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Animal Model |
From the Gastroenterology Unit,*
Institute of Child
Health, University College of London, London; the Department of
Histopathology,
Great Ormond Street Hospital
for Children, National Health Service Trust, London; and the
Nephro-Urology Unit,
Institute of Child
Health, University College of London, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-smooth muscle actin (-SMA).14
Such
overlapping expression domains suggest cooperation of these genes that
could be important for enteric neuromuscular development. Studying
RA-induced caudal agenesis in murine embryo can represent a useful
experimental model to investigate gut responsiveness to exogenous RA.
It may also give an insight into molecular pathogenesis of intestinal
malformations such as gut malrotations, atresia, and stenosis often
coexisting with enteric neuromuscular anomalies described in
humans.15,16 |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The influence of exogenous RA in vivo and in cultured
gut explants was studied using in situ hybridization and
immunohistochemistry for markers of neuronal and mesenchymal
development, c-ret, Hoxb5, and -SMA. The presence of
apoptotic cells in embryos and dissected guts after in vivo
and in vitro RA treatment was also monitored with
colorimetric terminal transferase-mediated dUTP nick-end labeling
(TUNEL) method.
Injections of Pregnant CD1 Mice with RA
Pregnant females from CD1 (Charles River Mouse Farms, Margate,
Kent, UK) crossings from natural overnight matings were used. The
morning of the vaginal plug was considered as 0.5 days post coitum
(dpc). All-trans RA (Fluka, Poole, Dorset, UK) was dissolved in
dimethyl sulfoxide (DMSO) as 100 mg/ml of stock solution and kept in
the dark at -20°C until use for up to 1 week. Before injection, RA
stock solution was diluted in DMSO and dispersed by vortexing in 1 ml
of peanut oil. The RA dose and stage of injection sufficient to induce
truncation of the caudal body axis and agenesis of the tail bud in
embryos were selected following a previously described
protocol.7
In four separate experiments, four pregnant
mice were injected intraperitoneally with 120 mg/kg of RA (Fluka,
Gillingham, Poole, Dorset, UK) at 9.5 dpc that coincides with the onset
of gut morphogenesis.17
A total of 39 embryos were
harvested at E10.5 (n = 9), E11.5
(n = 12), E12.5 (n = 10),
and E16.5 (n = 8) at a similar time point in the
morning into ice-cold diethyl pyrocarbonate-treated phosphate-buffered
saline (DEPC/PBS), preserved by incubating overnight in 4%
paraformaldehyde in PBS and dehydrated through the methanol/PBS grades
into absolute methanol and stored at -20°C until use. Four control
mice were injected intraperitoneally with DMSO dispersed in peanut oil
and 32 embryos were harvested at the same time points as those in the
RA-treated group E10.5 (n = 8), E11.5
(n = 10), E12.5 (n = 8),
and E16.5 (n = 6). Gut development was assessed
by the appearance of gut looping and the cecal bud that in
vivo occurs between E11.5 and E12.5.14
The expression
of neural and muscular markers such as c-ret, Hoxb5, and
-SMA served to assess differentiation as previously
described.14,17,18-20
In Vitro Culture of Gut Explants
Pregnant mice were sacrificed by spinal dislocation at similar time points in the morning at E10.5 and embryos removed into ice-cold L-15 Leibowitz medium (Life Technologies Ltd., Paisley, UK). The culture system was established as described previously.17 To parallel in vivo conditions, the earliest stage of the E10.5 guts consisting of a simple, straight tube was chosen as suitable for intact dissection. Guts were aseptically isolated from embryos and placed in pairs into wells of 24-well culture dishes with 500 µl of defined, serum-free, Optimem medium (Life Technologies Ltd.) supplemented with 1 mmol/L L-glutamine and L-amino acids with addition of 100 U/ml of antibiotic mixture (Life Technologies, Ltd). Intestines were kept floating in the medium and incubated for 3 days at 37°C in a humidified incubator in an atmosphere of 5% CO2 in air. The development and viability of gut explants was assessed according to the criteria used for embryos harvested from RA-treated mice such as the appearance of gut loops and the cecal bud and by the expression pattern of neural and muscular markers.
Retinoic Acid Treatment of Gut Explants in Culture
In the preliminary experiments, the dose of RA sufficient to
induce morphological changes in gut explants consistent with those
observed in vivo was established. The toxic effect of RA was
assessed by the appearance of apoptotic cells in gut explants using the
TUNEL method (see below). A total of 38 gut explants representing the
youngest developmental stage of the gut appearing as a simple unlooped
tube available for intact isolation at the age of 10.5 dpc were
dissected and cultured for 24 hours in RA-free medium. After checking
the explants viability manifest by a visible growth of the prececal gut
or attached lung buds, sets of 8 to 10 explants were chosen and
cultured with increasing concentrations of RA:
10-7 mol/L (n = 10),
10-6 mol/L (n = 10),
10-5 mol/L (n = 10), and
10-4 mol/L (n = 8). The
next day, RA medium was removed, the explants washed twice in fresh
medium and cultured for the next 24 hours in RA-free conditions. At the
end of 72 hours in culture, explants were washed in ice-cold PBS and
preserved in 4% paraformaldehyde for 20 minutes. The lowest RA
concentration (10-5 mol/L) that induced
morphological changes in gut looping and neuromuscular development in
the majority of tested explants comparable to those observed after
in vivo treatment, but without the toxic effect manifest by
the appearance of cell death, was chosen for the following experiments:
to investigate the expression pattern of -SMA, Hoxb5, and
c-ret in morphologically altered gut explants. Gut explants
dissected at E10.5 were cultured for 24 hours in RA-free medium. The
explant viability was examined on the next day as described above. A
total of 60 explants were chosen for further culture. Thirty gut
explants were RA-treated and the same number of control guts was
cultured in the absence of RA with the addition of the volume
equivalent of DMSO as vehicle and control. Gut explants were placed in
the fresh medium with the addition of 10-5 mol/L
RA dilution from 100 mg/ml of stock solution in DMSO and explants were
cultured for another 24 hours.21
The next day RA media
were removed, explants washed twice with fresh media, and cultured for
further 24 hours. At the end of culturing, explants were washed in
ice-cold PBS for 15 minutes and preserved in 4% paraformaldehyde in
PBS.
Detection of Apoptotic Cells
Whole embryos, dissected guts, or gut explants were assessed for the presence of apoptotic cells. TUNEL with Dead-End colorimetric detection system (Promega, Southampton, UK) was used according to the manufacturer’s instructions with minor modifications concerning blocking and permeabilization of the whole-mount specimens with 2% Marvel and 0.5% Triton X-100 in PBS. The final product of peroxidase coupled to biotin was detected with amino-ethyl carbazole. A separate set of gut explants treated overnight with 4 mg of anisomycin (Sigma, Poole, Dorset, UK) was used as a positive control for apoptosis22 according to the manufacturer’s suggestions.
Whole-Mount in Situ Hybridization and Immunohistochemistry
Prehybridization, hybridization, and posthybridization washes of
whole embryos and dissected gastrointestinal tracts were performed as
described before.14,23
Briefly, prehybridization,
hybridization, and blocking with blocking reagent (Roche Diagnostics,
Lewes, Beds, UK) were performed overnight. The probe for
c-ret19
was used at concentrations of 25 to 100
ng/ml of hybridization buffer. Prehybridization, hybridization, and
posthybridization washes were performed at 70°C. The probe was
visualized using alkaline phosphatase-conjugated anti-digoxigenin
antibody and BM Purple AP (both from Roche Diagnostics). The color
reaction was stopped by washing overnight in PBS/10 mmol/L
ethylenediaminetetraacetic acid (EDTA) and the signal was preserved by
overnight incubation in 4% paraformaldehyde in PBS. Control
experiments with probes omitted to check endogenous
alkaline-phosphatase activity were performed at each developmental
stage of the guts. Whole-mount embryos or gut primordia were
photographed after in situ hybridization using a Zeiss
Axiophot microscope with Nomarski optics (Jena, Germany). A rabbit
polyclonal antibody against Hoxb518
and monoclonal
antibody against -SMA (Sigma) in conjunction with c-ret in
situ hybridization were used in this study to monitor enteric
neuromuscular development. Detection was performed as previously
described.18
Control experiments with primary antibody
omitted were also performed.
Vibratome Sections
Some hybridized or immunostained whole-mount specimens were sectioned using a vibratome to determine the cellular distribution of the products in transverse or sagittal planes across the gut wall. Specimens were incubated in an embedding mix (gelatin, albumin, and sucrose) orientated and solidified with the addition of 25% glutaraldehyde. Sections were cut 30- to 50-µm thick and mounted in Citifluor (Agar Scientific, Stansted, Essex, UK) and sealed with nail varnish for a photography using a Zeiss Axiophot microscope with Nomarski Optics.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Maternal RA injection at E9.5 induced truncation of the posterior
body axis together with tail bud agenesis in all 39 harvested embryos
coexistent with stage-dependent morphological changes in
gastrointestinal tracts. RA induced ectopic expression of
c-ret and Hoxb5 in the central and peripheral nervous
system. In the gastrointestinal tracts of RA-treated embryos, impaired
cecal bud formation and delayed gut looping was observed in the first
half of gestation associated with malrotation of the caudal ileum
adjacent to the cecum in 31 embryos collected at the age of 10.5 to
E12.5 dpc. These changes were associated with a delayed onset of
-SMA expression and migration of c-ret-expressing enteric
neuronal precursors into the hindgut. In the rostral gut, RA induced
ectopic expression of c-ret and Hoxb5 in the esophagus.
Similarly, RA-treated E10.5 gut explants showed a lack of -SMA
immunoreactivity and delayed migration of c-ret-expressing
neural crest cells into the postcecal intestine. These changes were
coexistent with RA-induced ectopic c-ret and Hoxb5
expression in the esophagus in the explant. At E16.5 in five of eight
embryos morphological caudal gut anomalies in the putative anorectal
region were noted in vivo.
Morphological Anomalies in Embryos and Guts Treated in
Vivo with RA (Figure 1)
At E10.5, all 39 embryos harvested from RA-injected mice
demonstrated a truncated posterior body axis. Apoptotic cells were seen
in the degenerating tail and limb buds and in the whole prevertebral
region as assessed by whole-mount TUNEL staining. In vehicle-treated
embryos, by contrast, only a small proportion of apoptotic cells were
present in the caudal extremity of well-developed tail bud (Figure 1, A and B)
. No apoptotic cells were detected, however, in the gut (data not
shown). RA treatment resulted at E12.5 in a lack of tails and shorter
caudal body regions characteristic of the caudal regression syndrome,
as compared to control (Figure 1, C and D)
. In addition, at E16.5 there
were anorectal ring-like constrictions, in five of the eight (62%)
RA-treated embryos examined but these changes were not found in
controls (Figure 1
; E to J). Moreover in all 31 embryos with caudal
agenesis (100%) there were morphological gut anomalies at earlier
stages of gut development. These included impaired outgrowth of the
cecal bud observed in control E11.5 and E12.5 embryos (Figure 1
; K to
N) accompanied by delayed formation of the second intestinal loop at
E12.5 (Figure 1O)
and malrotation of the caudal ileum adjacent to the
cecum (Figure 1P)
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In controls at E10.5, -SMA immunoreactivity was absent in the
gut but was noted in the adjacent vitelline aorta (not shown).
Significant -SMA immunoreactivity was first noted at E11.5 in cells
located at the ventral site of rostral, prececal gut. In contrast,
RA-treated guts of the same age lacked -SMA expression although
attached vitelline aortas were positive (Figure 2, A and B)
. At E12.5
in controls -SMA was expressed in the whole small intestine up to
the cecal level (Figure 2C)
, whereas in RA-treated guts of the same
age, -SMA expression was confined to the rostral small intestine,
resembling the pattern observed 24 hours earlier (E11.5) in
vehicle-treated controls (Figure 2D)
. Vibratome sections showed that
the circular muscle layer expressing -SMA remained poorly
differentiated in RA-treated gut at E12.5 in comparison with the
controls (Figure 2, E and F)
. At E16.5, -SMA immunoreactivity was
present in the whole gut of the controls and RA-treated embryos (not
shown).
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. After RA treatment by E11.5, c-ret-positive cells were
only present in the prececal gut (Figure 2H)
. In E12.5 controls,
c-ret-expressing cells colonized almost the whole large intestine
(Figure 2I)
whereas in RA-treated embryos c-ret-positive cells were
noted in the cecum and adjacent rostral large intestine (Figure 2J)
. By
E16.5, c-ret-expressing cells were detected in the whole intestine both
in controls and RA-treated intestines (not shown).
In contrast to the migration pattern of c-ret-positive
cells, Hoxb5 immunoreactivity in control and RA-treated guts was noted
only in the prececal gut and small intestine at E10.5, E11.5, and E12.5
(Figure 2, K and L)
. However, in rostral gut Hoxb5 expression pattern
was different after RA treatment from that in controls. In controls,
Hoxb5 rostral expression boundary was confined to the stomach in all
developmental stages examined, whereas in RA-treated guts ectopic Hoxb5
expression was noted in the esophagus at E10.5 and E11.5 but not at
E12.5 (Figure 2, K and L
, and Figure 3, A and B
). This observation was
confirmed on vibratome sections (Figure 3, C and D)
. Similarly, in
contrast to controls at E10.5 and E11.5, ectopic c-ret expression was
found in the esophagus after RA treatment (Figure 3, E and F)
.
In addition, in whole control embryos at E10.5, the anterior Hoxb5
expression boundary in the hindbrain was between rhombomeres 7 and 8 at
the level between somites 4 and 5 (Figure 3G)
. In RA-treated embryos,
there was a rostral shift of expression up to the level of rhombomere 4
at the preotic hindbrain (Figure 3H)
. In control E10.5 embryos,
c-ret expression was observed in the cranial ganglia, and
also in dorsal root ganglia and the sympathetic chain of the peripheral
nervous system (Figure 3I)
. After RA treatment, ectopic c-ret
expression appeared in the midbrain, in the caudal branchial arches,
and in the dorsal rami of the spinal ganglia (Figure 3J)
.
RA-Induced Morphological Changes in Gut Explants (Figure 4)
The results of the preliminary experiments showed that 10-5 mol/L of RA induced the appearance of unlooped gut with an underdeveloped cecum in 10 of 10 (100%) of treated explants without stimulating apoptotic cell death, as compared to 10-4 mol/L of RA in which apoptosis was induced. RA (10-6 mol/L) produced morphological changes in 5 of 10 (50%) of explants whereas 10-7 mol/L of RA induced an altered gut appearance in 3 of 10 (30%) treated explants. No morphological changes or apoptotic cells were detected in control explants.
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; A to F). These morphological changes coexisted with
alterations in gut neuromuscular development as assessed by -SMA,
c-ret, and Hoxb5 expression.
All 10 control explants examined showed strong -SMA expression in
the whole gut up to the start of the cecum (Figure 4A)
that paralleled
observations in the controls in vivo at E11.5 to 12.5. After
RA treatment, all 10 explants of the same age showed a lack of -SMA
reactivity at the end of 72 hours of culture (Figure 4B)
.
In control explants, c-ret-expressing cells colonized the whole
postcecal gut after 72 hours of culture in 9 of 10 explants (90%)
whereas RA-treated explants showed impaired colonization of the
postcecal intestine in 8 of 10 explants (80%) (Figure 4, C and D)
. In
untreated gut explants the rostral limit of c-ret and Hoxb5
expression was in the dorsal stomach and the midgut (Figure 4, C and E)
. After RA treatment, Hoxb5 expression remained unchanged in the
midgut but appeared rostrally in the esophagus in all explants examined
(Figure 4F)
. Similar rostral expansion of c-ret expression into the
esophagus was observed in all gut explants cultured in the presence of
RA (Figure 4D)
. All these changes paralleled those noted in
vivo. In the RA-treated gut explants a few apoptotic cells were
noted. In contrast, control gut cultured overnight with anisomycin, a
known inducer of apoptosis, showed the presence of many apoptotic cells
(Figure 4, G and H)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The nature of the effects of RA on gut development is primarily unknown
and in particular the responsiveness of gut neuromusculature to
exogenous RA has not been previously studied. Gut morphogenesis
involves numerous interacting processes that are spatially and
temporally regulated and coordinated, which are concomitant with gut
elongation and looping and with the outgrowth of asymmetrical
structures such as the stomach and the cecum. These events coincide
with the colonization of the gut by neural crest-derived enteric
neuronal precursors. All these complex processes involve intense
proliferation and differentiation of gut layers because of inductive
signals produced by gut endoderm and mesoderm.29
The
presence of endogenous RA in the developing gut is indicated by the
expression of genes implicated in RA synthesis or metabolism that in
the gut coexist with the expression of RAREs, RARs, and retinol-binding
proteins.13,30-32
As teratogenic effects of RA are
transduced by specific retinoid receptors33
it would be
conceivable to conclude that increased RA levels both in
vivo and in vitro may interfere with natural
morphogenetic processes including gut looping, rotating, and growth of
asymmetrical structures such as the cecum resulting in the appearance
of an underdeveloped cecum and second gut loop coexistent with
malrotation of the caudal ileum adjacent to the cecum. The teratogenic
effect of RA persisted in a proportion of older embryos that showed
ring-like constrictions at the caudal gut extremity resulting in the
blind-ending rectum. These RA-induced gut malformations included
imperforate rectum, malrotation, or situs inversus associated with
changes in the gut neuromusculature are also reported in patients with
caudal regression syndrome or sirenomelia, VATER/VACTERL associations,
or Currarino triads.34-42
A number of studies have
previously demonstrated that either an excess or deficiency in the
retinoid signal results in laterality defects in vertebrate embryos. A
recognized anomaly of disturbed heart looping in mouse embryos is
caused by teratogenic doses of RA, and the development of uncoiled
intestine with delayed expression of intestinal -SMA has been also
described after stage-dependent RA treatment of Xenopus
embryos.43
The target genes of RA signaling in the gut are
unclear but misexpression of homeobox transcription factor, pitx2,
suggests that it could be a key factor in the process of heart and gut
looping/rotation in Xenopus.44
In the mouse,
heart looping involves temporally and spatially restricted activity of
transcription factors, extracellular matrix, and cytoskeleton proteins
including actin.44
These reports are in agreement with our
data that showed that RA treatment affects gut looping/rotation and
-SMA expression both in vivo and in vitro. A
previous study suggests that the initiation of enteric smooth muscle
differentiation in the gut, can be induced by putative diffusable
factors or their gradients produced by intestinal
endoderm.20
Interestingly, vascular -SMA expression can
be directly regulated by peptide growth factor, such as transforming
growth factor-ß (TGFß),45
which is also expressed by
the developing gut endoderm and is known to be involved in the
induction of mesodermal differentiation in early murine
embryos.29,46
Impaired gut looping and cecal development
associated with malrotation of the caudal ileum and transiently
perturbed determination and differentiation of enteric smooth muscle
cells by RA cannot be explained in terms of overall developmental delay
as the differentiation and morphogenesis of rostral structures such as
eyes, ears, and frontonasal mass structures in the affected embryos
appear to be normal when compared to control embryos. Similarly,
RA-induced caudal regression is associated with morphological
alterations in the more caudal gut whereas the esophagus and the
stomach remained unchanged. Moreover, -SMA expression in vitelline
artery associated with the gut appears to be the same as for control
embryos and therefore can serve as an internal control of development
and specificity of RA action in the gut. It can be concluded, that
differentiation of enteric smooth muscle cells at the correct time
could represent an essential event for the proper differentiation and
morphogenesis of the digestive tract.
The interference of RA with cecal bud formation suggests a role for RA
signaling in the regulation of putative genes involved in the
morphogenesis of this gut region. It is of interest that nested
Hox gene expression has been described in the cecum during
murine development.14,47
Defects in homeobox genes are
also found in patients with sacral agenesis48
and
in mutant mice where gene-targeted mutations of 5' Hox genes
such as Hoxd13 and Hoxa13/Hoxd13 cause severe
disorganization or lack of rectal musculature.49,50
In
addition, disorganization or missing muscle layers in the muscularis
propria and dysplasia of enteric nervous system have been described in
patients with congenital anal atresia and rectal
stenosis.15,16
These observations could be relevant to the
presence of ring-like constrictions in the caudal gut found in a
proportion of embryos after in vivo RA treatment. We found
that RA-induced changes in the gut appearance in vivo
paralleled those seen in in vitro experiments. The lack of
-SMA reactivity coexisted with delayed migration of
c-ret-positive neural crest cells to colonize the postcecal
gut indicates an important role of the differentiated mesoderm for the
migration of enteric neuronal precursors. After RA treatment in
vivo and in vitro, the appearance of the rostral gut
was not changed, however, a stage-specific, rostral shift of the
expression of Hoxb5 and c-ret, markers of the developing enteric
nervous system, was observed in the esophagus. These changes paralleled
the rostral shift of the expression boundary of these genes observed in
the embryonal hindbrain after in vivo RA treatment. Previous
studies have suggested that homeobox gene expression, including Hoxb5
can be regulated by RA in vivo and that RA induces their
ectopic expression that often leads to specific morphological
transformations.51
In the neural tube and vertebral
column, Hox genes are expressed with distinct anterior
boundaries set by RA-based signaling. They respond to the addition of
exogenous RA by a rostral shift in their expression domains often
associated with homeotic transformation of the hindbrain segments or
vertebrae.5,52,53
The involvement of RA in the regulation
of the expression of homeobox genes such as Hoxa1 and
Hoxb1 in the endoderm of the anterior intestinal portal has
been previously reported.54-56
It has been also shown
that some Hox genes contain RA-responsive elements in their
promoter regions.54-56
Exogenous RA also induced ectopic
expression of c-ret in the embryonic spinal nerves that
suggests RA involvement in the regulation of c-ret expression. This is
in agreement with previous reports describing RA involvement in the
regulation of c-ret expression in the cranial ganglia and in
the neural tube in chicken embryos57
and in the developing
visceral organs such as the kidney.58
Our results indicate
that the distribution of cells expressing markers of neural crest cells
in the esophagus is affected by RA treatment because it is still
unclear if the ectopic expression of c-ret and
Hoxb5 has been induced in cells that normally do not express
these markers or is the result of altered migration of cells that
usually express them.
The results presented in this study suggest that stage-specific
in vitro and in vivo treatment of the developing
gut with all-trans RA affects gut development by interfering with gut
looping/rotation and with the growth of asymmetrical structures such as
the cecum. These changes were coexistent with persistent teratogenic
changes in the caudal gut extremities. Moreover, differentiation of the
gut mesoderm and enteric nervous system as indicated by the expression
of specific markers such as -SMA, c-ret, and Hoxb5 was
also affected. These observations demonstrate that RA has a direct
effect on gut morphogenesis and innervation and that the RA-induced
caudal agenesis is a useful animal model to study gut responsiveness to
exogenous teratogenic agents such as RA.
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Acknowledgements |
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Footnotes |
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Supported by the Children Research Appeal Trust (ChRAT).
This work was partly undertaken by Great Ormond Street Hospital for Children National Health Service Trust who receive a proportion of its funding from the National Health Service Executive, London, England. The views expressed in this publication are those of the authors and not necessarily those of the National Health Service Executive.
Accepted for publication September 9, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-actin induction by TGFß in smooth muscle versus non-smooth muscle cells. Arterioscler Thromb Vasc Biol 1999, 19:2049-2058This article has been cited by other articles:
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