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First published online 19 December 2007
doi: 10.1242/dev.011783
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1 Max-Delbrück-Centrum for Molecular Medicine, Robert-Rössle-Strasse
10, 13125 Berlin, Germany.
2 UMR 8542 CNRS, Ecole Normale Supérieure, 46, rue d'Ulm, 75230 Paris
cedex 05, France.
Author for correspondence (e-mail:
cbirch{at}mdc-berlin.de)
Accepted 30 October 2007
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Sympatho-adrenal lineage, Chromaffin cells, Sympathetic neurons, Zn-finger transcription factor, Endocrine differentiation, Mash1 (Ascl1)
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Goridis and Rohrer, 2002;
Huber, 2006;
Unsicker et al., 2005).
Sympatho-adrenal precursor cells, sympathetic neurons and chromaffin cells
express a common set of genes essential for differentiation and catecholamine
biosynthesis (Howard, 2005).
The differentiation of these cells requires exogenous signals as well as an
endogenous network of transcription factors.
Migration of neural crest cells to the dorsal aorta depends on neuregulin 1
and the Erbb2/Erbb3 receptors (Britsch et
al., 1998). Upon arrival in the mesenchyme lateral of the dorsal
aorta, BMP signals induce the sympatho-adrenal differentiation of neural crest
cells, which results in the formation of the primary sympathetic ganglion
chain (Reissmann et al., 1996;
Schneider et al., 1999;
Shah et al., 1996). Initiation
of sympatho-adrenal differentiation can be assessed by the expression of
Phox2b and Mash1 (Ascl1 - Mouse Genome
Informatics), which encode homeobox and basic helix-loop-helix (bHLH)
transcription factors, respectively. During further maturation of
sympatho-adrenal precursor cells, Phox2a, the bHLH factor Hand2, the Zn-finger
factors Gata2/3 and pan-neuronal proteins appear in sympatho-adrenal
precursors. Finally, enzymes that are characteristic for noradrenergic neurons
and that are required for catecholamine synthesis, such as tyrosine
hydroxylase (Th) and dopamine-β-hydroxylase (Dbh), are produced
(Goridis and Rohrer, 2002). In
Phox2b mutant mice, Mash1 expression is correctly initiated, but none
of the other genes expressed in differentiating sympatho-adrenal cells appears
(Pattyn et al., 1999). In
Mash1 mutant mice, Phox2b expression is initiated correctly,
but pan-neuronal and neuronal subtype-specific genes are expressed delayed and
the sympathetic ganglia remain small
(Guillemot et al., 1993;
Pattyn et al., 2006). In
Gata3 mutant mice, the sympatho-adrenal differentiation is correctly
initiated, as assessed by the expression of Phox2a/b, Mash1 and pan-neuronal
genes. However, Gata2 and Th expression is severely downregulated, the size of
sympathetic ganglia is reduced, and Phox2b expression is not correctly
maintained (Lim et al., 2000;
Moriguchi et al., 2006;
Tsarovina et al., 2004).
Mutations of Phox2b, Mash1 and Gata3 also interfere with the
terminal differentiation of chromaffin cells in the adrenal gland
(Huber et al., 2002;
Huber et al., 2005;
Moriguchi et al., 2006).
Various lines of evidence indicate that a simple hierarchical model cannot
account for all aspects of the functions of Mash1, Phox2a/b, Hand2 and
Gata2/3. Instead, these factors crossregulate each other, forming a
transcriptional network that coordinately regulates differentiation of the
sympatho-adrenal lineage (Goridis and
Rohrer, 2002).
The insulinoma-associated 1 (Insm1, IA-1) gene encodes a
DNA-binding protein with five zinc-finger domains that is conserved in
evolution (Goto et al., 1992).
Insm1 is expressed in the developing central and peripheral nervous
system, in a large number of endocrine tumors, and in endocrine cells of the
developing pancreas and intestine (Gierl et
al., 2006; Goto et al.,
1992; Mellitzer et al.,
2006). Insm1 is required for differentiation of endocrine
cells in the pancreas and intestine, and its mutation affects the execution of
a gene expression program that comprises hormones and secretory proteins
(Gierl et al., 2006). Available
evidence indicates that expression of Insm1 can be controlled by
transcription factors of the bHLH family such as Mash1, Ngn1 or Ngn3
(Breslin et al., 2003;
Castro et al., 2006;
Mellitzer et al., 2006). We
investigate here the function of Insm1 in the peripheral nervous
system, and demonstrate that Insm1 is a crucial component of the
transcriptional network that coordinates the differentiation of
sympatho-adrenal cells. Our data indicate that Insm1 genetically acts
downstream of Mash1 and Phox2b, and that in addition
Insm1 represses Mash1. Furthermore, we show that the fetal
lethality of Insm1 mutant mice is caused by insufficient
catecholamine synthesis, highlighting the importance of Insm1 in
development of the sympatho-adrenal lineage.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Pattyn et al., 1999;
Wildner et al., 2006). We
observed a pronounced fetal lethality of
Insm1lacZ/Insm1lacZ mice on a 129/Ola/C57/BL6
genetic background. In order to rescue this fetal lethality pharmacologically,
pregnant Insm1lacZ/+ dams that had been mated to
Insm1lacZ/+ males received L-DOPA (1 mg/ml; 0.25% ascorbic
acid) in their drinking water (Thomas et
al., 1995). Rescued animals died shortly after birth. The
Insm1lacZ allele was also crossed for two to three
generations onto the CD1 strain, and the fetal lethality was less pronounced
on this outbred genetic background (Gierl
et al., 2006). However, changes in development of the
sympatho-adrenal lineage were comparable on both genetic backgrounds.
Noradrenaline levels were analyzed essentially as described
(Britsch et al., 1998;
Thomas et al., 1995). In
short, Insm1lacZ/+ and
Insm1lacZ/Insm1lacZ mice at E12.5 were
homogenized in 0.1 M perchloric acid, and the protein concentrations in the
homogenates were determined (Bio-Rad, Hercules, CA, USA). Catecholamines were
purified over alumina columns, and noradrenaline levels were determined by
HPLC chromatography (Haema Institute for Laboratory Medicine, Berlin,
Germany).
In situ hybridization, X-gal staining, BrdU labeling and immunohistochemistry
For in situ hybridization, embryonic or adult tissues were embedded either
in OCT compound or paraffin and sectioned. Hybridization was performed with
DIG-labeled riboprobes. Fragments amplified from cDNA were used to generate
the RNA probes for Insm1, Chromogranin A and B; other probes were generated
from plasmids obtained from various laboratories. Detection of
β-galactosidase activity by X-gal staining was performed as described
previously (Lobe et al.,
1999).
Immunohistochemistry was performed on 12 µm cryosections of mouse
embryos fixed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer,
pH7.4. The following primary antibodies were used: goat
anti-β-galactosidase (1:1000; AbD Serotec, Oxford, UK), rabbit
anti-β-galactosidase (1:1000; ICN Biochemical, Eschwege, Germany), mouse
anti-Mash1 (1:500 BD Biosciences, San Jose, CA, USA), rabbit anti-Th (1:200;
Pel-Freez, Rogers, AR, USA), rabbit anti-Phox2a, rabbit anti-Phox2b (both
1:1000; Christo Goridis and Jean-Francois Brunet, Ecole Normale Superieure,
Paris, France); rabbit anti-Npy (1:8000; Sigma, St Louis, MO, USA); rabbit
anti-p75 (1:200; Promega, Madison, WI, USA), rabbit and guinea-pig anti-Tlx3
(Muller et al., 2005); mouse
anti-Tuj1 (1:1000, Covance, Berkeley, CA, USA); rabbit anti-Pnmt (1:1000;
ImmunoStar, Hudson, WI, USA) and secondary antibodies conjugated with Cy2,
Cy3, or Cy5 (1:500; Jackson ImmunoResearch, West Grove, PA, USA). Cell death
was determined by TUNEL staining using an Apop-Tag fluorescein in situ
apoptosis detection kit (Millipore, Billerica, MA, USA).
For BrdU labeling, BrdU (75 µg/g body weight; Sigma) was injected intraperitoneally, and embryos were isolated at the indicated times. Sections were treated with primary antibodies that specifically detect various cell types, and subsequently labeled with anti-BrdU antibodies. Incorporated BrdU was detected with either mouse (1:200; Sigma) or rat anti-BrdU antibodies (1:200; AbD Serotec, Oxford, UK). Fluorescence was imaged on a Zeiss LSM 5 Pascal confocal microscope and images were processed using Adobe Photoshop software.
Cell counts
Cells in the anlage of the sympathetic nervous system were stained with
antibodies against β-galactosidase, BrdU, Phox2a, Phox2b, Th, Tuj1 or
Mash1, and the numbers of double-positive cells were counted on at least three
sections obtained from three or four heterozygous and homozygous
Insm1lacZ mice. TUNEL+ cells were counted in the adrenal
medulla on sections obtained from three heterozygous and three homozygous
Insm1lacZ mice at E18.5; at least three sections per
animal were counted. The numbers of β-galactosidase+ cells in the adrenal
gland were determined by counting β-galactosidase+ cells on cryosections
of adrenal glands that were obtained from three heterozygous and three
homozygous Insm1lacZ mice at E14.5, E16.5 and E18.5. The
entire adrenal gland was sectioned, and every second (E14.5) or forth section
(E16.5 and E18.5) was counted. To assess the statistical significance, a
Student's t-test for a two-tailed distribution and a two-sample
unequal variance was applied.
Microarray analysis
Adrenal glands were dissected from E18.5 wild-type and
Insm1lacZ/Insm1lacZ embryos and homogenized in
Trizol (Invitrogen, Carlsbad, CA, USA). RNA extraction, probe synthesis and
hybridization to Affymetrix MOE430 2.0 microarrays (Affymetrix, Santa Clara,
CA, USA) were performed according to the manufacturer's protocol. Data
processing and identification of differentially expressed genes was carried
out as described (Gierl et al.,
2006). Genes were considered differentially expressed if the
difference of their expression level had a P-value of 0.05.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Noradrenaline is the major catecholamine produced by
developing sympatho-adrenal cells and is required for fetal survival
(Britsch et al., 1998;
Lim et al., 2000;
Pattyn et al., 2000;
Thomas et al., 1995;
Zhou et al., 1995). We found
that noradrenaline concentrations in total extracts of
Insm1lacZ/Insm1lacZ embryos were significantly
lower than in control mice (2.5±1.0 and 0.5±0.2 ng
noradrenaline/mg protein in Insm1lacZ/+ and
Insm1lacZ/Insm1lacZ mice at E12.5,
respectively, n=5). The precursor of noradrenaline synthesis is
tyrosine, which is converted into L-DOPA by Th. Decarboxylation of L-DOPA by
aromatic amino acid decarboxylase produces dopamine, which is converted by Dbh
into noradrenaline. We observed that Th and Dbh transcripts
were markedly downregulated in the primary sympathetic ganglion chain of
Insm1lacZ/Insm1lacZ mice, indicating that
diminished Th and Dbh synthesis were responsible for the low noradrenaline
levels (see also below). Alterations in sympathetic activity in Phox2b,
Gata3, Th or Dbh mutant mice have been directly linked to fetal
lethality apparently caused by heart failure. Fetal lethality can be rescued
by feeding catecholamine intermediates or noradrenaline receptor agonists to
pregnant dams (Lim et al.,
2000; Pattyn et al.,
2000; Thomas et al.,
1995; Zhou et al.,
1995). We fed the catecholamine intermediate L-DOPA to pregnant
Insm1lacZ/+ females that had been crossed to
Insm1lacZ/+ males. Analysis of their offspring
demonstrated that homozygous Insm1 mutant mice could be recovered at
normal Mendelian ratios at birth, i.e. we observed 24.6% of the offspring to
display an Insm1lacZ/Insm1lacZ genotype at P0.
We conclude that a catecholamine deficiency causes the intrauterine death of
Insm1 mutant mice. Control of rescued mutant mice were comparable in
overall size and appearance at birth, but the Insm1lacZ
mutant mice were unable to breathe and died postnatally.
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; Gierl et al.,
2006). Here, we used mice carrying one copy of an
Insm1lacZ mutant allele to characterize Insm1
expression, and analyzed the appearance of the lacZ gene product,
β-galactosidase, in the sympatho-adrenal lineage. We did not detect
expression of β-galactosidase in migrating neural crest cells that were
visualized by anti-p75 antibodies (Fig.
1A). The first β-galactosidase+ cells in the peripheral
nervous system appeared around E9.5 and were detected in the anlage of the
primary sympathetic ganglion chain lateral of the dorsal aorta on interlimb
levels, and in differentiating neurons of the condensing dorsal root ganglia
(Fig. 1A; see also Fig. S1 in
the supplementary material). At E10.5, the entire primary sympathetic ganglion
chain expressed β-galactosidase, as assessed by X-gal staining
(Fig. 1B). Immunohistological
analysis showed that β-galactosidase+ cells co-expressed Phox2b,
indicating that sympatho-adrenal precursor cells express Insm1
(Fig. 1C). In addition, some
but not all β-galactosidase+ cells co-expressed Mash1
(Fig. 1D); the transient nature
of the expression of Mash1 might account for this. Insm1 expression
in sympathetic ganglia was observable at E18.5 (data not shown).
β-Galactosidase expression or Insm1 transcripts were also
detected in chromaffin cells of the adrenal gland
(Fig. 1E and data not shown).
Expression in the adrenal gland was detected at E13.5, and persisted in the
adult adrenal gland.
Insm1 is required for the differentiation of sympatho-adrenal precursor cells
We next investigated the formation and differentiation of the primary
sympathetic ganglion chain of Insm1 mutant mice. Neural crest cells
that initiate sympatho-adrenal differentiation were identified by in situ
hybridization using Phox2b as a probe, and we observed Phox2b+
sympatho-adrenal precursors lateral of the dorsal aorta in heterozygous and
homozygous Insm1lacZ mice
(Fig. 2). At E10.5, the size of
the primary ganglion chain was comparable when analyzed by Phox2b in situ
hybridization, by immunohistochemistry using anti-β-galactosidase
antibodies or by X-gal staining (Fig.
2A-F and data not shown). We observed comparable proportions of
β-galactosidase+ cells that expressed Phox2b in heterozygous and
homozygous Insm1lacZ mice at E10.5 (81.2±4.7% and
79.9±5.0% in Insm1lacZ/+ and
Insm1lacZ/Insm1lacZ mice, respectively,
n=3; see also Fig.
3A,B), indicating that the differentiation of sympatho-adrenal
progenitor cells is correctly initiated in Insm1 mutant mice.
However, at subsequent developmental stages we noted a pronounced reduction in
the size of the primary sympathetic ganglion chain in
Insm1lacZ/Insm1lacZ mice, regardless whether
Phox2b in situ hybridization, β-galactosidase
immunohistochemistry or X-gal staining were used
(Fig. 2G-N). Sympatho-adrenal
precursors possess proliferative capacities
(Rohrer and Thoenen, 1987). We
compared the proliferation in control and Insm1 mutant mice using
BrdU injections, and determined the proportions of β-galactosidase+
sympatho-adrenal precursor cells that incorporated BrdU. In control mice, we
observed considerable proliferation rates of sympatho-adrenal precursor cells
at E11.5 and E12.5, which were reduced in
Insm1lacZ/Insm1lacZ mice
(Fig. 2O). At E14.5,
proliferation rates were comparable in control and Insm1 mutant mice
(Fig. 2D). Apoptosis in cells
lateral of the dorsal aorta, as assessed by TUNEL staining, was not augmented
at E10.5 and E12.5 (data not shown). We therefore conclude that a reduced
proliferation of sympatho-adrenal precursor cells accounts for the reduced
size of the primary sympathetic ganglion chain in homozygous Insm1
mutant mice.
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Sympatho-adrenal precursor cells migrate from the primary ganglion chain to generate secondary sympathetic ganglia. The reduction in the overall size of the primary sympathetic ganglion chain in Insm1 mutant mice at E12.5 was accompanied by a reduced size of secondary sympathetic ganglia at E14.5 and E18.5. Thus, for instance the superior cervical and stellate ganglia were smaller in Insm1lacZ/Insm1lacZ than in Insm1lacZ/+ mice when assessed by whole-mount staining using X-gal (Fig. 5A,B). Similarly, immunohistochemical analyses of control and mutant stellate ganglia demonstrated a reduction in size when antibodies directed against Tuj1, Th or Phox2a were used (Fig. 5C-H). However, similar proportions of neurons expressed NPY, Tlx3 and Ret in the secondary sympathetic ganglia of control and mutant mice at E18.5 (Fig. 5C-H and data not shown), indicating that maturation of sympathetic neurons had occurred.
Differentiation of chromaffin cells in Insm1 mutant mice
Sympatho-adrenal precursor cells move to the anlage of the adrenal gland
around E12.5, and by E14.5 their derivatives, the chromaffin cells, can be
detected in great numbers in the adrenal medulla
(Britsch et al., 1998;
Huber et al., 2002).
Chromaffin cells that express β-galactosidase were present in comparable
numbers in the adrenal medulla of heterozygous and homozygous
Insm1lacZ mutant mice at E14.5, but these cells appeared
more dispersed in heterozygous mice (Fig.
6A,B; for a quantification see Q). At E18.5, the numbers of
β-galactosidase+ cells were reduced by 61% in homozygous Insm1
mutants (Fig. 6Q). Analysis of
cell proliferation using BrdU injection demonstrated comparable proliferation
rates of β-galactosidase+ cells in the medulla at E14.5 and E16.5
(24.1±8.3% and 20.7±6.3% at E14.5 in
Insm1lacZ/+ and
Insm1lacZ/Insm1lacZ mice, respectively,
n=3; 20.6±3.0% and 19.8±4.1% at E16.5 in
Insm1lacZ/+ and
Insm1lacZ/Insm1lacZ mice, respectively,
n=3). However, TUNEL staining indicated a marked increase in
apoptosis at E18.5 (0.2±0.1 and 2.0±0.8 TUNEL+
cells/mm2 of the adrenal medulla in Insm1lacZ/+
and Insm1lacZ/Insm1lacZ mice, respectively,
n=3). Thus, enhanced cell death accounts for the reduction in
chromaffin cell numbers.
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). We
analyzed Insm1 expression in Phox2b mutant mice at E10.5 and
E12.5, and detected no Insm1 expression lateral of the dorsal aorta
(Fig. 7A-D and data not shown).
This demonstrates that Insm1 expression is indeed restricted to the
neuronal population in the developing sympathetic nervous system, and
indicates that Phox2b is required to initiate Insm1
expression. In the primary sympathetic ganglion chain of Mash1 mutant
mice, Phox2b expression is correctly initiated, but other markers
appear delayed (Pattyn et al.,
2006). We observed no Insm1 expression in Mash1
mutant mice at E10.5 in sympatho-adrenal precursors, and at E12.5 expression
was downregulated in a pronounced manner
(Fig. 7E,F). Thus,
Mash1 is also required for correct Insm1 expression during
development of sympatho-adrenal precursors (see
Fig. 7G for a summary). |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Our
analyses indicate that Insm1 is dispensable for generic neurogenesis and
neuronal specification in spinal cord (H.W., M.S.G. and Thomas Müller,
unpublished). By contrast, neural crest-derived sympatho-adrenal precursors
express Insm1 for prolonged periods, and require Insm1 for
their differentiation. In particular, differentiation of sympatho-adrenal
precursors was delayed in Insm1 mutant mice, and we found that many
subtype-specific genes were expressed behind schedule. Sympatho-adrenal
precursors subsequently escaped the differentiation block in Insm1
mutant mice, and generated sympathetic neurons that matured correctly.
However, secondary sympathetic ganglia in Insm1 mutant mice remained
small, owing to a decreased proliferation of the precursors. Sympatho-adrenal
precursors also generate chromaffin cells in the adrenal medulla. We observed
a marked change in terminal differentiation of chromaffin cells, reduced
expression of genes whose protein products control catecholamine synthesis and
secretion, and low catecholamine levels in Insm1 mutant mice.
Catecholamines are essential for fetal survival and for heart function during
development (Lim et al., 2000;
Pattyn et al., 2000;
Thomas et al., 1995;
Zhou et al., 1995). We were
able to rescue the fetal lethality of Insm1 mutant embryos by the
administration of catecholamine intermediates. Thus, deficits in catecholamine
synthesis are responsible for the reduced viability of Insm1 mutant
mice during gestation.
|
). Changes in the differentiation of
sympatho-adrenal precursors cells were reported for Phox2b, Mash1 and
Gata3 mutant mice (Guillemot et
al., 1993; Lim et al.,
2000; Moriguchi et al.,
2006; Pattyn et al.,
2006; Pattyn et al.,
1999; Tsarovina et al.,
2004). We show here that several factors of the transcriptional
network (Phox2a, Gata3, Mash1) and subtype-specific genes (Th, Dbh)
are not correctly expressed during the differentiation of sympatho-adrenal
precursor cells in Insm1 mutant mice. Conversely, Insm1 expression is
not correctly initiated in sympatho-adrenal precursors of Phox2b and
Mash1 mutant mice. Insm1 was recently predicted to be a
direct Mash1 target (Castro et al.,
2006). Our data are in accordance with a function of Mash1 in the
control of Insm1 expression, and demonstrate that Insm1 acts downstream of
Phox2b and Mash1 during the development of sympatho-adrenal precursor
cells.
Similarities in the phenotypes of Mash1 and Insm1 mutant
mice are apparent. For example, in both mutant strains sympatho-adrenal
differentiation is correctly initiated, as assessed by Phox2b expression.
Phox2a, Gata3, Hand2 and Th transcripts appear behind
schedule in all sympatho-adrenal precursor cells of Mash1 mutant mice
(Guillemot et al., 1993;
Hirsch et al., 1998;
Pattyn et al., 2006). These
genes are also expressed delayed in Insm1 mutant mice, but this delay
is apparent in the majority, and not all sympatho-adrenal precursors. Finally,
in Mash1 mutant mice a block in the differentiation of enteric
neurons is present in the esophagus but not in other parts of the
gastrointestinal tract (Guillemot et al.,
1993; Hirsch et al.,
1998; Pattyn et al.,
1999). Similarly, differentiation of esophageal neurons is
severely impaired in Insm1 mutant mice, as assessed by the absence
Phox2a expression in the esophagus at E10.5 and E18.5; Phox2a is, however,
expressed in enteric neurons located in more posterior parts of the enteric
nervous system (H.W., M.S.G. and C.B., unpublished). The similarities in
phenotypes observed in Mash1 and Insm1 mutant mice suggest
that Insm1 mediates aspects of Mash1 functions in the differentiation of
catecholaminergic neurons. Mash1 controls generic and subtype-specific aspects
of neuronal differentiation of catecholaminergic neurons. Expression of
pan-neuronal markers is only mildly affected in Insm1 mutant mice, indicating
that Insm1 exerts its role primarily in the control of subtype-specific
neuronal differentiation.
In addition, our experiments revealed an upregulated expression of Mash1
and its direct target gene delta-like 1 (Dll1) in sympatho-adrenal
precursor cells of Insm1 mutant mice. During normal differentiation,
Mash1 is expressed transiently in sympatho-adrenal precursor cells. The
de-repression of Mash1 might interfere with differentiation of
sympatho-adrenal precursors of Insm1 mutant mice. It should be noted
that we did not observe upregulated expression of other Notch target genes
such as Hes1, Hes5, Hes1r and Nrarp, indicating that
upregulated Notch signaling is not responsible for the delayed
differentiation. It has previously been noted that the Mash1 promoter
is de-repressed in Mash1 mutant mice, but Mash1 does not directly
mediate this negative regulation (Meredith
and Johnson, 2000). De-repression of Mash1 was also
observed in chromaffin cells of Gata3 mutant mice
(Moriguchi et al., 2006),
indicating that the Zn-finger factors Gata3 and Insm1 participate in the
regulatory feedback loop that controls Mash1.
Mash1 and Phox2b are the first transcription factors that appear upon
initiation of differentiation of sympatho-adrenal precursors
(Tsarovina et al., 2004).
Hand2, Phox2a and Gata3 act genetically downstream of Mash1 and Phox2b, but
mis-expression of Hand2 and Phox2a induces Mash1 and Phox2b, and Gata3 is
required to maintain correct Phox2b expression
(Howard et al., 1999;
Howard et al., 2000;
Lucas et al., 2006;
Moriguchi et al., 2006;
Stanke et al., 1999;
Stanke et al., 2004;
Tsarovina et al., 2004). These
transcription factors seem thus to collaborate during specification of the
sympatho-adrenal lineage, and despite their sequential appearance during
development, they form a regulatory network rather than a linear cascade. We
report here changes in the expression of several of these transcription
factors in Insm1 mutant mice, which demonstrates that Insm1 is an
essential component of the transcriptional network that controls
differentiation of sympatho-adrenal precursor cells.
Timing of differentiation and execution of the differentiation program
Sympatho-adrenal precursors migrate in order to form secondary sympathetic
ganglia, as well as adrenal and extra-adrenal chromaffin cells
(Huber, 2006;
Unsicker et al., 2005). Mature
sympathetic neurons and chromaffin cells share characteristics, like the
expression of Th and Dbh, but they also display distinct features. Sympathetic
neurons extend axons and maintain typical neuronal markers such as
neurofilament 68 (Nefl), whereas neuronal markers are downregulated in
chromaffin cells. The presence of secretory granules and the expression of
Pnmt are typical for chromaffin cells and are further properties that
distinguish the two cell types. We report here a delayed differentiation of
sympatho-adrenal precursor cells in Insm1 mutant mice. Remaining
precursor cells of Insm1 mutant mice eventually escape the block, and
undergo sympatho-adrenal differentiation. During development of
sympatho-adrenal precursor cells, Insm1 is thus crucial for the correct timing
of differentiation.
Sympatho-adrenal precursors of Insm1 mutant mice subsequently form
sympathetic neurons, albeit at reduced numbers. In marked contrast, the
further development of chromaffin cells was significantly altered, and enzymes
of catecholamine biosynthesis (Th, Dbh, Pnmt) and components of secretory
granules (chromogranin A/B) were markedly downregulated, whereas neurofilament
(NF68) expression was increased. This was accompanied by an altered morphology
of the adrenal medulla, and by increased apoptosis of chromaffin cells. Insm1
is therefore required for the correct execution of the differentiation program
of chromaffin cells. Upregulated expression of Mash1 and neurofilament 68
indicate that chromaffin cells retain the character of immature
sympatho-adrenal precursors in Insm1 mutant mice. This represents a
further similarity in the phenotypes of Mash1 and Insm1
mutant mice [compare Huber et al. (Huber
et al., 2002) with this study]. It has previously been proposed
that two distinct types of sympatho-adrenal precursors exist in the primary
ganglion chain, one population destined to form sympathetic neurons and a
second destined to form chromaffin cells
(Huber et al., 2002). Such a
hypothesis is supported by the fact that the number of sympatho-adrenal cells
in the primary ganglion chain is markedly reduced in Insm1 mutant
mice, which affects the numbers of sympathetic neurons but not the numbers of
chromaffin precursors that arrive in the adrenal gland.
Insm1 is also essential for terminal differentiation of endocrine cells of
the pancreas and intestine (Gierl et al.,
2006; Mellitzer et al.,
2006). Genetic evidence indicates that Insm1 expression depends on
two bHLH transcription factors Ngn3 and Mash1 in the pancreas and
sympatho-adrenal cells, respectively, indicating that similar molecular
mechanisms function upstream of Insm1 in these distinct organs. Chromaffin
cells and endocrine cells of the pancreas and intestine produce different
hormones, but they share endocrine characteristics such as the expression of
granins. Chromogranin A/B are two genes whose expression is markedly
downregulated in chromaffin cells, as well as in endocrine cells of the
pancreas and intestine in Insm1 mutant mice. The identification of
direct target genes will reveal how Insm1 participates in the execution of a
gene expression program that controls endocrine features. During adrenergic
differentiation of neurons, Insm1 appears to mediate aspects of Mash1
functions in subtype-specific differentiation. Alternatively, Insm1 and Mash1
might co-operate to control expression of subtype-specific genes in developing
peripheral neurons.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/3/473/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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