Focus Article
Hirschsprung disease:
a developmental disorder
of the enteric nervous system
Sonja J. McKeown,1 Lincon Stamp,1,2 Marlene M. Hao1 and Heather
M. Young1∗
Hirschsprung disease (HSCR), which is also called congenital megacolon or
intestinal aganglionosis, is characterized by an absence of enteric (intrinsic)
neurons from variable lengths of the most distal bowel. Because enteric neurons
are essential for propulsive intestinal motility, infants with HSCR suffer from
severe constipation and have a distended abdomen. Currently the only treatment
is surgical removal of the affected bowel. HSCR has an incidence of around
1:5,000 live births, with a 4:1 male:female gender bias. Most enteric neurons arise
from neural crest cells that emigrate from the caudal hindbrain and then migrate
caudally along the entire gut. The absence of enteric neurons from variable lengths
of the bowel in HSCR results from a failure of neural crest-derived cells to colonize
the affected gut regions. HSCR is therefore regarded as a neurocristopathy.
HSCR is a multigenic disorder and has become a paradigm for understanding
complex factorial disorders. The major HSCR susceptibility gene is RET. The
penetrance of several mutations in HSCR susceptibility genes is sex-dependent.
HSCR can occur as an isolated disorder or as part of syndromes; for example,
Type IV Waardenburg syndrome is characterized by deafness and pigmentation
defects as well as intestinal aganglionosis. Studies using animal models have
shown that HSCR genes regulate multiple processes including survival, proliferation, differentiation, and migration. Research into HSCR and the development
of enteric neurons is an excellent example of the cross fertilization of ideas that
can occur between human molecular geneticists and researchers using animal
models. © 2012 Wiley Periodicals, Inc.
How to cite this article:
WIREs Dev Biol 2013, 2:113–129. doi: 10.1002/wdev.57
INTRODUCTION
I
n 1888, Harald Hirschprung described two unrelated boys who died with abdominal distension, congenital megacolon, and severe chronic
constipation.1 Although a number of studies from
the early 1900s had implicated defects in neurons,
the etiology of Hirschsprung disease (HSCR) was not
identified until the mid-1900s when it was found
that all postmortem samples of rectum from patients
∗ Correspondence
to: h.young@unimelb.edu.au
1
Department of Anatomy & Cell Biology, University of Melbourne,
Melbourne 3010, VIC, Australia
2
Murdoch Childrens Research Institute, Parkville 3052, VIC,
Australia
Volume 2, January/February 2013
with congenital megacolon lacked enteric (intrinsic)
neurons.2 Overall, HSCR has an incidence of around
1:5000 live births, but there are differences in incidence between ethnic groups and there is a 4:1
male:female sex bias.
HSCR can occur as an isolated disorder (70%
of patients) or as part of a syndrome in which other
neural crest derivatives are commonly affected. One
of these syndromes, Type IV Waardenburg syndrome
(WS4), is discussed in the section Type IV Waardenburg Syndrome (WS4)—An Example of a Syndrome
That Includes Aganglionic Megacolon of this article. Between 2 and 15% of HSCR patients have
the chromosomal abnormality, trisomy 21 (Down
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Focus Article
in ENS development. This review briefly describes
the ENS and the stages of ENS development relevant
to HSCR. We then discuss the etiology, diagnosis,
genetics, and current and potential treatments for
HSCR, and finally we briefly discuss one of the
syndromic forms of HSCR, WS4.
D
THE ENTERIC NERVOUS SYSTEM
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FIGURE 1 | Barium enema study, lateral view, of a 6-month-old
infant with HSCR. The descending colon is greatly dilated (‘megacolon’)
while the distal colon and rectum are constricted. (Reprinted with
permission from Ref 5. Copyright 1999 Radiological Society of North
America)
syndrome).3 HSCR is a multigenic disorder with
variable penetrance (see the section on Etiology below)
and severity. In over 80% of cases, aganglionosis is
restricted to the rectosigmoid colon (short-segment
HSCR) (Figure 1), but aganglionosis can also affect
significant lengths of the colon or even extend into the
distal small intestine (long segment HSCR). Extremely
rarely, the entire small and large intestines are aganglionic, which is termed total intestinal aganglionosis.
A region of bowel with reduced enteric neuron density, the transition zone, is always present oral to the
aganglionic region.4
The mechanisms controlling the development of
the enteric nervous system (ENS) are highly conserved.
Research into HSCR and ENS development has
consequently benefitted greatly from the exchange
of knowledge and ideas between human molecular
geneticists and researchers using animal models,
including mouse, chick, and zebrafish.6 Several HSCR
susceptibility genes were first identified following
studies on mice with targeted inactivation of particular
genes that resulted in HSCR-like phenotypes.
Likewise, genetic studies of HSCR patients identified
genes that were not previously known to be involved
114
The ENS is an extensive network of neurons and glia
within the wall of the bowel.7,8 The ENS is the largest
part of the peripheral nervous system, and there are
at least as many neurons in the gut as there are in
the spinal cord.9 In the small and large intestines,
enteric neurons are found in two main plexuses; the
myenteric plexus is located between the circular and
longitudinal muscle layers, and the submucosal plexus
is found within the connective tissue of the submucosa. In humans, some neurons are also found within
the mucosa.10 Neurons in myenteric ganglia are primarily involved in the control of motility whereas
most submucosal neurons regulate transport of ions
across the epithelium and blood flow.
There are many different types of enteric neurons that differ in their targets, inputs, direction of
projection, neurotransmitters, and electrophysiological characteristics. The neurons form circuits that
regulate a number of gut functions, including motility.
In the small and large intestines, enteric neurons are
essential for all coordinated motor patterns including
mixing and peristalsis. The essential role for enteric
neurons in peristalsis is exemplified by the bowel
obstruction that occurs in the aganglionic region of
patients with HSCR.
ORIGIN AND EARLY DEVELOPMENT
OF THE ENTERIC NERVOUS SYSTEM
Most enteric neurons arise from neural crest cells
that emigrate from the neural tube adjacent to somites
1–711 (Figure 2). This region of the neural axis is called
‘vagal’, and encompasses the most caudal hindbrain
(somites 1-5) and rostral trunk (somites 6-7). Sacral
level neural crest cells also contribute some enteric
neurons, but even in the most distal regions of the
bowel, the majority of enteric neurons arise from
vagal neural crest-derived cells.12–14
Studies in a variety of species, including human,
have shown that vagal neural crest-derived cells
enter the foregut and then migrate caudally along
the gut16,17 (Figure 2). The colonization of the
gastrointestinal tract by enteric neural crest-derived
cells (ENCCs) is notable both because of the distance
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FIGURE 2 | Immunostaining of E9.5 and E10.5 mice with antibodies to the neural crest cell marker, SOX10 (goat anti-SOX10, Santa Cruz). At
E9.5, vagal neural crest-derived cells from the post-otic hindbrain are migrating ventrally toward and into the foregut (arrow ). At E10.5, the most
caudal neural crest-derived cell is in the midgut (open arrow ). Neural crest cells that emigrate from the neural tube adjacent to somites 1–7 follow
the pathway (dotted line ) that is followed later by the vagus nerve (X). The ectoderm was removed from the E10.5 mouse to enable the gut to be
seen. OV, otic vesicle; BA1, branchial arch 1; BA2, branchial arch 2; asterisks, dorsal root ganglia; V, VII, IX, X—cranial nerves V, VII, IX and X.
(Reprinted with permission from Ref 15. Copyright 2012 Elsevier)
ENCCs migrate, and because of the length of time
it takes. Because the gut grows substantially while it
is being colonized, ENCCs probably migrate further
than any other embryonic cell population.18 For
example, in mice, the colon increases in length fivefold
between when ENCC first enter the proximal colon
and when they reach the distal end. In humans, it
takes 3 weeks for ENCCs to migrate from the foregut
to the anal end,17 and in mice it takes 5 days, which
is 25% of the gestation period.16
ENS development continues after the gut has
been colonized by ENCCs, and even beyond birth,
at least in laboratory animals.19,20 In this article,
however, we focus on the mechanisms involved in the
early development of the ENS as HSCR is caused by
a failure of ENCCs to colonize the entire gut.
ENCCs proliferate rapidly while migrating
toward and within the gut.21,22 Moreover, a subpopulation of ENCCs starts to undergo neuronal
differentiation as they are migrating within the
gut wall. The colonization of the gut by ENCCs
requires coordinated proliferation, migration, and
neuronal differentiation, as perturbations to cell
number,21–23 migratory behavior,24,25 or rate of neuronal differentiation26–29 can result in aganglionosis in
animal models. Although cell death is not prominent
Volume 2, January/February 2013
while ENCCs are colonizing the gut,30,31 inhibition
of cell death of chick vagal neural crest cells as
they are migrating from the hindbrain toward the
gut results in hyperganglionosis (increased density of
enteric neurons),32 and cell death is common when
RET signaling is conditionally abolished while ENCCs
are migrating along the colon in mice.33
Many molecules involved in the colonization
of the gut by ENCCs have been identified. These
include molecules secreted by the gut mesenchyme
that act on receptors expressed by ENCCs, as well
as transcription factors, adhesion molecules and other
molecules expressed by ENCCs themselves. Some of
the signaling pathways involved in ENS development
affect multiple processes including proliferation,
migration, differentiation, and/or survival. Mutations
in genes encoding components of many of these
signaling pathways have been associated with HSCR
(Table 1). Here we briefly summarize the main
pathways and molecules known to play a role in
the colonization of the gut by ENCCs.
GDNF-GFRA1-RET Signaling Pathway
This is a critical pathway for ENS development.
Glial cell line-derived neurotrophic factor (GDNF)
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Frequency of
Mutation in Isolated
HSCR
Isolated HSCR, Associated
Syndrome, Phenotype
© 2012 Wiley Periodicals, Inc.
∼5%48
Extremely rare (one
family only and
co-occurred with a
RET mutation)44
Heterozygous44
NRTN
HGNC:8007
Heterozygous
EDNRB
HGNC:3180
mutations47,48
Extremely rare (one
family only, low
penetrance,
significance
unknown)41
Very rare
Extremely rare
Heterozygous41
GFRA1
HGNC:4243
Heterozygous38
GDNF
HGNC:4232
Homozygous
missense36
Usually isolated,48
very occasional
WS449,50
Isolated44
Isolated41
Isolated38
Isolated. Total
intestinal
aganglionosis36
Non-coding
RET
Non-coding mutations: Mostly isolated but
HGNC:9967
mutations may be
very common. Coding can also be
homozygous or
mutations: ∼50% of
associated with a
heterozygous;
familial and 15–20%
syndrome1
coding mutations
of sporadic HSCR1
are heterozygous1
Gene
Zygosity of
Mutations in
HSCR Patients
TABLE 1 HSCR Susceptibility Genes and Mouse Models of HSCR
Required for the normal projection,
branching and/or survival of
axons of enteric excitatory
motor neurons45
EDNRB is a G
EDN3-EDNRB signaling promotes
protein-coupled receptor. proliferation and inhibits
Main receptor for EDN3
neuronal differentiation of
in ENS development.
enteric neural progenitors, and
Expressed by ENCCs and
regulates ENCC
some gut mesenchymal
migration.26,29,53 EDN3-EDNRB
52
signaling is also involved in
cells
melanocyte development.54
Neurons are present along the Neurturin is a secreted
entire gut of Nrtn−/−
molecule; member of
mice.45 Myenteric neuron
GDNF family ligands.
number is normal, but there
Expressed by the
are defects in submucosal
mesenchyme of the
neuron number and in the
embryonic gut46
density of excitatory nerve
fibers in Nrtn−/− mice30
Gfra1+/− mice have normal
GPI-linked protein to which
enteric neuron density;30
GDNF binds. Expressed
Gfra1−/− mice lack neurons
by ENCCs.43
42
distal to the stomach
Ednrb s/sl mice have reduced
neuron density in the colon
(note: s is a hypomorphic
mutation and sl is a null
mutation).51
Role in ENS
Development from
Animal Studies
RET tyrosine kinase.
Survival, proliferation, migration,
Expressed by ENCCs.34
and neuronal differentiation of
Expression is maintained
ENCCs6
in neurons but is
down-regulated in glia.35
Expression Pattern in
Mice Relevant to
ENS Development
Neurons are present along the Secreted molecule.
entire gut of Gdnf +/− mice,
Expressed by
but at reduced density30 ;
mesenchyme of
Gdnf −/− mice lack enteric
embryonic gut39 and
neurons distal to the
external muscle of
stomach39
mature gut.40
Ret −/− mice lack neurons
distal to the stomach37
Ret +/− mice have a normal
ENS30
ENS Phenotype of
Mouse Mutants
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Heterozygous
SOX10
HGNC:11190
mutations49,68 or
deletions69
Heterozygous SNPs Relatively
in the coding
common65
sequence that
increase the risk
of HSCR conferred
by RET 65
NRG1
HGNC:7997
Edn3 −/− mice lack of neurons
in the distal colon62
WS449,61
Sox10 homozygous null
Transcription factor expressed by
mutants lack neurons in the
migrating neural crest cells
entire gastrointestinal
including ENCCs.70,74 Also
70,71
+/−
Sox10
mice
expressed by enteric glia and
tract.
lack enteric neurons in the
glial progenitors, but is
distal bowel, with the
down-regulated upon enteric
severity and penetrance of
neuron differentiation.74
aganglionosis being
strain-dependent.72,73
WS468,69 or PCWH49
Are multiple isoforms of
neuregulin-1; type 1 is
expressed in the dorsal neural
tube at the time of neural crest
cell emigration.67 ErbB2/ErbB3
receptors are expressed by
migrating neural crest cells.67
Nrg1−/− mice die at E10.5
from heart defects.66
ECE1 is involved in the proteolytic
processing of big endothelins to
biologically active peptides.
Expression of ECE-1 does not yet
appear to have been examined
in the developing gut.
EDN3 is the main ligand for EDNRB
in ENS development. Expressed
by the mesenchyme of the
embryonic gut with highest
expression in the cecum27,60
Expression Pattern in
Mice Relevant to
ENS Development
Isolated65
No reports of ENS defects in
Ece1+/− mice. Ece1−/−
mice lack neurons in the
distal colon, lack epidermal
melanocytes, and also
exhibit craniofacial and
cardiac defects64
Edn3 +/− mice have normal
enteric neuron density59
Ednrb −/− mice lack of neurons
in the distal colon54
ENS Phenotype of
Mouse Mutants
Isolated56 except
one case of WS458
WS4; patients are
deaf, have piebald
pigment pattern,
HSCR and
neurological
deficits49,55
Isolated HSCR, Associated
Syndrome, Phenotype
Very rare (one case Cardiac, craniofacial
and autonomic
reported63 )
defects, and
HSCR63
<5%57
Heterozygous63
Mutations in both
alleles61
Heterozygous
mutations56
Mutations in both
alleles49,55
Frequency of
Mutation in
Isolated HSCR
ECE1
HGNC:3146
EDN3
HGNC:3178
Gene
Zygosity of
Mutations in
HSCR Patients
TABLE 1 Continued
Required for survival and
maintenance of neural
crest stem/progenitors
and the activation of
Phox2b and Ret.75–77
Also required later in
development for glial
and melanocyte
differentiation.78,79
ND
Role in ENS
Development from
Animal Studies
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118
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ND
ENCC migration along the
gut is slightly delayed in
L1cam null mutants, but
the entire gut is
colonized.94 L1cam+/−
mice have no reported
ENS phenotype
Goldberg–Shprintzen
syndrome; patients have
microcephaly, mental
retardation, and facial
dysmorphism. Most also
have HSCR.89
X-linked hydrocephalus.92
Only a small percentage
of patients also have
HSCR.93
Homozygous89
L1CAM is on the
X-chromosome
KIAA1279
HGNC:23419
L1CAM
HGNC:6470
Role in ENS
Development from
Animal Studies
L1CAM is a cell adhesion
molecule that is expressed
by most ENCCs94
KIAA1279 encodes kinesin
binding protein (KBP).
Expression pattern of KBP
not yet reported in mice.
Transcription factor expressed
by all ENCCs87 and in
differentiated neurons and
glia.88
Required for normal
migration of ENCCs94
KBP promotes neurite
formation and neuronal
differentiation in
zebrafish brain and
spinal axons,90 and in
CNS neurons in culture.91
Required for the activation
of Ret.86
Transcription factor that is
Required for the formation
expressed by enteric neural
of the vagal neural crest,
progenitors and then by
and also plays later roles
enteric glia but is not
in ENS development.83,84
expressed by enteric
neurons.84 Also expressed by
some mesenchymal cells in
the developing gut.84
Expression Pattern in
Mice Relevant to
ENS Development
CCHS, central congenital hypoventilation syndrome; ECE-1, endothelin-converting enzyme 1; EDN3, endothelin-3; EDNRB, endothelin receptor B; ENCC, enteric neural crest-derived cells; HGNC, HUGO Gene
Nomenclature Committee; ND, not determined; PCWH, peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, Waardenburg syndrome and Hirschsprung’s disease; WS4, Waardenburg
syndrome 4.
CCHS (Ondine’s curse). Only Homozygous null mutants
a small proportion of
lack neurons throughout
CCHS patients also have
entire gastrointestinal
HSCR.85
tract.86 Phox2b +/− mice
have no reported ENS
defects.
Zeb2 −/− mice die at E9.5
and the vagal neural
crest does not form.83
ENS Phenotype of
Mouse Mutants
Heterozygous for a
polyalanine
repeat expansion
mutation85
Mowat–Wilson syndrome;
patients have distinctive
facial features, epilepsy,
cardiac defects, mental
retardation, and a range
of other features
including HSCR82
Isolated HSCR, Associated
Syndrome, Phenotype
PHOX2B
HGNC:9143
Frequency of
Mutation in
Isolated HSCR
Heterozyous
nonsense or
frameshift
mutations80 or
truncating
mutations81
Zygosity of
Mutations in
HSCR Patients
ZEB2 (formerly
known as
ZFHX1B, SIP1)
HGNC:14881
Gene
TABLE 1 Continued
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belongs to a family of four secreted neurotrophic
factors that bind to GPI-anchored cell surface proteins called GDNF family receptor alphas (GFRAs).95
The GDNF family ligand–GFRA complex activates
the RET tyrosine kinase.96 GDNF preferentially binds
to GFRA1. GDNF is first expressed by the gut mesenchyme shortly before vagal neural crest cells enter
the foregut,97 while RET and GFRA1 are expressed
by ENCCs. Gdnf −/− , Gfra1−/− , and Ret−/− mice
lack neurons in the small and large intestines, and
from most of the stomach37,42,98 (Table 1). In vivo
and in vitro studies have shown that the GDNFGFRA1-RET signaling pathway is essential for the survival, proliferation, migration, and differentiation of
ENCCs.26,43,99–102 Increasing RET signaling by inactivation of negative regulators of RET signaling, or by
increasing GDNF availability, results in an increased
number of enteric neurons.103–105 There are two main
isoforms of RET, which differ in their C-terminal
domains, and are generated by alternate splicing,
RET9 and RET51. Although one study found that
RET9 was critical for colonization of the distal bowel
by ENCCs, but RET51 was not essential,106 another
group subsequently found that mice expressing only
RET9 did not exhibit aganglionosis.107,108 This disparity is probably due to differences in the transgenes
or strains used by the two different laboratories.
Neurturin-GFRA2-Ret Signaling Pathway
Neurturin (NRTN) belongs to the GDNF family ligands and binds to GFRA2 to activate RET. Neurons
are present along the entire gastrointestinal tract of
Nrtn and Gfra2 homozygous null mutant mice; the
number of myenteric neurons is normal, but the density of excitatory nerve fibers is reduced.30,109 Thus,
NRTN-GFRA2-Ret signaling promotes the projection
or branching of axons from some classes of enteric
neurons. Although studies in mice have not revealed a
role for NRTN signaling in colonization of the gut by
ENCCs, a mutation in NRTN has been found in one
family with HSCR (see the section on Genetics below).
Endothelin 3-Endothelin Receptor B
Signaling Pathway
Endothelin 3 (EDN3) is a ligand for the G proteincoupled receptor, endothelin receptor B (EDNRB).
Edn3 is expressed by the gut mesenchyme, with highest levels in the cecum.27,60 Ednrb is expressed by
ENCCs and some gut mesenchymal cells.52 Mice lacking EDN3 or EDNRB exhibit colonic aganglionosis62
(Figure 3) resulting from a delay in the entry of vagal
neural crest-derived cells into the gut as well as reduced
speed of colonization of the gut by ENCCs.25,27
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FIGURE 3 | Mouse model of HSCR. (a) Intestine from a 2-week-old
Edn3 −/− mouse. The mid-colonic region is distended and termed a
‘mega-colon’. (b–d) Whole mount preparations of the external muscle
of colonic wall from the regions indicated in (a) showing
immunostaining with an antibody to the pan-neuronal marker, Hu.
Neurons, which are clustered into ganglia, are present in the distended
region (b), but are totally absent from the distal colon (d). There is a
transition zone of reduced neuron density proximal to the aganglionic
region (c). Scale bar = 100 μm (applies to b–d).
Studies in vivo and in vitro have shown that signaling via EDNRB promotes the colonization of
the gut by ENCCs by promoting the proliferation
and inhibiting the neuronal differentiation of neural progenitors,26,28,29,110 and by directly promoting
ENCC migration.25 EDN3-EDNRB signaling is also
required for melanocyte development, as mice null
for Ednrb or Edn3 have pigmentation abnormalities as well as colonic aganglionosis (WS4 phenotype,
see the section on Type IV Waardenburg Syndrome
(WS4)—An Example of a Syndrome That Includes
Aganglionic Megacolon below), whereas Edn3 heterozygotes have no abnormalities and Ednrb heterozygous and hypomorphic mutants typically only have
pigmentation abnormalities.28,49 Hence melanocyte
precursors and ENCCs appear to have different
dosage requirements for EDN3/EDNRB signaling,
with melanocytes having a lower minimal threshold.
Transcription Factors
A network of transcription factors controls ENS
development.111 Here we very briefly review the
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roles of SOX10, PHOX2B, and ZEB2 (formerly
known as ZFHX1B and SIP1), as mutations in the
genes encoding these molecules are associated with
HSCR (Table 1). However, FOXD3, HAND2, ASCL1
(formerly known as MASH1), PAX3, HLX, and the
TFAP2 (formerly known as AP-2) family also play a
role in ENS development.
Sox10 is expressed prior to, and during, neural
crest migration.70 Sox10−/− mice die at birth, and
in null mutant embryos, vagal neural crest cells die
around E9, prior to their entry into the gut.71 SOX10
plays an essential role in ENS development most likely
because it is necessary for the expression of Ret and
Phox2b, and because it maintains the multipotency
of neural crest cell stem/progenitors.75,76,112,113 The
expression of Sox10 is down-regulated by ENCCs
that differentiate into neurons, but Sox10 expression is maintained in enteric glia and is required for
glial fate acquisition.35,78 In the neural crest-derived
melanocyte progenitors that migrate into the ectoderm, SOX10 regulates the expression of MITF, a key
transcription factor required for melanocyte development, as well as genes required for melanin synthesis
in melanocytes.79
Phox2b−/− mice do not have any peripheral
autonomic neurons, including enteric neurons, and
die at birth.86 During ENS development, Phox2b is
first expressed just prior to the entry of vagal neural
crest cells into the gut. Like SOX10, PHOX2B is
required for Ret expression in ENCCs.86 Although
PHOX2B is thought to be involved primarily in
neuronal specification, Phox2b is expressed by both
enteric neurons and glial cells.88
ZEB2 (Zinc finger E-box-binding homeobox 2)
is a transcription factor involved in neural specification and in epithelial–mesenchymal transition (EMT)
during early neural crest development. Zeb2−/− mice
die around E9.5 and exhibit a variety of cardiovascular and neural defects, including a failure of the
vagal neural crest to form.83 Mice with targeted ablation of Zeb2 in neural crest cells have craniofacial,
heart, pigment, and peripheral nervous system deformities as well as aganglionosis of the entire colon
that extends into the small intestine.114 Zeb2 has a
similar expression pattern to Sox10 in the developing
ENS,84 but the molecular mechanism by which ZEB2
regulates enteric neuron development has not yet been
elucidated.
Other Molecules and Interactions
ENCCs express cell surface molecules, including beta1-integrins and the cell adhesion molecule
L1CAM, which regulate interactions with the ECM,
120
TABLE 2 Genetic Interactions That Influence the Penetrance and
Severity of Aganglionosis in Mice
Interacting Genes
References
Ret and Ednrb
117
Ret and Edn3
27
Sox10 and Ednrb
72
Sox10 and Edn3
28
Sox10 and Sox8
118
Sox10 and L1cam
119
Sox10 and Zeb2
84
L1cam and Edrnb
120
L1cam and Edn3
120
other ENCCs and/or other cell types. Mice in which
ENCCs lack beta1-integrins exhibit aganglionosis
because beta1-integrins are required to overcome the
inhibitory effect of high levels of tenascin-C in the
hindgut.24 L1CAM is expressed by most migrating
ENCCs, and perturbation of L1CAM function in vivo
or in cultured explants of gut retards the migration of
ENCCs, although the entire length of the gut in L1cam
homozygous null mutants is eventually colonized.94
Other molecules shown to be involved in the development of the ENS from animal studies include
neurotrophin-3, sonic hedgehog, Indian hedgehog,
bone morphogenetic proteins (BMPs) 2 and 4, Notch,
small GTPases, neuregulin, microRNAs, serotonin,
the norepinephrine transporter, vitamin A, and the
axon guidance molecules, SEMA3A and netrin.15
The activity of the different pathways involved
in ENS development must be coordinated, and
there is substantial evidence for interactions between
pathways. The first evidence that interactions between
pathways influence ENS development came from
studies of humans with HSCR.115,116 Subsequent
studies in mice have shown that interactions between a
variety of signaling pathways influence the penetrance
and severity of aganglionosis (Table 2). Some of the
interactions are direct, for example, SOX10 binds
directly to Ednrb regulatory regions,112 whereas other
interactions may be indirect.
HIRSCHSPRUNG DISEASE
Etiology
Studies using animal models have shown that the
absence of enteric neurons from variable lengths of the
gastrointestinal tract in HSCR results from a failure
of ENCCs to colonize the affected gut regions during
development (Figures 3 and 4). Aganglionosis is associated with a delay in the entry of neural crest-derived
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S
C
C
S
A
S
FIGURE 4 | Diagram showing the location of ENCCs along the gut
(green) in E10.5 and E12.5 wild-type and Edn3 −/− mice. Edn3
homozygous null mutants are a mouse model of Hirschsprung’s disease.
Already at E10.5, ENCCs are not as caudally advanced along the gut in
Edn3 null mice compared to wild-type mice.27 There is a similar delayed
entry into the gut in Ednrb homozygous null mutants.25 (Reprinted with
permission from Ref 122. Copyright 2001 John Wiley and Sons)
cells into the foregut, as well as a delayed progression
of ENCCs along the gut.25,27 The delay is exacerbated
by changes to the gut microenvironment with age that
make it less permissive for ENCC migration.25 Defects
in multiple processes can contribute to the delay in
the colonization of the gut by ENCC (see the section
Origin and Early Development of the Enteric Nervous
System above), but ENCC proliferation is critical, as
a minimal number of ENCCs are required for normal
ENS formation.21,23 Although cell death does not play
a major role during normal ENS development, it may
contribute to the etiology of HSCR.121
Genetics
The genetics of HSCR are complex and are reviewed
in detail by Amiel et al.1 Mutations in genes encoding
members of a variety of signaling pathways are
associated with HSCR (Table 1), but mutations in
the coding sequence of these known genes account
for only about 50% of familial cases of HSCR and
15% of sporadic cases of HSCR.1 RET is the major
susceptibility gene as more than 80% of identified
mutations associated with HSCR are in RET; these
include both coding and noncoding mutations.123,124
RET coding sequence mutations have been identified
in 20–30% of HSCR cases, and alone, can result in
HSCR by haploinsufficiency.1 Noncoding mutations
are more common and result in reduced amounts of
wild-type RET protein due to a reduction in RET
transcription; these mutations probably only result in
Volume 2, January/February 2013
HSCR when combined with other mutations.1,124 For
example, a common noncoding mutation, a T>C SNP
lying within an intronic RET enhancer, has a 20-fold
greater contribution to HSCR susceptibility than RET
coding sequence mutations.123 This mutation appears
to account for some, but not all, of the sex ratio in
HSCR.1,123 Like mutations in most genes associated
with HSCR, the penetrance of RET mutations,
even coding sequence mutations, is incomplete. For
instance, a missense mutation in RET was found in
four members of one family, of which one had long
segment HSCR, two had short segment HSCR, and
one was unaffected.125
There appear to be some phenotypic differences
in mice and humans with reduced RET signaling.
Unlike humans, Ret heterozygous mice do not show
an HSCR-like phenotype30,37 (Table 1). However,
colonic aganglionosis can be observed in mice when
the levels of Ret are reduced to around 40% of
wild-type levels.126 In addition to intestinal aganglionosis, Ret null homozygous mutant mice lack
kidneys and have sympathetic and parasympathetic
neuron defects. In contrast, infants with HSCR only
extremely rarely have kidney defects. Studies in mice
have shown that different tissues appear to require
distinct RET-stimulated signaling pathways, which
might partly explain why intestinal aganglionosis can
occur as an isolated defect in HSCR patients with
RET mutations.108
In addition to RET, mutations in a dozen
or more genes have been associated with HSCR
(Table 1). Although mutation in a single gene (RET,
EDNRB, EDN3) can be sufficient for the development
of HSCR, patients with mutations in two genes
have been identified, and complex genetic interactions
contribute to the variability in penetrance and severity
(length of the aganglionic region). Most of the genes
associated with HSCR in humans also result in
aganglionosis in mice following inactivation (Table 1).
However, a heterozygous mutation in NRTN that cooccurred with a RET mutation was found in one
family with HSCR,44 but mice lacking NRTN do
not have aganglionosis.30 L1CAM is the only known
X-linked gene associated with HSCR, but as only
3% of patients with mutations in L1CAM also have
HSCR, L1CAM is thought to act as an X-linked HSCR
modifier gene.127 Most of the syndromes involving an
HSCR phenotype are each associated with mutations
in one or a small number of genes (Table 1).
Copy number variants (CNVs), which are structural variations in DNA that result in an abnormal
number of copies of sections of DNA, have been
associated with susceptibility or resistance to some
human diseases. Data from a recent study of 67
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Focus Article
proven or candidate HSCR genes from human and
mice studies suggest that CNVs, particularly in regulatory sequences, can play a role in HSCR.128
HSCR has become a paradigm for understanding
complex multigenetic disorders. For example, studies
of HSCR have shown that small structural variants in
a gene can act as powerful genetic modifiers in human
disease. Furthermore HSCR studies have blurred the
definition of modifier gene, as depending on the
mutation, the same gene can act as a principal gene or
a modifier gene. For example, large genomic mutations
in RET, alone, can result in HSCR (‘principal gene’)
but small structural variants in RET probably only
result in HSCR when combined with other mutations
(‘modifier gene’).124 Humans appear to be more
sensitive to decreased levels of RET and EDNRB
signaling than mice, which might be due, at least
in part, to the larger area of bowel that has to be
colonized by ENCCs in humans compared with mice.
Although it has been assumed that all cases
of HSCR have a genetic basis, vitamin A deficiency
increases the penetrance and severity of aganglionosis
in a mouse model of HSCR.129 Thus, environmental
factors could contribute to susceptibility to HSCR.
is predominantly excitatory (mediates muscle contraction), and may even be hypertrophied in some
HSCR patients.4,130 Relaxation of the gut wall during
propulsive motility is normally mediated by inhibitory
enteric (intrinsic) motor neurons, and so an absence of
intrinsic inhibitory neurons may also contribute to the
aganglionic segment being persistently constricted.130
Muscle hypertrophy also occurs in the aganglionic
segment in mouse and rat models of HSCR,131,132
probably as a secondary effect.
Perturbation of intestinal motility is not limited to the aganglionic gut segment. HSCR patients
often have motility disturbances following surgical
resection. In mouse models of HSCR, spontaneous
propagating motility patterns are absent or abnormal
along the entire colon, including the proximal colon
(oral to the transition zone) where enteric ganglia are
present.51,59 The most likely cause is a reduction in
enteric neuron density, although inflammation and
damage to the interstitial cells of Cajal (ICC), a population of cells involved in gut motility, in regions
proximal to the aganglionic bowel could also contribute to abnormal motility.133
Current Treatment and Potential Therapies
Diagnosis, Pathology, and Physiology
Healthy infants normally pass meconium (the earliest
stools) within the first 24–48 h following birth. Most
infants with HSCR, however, fail to pass meconium
and suffer from severe constipation. Other symptoms
include gradual distension of the abdomen, vomiting,
and fever. Although infants with HSCR normally
show symptoms within the first weeks after birth,
other children with HSCR may not exhibit symptoms
for several months.
HSCR is usually diagnosed by a barium enema
(Figure 1), anorectal manometry, and a biopsy of the
rectum. The pathological evaluation of rectal biopsies
for HSCR is reviewed by Kapur.4 Most laboratories rely on hematoxylin and eosin-stained paraffin
sections to identify neuronal cell bodies, although histochemical and immunohistochemical techniques are
also used by some pathologists to assist the diagnosis.
During surgery to remove the aganglionic region of
bowel, frozen sections are taken to localize the boundary between the ganglionic and aganglionic regions
prior to resection.4
The megacolon and severe constipation experienced by patients with HSCR are caused by a lack
of propulsive motility patterns in the distal bowel
due to the lack of intrinsic enteric neurons. The
persistent constriction of the aganglionic segment is
probably mediated by the extrinsic innervation, which
122
If left untreated, HSCR can be fatal due to enterocolitis, perforation of the bowel, or malnutrition. Current
treatment for HSCR involves the surgical removal of
the defective, aganglionic bowel, and reanastomosis
of the distal-most, normal (ganglionated) bowel to
the anus. Depending on the severity of the disease
(length of aganglionosis) and the general health of the
patient, the surgery may be performed in one or two
stages; for patients with long-segment aganglionosis or
major health problems, an initial primary colostomy
is often performed with the resection of the aganglionic bowel performed later. While these operative
procedures are lifesaving, they can be associated with
acute (anastomotic stricture, enterocolitis) and chronic
(constipation, fecal incontinence) complications.
Cell transplantation therapy has been proposed
as an alternative to surgical resection of aganglionic
bowel for the treatment of HSCR.10,57,134,135
This would involve the transplantation of neural
stem/progenitor cells into the aganglionic bowel,
which would colonize the defective region, generate
neurons, and form the neuronal circuits required for
propulsive motility. Neural stem/progenitor cells can
be isolated from the bowel of postnatal and adult
laboratory animals and humans, including HSCR
patients.10,136 These cells are capable of both selfrenewal and of giving rise to multiple lineages in vitro
including neurons and glia (Figure 5). Pluripotent stem
© 2012 Wiley Periodicals, Inc.
Volume 2, January/February 2013
WIREs Developmental Biology
Hirschsprung disease
(a)
(b)
N
N
FIGURE 5 | Enteric neural crest stem/progenitor cells form neurospheres in vitro. (a) Neural crest stem/progenitors were isolated from the gut of
an E14.5 mouse and cultured in suspension conditions. Neurospheres formed, which were then plated onto fibronectin for 48 h to allow for migration
of neural crest-like cells (identified using an antibody to SOX10, green, Santa Cruz) and neurite outgrowth (identified using the neuronal marker,
TUBB3, formerly known as TuJ1, red, Covance). (b) Higher magnification image of the outgrowth of TUBB3+ neurites and SOX10+ cells that have
migrated away from the neurosphere. There are also a small number of TUBB3+ cell bodies in the outgrowth (arrows ). Scale bars: 200 μm (a);
50 μm (b).
cells, such as embryonic stem (ES) cells and induced
pluripotent stem (iPS) cells, are also a potential source
of cells to generate enteric neurons as ES cells can
give rise to neural crest-like cells and to neurons when
co-cultured with explants of embryonic gut.137,138 iPS
cells and ‘adult’ stem cells have the added benefit of
being patient-derived and thus have reduced chance
of immunological rejection after engraftment. Cell
therapy to treat HSCR is currently a very active field
of research.10,57,134,135
TYPE IV WAARDENBURG SYNDROME
(WS4)—AN EXAMPLE OF A
SYNDROME THAT INCLUDES
AGANGLIONIC MEGACOLON
There are four types of Waardenburg syndrome,
all of which involve sensorineural hearing loss and
pigmentation abnormalities.49 Type IV Waardenburg
syndrome (WS4) also involves HSCR and sometimes
neurological features. The sensorineural hearing loss
and depigmentation are due to abnormal development
of neural crest-derived melanocytes in the stria
vascularis of the cochlea, skin, hair, or eyes.139
Mutations in three genes, SOX10, EDNRB, and
EDN3, account for approximately 60–80% of cases
of WS4.69 These three genes are required for the
development of both the ENS and melanocytes (see
the section Origin and Early Development of the
Enteric Nervous System above).
Heterozygous mutations in SOX10 account for
approximately 50% of WS4 cases, mostly occurring de novo.49 The Sox10 ‘Dom’ mutation in mice
Volume 2, January/February 2013
occurred spontaneously and is a single base pair
insertion that results in loss of the SOX10 protein transcriptional activation domain.70 Sox10Dom/+
mice exhibit aganglionosis and pigmentation defects
and therefore exhibit a similar phenotype to humans
with WS4. The severity and penetrance of aganglionosis in Sox10Dom/+ mice varies with the genetic
background.72 SOX10 mutations in humans can
also result in a more severe phenotype that also
includes central and peripheral demyelinating neuropathies, which is called PCWH syndrome (peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, Waardenburg syndrome, and
Hirschsprung disease).69 This neurological phenotype
has been mostly associated with SOX10 mutations
resulting in a dominant negative effect, rather than
haploinsufficiency.69
Of the approximately 20–30% cases of WS4 due
to mutations in EDNRB or EDN3, about 70% are
recessive (homozygous or compound heterozygous)
mutations of EDNRB or EDN3.49 Heterozygous
mutations in EDNRB or EDN3 are most commonly
associated with isolated HSCR, although rare cases of
WS4 with heterozygous EDNRB mutations have been
reported.49
Phenotypes associated with SOX10, EDNRB
and EDN3 mutations show a large degree of
variability.49 Moreover, heterozygous disruptions in
SOX10 account for approximately 15% of cases of
Type II Waardenburg syndrome (WS2), which lacks
HSCR,69 while heterozygous mutations in EDNRB
or EDN3 have also been associated with WS2.49
These findings further highlight the genetic complexity
of HSCR and Waardenburg syndromes, and the
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Focus Article
importance of the genetic background for the severity
of the phenotype.
CONCLUSION
Molecular genetics of HSCR patients and studies using
animal models of HSCR have, in parallel, revealed
much about the pathogenesis of HSCR. However,
there are many outstanding questions. For example,
the molecular mechanisms underlying the interactions
between pathways, and how particular mutations
impact on those interactions, are not well understood.
The 4:1 male:female bias in the incidence of HSCR
is only partly explained by current knowledge. The
transplantation of neural stem/progenitor cells into
the aganglionic region of HSCR patients, as an alternative treatment to surgical removal, is an exciting,
but currently untested possibility.
ACKNOWLEDGMENTS
The authors’ work is supported by NHMRC (Australia) Senior Research Fellowship #1002506, CJ Martin
Fellowship #400433 and Project grant #546473. We thank Annette Bergner for assistance with obtaining the
images shown in Figures 2 and 3, and Lauren Young and John Stephenson for assistance with processing the
embryos shown in Figure 2.
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