecules to directly pass through (Evans & Martin, 2002).
In vertebrates, gap junctions are composed of a family of proteins called connexins and
pannexins. There are 21 connexins and 3 pannexins in the human genome. Invertebrates do
not encode connexins but they encode a family of proteins called innexins. Like connexins,
innexins are transmembrane proteins that have four transmembrane domains, two
extracellular loops, and amino and carboxyl terminus in the cytoplasmic region (Simonsen,
Moerman, & Naus, 2014). Although innexins, connexins, and pannexins share similar
topology, the pannexin family has been demonstrated to be homologous to the innexin gap
junction proteins (Baranova et al., 2004), while connexins show more limited sequence
homology with innexins (T. A. Starich, Lee, Panzarella, Avery, & Shaw, 1996). Previous
studies in C. elegans have demonstrated a role for innexins in embryogenesis, pharyngeal
development, intestinal muscle contraction, oocyte maturation and fertilization,
determination of neuronal cell fate, and electrical coupling in body wall muscles (Chuang,
VanHoven, Fetter, Verselis, & Bargmann, 2007; Q. Liu, Chen, Gaier, Joshi, & Wang, 2006;
Peters et al., 2007; T. A. Starich et al., 1996; Todd A. Starich, Miller, Nguyen, Hall, & Shaw,
2003; Whitten & Miller, 2007). However, the function of over half the innexins is unknown.
Here in our study, for the first time, we identify the function of an innexin family member,
inx-1 that negatively regulates the expulsion step of the defecation motor program in C.
elegans.
6

Chapter 2
Materials and Methods
2.1 C. elegans Strains
Strains were maintained under standard conditions. The wild type strain was N2
Bristol. Mutant strains were: OJ794 nlp-40(tm4085) I, OJ2446 inx-1(tm3524) X, OJ2323 nlp-
40(tm4085) I;inx-1(tm3524) X, OJ2291 snt-2(tm1711) III, snt-2(tm1711) III;inx-1(tm3524)
X, OJ1540 aex-2(sa3) X, OJ2327 aex-2(sa3) X inx-1(tm3524) X, OJ1603 vjIs76 V, OJ2319
vjIs76 V;inx-1(tm3524) X, OJ1526 unc-2(lj1) X, OJ2290 unc-2(lj1) X inx-1(tm3524) X,
OJ2165 vjIs119 III, OJ2316 vjIs119 III;inx-1(tm3524) X, OJ1218 unc-25(e156) III, unc-
25(e156) III;inx-1(tm3524) X, OJ1441 exp-1(sa6) II, OJ2157 exp-1(sa6) II;inx-1(tm3524) X,
KP3948 eri-1(mg366) IV;lin-15B(n744) X, OJ2600 inx-1(tm3524) lin-15B(n744) X;eri-
1(mg366) IV, OJ1443 unc-13(s69) I;vjIs58 IV, OJ1467 nlp-40(tm4085) X unc-13(s69)
I;vjIs58 IV, OJ2341 unc-13(s69) I;vjIs58 IV;inx-1(tm3524) X, nlp-40(tm4085) unc-13(s69)
I;vjIs58 IV;inx-1(tm4085) X, OJ2451 inx-10(ok2714) V, OJ2326 nlp-40(tm4085) I;inx-
10(ok2714) V, inx-7(ok2319) IV, OJ2447 nlp-40(tm4085) I;inx-7(ok2319) IV, inx-11(ok2783)
V, OJ2448 nlp-40(tm4085) I;inx-11(ok2783) V, unc-7(e5) X, nlp-40(tm4085) I;unc-7(e5) X,
unc-9(e101) X, nlp-40(tm4085) I;unc-9(e101) X, JT577 egl-36(n2332) X, OJ1897 egl-
36(gf)(n728) X, nlp-40(tm4085) I;egl-36(n2332) X, OJ1920 vjIs77 IV;egl-36(n2332) X, egl-
36(gf)(n728) inx-1(tm3524) X, OJ1858 vjIs103 I, OJ1898 vjIs103 I:egl-36(gf)(n728) X.

7

2.2 Molecular Biology
The plasmids were constructed using the backbone pPD49.26 (A. Fire). Promoter
regions were amplified from genomic DNA of C. elegans and cDNA was used to clone genes
using standard molecular biological techniques. The following plasmids were constructed:
pHW175[Punc-47(FL)::inx-1a], pUC07[Pmyo-3::inx-1a], pUC08[Pnlp-40::inx-1a],
pUC09[Phsp-16.2::inx-1a], pUC05[Punc-47(FL)::inx-1a::GFP], pUC19[Punc-47(FL)::inx-
1b::GFP], pUC20[Punc-47(FL)::inx-1a(1~307)::linker::GFP], pUC21[Punc-47(FL)::inx-
1a(1~338)::linker::GFP], pUC25[Punc-47(FL)::inx-1(1~372)::linker::GFP], pUC26[Punc-
47(FL)::inx-1(1~356)::linker::GFP], pUC31[Punc-47(FL)::inx-1(1~307)::inx-
1a(339~428)::GFP], pUC32[Punc-47(FL)::inx-1(1~307)::inx-1a(357~428)::GFP],
pUC30[Pflp-22(delta)::inx-1a::GFP], pUC33[Pflp-10::aex-2], pUC34[Pflp-10::inx-
1a::GFP], pMH44[Pflp-22(delta)::aex-2]
2.2 Transgenic Strains
Transgenic strains were generated by injecting into N2, nlp-40(tm4085);inx-
1(tm3524), aex-2(sa3), or aex-2(sa3)inx-1(tm3524) with expression plasmids together with
co-injection markers KP708(Pttx-3::RFP at 40ng/μl) or pZB07(Pttx-3::GFP at 40ng/μl) or
KP1106 (Pmyo-2::NLS::GFP at 5ng/μl) or KP1368 (Pmyo-2::NLS::mCherry at 5ng/μl).
Microinjection was performed using standard procedure. Generally, three lines were
generated and examined representatively. The following arrays were generated:
vjEx912[pHW175], vjEx913[pUC05], vjEx915[pUC07], vjEx916[pUC09],
vjEx917[pUC08], vjEx1067[pUC19], vjEx1068[pUC20], vjEx1069[pUC21],
vjEx1066[pUC25], vjEx1062[pUC26], vjEx1070[pUC31], vjEx1071[pUC32],
8

vjEx1064[pUC30], vjEx1095[pUC34], vjEx1080[pUC30, pUC34], vjEx1028[pMH44],
vjEx1090[pUC33].
2.3 Behavioral assay
The defecation motor program was performed as previously described (D. Liu &
Thomas, 1994; Thomas, 1990). Ten to fifteen worms were moved to fresh NGM plate seeded
with OP50 bacterial lawn and were allowed to settle for at least 10 min for recovery. Ten
constitutive defecation cycles were observed from each worm. The pBoc and Exp steps were
recorded by the Etho program (James Thomas Lab website:
http://depts.washington.edu/jtlab/software/otherSoftware.html) (Liu and Thomas, 1994;
Mahoney et al., 2008). Five to ten worms were assayed, and the mean and standard deviation
was calculated for each genotype. Statistical significance was determined between two
samples by using an unpaired two-tail Student’s t test with unequal variance.
2.4 Fluorescence imaging
Fluorescence imaging was done by using a Nikon eclipse 90i microscope equipped
with a Nikon Plan Apo 40x oil objective (N.A.=1.0), Nikon Plan Apo 100x oil objective
(N.A.=1.40), and a Photometrics Coolsnap ES
2
camera. Young adult worms were paralyzed
with 30mg/ml 2, 3-Butanedione monoxime (BDM, Sigma) in M9 buffer, and then mounted
on 2% agarose imaging pad. Metamorph 7.0 software (Universal Imaging) was used to
capture image stacks and to obtain maximum intensity projections. Fluorescence imaging in
the GABAergic motor neurons were performed in N2 or inx-1 mutant background. The
images were captured from the synaptic region of the DVB.
9

2.5 In vivo calcium imaging
The calcium imaging experiment was performed as previously described (Han 2013).
We used an integrated transgenic line vjIs58 (Pmyo-2::NLS::mCherry, Punc-
47mini::GCaMP3.0) which specifically expresses GCaMP3.0 in AVL and DVB neurons to
perform in vivo calcium imaging at the synaptic region. vjIs58 was used in unc-13(s69)
mutant background to immobilize animals because unc-13 mutants are almost completely
paralyzed. Both vjIs58 and unc-13 have normal Exp steps. Young adult worms were
transferred to NGM-agarose plate with the food OP50 and the plate was topped with a cover
slide. Live imaging was done using a Nikon eclipse 90i microscope equipped with a Nikon
Plan Apo 40x oil objective (N.A.=1.0), a standard GFP filter and a Photometrics Coolsnap
ES
2
camera. The worms that were keep pumping and positioned laterally with the left side
facing the objective were selected for imaging. Metamorph 7.0 software (Universal Imaging)
was used to obtain time lapse imaging. For each worm, the neuromuscular junction of the
GABAergic neurons (AVL and DVB) was recorded for 250 s at 4 frames per second (2x2 or
3x3 binning with 30-90 ms exposure time depending on the baseline of GCaMP3.0
fluorescence in the neuromuscular junction region in each worm).
The GCaMP3.0 fluorescence intensity in the synaptic region of DVB neurons was
quantified using Metamorph 7.0 software (Universal Imaging). The average fluorescence (F)
of GCaMP3.0 was calculated by the average fluorescence of a region of interest (ROI) in the
synaptic region of the DVB neuron minus the background fluorescence of a similar region
near the tail. The baseline fluorescence (F0) was defined by the average GCaMP3.0
fluorescence in the first 10 frames before the initiation of pBoc. The fluorescent change of
the GCaMP3.0 for each frame was defined as ΔF/F0 = (F-F0)/F0.
10

Chapter 3
Results
3.1 inx-1 mutations restore Exp defect of nlp-40 mutants
To identity proteins that regulate the defecation motor program, we conducted a
suppressor screen for nlp-40 mutants. nlp-40 is a neuropeptide-like protein 40 (NLP-40)
secreted from the intestine which activates the downstream GABAergic neurons that controls
the Exp step. nlp-40 mutants have a normal pBoc frequency but lack Exp and calcium entry
in GABAergic neurons nearly all cycles (Wang et al., 2013). We reasoned that suppressors
should bypass the need for nlp-40 signaling and therefore, should represent genes that
function in parallel to the nlp-40 signaling pathway. We imagine that these genes would
negatively regulate the excitability of the circuit downstream of nlp-40, either in the motor
neurons or in the muscle.
We identified three suppressors vj46, vj48 and vj49 of the expulsion defect of nlp-40
null mutants. Each suppressor suppressed the constipated phenotype of nlp-40 mutants as
well as the Exp defect (Figure 3A). We mapped the suppressors and conducted whole
genome sequencing (Table 1). The vj46 suppressor maps to LG X and carries a G to A
transition at the splice donor site of the inx-1 gene. inx-1 encodes two isoforms, a and b. inx-
1a differs from inx-1b in that it is transcribed from a start site that is upstream of inx-1b, and
also has a unique C terminus generated by differential splicing. vj46 encodes a G to A
transition in intron 7 of the inx-1a gene (intron 5 of the inx-1b gene). This mutation is
predicted to lead to splicing defects of inx-1 that result in the truncation of both inx-1
isoforms. We obtained an independently isolated inx-1 allele, tm3524, which contains a
11

238bp deletion that removes all of exon 8 of the coding region (Table 2)(Figure 3B). Both
inx-1 mutants restored Exp to nlp-40 mutants from a frequency of less than 5% to about 50%
(Figure 3A). Also, nlp-40;inx-1 double mutants displayed decreased distended intestinal
lumens compare to nlp-40 mutants (data not shown). Examination of the timing of the
restored expulsions revealed that they occurred at random times. In wild type animals, the
normal pattern of Exp occurs about 3-4 seconds after pBoc. However, in nlp-40;inx-1 the
Exp occurred at random times within the 50 second cycle after pBoc (Figure 3C). Thus, inx-1
is a suppressor of the Exp defects of nlp-40 mutants during the defecation motor program,
but inx-1 mutations do not restore the proper timing of expulsion to nlp-40 mutants.
Examination of inx-1 mutants alone reveals that they have normal Exp frequency and
timing. inx-1 mutants also have superficially normal locomotion and growth rates. However,
they display a mild egg-laying defect that results in accumulation of eggs in the uterus
compare to wild type animals.

12

Figure 3. inx-1 mutants restore Exp defects
(A) Quantification of the number of Exp per defecation cycle in adult worms of the indicated
genotypes. “Exp per cycle” is defined as the ratio Exp per pBoc. (B) The gene structure of
inx-1a and b isoforms and the location of tm3524 deletion and vj46 substitution are indicated.
(C) Classification of different patterns of Exp in worms with the indicated genotypes. <5 sec
denotes Exp occurring less than 5 seconds after pBoc which is defined as the normal pattern
of Exp, and >5 sec denotes Exp occurring more than 5 seconds after pBoc, which is defined
as ectopic Exp. Means and standard errors are shown. Asterisks indicate significant
differences: ** P<0.005; *** P<0.0005 in Student’s t-test.

13

3.2 inx-1 functions in the GABAergic neurons to negatively regulate the Exp step
To examine which of the three tissues in the Exp circuit inx-1 functions in, we
conducted tissue-specific rescue experiments. To do this, we expressed the full length inx-1a
or b cDNA under GABAergic neuron-specific (Punc-47), muscle-specific (Pmyo-3), and
intestine-specific (Pnlp-40) promoters. The phenotype of nlp-40;inx-1 double mutants were
only reverted to the nlp-40 phenotype when inx-1 cDNA is expressed under the GABAergic
neuron-specific promoter, while muscle-specific and intestine-specific promoters did not
revert the nlp-40;inx-1 phenotype (Figure 4). Thus, inx-1 functions specifically in the
GABAergic neurons to negatively regulate Exp.
In principal, inx-1 could restore Exp to nlp-40 mutants by impacting either the
development of the motor neurons or their function. To distinguish between these
possibilities, we selectively introduced inx-1 activity to nlp-40;inx-1 mutants after motor
neuron development using a heat shock promoter. We generated transgenic nlp-40;inx-1
mutants that express inx-1a cDNA under the heat shock (Phsp16.2) promotor. Before heat
shock, the transgenic animals displayed 63% Exp, similar to non-transgenic controls.
Immediately following a one-hour heat shock, the transgenic animals displayed 10% Exp,
which is a frequency that is similar to nlp-40 mutants (Figure 5). Together, these results
indicate that inx-1 does not appear to be required for motor neuron development, but instead
inx-1 functions in the GABAergic neurons to negatively regulate their activity during the Exp
step.

14

Figure 4. inx-1 functions in GABAergic neurons
Quantification of the number of Exp per defecation cycle in adults with the indicated
genotypes. Full length inx-1 cDNA was expressed under tissue-specific promoters which are
GABAergic neuron-specific (Punc-47), muscle-specific (Pmyo-3), and intestine-specific
(Pnlp-40). The restored Exp of nlp-40;inx-1 double mutants are only reverted to nlp-40
phenotype when inx-1 cDNA is expressed under the GABAergic neuron-specific promoter.
Means and standard errors are shown. Asterisks indicate significant differences: ***
P<0.0005 in Student’s t-test.

15

Figure 5. inx-1 functions post-developmentally
Expressing inx-1 cDNA using heat shock promoter (phsp-16.2) in adults reverts the Exp
frequency of nlp-40;inx-1 double mutants back to nlp-40 phenotype. Means and standard
errors are shown. Asterisks indicate significant differences: *** P<0.0005 in Student’s t-test.

3.3 inx-1 expresses in DVB
To confirm that inx-1 is indeed expressed in AVL and DVB, and to determine in
which other tissues inx-1 is expressed, we generated inx-1 transcriptional reporters, in which
the inx-1 promoter drives the expression of GFP. We first generated a reporter using a 5kb
fragment upstream of the inx-1a transcriptional start site. This reporter resulted in robust
expression in several neurons as well as the muscle. However, we were unable to detect
fluorescence in the DVB neuron. One possible reason is that the entire promoter region was
not included within this 5kb fragment. Therefore, we decided to use CRISPR Cas9 system to
insert gfp into the inx-1 locus. We generated GFP knock-in strains by inserting the gfp
sequence downstream from the start codon of endogenous inx-1 coding region (Figure 6A).
16

We obtained a single knock-in strain and we observed GFP expression in several neurons
including in a single cell in the tail. The identity of this cell was confirmed as being DVB by
co-localization with mCherry expressed in a DVB-expressing neuron (using the unc-47
promoter) (Figure 6B). We could not conclusively determine whether inx-1 was also
expressed in AVL because expression was seen in multiple neurons in the head all near AVL,
making resolving AVL difficult.

Figure 6. CRISPR-Cas9 mediated GFP knock-in at endogenous inx-1 loci
(A) Schematic view of Cas9 target region, donor plasmid for GFP knock-in, and process of
homologous recombination. The donor plasmid contains the gfp coding sequence inserted
right after the start codon of inx-1, and also a stop codon was introduced in the gfp sequence
to generate a transcriptional reporter. (B) GFP expression in DVB and co-localization with
mCherry. mCherry is expressed under GABAergic neuron-specific promoter. DVB cell body
is indicated by white arrow.

17

3.4 inx-1 acts downstream of PKA and upstream of calcium channels
To further determine where inx-1 functions in the AEX-2/GPCR-KIN-1/PKA
signaling pathway in DVB, we generated double mutants of the genes in this signaling
pathway with inx-1. We selected nlp-40 from the intestine and aex-2, gas-1, acy-1, dominant
negative PKA (vjIs76), unc-2, dominant negative ORAI-1 (vjIs119), and unc-25 from
GABAergic neurons, exp-1 from enteric muscle. snt-2 encodes synaptotagmin which is a
calcium sensor on the dense core vesicles (DCVs), and snt mutants are 55% Exp. aex-2
encodes a GPCR for nlp-40 and mutants are 4% Exp. Dominant negative PKA (vjIs76) is a
Gly to Asp (G310D) substitution in the B site of the regulatory subunit of the KIN-2a results
in inhibition of cAMP binding which prevents PKA activation (Wang & Sieburth, 2013), and
vjIs76 shows 2% Exp. unc-2 encodes a voltage-gated calcium channel, and unc-2 mutants
show 68% Exp. oria-1 encodes a calcium release-activated calcium (CRAC) channels and
vjIs119 which is a dominant negative ORIA-1 shows 10% Exp. unc-25 encodes a GABA
neurotransmitter biosynthetic enzyme, glutamic acid decarboxylase (GAD), and mutants are
20% Exp. exp-1 encodes an excitatory, cation-selective GABA receptor, and mutants show
17% Exp. inx-1 mutation restores the Exp defect of snt-2 mutants to 77%, aex-2 mutants to
46%, and vjIs76/PKA[DN] to 48%, but failed to restore the Exp defect of calcium channels,
unc-25/GABA, or exp-1/EXP-1 (Figure 7A). Consistent with these results, the Exp defect of
acy-1 RNAi is also restored in inx-1 background (Figure 7B). For acy-1, we used RNAi since
acy-1 mutants are lethal. Together, the results indicate that inx-1 acts downstream of PKA
and upstream of calcium channels in the GABAergic neurons to negatively regulate Exp.

18

Figure 7. inx-1 functions downstream of PKA and upstream of calcium channels
(A and B) Quantification of the Exp frequency in adults with the indicated genotypes (A) and
RNAi-treated adults (B). Means and standard errors are shown. Asterisks indicate significant
differences: * P<0.05; *** P<0.0005 in Student’s t-test.

3.5 INX-1 localizes to synaptic regions
DVB and AVL are connected to each other by gap junctions (J. G. White, Southgate,
Thomson, & Brenner, 1986). Gap junctions are also found at synapses, connecting the pre-
and post-synaptic cells. Therefore, inx-1 could potentially function in either capacity. To
19

determine whether inx-1 forms gap junctions at synapses or not, we determined the
subcellular localization of INX-1 in GABAergic motor neurons. To do this, we expressed a
INX-1::GFP fusion protein under the GABAergic neuron-specific promoter (Figure 8A).
Expressing full-length inx1a cDNA tagged with GFP at the C-terminus under GABAergic
neuron-specific promoter rescued nlp-40;inx-1 phenotype to nlp-40 phenotype, indicating the
fusion protein is functional (Figure 8B). GFP-tagged INX-1 adopted a punctate pattern in the
synaptic region of DVB neuron (Figure 8C). Generally, one puncta was observed at the
synaptic region, but two or three puncta were occasionally observed, and INX-1::GFP
sometimes showed one to three puncta in the cell body of DVB. Although, we can also see a
lot of puncta in the ventral nerve cord, we do not know whether the puncta are coming from
AVL or DVB motor neurons, or other GABAergic neurons since the promoter we are using
expresses inx-1 in all GABAergic neurons along the ventral nerve cord.
Next, to determine the INX-1 motifs required for INX-1 localization at synapses, we
generated different versions of truncated INX-1 and expressed them under a GABAergic
neuron specific promotor in nlp-40;inx-1 mutants. INX-1a and INX-1b proteins have
identical N termini, but differ in their C termini. The C terminal region of INX-1a is 45
amino acids longer than INX-1b(Figure 9A). Both inx-1a and inx-1b expressing under
GABAergic neuron-specific promoter rescued the nlp-40;inx-1 phenotype to inx-1 phenotype
(Figure 9B), indicating the extra C terminal region in INX-1a is not important for inx-1
function. We found only truncated INX-1(1~372) reverted the Exp frequency of nlp-40;inx-1
mutants to nlp-40 mutants (Figure 9B) and found that the INX-1(357~372) region is
necessary for inx-1 function. However, we could not identify the sufficient region in this
study. Consistent with the behavioral assays, fluorescence imaging results showed punctate
20

pattern only in inx-1a, inx-1b, and inx-1(1~372) (Figure 9C). Together, these results indicate
inx-1 must localize to synapses to function.

Figure 8. Functional inx-1 fusion protein and expression in GABAergic neurons
(A) INX-1::GFP fusion protein under GABAergic neuron-specific promoter. (B) INX-
1::GFP fusion protein rescues nlp-40;inx-1 to nlp-40 phenotype, indicating the fusion protein
is functional. (C) INX-1::GFP fusion protein localized to the neuromuscular junction (NMJ,
white arrow) of DVB. DVB is expressing mCherry under GABAergic neuron-specific
promoter. Means and standard errors are shown. Asterisks indicate significant differences:
*** P<0.0005 in Student’s t-test.

21

Figure 9. Searching for the functional motif within inx-1
(A) Sequence variance of inx-1 isoforms. (B) Quantification of the Exp frequency in adults
with the indicated truncated INX-1 proteins. (C) Differential expression patterns of various
truncated INX-1::GFP fusion proteins in the ventral nerve cord (VNC). Means and standard
errors are shown. Asterisks indicate significant differences: ** P<0.005 in Student’s t-test.

22

3.6 inx-1 mutation disrupts rhythmic calcium influx in the GABAergic neurons
Our data supports a model whereby inx-1 inhibits expulsion by blocking the rhythmic
release of GABA from the GABAergic motor neurons. We speculate that inx-1 could either
regulate the activity of the GABAergic motor neurons or inx-1 could regulate the SV
recycling at the neuromuscular junction. To distinguish between these two possibilities, we
examined calcium influx into the GABAergic motor neurons using in vivo calcium imaging.
GCaMP3.0 is a genetically-encoded calcium indicator whose fluorescence intensity is a
function of calcium concentration (Tian et al., 2009). Expression of GCaMP3.0 in the
GABAergic motor neurons enables visual observation of the calcium waves in the DVB cell
body and the synaptic region in behaving animals. In the wild type worms, we observed a
single fluorescent spike in the synaptic region of the DVB neuron three seconds after each
pBoc step. The fluorescence levels return to baseline about two seconds after initiation of the
spike (Figure 10B). The calcium spikes initiated at the synaptic region of DVB and spread
out anteriorly along the axon and posteriorly to the DVB cell body. Immediately after the
peak of the fluorescent spike, an Exp can be observed. In inx-1 mutants, we observed normal
calcium transients that were accompanied by Exp step about three seconds after pBoc,
similar to wild type controls. However, we also observed additional (“ectopic”) calcium
transients in most cycles that occurred at random times following the first calcium spike
(Figure 10D). These ectopic calcium spikes were associated with an Exp about 40% of the
time. The remaining calcium spikes were not associated with an Exp. Together these results
suggest that inx-1 functions to inhibit calcium influx into the GABAergic motor neurons at
the wrong time in between cycles.
23

inx-1 also restored calcium entry in GABAergic motor neurons to nlp-40 mutants. In
nlp-40;inx-1 mutants, we observed either one or two calcium spikes that were accompanied
by Exp in 70% of cycles. However, the calcium spikes and Exp were rarely observed at the
correct time (three seconds after the pBoc), but instead occurred at random times during the
cycle. In addition, we occasionally observed additional (second or third) calcium transients in
each cycle that were usually not accompanied by an Exp (Figure 10E). The random timing of
the ectopic calcium transients seen in the inx-1 mutants as well as the nlp-40;inx-1 mutants
suggests that inx-1 functions independently of the pacemaker to negatively regulate
expulsion by inhibiting ectopic calcium entry into the GABAergic motor neurons.

24

Figure 10. inx-1 mutation causes ectopic calcium influx in DVB neuron
(A) Expression of the genetically encoded calcium indicator, GCaMP 3.0 in DVB neuron
(vjIs58). Two snapshots from a real-time imaging video of wild type animals showing
increased fluorescence in the synaptic region of DVB (white arrow) right before the Exp
step. (B) A representative trace of the GCaMP 3.0 fluorescence in the synaptic region of
DVB neuron in wild type animals showing DVB neuron is rhythmically activated about 3
seconds after pBoc during the defecation motor program. (C-E) Representative traces of the
GCaMP3 fluorescence in the synaptic region of DVB neurons in worms with the indicated
genotypes. The observed pBoc step and Exp step are indicated by arrows and arrowheads,
respectively.

25

3.7 inx-1 requires expression in both AVL and DVB neurons to function
Because inx-1 shares sequence similarity to both connexins and pannexins, it is
possible that inx-1 may either form gap junctions or channels. To test whether inx-1 might
form gap junctions, we determined whether inx-1 function is required in both DVB and AVL
or whether it can function in one cell independently of the other cell. Both AVL and DVB
motor neurons contribute to the expulsion step of the defecation motor program because laser
ablation of the both AVL and DVB neurons causes a severe Exp defect, and ablation of AVL
or DVB alone results in partial Exp defects (McLntire et al., 1993). To address whether INX-
1 functions in one of these neurons or both to negatively regulate the Exp step, we expressed
inx-1 cDNA under an AVL neuron-specific (Pflp-22(delta)) or DVB neuron-specific (Pflp-
10) promoters in nlp-40;inx-1 mutants. Surprisingly, transgenic nlp-40;inx-1 animals
expressing inx-1 cDNA under either the AVL or DVB-specific promoter displayed expulsion
phenotypes that were similar to nlp-40;inx-1 mutants (Figure 11). Based on the results, we
speculated that inx-1 may be required in both neurons to regulate Exp. To test this idea, we
co-expressed inx-1 cDNA in AVL and DVB by co-injecting both plasmids in nlp-40;inx-1
mutants. Transgenic nlp-40;inx-1 animals co-expressing inx-1 in both AVL and DVB
displayed a 5% Exp phenotype, similar to the nlp-40 phenotype (Figure 11), indicating inx-1
must be expressed in both AVL and DVB motor neurons in order to function. These results
are consistent with the idea that inx-1 may be a constituent of hemichannels in AVL and
DVB that come together to form functional gap junctions that connect the two cells. The
results are less consistent with the idea that inx-1 is a channel since in this case we would
expect partial rescue when inx-1 is expressed in one or the other cell.
26

Figure 11. inx-1 requires both AVL and DVB expression
Quantification of the number of Exp per defecation cycle in adults with indicated genotypes.
Full length inx-1 cDNA was expressed under cell specific promoters which are AVL neuron-
specific (Pflp-22(delta)) and DVB neuron-specific (Pflp-10). AVL rescue denotes expressing
inx-1 under AVL neuron-specific promoter and DVB rescue denotes expressing inx-1 under
DVB neuron-specific promoter. AVL, DVB rescue denotes co-expression of inx-1 under
both AVL and DVB promoters. GABA neuronal rescue denotes expressing inx-1 under
GABAergic neuron-specific (Punc-47) promoter. Means and standard errors are shown.
Asterisks indicate significant differences: *** P<0.0005 in Student’s t-test.

Gap junction proteins have three forms that are heteromeric, heterotypic, and
homotypic (T. W. White & Paul, 1999). Since our data showed inx-1 requires expression in
both AVL and DVB for its function, we speculated inx-1 forms either heteromeric or
homotypic gap junctions. To test if inx-1 forms gap junctions with other innexin subunits, we
generated double mutants between nlp-40 and other innexins. We chose to test innexins that
are known to express in DVB or that have been reported to genetically interact with inx-1 (P.
Liu et al., 2013) (Figure 12A). We tested mutants of inx-10, inx-11, inx-7, unc-7, and unc-9.
inx-3 and inx-13 were not tested since the mutants are reported to have a lethal phenotype
27

(Simonsen et al., 2014). Also, inx-16 was not tested because it already had a constipated
phenotype making assaying it difficult (Peters et al., 2007). None of the innexin mutants
tested had defects in Exp, or restored the Exp defect of nlp-40 mutants (Figure 12B). These
results suggest that inx-1 does not function together with these innexins to regulate Exp.
Instead, inx-1 may form a homotypic gap junction, or may form a heterotypic gap junction
with one of the other 16 innexins that we have not tested.

Figure 12. Phenotype of other innexins in the Exp step
(A) List of innexins expressed in muscle that are predicted to form a gap junction in pair with
INX-1, and innexins expressed in DVB. (B) Quantification of the number of Exp per
defecation cycle in adults with indicated nlp-40 inx- double mutants. Means and standard
errors are shown. Asterisks indicate significant differences: *** P<0.0005 in Student’s t-test.
28

3.8 AVL neuron negatively regulates DVB neuron through INX-1
We wanted to further identify the role of INX-1 in the GABAergic neurons. First, we
expressed aex-2 cDNA under AVL specific (Pflp-22(delta)) and DVB specific (Pflp-10)
promoters respectively in aex-2 mutants. aex-2/GPCR is the receptor for NLP-40. Therefore,
aex-2 mutants show the Exp defect similar to nlp-40 mutants. The AVL specific promoter
driving aex-2 cDNA showed restore of the Exp defect from a frequency less than 5% to
about 10% and aex-2 cDNA expressing under DVB specific promoter showed about 15%.
Co-expression of aex-2 cDNA in AVL and DVB restored to 80% (Figure 13A). Previous
study has shown that aex-2 expression under GABAergic specific (Punc-47) promoter
rescued the Exp defect to about 65% (Mahoney et al., 2008). The discrepancy between
respective expression and co-expression is due to partial rescue by mosaic expression.
Therefore, to further reveal the cell-cell communication between AVL and DVB by INX-1,
we analyzed the pattern of Exp after pBoc in inx-1 mutant background. Wild type shows
normal pattern of the Exp which occurs 3-4 seconds after pBoc (96%, 45 out of 47 cycles, 5
animals), as well as inx-1 mutants (95%, 81 out of 85 cycles, 10 animals). In aex-2;inx-1
double mutants, only 15% showed a normal pattern (15%, 7 out of 46 cycles, 10 animals),
which is consistent with the Exp pattern of nlp-40;inx-1 (Figure 3C). However, aex-2 cDNA
rescue under DVB specific promoter in aex-2;inx-1 double mutant showed higher normal
pattern of Exp (35%, 26 out of 74 cycles, 10 animals) than rescue under AVL specific
promoter (22%, 16 out of 73 cycles, 10 animals) (Figure 13B), indicating aex-2 rescue under
DVB specific promoter has less ectopic Exp than rescue under AVL specific promoter. The
difference in the frequency of normal pattern of Exp in AVL compare with DVB indicate
that AVL suppress DVB through INX-1.
29

Figure 13. AVL negatively regulates DVB through INX-1
(A) Quantification of the number of Exp per defecation cycle in adults with indicated
genotypes. Full length aex-2 cDNA was expressed under cell specific promoters which are
AVL neuron-specific (Pflp-22(delta)) and DVB neuron-specific (Pflp-10). AVL rescue
denotes expressing aex-2 under AVL neuron-specific promoter and DVB rescue denotes
expressing aex-2 under DVB neuron-specific promoter. AVL, DVB rescue denotes co-
expression of aex-2 under both AVL and DVB promoters. (B) Classification of different
patterns of Exp in worms with the indicated genotypes. <5 sec denotes Exp occurring less
than 5 seconds after pBoc which is defined as normal pattern of Exp, and >5 sec denotes Exp
occurring more than 5 seconds after pBoc, which is defined as ectopic Exp.

30

3.9 inx-1 positively regulates potassium channels
Our genetic results suggest that inx-1 may function downstream of PKA to inhibit
calcium influx in GABAergic neurons. However, the question whether inx-1 directly inhibits
calcium influx or interacts with other effectors between of PKA and calcium channels still
remains unclear. To open the voltage-gated calcium channels such as unc-2, neuronal cells
have to undergo depolarization. Studies have shown depolarization of the membrane is
driven by voltage-gated calcium and voltage-gated potassium channels due to the lack of
voltage-gated sodium channels in C. elegans (Gao & Zhen, 2011). egl-36 encodes a Shaw-
type voltage-gated potassium channel that is expressed broadly in the nervous system
including in AVL and DVB GABAergic motor neurons (Johnstone, Wei, Butler, Salkoff, &
Thomas, 1997). egl-36(n728) gain-of-function mutants display severe expulsion defects
(35%) whereas and egl-36(n2332) loss of function mutants have wild type expulsion (Figure
14A). This suggests that constitutively opened potassium channels lead to hyperpolarization,
causing decreased excitability in GABAergic neurons. We found that constitutively active
PKA (vjIs103) failed to restore the Exp defect of egl-36(gf) (Figure 14B), confirming that
egl-36 functions downstream of PKA in the GABAergic motor neurons to inhibit their
activation. In contrast, we found that inx-1 mutations restored the Exp defect of egl-36(gf)
mutants from 35% to 63%. These results indicate that inx-1 may function either downstream
of the egl-36 potassium channel or directly regulates egl-36 activity to inhibit expulsion.
31

Figure 14. inx-1 mutation restores Exp defect of gain of function potassium channels
(A, B) Quantification of the number of Exp per defecation cycle in adults with indicated
genotypes. egl-36 is defined as loss of function mutant, egl-36(gf) is defined as gain of
function mutant, vjIs76 is used for dominant negative PKA, and vjIs103 is used for
constitutively active PKA. Means and standard errors are shown. Asterisks indicate
significant differences: * P<0.05 in Student’s t-test.
32

Chapter 4
Discussion
Emerging evidence from previous mammalian studies have suggested that gap
junctions are involved in neuronal diseases including brain development, neuropathies,
epilepsy, brain trauma, and that gap junctions can play a role in synchronization and
oscillation of neurons, perhaps crucial for formation and consolidation of memory (Dere &
Zlomuzica, 2012). We have shown that 1) inx-1 mutations lead to ectopic calcium influx in
GABAergic neurons that occurs at random times during the cycle, 2) inx-1 is required in both
DVB and AVL motor neurons, and 3) inx-1 is localized to synapses. Together, these results
suggest that inx-1 may play role in synchronization and oscillation between AVL and DVB
motor neurons by inhibiting generation of calcium spikes in these neurons. However, several
questions remain regarding the mechanism by which inx-1 regulates calcium influx.
Does inx-1 directly regulate potassium channels or other effectors?
Although inx-1 mutations trigger calcium influx in the GABAergic neurons, it is
unclear whether inx-1 directly regulates calcium channels, or whether regulates calcium entry
via a less direct mechanism, such as by regulating the activity of potassium channels. Gain of
function egl-36(gf) mutants show the Exp defect, while the loss of function egl-36 mutants
show normal Exp frequencies (Johnstone et al., 1997). This suggests a redundancy of
potassium channels in the GABAergic neurons. We show that inx-1 mutants restore the Exp
defect of egl-36(gf) mutants. The result suggests that inx-1 can positively regulate potassium
channels, possibly by regulating the expression, localization, stability, or gating of the egl-36
channels themselves.
33

Murine studies have shown that Panx1 which is a member of the vertebrate gap
junction family, interacts with potassium channels to attenuate changes in redox potentials
(Bunse et al., 2009). Thus, inx-1 may interact with potassium channels directly to attenuate
calcium influx in GABAergic motor neurons to inhibit its activation. To further demonstrate
whether inx-1 directly interacts with potassium channels, we will need to utilize fluorescence
imaging to examine the localization of EGL-36::GFP fusion protein in wild type background
and in inx-1 mutant background. We speculate that if inx-1 regulates localization or stability
of potassium channels, we should observe changes in the localization of EGL-36::GFP fusion
protein at the NMJ inx-1 mutant background when compared to wild type controls.
Alternatively, inx-1 may function downstream of egl-36 but upstream of voltage-gated
calcium channels. In this case, we expect inx-1 mutations to have little impact on the
expression or localization of EGL-36::GFP.
Does inx-1 form electrical coupling between AVL and DVB?
Vertebrate studies in the CNS and retinal neurons have shown that gap junctions
mediate electrical coupling between neurons. In retinal neurons, gap junctions are formed in
amacrine, rod, and cone cells to transfer electrical currents carried by ions for day and night
vision (Sohl et al., 2005). Particularly, connexin 36 (Cx36) has been reported to be expressed
broadly in these cells. Therefore, inx-1 may transfer electrical impulses to regulate
GABAergic neuron activation. To test this idea, electrophysiology in AVL and DVB neurons
can be utilized. Also, phenotypic rescue using mammalian DNA can be applied. For
example, if we see a rescue of the nlp-40;inx-1 phenotype to the nlp-40 phenotype when
Cx36 is expressed in nlp-40;inx-1 double mutants, we can speculate that inx-1 and Cx36 have
a similar function. However, to express a mammalian DNA in C. elegans, first one needs to
34

identify the motif necessary and sufficient for transport of protein to the synaptic region as
inx-1. In this study, we have generated various truncated INX-1 proteins to find the
functional motif and have found a region that is necessary for transportation to synapses
(Figure 9).
Does inx-1 allow metabolites and small molecules to pass through?
Gap junctions are also known to enable the passage of metabolites and second
messengers such as calcium, cAMP, and IP3 (Zoidl & Dermietzel, 2010). Studies have
shown that calcium can activate calcineurin, which is a phosphatase that can dephosphorylate
potassium channels inducing hyperpolarization that causes inhibition of neuronal excitability
(Park, Mohapatra, Misonou, & Trimmer, 2006). C. elegans encoded a single calcineurin
homolog, tax-6. Interestingly, constitutive activation of tax-6 results in a severe defect in Exp
(Lee, Song, Jee, Vanoaica, & Ahnn, 2005). This defect can be explained in part by a defect in
the activation of enteric muscles. However, our preliminary results have shown that
expression of tax-6(gf) transgene specifically in AVL and DVB neurons leads to expulsion
defects, suggesting a function for calcineurin in negatively regulating AVL and DVB
activation. Thus, tax-6/calcineurin is a candidate for regulation by inx-1. In this model, inx-1
would positively regulate tax-6/calcineurin, which in turn could positively regulate egl-
36/potassium channel. Thus, calcium may pass through inx-1 to regulate calcineurin thereby,
activating potassium channels which lead to hyperpolarization causing inhibition of calcium
influx in GABAergic neurons. To confirm such a mechanism of regulation and calcium
influx, a comprehensive genetic analysis of interactions between calcineurin, PKA,
potassium channels, and inx-1 will be conducted. Furthermore, expressing the genetically
encoded calcium indicator GCaMP under AVL- and DVB-specific promotors can help to
35

demonstrate how inx-1 might impact calcium wave propagation between AVL and DVB
motor neurons.
36

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Archiv - European Journal of Physiology, 460(2), 451-466.

Abstract (if available)

Abstract Gap junctions are specialized connections between cells that regulate the intercellular transfer of chemical and electrical signals. Gap junctions play important roles during development and in the functions of mature tissues such as neurons and muscles. Gap junctions are composed of two hemi-channels, each made up of hexamers of connexin subunits (or innexin subunits in invertebrates). There are over two dozen connexin (and over one dozen innexin) family members that can combine to form either homo- or hetero hemi-channels, and the composition of the hemichannels is thought to contribute to the structural and functional diversity of gap junctions. Here, we identify a novel role for a putative gap junction protein, inx-1/innexin in negatively regulating the contraction of enteric muscles during a pacemaker-controlled motor program in C. elegans. inx-1 mutants restore enteric muscle contraction and presynaptic calcium influx to animals with defects in pacemaker-induced activation of the enteric muscle neuromuscular junction. Our genetic analysis reveals that that inx-1 functions in the motor neurons that innervate the enteric muscles and inx-1 regulates calcium influx at presynaptic terminals possibly by regulating the activity of presynaptic potassium channels. We find that the INX-1 protein is concentrated at presynaptic terminals where it may form gap junctions that connect two motor neurons that control the enteric muscles contraction. We propose that INX-1 functions to negatively regulate motor neuron activation by inhibiting the generation of rhythmic calcium spikes in motor axons at this synapse.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Choi, Ukjin (author)

Core Title inx-1 is a negative regulator of the expulsion step of the defecation motor program in C. elegans

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 01/15/2017

Defense Date 06/09/2016

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Exp,GABAergic motor neurons,gap junction,inx-1,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Tokes, Zoltan (committee chair), Patel, Pragna I. (committee member), Sieburth, Derek (committee member)

Creator Email exnbac@gmail.com,ukjincho@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-268012

Unique identifier UC11279479

Identifier etd-ChoiUkjin-4553.pdf (filename),usctheses-c40-268012 (legacy record id)

Legacy Identifier etd-ChoiUkjin-4553.pdf

Dmrecord 268012

Document Type Thesis

Format application/pdf (imt)

Rights Choi, Ukjin

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA