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Neurogenic placodes provide migratory enteric sensory neural progenitors in response to endothelin signaling pathway

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Neurogenic placodes provide migratory enteric sensory neural progenitors in response to endothelin signaling pathway

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NEUROGENIC PLACODES PROVIDE MIGRATORY
ENTERIC SENSORY NEURAL PROGENITORS IN
RESPONSE TO ENDOTHELIN SIGNALING PATHWAY
By
Paul Chung

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 2013

Paul Chung

i

Acknowledgements

I would like to express my deepest appreciation to all those who provided me the
possibility to complete this report. A special gratitude I would give to is on my
committee chair Dr. Zoltan Tokes, whose contribution in stimulating suggestions and
encouragement helped me organize my project. Without his guidance and persistent help,
this thesis would not have been possible. I would also like to acknowledge with much
appreciation to Dr. Takako Makita who gave me the golden opportunity to do this
wonderful project on the topic, which also helped me in doing a lot of research and I
came to know about so many new things I am really excited in. Furthermore, I would like
to thank to Dr. Ruchi Bajpai for providing me with a lot of valuable insights and critical
scientific feedback towards this present work and career path for the future.

Finally, I would like to thank to my parents, Kangmin Chung and Suncho Choi, to my
sister Susan and my brother Mathew, and to all of my friends who supported and
encouraged me every step of the way. And I would like to thank my best friend, Ja Yeon
Kim for her endless support and help throughout my graduate studies.

ii

Abstract

The enteric nervous system (ENS) is the neuronal regulatory network, which maintains
homeostasis and peristalsis of the gastrointestinal (GI) tract. Abnormal development of
ENS may cause the congenital aganglionic megacolon phenotype as Hirschsprung’s
disease (HSCR). Previous studies showed that enteric neural crest cells migrate to and
colonize the GI tract for the development of ENS that is dependent on the endothelin
signaling pathway. However, the contribution of non-neural crest cells to ENS is largely
unknown. Since the X
th
cranial nerve (CNX), where the enteric neural crest cells emigrate
from, contains epibranchial placode-derived cells as well, it was suggested that
epibranchial placode provides another neurogenic cell population for ENS development.
Interestingly, the visualization of placode-derived cells in Pax2cre/R26R
LacZ
mice
uncovered that migratory placodal cells reached the distal colon by E12.5. In addition, the
ablation of endothelin signaling in placodal cells, using conditional knockout mouse
model (Pax2cre/ET
B
), caused aberrant formation of myenteric ganglions and disrupted
sensory neuronal circuit. All together, these new findings suggest that migratory
neurogenic placode provides enteric neural precursors in the endothelin signaling-
dependent manner, and placode-derived enteric sensory neurons are essential elements
for proper ENS establishment. These observations provide the insights to the
developmental mechanism underlying ENS, and to the therapeutic approaches for HSCR.

iii

List of Figures
Figure 1. Enteric Neural Crest Cells for ENS
Figure 2. Enteric Neurons of Plexus from Neurogenic Placodes
Figure 3. Dual Origin of Enteric Nervous System
Figure 4. Abnormal organization of ENS in placode-specific ET
B

mutants
Figure 5. HSCR disease phenotype in neural crest- and placode-specific
ET
B
mutants
Figure 6. Abnormal sensory neural network in placode-specific ET
B

mutants
Figure 7. Distinctive delamination of neurogenic placode
Figure 8. Expression of RhoB and Snail2 for delamination processes in
Neural Crest and Placode
Figure 9. Modulation of the migratory process of neural crest and
placode by endothelin signaling
Figure 10. Activities of PKA and Rac1 regulated by GDNF and ET3
Figure 11. Inhibitory function of endothelin signaling on neural/ glial
differentiation
Figure 12. Schematic summary of ENS development from dual origin,
neural crest and placode in response to endothelin signaling
pathway

4
11
13
15

17

18

19
21

23

24
26

27

iv

Figure 13. Knockdown of Robo2 or Slit1 causes abnormal
trigeminal ganglion formation

28

v

Table of Contents
Acknowledgements
Abstract
List of Figures
Chapter. 1 Introduction
1.1 Enteric Nervous System for Normal Gut Physiology
1.2 Cellular Origins for ENS Development
1.2.1 Enteric Neural Crest Cells
1.2.2 Neurogenic Placodes
1.3 Endothelin Signaling Pathway in ENS Development
Chapter. 2 Materials and Methods
2.1 Mice
2.2 X-gal Staining and Immunohistochemistry
2.3 in Situ Hybridization
2.4 FRET Time-lapse Imaging
Chapter. 3 Results
3.1 Neurogenic Placodes as a Novel Cellular Origin of ENS
3.2 Developmental Processes of Neurogenic Placodes for ENS
3.2.1 Delamination of Neurogenic Placodes for ENS
3.2.2 Migration of Neurogenic Placodes for ENS

i
ii
iii
1
1
3
3
5
6
8
8
8
9
10
11
11
18
18
21

vi

3.3 The role of endothelin signaling pathway in migration and neural
differentiation of enteric progenitors
Chapter. 4 Discussion
4.1 Discussion
4.2 Future Opportunities
References

24

27
27
32
34

1

Chapter 1
Introduction
1.1 Enteric nervous system for normal gut physiology
During the early developmental process, the peripheral nervous system (PNS) establishes
neural networks for proper communication between the central nervous system (CNS)
and peripheral tissues. As one of the most complex neural networks in the periphery, the
enteric nervous system (ENS) plays multiple roles in regulating peristalsis and
maintaining homeostasis by modulation of muscle contraction, nutrient absorption, fluid
exchange, and immune response inside the gut (1-3). For decades of developmental
studies of ENS, it has been reported that enteric neural crest cells provide the migratory
neurogenic progenitors of enteric neurons, and ablation of neural crest induces the
aganglionosis in the GI tract (4). Interestingly, however, there are growing evidences that
subsets of enteric neurons are derived from non-neural crest origin, neurogenic placodes,
which supports the idea of multiple origins for ENS.

Enteric nervous system consists of extrinsic nerve fibers, which innervate into the
gastrointestinal tract from outside, and intrinsic enteric neurons, which form the
ganglionic web-like network of various enteric neurons and glia. The enteric ganglia are
interconnected to convey the information through the myenteric plexus, which is placed
in between the circular and longitudinal smooth muscle layers, and the submucosal
plexus, which is located inside the submucosa layer. Both myenteric plexus and

2

submucosal plexus form the neuro-functional unit to deliver mechano- and chemo-
sensation from the gut lumen and to initiate the contractile movement of smooth muscle
for peristaltic activity (5, 6). The axons of enteric sensory neurons reach enteroendocrine
(EE) cells and enterochromaffin (EC) cells, which function as signal transducing cells, at
the gut epithelium of the lumen. Afferent enteric sensory neurons have synaptic
connectivity to the interneurons, which transmit the sensational information to efferent
excitatory- or inhibitory- enteric motor neurons, innervated into the smooth muscle layer
to induce peristaltic muscle contraction.
In order to control physiological functions of the gut, each neuronal plexus consists of
specific neuronal subsets and glial cells that are distinguishable by molecular markers.
For example, enteric sensory neurons express calcitonin gene-related protein (CGRP) as a
sensory neurotransmitter, sympathetic motor neurons/ interneurons express dopaminergic
neuronal marker, tyrosine hydroxylase (TH), and parasympathetic motor neurons/
interneurons express cholinergic neuronal markers, choline acetyltransferase (ChAT) or
acetylcholine esterase (AChE). Additionally, glial fibrillary acidic protein (GFAP) is the
marker for identification of glial cells (7). These cell fates of enteric neurons are
determined by neural differentiation process of enteric progenitors during development of
ENS.
The defects in the development of ENS by the lack of migration or differentiation of
enteric neural progenitors may cause the absence of the ganglions in certain regions of
the gut, which leads to Hirschsprung’s disease (HSCR) (8). Since the functional unit of
ENS is absent in the affected segment, loss of peristalsis causes accumulation of fecal

3

contents in large intestine and congenital megacolon phenotype. Clinical studies on
HSCR revealed that the majority of HSCR patients are sporadic ‘short-segment HSCR’
cases, however, HSCR patients can also be familial cases (8, 9). In familial cases, HSCR
shows non-Mendelian inheritance, which is affected by complex genetic and
environmental factors, leading to variable penetrance. As one of the congenital gut
motility disorders, HSCR has been reported to occur in approximately 1 in 5000 infants.
According to the HSCR mouse model studies, HSCR-developing mice died in 3-4 weeks.
However, even though significantly large numbers of patients are suffered from the
HSCR disease, and there are potential life-threatening risks, there is no currently
available cure for the disease other than physical excision of the affected gut segment.
Moreover, the long-term consequences of HSCR, or repeated surgeries, may generate
lifelong gastrointestinal problems, including constipation and enterocolitis, and other
complications. Therefore, there are continuous efforts to elucidate the mechanism
underlying HSCR disease for alternative therapies.

1.2 Cellular origins for ENS development
1.2.1 Enteric neural crest cells
The majority of neurons in the ENS are derived from neural crest cells that undergo
epithelial-to-mesenchymal transition (EMT) and delaminate from neural tube (10). As the
migratory population of enteric neural crest-derived cells (ENCCs) that become the
enteric progenitors, vagal neural crest cells from X
th
cranial nerve region enter the foregut
around E9 in mice, and migrate in the rostral to caudal direction until they reach distal

4

colon region approximately by E15 (Fig. 1). On the other hand, sacral neural crest cells
provide a minor population of ENS progenitors, which enter the hindgut at the later stage
around E13.5 and migrate in the caudal to rostral direction to colonize up to the proximal
colon boundary (11). A previous study has demonstrated that enteric neurons were absent
from the esophagus, stomach, small and large intestine if neural crest cells were ablated
from post-otic hindbrain of the chick (12). In addition, previous studies have shown that
the functional processes among ENCCs including cell clustering, proliferation, and
extending strand-like migration are critical for the migratory enteric progenitors to
advance the wave-front throughout the developing gut (13, 14). To address how enteric
neurons are differentiated out of the neural crest progenitors, it has been shown that

5

neurogenesis and gliogenesis occur predominantly behind the ENCC wave-front,
detected by pan-neuronal markers (neurofilament M or Hu) and glial markers (S100b)
(15). Therefore, these results support the idea that neural crest cells provide the migratory
enteric progenitors that are essential for enteric neurons and glia differentiation.

1.2.2 Neurogenic placodes
Placode is the transient ectodermal thickening, which gives rise to sensory neurons for
craniofacial region and sensory organs of the head. The developmental process of
placode is initiated after gastrulation, when a pre-placodal field of naive ectoderm is
established at the border between the future epidermis and the anterior neural plate/
neural crest forming regions. Then, it undergoes further specification process so that it
forms lens, otic, olfactory, and epibranchial placodes (16, 17). It has been also reported
that each specified placode is essential for normal development of surrounding structure
(17). Especially, epibranchial placode resides in the proximity of vagal X
th
cranial
ganglia, which suggests that vagal placode cells are derived from epibranchial placode.
Notably, both neural crest and placode are developmentally originated from the close
vicinity in between non-neural ectoderm and the neural plate. In addition, previous
studies have indicated that the communication between neural crest and placode is
required for proper development of neural organization. Even though they share
important characteristics in terms of neurogenesis, it has been reported that neural crest
cells possess the migratory capability along the gut during embryonic developmental
process (13), whereas placodal cells have never been appreciated whether they are

6

migratory population as well. Based on the current understanding, the migratory neural
crest cell is the developmental origin for enteric nervous system of the gut. However,
there are cumulative evidences including the new findings in this project that show that
enteric neurons are derived from multiple origins, and the neurogenic placode provides
long-range migratory neuronal progenitor cell population for enteric nervous system (28).

1.3 Endothelin signaling pathway, required for ENS development
Endothelin signaling pathway has been studied for decades in various biological contexts,
including cardiovascular, renal, retina, and neuronal system (18, 19, 20, 21). Depending
on the context, the interaction between three ligands (ET-1, -2, -3) and two receptors
(ET-A, -B receptor) mediate the intracellular signaling cascade. Endothelins are
expressed as inactive precursors, and two endothelin converting enzymes (Ece-1, -2) are
involved in proteolytic processing of endothelins. Activated endothelin ligands bind to
their preferred receptors that are G-protein coupled receptors. There are two principal
signaling transduction pathways involving the GPCRs: the cAMP signaling pathway and
the phosphatidylinositol-signaling pathway. The G-protein subunits activate downstream
molecules, including adenylyl cyclase and phospholipase C to modulate mitogen-
activated protein kinases (MAPKs) signaling cascade as well as intracellular calcium
concentration to trigger target gene expression.
The endothelin-3 ligand (ET3) and endothelin receptor-B (ET
B
) constitute the essential
signaling pathway for ENS development. ET
B
is expressed in the migrating vagal and
sacral ENCCs, whereas ET3 is expressed in the midgut, hindgut, cecum and proximal

7

colon mesenchyme, as ENCCs migrate and colonize the entire gut. The ET
B
gene
ablation studies have shown that ET3-ET
B
signaling regulates the migration of ENCCs
along the developing gut (22, 23).
Patients with HSCR have been identified to carry heterozygous mutations in ET3, ET
B
or
the endothelin converting enzyme-1 (Ece1), and these patients comprises approximately
5% of human HSCR cases (8). In addition, mice carrying genetic mutations in ET3, ET
B
,
or Ece1 exhibit the consistent disease phenotype, aganglionosis in the GI tract and
pigmentation defects. Even though apparently small portions of human HSCR cases are
familial cases, and possess the mutations in endothelin-related genes, it is worthwhile to
note that heterozygous mutations in ET3, ET
B
or Ece1 are associated with autosomal
recessive HSCR disease. Taken together, endothelin signaling provides the key
mechanism in the development of enteric nervous system.

It has been demonstrated that ET3-ET
B
signaling is required for the migration and
colonization processes of enteric neural crest cells along the gut (20). However, the
contribution of non-neural crest cells to the ENS in response to endothelin signaling
pathway has never been characterized. Here, it is discussed that epibranchial placode may
serve another neurogenic source for ENS development, and ET3-ET
B
signaling is also
required for migration and neural differentiation, through downstream effector molecules,
in enteric neural crest cells and placodal enteric progenitors, as dual neurogenic origins
for ENS.

8

Chapter 2
Materials and Methods
2.1 Mice
Pax2-Cre and Wnt1-Cre transgenic line were used to trace the lineage of placode and
neural crest, respectively (24, 25). For lineage analysis, mice carrying Cre reporter allele
R26R
LacZ
were incorporated into crosses to generate Pax2Cre/R26R
LacZ
and
Wnt1Cre/R26R
LacZ
mice. ET
B
conditional knockout allele was used for tissue-specific
deletion (ET
B
flox/-
for placode and ET
B
flox/flox
for neural crest). Mouse lines were interbred
to generate Pax2Cre/ET
B
and Wnt1Cre/ET
B
(without Cre reporter allele) or
Pax2Cre/ET
B
/R26R
LacZ
and Wnt1Cre/ET
B
/R26R
LacZ
(with Cre reporter allele), the
lineage-specific mutant embryos or mice and their littermate controls. All mouse lines are
maintained in accordance with protocols approved by the Saban Animal Care Facility at
the Childrens Hospital Los Angeles. This facility is accredited by AAALAC and
conforms to the PHS guidelines for animal care.

2.2 X-gal staining and immunohistochemistry
The guts, isolated from the mice of Pax2Cre/R26R
LacZ
and Wnt1Cre/R26R
LacZ
to
visualize each lineage specific domain, were treated with X-gal substrate for the β-
galactosidase enzymatic reaction. The isolated guts from the mice of
Pax2Cre/ET
B
/R26R
LacZ
and Wnt1Cre/ET
B
/R26R
LacZ
were also used to visualize the
affected region by X-gal staining.

9

For the wholemount staining on the embryonic or postnatal guts, the isolated guts were
bleached overnight after fixation and blocked for 1 hour. Then, tissues were incubated in
primary antibodies for 3 days at 4°C, washed in PBS with 1% Tween-20, incubated with
secondary antibodies conjugated with the horseradish peroxidase (HRP) for 3 days at 4°C.
The substrate for HRP, Diaminobenzidine (DAB, from Sigma, St. Louis, MO), was
treated for color development reaction.
For the immunofluorescence staining on the frozen section of the embryonic or postnatal
guts, sections of the fixed guts on the slides were treated with hydrogen peroxide to
quench the endogenous peroxidases. After blocking, the sections were incubated with the
primary antibodies at 4°C overnight, washed three times in PBS with 0.1% Tween-20,
incubated with secondary antibodies conjugated with the fluorophore for 3 hours at room
temperature. Dilution of antibodies was used as following; CGRP (1:500, Abcam,
Cambridge, MA), Tuj-1 (1:1000, Sigma, St. Louis, MO).

2.3 in situ hybridization
To detect ET3 and ET
B
gene expression on the wholemount gut tissue and tissue section
slides, complementary RNA oligos are designed and currently available as described (26).
After post-fixation with 4% PFA, cRNA oligo probes (400ng/ul) were incubated with the
samples at 65’C overnight, and then washed out with SSC solution. Next, samples were
incubated with anti-DIG-HRP antibody for three hours at room temperature. DAB color
development reaction was followed to visualize the amount and location of ET3 and ET
B

expression.

10

2.4 Fluorescence Resonance Energy Transfer (FRET) time-lapse imaging
Transgenic mice expressing a PKA biosensor AKAR3EV, a negative control FRET
biosensor AKAR3EV-NC, FRET biosensor for Rac1, EaichuEV-Rac1, were prepared as
described (27). FRET imaging of ENCCs was taken by Two-photon excitation
microscopy and confocal laser scanning microscopy as described (27).
For confocal time-lapse imaging, the imaging core facility has a fluorescent microscope
equipped with an incubation chamber with a CO
2
supply and temperature control. A
time-lapse sequence was obtained for up to 30min to analyze the speed and directionality
of ENCCs on the guts and the activities of PKA and Rac1 in response to GDNF and ET-3
as described (27).

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Chapter 3
Results
3.1 Neurogenic Placodes as a Novel Cellular Origin of ENS
The discussion that ENS is originated from neurogenic placodes was initiated in early 90s.
A previous study has shown that neural cells reside in the enteric plexus are generated
from placodal lineage in the insect model system (28). By the traditional lineage tracing
method, injection of fluorescence dye into individual cells of developing ENS, they have
found that enteric
neurons of each
plexus are largely
derived from
neurogenic
placodes (Fig.2). In
addition, according
to their findings,
post-mitotic
placodal cells leave
the epithelium
while their final
differentiation is

12

delayed until they reach the gut. These series of observations provided the insights of
generation of pre-migratory enteric neurons from the epithelial placode.

Intensive studies on the contribution of neural crest to the ENS have been performed for
several decades, whereas studies on the neurogenic placode for ENS development have
been rarely conducted due to the difficulties of placodal lineage specification and
limitations in the resolution of lineage-specific markers.
In 2004, Pax2Cre mice have been generated, and it has been defined that the otic placode
develops from the ectodermal field, characterized by the expression of the transcriptional
factor, Pax2 (24). Interestingly, based on the preliminary observations from Dr. Makita’s
lab, expression of Pax2 is not only limited to the otic placode, but also activated in the
region of other placodes, including epibranchial placode near the X
th
cranial nerve (CNX,
also known as the vagus nerve). To rule out the possibility that Pax2cre may be also
activated in the neural crest region, X-gal staining profile of the whole embryo was
assessed. It turned out that neural crest-derived regions, including the dorsal root
ganglion, were not stained, which suggest that the activity of Pax2 is localized in
placode-derived region (data not shown).

The cell bodies of vagus nerve fibers form a ganglion, which may be originated from
multiple neurogenic lineages, vagal neural crest cells and epibranchial placodes (Fig. 3a).
Although it is still unclear how enteric neural progenitors are delaminated from CNX, it

13

has been reported that they enter the foregut at E9.5 and migrate along the entire GI tract
by E13.5, where they contribute to the intrinsic neurons of ENS, which form myenteric
plexus and submucosal plexus (Fig.3b). It has been suggested that enteric neural crest
cells from vagal and sacral region is responsible for colonization of the whole GI tract.
However, it is worthwhile to note that, based on the lineage-tracing observations using
Pax2Cre mice from Dr. Makita’s lab, neural crest derived neurons (brown only) and

14

placode-derived neurons (blue and brown) together form CNX by E10.5 (Fig.3c), and
also placode-derived cells advance along the gut and differentiate into neurons in the
myenteric plexus (Fig.3d), which suggests that not only enteric neural crest cells but also
placodal enteric neurons indeed contribute to the ENS.
Previously, the positioning of placode-derived neurons in the myenteric plexus was
visualized (Fig 3d). Next, it is questioned whether enteric progenitors derived from
placode migrate along the gut in response to ET3-ET
B
signaling, the same as those from
neural crest does. Since it has been reported that ET3-ET
B
signaling pathway induces the
migration of vagal neural crest cells, enteric neural progenitors from the placodes may
share the same mechanism of endothelin signaling pathway for migration and
colonization process along the GI tract. To test this possibility, conditional knockout mice
model was introduced. Compared to the guts isolated from ET
B
wild-type control mice,
placode-specific ET
B
mutant guts from Pax2cre/ET
B
mice and neural crest-specific ET
B

mutant guts from Wnt1cre/ET
B
mice show defective ENS establishment, as visualized by
wholemount Tuj1 immunostaining (Fig.4a-c). The guts isolated from ET
B
conventional
knockout mice were aganglionic as expected, and only extrinsic nerve fibers were present
(Fig.4d). Based on these observations, placode-specific ET
B
mutation causes defective
ENS network, which suggests that indeed enteric neurons derived from placodal lineage
contribute to the ENS organization. Neural crest-specific ET
B
mutation also induces
significantly decreased amount of ganglions and the number of neurons of each ganglion.
Interestingly, even though enteric neural crest cells are not present in the distal colon
region of Wnt1cre/ET
B
mice, Tuj1
+
ganglions are observed, which means

15

that those neurons are likely originated from other than neural crest, the placodal lineage.
Taken together, even though affected phenotypes are not identical in Wnt1cre/ET
B
and
Pax2cre/ET
B
mice, both of them show defective ENS, which may suggest that neural
crest-derived and placode-derived enteric neurons take effects in an additive manner.

These observations may be explained as two different neurogenic lineages, neural crest
and placode are responsible for distinct neuronal subsets. Since the supporting evidences
have shown that neurogenic placodes in the craniofacial region exclusively give rise to
sensory neuronal population (16), the enteric neurons derived from the placode may
contribute to the sensory neuronal subsets of the ENS. As previously described, CGRP,

16

the sensory neuronal marker, was used to visualize enteric sensory neurons and their
axons in the distal colon from adult mice before they died, P30 for placode-specific
mutant, and P21 for neural crest-specific mutant (Fig.4e-g). Indeed, CGRP
+
neurons were
entirely absent within the myenteric plexus as well as their axonal projection in the
mucosa of the Pax2Cre/ET
B
distal colon (Fig.4e). In contrast, CGRP
+
neurons were
completely normal in the Wnt1Cre/ET
B
distal colon (Fig.4g). These results suggest that
neurogenic placodes may contribute to the enteric sensory neurons, which supports the
extended idea that lack of enteric sensory neurons from the placode lineage may cause
the disrupted establishment of ENS, which possibly results in the functional defects
including abnormal peristaltic activity.
To address the question whether the absence of enteric neurons from placodal lineage
may cause the abnormal peristaltic activity, mice at the survival limit of each lineage-
tracing line have been examined to monitor the pathological phenotype and their body
weights (Fig.5). As it is expected, compared to the control, Wnt1Cre/ET
B
mice had the
affected gut similar to HSCR disease phenotype, filled up with the fecal contents due to
the lack of peristalsis. Their growth was severely retarded, and their body weights were
significantly lower than normal mice at P21 (Fig.5a, c, e).
Consistent with the previous findings, Pax2Cre/ET
B
mice had the obvious megacolon
phenotype compared to the control, even though it is not as severe as Wnt1Cre/ET
B
mice.
However, the outcome from the malfunction of gut was almost equally severe to
Wnt1Cre/ET
B
based on the body weights measurement at P30 (Fig.5b, d, f). These

17

observations collectively imply that the absence of placode-derived neurons is enough to
induce malfunction of the gut.
Since there is a possibility that the lack of enteric sensory neurons is not caused by the
migration defect but the neurodegenerative process of the non-functional neurons when
they reach the time point of survival limit, the observation of enteric sensory neurons was
extended to the guts isolated from P0, neonates. In consistent with the previous
observation, placode-specific ET
B
mutant gut shows absence of CGRP
+
neuronal cell

18

bodies and their axons (Fig.6d-f) compared to the wild type control (Fig.6a-c), whereas
neural crest-specific ET
B
mutant gut shows relatively normal enteric sensory neuronal
network (data not shown). Collectively, these results strongly support that placode as an
unsuspected neurogenic origin for ENS contributes to the functional units of the sensory
neuronal circuit in the distal colon region.

3.2 Developmental Processes of Neurogenic Placodes for ENS
3.2.1 Delamination of Neurogenic Placodes for ENS development
Delamination process of both neural crest and neurogenic placode is required for
migratory neuronal precursor to leave the epithelium. Neural crest cells delaminate from

19

the neural tube in a ventral-to-dorsal direction toward X
th
cranial nerve (CNX), whereas
the epibranchial placodes delaminate from the transient ectodermal thickening of
pharyngeal arches in a dorsal-to-ventral direction. Although, it has been shown that
neurogenic placode undergoes delamination process, the detail mechanism underlying
how it delaminates is not clearly described yet (26).
As shown in st17 chick embryo (Fig.7), the placodal neuroblast (green) from ectodermal
thickening penetrates through a break in basal lamina for delamination process (Fig. 7a,
e). Interestingly, when it buds out of the basal lamina, the cell membrane becomes

20

disorganized to allow the neuroblast to exit, while non-placodal ectoderm maintains its
integrity as shown in the staining of β-catenin (magenta), which is localized on the cell
membrane (Fig. 7b, c). Similarly, the staining of laminin (magenta) shows the
discontinuous cellular boarder-line, where the placodal neuroblast escapes (Fig. 7f, g, h).
These observations collectively suggest that neurogenic placode forms the neuroblast and
delaminates from the thickened ectodermal barrier in st17 chick embryo, which
potentially develops into enteric neural progenitors as it reaches the foregut region.

Previously, it has been discovered that neural crest cells undergo epithelial-to-
mesenchymal transition (EMT) during delamination process (29). Expression of EMT-
regulating genes, such as Snail family zinc-finger transcription factors and Rho
superfamily of GTPases are involved delamination process of neural crest cells. However,
there is limited knowledge of what is involved in the delamination process of neurogenic
placodes out of the epithelium. Even though the neural crest and the placode share
multiple neurogenic characteristics, a previous study has proposed that delamination
process of the placode is independent on EMT (26). Based on the in situ hybridization
assay, high level of RhoB expression was visualized in the neural crest region of the
hindbrain and the trunk (Fig.8a, c). On the contrary, expression of RhoB was barely
detected in the placodal ectoderm region, even though placode actively delaminated at
st17-18 in chicken (Fig.8b).

21

Consistent with RhoB expression, Snail2 expression was visualized in the delaminating
neural crest (Fig.8d, f), whereas it was absent in the placodal ectoderm (Fig. 8e). All
together, EMT is not required for delamination process of neurogenic placodes.

3.2.2 Migration of Neurogenic Placodes for ENS development
Migration of enteric neural crest cells is one of the key features for ENS development. As
described before, neural progenitors from enteric neural crest cells advance along the gut,
and start the neural differentiation process to generate enteric neurons for normal gut

22

function. If the neurogenic placode provides enteric neural precursors, migration of those
precursor cells is the critical process to be addressed. As described above, it has been
studied that ET3-ET
B
signaling is involved in the migratory process. Thus, it is
questioned whether ET3-ET
B
signaling regulates the migration process of the enteric
progenitors from both lineages of neural crest and neurogenic placode. A previous study
has visualized the migratory enteric progenitors with taking advantages of the mouse
model, ET
B
LacZ
mice (23). Since the mutation in ET
B
presumably affects the enteric
progenitors from both neural crest and placode lineages, if endothelin signaling pathway
is not irrelevant to the migration process of either one of those lineages, at least some
extent of normal migratory pattern from one lineage should be observed. Compared to the
gut from ET
B
LacZ/+
control, the gut from ET
B
LacZ/LacZ
mouse showed delayed migration of
enteric progenitors at E11.5 (Fig.9a) and halted migration before cecum at E12.5 (Fig.9b).
Therefore, these observations support the idea that ET3-ET
B
signaling is involved in the
migration process of enteric neural crest cells and placodal enteric progenitors.
Since Pax2 is exclusively activated in the placodal lineage cells, the mouse model with
lineage tracer Pax2Cre/R26R
LacZ
has been used to visualize placodes-derived cell
population by X-gal staining. The migration profile has been examined on the isolated
gut from Pax2Cre/R26R
LacZ
mouse embryos at E11.5 and E12.5 (Fig.9c, d). At the wave-
front of the X-gal staining, extending strand-like structure (white arrows) indicates that
placode-derived cell population just passed the cecum region at E11.5, and it approaches
to the border of proximal colon and distal colon at E12.5. For the further analysis,
Pax2Cre/ET
B
/R26R
LacZ
mouse embryos have been used to address whether the ablation

23

of ET3-ET
B
signaling affects placodal cell migration. As expected, ET
B
mutation causes
the defective migration, so that extending strands have reached the cecum region, but
could not proceed toward the proximal colon (Fig.9e). As a result, placodal enteric
progenitors with abnormal ET
B
receptor expression fail to colonize the distal colon. Thus,

24

these observations suggest that both placode-derived cells and neural crest cells share the
ET3-ET
B
signaling for their migration process.

3.3 The role of endothelin signaling pathway in migration and neural differentiation
of enteric progenitors
Based on the previous genetic studies, it has been discovered that migration of enteric
neural crest-derived cells and establishment of ENS are governed predominantly by
GDNF-RET signaling and ET3-ET
B
signaling (30, 31). However, it is unclear that how
these signaling pathways modulate downstream molecules to initiate migration and

25

neural differentiation. Recently, one possibility has been discovered that both GDNF-
RET and ET3-ET
B
signaling regulate downstream Protein Kinase A (PKA) and Rac1in
the reciprocal way (27). They have used time-lapse imaging of fluorescence resonance
energy transfer (FRET) approach to visualize the activity of PKA and Rac1. According to
the intensity measurement of the energy transfer, GNDF-RET signaling activates Rac1,
and inhibits PKA, whereas ET3-ET
B
signaling activates PKA, and inhibits Rac1
(Fig.10a). They have recorded the time-lapse imaging up to 30 minutes, and the changes
in the PKA and Rac1 activities are quantified in response to GDNF-RET signaling and
ET3-ET
B
signaling (Fig.10b, c). Including these observations, they have found that PKA
is less activated in the fast-migrating neural crest cells than the slow-migrating neural
crest cells, and PKA activity down-regulates the activity of Rac1 to reduce the mobility
of neural crest cells. Since Rac1 is the member of Rho GTPase family, which is the
critical molecules for cytoskeletal dynamics, PKA may modulate the cytoskeletal
structure of migratory enteric progenitor cells from both neural crest and the placode.

One of the key questions would be how the cell fate of enteric progenitors is regulated
between cell proliferation and neural differentiation. When they reach the foregut, enteric
progenitors may undergo intensive proliferation to generate enough number of enteric
neurons, while the wavefront of migratory progenitors proceed rostral to caudal direction.
At the same time, those enteric progenitors left behind should initiate the neural
differentiation process to colonize the gut. A previous research has shown that ET3 has

26

the inhibitory effects on the neural differentiation of multi-lineage ENS progenitors (32).
It has been reported that, if the ET3-ET
B
signaling is present, the neural differentiation
(Tuj1), and glial differentiation (B-FABP, GFAP) are significantly reduced as visualized
by in vitro enteric progenitor cell culture isolated from the mouse embyo (Fig.11).
Therefore, the neural differentiation process may require another signaling molecules or
inhibitory pathways against ET3-ET
B
signaling to initiate the generation of enteric
neurons and the formation of functional units of ENS.

27

Chapter 4
Discussion
4.1 Discussion
As one of the most complex peripheral nervous systems (PNS), the enteric nervous
system (ENS) performs the autonomic regulatory functions to maintain the integrity of
the normal gut. During the embryonic developmental process, ENS is established through
the migration and neural differentiation processes of enteric progenitors. As summarized
in Fig.12, dual lineages, the well-known vagal neural crest and the previously
unsuspected epibranchial placode, provide enteric precursors, which migrate along the
gut and give rise to enteric neurons in response to ET3-ET
B
signaling pathway.

28

It is worthwhile to note that neural crest cells and placodal cells may have the mutual
dependency for the proper gangliogenesis. Even though, some extents of ganglionic
organization in the gut were observed in either neural crest- or placode-lineage ET
B

conditional knockout mice in Fig.4, it is unclear that whether those ganglions are
properly organized. Previous studies have shown that Robo2-Slit1 dependent cell-cell
interaction is required to mediate the assembly of the trigeminal ganglion (33, 34). Robo2
is the receptor expressed on the placodal cells, whereas Slit1 is the ligand expressed on
the neural crest cells. They have found that Robo2-Slit1 interaction is required for the

29

proper formation of cranial sensory ganglions (Fig.13). Therefore, when enteric neurons
form ganglions in the myenteric plexus enteric neurons from neural crest and the placode,
presumably sensory neuronal population, Robo2-Slit1 dependent cell-cell interaction may
be required for proper enteric gangliogenesis as well. Additionally, it has been reported
that N-cadherin, the cell adhesion molecule on the neurons, cooperates with Robo2-Slit1
signaling in regulating aggregation of placode-derived sensory ganglia (33). Based on
their observations, N-cadherin is localized on the placodal neurons, and the lack of N-
cadherin expression causes the loss of the proper trigeminal ganglions as similar as the
lack of Robo2 on the surface. Collectively, these observations suggest that proper
communication between neural crest and placode is required for the normal
gangliogenesis. Therefore, inter-communication between enteric neural crest cells and
placodal enteric progenitors may be required for the proper gangliogenesis in the
myenteric plexus in the gut.

There has been research updates in the signaling pathways involved in the migration
process. Previously, it has been shown that GDNF-RET signaling and ET3-ET
B
signaling
are essential for the migration of enteric neural crest cells. However, it is still unclear that
how they modulate downstream molecules to induce the directional migration. As
discussed above, PKA activity is affected by GDNF-RET signaling and ET3-ET
B

signaling to modulate the activity of Rac1, which may crosstalk to Rho-ROCK signaling
that is responsible for cytoskeletal dynamics. Interestingly, it has been recently suggested
that Phactr4, a member of phosphatase and actin regulator family, regulates directional

30

migration of enteric neural crest cells through protein phosphatase 1 (PP1), integrin
signaling, and cofilin activity (35). They have found that Phactr4 modulates Rho/ ROCK
GTPase family to modify cytoskeletal dynamics, which may provide the driving force for
the directional migration of enteric progenitors from both enteric neural crest and the
placode.

As it is discussed that ET3-ET
B
signaling may have the inhibitory function on the neural
differentiation process of enteric progenitors, there is high possibility that other signaling
pathways are coupled to overpower the endothelin signaling and induce the neural
differentiation. To address this possibility, a previous study showed that the regulation of
progenitor cell proliferation and neural differentiation in ENS progenitor cell culture (36).
The dissociated enteric progenitor cells from in vitro ENS neurosphere culture are
dissociated, and then treated with DAPT, a chemical inhibitor of Notch signaling, or
siRNA against RBPjκ, a key component of Notch signaling. They have found that
disruption of Notch signaling inhibits the ENS cell proliferation, but enhances the neural
differentiation. Taken together, Notch signaling is potentially required to initiate the
neural differentiation process for enteric progenitors to generate the various neuronal
subsets of ENS.

Previously, it has been discussed that endothelin signaling modulates several processes
including cellular migration and neural differentiation for enteric nervous system
development. It also needs to be addressed how endothelin signaling modulate the cross

31

talks among downstream signaling pathways for complicated physiological phenomenon.
Interestingly, once GPCR including ET
B
is stimulated by the ligand, GPCR-kinase
phosphorylates the intracellular loops of the receptor, which generate the docking site for
β-Arrestin (37). β-Arrestin is reported to play a role in desensitization and internalization
of the receptor for recycling. When β-Arrestin binds to the GPCR, it sterically interfere
further interaction of G proteins, and it also associates with the clathrin adaptor AP-2 for
the β-Arrestin-mediated targeting of GPCRs to clathrin-coated pits. However, in case of
ET
B
, previous studies have shown that ET
B
recruits β-Arrestin upon agonist stimulation,
but fails to promote the interaction between β-Arrestin and AP-2, which suggests that
downstream signaling of ET
B
may be β-Arrestin-dependent without formation of AP-2-
dependent clathrin-coated pits (38).
At the same time, β-Arrestin functions as a scaffold protein to recruit diverse mediator
molecules for ligand-dependent downstream signaling pathway, including extracellular
signal-regulated kinase (ERK) signaling cascade (39). ERK plays a diverse role
depending on the biological contexts, such as cell proliferation and neural differentiation,
by modulating phosphorylation status of key transcription factors for target gene
expression. For example, it has been reported that ERK activates p35, a neuron specific
activator of Cdk5, which is essential for neural differentiation and neurite outgrowth (40).
Thus, coupling of endothelin signaling and β-Arrestin – ERK cascade may suggest that
activation of endothelin signaling pathway in enteric progenitors directly switches on
their own mechanism to initiate neuronal differentiation process.

32

4.2 Future Opportunities
Characterization of neurogenic placode as the novel developmental origin for enteric
nervous system provides the insights for the treatment to the developmental disorder,
including Hirschsprung’s disease (HSCR). It is known that symptoms of HSCR usually
show up during the first week of life, and can be diagnosed in 48 hrs after birth by rectal
suction biopsy. Based on the new findings from this project, CGRP+ enteric sensory
neurons would be the potential markers to determine whether enteric neurons, missing in
the gut, are from the placode in HSCR patients. Without enteric sensory neurons
assessment, it would be inconclusive after histochemical approaches using neural crest
markers. Furthermore, currently developing strategies utilizing neural crest cells would
not be effective, if HSCR patients do not have enteric sensory neurons from the placodal
lineage.

Currently, physical diagnosis followed by surgical excision of aganglionic region is the
only way to cure HSCR patients. However, success rate of the operation depends on how
early it is diagnosed, which affects the type and number of procedures, and reoccurrence
of the disease. If the affected region is significantly long enough to threaten the wellness
of patients, then transplantation of a gut segment is required. The total number of donors
is inadequate to meet the needs, and unfortunately, there is a high chance of immuno-
rejection between donor and recipient. To resolve this issue, one of the developing
strategies is the stem cell therapy. Since patients with surgical treatment tend to suffer
from distension and abnormal gut motility function, regeneration of enteric neurons in the

33

aganglionic region of GI tract would be a promising option for HSCR disease. Currently,
clinical application of stem cells has been applied into wide range of diseases and
developmental disorders. A recent study has shown that isolation of neural stem cells
from the gut of embryo and early stage of postnatal mice and neurosphere culture of
those neural stem cells may provide a therapeutic source of enteric nervous system (36).
They have found that neurosphere-derived cells from cultured neural stem cells migrated
over three weeks along the GI tract. It would be the next step to prove that neurosphere-
derived cells account for different neuronal subsets that are properly functional.

Another strategy is the tissue engineering approach. There has been a great advancement
in bioengineering field, and it would be available to take advantages of tissue engineered
gastrointestinal tract in the near future. It has been proven that culturing of organoid, a
mixture of precursor cells for the intestinal development, in the specifically designed bio-
scaffold successfully generated fully functional intestine that contains villi structure,
smooth muscle, and neurons (41). One thing we need to confirm is that those each of
components properly communicates with each other. For example, since lack of
peristalsis in the tissue engineered intestine should be avoided for therapeutic purposes,
the enteric motor neurons, interneurons and sensory neurons have to be organized into the
enteric nervous system. It is anticipated that organoid mixture should include the enteric
neural crest cells and placode-derived enteric progenitors for the proper enteric neural
network formation during the intestinal development.

34

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Abstract (if available)

Abstract The enteric nervous system (ENS) is the neuronal regulatory network, which maintains homeostasis and peristalsis of the gastrointestinal (GI) tract. Abnormal development of ENS may cause the congenital aganglionic megacolon phenotype as Hirschsprung’s disease (HSCR). Previous studies showed that enteric neural crest cells migrate to and colonize the GI tract for the development of ENS that is dependent on the endothelin signaling pathway. However, the contribution of non-neural crest cells to ENS is largely unknown. Since the Xᵗʰ cranial nerve (CNX), where the enteric neural crest cells emigrate from, contains epibranchial placode-derived cells as well, it was suggested that epibranchial placode provides another neurogenic cell population for ENS development. Interestingly, the visualization of placode-derived cells in Pax2cre/R26RLacZ mice uncovered that migratory placodal cells reached the distal colon by E12.5. In addition, the ablation of endothelin signaling in placodal cells, using conditional knockout mouse model (Pax2cre/ETB), caused aberrant formation of myenteric ganglions and disrupted sensory neuronal circuit. All together, these new findings suggest that migratory neurogenic placode provides enteric neural precursors in the endothelin signaling-dependent manner, and placode-derived enteric sensory neurons are essential elements for proper ENS establishment. These observations provide the insights to the developmental mechanism underlying ENS, and to the therapeutic approaches for HSCR.

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Asset Metadata

Creator Chung, Paul J. (author)

Core Title Neurogenic placodes provide migratory enteric sensory neural progenitors in response to endothelin signaling pathway

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 08/01/2013

Defense Date 06/18/2013

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

Tag endothelin signaling,neurogenic placode,OAI-PMH Harvest

Format application/pdf (imt)

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Contributor Electronically uploaded by the author (provenance)

Advisor Tokes, Zoltan A. (committee chair), Bajpai, Ruchi (committee member), Chen, Jeannie (committee member)

Creator Email joon.paul@gmail.com,paul0629@hotmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-310482

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