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First published online 16 May 2007
doi: 10.1242/dev.003814
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1 Department of Pathology and Cell Biology, Columbia University, P&S, New
York, NY 10032, USA.
2 Department of Cell and Molecular Biology, Tulane University, New Orleans, LA
70118, USA.
* Author for correspondence (e-mail: mdg4{at}columbia.edu)
Accepted 3 April 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Autonomic nervous system, bHLH transcription factor, Conditional knockout, Enteric nervous system, Gene knockout, Gut development, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
Hand genes Hand1 (eHand) and Hand2 (dHand)
are members of the twist family of bHLH transcription factors
(Firulli, 2003). Hand genes
are expressed in the developing heart, in lateral mesoderm and in subsets of
neural crest-derived cells (Cserjesi et
al., 1995). Hand genes are best-known for their roles in cardiac
(Firulli et al., 1998;
Srivastava et al., 1995;
Srivastava et al., 1997),
vascular (Dai et al., 2004;
Yamagishi et al., 2000) and
limb development (Cai and Jabs,
2005). They also play crucial roles in the development of
sympathetic neurons, where they are necessary for catecholamine expression
(Howard, 2005;
Lucas et al., 2006). Partial
redundancy might occur between Hand1 and Hand2 functions in
heart (McFadden et al., 2005)
and sympathetic (Howard et al.,
1999) ganglia.
Although Hand2 expression is linked to the specification of the
noradrenergic phenotype of sympathetic neurons
(Howard, 2005), Hand2
is also expressed in parasympathetic ganglia
(Morikawa et al., 2005) and
gut (Cross et al., 1995;
Cserjesi et al., 1995;
Hollenberg et al., 1995;
Srivastava et al., 1995),
which do not contain noradrenergic neurons
(Brookes and Costa, 2006;
Furness, 2000;
Lomax and Furness, 2000). The
function of Hand genes in the autonomic nervous system is thus not limited to
noradrenergic phenotypic expression. Because Hand2 expression in
postnatal day (P)19 embryonic carcinoma cells causes the expression of
peripheral neural markers, such as peripherin 1, and its expression occurs
before neurons arise (Morikawa et al.,
2005), crest-derived precursors may require Hand2 to
become neurons rather than only for the specification of their
neurotransmitter-defined identity
(Hendershot et al., 2007;
Howard, 2005).
Hand genes are expressed in developing mouse
(Cserjesi et al., 1995;
Hendershot et al., 2007;
Hollenberg et al., 1995) and
chick (Wu and Howard, 2002)
gut. In situ hybridization has suggested that Hand2 is expressed in
the presumptive enteric nervous system (ENS) in mice
(Dai et al., 2004) and chicks
(Wu and Howard, 2002). We now
report that Hand2, but not Hand1, is necessary for
crest-derived cells to become enteric neurons. We found that crest-derived
cells selectively express Hand2, whereas mesodermal derivatives
express Hand1, in mouse gut. Additionally, our results show that
crest-derived precursors from Hand2-/- mouse gut survive
and give rise to glia in vitro but fail to develop as neurons. Hand2
expression was also found to be developmentally regulated, but was found to
continue at a low level in mature neurons. We also show that Hand2 is
intranuclear during differentiation, but cytoplasm-restricted in mature
neurons. Although the conditional knockout of Hand2 in neural crest
cells did not prevent the limited expression of early neural markers, it did
block the terminal differentiation of enteric neurons.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Mice were considered wild type only when verified to be
Hand2+/+ and not Hand2+/-. The
generation and characterization of transgenic mice bearing a lacZ
reporter gene encoding ß-galactosidase under the control of the
Hand1 promoter have previously been described
(Dai et al., 2004). To
generate mice carrying `floxed' Hand2 alleles, the targeting
construct of the Hand2 gene included loxP sites placed 5' of
the start of transcription and within the first intron. Recombination between
the loxP sites deletes the first intron, which includes the bHLH domain and
most of the coding region. A manuscript describing the generation of this
mouse line will be presented elsewhere
(Morikawa et al., 2007). Mice
lacking Hand2 in crest-derived cells (Wnt1-Cre-H2
)
were generated by crossing males carrying the Wnt1-Cre transgene and
heterozygous for the null allele of Hand2
(Srivastava et al., 1997) with
females homozygous for the `floxed' Hand2 allele. Mutant embryos were
identified by PCR genotyping of extraembryonic membranes. Mice with the
Wnt1-Cre transgenes (Danielian et
al., 1998) were contributed by Henry M. Sukov (University of
Southern California, Los Angeles, CA; permission from Andrew MacMahon,
University of Illinois, Urbana, IL).
Isolation of crest-derived cells
To obtain enteric crest-derived cells, fetal mouse or rat gut was
dissociated with collagenase. The resulting cellular suspension was cultured
or purified by immunoselection with rabbit polyclonal antibodies to the common
neurotrophin receptor p75NTR (gift from Moses Chao, New York
University, NY) as described previously
(Chalazonitis et al., 1994;
Chalazonitis et al.,
1997).
Cell and organotypic tissue culture
Suspensions of dissociated or immunoselected cells were plated
(2x105 cells/ml) on laminin-coated charged glass chamber
slides (NUNC, Denmark). The maintenance medium consisted of Neurobasal medium
(Invitrogen, Carlsbad, CA) supplemented with L-glutamine (2 mM; Invitrogen),
neurotrophin 3 (1.4 nM), glial cell-line-derived neurotrophic factor (0.3 nM),
epithelial growth factor (1.7 nM), and basic fibroblast growth factor (0.2
nM). Cells were plated in the maintenance medium enriched with 10% fetal
bovine serum (FBS) and 20% horse serum (plating medium), and were transferred
to the maintenance medium with B-27 Supplement (Invitrogen) 1 day later.
Explanted foregut was cultured in a three-dimensional collagen gel as
previously described (Natarajan et al.,
1999; Tessier-Lavigne et al.,
1988). Foregut explants were cultured in the plating medium for 2
days, then transferred to maintenance medium supplemented with 2% FBS. Media
were changed at 2-day intervals. Explants of Hand2-/- gut
were transfected with a pcDNA3.1 construct containing the coding sequence of
Hand2 with an in frame histidine tag in a transfection mixture (50
µl; plasmid DNA 0.2 µg, 0.5 µl lipofectamine 2000) that was
microinjected directly into the explants.
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). The cultures were transfected twice with small interfering
(si)RNA, once at 2 hours after explantation and the second time at 2 days
after explantation. A commercial kit (Silencer siRNA construction; Ambion,
Austin, TX), used according to the manufacturer's directions, was employed to
prepare siRNA directed against Hand2. A control siRNA was directed
against Gapdh. Three different Cy3-labeled siRNAs (Silencer siRNA
labeling kit; Ambion) were made to target Hand2 mRNA sequences
(siRNA1: 5'-AAGATCAAGACACTGCGCCTG-3'; siRNA2:
5'-AAGGCGGAGATCAAGAAGACC-3'; and siRNA3:
5'-AAGAAGACCGACGTGAAAGAG-3') and pooled. Transfection was carried
out, using transfection reagent (Siport; Ambion). The transfection mixture (75
µl; siRNA mixture 60 pM, 1.5 µl transfection reagent) was added directly
to the culture medium (300 µl total volume) in four well plates (NUNC).
Lentivirus-based vectors (pFIV-H1/U6-copGFP; System Biosciences, Mountain
View, CA) were engineered to express siRNA1, siRNA2 or siRNA3 (see above;
siRNAHand2/GFP), or a control siRNA (siRNAscrambled/GFP)
with a scrambled target sequence (siScrambled:
5'-AAGTAAAGCCAATCGCCGCGT-3'), and were then transfected into
cultured immunoselected or dissociated intestinal cells using lipofectamine
2000 (Invitrogen). The transfection mixture (50 µl; plasmid DNA 0.2 µg,
0.5 µl lipofectamine 2000) was added directly to the culture medium (200
µl total volume) in eight-well chamber slides (NUNC). The ability of
siRNAHand2/GFP transfection to silence Hand2 was evaluated
in stably transfected Hand2-expressing HeLa cells. The control level
of Hand2 expression was taken as that found in cells exposed to
siRNAscrambled/GFP. Each of the three different
siRNAHand2/GFP constructs were found to silence Hand2
expression; one of these constructs (siRNA3), which reduced Hand2
expression by 77% (P<0.001), was selected for subsequent
experiments. Information on methods used to select the target sequences can be
found at
http://www.rockefeller.edu/labheads/tuschl/sirna.html
(revised May 2006).
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).
All PCR products were sequenced and found to match the appropriate sequences
in the GenBank. Transcripts encoding Hand2 (sense:
5'-TACCAGCTACATCGCCTACCT-3'; antisense:
5'-TCACTGCTTGAGCTCCAGGG-3'), Hand1 (sense:
5'-TTGAAGGCTCGACTCAAGGT-3'; antisense:
5'-AGCGACAAGAAGGAAAACCA-3'), Phox2b (sense:
5'-CCTCAATTCCTCTGCCTACG-3'; antisense:
5'-AGTTTGTATGGAACTGCGGC-3'), Mash1 (Ascl1;
sense: 5'-GACTTGAACTCTATGGCGG-3'; antisense:
5'-AGATGCAGGATCTGCT-3'), and ß-actin (sense:
5'-TGTTTGAGACCTTCAACAC-3'; antisense:
5'-CAGTAATCTCCTTCTGCATCC-3') were quantified in fetal and mature
mouse gut by using real-time PCR (SYBR Green I; LightCycler, Roche Molecular
Biochemicals, Indianapolis, IN). Three independent experiments were carried
out and a standard curve was obtained for each gene product. Transcripts
encoding rat Hand1 (sense: 5'-CCAACATGAACCTCGT-3';
antisense: 5'-CCTGAACCTTTTCGCC-3'), rat p75NTR
(sense: 5'-GAGGGCACATACTCAGACGAAGCC-3'; antisense:
5'-GTCTATATGTTCAGGCTGGTAA-3') and rat ß-actin (see
above) were amplified by using reverse transcriptase (RT)-PCR.
In situ hybridization and immunocytochemistry
Details of the procedures used have been presented previously
(Li et al., 2006). Briefly,
tissues were fixed with 4% formaldehyde (from paraformaldehyde) in 0.2 M
phosphate buffer at pH 7.4. Fetuses or dissected bowel were fixed overnight at
4°C, whereas cultures were fixed for 30 minutes at ambient temperature.
Fixed preparations were dehydrated, cleared and paraffin sectioned or
cryoprotected (30% sucrose; 4°C), embedded in Neg50 (Richard Allan
Scientist, Kalamazoo, MI), frozen (liquid N2), and cryosectioned.
Sections were collected on Superfrost slides (Fisher Scientific, UK).
Digoxygenin (Dig)-labeled full-length cRNA probes encoding mouse Hand2 (1.2
kb) and Hand1 (1.7 kb) were synthesized and used for in situ hybridization.
Pre-hybridization and hybridization were carried out for 2 hours at 70°C
and for 18-22 hours at 70°C, respectively.
For immunocytochemical detection of markers, sections were washed with
phosphate-buffered saline with Tween (PBST) and, if horseradish peroxidase
(HRP) was to be used to visualize antigens, they were exposed for 20 minutes
to 0.3% H2O2 to quench endogenous peroxidase activity.
Primary and secondary antibodies (Table
1) were applied as described previously
(Li et al., 2006). Sections
were blocked overnight with monovalent goat Fab fragments against mouse IgG
(Jackson Laboratories, West Grove, PA) before applying primary antibodies of
mouse origin. DNA in sections was stained with bisbenzimide to enable cell
density to be determined. Coverslips were mounted in 50% glycerol in 0.5 M
bicarbonate buffer (pH 8.6).
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Quantitative imaging
Immunocytochemically labeled cells or those expressing GFP were counted
when cells could individually be discerned. Counts were normalized to the
total number of cells, which was determined by counting bisbenzamide-stained
nuclei. Alternatively, the density of labeled cells was ascertained by
computer-assisted morphometry (Openlab software; Improvision, Lexington, MA).
Images were acquired using a cooled CCD camera (Retiga; Q Imaging) installed
on a Leica DMRXA2 microscope. Where indicated in the text, confocal images
were obtained with a Zeiss LSM 510 NLO Multiphoton Confocal Microscope.
Statistical analyses
Student's t-test was used to compare sample means. Equality of
variances was analyzed with an F test and Welch's correction was employed when
variances of populations was significantly different.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), and to that of Mash1 (also known as Ascl1
- Mouse Genome Informatics), which is required later by a subset of neural
precursors (Blaugrund et al.,
1996). Hand2 expression was developmentally regulated in
the mouse gut (Fig. 1A, red
curve). Transcripts encoding Hand2 were detected at E9.5, when the
first émigrés from the crest begin to colonize the bowel
(Rothman et al., 1984;
Young et al., 1999).
Hand2 expression was later upregulated and peaked at E12.5. By E14,
Hand2 expression declined to a lower level, which was maintained
throughout adult life. Hand1 expression was detected by E9.5 but,
unlike that of Hand2, was bimodal
(Fig. 1A, green curve), peaking
at E12.5, declining by E14, and being upregulated again during adult life.
Phox2b expression was upregulated before that of Hand2
(Fig. 1A, blue curve). Although
transcripts encoding Hand2, Hand1 and Phox2b were detectable
at all ages tested, transcripts encoding Mash1
(Fig. 1A, purple curve) were
not detected prior to E11 or after E17.
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)
were analyzed to identify Hand1-expressing cells.
ß-galactosidase was detected in smooth muscle cells in the stomach, and
in the small and large intestines, but not in ganglia
(Fig. 1B). Interstitial cells
of Cajal (ICCs), identified by coincident immunostaining with antibodies to
Kit, also contained ß-galactosidase
(Fig. 1C). This localization
was confirmed by the reciprocal in situ localization of transcripts encoding
Hand1 in muscle layers, but not in neural crest derivatives, and encoding
Hand2 in neural crest derivatives, but not in smooth muscle
(Fig. 1D). Further confirmation
was obtained by separating crest and non-crest-derived cells from dissociated
preparations of rat fetal bowel by positive and negative immunoselection with
antibodies to p75NTR. Transcripts encoding Hand1 were not detected
in preparations of immunoselected crest-derived cells, but were abundant in
preparations of non-crest-derived cells
(Fig. 1E).
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), mucosal expression of Hand2 was
investigated further. RNA was extracted separately from the mucosa and from
the ganglion-containing layer. Transcripts encoding Hand2 were detected, by
RT-PCR, in the ganglion-containing bowel wall but not in the mucosa
(Fig. 1F). Different lineages
of cells in the developing gut thus express Hand2 and
Hand1.
Hand2 is expressed in the gut by cells in neuronal and glial lineages
Double labeling was employed with antibodies to the neuronal markers HuC
and HuD (collectively referred to here as Hu), and a riboprobe to detect
transcripts encoding Hand2 (Fig.
1G). At E14, all Hu-immunoreactive (Hu+) cells contained
transcripts encoding Hand2. Hand2 is thus expressed at E14 by all
cells developing as neurons; however, the presence of
Hand2-expressing cells that lack Hu
(Fig. 1G, arrows) is consistent
with the possibility that Hand2 is also expressed by glia. The
immunoreactivity of the early glial marker brain-specific fatty acid-binding
protein (B-FABP, also known as Fabp7 - Mouse Genome Informatics) was thus
demonstrated simultaneously with transcripts encoding Hand2
(Fig. 1H). A subset of
Hand2-expressing cells was B-FABP+. Transcripts encoding
Hand2, therefore, are found in cells developing as neurons and glia.
The pattern and timing of Hand2 expression are consistent with its
involvement in the development of enteric neurons and/or glia.
The intracellular distribution of Hand2 changes during neuronal development
The persistence of Hand2 expression in mature enteric neurons
raises the possibility that Hand2 acquires another function following
the differentiation of neurons and/or glia. Such a change in function might be
associated with a change in its intracellular compartmentation. Hand2 protein
was thus located immunocytochemically by using a rabbit polyclonal antibody
raised against a synthetic peptide corresponding to the sequence
VKEEKRKKELNEILK, which is found in the amino terminal domain of Hand2
(Dai and Cserjesi, 2002). The
antibodies showed Hand2 protein in the nuclei of transfected
Hand2-expressing HELA cells, but it did not react with parental HELA
cells (Fig. 2A). More
importantly, the antibodies immunostained cells in the presumptive ENS and
sympathetic ganglia of wild-type mice, but did not do so in those of
Wnt1-Cre-H2 animals (Fig.
2B).
Enteric neurons, differentiating in vitro from cultures of dissociated gut at E11.5 or E14, were Hand2+ within 24 hours of plating. Only nuclei were Hand2+ at 24 hours in cells dissociated at E11.5 (Fig. 2C); however, by 6 days after plating, both nuclei and cytoplasm were Hand2+. In contrast to neurons developing from E11.5 gut, the cytoplasm as well as the nuclei of neurons in cultures of bowel dissociated at E14 was already Hand2+ by 24 hours post-dissociation (Fig. 2D). Hand2 immunoreactivity was restricted to the cytoplasm in essentially all enteric neurons of adult mice, in situ, in whole mounts of laminar preparations of the bowel wall, and in culture (Fig. 2E). These observations are consistent with the idea that Hand2 is intranuclear and thus able to influence transcription during differentiation; however, after differentiation is completed, cytoplasmic sequestration may inactivate Hand2 as a transcription factor.
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; Srivastava et al.,
1995), and enteric neurons cannot be recognized prior to E10.5-E12
(Rothman and Gershon, 1982;
Young et al., 2003). ENS
precursors, however, enter the foregut at E9-E9.5, and give rise to neurons
and glia in cultured explants (Rothman et
al., 1984). Transcripts encoding the crest marker Phox2B
are detectable by RT-PCR in bowel of Hand2-/- mice at E9.5
(Fig. 3A), suggesting that
crest-derived cells do enter this gut. To confirm their presence, the
immunoreactivities of four crest markers - Phox2B
(Pattyn et al., 1999),
p75NTR (Anderson et al.,
2006; Baetge et al.,
1990), Ret (Durbec et al.,
1996; Pachnis et al.,
1993; Schuchardt et al.,
1994) and Sox10 (Herbarth et
al., 1998; Kapur,
1999; Southard-Smith et al.,
1998) - were compared in sections of bowel cut from E9.5
Hand2-/- and wild-type littermates. Phox2B, Ret, Sox10
(Fig. 3B) and p75NTR
(Fig. 3C) immunoreactivities
were each found, distributed similarly in the outer gut mesenchyme, and
comparable in abundance in Hand2-/- and wild-type animals.
These observations suggest that the Hand2-/- gut is
normally colonized by crest-derived precursors and that Hand2
expression is not required for the initial expression of Phox2B, Ret, Sox10 or
p75NTR.
Neurogenesis, but not gliogenesis, fails in explants of Hand2-/- gut
Because crest-derived precursors are present in the foregut of
Hand2-/- fetuses, it was possible to explant the bowel
before the death of the animals, culture the explants, and evaluate the
development of neurons and glia in vitro. Foregut was explanted from wild-type
and Hand2-/- mice at E9.5 and cultured for 6-10 days in
three-dimensional collagen gels (Natarajan
et al., 1999). Markers used to demonstrate the development, if
any, of neurons were Hu (Fairman et al.,
1995; Phillips et al.,
2004), PGP9.5 (also known as Uchl1 - Mouse Genome Informatics)
(Sidebotham et al., 2001;
Wilkinson et al., 1989) and
neuron-specific enolase (NSE; also known as Eno2 - Mouse Genome Informatics)
(Bishop et al., 1985;
Hearn et al., 1999).
Antibodies to B-FABP were employed to demonstrate glia
(Simon et al., 1993;
Young et al., 2003). Hu+,
PGP9.5+ and NSE+ neurons developed reproducibly in cultures from wild-type
mice (n=20) (Fig. 3D).
By contrast, cultures obtained from Hand2-/- mice
(n=9) never contained Hu+ cells
(Fig. 3E,I), although they did
contain B-FABP+ cells, which were comparable in form and abundance to B-FABP+
cells in cultures from wild-type mice (Fig.
3E). Despite the absence of Hu+ neurons in cultures of
Hand2-/- bowel, these cultures contained many Sox10+
(Fig. 3F), Phox2b+
(Fig. 3G), Ret+
(Fig. 3H) and
p75NTR+ (Fig. 3I)
cells; therefore, the knockout of Hand2 evidently prevented neither
the colonization of the bowel by émigrés from the neural crest,
nor their survival in vitro.
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Silencing of Hand2 expression prevents neuronal expression in cultures of enteric crest-derived cells
To verify independently that Hand2 expression is necessary for the
development of neurons, Hand2 expression was silenced with small
interfering RNA (siRNA). Experiments were carried out with crest-derived
cells, immunoselected with antibodies to p75NTR, from E13 bowel
(Fig. 4A,B). The percentage of
Hu+ neurons developing in cultures transfected with siRNAHand2/GFP
was significantly less than that developing in control cultures transfected
with siRNAscrambled/GFP (Fig.
4B, top left). More strikingly, the percentage of GFP+ cells
(Fig. 4B, top right) and, even
more, the percentage of GFP+/Hu+ doubly labeled cells were selectively reduced
by transfection with siRNAHand2/GFP
(Fig. 4B, bottom left). By
contrast, the numbers of GFP+ cells that did not co-express Hu were similar in
cultures transfected with siRNAHand2/GFP and
siRNAscrambled/GFP (Fig.
4B, bottom right), suggesting that the transfection efficiency of
siRNAHand2/GFP and siRNAscrambled/GFP is comparable.
Silencing of Hand2 expression, therefore, appears to interfere with
the development and/or survival of neurons. Comparable results were obtained
when siRNAHand2 was used to transfect organotypic cultures of gut
explanted at E10 or cultures of cells dissociated from fetal bowel at E14
(data not shown). Again, siRNAHand2-transfected crest-derived cells
failed to give rise to neurons, but neurons did arise from crest-derived cells
transfected with control siRNA. Together, these data suggest that interference
with Hand2 expression inhibits enteric neurogenesis and does so no
matter whether Hand2 is silenced prior to the in situ appearance of
neurons (E10) or after neuronal differentiation has already begun
(E13-E14).
Conditional knockout of Hand2 in the neural crest selectively blocks the terminal differentiation of enteric neurons
The in vitro data described above are consistent with the hypothesis that
Hand2 expression is required for the development of enteric neurons
but not glia. To test this hypothesis in vivo, experiments were carried out
with Wnt1-Cre-H2 mice, in which the knockout of
Hand2 is restricted to crest-derived cells. This restriction would
not be expected to prevent the development of cardiac abnormalities because
Hand2 is expressed in the cardiac crest; nevertheless, the
restriction of the knockout of Hand2 to crest-derived cells would be
anticipated to mitigate the resulting defect because the restricted knockout
would not interfere with Hand2 expression by cardiomyocytes. Because
the Wnt1-promoted expression of Cre occurs in premigratory crest cells, the
postmigratory crest-derived cells in the sympathetic ganglia and bowel of
Wnt1-Cre-H2 mice do not express Hand2 and thus lack
Hand2 immunoreactivity (see above; Fig.
2B). Wnt1-Cre-H2 mice hemorrhage in the cardiac
outflow tract and die at E12.5; nevertheless, fetuses survive long enough to
permit enteric neurogenesis to be analyzed. Wnt1-Cre-H2 mice
were compared with wild-type littermates at E12, when neurons are
morphologically recognizable in the wild-type mouse gut
(Rothman and Gershon, 1982;
Young et al., 2003). Sox10+,
Phox2b+ (Fig. 5A) and
p75NTR+ cells (Fig.
5B) were each present in the gut of Wnt1-Cre-H2
mice and were distributed identically to that observed in their wild-type
littermates; moreover, their densities in Wnt1-Cre-H2 and
wild-type bowel also did not differ significantly
(Fig. 5C). These observations
confirm that deletion of Hand2 does not interfere with the
colonization of the gut by crest-derived cells. By striking contrast, Hu+
cells were greatly diminished in the Wnt1-Cre-H2 stomach and
small intestine (Fig. 5D,E);
moreover, cells expressing MAP2 (also known as Mtap2 - Mouse Genome
Informatics; not illustrated) and type-specific neuronal markers, such as Dbh
and nNOS (Nos1 - Mouse Genome Informatics), were virtually absent in the
stomach and small intestine of Wnt1-Cre-H2 mice
(Fig. 6A). Despite this
reduction in enteric Hu+ cells in the Wnt1-Cre-H2 gut, there
were no changes from wild type in the Hu+ cells of DRG or prevertebral
sympathetic ganglia, or in the spinal cord of Wnt1-Cre-H2
fetuses (Fig. 5F). Very few
tyrosine hydroxylase (TH)+ or Dbh+ neurons, however, were observed in
paravertebral and prevertebral sympathetic neurons of
Wnt1-Cre-H2 mice (not illustrated). These observations
confirm that the knockout of Hand2 interferes specifically with the
terminal differentiation of enteric neurons.
In contrast to Hu+, Dbh+ and nNOS+ neurons, B-FABP+ glia were present in
Wnt1-Cre-H2 bowel and were distributed in a manner that
could not be distinguished from that in wild-type animals
(Fig. 6B). The density of
B-FABP+ glia, furthermore, did not differ significantly between
Wnt1-Cre-H2 and wild-type mice
(Fig. 6C). It is noteworthy
that the failure of neuronal differentiation in the gut in the absence of
Hand2 expression did not lead to a shift of crest-derived precursors
towards the glial lineage. The TUNEL procedure and activated caspase-3
(caspase 3) immunostaining were thus employed to test the hypothesis that the
knockout of Hand2 causes the death of crest-derived precursors that
would otherwise have developed as neurons. This hypothesis was not confirmed
because the abundance of cells in the gut demonstrated by TUNEL
(Fig. 6D) or activated
caspase-3 immunoreactivity (not illustrated), which were very low, did not
detectably differ in wild-type and Wnt1-Cre-H2 mice. In
contrast to gut, where apoptosis is uncommon during development
(Gianino et al., 2003), many
TUNEL- and activated caspase-3-demonstrable cells were found in the E12 DRG of
wild-type (Ernfors, 2001) and
Wnt1-Cre-H2 animals (Fig.
6D).
Because no evidence suggested that, in the absence of Hand2
expression, crest-derived enteric precursors shift development towards the
glial lineage or die, experiments were carried out to test the hypothesis that
these cells begin to develop as neurons but fail to terminally differentiate.
Neuronal markers, other than Hu, which are expressed by still-proliferating
neuronal precursor cells were thus investigated, including ß3-tubulin
(Fig. 7A,B), GAP-43 (Gap43;
Fig. 7C,D) and PGP9.5
(Fig. 7E,F). The densities of
cells expressing each of these markers were decreased in the
Wnt1-Cre-H2 bowel, although not to the same extent as was Hu
(Fig. 5E). In general, the
decreases in cells expressing ß3-tubulin
(Fig. 7B), GAP-43
(Fig. 7D) and PGP9.5
(Fig. 7F) were more severe in
the primordial stomach than in the small intestine. The marker least affected
by the knockout of Hand2 was PGP9.5; therefore, subsets of cells were
found in Wnt1-Cre-H2, but not wild-type, mice that were
PGP9.5+ but not GAP-43+ (Fig.
7G). Another test of the hypothesis that cells begin to develop as
neurons despite the knockout of Hand2 was to examine the coincidence
of Sox10 and Phox2b immunoreactivities. These transcription factors are
co-expressed in uncommitted precursor cells, but enteric neurons maintain
expression of Phox2b while downregulating Sox10; glia downregulate Phox2b
while maintaining Sox10. Many strongly Phox2b+ cells, which co-expressed
little or no Sox10, were observed in the gut of both wild-type and
Wnt1-Cre-H2 mice (Fig.
7H), suggesting that the inactivation of Hand2 does not
prevent precursors from entering the neuronal lineage.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Young et al.,
2003). Hand2 immunoreactivity is intranuclear in early neuronal
precursors; however, in mature neurons, Hand2 is predominantly cytoplasmic.
This subcellular localization is compatible with the ideas that Hand2 affects
the transcription of genes involved in neuronal differentiation but, when that
process is completed, Hand2 is both downregulated and sequestered in the
cytoplasm. Cytoplasmic sequestration might inactivate Hand2 but, if
reversible, might permit Hand2 to regain transcriptional activity when
plasticity is required. That remains to be demonstrated; however, the rapid
downregulation (between E12-E14; see Fig.
1A) and cytoplasmic sequestration of Hand2 suggests that it might
not play the role previously postulated for it as a determinant of
neurotransmitter choice (Hendershot et
al., 2007; Howard,
2005), which, for neurons using neurotransmitters, such as the
neurotransmitters vasoactive intestinal polypeptide (Vip) and calcitonin
gene-related peptide, does not occur until after E15-E16
(Blaugrund et al., 1996;
Pham et al., 1991). The
universal expression of Hand2 by all Hu+ neurons also suggests that
Hand2 expression is not sufficient for specifying any particular
neurotransmitter.
|
;
Young et al., 2003;
Young et al., 1999). Because
crest-derived cells are present, neurons and glia develop in cultures of
wild-type gut explanted at E9.5; however, only glia arise in similar explants
from Hand2-/- mice. The conclusion that enteric
neurogenesis is Hand2-dependent was further supported by the demonstration
that siRNA silencing of Hand2 expression in crest-derived cells from
wild-type gut also specifically blocked neuronal differentiation. In
Hand2-expressing HeLa cells, siRNA reduced expression by
approximately 77%. It is likely, therefore, that siRNA did not totally
extinguish Hand2 expression in enteric crest-derived cells. Because
neurogenesis was nevertheless abolished in siRNA-transfected cells, there is
probably a threshold of Hand2 expression below which neuronal
differentiation fails.
The conditional inactivation of Hand2 in
Wnt1-Cre-H2 mice permitted the in vivo verification of its
role in enteric neurogenesis. Virtually no neurons expressing Hu, nNOS, Dbh or
MAP2 develop in the Wnt1-Cre-H2 bowel. This failure of
neurons to develop raises the question of what happens to the crest-derived
cells that colonize the Wnt1-Cre-H2 gut? In contrast to
neurons, glia develop, but glial abundance does not increase. Hand2
deletion thus does not appear to cause common neural-glial progenitors to
generate glia instead of neurons. TUNEL- or activated caspase-detectable
apoptosis also fails to account for the progenitors that do not develop as
neurons. Development of neurons from migrating crest-derived precursors has
been proposed to occur in stages (Sommer
et al., 1995). Sympathetic neuronal precursors, for example,
become noradrenergic while still proliferating
(Rothman et al., 1978).
Mash1-dependent enteric neuronal precursors also proliferate, but contain
neurofilament proteins, GAP-43 and peripherin 1; these cells are
catecholaminergic from E10-E13 but terminally differentiate as
non-catecholaminergic neurons (Baetge and
Gershon, 1989; Baetge et al.,
1990; Blaugrund et al.,
1996). We thus looked at early markers in the E12
Wnt1-Cre-H2 gut to determine whether neuronal precursors
might be present but unable to complete development. The
Wnt1-Cre-H2 bowel contained GAP-43+, ß3-tubulin+ and
PGP9.5+ cells, albeit at a lower density than that found in wild-type gut.
These markers are expressed by still-proliferating neuronal precursor cells
(Baetge et al., 1990;
Sidebotham et al., 2001;
Sommer et al., 1995;
Young et al., 2003).
Hand2 expression is thus not essential for enteric crest-derived
cells to enter the neuronal lineage; however, it is required to enable them to
complete neuronal differentiation.
The effects on sympathetic and enteric neuronal development of the
conditional knockout of Hand2 appear to differ. Whereas Hu
immunoreactivity was not expressed in the primordial ENS of
Wnt1-Cre-H2 mice, it was expressed in developing sympathetic
neurons of the same animals; nevertheless, Th and Dbh in sympathetic ganglia
were virtually absent. Similar observations have recently been made on the
role of Hand2 in developing sympathetic neurons of zebrafish
(Lucas et al., 2006). In
hands off (also known as hand2 - Zebrafish Information
Network) mutant embryos, which lack hand2, crest-derived cells
migrate to presumptive ganglia and express the generic neuronal marker Elavl3
(HuC). They fail, however, to express Th and Dbh and later genes, such as
gata2 and tfap2a. A common sympathoadrenal-enteric
progenitor has been proposed (Blaugrund et
al., 1996; Carnahan et al.,
1991); it is thus interesting that Hand2 deletion
interferes with terminal differentiation in both lineages. The
Hand2-independent expression of Hu orthologs in mouse and zebrafish
sympathetic neurons suggests that Hand2 may function at different
stages in enteric and sympathetic differentiation.
On the basis of a study of the effects of the Wnt1-Cre-mediated conditional
knockout of Hand2 in mice, Hendershot et al. have recently postulated
that Hand2 expression is sufficient and required specifically for the
generation of Th+ and Vip+ neurons, the choice of these cell-type-specific
markers, and the migration of precursors to pattern the ENS
(Hendershot et al., 2007). By
contrast, our data indicate that Hand2 is required for precursors that have
entered the neuronal lineage to become neurons. Failure of development into
neurons implies that events downstream of cells becoming neurons, including
the acquisition of subtype-specific neurotransmitters or markers, will be
affected and not limited to specific enteric neuronal subsets. We also found
that Hand2 is not necessary for the migration of neuronal and glia precursors
to and within the gut, although the pattern of ganglia in which neurons cannot
terminally differentiate might appear abnormal. The animals studied by
Hendershot et al. survived to birth, whereas all of the
Wnt1-Cre-H2 animals that we analyzed died by E12.5. We
anticipated fetal lethality in Wnt1-Cre-H2 mice because of
the excision of Hand2 in crest-derived cells of the cardiac outflow
tract and sympathetic nervous system. Norepinephrine (NE) is essential for
fetal survival (Kobayashi et al.,
1995; Thomas et al.,
1995; Zhou et al.,
1995) and Hand2 expression is necessary for the
acquisition of the noradrenergic sympathetic phenotype
(Howard et al., 1999;
Howard, 2005;
Lucas et al., 2006;
Xu et al., 2003). Indeed, we
found that Th and Dbh were almost absent in sympathetic ganglia of
Wnt1-Cre-H2 mice. By contrast, Th expression was
observed in the conditional knockout animals studied by Hendershot et al.,
which might account for the ability of those mice to survive to gestation
(Hendershot et al., 2007). At
E14, the age at which Hendershot et al. observed enteric Th, the wild-type gut
is known to contain no Th+ cells (Baetge
and Gershon, 1989; Blaugrund et
al., 1996; Teitelman et al.,
1981). Such cells do not appear until dopaminergic neurons develop
from non-catecholaminergic Mash1-independent progenitors at the end of
gestation (Li et al., 2004).
The gut, however, receives a Th+ sympathetic innervation, which is extensive
in the stomach at E14, and is made up of axons with large varicosities that
can be misidentified as nerve cell bodies. Differences between our
observations and those of Hendershot et al. might be explained by differences
in the design or configuration of loxP-Hand2 alleles. Hendershot et
al. studied Hand2loxP/loxP;Wnt1-Cre mice, whereas the
Wnt1-Cre-H2 mice that we investigated were
Hand2loxP/null;Wnt1-Cre. When two floxed alleles, instead
of one, are used for Cre-mediated recombination, a mosaic often results
(Kwan, 2002). The presence of
two floxed alleles might also delay the deletion of Hand2 beyond the
time when Hand2 is required for early enteric and sympathetic
neurogenesis.
The hypothesis that Hand2 is required for terminal differentiation
of enteric neurons, but not glia, is strongly supported by the concordance we
report of in vitro and in vivo observations; nevertheless, because enteric
neurons of different phenotypes are born at different times of development
(E8.5 to P21) (Pham et al.,
1991), it remains formally possible that only early-born subsets
of enteric neurons are Hand2-dependent. The observation that late-appearing
neurons develop in explants of bowel from Hand2+/+, but
not Hand2-/-, mice when the explants were maintained for
up to 10 days is consistent with the idea that the terminal differentiation of
all enteric neurons requires Hand2. Still, it will be necessary in
the future to investigate the differentiation of enteric neurons in mice that
lack Hand2 expression in crest-derived cells but survive past the age
when late-born neurons are generated.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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